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1

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Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion (1997)

Lu, Hong-Mei ; Motley, S.Timothy ; Lory, Stephen

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 25 (1997), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The general secretion pathway (GSP), found in a wide range of bacteria, is responsible for extracellular targeting of a subset of proteins from the periplasm. In Pseudomonas aeruginosa, the GSP requires the participation of 12 proteins, of which XcpT, XcpU, XcpV, XcpW are homologues of PilA, the major subunit of type IV pili. The interaction between the pilin-like Xcp proteins was investigated using bifunctional cross-linking reagents. Cross-linking analysis of whole cells of wild-type P. aeruginosa, followed by immunoblot analysis, revealed a 34-kDa XcpT-containing complex. This complex was shown to consist of XcpT/PilA heterodimers. The role of PilA in the GSP was examined, using P. aeruginosa mutants in the pilA gene, or in rpoN, a gene regulating pilA expression. Each mutant showed a significant reduction in the efficiency of extracellular protein secretion, and this defect could be restored by expression of the cloned pilA gene in the mutant cells. The formation of the PilA/XcpT complex did not require XcpR or XcpQ, two other components of the secretion machinery, nor did it require the pilus biogenesis factors PilB and PilC. The dimeric XcpT/PilA complex was also formed in a pilD mutant, which lacks the leader peptidase enzyme, demonstrating that the leader peptide at the N-terminus of PilA or XcpT did not have to be removed for the dimerization to occur. XcpW and XcpU can also be cross-linked to form dimeric complexes with PilA. When expression of XcpT is increased, its homodimers, as well as XcpT/XcpW heterodimers, can be detected. Finally, an oligohistidine-tagged XcpT was shown to form stoichiometric complexes with PilA, and with XcpT, U, V and W. These dimers were co-purified by nickel-affinity chromatography. The results of this study suggest that XcpT can form heterodimers with PilA, and Xcp U, V and W, which may be assembly intermediates of the secretion apparatus. Alternatively, these may represent dynamic intermediates that facilitate protein secretion by continuous association and dissociation. The requirement for PilA for efficient protein secretion argues for a critical role played by PilA in two related processes during P. aeruginosa infections: formation of an adhesive pilus organelle and secretion of exoenzymes.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1997.4561818.x

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2

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A second prepilin peptidase gene in Escherichia coli K-12 (1998)

Francetić, Olivera ; Lory, Stephen ; Pugsley, Anthony P.

Oxford BSL : Blackwell Science Ltd, UK

Molecular microbiology 27 (1998), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Escherichia coli K-12 strains grown at 37°C or 42°C, but not at 30°C, process the precursors of the Neisseria gonorrhoeae type IV pilin PilE and the Klebsiella oxytoca type IV pseudopilin PulG in a manner reminiscent of the prepilin peptidase-dependent processing of these proteins that occurs in these bacteria. Processing of prePulG in Escherichia coli requires a glycine at position −1, as does processing by the cognate prepilin peptidase (PulO), and is unaffected by mutations that inactivate several non-specific proteases. These data suggested that E. coli K-12 has a functional prepilin peptidase, despite the fact that it does not itself appear to express either type IV pilin or pseudopilin genes under the conditions that allow prePilE and prePulG processing. The E. coli K-12 genome contains two genes encoding proteins with significant sequence similarity to prepilin peptidases: gspO at minute 74.5 and pppA (f310c) at minute 67 on the genetic map. We have previously obtained evidence that gspO encodes an active enzyme but is not transcribed. pppA was cloned and shown to code for a functional prepilin peptidase capable of processing typical prepilin peptidase substrates. Inactivation of pppA eliminated the endogenous, thermoinducible prepilin peptidase activity. PppA was able to replace PulO prepilin peptidase in a pullulanase secretion system reconstituted in E. coli when expressed from high-copy-number plasmids but not when present in a single chromosomal copy. The analysis of pppA–lacZ fusions indicated that pppA expression was very low and regulated by the growth temperature at the level of translation, in agreement with the observed temperature dependence of PppA activity. Polymerase chain reaction and Southern hybridization analyses revealed the presence of the pppA gene in 12 out of 15 E. coli isolates.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1998.00723.x

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3

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Isolation and characterization of Pseudomonas aeruginosa genes inducible by respiratory mucus derived from cystic fibrosis patients (1996)

Wang, Jingyi ; Lory, Stephen ; Ramphal, Reuben ; [et al.]

Oxford BSL : Blackwell Science Ltd

Molecular microbiology 22 (1996), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Pseudomonas aeruginosa, an opportunistic human pathogen, is a major causative agent of mortality and morbidity in immunocompromised individuals and those with cystic fibrosis (CF). In CF patients, the secretion of abnormally high amounts of mucus into the airways contributes to their susceptibility to infection by P. aeruginosa. To identify virulence genes of P. aeruginosa that are important in infection of CF patients, an in vivo selection system (IVET) was used to identify promoters that are specifically inducible by respiratory mucus derived from CF patients. Three genetic loci that are highly inducible by the mucus were identified. One of them is a well-characterized virulence gene (fptA), encoding the receptor for pyochelin, which is a P. aeruginosa iron siderophore. Induction of the fptA gene by mucus is suppressed by the addition of exogenous iron, demonstrating that the mucus is an iron chelator and generates an iron-deficient environment in CF lungs. Therefore, as a part of the host-defence mechanism, the mucus could also be responsible for induction of iron-regulated virulence factors of bacterial pathogens. The second locus, np20, encodes a peptide that shares sequence homology to a number of transcriptional regulators. An identical locus was previously identified to be inducible in vivo during infection of mice and was shown to be important in bacterial virulence in a neutropenic-mouse infection model. The third locus, designated migA (mucus inducible gene), was sequenced and found to encode a 299-amino-acid peptide which is homologous to glycosyltransferases of other bacteria, and is involved in the biosynthesis of lipopolysaccharides or exopolysaccharides. Inducibilities of the np20 and migA genes are not affected by iron and the exact nature of the inducing signals in the mucus is not known. The possible implications of the migA inducibility by respiratory mucus is discussed in relation to the P. aeruginosa infection in CF.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1996.01533.x

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4

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PilR, a transcriptional regulator of piliation in Pseudomonas aeruginosa, binds to a cis-acting sequence upstream of the pilin gene promoter (1994)

Jin, Shouguang ; Ishimoto, Karyn S. ; Lory, Stephen

Oxford, UK : Blackwell Publishing Ltd

Molecular microbiology 14 (1994), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The PilR protein of Pseudomonas aeruginosa is a transcriptional activator of the pilin gene and belongs to a two-component sensor–regulator family. PilR was overproduced by fusing pilR to the gene for the maltose-binding protein (malE), yielding a MalE–PilR hybrid protein. The plasmid with the malE–pilR fusion, when introduced into a non-piliated pilR mutant strain of P. aeruginosa, restored piliation, indicating that the hybrid protein retains PilR function in vivo. The MalE-PilR protein was purified from Escherichia coli and used in a series of DNA-binding studies. A specific pilin promoter-binding activity of MalE-PilR was observed in a gef retardation assay. Subsequent DNase I footprinting analysis revealed a 40bp PilR-binding site located at the −120 to −80 region, relative to the transcriptional start site of the pilin gene. This PilR-binding region consists of a nine-base sequence and three consensus sequences of 5-(N)4–6C/GTGTC-3′, in a tandem array in which the first 7–9 bp are bound by the PilR on the non-Goding strand, leaving the last two nucleotides (TC) unbound. On the coding strand, PilR binds of sequences complementary to the two middle consensus sequences of the non-coding strand. A sequence similar to the NifA recognition site (5-TGT-(N)11-ACA-3′) is also found within the PilR-binding region. Deletion analysis and disruption of the individual consensus PilR-binding sequences by site-directed mutagenesis revealed that all four PilRbinding sites are absolutely required for the PilS/PilR-mediated pilin gene expression. The presence of four PilR-binding repeat sequences suggests that PilR protein may bind co-operatively or as a multimer.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2958.1994.tb01338.x

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5

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A novel two-component system controls the expression of Pseudomonas aeruginosa fimbrial cup genes (2005)

Kulasekara, Hemantha D. ; Ventre, Isabelle ; Kulasekara, Bridget R. ; [et al.]

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 55 (2005), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa requires the expression of a number of surface adhesive components. The expression of surface organelles facilitating biofilm formation is controlled by environmental signals acting through transcriptional regulatory networks. We analysed the expression of a family of P. aeruginosa adhesins encoded by three distinct fimbrial gene clusters (cupA, cupB and cupC). Using transposon mutagenesis, we have identified several regulatory loci that upregulated cupB and cupC transcription. One such locus contains three components, RocS1, RocR and RocA1, which represent a variant of a classical two-component signal transduction pathway. RocS1 is a sensor kinase, RocA1 is a DNA binding response regulator that activates cup genes, and RocR is an antagonist of RocA1 activity. Using a two-hybrid assay, we have shown that RocS1 interacts with receiver domains of both RocA1 and RocR. Expression of the Cup system in response to environmental stimuli is accomplished by a novel mechanism in which the sensor kinase activates its cognate response regulator through a phosphorelay pathway, while an additional repressor protein modulates this interaction.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04402.x

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6

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A four-tiered transcriptional regulatory circuit controls flagellar biogenesis in Pseudomonas aeruginosa (2003)

Dasgupta, Nandini ; Wolfgang, Matthew C. ; Goodman, Andrew L. ; [et al.]

Oxford, UK : Blackwell Publishing Ltd.

Molecular microbiology 50 (2003), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The single polar flagellum of Pseudomonas aeruginosa is an important virulence and colonization factor of this opportunistic pathogen. In this study, the annotation of the genes belonging to the fla regulon was updated and their organization was analysed in strains PAK and PAO1, representative type-a and type-b strains of P. aeruginosa respectively. The flagellar genes are clustered in three non-contiguous regions of the chromosome. A polymorphic locus flanked by flgJ and fleQ in Region I contains a glycosylation island in PAK. The expression and ordered assembly of the complex multicomponent flagellum is intricately regulated. Dedicated flagellar genes fleQ, fleS, fleR, fliA, flgM and fleN encode proteins that participate in the regulation of the flagellar transcriptional circuit. In addition, expression of the flagellum is coordinately regulated with other P. aeruginosa virulence factors by the alternative sigma factor σ54, encoded by rpoN. In order to gain insight into the hierarchical regulation of flagellar genes, deletion mutations were constructed in fleQ, fleR, fliA and rpoN. The transcriptional impact of these mutations was examined by transcriptional profiling using a P. aeruginosa whole genome microarray. Analysis of the transcriptomes generated for each of these mutants indicates a four-tiered (Classes I-IV) hierarchy of transcriptional regulation. Class I genes are constitutively expressed and include the transcriptional regulator fleQ and the alternative sigma factor fliA (σ28). Class II genes including fleSR, encoding a two-component regulatory system require FleQ and RpoN (σ54) for their transcriptional activation. Class III genes are positively regulated by the activated response regulator FleR in concert with RpoN. The transcription of Class IV genes is dependent on the availability of free FliA following the export of the FliA specific antisigma factor FlgM through the basal body rod-hook structure (assembled from Class II and III gene products). Two previously uncharacterized genes, which are coordinately regulated with known flagellar genes have been identified by genome-wide analysis and their role in flagellar biogenesis was analysed.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2003.03740.x

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7

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The XcpR protein of Pseudomonas aeruginosa dimerizes via its N-terminus (1997)

Turner, Leah R. ; Olson, John W. ; Lory, Stephen

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 26 (1997), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Extracellular protein secretion by the main terminal branch of the general secretory pathway in Pseudomonas aeruginosa requires a secretion machinery comprising the products of at least 12 genes. One of the components of this machinery, the XcpR protein, belongs to a large family of related proteins distinguished by the presence of a highly conserved nucleotide binding domain (Walker box A). The XcpR protein is essential for the process of extracellular secretion and amino acid substitutions within the Walker A sequence result in inactive XcpR. The same mutations exert a dominant negative effect on protein secretion when expressed in wild-type bacteria. Transdominance of XcpR mutants suggests that this protein is involved in interactions with other components of the secretion machinery or that it functions as a multimer. In this study, the amino-terminal portion of the cI repressor protein of phage λ was used as a reporter of dimerization in Escherichia coli following fusion to full-length as well as a truncated form of XcpR. The cI–XcpR hybrid proteins were able to dimerize, as demonstrated by the immunity of bacteria expressing them to killing by λ phage. The full-length XcpR as well as several deletion mutants of XcpR were able to disrupt the dimerization of the chimeric cI–XcpR protein. The disruption of cI–XcpR dimers using the deletion mutants of XcpR, combined with the analysis of their dominant negative effects on protein secretion, was used to map the minimal dimerization domain of XcpR, which is located within an 85 amino acid region in its N-terminal domain. Taken together, the data presented in this paper suggest that the XcpR protein dimerizes via its N-terminus and that this dimerization is essential for extracellular protein secretion.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1997.6201986.x

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8

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Gene expression in Pseudomonas aeruginosa biofilms (2001)

Whiteley, Marvin ; Bangera, M. Gita ; Bumgarner, Roger E. ; [et al.]

[s.l.] : Macmillian Magazines Ltd.

Nature 413 (2001), S. 860-864

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ISSN: 1476-4687

Source: Nature Archives 1869 - 2009

Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics

Notes: [Auszug] Bacteria often adopt a sessile biofilm lifestyle that is resistant to antimicrobial treatment. Opportunistic pathogenic bacteria like Pseudomonas aeruginosa can develop persistent infections. To gain insights into the differences between free-living P. aeruginosa cells and those in biofilms, ...

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1038/35101627

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9

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Identification and characterization of PilS, an essential regulator of pilin expression in Pseudomonas aeruginosa (1994)

Boyd, Jessica M. ; Koga, Tetsuro ; Lory, Stephen

Springer

Molecular genetics and genomics 243 (1994), S. 565-574

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ISSN: 1617-4623

Keywords: Pseudomonas aeruginosa ; Two-component regulatory systems ; Pili ; pilS ; pilR

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract Expression of the pilin gene, pilA, of Pseudomonas aeruginosa requires the alternative sigma factor, σ54, and also two other transcriptional regulators encoded by the pilS and pilR genes. These two linked genes, which have been identified by transposon insertion mutagenesis, share significant amino acid sequence homology with members of the two-component family of regulators. The transcriptional regulator, PilR, has been described previously. PilS, a 37285 Dalton protein, shares significant homology with the protein kinase sensors of the two-component regulatory family. PilS, however, has no hydrophobic domains which might be membrane-spanning alpha-helices, suggesting that PilS is a cytoplasmic protein. Characterization of the pilS gene revealed that when overexpressed in Escherichia coli by the bacteriophage T7 promoter it specifies a protein of approximately 40000 daltons, corresponding to the molecular weight of Pi1S predicted from the deduced amino acid sequence. Deletion analysis of the pilS promoter fused to a promoterless lacZ gene further showed that a significant region upstream of pilS is essential for expression of pilS and pilR, suggesting a need for transcriptional activation. The pilA promoter can be activated in E. coli but only when PilR and σ54 are present. This work suggests that the Pi1S activation signal is received in the bacterial cytoplasm, and that the mechanism of Pi1S/PiIR-mediated signal transduction resulting in activation of the pilin gene promoter is likely to be similar to that of other two-component systems.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00284205

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