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  • Articles  (180,153)
  • Elsevier  (173,905)
  • Blackwell Science Ltd  (6,248)
  • American Institute of Physics (AIP)
  • 2005-2009  (96,348)
  • 2000-2004  (83,805)
  • Biology  (171,978)
  • Process Engineering, Biotechnology, Nutrition Technology  (11,824)
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  • Articles  (180,153)
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  • 1
    Electronic Resource
    Electronic Resource
    Lack of association between pro-inflammatory cytokine (IL-6, IL-8 and TNF-α) gene polymorphisms and Graves’ disease (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Graves’ disease (GD) is a common, autoimmune disease involving the thyroid gland, and it has been previously suggested that pro-inflammatory cytokines are involved in the disease's pathogenesis. The aim of this study was to test whether the interleukin (IL)-6 gene promoter region, or tumour necrosis factor (TNF)-α or IL-8 gene 3′-untranslated region (3′-UTR) polymorphisms could provide useful genetic markers for an individual's susceptibility to GD. A normal control group of 60 healthy people and 95 patients featuring GD were examined. Polymerase chain reaction (PCR)-based restriction analysis was performed for the three gene polymorphisms using endonucleases BsrBI, NcoI and ApaLI, respectively. We found no significant difference between the frequencies of genotype and allelic variants for the IL-6 gene promoter (−572 G/C), the TNF-α gene promoter (−308 A/G) and the IL-8 gene 3′-UTR (2767 A/G) for GD patients and for normal controls. Cytokines are a large group of proteins that may elicit multiple effects upon immunological reactions. It still appears to be very worthwhile to continue to aggressively search for cytokine gene polymorphisms in order to predict the development of such disease.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00536.x
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  • 2
    Electronic Resource
    Electronic Resource
    Alternative splicing of IL-24 in melanocytes by deletion of exons 3 and 5 (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Two novel interleukin-24 (IL-24) splice variants were identified in normal human melanocytes by sequencing cloned polymerase chain reaction (PCR) products that are not expressed in metastatic melanoma. These gene products have been generated by differential skipping of exons 3 (IL-24 delE3) and 5 (IL-24 delE5). IL-24 delE3 has limited sequence identity to the IL-24-interacting protein mda-7s, and IL-24 delE5 is homologous to IL-24.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00540.x
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  • 3
    Electronic Resource
    Electronic Resource
    Nomenclature for factors of the HLA system, updated July 2005 (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00544.x
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  • 4
    Electronic Resource
    Electronic Resource
    HLA and eye disease: a synopsis (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Human leukocyte antigen (HLA) gene products have been implicated in the pathogenesis of an increasing number of eye diseases, mainly inflammatory in nature. This perspective reviews the current hypotheses for why HLA polymorphisms are associated with specific eye diseases. Statistical problems in studies involving HLA associations are discussed, and possible solutions outlined. The relevance of HLA testing in routine ophthalmic practice, its practical and cost implications is also assessed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00548.x
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  • 5
    Electronic Resource
    Electronic Resource
    A NFKB1 promoter polymorphism is involved in susceptibility to ulcerative colitis (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Nuclear factor κB (NF-κB) designates a group of critical transcription factors involved in a variety of immunologic and/or inflammatory processes. Conceivably, genes involved in the NF-κB pathway make interesting candidate genes for chronic inflammatory disorders, including the inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC). In two mouse models of colitis, strong linkage has been observed with a locus on chromosome 3 that harbours the Nfkb1 gene. In addition, a polymorphism in the promoter region of the human NFKB1 gene was found to be associated with susceptibility to UC. In this study, we searched to confirm this previously found association in IBD in a different population. Allele and genotype frequencies of the −94 ins/delATTG polymorphism were determined in 266 unrelated Dutch Caucasian IBD patients (127 UC, 139 CD), and 155 matched healthy controls. The allele frequency of the deletion was significantly higher in UC patients (P = 0.019), but not in CD patients, compared to healthy controls, and the UC patients homozygous for the −94 ATTG deletion had a younger age of onset. Our findings confirm the previously found association between this polymorphism and susceptibility to UC in an independent study population and adds further evidence for the role of this gene in disease susceptibility.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00546.x
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  • 6
    Electronic Resource
    Electronic Resource
    Nomenclature for factors of the HLA system, update August 2005 (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00555.x
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  • 7
    Electronic Resource
    Electronic Resource
    Variability in TRBV haplotype frequency and composition in Caucasian, African American, Western African and Chinese populations (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The polymorphic T-cell receptor Vβ (TRBV) genes encode much of the variable region of the T-cell receptor β chain. Analysis of allele frequencies of three closely linked polymorphic TRBV genes, TRBV7-3, TRBV9 and TRBV6-4, was undertaken in several populations. The frequencies of these alleles are not significantly different in populations of Caucasians, African Americans and Western Africans. However, Chinese population is extremely homogenous at all three loci. The current study identifies the existence of haplotypic relationships between alleles of these genes in the Caucasian population. The ORF allele TRBV7-3*A3 is found exclusively on chromosomes bearing TRBV9*A2 and TRBV6-4*A2 in this cohort. In contrast, TRBV7-3*A1 and the null allele TRBV7-3*A2 are associated only with TRBV9*A1 and TRBV6-4*A1. This pattern of linkage disequilibrium (LD) is altered in the African American and Western African populations. In these cohorts, there is a marked reduction in LD between alleles of TRBV7-3 and TRBV9. This study is consistent with previous population genetic studies wherein African-derived samples have a greater level of genetic diversity compared to Caucasians. These data also demonstrate that patterns of LD are not consistent across the entire TRBV locus.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00550.x
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  • 8
    Electronic Resource
    Electronic Resource
    Nomenclature for factors of the HLA system, update September 2005 (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00556.x
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  • 9
    Electronic Resource
    Electronic Resource
    A note of thanks to referees Volume 32, 2005 (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00558.x
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  • 10
    Electronic Resource
    Electronic Resource
    MMP-7 promoter polymorphisms do not influence CD4+ recovery and changes in plasma viral load during antiretroviral therapy for HIV-1 infection (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Matrix metalloproteinase-7 (MMP-7) generates soluble Fas Ligand (FasL), which is involved in the apoptotic loss of CD4+ T cells during HIV infection. We evaluated whether two polymorphisms in MMP-7 promoter could influence CD4+ recover in response to antiretroviral therapy, and found that these polymorphisms are ineffective.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00523.x
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  • 11
    Electronic Resource
    Electronic Resource
    Single nucleotide polymorphisms in four functionally related immune response genes in the horse: CD14,TLR4, Cɛ, andFcɛ R1 alpha (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The objective of this study was to identify single nucleotide polymorphisms (SNPs) within four functionally related immune response genes in the horse, and to develop genotyping techniques that could be useful for future genomic studies of horse infectious and allergic diseases. The genes analysed were: the lipopolysaccharide (LPS) receptor gene CD14, the toll-like receptor 4 gene TLR4, the gene Cɛ encoding the IgE heavy chain molecule and the gene FcɛR1 alpha coding for the alpha subunit of the IgE receptor molecule. Horse-specific primers amplifying selected gene regions were designed and SNPs were searched by selective resequencing and/or by PCR-SSCP (polymerase chain reaction-sequence specific conformational polymorphism) or PCR-RFLP (PCR-restriction fragment length polymorphism). Gene expression was analysed by RT-PCR (reverse transcriptase-PCR) of all four genes examined. For CD14, the cDNA sequence was determined and a novel sequence of the 5′UTR region was identified. The protein-coding sequence was identical to that previously deposited in GenBank. 5′UTR, intronic and both synonymous and non-synonymous exonic SNPs were identified. Three SNPs were found in the CD14 gene, four in the TLR4 gene; two SNPs were identified in the Cɛ gene, and one SNP was found in the FcɛR1 alpha gene. PCR-RFLP was developed for genotyping eight of the SNPs identified. The RT-PCR assay showed that all the SNPs reported here are parts of expressed genes. The results showed that important immunity-related genes in horses are polymorphic and that even non-synonymous SNPs with potential functional impact may occur. The methods developed for genotyping and haplotyping the SNPs identified represent, along with markers described previously, a potentially useful tool for genomic analysis of the function and role of these genes in immunity and in mechanisms of disease.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00522.x
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  • 12
    Electronic Resource
    Electronic Resource
    No allelic variant associations of the IL-1 and TNF gene polymorphisms in the susceptibility to duodenal ulcer disease (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Recent studies have reported the association of a pro-inflammatory profile of genetic polymorphisms in IL-1B, IL-1RN, TNF-A, and IL-10 genes with an increased risk of non-cardia gastric cancer. Because gastric cancer and duodenal ulcer are mutually exclusive outcomes of Helicobacter pylori infection, we aimed to investigate possible allelic variant associations of several functional polymorphisms in the IL-1B, IL-1RN, TNFA, and LTA genes in the susceptibility to duodenal ulcer. Genomic DNA from 118 patients with duodenal ulcer and 97 healthy controls was typed for the IL-1B polymorphisms at positions −511, −31, and +3954, the VNTR polymorphism in intron 2 of the IL-1RN gene, the TNFA−308, TNFA −238, and the NcoI and BsI LTA polymorphisms by PCR, SSCP and TaqMan assays. H. pylori infection and non-steroidal anti-inflammatory drugs (NSAIDs) use was investigated in patients and controls. Logistic regression analysis identified H. pylori infection (OR: 12.86; 95%CI: 3.85–43), NSAID use (OR: 11.95; 95%CI: 4.19–34.05), and family history-ulcer (OR: 3.79; 95%CI: 1.68–8.54) as independent risk factors for duodenal ulcer. When the effect of the combinations of IL-1 and TNF genotypes was studied we found that the distribution of all possible combinations of these eight polymorphisms was similar in duodenal ulcer patients and controls. The simultaneous carriage of alleles IL-1RN*2/IL-1B −31T/IL-1B −511C/IL-IB +3954C/TNF-HaplotypeE negative (termed in some studies as ‘low-producing’ alleles) was increased in H. pylori-positive duodenal ulcer patients compared to H. pylori-infected healthy controls (10.5% vs. 5.9%) although the difference did not reach statistical significance (OR: 1.85; 95%CI: 0.57–5.99, P = 0.41). Moreover, no differences were found with respect to H. pylori status, NSAID use, age, gender, smoking habit, type of complication, recurrence of the ulcer, and need for surgical treatment. Our data show no association between allelic variants of IL-1 and TNF gene polymorphisms in the susceptibility to and final outcome of duodenal ulcer.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00528.x
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  • 13
    Electronic Resource
    Electronic Resource
    Nomenclature for factors of the HLA system, update June 2005 (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00535.x
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  • 14
    Electronic Resource
    Electronic Resource
    A novel HLA-DRB1*15 allelic sequence isolated from the Han population of Guangdong, China (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A novel DRB1*15 allele, DRB1*1516, has been identified in a Guangdong Han individual. Its sequence was confirmed by sequencing of polymerase chain reaction products and clones. This allele differed by one nucleotide from DRB1*150101 at position 220 (G→A), resulting in an amino acid substitution from Gly to Arg at codon 45.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00514.x
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  • 15
    Electronic Resource
    Electronic Resource
    Abstracts of Papers Presented at the Sixteenth Annual BSHI Conference, Plymouth, 7th−9th September 2005 (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00525.x
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  • 16
    Electronic Resource
    Electronic Resource
    Genetic contribution of the tumour necrosis factor (TNF) B + 252*2/2 genotype, but not the TNFa,b microsatellite alleles, to systemic lupus erythematosus in Japanese patients (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The contribution of the tumour necrosis factor (TNF) B + 252 (TNFB) dimorphism and microsatellite polymorphisms of TNFa and TNFb to the pathogenesis of systemic lupus erythematosus (SLE) was studied in Japanese patients. The TNFB dimorphism was determined using the restriction fragment length polymorphism (RFLP) method with NcoI digestion followed by specific polymerase chain reaction (PCR) amplification. TNFa and TNFb microsatellite polymorphisms were determined using the DNA sequencer and GeneScan program (Applera Corporation, Foster City, CA) followed by specific PCR amplification. HLA-DRB1*15 typing was carried out by the PCR-sequence specific conformational polymorphism (SSCP) method. In SLE, the allele frequency of TNFB*2 significantly increased (68.9%, P 〈 0.05) and the genotype frequency of TNFB*2/2 also increased (52.8%, P 〈 0.05). TNFB*2 showed no significant linkage disequilibrium with HLA-DRB1*1501. The prevalence of TNFa13 and TNFb4 showed very slight increases, but these increases were not significant. An association analysis indicated that TNFB*2/2 conferred greater, or at least equal, susceptibility to SLE in Japanese patients in comparison with HLA-DRB1*1501. The TNFB*2/2 genotype may contribute additively with DRB1*1501 to SLE in Japanese patients. No association was observed between auto-antibodies and TNF. TNFB*2 is a genetic marker for SLE in Japanese patients, while TNFa and TNFb microsatellites are not associated with SLE.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00504.x
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  • 17
    Electronic Resource
    Electronic Resource
    Analysis of a biomarker for Wegener's granulomatosis (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: This molecular epidemiology study integrated questionnaire and genotype information to examine a disease susceptibility hypothesis. The study was based on a previously reported association demonstrated between a single nucleotide polymorphism (SNP) identified as A-564G within the promoter of the proteinase-3 gene (PRTN3) and the autoimmune disease Wegener's granulomatosis (WG). To further examine the strength of this association, we employed a family-based design in which the inheritance of alternate alleles could be ascertained from the parents of affected and unaffected progeny. Genotype information for the study participants was derived from DNA samples from participants who collected buccal cells using a harvesting method that was non-invasive and self-administered. A brief questionnaire captured demographic data on the participants, the family relationships between participants, and the prevalence of autoimmune disease among family members. Samples were obtained on 132 individuals representing 43 WG cases and 89 unaffected controls. Thirty-four nuclear families containing at least one unaffected sibling or parent of a WG case were represented in this sample. We found no evidence for an association between A-564G and the likelihood of a WG diagnosis. We examined five additional SNPs and a sixth SNP haplotype within the PRTN3 promoter region in a family-based association analysis and found no evidence that mutations within PRTN3 are associated with WG diagnosis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00519.x
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  • 18
    Electronic Resource
    Electronic Resource
    Mitochondrial mutation detection using enhanced multiplex denaturing high-performance liquid chromatography (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In this study, we investigated the presence of mutations within the mitochondrial genome in 40 Caucasian subjects using an enhanced multiplex denaturing high-performance liquid chromatography (DHPLC) approach. The enhanced DHPLC approach has increased sensitivity and throughput, and reduced analysis time per individual sample compared to conventional methods. This technique involved amplifying the mitochondrial genome in 18 fragments ranging in size from 300 to 2000 bp using a novel proofreading polymerase (OptimaseTM, Transgenomic Inc., Omaha, NE) with a low misincorporation rate. Fourteen of these fragments underwent subsequent restriction digestion using a combination of five restriction enzymes to enable multiplex DHPLC analysis; the remaining four underwent conventional DHPLC. Using this complete mitochondrial genome-screening approach, we confirmed a number of previously reported mutations and additionally identified a large number of novel mutations using an enhanced DHPLC technique.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00508.x
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  • 19
    Electronic Resource
    Electronic Resource
    Promoter and intron-1 region polymorphisms in the IFNG gene in patients with hepatitis E (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Allelic and genotype variations in the promoter region and the dinucleotide (CA)n repeat region in intron 1 of the interferon-g (IFNG) gene were analysed by direct sequencing and simple sequence length polymorphism (SSLP), respectively, in patients with acute hepatitis, and the prevalence was compared with that in healthy controls. Our results showed a significant association of heterozygous genotypes (CA)12/(CA)14 and (CA)12/(CA)16 in intron 1 of the IFNG gene in all categories of patients with acute hepatitis, classified on the basis of presence or absence of hepatitis E virus (HEV), in comparison with healthy controls. A novel polymorphism, −288 A→T [from the translational start site, as per Human Genome Organization (HUGO) nomenclature], in the promoter region of the IFNG gene leading to a loss of the consensus domain for the interferon-stimulated response element (ISRE), as predicted by in silico analysis, was observed in 12.5% of patients with acute HEV infection. However, no significant difference in allele or genotype frequency was observed for the −288 promoter polymorphism, although the heterozygous −288 A/T genotype showed a moderate risk in patients with acute HEV infection alone (P = 0.29, odds ratio = 1.964, confidence interval = 0.46–8.45). The data suggest that the genotype at intron 1 of IFNG might affect susceptibility to acute hepatitis in HEV infection, which warrants further elucidation in a larger sample and also functional studies.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00512.x
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  • 20
    Electronic Resource
    Electronic Resource
    Nomenclature for factors of the HLA system, update March 2005 (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00520.x
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  • 21
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    Polymorphisms of the upstream regulatory region of the major histocompatibility complex DRB genes in domestic horses (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Sequence information was obtained on the variation of the ELA-DRB upstream regulatory region (URR) after polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) cloning and sequencing of ≈ 220 bp upstream of the first exon of horse DRB genes. The sequence of the proximal URR of equine DRB is composed of highly conserved sequence motifs, showing the presence of the W, X, Y, CAAT and TATA conserved boxes of major histocompatibility complex (MHC) class II promoters. Five different polymorphic horse DRB promoter sequences were detected in five horse breeds. The results demonstrate the existence of polymorphism in the nucleotide sequences of the ELA-DRB URR, located in the functionally important conserved consensus sequences, the X2 box, the Y box and the TATA box, while conservation were observed in X1 and CAAT boxes. The nucleotide diversity among horse URRs was intermediate between that seen within human and mouse DRB promoters, suggesting the existence of another important source of variability in ELA-DRB genes. In addition, phylogenetic comparisons, identity analysis and sequence organization suggested that the reported sequences would correspond to an expressed ELA-DRB locus. However, further information about the functional significance of these promoter polymorphisms will probably be acquired through expression studies on the different sequences.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00496.x
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  • 22
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    Nomenclature for factors of the HLA system, update November 2004 (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00500.x
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  • 23
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    Nomenclature for factors of the HLA system, 2004 (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
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    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00509.x
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  • 24
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    The HLA Dictionary 2004: a summary of HLA-A, -B, -C, -DRB1/3/4/5 and -DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR and -DQ antigens (2005)
    Oxford, UK : Blackwell Science Ltd
    International journal of immunogenetics 32 (2005), S. 0 
    Details
    ISSN: 1744-313X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: This report presents serological equivalents of HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5 and -DQB1 alleles. The dictionary is an update of that published in 2001. The data summarize equivalents obtained by the World Health Organization Nomenclature Committee for Factors of the HLA System, the International Cell Exchange (UCLA), the National Marrow Donor Program (NMDP), recent publications and individual laboratories. This latest update of the dictionary is enhanced by the inclusion of results from studies performed during the 13th International Histocompatibility Workshop and from neural network analyses. A summary of the data as recommended serological equivalents is presented as expert assigned types. The tables include remarks for alleles, which are or may be expressed as antigens with serological reaction patterns that differ from the well-established HLA specificities. The equivalents provided will be useful in guiding searches for unrelated haematopoietic stem cell donors in which patients and/or potential donors are typed by either serology or DNA-based methods. The serological DNA equivalent dictionary will also aid in typing and matching procedures for organ transplant programmes whose waiting lists of potential donors and recipients comprise mixtures of serological and DNA-based typings. The tables with HLA equivalents and a questionnaire for submission of serological reaction patterns for poorly identified allelic products will be made available through the WMDA web page () and, in the near future, also in a searchable form on the IMGT/HLA database.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1744-313X.2005.00497.x
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    Predicting mutation frequencies in stem–loop structures of derepressed genes: implications for evolution (2003)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 48 (2003), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: This work provides evidence that, during transcription, the mutability (propensity to mutate) of a base in a DNA secondary structure depends both on the stability of the structure and on the extent to which the base is unpaired. Zuker's DNA folding computer program reveals the most probable stem–loop structures (SLSs) and negative energies of folding (–ΔG) for any given nucleotide sequence. We developed an interfacing program that calculates (i) the percentage of folds in which each base is unpaired during transcription; and (ii) the mutability index (MI) for each base, expressed as an absolute value and defined as ­follows: MI = (% total folds in which the base is unpaired) × (highest –ΔG of all folds in which it is unpaired). Thus, MIs predict the relative mutation or reversion frequencies of unpaired bases in SLSs. MIs for 16 mutable bases in auxotrophs, selected during starvation in derepressed genes, are compared with 70 background mutations in lacI and ebgR that were not derepressed during mutant selection. All the results are consistent with the location of known mutable bases in SLSs. Specific conclusions are: (i) Of 16 mutable bases in transcribing genes, 87% have higher MIs than the average base of the sequence analysed, compared with 50% for the 70 background mutations. (ii) In 15 of the mutable bases of transcribing genes, the correlation between MIs and relative mutation frequencies determined experimentally is good. There is no correlation for 35 mutable bases in the lacI gene. (iii) In derepressed auxotrophs, 100% of the codons containing the mutable bases are within one codon's length of a stem, compared with 53% for the background mutable bases in lacI. (iv) The data suggest that environmental stressors may cause as well      as     select     mutations     in     derepressed     genes. The implications of these results for evolution are discussed.
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    URL: http://dx.doi.org/10.1046/j.1365-2958.2003.t01-1-03436.x
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    Cytochrome c oxidase genes required for nitrogenase activity and diazotrophic growth in Anabaena sp. PCC 7120 (2003)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 47 (2003), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: N2 fixation is an O2-sensitive process and some filamentous diazotrophic cyanobacteria that grow performing oxygenic photosynthesis confine their N2 fixation machinery to heterocysts, specialized cells that maintain a reducing environment adequate for N2 fixation. Respiration is thought to contribute to the diazotrophic metabolism of heterocysts and the genome of the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 bears three gene clusters putatively encoding cytochrome c oxidases. Transcript analysis of these cox gene clusters through RNA/DNA hybridization identified two cox operons, cox2 and cox3, that are induced after nitrogen step-down in an NtcA- and HetR-dependent manner and appear to be expressed specifically in heterocysts. In contrast, cox1 was expressed only in vegetative cells. Expression of cox2 and cox3 occurred at an intermediate stage (about 9 h) during the process of heterocyst development following nitrogen step-down. Inactivation of genes in the two inducible cox operons, but not separately in either of them, strongly reduced nitrogenase activity and prevented diazotrophic growth in aerobic conditions. These results show that the nitrogen-regulated cytochrome c oxidase-type respiratory terminal oxidases Cox2 and Cox3 are essential for heterocyst function in Anabaena sp. PCC 7120.
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    Characterization of a novel inhibitory feedback of the anti-anti-sigma SpoIIAA on Spo0A activation during development in Bacillus subtilis (2003)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 47 (2003), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Compartmentalized gene expression during sporulation is initiated after asymmetric division by cell-specific activation of the transcription factors σF and σE. Synthesis of these σ factors, and their regulatory proteins, requires the activation (phosphorylation) of Spo0A by the phosphorelay signalling system. We report here a novel regulatory function of the anti-anti-σF SpoIIAA as inhibitor of Spo0A activation. This effect did not require σF activity, and it was abolished by expression of the phosphorelay-independent form Spo0A-Sad67 indicating that SpoIIAA directly interfered with Spo0A∼P generation. IPTG-directed synthesis of the SpoIIE phosphatase in a strain carrying a multicopy plasmid coding for SpoIIAA and its specific inhibitory kinase SpoIIAB blocked Spo0A activation suggesting that the active form of the inhibitor was SpoIIAA and not SpoIIAA-P. Furthermore, expression of the non-phosphorylatable mutant SpoIIAAS58A (SpoIIAA-like), but not SpoIIAAS58D (SpoIIAA-P-like), completely blocked Spo0A-dependent gene expression. Importantly, SpoIIAA expressed from the chromosome under the control of its normal spoIIA promoter showed the same negative effect regulated not only by SpoIIAB and SpoIIE but also by septum morphogenesis. These findings are discussed in relation to the potential contribution of this novel inhibitory feedback with the proper activation of σF and σE during development.
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    Genes encoding synthetases of cyclic depsipeptides, anabaenopeptilides, in Anabaena strain 90 (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Anabaena strain 90 produces three hepatotoxic heptapeptides (microcystins), two seven-residue depsipeptides called anabaenopeptilide 90A and 90B, and three six-residue peptides called anabaenopeptins. The anabaenopeptilides belong to a group of cyanobacterial depsipeptides that share the structure of a six-amino-acid ring with a side-chain. Despite their similarity to known cyclic peptide toxins, no function has been assigned to the anabaenopeptilides. Degenerate oligonucleotide primers based on the conserved amino acid sequences of other peptide synthetases were used to amplify DNA from Anabaena 90, and the resulting polymerase chain reaction (PCR) products were used to identify a peptide synthetase gene cluster. Four genes encoding putative anabaenopeptilide synthetase domains were characterized. Three genes, apdA, apdB and apdD, contain two, four and one module, respectively, encoding a total of seven modules for activation and peptide bond formation of seven l-amino acids. Modules five and six also carry methyltransferase-like domains. Before the first module, there is a region similar in amino acid sequence to formyltransferases. A fourth gene (apdC), between modules six and seven, is similar in sequence to halogenase genes. Thus, the order of domains is co-linear with the positions of amino acid residues in the finished peptide. A mutant of Anabaena 90 was made by inserting a chloramphenicol resistance gene into the apdA gene. DNA amplification by PCR confirmed the insertion. Mass spectrometry analysis showed that anabaenopeptilides are not made in the mutant strain, but other peptides, such as microcystins and anabaenopeptins, are still produced by the mutant.
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    Characterization of the A2–A2rel gene cluster in Leishmania donovani: involvement of A2 in visceralization during infection (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The A2 gene family is present in Leishmania donovani, which causes fatal visceral leishmaniasis in human patients, but is not present in Leishmania major, which causes cutaneous leishmaniasis infections. The A2 genes in L. donovani are stage specific and are expressed at high levels in the amastigote stage in the mammalian host, but are not expressed in the promastigote stage in the insect sandfly vector. The A2 genes are tandem repeated with a distinct gene family termed the A2rel genes. In order to characterize the structure and function of the A2–A2rel gene clusters, the 5′ and 3′ DNA sequences flanking the A2–A2rel cluster were isolated, sequenced and used to generate mutants through gene targeting. Although it was possible to generate partial A2–A2rel gene clusters knock-out mutants, it was not possible to delete all the A2–A2rel gene clusters completely from the L. donovani genome, suggesting that, within this cluster, there are genes that are essential for survival in culture. Characterization of these mutants revealed that A2 and A2rel gene expression was compensated by amplifying the remaining intact A2 and A2rel genes, and the proliferation of these mutants in culture and their virulence in BALB/c mice were compromised. In order to explore further the biological role of A2, the L. donovani A2 gene was introduced into L. major. In comparison with the control L. major, the A2-expressing L. major parasites demonstrated an increased ability to survive in the spleen of BALB/c mice. These data suggest that A2 plays a role in the visceralization of infection associated with L. donovani.
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    URL: http://dx.doi.org/10.1046/j.1365-2958.2001.02286.x
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    Plasmodium protein phosphatase 2C dephosphorylates translation elongation factor 1β and inhibits its PKC-mediated nucleotide exchange activity in vitro (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The elongation step of protein synthesis involves binding of aminoacyl-tRNA to the ribosomal A site, formation of a peptide bond and translocation of the newly formed peptidyl-tRNA to the P site. The nucleotide exchange factor EF-1β plays a major role in the regulation of this process by regenerating a GTP-bound EF-1α necessary for each elongation cycle. EF-1β has been shown to be phosphorylated and its phosphorylation is critical for optimal activity. We have previously identified a serine/threonine protein phosphatase 2C (PP2C) from the human malaria parasite Plasmodium falciparum. In the current work, we performed Far-Western analysis to identify PfPP2C substrates. Several components of the translation and transcription machinery were identified, including translation elongation factor 1-beta (PfEF-1β). PfEF-1β is efficiently phosphorylated by protein kinase C and this phosphorylation results in a 400% increase in its nucleotide exchange activity. PKC-phosphorylated PfEF-1β is readily and selectively dephosphorylated by recombinant and native PfPP2C, which downregulates the nucleotide exchange activity to its basal level. The identification of a translation elongation component as substrate for PP2C suggests an important regulatory function for this enzyme and suggests that it may be a good target for drug design in the fight against malaria.
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    Regulation of immunity to the two-component lantibiotic, lacticin 3147, by the transcriptional repressor LtnR (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Lacticin 3147 is a membrane-active, two-component lantibiotic produced by Lactococcus lactis ssp. lactis DPC3147. In this study, the promoters of the lacticin 3147 gene cluster were mapped to the intergenic region between ltnR and ltnA1 (the genes encoding the regulatory protein LtnR and the first structural gene, LtnA1), and Northern analyses revealed that the biosynthetic and immunity genes are divergently transcribed in two operons, ltnA1A2M1TM2D and ltnRIFE respectively. Although the promoter controlling biosynthesis (Pbac) appears to be constitutive, characterization of a downstream β-galactosidase (β-gal) fusion beyond an intragenic stem–loop structure in ltnM1 confirmed that this putative transcriptional attenuator allows limited readthrough to the downstream biosynthetic genes, thus maintaining the correct stoichiometry between structural peptides and biosynthetic machinery. The promoter of the ltnRIFE operon (Pimm) was shown to be regulated by the transcriptional repressor LtnR. A mutant with a truncated ltnR gene exhibited a hyperimmune phenotype, whereas overexpression of ltnR resulted in cells with increased sensitivity to lacticin 3147. Gel mobility shift analysis indicated that LtnR binds to the Pimm promoter region, and fusion of this promoter to the β-gal gene of pAK80 revealed that expression from Pimm is significantly reduced in the presence of LtnR. Thus, we have demonstrated that lacticin 3147 uses a regulatory mechanism not previously identified in lantibiotic systems.
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    SoxR-dependent response to oxidative stress and virulence of Erwinia chrysanthemi: the key role of SufC, an orphan ABC ATPase (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Erwinia chrysanthemi causes soft-rot disease in a great variety of plants. In addition to the depolymerizing activity of plant cell wall-degrading enzymes, iron acquisition and resistance to oxidative stress contribute greatly to the virulence of this pathogen. Here, we studied the pin10 locus originally thought to encode new virulence factors. The sequence analysis revealed six open reading frames that were homologous to the Escherichia coli sufA, sufB, sufC, sufD, sufS and sufE genes. Sequence similarity searching predicted that (i) SufA, SufB, SufD, SufS and SufE proteins are involved in iron metabolism and possibly in Fe–S cluster assembly; and (ii) SufC is an ATPase of an ABC transporter. The reverse transcription–polymerase chain reaction procedure showed that the sufABCDSE genes constitute an operon. Expression of a sufB::uidA fusion was found to be induced in iron-deficient growth conditions and to be repressed by the iron-sensing Fur repressor. Each of the six suf genes was inactivated by the insertion of a cassette generating a non-polar mutation. The intracellular iron level in the sufA, sufB, sufC, sufS and sufE mutants was higher than in the wild type, as assessed by increased sensitivity to the iron-activated antibiotic streptonigrin. In addition, inactivation of sufC and sufD led to increased sensitivity to paraquat. Virulence tests showed that sufA and sufC mutants exhibited reduced ability to cause maceration of chicory leaves, whereas a functional sufC gene was necessary for the bacteria to cause systemic invasion of Saintpaulia ionantha. The E. coli sufC homologue was inactivated by reverse genetic. This mutation was found to modify the soxR-dependent induction of soxS gene expression. We discuss the possibility that SufC is a versatile ATPase that can associate either with the other Suf proteins to form a Fe–S cluster-assembling machinery or with membrane proteins encoded elsewhere in the chromosome to form an Fe–S ABC exporter. Overall, these results stress the importance of the connection between iron metabolism and oxidative stress during the early steps of infection by E. chrysanthemi.
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    The cold-shock stress response in Mycobacterium smegmatis induces the expression of a histone-like protein (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The response of Mycobacterium smegmatis to a cold shock was investigated by monitoring changes in both growth and cellular protein composition of the organism. The nature of the cellular response was influenced by the magnitude of the temperature reduction, with the shock from 37°C to 10°C having the most widespread effect on growth, metabolism and protein composition. This 27°C temperature reduction was associated with a lag period of 21–24 h before increases were seen in all the measured cellular activities. The response to cold shock was adaptive, with growth resuming after this period, albeit at a 50-fold slower rate. The synthesis of at least 15 proteins was induced during the lag period. Two distinct patterns of cold-induced synthesis were apparent, namely transient and continuous, indicating the production of both cold-induced and cold-acclimation proteins. One of these cold-shock proteins, CipMa, was identified as the histone-like protein, Hlp, of M. smegmatis, which is also induced during anaerobic-induced dormancy. The corresponding gene demonstrated transient, cold-inducible expression with a five- to sevenfold increase in mRNA occurring 9–12 h after temperature shift. Although bacterial survival was unaffected, CipMa/Hlp knock-out mutants were unable to adapt metabolically to the cold shock and resume growth, thus indicating a key role for CipMa in the cold-shock response.
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    FAP1, a homologue of human transcription factor NF-X1, competes with rapamycin for binding to FKBP12 in yeast (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
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    Analysis of the replicon region and identification of an rRNA operon on pBM400 of Bacillus megaterium QM B1551 (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: An 18 633 bp region containing the replicon from the ≈ 53 kb pBM400 plasmid of Bacillus megaterium QM B1551 has been sequenced and characterized. This region contained a complete rRNA operon plus 10 other potential open reading frames (ORFs). The replicon consisted of an upstream promoter and three contiguous genes (repM400, orfB and orfC) that could encode putative proteins of 428, 251 and 289 amino acids respectively. A 1.6 kb minimal replicon was defined and contained most of repM400. OrfB was shown to be required for stability. Three 12 bp identical tandem repeats were located within the coding region of repM400, and their presence on another plasmid caused incompatibility with their own cognate replicon. Nonsense, frameshift and deletion mutations in repM400 prevented replication, but each mutation could be complemented in trans. RepM400 had no significant similarity to sequences in the GenBank database, whereas five other ORFs had some similarity to gene products from other plasmids and the Bacillus genome. An rRNA operon was located upstream of the replication region and is the first rRNA operon to be sequenced from B. megaterium. Its unusual location on non-essential plasmid DNA has implications for systematics and evolutionary biology.
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    Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12–FK506 in Cryptococcus neoformans (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Calcineurin is a Ca2+–calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 37°C and hence for virulence of the pathogenic fungus Cryptococcus neoformans. Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12–FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12–FK506–calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.
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    Molecular basis of Salmonella-induced enteritis (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Salmonella pathogenesis is a complex and multifactorial phenomenon. Many genes required for full virulence in mice have been identified, but only a few of these have been shown to be necessary for the induction of enteritis. Likewise, at least some of the Salmonella virulence factors affecting enteritis do not appear to be required for infection of systemic sites in mice. This suggests that subsets of virulence genes influence distinct aspects of Salmonella pathogenesis. Recently, considerable progress has been made in characterizing the virulence mechanisms influencing enteritis caused by non-typhoid Salmonella spp. The Salmonella pathogenicity island-1-encoded type III secretion system mediates the translocation of secreted effector proteins into target epithelial cells. These effector proteins are key virulence factors required for Salmonella intestinal invasion and the induction of fluid secretion and inflammatory responses.
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    Host adaptation and the emergence of infectious disease: the Salmonella paradigm (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The recent emergence of food-borne pathogens, such as Salmonella enterica serotype Enteritidis (S. enteritidis) and Escherichia coli O157:H7, has generated increasing interest in how infectious diseases can invade, persist and spread within new host populations. To alter their host range pathogens require adaptations, which ensure their circulation in a new animal population. Adaptations for circulation in different populations of vertebrate hosts seem to have been acquired multiple times within the genus Salmonella because extant Salmonella serotypes differ greatly with regard to host range. In this article, mechanisms involved in host adaptation are deduced by considering the influence of the host immune response on circulation of Salmonella serotypes within populations of vertebrate animals. This approach contributes to the identification of genes involved in host adaptation and provides new insights into the emergence of food-borne pathogens.
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    Is gene expression in Halobacterium NRC-1 regulated by multiple TBP and TFB transcription factors? (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
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    Saturation mutagenesis of the haloarchaeal bop gene promoter: identification of DNA supercoiling sensitivity sites and absence of TFB recognition element and UAS enhancer activity (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Transcription from the bop promoter in the haloarchaeon Halobacterium NRC-1, is highly induced under oxygen-limiting conditions. A DNA gyrase inhibitor, novobiocin, was previously shown to block bop gene induction and suggested that DNA supercoiling mediates transcriptional induction. A region of non-B structure was found 3′ to the TATA box within an 11 bp alternating purine–pyrimidine sequence (RY box), which correlated to both increased DNA supercoiling and transcriptional induction. Here, saturation mutagenesis of the RY box region has been used to show that single-base substitutions of A(r)G either 23 or 19 bp 5′ to the transcription start site temper the effect of DNA supercoiling based on novobiocin insensitivity of transcription. Mutagenesis of the region 5′ to the TATA box showed its involvement in DNA supercoiling modulation of transcription, defined the 3′ end of the upstream activator sequence (UAS) regulatory element, and ruled out the requirement for a TFB (TFIIB) Recognition Element. Spacing between the TATA box and UAS was found to be critical for promoter activity because insertion of partial or whole helical turns between the two elements completely inhibited transcription indicating that the UAS element does not function as a transcriptional enhancer. The results are discussed in the context of DNA melting and flexibility around the TATA box region and the involvement of multiple regulatory and transcription factors in bop promoter activity.
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    Identification of the urease operon in Helicobacter pylori and its control by mRNA decay in response to pH (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We investigated the transcription of the urease gene cluster ureABIEFGH in Helicobacter pylori to determine the regulation of gene expression of the highly produced enzyme urease. Northern blot hybridization analysis demonstrated that cells of the wild-type strain grown in an ordinary broth had transcripts of ureAB, ureABI, ureI, ureIE′ and ure′FGH, but cells of a ureI-disrupted mutant had only the ureAB transcript. When the wild-type cells were exposed to pH 8 for 30 min, very little mRNA was detected. However, when exposed to pH 6, a large amount of the ureIE′′ transcript, which was longer than the ureIE′ transcript, together with the additional transcripts ureABIEFGH and ure′EFGH were detected. Rifampicin addition experiments demonstrated that urease mRNAs, and the ureIE′ transcripts in particular, are more stable at pH 5.5 than at pH 7. In accord with these results, urease activity in the crude cell extract of the pH 5.5 culture was twice as much as that of the pH 7 culture, although the amounts of UreA and UreB detected by immunoblot analysis were similar. The transcription start point of ureI was identified by primer extension using a ureA promoter-deleted mutant, and a consensus sequence of RpoD-RNA polymerase was found in the ureI promoter. The 3′ end of the ureIE′′ mRNA, determined using S1 nuclease mapping, revealed that the transcript is able to cover the majority of the ureE open reading frame (ORF) that might be sufficient for UreE activity. Based on the above results, we conclude that the urease gene cluster of H. pylori consists of two operons, ureAB and ureIEFGH, and that primary transcripts of the latter as well as the read-through transcript, ureABIEFGH, are cleaved to produce several species of mRNA. It has been suggested that the ureIEFGH operon is regulated post-transcriptionally by mRNA decay in response to environmental pH. We are tempted to speculate that the ureE′′ transcript present in acidic pH may contribute to produce an active product that can proceed the nickel incorporation to the active centre, the final step of urease biosynthesis.
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    Rst1 and Rst2 are required for the a/α diploid cell type in yeast (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 46 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In the budding yeast Saccharomyces cerevisiae, the preservation of the mating competent haploid (a or α) and the mating incompetent diploid (a/α) is necessary to prevent aneuploidy. Once haploid cells respond to pheromone, the mating-specific signal transduction pathway is activated, and the MAP kinase Fus3 phosphorylates two specific repressor proteins Rst1 and Rst2 (also known as Dig1 and Dig2) to promote Ste12-dependent transcription of mating-specific genes. In contrast, diploid cells cannot mate because genes that encode components of the mating pathway are repressed through the combined action of the Mata1–Matα2 and Matα2–Mcm1 repressors. Surprisingly, repression of Ste12 by Rst1 and Rst2 is essential for diploid sterility. Homozygous deletion of both RST1 and RST2 (rst–) causes a/α diploid cells constitutively to express a-specific genes and mate preferentially as a-cells. This phenotype is sensitive to Ste12 dosage, as removal of one copy of STE12 completely reduces the ectopic activation of a-specific genes. The Matα2–Mcm1 complex, which normally represses a-specific genes, is defective in rst– diploids because Matα2 is destabilized in rst– diploids, possibly as a consequence of its relocalization from the nucleus to the cytoplasm. This study finds that Rst1 and Rst2 are necessary for the a/α diploid cell type. Rst1 and Rst2 are required in order to prevent the amplification of a robust Ste12 transcriptional programme that appears to over-ride Matα2-dependent repression of haploid and a-specific genes.
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    Thioredoxins are required for protection against a reductive stress in the yeast Saccharomyces cerevisiae (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 46 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Thioredoxins are small, highly conserved oxidoreductases that are required to maintain the redox homeostasis of the cell. They have been best characterized for their role as antioxidants in protection against reactive oxygen species. We show here that thioredoxins (TRX1, TRX2) and thioredoxin reductase (TRR1) are also required for protection against a reductive stress induced by exposure to dithiothreitol (DTT). This sensitivity to reducing conditions is not a general property of mutants affected in redox control, as mutants lacking components of the glutathione/glutaredoxin system are unaffected. Furthermore, TRX2 gene expression is induced in response to DTT treatment, indicating that thioredoxins form part of the cellular response to a reductive challenge. Our data indicate that the sensitivity of thioredoxin mutants to reducing stress appears to be a consequence of elevated glutathione levels, which is present predominantly in the reduced form (GSH). The elevated GSH levels also result in a constitutively high unfolded protein response (UPR), indicative of an accumulation of unfolded proteins in the endoplasmic reticulum (ER). However, there does not appear to be a general defect in ER function in thioredoxin mutants, as oxidative protein folding of the model protein carboxypeptidase Y occurs with similar kinetics to the wild-type strain, and trx1 trx2 mutants are unaffected in sensitivity to the glycosylation inhibitor tunicamycin. Furthermore, trr1 mutants are resistant to tunicamycin, consistent with their high UPR. The high UPR seen in trr1 mutants can be abrogated by the GSH-specific reagent 1-chloro-2,4-dinitrobenzene. In summary, thioredoxins are required to maintain redox homeostasis in response to both oxidative and reductive stress conditions.
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    On the mechanism of substrate specificity by resistance nodulation division (RND)-type multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 46 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Tripartite efflux systems of Gram-negative bacteria that contain an inner membrane transporter belonging to the resistance nodulation division (RND) superfamily can extrude a large variety of structurally diverse compounds. To gain an insight into the molecular mechanisms of substrate recognition by these multidrug resistance (MDR) transporters, we isolated spontaneous mutations that altered the substrate specificity of the MexCD–OprJ pump from Pseudomonas aeruginosa. These mutations enabled the pump to extrude the normally non-transported β-lactam antibiotic carbenicillin. All amino acid substitutions were mapped to the large periplasmic loops (LPLs) of the RND proper, MexD. Q34K, E89K, A292V and P328L were found in the first LPL, located between transmembrane domains (TMD) 1 and 2, whereas F608S and N673K were contained in the second LPL, located between TMD7 and TMD8. These mutations also had a substantial impact on the MexCD–OprJ-mediated transport of numerous other substrates. Subsequent replacement of amino acid residues identified above by cysteines rendered MexCD–OprJ susceptible to inhibition by a thiol-reactive agent, MIANS. Interestingly, MIANS inhibited the transport of some (pyronin, EtBr) but not other (ANS, Leu–Nap) substrates of the pump. Our results suggest that the precise structure of the periplasmic loops of MexD determines the rate of transport of individual substrates. These results are consistent with the hypothesis that, in the case of RND transporters, the LPLs are directly implicated in substrate recognition and contain multiple sites of interaction for various structurally diverse compounds.
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    The Escherichia coli histone-like protein HU regulates rpoS translation (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Escherichia coli HU protein is a major component of the bacterial nucleoid. HU stabilizes higher order nucleoprotein complexes and belongs to a family of DNA architectural proteins. Here, we report that HU is required for efficient expression of the sigma S subunit of RNA polymerase. This rpoS-encoded alternative σS factor induces a number of genes implicated in cell survival in stationary phase and in multiple stress resistance. By analysis of rpoS–lacZ fusions and by pulse-chase experiments, we show that the efficiency of rpoS translation is reduced in cells lacking HU, whereas neither rpoS transcription nor protein stability is affected by HU. Gel mobility shift assays show that HU is able to bind specifically an RNA fragment containing the translational initiation region of rpoS mRNA 1000-fold more strongly than double-stranded DNA. Together with the in vivo data, this finding strongly suggests that, by binding to rpoS mRNA, HU directly stimulates rpoS translation. We demonstrate here that HU, an abundant DNA-binding, histone-like protein, is able specifically to recognize an RNA molecule and therefore play a role in post-transcriptional regulation.
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    Blue light adaptation and desensitization of light signal transduction in Neurospora crassa (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The ascomycete Neurospora crassa has the capacity of adapting to a given light quantity, leading to transient blue light responses under continuous light conditions. Here, we present an investigation of this photoadaptation phenomenon. We demonstrated previously that two proteins of the Neurospora blue light signal transduction chain, WC1 and WC2, are subject to light-dependent phosphorylation. WC1 was phosphorylated in parallel with the transient increase in transcript levels of light-regulated genes. Using the light-dependent phosphorylation of WC1 as a marker for an active signalling state of WC1, we show that the transiency of Neurospora blue light responses results from desensitization of the photoreceptor and/or the signalling cascade. Furthermore, a Neurospora mutant was characterized that revealed a specific defect in photoadaptation. In this mutant, the transient expression of light-regulated genes under continuous light, the temporary insensitivity after a light pulse and the capability of differentiating between and adapting to low and high light intensities were abolished. The corresponding protein seems to represent a central component of a negative feedback desensitization mechanism. This negative feedback regulation requires continuous and light-dependent protein de novo biosynthesis.
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    The NAT L. Sternberg Thesis Prize (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
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    Two atypical mobilization proteins are involved in plasmid CloDF13 relaxation (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The mobilization region of plasmid CloDF13 was localized to a 3.6 kb DNA segment that was analysed by transposon mutagenesis and DNA sequencing. Analysis of the DNA sequence allowed us to identify two mobilization genes and the CloDF13 origin of conjugative transfer (oriT), which was localized to a 661 bp segment at one end of the mobilization (Mob) region. Thus, the overall organization was oriT–mobB–mobC. Plasmid CloDF13 DNA was isolated mainly as a relaxed form that contained a unique strand and site-specific cleavage site (nic). The position of nic was mapped to the sequence 5′-GGGTG/GTCGGG-3′ by primer extension and sequencing reactions. Analysis of Mob− insertion mutants showed that mobC was essential for CloDF13 relaxation in vivo. The sequence of mobC predicts a protein (MobC) of 243 amino acids without significant similarity to previously reported relaxases. In addition to MobC, the product of mobB was also required for CloDF13 mobilization and for oriT relaxation in vivo. mobB codes for a protein (MobB) of 653 amino acids with three predicted transmembrane segments at the N-terminus and the NTP-binding motifs characteristic of the TraG family of conjugative coupling proteins. Membership of the TraG family was confirmed by the fact that CloDF13 mobilization by plasmid R388 was independent of TrwB and only required PILW. However, contrary to the activities found for other coupling proteins, MobB was required for efficient oriT cleavage in vivo, suggesting an additional role for this particular protein during oriT processing for mobilization. Additionally, the cleavage site produced by the joint activities of MobB and MobC was shown to contain unblocked ends, suggesting that no stable covalent intermediates between relaxase and DNA were formed during the nic cleavage reaction. This is the first report of a conjugative transfer system in which niccleavage results in a free nicked-DNA intermediate.
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    Global and cognate regulators control the expression of the organic solvent efflux pumps TtgABC and TtgDEF of Pseudomonas putida (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Pseudomonas putida DOT-T1E grows on a water–toluene double liquid phase. Toluene tolerance in this microorganism is mainly achieved by at least two efflux pumps that belong to the RND family. The TtgDEF efflux pump is induced by toluene, whereas the other efflux pump, called TtgABC, is expressed at a high level in cells not exposed to toluene and at a lower level in cells grown with toluene. The ttgR gene is adjacent to the ttgABC operon and is transcribed divergently from ttgA. The expression level of ttgR was fourfold higher in cells growing in the presence of toluene than in its absence. In a TtgR-deficient background, expression from the ttgA promoter increased about 20-fold, suggesting that TtgR represses expression from the ttgA promoter. In this mutant, background expression of the ttgR gene was also much higher than in the wild-type background; however, its level of expression increased in the presence of toluene. In a ttgR mutant background, expression from the ttgD promoter followed the same pattern of expression as in the wild type. Analysis of a P. putida pTn5cat mutant that exhibited increased sensitivity to a sudden toluene shock, regardless of whether or not it was previously exposed to low toluene concentrations, revealed that pTn5cat had interrupted an lrp-like gene. The ttgR gene was expressed at very high levels in this mutant, with concomitant repression of expression of the ttgABC operon. The second ttgDEF efflux pump was expressed at low levels in this mutant strain, suggesting that the Lrp-like protein is a global regulatory protein involved in the solvent-tolerant response of this strain.
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    Salmonella Pathogenicity Island 2 (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Systemic infections by Salmonella enterica, such as typhoid fever, are a significant threat to human health. Recent studies indicate that the function of a type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2) is central for the ability of S. enterica to cause systemic infections and for intracellular pathogenesis. This review summarizes approaches leading to the identification of SPI2, the molecular genetics and evolution of SPI2, and the current understanding of the regulation of gene expression. Recent studies have indicated that SPI2 is used by intracellular Salmonella to actively modify functions of the host cells. The role of SPI2 during pathogenesis of salmonellosis and current models regarding function will be discussed.
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    Frequent interspecific genetic exchange between commensal neisseriae and Neisseria meningitidis (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Natural sequence variation was investigated among serogroup A subgroup IV-1 Neisseria meningitidis isolated from diseased patients and healthy carriers in The Gambia, West Africa. The frequencies of DNA import were analysed by sequencing fragments of four linked genes encoding the immunogenic outer membrane proteins TbpB (transferrin binding protein B) and OpaA (an adhesin) plus two housekeeping enzymes. Seventeen foreign tbpB alleles were independently imported into the 98 strains tested, apparently due to immune selection. The median size of the imported DNA fragments was 5 kb, resulting in the occasional concurrent import of linked housekeeping genes by hitchhiking. Sequences of tbpB from other strains of N. meningitidis as well as commensal Neisseria lactamica and Neisseria spp. isolated from the same geographical area revealed that these species share a common tbpB gene pool and identified several examples of interspecific genetic exchange. These observations indicate that recombination can be more frequent between related species than within a species and indicate that effective vaccination against serogroup B meningococcal disease may be difficult to achieve.
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    Interacting interfaces of the P4 antirepressor E and the P2 immunity repressor C (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Antirepressors have been identified as proteins interacting with transcriptional repressors leading to expression of the repressed genes. The defective satellite phage/plasmid P4 has the capacity to derepress the unrelated prophage P2 after infection, thereby getting access to the late functions of the helper that are required for P4 lytic growth. The derepression of prophage P2 is mediated by the P4 E protein that function as an antirepressor by binding to the P2 immunity repressor C. A P2 mutant, sos, has been isolated that is insensitive to the action of the P4 E protein. In the present study, we show that sos is a point mutation in the P2 immunity repressor gene C and that it makes P4 E unable to turn the transcriptional switch of P2 from the lysogenic state to the lytic mode in a two plasmid reporter system. Furthermore, the interaction between C and E, when analysed in the yeast two-hybrid system, is blocked by the sos mutation. An analysis of C mutants indicates that the dimerization function of C is located in the C-terminal part of the protein and the dimerization defective mutants are unable to bind to their operator DNA. The sos mutation does not affect the capacity of the protein to dimerize. Using the yeast two-hybrid system, compensatory E mutants have been isolated that can interact with Sos, but they are unable to turn the transcriptional switch controlled by the Sos repressor. However, one point mutation in the E protein is shown to be unable to turn the transcriptional switch controlled by the wild-type C repressor.
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    The proteolytic control of restriction activity in Escherichia coli K-12 (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The endonuclease activity of EcoKI is regulated by the ClpXP-dependent degradation of the subunit that is essential for restriction, but not modification. We monitored proteolysis in mutants blocked at different steps in the restriction pathway. Mutations that prevent DNA translocation render EcoKI refractory to proteolysis, whereas those that permit DNA translocation, but block endonuclease activity, do not. Although proteolysis alleviates restriction in a mutant that lacks modification activity, some restriction activity remains; our evidence indicates residual EcoKI associated with the membrane fraction. ClpXP protects the bacterial chromosome, but little effect was detected on unmodified foreign DNA within the cytoplasm of a restriction-proficient cell. The molecular basis for the distinction between unmodified resident and foreign DNA remains to be determined.
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    Analysis of the oxidative stress regulation of the Candida albicans transcription factor, Cap1p (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: CAP1 encodes a basic region-leucine zipper (bZip) transcriptional regulatory protein that is required for oxidative stress tolerance in Candida albicans. Cap1p is a homologue of a Saccharomyces cerevisiae bZip transcription factor designated Yap1p that is both required for oxidative stress tolerance and localized to the nucleus in response to the presence of oxidants. Oxidant-regulated localization of Yap1p to the nucleus requires the presence of a carboxy-terminal cysteine residue (C629) that is conserved in Cap1p as C477. To examine the role of this conserved cysteine residue, C477 was replaced with an alanine residue. This mutant protein, C477A Cap1p, was analysed for its behaviour both in S. cerevisiae and C. albicans. Wild type and C477A Cap1p were able to complement the oxidant hypersensitivity of a Δyap1 S. cerevisiae strain. Whereas a Yap1p-responsive lacZ fusion gene was oxidant inducible in the presence of YAP1, the C. albicans Cap1p derivatives were not oxidant responsive in S. cerevisiae. Introduction of wild type and C477A Cap1p-expressing plasmids into C. albicans produced differential resistance to oxidants. Glutathione reductase activity was found to be inducible by oxidants in the presence of Cap1p but was constitutively elevated in the presence of C477A Cap1p. Western blot assays indicate Cap1p is post-translationally regulated by oxidants. Green fluorescent protein fusions to CAP1 showed that this protein is localized to the nucleus only in the presence of oxidants while C477A Cap1p is constitutively nuclear localized. Directly analogous to S. cerevisiae Yap1p, regulated nuclear localization of C. albicans Cap1p is crucial for its normal function.
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    Analysis of the DNA-binding domain of Escherichia coli DnaA protein (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The DNA-binding domain of the Escherichia coli DnaA protein is represented by the 94 C-terminal amino acids (domain 4, aa 374–467). The isolated DNA-binding domain acts as a functional repressor in vivo, as monitored with a mioC::lacZ translational fusion integrated into the chromosome of the indicator strain. In order to identify residues required for specific DNA binding, site-directed and random PCR mutagenesis were performed, using the mioC::lacZ construct for selection. Mutations defective in DNA binding were found all over the DNA-binding domain with some clustering in the basic loop region, within presumptive helix B and in a highly conserved region at the N-terminus of presumptive helix C. Surface plasmon resonance (SPR) analysis revealed different binding classes of mutant proteins. No or severely reduced binding activity was demonstrated for amino acid substitutions at positions R399, R407, Q408, H434, T435, T436 and A440. Altered binding specificity was found for mutations in a 12 residue region close to the N-terminus of helix C. The defects of the classical temperature sensitive mutants dnaA204, dnaA205 and dnaA211 result from instability of the proteins at higher temperatures. dnaX suppressors dnaA71 and dnaA721 map to the region close to helix C and bind DNA non-specifically.
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    A third envelope stress signal transduction pathway in Escherichia coli (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 45 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Escherichia coli uses overlapping envelope stress responses to adapt to insults to the bacterial envelope that cause protein misfolding. The σ E and Cpx envelope stress responses are activated by both common and distinct envelope stresses and respond by increasing the expression of the periplasmic protease DegP as well as target genes unique to each response. The σ E pathway is involved in outer membrane protein (OMP) folding quality control whereas the Cpx pathway plays an important role in the assembly of at least one pilus. Previously, we identified the spy gene as a new Cpx regulon member of unknown function. Interestingly, induction of spy expression by severe envelope stresses such as spheroplasting is only partially dependent on an intact Cpx signalling pathway, unlike other Cpx-regulated genes. Here we show that the BaeS sensor kinase and BaeR response regulator also control expression of spy in response to envelope stress. BaeS and BaeR do not affect expression of other known Cpx-regulated genes, however, baeR cpxR double mutants show increased sensitivity to envelope stresses relative to either single mutant alone. We propose that the Bae signal transduction pathway controls a third envelope stress response in E. coli that induces expression of a distinct set of adaptive genes.
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    OhrR, a transcription repressor that senses and responds to changes in organic peroxide levels in Xanthomonas campestris pv. phaseoli (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 45 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We report the physiological role of OhrR as an organic peroxide sensor and transcription repressor in Xanthomonas campestris pv. phaseoli. In vivo exposure of X. campestris pv. phaseoli to either tert-butyl or cumene hydroperoxides efficiently neutralized OhrR repression of expression from the OhrR-regulated P1 promoter. H2O2 was a weak and non-physiological inducer of the system while other oxidants and metabolites of organic peroxide metabolism did not induce the expression from the P1. Northern blotting results indicated a correlation between concentrations of tert-butyl hydroperoxide used in the treatment and the induction of ohr (an OhrR-regulated gene) expression. In addition, the levels of ohr mRNA in cultures induced by various concentrations of tert-butyl hydroperoxide were reduced in cells with high levels of an organic peroxide metabolising enzyme (AhpC-AhpF) but not in cells with high catalase levels suggesting that organic peroxide interacts with OhrR. DNA band shift experiments using purified OhrR and the P1 promoter fragment showed that organic peroxide treatment prevented binding of the protein to the P1 promoter by oxidation of OhrR, as the inhibition of binding to the P1 promoter was reversed by addition of a reducing agent, DTT. The highly conserved cysteine residue C22 of OhrR is required for organic peroxide inducible gene expression. A mutant protein, OhrRC22S can repress the P1 promoter activity but is insensitive to organic peroxide treatment. Thus, OhrR is the first transcription repressor characterized that appeared to evolve to physiologically sense organic peroxides.
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    Two pathways of homologous recombination in Trypanosoma brucei (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 45 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: African trypanosomes are unicellular parasites that use DNA recombination to evade the mammalian immune response. They do this in a process called antigenic variation, in which the parasites periodically switch the expression of VSG genes that encode distinct Variant Surface Glycoprotein coats. Recombination is used to move new VSG genes into specialised bloodstream VSG transcription sites. Genetic and molecular evidence has suggested that antigenic variation uses homologous recombination, but the detailed reaction pathways are not understood. In this study, we examine the recombination pathways used by trypanosomes to integrate transformed DNA into their genome, and show that they possess at least two pathways of homologous recombination. The primary mechanism is dependent upon RAD51, but a subsidiary pathway exists that is RAD51-independent. Both pathways contribute to antigenic variation. We show that the RAD51-independent pathway is capable of recombining DNA substrates with very short lengths of sequence homology and in some cases aberrant recombination reactions can be detected using such microhomologies.
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    A Salmonella SipB-derived polypeptide blocks the ‘trigger’ mechanism of bacterial entry into eukaryotic cells (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 45 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Entry into non-phagocytic mammalian cells by the invasive pathogens Salmonella and Shigella is triggered by the delivery of bacterial virulence effector proteins into the host cell. This is dependent upon Salmonella SipB or its Shigella homologue IpaB, which insert into the eukaryotic cell plasma membrane. Here we show that a SipB-derived 166 residue α-helical polypeptide is a potent inhibitor of SipB-directed liposome fusion in vitro, preventing the membrane-associated form of SipB from inserting deeply into the bilayer. This polypeptide blocks Salmonella entry into cultured mammalian cells at 10−10 M, and is a heterologous inhibitor of analogous IpaB activity and Shigella cell entry. These findings reveal a potential strategy to identify inhibitors of the ‘trigger’ mechanism underlying cell entry by these major invasive pathogens.
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    The gene bolA regulates dacA (PBP5), dacC (PBP6) and ampC (AmpC), promoting normal morphology in Escherichia coli (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 45 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The gene bolA has been shown to trigger the formation of osmotically stable round cells when overexpressed in stationary phase. We show that in poor growth conditions bolA is essential for normal cell morphology in stationary phase and under conditions of starvation. During exponential growth bolA promotes round morphology through a mechanism that is strictly dependent on the two main Escherichia colid,d-carboxypeptidases, PBP5 and PBP6. The results show that bolA controls the levels of transcription of dacA (PBP5), dacC (PBP6) and ampC (AmpC), a class C β-lactamase, thus connecting for the first time penicillin binding proteins (PBPs) and β-lactamases at the level of gene regulation. Furthermore, PBP5 and PBP6 are shown to be independently regulated and to have distinct effects on the peptidoglycan layer. The evidence presented demonstrates that bolA is a regulator of cell wall biosynthetic enzymes with different roles in cell morphology and cell division.
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    Novel Mycobacterium tuberculosis anti-σ factor antagonists control σF activity by distinct mechanisms (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 45 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The aetiological agent of tuberculosis, Mycobacterium tuberculosis, encodes 13 σ factors, as well as several putative anti-, and anti-anti- σ factors. Here we show that a σ factor that has been previously shown to be involved in virulence and persistence processes, σF, can be specifically inhibited by the anti-σ factor UsfX. Importantly, the inhibitory activity of UsfX, in turn, can be negatively regulated by two novel anti-anti-σ factors. The first anti-anti-σ factor seems to be regulated by redox potential, and the second may be regulated by phosphorylation as it is rendered non-functional by the introduction of a mutation that is believed to mimic phosphorylation of the anti-anti-σ factor. These results suggest that σF activity might be post-translationally modulated by at least two distinct pathways in response to different possible physiological cues, the outcome being consistent with the bacteria's ability to adapt to diverse host environments during disease progression, latency and reactivation.
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    Pieter W. Postma 1942–2002 (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 45 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
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    URL: http://dx.doi.org/10.1046/j.1365-2958.2002.03157.x
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    Author Index (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 45 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
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    URL: http://dx.doi.org/10.1046/j.1365-2958.2002.t01-1-03221.x
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    Agrobacterium type IV secretion is a two-step process in which export substrates associate with the virulence protein VirJ in the periplasm (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 45 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Type IV secretion systems are virulence determinants in many bacteria and share extensive homology with many conjugal transfer systems. Although type IV systems and their homologues have been studied widely, the mechanism by which substrates are secreted remains unclear. In Agrobacterium, we show that type IV secretion substrates that lack signal peptides form a soluble complex in the periplasm with the virulence protein VirJ. Additionally, these proteins co-precipitate with constituents of the type IV transporter: the VirB pilus and the VirD4 protein. Our findings suggest that the substrate proteins localized to the periplasm may associate with the pilus in a manner that is mediated by VirJ, and suggest a two-step process for type IV secretion in Agrobacterium. Our analyses of protein–protein interactions in a variety of mutant backgrounds indicate that substrates are probably secreted independently of one another.
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    Function in Escherichia coli of the non-catalytic part of RNase E: role in the degradation of ribosome-free mRNA (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 45 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Summary RNase E contains a large non-catalytic region that binds RNA and the protein components of the Escherichia coli RNA degradosome. The rne gene was replaced with alleles encoding deletions in the non-catalytic part of RNase E. All the proteins are stable in vivo. RNase E activity was tested using a PT7–lacZ reporter gene, the message of which is particularly sensitive to degradation because translation is uncoupled from transcription. The non-catalytic region has positive and negative effectors of mRNA degradation. Disrupting RhlB and enolase binding resulted in hypoactivity, whereas disrupting PNPase binding resulted in hyperactivity. Expression of the mutant proteins in vivo anticorrelates with activity showing that autoregulation compensates for defective function. There is no simple correlation between RNA binding and activity in vivo. An allele (rne131), expressing the catalytic domain alone, was put under Plac control. In contrast to rne+, low expression of rne131 severely affects growth. Even with autoregulation, all the mutants are less fit when grown in competition with wild type. Although the catalytic domain of RNase E is sufficient for viability, our work demonstrates that elements in the non-catalytic part are necessary for normal activity in vivo.
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    Molecular analysis of tyrosine- and phenylalanine-mediated repression of the tyrB promoter by the TyrR protein of Escherichia coli (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 45 (2002), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The mechanism of repression of the tyrB promoter by TyrR protein has been studied in vivo and in vitro. In tyrR+ strains, transcription of tyrB is repressed by either tyrosine or phenylalanine. Both of the TyrR binding sites (strong and weak TyrR boxes) lie downstream of the tyrB transcription start site and are required for tyrosine- or phenylalanine-mediated repression. Our results establish that the binding of the TyrR protein to the weak box, induced by cofactor tyrosine or phenylalanine, is critical for repression to occur. Neither the binding of the TyrR protein dimer formed in the presence of phenylalanine, nor the binding of the hexamer formed in the presence of tyrosine, blocks the binding of RNA polymerase to the promoter. Instead, open complex formation is inhibited in the presence of tyrosine whereas a step(s) following open complex formation is inhibited in the presence of phenylalanine. Moving the TyrR boxes 3 bp or more further away from the promoter affects tyrosine-mediated repression without affecting phenylalanine-mediated repression which remains unaltered until 6 bp are inserted between the TyrR boxes and the promoter. Analysis of deletion and insertion mutants fails to reveal any face of the helix specificity for either tyrosine- or phenylalanine-mediated ­repression.
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    Specificity determinants for bacteriophage Hong Kong 022 integrase: analysis of mutants with relaxed core-binding specificities (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The integrase (Int) proteins encoded by bacteriophages HK022 and λ catalyse similar site-specific integration and excision reactions between specific DNA regions known as attachment (att) sites. However, the Int proteins of HK022 and λ are unable to catalyse recombination between non-cognate att sites. The att sites of both phages contain weak binding sites for Int, known as ‘core-type’ sites. Negatively acting nucleotide determinants associated with specific core sites (λ B′, HK022 B′, HK022 C) are responsible for the barrier to non-cognate recombination. In this study, we used challenge phages to demonstrate that the λ and HK022 Ints cannot bind to core sites containing non-cognate specificity determinants in vivo. We isolated mutants of the HK022 Int, which bind the λ B′ core site. Two mutants, D99N and D99A, have changed a residue in the core-binding (CB) domain, which may be directly contacting the core site DNA. We suggest that binding to the λ B′ site was accomplished by removing the negatively charged aspartate residue, which normally participates in a conflicting interaction with the G4 nucleotide of the λ B′ site. We showed that, although our mutants retain the ability to recombine their cognate att sites, they are unable to recombine λatt sites.
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    Effects of local transcription and H-NS on inversion of the fim switch of Escherichia coli (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The fim switch of Escherichia coli is responsible for phase-variable expression of type 1 fimbriae. Switching in the ON-to-OFF and OFF-to-ON directions is promoted by the FimB recombinase, while the FimE recombinase directs switching predominantly in the ON-to-OFF direction. The effects of local promoter activity and the H-NS nucleoid-associated protein on inversion of the switch were assessed. In contrast to FimB-mediated inversion, inversion of the switch by the FimE recombinase was unaffected by the H-NS status of the cell. Transcription towards the switch from within a translationally inactivated fimE gene was found to bias the switch strongly in the OFF direction, creating a FimE+-like phenotype in the absence of the FimE protein. This biasing was H-NS dependent and was also contingent on transcription from within the switch. These data show that local transcription and a nucleoid-associated protein both contribute to the modulation of a site-specific recombination event on the bacterial chromosome.
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    Membrane-fusion protein homologues in Gram-positive bacteria (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
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    A novel autoregulation mechanism of fnrN expression in Rhizobium leguminosarum bv viciae (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The fnrN gene from Rhizobium leguminosarum UPM791 controls microaerobic expression of both nitrogen fixation and hydrogenase activities in symbiotic cells. Two copies of fnrN are present in this strain, one chromosomal (fnrN1) and the other located in the symbiotic plasmid (fnrN2). Their expression was studied by cloning the regulatory regions in lacZ promoter-probe vectors. The fnrN genes were found to be autoregulated: they are expressed only at basal levels under aerobic conditions; they are highly expressed under microaerobic conditions; and they are expressed at basal levels in the double mutant DG2 (fnrN1 fnrN2) under any condition. The promoters of both genes contain two FnrN-binding sequences (anaeroboxes), centred at positions −12.5 (proximal anaerobox) and −44.5 (distal anaerobox). Expression analysis and gel retardation experiments with fnrN1-derivative promoter mutants altered in key bases of the anaerobox sequences demonstrated that binding of FnrN1 to the distal anaerobox is necessary for microaerobic activation of transcription, and that binding of FnrN1 to the proximal anaerobox results in transcriptional repression. The apparent affinity of FnrN1 for the proximal anaerobox was fivefold lower than for the distal anaerobox, resulting in repression of transcription of fnrN1 only at high-FnrN1 concentrations. This positive and negative autoregulation mechanism ensures an equilibrated expression of fnrN in response to microaerobic conditions.
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    The use of listeriolysin to identify in vivo induced genes in the Gram-positive intracellular pathogen Listeria monocytogenes (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Listeria monocytogenes is capable of growth within the cytoplasm of infected host cells. Escape from the host cell phagosome is mediated primarily through secretion of listeriolysin, a haemolytic factor which functions to actively lyse the phagosomal membrane. Listeriolysin negative mutants of L. monocytogenes are non-haemolytic on blood agar plates and demonstrate a significant reduction of virulence in the mouse model of infection. We have developed a system for the identification of in vivo induced genes in L. monocytogenes which utilizes the listeriolysin gene, hly, as both a reporter of gene expression and as a means of selection of promoter elements expressed in vivo. The system is analogous to in vivo expression technology (IVET) first reported for Salmonella, however, as listeriolysin functions in the environment of the host phagosome the loci identified in this study are most likely expressed during residence in the phagosome. The system was successfully tested using the promoter of the inducible virulence gene plcA. A bank was created by fusing a promoterless copy of hly to random promoter elements in a listeriolysin negative IVET host. Sequential inoculations of mice with this bank resulted in the isolation of clones with increased survival potential in the mouse model relative to a negative control, but which remained haemolysin negative on blood agar plates. Nine in vivo induced loci were identified including genes encoding a DNA topoisomerase III, a cellobiose transporter and a fumarase. Two isolates represented fusions to proteins of unknown function and three isolates contained no significant homologues in the database. A mutant in the fumarase gene demonstrated reduced virulence for mice and an inability to grow in cultured mouse phagocytes.
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    Aspergillus SteA (Sterile12-like) is a homeodomain-C2/H2-Zn+2 finger transcription factor required for sexual reproduction (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Saccharomyces cerevisiae Ste12p plays a key role in coupling signal transduction through MAP kinase modules to cell-specific or morphogenesis-specific gene expression required for mating and pseudohyphal (PH)/filamentous growth (FG). Ste12p homologues in the pathogenic yeasts Candida albicans and Filobasidiela neoformans apparently play similar roles during dimorphic transitions. Here we report the isolation and characterization of the first Ste12 protein from a true filamentous fungus. Aspergillus nidulans steA encodes a protein with a homeodomain 63–75% identical to those of other Ste12 proteins, with greatest similarity to FnSte12αp. SteAp and Ste12αp lack the pheromone induction domain found in budding yeast Ste12p, but have C-terminal C2/H2-Zn+2 finger domains not present in the other Ste12 proteins. A ΔsteA strain is sterile and differentiates neither ascogenous tissue nor fruiting bodies (cleistothecia). However, the development of sexual cycle-specific Hülle cells is unaffected. Filamentous growth, conidiation and the differentiation of PH-like asexual reproductive cells (metulae and phialides) are normal in the deletion strain. Northern analysis of key regulators of the asexual and sexual reproductive cycles support the observation that although SteAp function is restricted to the sexual cycle, cross regulation between the two developmental pathways exists. Our results further suggest that while several classes of related proteins control similar morphogenetic events in A. nidulans and the dimorphic yeasts, significant differences must exist in the regulatory circuitry.
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    The carbon metabolism-controlled Synechocystis gap2 gene harbours a conserved enhancer element and a Gram-positive-like −16 promoter box retained in some chloroplast genes (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The two glyceraldehyde-3-phosphate dehydrogenase-encoding genes (gap) of Synechocystis were shown to be expressed as monocistronic transcripts. Whereas gap1 expression is slow and weak, gap2 gene induction is rapid and strong. Transcription of the gap2 gene was shown to depend on functional photosynthetic electron transport and on active carbon metabolism. The basal promoter of gap2 (P, −45 to +34, relative to the transcription start site) is controlled by three cis-acting elements designated A (−443 to −45), B (+34 to +50, in the untranslated leader region) and C (+50 to +167, in the coding region) that, together, promote a 100-fold stimulation of P activity. Element B was found to behave as a transcriptional enhancer, in that it was active regardless of its position, orientation and distance relative to P. All three cis-acting stimulatory elements exhibit a common 5′-agaTYAACg-3′ nucleotide motif that appears to be conserved in cyanobacteria and may be the target for a transcriptional enhancer. We also report that gap2 transcription depends on a Gram-positive-like −16 promoter box (5′-TRTG-3′) that was obviously conserved throughout the evolution of chloroplasts. This is the first report on the occurrence of a −16 promoter element in photoautotrophic organisms.
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    Developmental arrest of the human malaria parasite Plasmodium falciparum within the mosquito midgut via CTRP gene disruption (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Apicomplexan protozoa possess a family of micronemal and cell surface-associated proteins, each comprised a combination of cell-adhesive vertebrate von Willebrand factor (vWF)-like A domains and thrombospondin (TSP) type 1-like domains. The human malaria parasite Plasmodium falciparum has in the extracellular portion of the CS protein TRAP-related protein (CTRP) six tandemly arrayed A domains followed by seven TSP type 1-like domains, whereas a second member of this family, thrombospondin-related anonymous protein (TRAP), contains a single vWF-like A domain and a single TSP type 1-like domain. Here we show that CTRP transcripts are present within the infected mosquito midgut and that CTRP protein is expressed with a punctate distribution and a predominance at the apical end of mosquito midgut-stage ookinetes. This expression pattern is analogous to micronemal expression of TRAP in Plasmodium sporozoites. Disruption of the CTRP gene by homologous recombination in cultures of the human malaria parasite P. falciparum demonstrates that CTRP is essential for mosquito midgut development. Oocyst formation was never observed following membrane feeds of CTRP disruptant lines to Anopheline mosquitoes, despite the development of mature ookinetes. We propose that CTRP is involved in essential recognition or motility processes at the ookinete cell surface within the mosquito midgut.
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    Differential functionalities of amphiphilic peptide segments of the cell-septation penicillin-binding protein 3 of Escherichia coli (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The class B M1-V577 penicillin-binding protein (PBP) 3 of Escherichia coli consists of a M1–L39 membrane anchor (bearing a cytosolic tail) that is linked via a G40–S70 intervening peptide to an R71–I236 non-catalytic module (containing the conserved motifs 1–3) itself linked via motif 4 to a D237–V577 catalytic module (containing the conserved motifs 5–7 of the penicilloyl serine transferases superfamily). It has been proposed that during cell septation the peptidoglycan crosslinking activity of the acyl transferase module of PBP3 is regulated by the associated M1–I236 polypeptide itself in interaction with other components of the divisome. The fold adopted by the R71–V577 polypeptide of PBP3 has been modelled by reference to the corresponding R76–S634 polypeptide of the class B Streptococcus pneumoniae PBP2x. Based on these data and the results of site-directed mutagenesis of motifs 1–3 and of peptide segments of high amphiphilicity (identified from hydrophobic moment plots), the M1–I236 polypeptide of PBP3 appears to be precisely designed to work in the way proposed. The membrane anchor and the G40–S70 sequence (containing the G57–Q66 peptide segment) upstream from the non-catalytic module have the information ensuring that PBP3 undergoes proper insertion within the divisome at the cell septation site. Motif 1 and the I74–L82 overlapping peptide segment, motif 2 and the H160–G172 overlapping peptide segment, and the G188–D197 motif 3 are located at or close to the intermodule junction. They contain the information ensuring that PBP3 folds correctly and the acyl transferase catalytic centre adopts the active configuration. The E206–V217 peptide segment is exposed at the surface of the non-catalytic module. It has the information ensuring that PBP3 fulfils its cell septation activity within the fully complemented divisome.
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    Restriction–modification system differences in Helicobacter pylori are a barrier to interstrain plasmid transfer (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Helicobacter pylori cells are naturally competent for the uptake of both plasmid and chromosomal DNA. However, we demonstrate that there are strong barriers to transformation of H. pylori strains by plasmids derived from unrelated strains. We sought to determine the molecular mechanisms underlying these barriers. Transformation efficiency was assessed using pHP1, an Escherichia coli–H. pylori shuttle vector conferring kanamycin resistance. Transformation of 33 H. pylori strains was attempted with pHP1 purified from either E. coli or H. pylori, and was successfully introduced into only 11 strains. Digestion of H. pylori chromosomes with different restriction endonucleases (REs) showed that DNA methylation patterns vary substantially among strains. The strain most easily transformed, JP26, was found to have extremely low endogenous RE activity and to lack a restriction–modification (R–M) system, homologous to MboI, which is highly conserved among H. pylori strains. When we introduced this system to JP26, pHP1 from MboI.M+ JP26, but not from wild-type JP26, transformed MboI R−M+ JP26 and heterologous MboI R−M+ wild-type H. pylori strains. Parallel studies with pHP1 from dam+ and dam−E. coli strains confirmed these findings. These data indicate that the endogenous REs of H. pylori strains represent a critical barrier to interstrain plasmid transfer among H. pylori.
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    Iron availability regulates DNA recombination in Neisseria gonorrhoeae (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The pilus of Neisseria gonorrhoeae (the gonococcus Gc), the causative agent of gonorrhoea, promotes attachment of the gonococcus to the host epithelium and is essential for the establishment of disease. The ability of N. gonorrhoeae to infect previously exposed individuals is partially due to pilus antigenic variation. In addition, variation of the pilus has been proposed to function in the adaptation of the gonococcus to host environments. Previously, we described the development of a competitive reverse transcriptase (RT)-PCR assay that quantifies the frequency of pilin antigenic variation within a gonococcal population. Using this assay, the effect of different biologically relevant environmental conditions on the frequency of pilin antigenic variation was tested. Of the environmental conditions examined in vitro, only limited iron affected a significant change in the frequency of antigenic variation. Further investigation revealed that an observed increase in pilin antigenic variation reflected an increase in other DNA recombination and DNA repair processes within iron-starved cultures. In addition, this low iron-induced increase was determined to be independent of changes in RecA expression and was observed in a Fur mutant strain. As gonococci encounter conditions of low iron during infection, these data suggest that iron-limitation signals for increased recombinational events that are important for gonococcal pathogenesis.
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    Rifampicin-resistant initiation of chromosome replication from oriC in ihf mutants (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: IHF (integration host factor) mutants exhibit asynchronous initiation of chromosome replication from oriC as determined from flow cytometric analysis of cultures where RNA synthesis was inhibited with rifampicin. However, the run-out kinetics of chromosome replication in ihf mutants shows that they continue to produce oriCs for some time in the absence of RNA synthesis resulting in a twofold increase in the oriC per mass ratio. An ihf dnaA double mutant did not exhibit this continued increase of the oriC per mass ratio. This indicates that ihf mutants can initiate replication from oriC in a rifampicin-resistant initiation mode but requires fully functional DnaA protein. The origin per mass ratio, determined by a quantitative Southern blotting technique, showed that the ihf mutants had an origin per mass ratio that was 60% of the wild type although it had a normal DnaA protein concentration. This shows that the initiation mass was substantially higher in the ihf mutants. The oriC per terminus ratio, which was also determined by Southern blotting, was very low in the ihf mutant, although it grew with the same doubling times as the wild-type strain. This indicates that cells lacking IHF replicate their chromosome(s) very fast.
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    Elucidating the molecular mechanisms of bacterial virulence using non-mammalian hosts (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Several strains of the human opportunistic pathogen Pseudomonas aeruginosa infect plants, nematodes and insects. Our laboratory has developed a multihost pathogenesis system based on the P. aeruginosa clinical isolate PA14, in which non-mammalian hosts are used to screen directly for virulence-attenuated mutants. The majority of PA14 mutants isolated using non-mammalian hosts also displayed reduced virulence in a burned mouse model. Surprisingly, only a few host-specific virulence factors were identified, and many of the P. aeruginosa mutants were attenuated in virulence in all the hosts. These studies illustrate the extensive conservation in the virulence mechanisms used by P. aeruginosa to infect evolutionarily diverged hosts, and validate the multihost method of screening for virulence factors relevant to mammalian pathogenesis. Through the use of genetically tractable hosts, the multihost pathogenesis model also provides tools for elucidating host responses and dissecting the fundamental molecular interactions that underlie bacterial pathogenesis.
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    Auto-transporter A protein of Neisseria meningitidis: a potent CD4+ T-cell and B-cell stimulating antigen detected by expression cloning (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A meningococcal genomic expression library was screened for potent CD4+ T-cell antigens, using patients' peripheral blood lymphocytes (PBLs). One of the most promising positive clones was fully characterized. The recombinant meningococcal DNA contained a single, incomplete, open reading frame (ORF), which was fully reconstructed with reference to available genomic sequence data. The gene was designated autA (auto-transporter A) as its peptide sequence shares molecular characteristics of the auto-transporter family of proteins. Only a single copy of this gene was detected in the meningococcal, and none in the gonococcal, genomic sequence databases. The complete autA gene, when cloned into an expression vector, expressed a protein of approximately 68 kDa. Purified rAutA recalled strong secondary T-cell responses in PBLs of patients and some healthy donors, and induced strong primary T-cell responses in healthy donors. The human B-cell immunogenicity and cross-reactivity of AutA, purified under native conditions, was confirmed in dot immunoblot experiments. Immunoblots with rabbit polyclonal antibodies to rAutA demonstrated the conserved nature, antigenicity and cross-reactivity of AutA amongst meningococci of different serogroups and strains representing different hypervirulent lineages. AutA showed homology with another meningococcal and gonococcal ORF (designated AutB). AutB was cloned and expressed and used to raise an autB-specific antiserum. Immunoblot experiments indicated that AutB is not expressed in meningococci and does not cross-react with AutA. Thus, AutA, being a potent CD4+ T-cell and B-cell-stimulating antigen, which is highly conserved, deserves further investigation as a potential vaccine candidate.
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    The spvB gene-product of the Salmonella enterica virulence plasmid is a mono(ADP-ribosyl)transferase (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A number of well-known bacterial toxins ADP-ribosylate and thereby inactivate target proteins in their animal hosts. Recently, several vertebrate ecto-enzymes (ART1–ART7) with activities similar to bacterial toxins have also been cloned. We show here that psiblast, a position-specific-iterative database search program, faithfully connects all known vertebrate ecto-mono(ADP-ribosyl)transferases (mADPRTs) with most of the known bacterial mADPRTs. Intriguingly, no matches were found in the available public genome sequences of archaeabacteria, the yeast Saccharomyces cerevisiae or the nematode Caenorhabditis elegans. Significant new matches detected by psiblast from the public sequence data bases included only one open reading frame (ORF) of previously unknown function: the spvB gene contained in the virulence plasmids of Salmonella enterica. Structure predictions of SpvB indicated that it is composed of a C-terminal ADP-ribosyltransferase domain fused via a poly proline stretch to a N-domain resembling the N-domain of the secretory toxin TcaC from nematode-infecting enterobacteria. We produced the predicted catalytic domain of SpvB as a recombinant fusion protein and demonstrate that it, indeed, acts as an ADP-ribosyltransferase. Our findings underscore the power of the psiblast program for the discovery of new family members in genome databases. Moreover, they open a new avenue of investigation regarding salmonella pathogenesis.
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    Regulation of the CYB2 gene expression: transcriptional co-ordination by the Hap1p, Hap2/3/4/5p and Adr1p transcription factors (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Expression of the Saccharomyces cerevisiae nuclear gene CYB2 encoding the mitochondrial enzyme l-(+)-lactate–cytochrome c oxidoreductase (EC 1.2.2.3) is subject to several strict metabolic controls at the transcriptional level: repression due to glucose fermentation, derepression by ethanol, induction by lactate and inhibition under anaerobic conditions or in response to deficiency of haem biosynthesis. In this respect, the data obtained from the transcriptional analysis of the CYB2 gene contribute to a better understanding of the control of mitochondrial biogenesis. In this study, we show that Hap1p is the main transcriptional activator involved in the control of CYB2 transcription. We found that Hap1p activity, known to be oxygen dependent, is effected by DNA–protein interaction with two binding sites present in the CYB2 promoter. Control is moreover dependent on carbon sources. This regulation by the carbon substrates is subordinate to the activity of the complex Hap2/3/4/5p, which counteracts the negative effect of the URS1 element. Finally, our results suggest that the Adr1p transcriptional activator is also required in CYB2 transcription control. This work provides new data which allows a better understanding of the molecular mechanisms implicated in the co-regulation at the transcriptional level of the genes encoding proteins involved in various aspects of oxidative metabolism.
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    The Salmonella type III secretion translocon protein SspC is inserted into the epithelial cell plasma membrane upon infection (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Salmonella species translocate effector proteins into the host cell cytoplasm using a type III secretion system (TTSS). The translocation machinery probably contacts the eukaryotic cell plasma membrane to effect protein transfer. Data presented here demonstrate that both SspB and SspC, components of the translocation apparatus, are inserted into the epithelial cell plasma membrane 15 min after Salmonella typhimurium infection. In addition, a yeast two-hybrid interaction between SspC and an eukaryotic intermediate filament protein was identified. Three individual carboxyl-terminal point mutations within SspC that disrupt the yeast two-hybrid interaction were isolated. Strains expressing the mutant SspC alleles were defective for invasion, translocation of effector molecules and membrane localization of SspC. These data indicate that insertion of SspC into the plasma membrane of target cells is required for invasion and effector molecule translocation and that the carboxyl terminus of SspC is essential for these functions.
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    Molecular models accounting for the gene conversion reactions mediating gonococcal pilin antigenic variation (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The pilus antigenic variation (Av) system of Neisseria gonorrhoeae is one of several high-frequency variation systems that utilize gene conversion to switch between numerous forms of an antigen on the cell surface. We have tested three predictions of the first models that explain the movement of DNA during pilin Av: (i) Av requires two recombinations at short regions of identity, (ii) circular intermediates exist that carry pilE/pilS hybrid loci and (iii) these pilE/pilS hybrid loci target the pilS sequences to a recipient pilE gene. We confirm that normal pilin Av utilizes recombination at very short regions of DNA sequence identity and that these recombination events can occur independent of homologous recombination functions. We have isolated covalently closed circular DNA molecules carrying hybrid pilin loci, but propose that an alternative hybrid molecule is the intermediate of pilin Av. Our most striking finding is that transformation of isolated pilE/pilS hybrid loci targets the pilS sequences of the hybrid to a recipient pilE at frequencies much higher than normal recombination frequencies. These results show that the different steps of a model that explains pilin Av can be separately tested to support the validity of these novel models that account for the high-frequency gene conversions that mediate pilin Av.
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    Divergent structure of the ComQXPA quorum-sensing components: molecular basis of strain-specific communication mechanism in Bacillus subtilis (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In Bacillus subtilis, the ComQXPA quorum-sensing system controls cell density-dependent phenotypes such as the production of degradative enzymes and antibiotics and the development of genetic competence. Bacillus subtilis (natto) NAF12, a mutant defective in poly-γ-glutamate (γ-PGA) production, was derived from B. subtilis (natto) NAF4 by Tn917-LTV1 insertional mutagenesis. Determination of the mutant DNA sequences flanking the Tn917-LTV1 insert revealed that the insertion had inactivated comP in this mutant, indicating that γ-PGA synthesis in B. subtilis (natto) is under the control of the ComP–ComA signal transduction system. A comparison of the amino acid sequences revealed striking variation in the primary structures of ComQ (44% identity), ComX (26%) and the sensor domain of ComP (36%) between B. subtilis (natto) NAF4 and B. subtilis 168. In contrast, the amino acid and nucleotide sequences of the kinase domains of ComP and of the ComA response regulator share 95% and 100% identity respectively. The comP genes of NAF4 and 168 restored the impaired competence of B. subtilis BD1658 (comP::cat) and γ-PGA production of B. subtilis (natto) NAF12 (comP::Tn917-LTV1) to only 15% of the level achieved by the respective parent comP genes. However, when introduced together with the cognate comQ and comX genes, the comP genes restored the relevant defect of the heterologous comP mutants nearly to wild-type levels. Analogous to the comCDE system of Streptococcus strains and the agrBCDE system of Staphylococcus aureus, the concerted variation in the comQXP genes appears to establish specific intercellular communication between B. subtilis strains sharing the same pheromone system.
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    Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The spo0A genes of Clostridium beijerinckii NCIMB 8052 and Clostridium cellulolyticum ATCC 35319 were isolated and characterized. The C-terminal DNA-binding domains of the predicted products of spo0A from these two organisms, as well as 16 other taxonomically diverse species of Bacillus and Clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues). A 12-amino-acid motif (SRVERAIRHAIE) that forms the putative DNA recognition helix is particularly highly conserved, suggesting a common DNA target. Insertional inactivation of spo0A in C. beijerinckii blocked the formation of solvents (as well as spores and granulose). Sequences resembling Spo0A-binding motifs (TGNCGAA) are found in the promoter regions of several of the genes whose expression is modulated at the onset of solventogenesis in Clostridium acetobutylicum and C. beijerinckii. These include the upregulated adc gene, encoding acetoacetate decarboxylase (EC 4.1.1.4), and the downregulated ptb gene, encoding phosphotransbutyrylase (EC 2.3.1.c). In vitro gel retardation experiments using C. acetobutylicum adc and C. beijerinckii ptb promoter fragments and recombinant Bacillus subtilis and C. beijerinckii Spo0A suggested that adc and ptb are directly controlled by Spo0A. The binding affinity was reduced when the 0A boxes were destroyed, and enhanced when they were modified to conform precisely to the consensus sequence. In vivo analysis of wild-type and mutagenized promoters transcriptionally fused to the gusA reporter gene in C. beijerinckii validated this hypothesis. Post-exponential phase expression from the mutagenized adc promoter was substantially reduced, whereas expression from the mutagenized ptb promoter was not shut down at the end of exponential growth.
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    Tethering of CpxP to the inner membrane prevents spheroplast induction of the Cpx envelope stress response (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Cpx envelope stress response of Escherichia coli is controlled by a two-component regulatory system that senses misfolded proteins in extracytoplasmic compartments and responds by inducing the expression of envelope protein folding and degrading factors. We have proposed that in the absence of envelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and the sensing domain of the histidine kinase CpxA. In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response. Further, removal of CpxP is an important component of this induction because tethering an MBP–CpxP fusion protein to the spheroplast inner membranes prevents full activation by this treatment. Spheroplast formation has previously been demonstrated to induce the expression of a periplasmic protein of unknown function, Spy. Analysis of spy expression in response to spheroplast formation by Western blot analysis and by lacZ operon fusion in various cpx mutant backgrounds demonstrated that spy is a member of the Cpx regulon. Interestingly, although the only known spy homologue is cpxP, Spy does not appear to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response. Rather, deletion of spy leads to activation of the σE stress response. Because the σE response is specifically affected by alterations in outer membrane protein biogenesis, we think it possible that Spy may be involved in this process.
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    Interaction of ResD with regulatory regions of anaerobically induced genes in Bacillus subtilis (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The two-component regulatory proteins ResD and ResE are required for anaerobic nitrate respiration in Bacillus subtilis. ResD, when it undergoes ResE-dependent phosphorylation, is thought to activate transcriptionally anaerobically induced genes such as fnr, hmp and nasD. In this report, deletion analysis of the fnr, hmp and nasD promoter regions was carried out to identify cis-acting sequences required for ResDE-dependent transcription. The results suggest that the hmp and nasD promoters have multiple target sequences for ResDE-dependent regulation and that fnr has a single target site. Gel mobility shift assays and DNase I footprinting analyses were performed to determine whether ResD interacts directly with the regulatory regions of the three genes. Our results indicate that ResD specifically binds to sequences residing upstream of the hmp and nasD promoters and that phosphorylation of ResD significantly stimulates this binding. In contrast, a higher concentration of ResD is required for binding to the fnr promoter region and no stimulation of the binding by ResD phosphorylation was observed. Taken together, these results suggest that ResD activates transcription of fnr, hmp and nasD by interacting with DNA upstream of these promoters. Our results suggest that phosphorylation of ResD stimulates binding to multiple ResD binding sites, but is much less stimulatory if only a single binding site exists.
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    ResD signal transduction regulator of aerobic respiration in Bacillus subtilis: ctaA promoter regulation (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A two-component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, encoded by resD and resE genes of the res operon (resABCDE), has a regulatory role in both aerobic and anaerobic respiration. In terms of aerobic respiration, resD functions upstream of ctaA, a gene required for haem A biogenesis and hence for the synthesis of haem A-containing cytochrome terminal oxidases. Although ResD is probably a transcription factor, there was no direct evidence that ResD protein, either phosphorylated or unphosphorylated, interacts directly with regulatory regions of ResD-controlled genes. Here, we report the overexpression and purification of ResD and ResE and their role in gene activation. ResD can be phosphorylated by ResE in vitro and is a monomer in solution in either the phosphorylated or unphosphorylated state. The binding activity of ResD to the ctaA promoter was examined by gel shift assays and DNase I footprinting assays. DNase I footprinting showed both unphosphorylated and phosphorylated ResD binding to the ctaA promoter and showed that there are three binding sites (A1, A2 and A3), two (A1 and A2) upstream of the −35 promoter region and one (A3) downstream of the −10 of the promoter. The role of each site in ctaA promoter activity and ResD binding was characterized using deletion analysis, followed by the DNase I footprinting and in vivo transcription assays of promoter–lacZ fusions. Our results showed that the concentration of ResD required to bind at each site is different and that ResD binding at the A1 site is independent of the other two ResD binding sites, but that the concentration of ResD∼P required to protect site A2 is reduced when site A3 is present. In vivo transcription assays from promoter–lacZ fusion constructs showed that DNA containing ResD-binding site A2 was essential for promoter activity and that promoter constructs containing both binding sites A2 and A3 were sufficient for full promoter activity.
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    Completion of the hook–basal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The flhDC operon of Salmonella typhimurium is the master control operon required for the expression of the entire flagellar regulon. The flagellar master operon was placed under the tetracycline-inducible promoter PtetA using the T-POP transposon. Cells containing this construct are motile in the presence of tetracycline and non-motile without inducer present. No flagella were visible under the electron microscope when cells were grown without inducer. The class 1, class 2 and class 3 promoters of the flagellar regulon are temporally regulated. After addition of tetracycline, the class 1 flhDC operon was transcribed immediately. Transcription of flgM (which is transcribed from both class 2 and class 3 promoters) began 15 min after induction. At 20 min after induction, the class 2 fliA promoter became active and intracellular FliA protein levels increased; at 30 min after induction, the class 3 fliC promoter was activated. Induction of fliC gene expression coincides with the appearance of FlgM anti-sigma factor in the growth medium. This also coincides with the completion of hook–basal body structures. Rolling cells first appeared 35 min after induction, and excess hook protein (FlgE) was also found in the growth medium at this time. At 45 min after induction, nascent flagellar filaments became visible in electron micrographs and over 40% of the cells exhibited some swimming behaviour. Multiple flagella assemble and grow on individual cells after induction of the master operon. These results confirm that the flagellar regulatory hierarchy of S. typhimurium is temporally regulated after induction. Both FlgM secretion and class 3 gene expression occur upon completion of the hook–basal body structure.
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    A defined sequence within the 3′ UTR of the areA transcript is sufficient to mediate nitrogen metabolite signalling via accelerated deadenylation (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Nitrogen metabolism in Aspergillus nidulans is regulated by AREA, a member of the GATA family of transcription factors. One mechanism that modulates AREA activity involves the rapid degradation of the areA transcript when sufficient NH4+ or Gln are available. This signalling mechanism has been shown to require a region of 218 nucleotides within the 3′ untranslated region of areA mRNA. We demonstrate that this region functions independently in a heterologous transcript and acts to accelerate degradation of the poly(A) tail, which in turn leads to rapid transcript degradation in response to the addition of NH4+ or Gln to the growth medium. areA transcript degradation is inhibited by cycloheximide, but this is not a general consequence of translational inhibition. We believe that this is the first reported example in which specific physiological signals, acting through a defined sequence within a transcript, have been shown to promote accelerated poly(A) degradation, which in turn triggers transcript degradation.
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    Virulent aggregates of Streptococcus pyogenes are generated by homophilic protein–protein interactions (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Many strains of the important human pathogen Streptococcus pyogenes form aggregates when grown in vitro in liquid medium. The present studies demonstrate that this property is crucial for the adherence, the resistance to phagocytosis and the virulence of S. pyogenes. A conserved sequence of 19 amino acid residues (designated AHP) was identified in surface proteins of common S. pyogenes serotypes. This sequence was found to promote bacterial aggregation through homophilic protein–protein interactions between AHP-containing surface proteins of neighbouring bacteria. A synthetic AHP peptide inhibited S. pyogenes aggregation, reduced the survival of S. pyogenes in human blood and attenuated its virulence in mice. In contrast, mutant bacteria devoid of surface proteins containing AHP-related sequences did not aggregate or adhere to epithelial cells. These bacteria are also rapidly killed in human blood and show reduced virulence in mice, underlining the pathogenic significance of the AHP sequence and S. pyogenes aggregation.
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    Localization of the histidine kinase PilS to the poles of Pseudomonas aeruginosa and identification of a localization domain (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Transcription of the type IV pilus subunit gene of Pseudomonas aeruginosa is controlled by a two-component signal transduction system. PilS, the histidine kinase, is membrane bound and PilR, its cognate response regulator, is cytoplasmic. The signal that activates PilS is unknown. PilS has three domains: (i) The N-terminus, predicted to form six transmembrane (TM) helices; (ii) a central linker domain; and (iii) the C-terminal transmitter domain containing all the conserved residues of sensor kinases. A translational fusion of the gfp gene (green fluorescent protein) to the 3′ end of pilS was used to determine the position of PilS in the bacterial cell. Epifluorescence microscopy revealed that PilS is retained to the poles of P. aeruginosa but is distributed evenly about the membrane of Escherichia coli. Deletions of the PilS–GFP fusion revealed that the TM domain was sufficient and necessary to bring GFP to the membrane of P. aeruginosa and E. coli but was not sufficient to confine GFP to the poles. Retention to the poles of P. aeruginosa required both the TM and linker domains. Replacement of the PilS TM domain with an E. coli membrane protein, MalG, still allowed polar localization. Therefore, the PilS TM domain positions the linker domain close to the membrane allowing it to interact with the putative polar anchor which is specific to P. aeruginosa.
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    The NC2 repressor is dispensable in yeast mutated for the Sin4p component of the holoenzyme and plays roles similar to Mot1p in vivo (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: NC2 (Dr1/DRAP1) and Mot1p are global repressors of transcription that have been isolated in both Saccharomyces cerevisiae and humans. NC2 is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TBP and inhibition of TFIIA and TFIIB. Mot1p is an ATPase that removes DNA-bound TBP upon ATP hydrolysis. In this work, we studied the core promoter specificity of NC2 in vivo using a strain that carries mutated NC2β activity. We show that NC2, like Mot1p, is required for transcription of the HIS3 and HIS4 TATA-less core promoters. Furthermore, whereas neither Mot1p nor NC2 appear to function as repressors of the HIS3 gene in cells growing exponentially in glucose, we find that both are required for repression of the HIS3 TATA promoter when cells go through the diauxic shift. Thus, the activity of these factors is similarly regulated depending upon the physiological conditions, and it appears that core promoters activated or repressed by them in vivo might be distinguishable by whether or not they contain a canonical TATA sequence. Finally, although NC2 is an essential factor for yeast viability, we isolated a mutation in a non-essential component of the holoenzyme, Sin4p, that bypasses the requirement for NC2.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2000.01839.x
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    The Aspergillus niger transcriptional activator XlnR, which is involved in the degradation of the polysaccharides xylan and cellulose, also regulates d-xylose reductase gene expression (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Screening of an Aspergillus niger differential cDNA library, constructed by subtracting cDNA fragments of a xlnR loss-of-function mutant from wild-type cDNA fragments, resulted in the cloning of the gene encoding d-xylose reductase (xyrA). Northern blot analysis using an A. niger wild-type strain, a xlnR multiple-copy strain and a xlnR loss-of-function mutant confirmed that the xyrA gene is regulated by XlnR, the transcriptional activator of the xylanolytic enzyme system in A. niger. d-xylose reductase catalyses the NADPH-dependent reduction of d-xylose to xylitol, which is the first step in d-xylose catabolism in fungi. Until now, XlnR was shown to control the transcription of genes encoding extracellular hydrolytic enzymes involved in cellulose and xylan degradation. In the present study, we show that A. niger is able to harmonize its sugar metabolism and extracellular xylan degradation via XlnR by regulating the expression of XyrA.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2000.01843.x
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  • 96
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    Repression of the Escherichia coli melR promoter by MelR: evidence that efficient repression requires the formation of a repression loop (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Escherichia coli MelR protein is a transcription activator that, in the presence of melibiose, activates expression of the melAB operon by binding to four sites located just upstream of the melAB promoter. MelR is encoded by the melR gene, which is expressed from a divergent transcript that starts 237 bp upstream of the melAB promoter transcript start point. In a recent study, we have identified a fifth DNA site for MelR that overlaps the melR promoter transcript start and −10 region. Here we show that MelR binding to this site can downregulate expression from the melR promoter; thus, MelR autoregulates its own expression. Optimal repression of the melR promoter is observed in the absence of melibiose and requires one of the four other DNA sites for MelR at the melAB promoter. The two MelR binding sites required for this optimal repression are separated by 177 bp. We suggest that, in the absence of melibiose, MelR forms a loop between these two sites. We argue that, in the presence of melibiose, this loop is broken as the melAB promoter is activated. However, in the presence of melibiose, the melR promoter can still be partially repressed by MelR binding to the site that overlaps the transcript start and −10 region. Parallels with the Escherichia coli araC–araBAD regulatory region are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2000.01850.x
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  • 97
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    Low levels of Ypt protein prenylation cause vesicle polarization defects and thermosensitive growth that can be suppressed by genes involved in cell wall maintenance (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 35 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast. The vesicle-docking process requires that Ypt proteins are located in the vesicle membrane. C-terminal geranylgeranyl anchors mediate the membrane attachment of these proteins. The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes. What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown. Here, we report the characterization of a yeast REP mutant, mrs6-2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature. A shift to the restrictive temperature does not alter exponential growth during the first 3 h. The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild-type or mrs6-2 cells incubated at 25°C. In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point. Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6-2 thermosensitivity. Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization.
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    URL: http://dx.doi.org/10.1046/j.1365-2958.2000.01782.x
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    The role of tandem IS dimers in IS911 transposition (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 35 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Using a combined in vivo and in vitro approach, we demonstrated that the transposition products generated by IS911 from a dimeric donor plasmid are different from those generated from a plasmid monomer. When carried by a monomeric plasmid donor, free IS911 transposon circles are generated by intra-IS recombination in which one IS end undergoes attack by the other. These represent transposition intermediates that undergo integration using the abutted left (IRL) and right (IRR) ends of the element, the active IRR–IRL junction, to generate simple insertions. In contrast, the two IS911 copies carried by a dimeric donor plasmid not only underwent intra-IS recombination to generate transposon circles but additionally participated in inter-IS recombination. This also creates an active IRR–IRL junction by generating a head-to-tail IS tandem dimer ([IS]2) in which one of the original plasmid backbone copies is eliminated in the formation of the junction. Both transposon circles and IS tandem dimers are generated from an intermediate in which two transposon ends are retained by a single strand joint to generate a figure 8 molecule. Inter-IS figure 8 molecules generated in vitro could be resolved into the [IS]2 form following introduction into a host strain by transformation. Resolution did not require IS911 transposase. The [IS]2 structure was stable in the absence of transposase but was highly unstable in its presence both in vivo and in vitro. Previous studies had demonstrated that the IRR–IRL junction promotes efficient intermolecular integration and intramolecular deletions both in vivo and in vitro. Integration of the [IS]2 derivative would result in a product that resembles a co-integrate structure. It is also shown here that the IRR–IRL junction of the [IS]2 form and derivative structures can specifically target one of the other ends in an intramolecular transposition reaction to generate transposon circles in vitro. These results not only demonstrate that IS911 (and presumably other members of the IS3 family) is capable of generating a range of transposition products, it also provides a mechanistic framework which explains the formation and activity of such structures previously observed for several other unrelated IS elements. This behaviour is probably characteristic of a large number of IS elements.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2000.01800.x
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    The role of lateral gene transfer in the evolution of isoprenoid biosynthesis pathways (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 37 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Lateral gene transfer (LGT) is a major force in microbial genome evolution. Here, we present an overview of lateral transfers affecting genes involved in isopentenyl diphosphate (IPP) synthesis. Two alternative metabolic pathways can synthesize this universal precursor of isoprenoids, the 1-deoxy-d-xylulose 5-phosphate (DOXP) pathway and the mevalonate (MVA) pathway. We have surveyed recent genomic data and the biochemical literature to determine the distribution of the genes composing these pathways within the bacterial domain. The scattered distribution observed is incompatible with a simple scheme of vertical transmission. LGT (among and between bacteria, archaea and eukaryotes) more parsimoniously explains many features of this pattern. This alternative scenario is supported by phylogenetic analyses, which unambiguously confirm several cases of lateral transfer. Available biochemical data allow the formulation of hypotheses about selective pressures favouring transfer. The phylogenetic diversity of the organisms involved and the range of possible causes and effects of these transfer events make the IPP biosynthetic pathways an ideal system for studying the evolutionary role of LGT.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2000.02004.x
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  • 100
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    Characterization of the Lactococcus lactis transcription factor FlpA and demonstration of an in vitro switch (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 35 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The commercially important bacterium Lactococcus lactis contains two FNR-like proteins (FlpA and FlpB) which have a high degree of identity to each other and to the FLP of Lactobacillus casei. FlpA was isolated from a GST–FlpA fusion protein produced in Escherichia coli. Like FLP, isolated FlpA is a homodimeric protein containing both Zn and Cu. However, the properties of FlpA were more like those of the E. coli oxygen-responsive transcription factor FNR than the FLP of L. casei. As prepared FlpA recognized an FNR site (TTGAT-N4-ATCAA) but not an FLP site (CCTGA-N4-TCAGG) in band-shift assays. In contrast to FLP, DNA binding by FlpA did not require the formation of an intramolecular disulphide bond. However, despite containing only two cysteine residues per monomer, FlpA was able to acquire an FNR-like, oxygen-labile [4Fe 4S] cluster. But, whereas the incorporation of a [4Fe 4S] cluster into FNR enhances interaction with target DNA, it abolished DNA binding by FlpA. An FlpA variant (FlpA′) with an N-terminal region designed to be more FLP-like failed to incorporate an iron–sulphur cluster but could now form an intramolecular disulphide. This simple example of protein engineering, converting an oxygen-labile [4Fe 4S] containing FNR-like protein into a dithiol–disulphide FLP-like redox sensor demonstrates the versatility of the basic CRP structure. Attempts to demonstrate an FlpA-based aerobic–anaerobic switch in the heterologous host E. coli were unsuccessful. However, studies with a series of FNR-dependent lac reporter fusions in strains of E. coli expressing flpA or flpB revealed that both homologues were able to activate expression of FNR-dependent promoters in vivo but only when positioned 61 base pairs upstream of the transcription start.
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    URL: http://dx.doi.org/10.1046/j.1365-2958.2000.01799.x
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