ALBERT

Notizen: We describe a rapid non-radioactive DNA typing of the serological types DR1-DRw10 using polymerase chain reaction (PCR)-amplified DNA and 15 sequence-specific oligonucleotides (SSO) which are labelled enzymatically at their 3’end with one digoxigenin (DIG). The hybridized SSOs were detected using anti-DIG alkaline phosphatase and Fab fragments and visualization was obtained with the chemilumine-scent substate 3-(2‘-spiroadamantan)-4-(3′-phosphoryloxy)-phenyl-1,2-dioxetan (AMPPD). The results were identical with those of the previously used 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/4-nitrobluetetrazolium chloride (NBT) system. The use of AMPPD is more rapid and allows the repeated rehybridization of the membrane-bound DNA.

Notizen: Cell lines and a clone established from the C57BL/6 (H-2b) Lewis lung (3LL) tumour were previously characterized with respect to tumour growth and metastatic spread in vivo, and to the expression of a 3LL tumour-specific antigen (3LL TA) using a monoclonal antibody raised in syngeneic mice immunized with 3LL cells. No correlation was observed between the presence of 3LL TA and the prevention of metastatic spread which suggests that the immune recognition of this tumour antigen requires the presence of a self H-2 molecule absent from these tumour cells. Indeed, radioimmunoassay (RIA) and cytofluorometric analysis using specific monoclonal antibodies have shown that the H-2Kb molecule was not expressed at the cell surface of all 3LL cell lines and clones, while the H-2Db molecule was present at normal levels. This defect, which was not the consequence of a lack Of (32m expression, was accompanied by an absence or a marked reduction of the H-2K mRNA level (which has been reversed in the M4 cell line by in vitro gamma interferon treatment), while the H-2D class I gene was normally transcribed. Another defective transcription was also observed for a gene in the Tla region (gene 37). This low‘37’phenotype was corrected by in vitro treatment of the M4 cell line with gamma interferon, which indicates that this class I gene of the Qa/Tla region has an interferon response sequence in the promoter.

Notizen: Using H-2 recombinant congenic strains of mice, genetic analysis of resistance to murine hepatitis virus type 3 (MHV3)-induced paralysis was performed. It appeared that both H-2K and H-2D, two class I gene regions of the mouse major histocompatibility complex (MHC), can play independent significant roles in the establishment of such resistance.

Notizen: The HLA-DQw2 specificity, homogeneous in serology, is strongly associated to two HLA-DR specificities: DR3 and DR7. These alleles are found mainly on DQw2 bearing extended haplotypes with strong linkage disequilibrium. We describe, with BamHI, HindIII and RsaI, two restriction fragments length polymorphisms (RFLP) for the A gene of DQw2. These two subtypes correlated with the DR3 and DR7 specificities. Interestingly, by non-equilibrium pH gradient electrophoresis (NEPHGE), two DQα chains were also found, respectively correlated with the same DR specificities. In addition, Hindi polymorphism allowed us to distinguish several patterns of B genes for (DR7) DQw2 haplotypes but without any detectable association with another HLA marker. However, only one DQ(3 chain was found by NEPHGE in the (DR7) DQw2 haplotype. Furthermore, MncII discriminated the B genes of the two extended haplotypes: (B8, DR3) DQw2 and (B18, DR3) DQw2. The same result was found by NEPHGE: two DQβ chains were described, corresponding to the same extended haplotypes. The use of exon-specific DQB probes showed that the genomic polymorphism in DOw2 haplotypes is located, at least, at the 3’end of the gene. These data add new characteristics to the different DQw2 extended haplotypes.

Notizen: Differences between the mouse Ly5a and Ly5b alleles can be distinguished on the basis of polymerase chain reaction (PCR)-restriction enzyme analysis and differential monoclonal antibody reactivities. To more precisely map the Ly5 gene on the mouse chromosome 1, analytical DNA and protein tests were performed on recombinant inbred strains of mice prepared from SJL/J (Ly5a) and B ALB/cke (Ly5b) progenitor strains. Each recombinant inbred strain was characterized to determine whether it carried the Ly5a or Ly5b allele. Both assays, DNA-PCR and protein-immunofluorescence, yielded identical results for each strain examined. Placement of the Ly5 gene with respect to other characterized markers of mouse chromosome 1 for these recombinant inbred mouse strains shows a gene order of Idh-1.Ity:Pep3:/Ly5, CfhJ.

Notizen: Polymerase chain reaction/oligonucleotide typing was used to identify HLA-Cw*0601 (Cw6) in patients with psoriasis and psoriatic arthritis. The assignment of HLA-Cw*0601 was established by the concordant presence of codons for alanine (position 73), lysine (position 80) and tryptophan (position 97). The frequencies of all three codons were increased in the patient groups.

Notizen: Associations between a large number of diseases and markers within the major histocompatibility complex (MHC) have been described. In particular, susceptibility to several autoimmune disorders, including type I diabetes mellitus and rheumatoid arthritis, is linked to genes within the MHC and strong population associations are demonstrable between certain HLA class II alleles and these conditions. Genetic mapping of HLA susceptibility loci has traditionally relied on the use of phenotypic markers defined by alloantisera, cellular typing reagents and biochemical analysis of histocompatibility antigens. Polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) typing combines the ability to define the finest of HLA specificities, by analysis of the corresponding DN A sequences, with the possibility of studying large populations of normal and affected individuals. The applications of this technology to characterizing precisely the MHC loci associated with susceptibility to autoimmune diseases such as rheumatoid arthritis, type I diabetes mellitus, coeliac disease and pemphigus vulgaris are reviewed here.

Notizen: The protein kinase C (PKC) family comprises calcium- and phospholipid-dependent kinases whose activity is stimulated by diacylglycerol and tumour-promoting phorbol esters such as 12-tetradecanoyl phorbol-13-acetate (TPA). In the Gram-negative bacterium Escherichia coli, functional similarity to PKC was demonstrated in crude extracts by calcium and phospholipid-dependent, TPA-stimulated phosphorylation of a small number of endogenous substrates. Activity was reduced by sphingosine, a known inhibitor of eukaryotic PKC. Structural similarity to PKC was demonstrated in crude and partially purified bacterial extracts by cross-reactivity with several monoclonal antibodies. This revealed isozyme-specific homology between a protein(s) of relative molecular mass 80–85000 in E. coli and the α-and γ-isozymes, but probably not the β-isozyme, of eukaryotic PKC.

Notizen: Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4 and MAL6. Each MAL locus is complex consisting of at least three genes: a trans-acting activator, a maltose permease, and maltase. All the MAL loci show homology to each other both at the sequence level as determined by Southern transfer analysis and at the functional level as determined by complementation. We describe the organization of the MAL loci in yeast and the basic features of their regulation. The analysis of MAL has contributed to our understanding of the evolution of multigenic families. The global integration of carbohydrate metabolism, and gene regulation.

Notizen: Both ATP and an electrochemical potential play roles in translocating proteins across the Inner membrane of Escherichia coli. Recent discoveries have dissected the overall transmembrane movement into separate subreactions with different energy requirements, identified a translocation ATPase, and reconstituted both energy-requiring steps of the reaction from purified components. A more refined understanding of the energetics of this fundamental process is beginning to provide answers about the basic issues of how proteins move across the hydrophobic membrane barrier.

Notizen: Electrotransformation of Rhodococcus fascians by non-replicating plasmids containing a suitable resistance marker resulted in stable transformants by integration of these constructs at various sites in the genome, thereby generating different mutations. Tagged genes could be isolated in Escherichia coli owing to the presence of a CoIE1 replicon and an ampicillin resistance gene in the inserted sequences.Southern analysis and nucleotide sequencing revealed that recombination can occur at defined locations in the plasmid, while no site preference for target sequences could be detected. Low homology between the recombining sequences indicates illegitimate recombination. The specificity of the plasmid sites could be explained by assuming a linear recombination intermediate, generated by cleavage of the transformed plasmid.

Notizen: A group of four genes of Erwinia chrysanthemi involved in pectin degradation has been characterized. These four genes form independent transcription units and are regulated by the negative regulatory gene, kdgR. The functions of two of these genes are known: kduD codes for the 2-keto-3-deoxygluconate oxydoreductase and kdul for the 5-keto-4-deoxyuronate isomerase, two enzymes of the pectin degradation pathway. kdgC has 36% homology with pectate lyase genes of the periplasmic family but its product does not seem to have pectinolytic activity. The fourth gene, kdgF, could have a role in the pathogenicity of E. chrysanthemi. A comparison of the regulatory regions of all the genes controlled by kdgR allowed better definition of the KdgR-binding-site consensus.

Notizen: To establish the molecular basis of the chromosomal virulence genes of Shigella flexneri 2a (YSH6000), a Notl restriction map of the chromosome was constructed by exploiting Notl-linking clones, partial Notl digestion and DNA probes from various genes of Escherichia coli K-12. The map revealed at least three local differences in the placements of genes between YSH6000 and E. coli K-12. Using the additional Notl sites introduced by Tn5 insertion, nine virulence loci Identified previously by random Tn5 insertions were physically mapped on the chromosome. To demonstrate the versatility of the Notl map in direct assignment of the virulence loci tagged by Tn5 to a known genetic region in E. coli K-12, the major class of avirulent mutants defective in the core structure of lipopolysaccharide (LPS) was examined for the sites of Tn5 insertions. The two Notl segments created by the Tn5 insertion in the Notl fragment were analysed by Southern blotting with two DNA probes for the 5′ and 3’flanking regions of the rfa region, and shown to hybridize separately with each of them, confirming the sites of Tn5 in the rfa locus. This approach will facilitate direct comparison of genetically mapped Tn5 insertion mutations of S. flexneri with genes physically determined in E. coli K-12.

Notizen: The O antigen of Escherichia coli O111 is identical in structure to that of Salmonella enterics serovar adelaide. Another O-antigen structure, similar to that of E. coli O111 and S. enterica serovar adelaide is found in both E. coli O55 and S. enterica serovar greenside. Both O-antigen structures contain colitose, a 3,6 dideoxyhexose found only rarely in the Enterobacteriaceae. The O-antigen structure is determined by genes generally located in the rfb gene cluster. We cloned the rfb gene cluster from an E. coli O111 strain (M92), and the clone expressed O antigen in both E. coli K-12 and a K-12 strain deleted for rfb. Lipopoly-saccharide analysis showed that the O antigen produced by strains containing the cloned DNA is polymerized. The chain length of O antigen was affected by a region outside of rfb but linked to it and present on some of the plasmids containing rfb. The rfb region of M92 was analysed and compared, by DNA hybridization, with that of strains with related O antigens. The possible evolution of the rfb genes in these O antigen groups is discussed.

Notizen: A 2 kb DNA fragment isolated from a cosmid library of Aquaspirillum magnetotacticum strain MS-1 complements the aromatic-metabolite requirements and iron-uptake deficiencies of Escherichia coli and Salmonella typhimurium strains that lack a functional aroD (biosynthetic dehydrodquinase) sequence. All recombinant cosmids selected for their aroD complementation property carry this sequence. No DNA sequence homology has, however, been detected by Southern hybridization between the cloned fragment and the aroD gene of E. coli or the qa2 (catabolic dehydroquinase) gene of Neurospora crassa.

Notizen: Evidence for ptelotropic activation of virulence genes in Listeria monocytogenes is presented. A complementation study of a spontaneous prfA-deletion mutant and analysis of cassette and transposon insertion mutants showed that the gene prfA activates the transcription of four independent genes which code for a phosphatidyl-inositol-specific phospholipase C (gene plcA), listeriolysin O (gene hlyA), a metallo-protease (gene prtA) and a lecithinase (gene prtC). Transcription of prfA is not constitutive. During the growth phase, two peaks of prfA transcript accumulation were observed: the first was during exponential growth, and the second was at the beginning of the stationary phase. In addition, two prf4-specific transcripts of 2.2 kb and 1 kb are detected. Early in exponential growth, prfA is co-transcribed with plcA which lies upstream prfA, giving rise to the 2.2 kb plcA-prfA transcript. In late-exponential growth and at the beginning of the stationary phase, prfA transcripts of 1 kb are predominantly detected. Our results demonstrate that since prfA controls plcA transcription, it also regulates its own synthesis.

Notizen: It is now well documented that some enteric bacteria which cause diarrhoeal and/or dysenteric disease produce, at high levels, one or more of a family of protein toxins referred to as Shiga toxin and Shiga-like toxins (SLTs; alternatively called verocytotoxins or VTs). Within the past few years, there have been considerable advancements made in our understanding of the biochemistry and molecular biology of Shiga toxin and SLTs. However, the precise role of the toxins in mediating colonic disease, as well as their contribution to the development of extra-intestinal sequelae (e.g. the haemolytic uraemic syndrome and neurological disorders), remain less clear. In this MicroReview, we will briefly summarize recent progress in Shiga toxin- and SLT-related research and present evidence supporting the concept that these toxins contribute to pathogenesis by directly damaging vascular endothelial cells, thereby disrupting the homeostatic properties of these cells. We will also discuss data which suggest that toxin-mediated damage in the kidney may not be limited to glomerular endothelial cells but may include tubular epithelial cells. Thus, the role of the toxins in renal disease may not be limited to the glomeruli, as was initially hypothesized when the association of infection with toxin-producing strains and the development of acute renal failure was established.

Notizen: Retrotransposons are a widely distributed group of eukaryotic mobile genetic elements that transpose through an RNA intermediate. The element Ty (Transposon yeast), found in the yeast Saccharomyces cerevisiae, is a model system for the study of retrotransposons because of the experimental tools that exist to manipulate and detect transposition. Ty transposition can be elevated to levels exceeding one transposition event per cell when an element is expressed from an inducible yeast promoter. In addition, individual genomic Ty elements can be tagged with a retrotransposition indicator gene that allows transposition events occurring at at a rate of 10−5 to 10−7 per element per cell division to be detected phenotypically. These systems are being used to elucidate the mechanism of Ty transposition and clarify how Ty transposition is controlled.

Notizen: Oligonucleotide primers derived from the ipaH7.8 sequence have been used to determine the boundaries of DNA sequence homology among five lpaH genes on the invasion plasmid (pWR100) of Shigella flexneri 5, strain M9OT-W. The primary structure of lpaH4.5 has been established from DNA sequence analysis. The first 197 amino acids in lpaH7.8 were replaced in lpaH4.5 by a unique set of 251 amino acids, generating two related proteins with variable and conserved sequences. The amino-terminal region of lpaH4.5 displayed an internal repeat structure, also seen in lpaH7.8, characteristic of members of the leucine-rich glycoprotein (LRG) family. The DNA sequences of ipaH2.5 and ipaH1.4 indicate that these genes are truncated versions of lpaH7.8. Western blot analysis of a λgt11 ipaH recombinant (W7) subclone demonstrated that the antigenicity of lpaH7.8 resides outside the leucine-rich repetitive region.

Notizen: The meta operon of the Pseudomonas putida TOL plasmid (pWW0) encodes all enzymes of a meta-cleavage pathway for the metabolism of benzoic acids to Krebs-cycle intermediates. We have determined and analysed the nucleic acid sequence of a 3442 bp region of the meta operon containing the xylGFJ genes whose products are involved in the post meta-ring fission transformation of catechols. Homology analysis of the xylGFJ gene products revealed evidence of biochemical relatedness, suggested enzymatic mechanisms, and permitted us to propose evolutionary events which may have generated the current variety of aromatic degradative pathways.The xylG gene, which specifies 2-hydroxymuconic semialdehyde dehydrogenase (HMSD), was found to encode a protein of 51.7 kDa. The predicted protein sequence exhibits significant homology to eukaryotic aldehyde dehydrogenases (ADHs) and to the products of two other Pseudomonas catabolic genes, i.e. xylC and alkH. Expansion of the ADH superfamily to include these prokaryotic enzymes permitted a broader analysis of functionally critical ADH residues and phylogenetic relationships among superfamily members. The importance of three regions of these enzymes previously thought to be critical to ADH activity was reinforced by this analysis. However glutamine-487, also thought to be critical, is less well conserved. The revised ADH phylogeny proposed here suggests early catabolic ADH divergence with subsequent interkingdom gene exchange.The xylF gene, which specifies 2-hydroxymuconic semialdehyde hydrolase (HMSH), was delineated by N-terminal sequence analysis of the purified gene product and is shown to encode a protein of 30.6 kDa. Homology analysis revealed sequence similarity to a chromosomally encoded serine hydrolase, especially in the region of the previously identified active-site serine residue, suggesting that HMSH may also possess a serine hydrolytic enzymatic mechanism.Likewise, the xylJ gene, which specifies 2-hydroxypent-2,4-dienoate hydratase (HPH), was delineated by N-terminal sequence analysis of purified HPH, and was found to encode a 23.9 kDa protein.Sequence comparisons revealed that both HMSH and HPH have analogues in the tod gene cluster, which specifies a toluene/benzene degradative pathway. Although the newly identified todF and todJ genes had been at least partially sequenced (Zylstra and Gibson, 1989), the open reading frames had not been positively identified. The presence of todJ provides strong evidence that the reactions following ring fission in the tod pathway are identical to those of the TOL pathway.The meta operon is one of the longest prokaryotic operons known. The structure of the xylGFJ genes displays short intergenic regions and overlapping translational signals. This genetic organization suggests a mechanism of post-transcriptional regulation whereby translational competency of downstream genes might be enhanced and lengthy transcripts stabilized against ribonucleolytic attack. Such an operon structure may have evolved to facilitate adequate expression of promoter distal genes of long operons.

Notizen: Vibrio cholerae is known to secrete DNase(s) into the extracellular environment. These proteins have been thought to be responsible for the difficulties in transforming this organism. In this work we demonstrate that the dns and xds genes differ and that their products are solely responsible for the extracellular DNase activity. By site-directed mutagenesis, strains have been constructed which are mutant in one or both genes. These strains have been assessed for their ability to be transformed with plasmid DNA and for their virulence in the infant mouse cholera model. DNase-deficient mutants can be readily transformed and the product of dns appears to be the more significant barrier. No effect on virulence was observed with the mutants.

Notizen: As an additional system for analysing mutations that appear to be specifically induced or directed, we have used a plasmid that contains the mnt repressor gene inserted as an operon fusion with the tet gene of the plasmid pBR322. Thus, the mnt gene product acts as a negative transcriptional regulator of tet gene expression. Mutations inactivating the Mnt repressor are recessive while those destroying operator recognition (Oc) are dominant in conferring tetracycline resistance on the host. When resistance mutations were isolated on plates with high levels of tetracycline they were preferentially mnt- and the plasmids were monomers. Pre-exposure to low concentrations increased the frequency of resistant mutants by 100- to 1000-fold, and the mutations were now mostly Oc, located on one unit of a plasmid multimer. Recessive repressor mutations on one unit would not have been selected. We suggest that the high frequency of mutation in tandem multimeric plasmids may be caused by the formation of single-stranded and hence highly mutable regions by homologous pairing out of register. The role of tetracycline in promoting mutations is discussed.

Notizen: At the onset of starvation Escherichia coli undergoes a temporally ordered program of starvation gene expression involving 40–80 genes which some four hours later yields cells possessing an enhanced general resistance. Two classes of genes are induced upon carbon starvation: the cst genes, requiring cyclic AMP, and the pex genes, not requiring this nucleotide for induction. The cst genes are not involved in the development of the resistant state and are concerned with escape from starvation, while the pex gene induction appears to be associated with resistance. Many of the latter are induced in response to a variety of starvation conditions. They include heat shock and oxidation resistance genes, and some utilize minor, stationary-phase-specific sigma factors for induction during starvation. The protective role of stress proteins may be due to their ability to rescue misfolded macromolecules. The starvation promoters can be potentially useful for selective expression of desired genes in metabolically sluggish populations, e.g. in high-density industrial fermentations and in situ bioremediation.

Notizen: During carbon-starvation-induced entry into stationary phase, Escherichia coli cells exhibit a variety of physiological and morphological changes that ensure survival during periods of prolonged starvation. Induction of 30–50 proteins of mostly unknown function has been shown under these conditions. In an attempt to identify C-starvation-regulated genes we isolated and characterized chromosomal C-starvation- induced csi::lacZ fusions using the λplac Mu system. One operon fusion (csi 2::lacZ) has been studied in detail. csi 2::lacZ was induced during transition from exponential to stationary phase and was negatively regulated by cAMP. It was mapped at 59 min on the E. coli chromosome and conferred a pleiotropic phenotype. As demonstrated by two-dimensional gel electrophoresis, cells carrying csi 2::lacZ did not synthesize at least 16 proteins present In an isogenic csi 2+ strain. Cells containing csi 2::lacZ or csi 2::Tn10 did not produce glycogen, did not develop thermo-tolerance and H2O2 resistance, and did not induce a stationary-phase-specific acidic phosphatase (AppA) as well as another csi fusion (csi5::lacZ). Moreover, they died off much more rapidly than wild-type cells during prolonged starvation. We conclude that csi 2::lacZ defines a regulatory gene of central importance for stationary phase E. coli cells. These results and the cloning of the wild-type gene corresponding to csi 2 demonstrated that the csi 2 locus is allelic with the previously identified regulatory genes katF and appR. The katF sequence indicated that its gene product is a novel sigma factor supposed to regulate expression of catalase HPII and exonuclease III (Mulvey and Loewen, 1989). We suggest that this novel sigma subunit of RNA polymerase defined by csi 2/katF/appR is a central early regulator of a large starvation/stationary phase regulon in E. coli and propose ‘rpoS’ (‘σs’) as appropriate designations.

Notizen: Rhizobium leguminosarum biovar phaseol CFN23 loses its ability to nodulate beans at a high frequency because of a deletion of part of its symbiotic (pSym) plasmid (Soberón-Chávez et al., 1986). We report here that at least 80kb of pSym are deleted upon loss of the symbiotic phenotype; the deletion removes the nitrogenase structural nifHDK and the common nod ABC genes. The size of the deleted pSym is not reduced, since it is accompanied by an amplification of other pSym plasmid sequences. This genetic rearrangement is similar to the deletion and amplification of yeast mitochondrial DNA leading to‘petite’mutations.

Notizen: The 58/59 min region of the Escherichia coli chromosome contains two divergently oriented gene clusters coding for proteins with a function in hydrogenase formation. One cluster (the hyc operon), transcribed counterclockwise with respect to the E. coli chromosome, codes for gene products with a structural role in hydrogenase 3 formation (Böhm et al., 1990). The nucleotide sequence of the divergently transcribed operon (hyp) has been determined. It contains five genes, all of which are expressed in vivo in a T7 promoter/polymerase system, and the sizes of the synthesized products correspond with those predicted from the amino acid sequence. Complementation analysis of previously characterized mutants showed that the hypB, hypC and hypD genes have a function in the formation of all three hydrogenase isoenzymes, lesions in hypB being complemented by high nickel ion concentration in the medium. Prevention of hypBCDE gene expression led to an altered electrophoretic pattern of hydrogense 1 and 2 constituent subunits, indicating increased chemical or proteolytic susceptibility. Under fermentative growth conditions, operon expression was governed by an NtrA-dependent promoter lying upstream of hypA working together with an fnr gene product-dependent promoter which was localized within the hyp A gene. The latter (operon-internal) promoter is responsible for hypBCDE transcription under non-fermentative conditions when the -24/-12 NtrA-dependent promoter upstream of hyp A is silent.

Notizen: In order to investigate the basis of functional diversity among the pyridine nucleotide-oxidoreductases the gor gene from Pseudomonas aeruginosa PAO, which encodes glutathione reductase, was analysed. The P. aeruginosa gor gene was identified by hybridization with a short DNA sequence from the gene encoding mercuric reductase in transposon Tn501. The gene was cloned, sequenced and overexpressed in Escherichia coli. Expression of the gene enabled rescue of an E. coli gor- mutant, confirming the identity of the cloned gene. The predicted sequence of the gene product showed homology with other members of the pyridine nucleotide-disulphide oxidoreductase family, and allowed determination of positions that may be involved in substrate specificity. These predictions provided information on the relationship of sequence to function, independently of structural data used in previous studies.

Notizen: A 1.3kb cDNA (cDNA52) was derived from Trypanosoma cruzi trypomastigote mRNA. Using single stranded probes in Northern blots, we identified the putative coding strand of cDNA52. In addition, a minor band was detected in RNA from epimastigotes that was absent in RNA from trypomastigotes. Nucleotide sequence analysis revealed that cDNA52 was highly homologous to T. cruzi kinetoplast DNA minicircle sequences. All four conserved regions of T. cruzi minicircles were identified in cDNA52. Using several criteria, we demonstrated that the hybridization signals were not caused by contaminating minicircle DNA in the RNA preparations. The data provide direct evidence for the unprecedented finding that the entire length of a kDNA minicircle is transcribed in T. cruzi.

Notizen: Transcription of the Vibrio cholerae hlyA gene, which encodes a cytotoxic haemolysin, has been investigated. The hlyA transcript initiates 430 nucleotides (nt) upstream of the translational start site, hlyA–cat transcriptional fusion constructs were active in V. cholerae but not in Escherichia coli. An hlyA–cat fusion was used to select, from a V. cholerae O17 plasmid library, a clone that could activate the hlyA promoter in E coli. This regulatory locus has been designated hlyU. hlyU appears to be distinct from the previously described hlyR locus.

Notizen: The ctaBCDEF genes coding for cytochrome c oxidase were found to reside adjacent to a regulatory gene ctaA at 127° on the Bacillus subtilis chromosome. The structural genes for subunits I and II, ctaD and ctaC, were deleted by gene-replacement using a phleomycin-resistance marker. The mutant was unable to oxidize N,N,N′,N′-tetramethyl-p-phenylene-diamine and oxidized cytochrome c at a significantly lower rate. Absorption spectra of the mutant and wild-type membranes confirmed the presence of two haem A-containing enzymes in B. subtilis. Another mutant, with a spontaneous deletion upstream from CtaC, was found to express neither of these enzymes. Radioactive haem-labelling was used to identify sub-unit ii, which contains a haem C, and cytochrome c-550 among the membrane-bound c-type cytochromes of B. subtilis.

Notizen: Synthesis of the capsular polysaccharide colanic acid in Escherichia coli K12 Is regulated by a complex network of regulatory proteins. This regulation is expressed at the level of transcription of the cps (capsular polysaccharide synthesis) genes. Two positive regulators, RcsA and RcsB, are necessary for maximal capsule expression. The availability of RcsA is normally limited because the RcsA protein is rapidly degraded by the Lon ATP-dependent protease. Therefore Lon acts, indirectly, as a negative regulator of capsule synthesis. The sequence predicted for RcsB suggests that it is the effector component of a two-component system; a protein with homology to sensors, RcsC, also plays a role in capsule regulation. We propose a model for capsule synthesis in which RcsA interacts with RcsB to stimulate transcription of the cps genes. The mechanism of regulation of colanic acid synthesis in E. coli may apply to other capsules in a variety of Gram-negative bacteria.

Notizen: Filamentous phages present a genetically well-defined system for studying the ordered membrane assembly of five different phage-encoded proteins around the circular single-stranded DNA phage genome. Assembly occurs at high efficiency in vivo, catalysed by two phage-encoded membrane proteins and al least one host protein, thioredoxin. This review presents a description of the virion and its cytoplasmic precursor and summarizes the results of genetic and biochemical experiments that are beginning to elucidate the rote of the three morphogenetic proteins. The recent discovery of bacterial transport proteins with homology to a phage morphogenetic protein located in the outer membrane suggests the existence of a common mechanism for moving complex macromolecules across bacterial membranes.

Notizen: The chromosomal gef gene of Escherichia coli is a member of the gef gene family which encodes strongly toxic proteins of about 50 amino acids. We demonstrate here that the Gef protein is detectable by anti-peptide antibodies. Furthermore, we show that Gef is anchored in the cytoplasmic membrane by the N-terminal part of the protein, and that the C-terminal part is localized in the periplasm in a dimeric form with at least one disulphide bond. By mutagenesis of gef it is shown that the periplasmic portion of Gef encodes the toxic domain and that the dimerization of Gef is not essential for the toxic effect.

Notizen: We have cloned a gene encoding a polygalacturonase (PG) in the filamentous fungus Aspergillus niger RH5344. The structural gene comprises 1141 bp codIng for 362 amino acids and the open reading frame is disrupted by one intron of 52 bp. Eukaryotic consensus sequences for transcription regulation are found only in deviated forms. The biological functionality of the isolated PG gene was established by retransformation in A. niger and Aspergillus awamori. In addition, we have found that the PG protein of A. niger shares significant similarities with PG proteins from tomato and Erwinia carotovora. Comparison of the three enzymes revealed a highly conserved region in their C-terminal region probably comprising the elements of substrate binding and the catalytic centre.

Notizen: The plasmid pBRD026, which directs expression of the B subunrt of the Escherichia coli heat-labile toxin (LTB), was modified so that DNA encoding epitopes could be inserted at the 3 end of the gene. An oligonucleotide linker containing restriction sites for BglH and Spel was inserted at the Spel site at the 3′ end of the LTB gene to form plasmid pFV1. This linker also encodes the amino acid sequence Gly–Pro–Gly–Pro which we propose acts as a ‘hinge’ between the LTB and the foreign epitope. Oligonucleotides specifying an epitope from the Bordetelia pertussis P.69 outer membrane protein were cloned into pFV1 to form pFV169. The resultant fusion protein (LTB69) was partially purified from the periplasm of E. coli strains in a soluble pentameric form which could bind GM1 gangliosides. Mice immunized intranasally with purified LTB69 produced antibodies against both LTB and the P.69 protein. In addition, ELISPOT assays demonstrated the presence of LTB-specific and P.69-specific antibody-secreting cells in the lungs of immunized mice.

Notizen: Class 5 outer membrane proteins of Neisseria meningitidis show both phase- and antigenic variation of expression. The proteins are encoded by a family of opa genes that share a conserved framework interspersed with three variable regions, designated the semivariable (SV) region and hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of all of the opa genes of meningococcal strain FAM18, to assess the structural and antigenic variability in the family of proteins made by one strain. Pulsed field electrophoresis and Southern blotting showed that there are four opa genes in the FAM18 chromosome, and that they are not tightly clustered. DNA sequence analysis of the four cloned genes showed a modest degree of diversity in the SV region and more extensive differences in the HV1 and HV2 regions. There were four versions of HV1 and three versions of HV2 among the four genes. Each of the FAM18 opa loci contained a gene with a unique combination of SV, HV1, and HV2 sequences. We used λgt11 cloning and synthetic peptides to demonstrate that HV2 sequences completely encode the epitopes for two monoclonal antibodies specific for different class 5 proteins of FAM18.

Notizen: The population of capsulate Haemophilus influenzae is divided into two phylogenetic divisions. Here we show that in division I strains the capsulation (cap) gene cluster lies between direct repeats of a novel insertion sequence (IS)-like element, IS 1016. cap has apparently been mobilized in the chromosome as a compound transposon by IS 1016, and the repeats have provided a molecular substrate for reversible cap gene amplification, with augmentation of capsule production, through unequal homologous recombination. Such amplification has occurred in serotype b strains, but in these a large direct repeat of cap genes has become fixed in the population. We have found a 1.2kb deletion at one end of this duplicated cap b locus, removing most of one copy of the polysaccharide export gene bexA. We have shown that this makes capsulation dependent on preservation of the direct repeat structure in order to avoid recombination-mediated loss of the other copy of bexA. Type b strains with this cap configuration are disseminated worldwide and currently cause nearly all invasive Haemophilus infections, leading us to speculate that the 1.2 kb deletion occurred in an ancestral type b strain and conferred significant biological advantage.

Notizen: Genetic studies have identified a number of genes whose products appear to be required for the transport of the group A colicins and the single-stranded DNA of certain filamentous bacteriophages into Escherichia coli. Mutations in these genes allow normal binding of the colicins to their outer-membrane receptors and of the bacteriophage to the tip of specific conjugative pili, but do not allow translocation of the macromolecules to their target. These mutations have been designed‘tolerant’(tol) mutations and the protein products specified by these genes appear to comprise part of a transport system known as the Tol import system. Some of these genes have been isolated, sequenced and their protein products localized to the membranes or periplasm of E. coli. Information is also available regarding the domains of the colicins or phage proteins which interact with the Tol proteins. A preliminary model of the location and possible interactions of the Tol proteins is presented.

Notizen: Previous work in our laboratory suggested that DNA topology could be implicated in the regulation of the division gene ftsZ. To settle this question, we have selected and characterized mutants in the gyrB gene able to phenotypically suppress the defects of the ftsZ84 mutation. No strict correlation was found between the degree of plasmid DNA relaxation and the level of suppression of the thermosensitivity of the ftsZ84 strain. Interestingly, the class of mutants that shows maximal suppression is substantially unaffected in DNA topology. In addition, the amount of ffsZ-specific mRNA in this class of mutants is comparable to that present in the ftsZ84 strain. These results hint that the ability of these gyrB mutants to correct the effects of the ftsZ84 mutation is largely unrelated to the function of the GyrB (as a part of DNA gyrase) in the control of DNA superhelicity and suggest hitherto unsuspected interaction between the ftsZ and gyrB gene products.

Notizen: A previous study in our laboratory identified a surface-exposed peptidoglycan-associated protein of Neisseria gonorrhoeae which had an apparent molecular mass of 44000 daltons (44kDa) (Hill and Judd, 1989). This paper reports results which confirm that the 44 kDa protein is surface-exposed, and that the protein is expressed in, and is structurally invariant among, 14 strains of N. gonorrhoeae. The fact that the 44kDa outer-membrane protein is found in a conserved form in all gonococci examined strongly suggests that ft is crucial to the bacterium's survival. Moreover, it appears that this protein is a penicillin-binding protein (PBP3) (Shafer and Judd, 1991). This invariant, surface-exposed, peptidoglycan-associated outer-membrane protein deserves further investigation to elucidate its role in the immunobiology of N. gonorrhoeae, and its possible use as an immunoprophylactic reagent.

Notizen: A 34 kb fragment of the Nocardia lactamdurans DNA carrying the cluster of early cephamycin biosynthetic genes was cloned in λ EMBL3 by hybridization with probes internal to the pcbAB and pcbC genes of Penicillium chrysogenum and Streptomyces griseus. The pcbAB and pcbC genes were found to be closely linked together in the genome of N. lactamdurans. The pcbAB gene of N. lactamdurans showed the same orientation as the pcbC gene, in contrast to the divergent expression of the genes in the pcbAB-pcbC cluster of P. chrysogenum and Acremonium chrysogenum. The pcbAB gene encodes a large (3649 amino acids) multidomain δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase with a deduced Mr, of 404134. This enzyme contains three repeated domains and a consensus thioesterase active-site sequence. The pcbC gene encodes a protein of 328 amino acids with a deduced Mr of 37469, which is similar to other isopenicillin N synthases except that it lacks one of two cysteine residues conserved in all other isopenicillin N synthases. The different organization of the pcbAB-pcbC gene cluster in N. lactamadurans and Streptomyces clavuligerus relative to P. chrysogenum and A. chrysogenum is intriguing in relation to the hypothesis of horizontal transference of these genes from actinomycetes to filamentous fungi by a single transfer event.

Notizen: Selenocysteyl-tRNAs that decode UGA were identified previously in animal and bacterial cells and the genes for these tRNAs have been shown to be widespread in animals and eubacteria. In the present study, we identify a selenocysteyl-tRNA that codes for UGA in Thalassiosira pseudonana, which is a diatom, and in Tetrahymena borealis, which is a ciliate. The fact that these very diverse unicellular organisms also contain a selenocysteyl-tRNA suggests that selenocysteine-containing proteins and the use of UGA as a codon for selenocysteine are widespread, if not ubiquitous, in nature.

Notizen: OmpC, a major outer-membrane protein, is highly expressed when Salmonella typhi is grown in nutrient broth (NB) of either low (NB + 0% sucrose) or high (NB + 20% sucrose) osmolarity. This contrasts with the expression of Escherichia coli OmpC, which is inhibited in low osmolarity and enhanced in high osmolarity, as has been described previously (van Alphen and Lugtenberg, 1977; Verhoef et al., 1979; Kawaji et al., 1979). Nevertheless, expression of S. typhi OmpC is dependent on the E. coli OmpR transcriptional activator. These findings suggest differences between the mechanisms of osmoregulation of gene expression in both bacteria, although common effectors appear to be shared.

Notizen: Glucose is taken up in Bacillus subtilis via the phosphoenolpyruvate:glucose phosphotransferase system (glucose PTS). Two genes, orfG and ptsX, have been implied in the glucose-specific part of this PTS, encoding an Enzyme IIGlc and an Enzyme IIIGlc, respectively. We now show that the glucose permease consists of a single, membrane-bound, polypeptide with an apparent molecular weight of 80000, encoded by a single gene which will be designated ptsG. The glucose permease contains domains that are 40-50% identical to the IIGlc and IIIGlc proteins of Escherichia coli. The B. subtilis IIIGlc domain can replace IIIGlc in E. coli crr mutants in supporting growth on glucose and transport of methyl α-glucoside.Mutations in the IIGlc and IIIGlc domains of the B. subtilis ptsG gene impaired growth on glucose and in some cases on sucrose. ptsG mutants lost all methyl α-glucoside transport but retained part of the glucose-transport capacity. Residual growth on glucose and transport of glucose in these ptsG mutants suggested that yet another uptake system for glucose existed, which is either another PT system or regulated by the PTS.The glucose PTS did not seem to be involved in the regulation of the uptake or metabolism of non-PTS compounds like glycerol. In contrast to ptsl mutants in members of the Enterobacteriaceae, the defective growth of B. subtilis ptsl mutants on glycerol was not restored by an insertion in the ptsG gene which eliminated IIGlc. Growth of B. subtilis ptsG mutants, lacking IIGlc, was not impaired on glycerol. From this we concluded that neither non-phosphorylated nor phosphorylated IIGlc was acting as an inhibitor or an activator, respectively, of glycerol uptake and metabolism.

Notizen: In the life cycle of a bacterium there are several key processes: cellular growth, chromosome replication and decatenation, nucleoid partition, septum formation, and cell division. These processes have to be carefully controlled and co-ordinated both with respect to each other and to the growth of the cell, and could be viewed as parts of a single cycle in which each step is dependent upon the previous one. Alternatively, they could be independently controlled and carefully tuned to each other without actually constituting a true cycle. In this review, using Escherichia coli as model system, we discuss these two ways of describing the bacterial life cycle. The evidence supporting independent control of the processes is presented, and some of the key questions in the elucidation of the regulation of the bacterial life cycle are discussed.

Notizen: Calcium and calcium-binding proteins including those resembling calmodulin are implicated in numerous diverse processes in bacteria. These processes include chemotaxis, sporulation, virulence, the transport of sugars and proteins, phosphorylation, heat shock, the initiation of DNAS replication, septation, nucleoid structure, nuclease activity and recombination, the stability of the envelope, and phospholipids synthesis and configuration. That such varied processes should have a common factor, calcium, suggests major underlying principles of calcium metabolism metabolism which have yet to be discovered.

Notizen: We describe a family of highly conserved, Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences, 14 of which have been identified in Escherichia coli and Salmonella typhimurium and a further three in other enterobacterial species (Yersinia pseudotubercuiosis, Kiebsiella pneumoniae and Vibrio cholerae). ERIC sequences are 126 bp long and appear to be restricted to transcribed regions of the genome, either in intergenic regions of polycistronic operons or in untranslated regions upstream or downstream of open reading frames. ERIC sequences are highly conserved at the nucleotide sequence level but their chromosomal locations differ between species. Several features of ERIC sequences resemble those of REP sequences (Stern et al., 1984) although the nucleotide sequence is entirely different. The question of whether ERICs have a specific function, or represent a form of 'selfish’DNA, is discussed.

Notizen: Two distinct steps in the secretion of the extracellular, cell-surface-anchored lipoprotein pullulanase by Escherichia coli were uncoupled by allowing export of the enzyme to the cytoplasmic membrane via the signal peptide sec-gene-dependent general export pathway, and then inducing the pulC—0 operon of genes required for translocation to the cell surface. The secretion intermediate cofractionated mainly with intermediate-density vesicles when cells were gently lysed and the resulting vesicles were separated by isopycnic sucrose density centrifugation. Cytoplasmic forms of pullulanase (which are not exported because they lack a functional signal peptide) are more sensitive to heat inactivation, denaturation by sodium dodecyl sulphate and carboxymethylation than the intermediate and cell-surface forms. The latter are distinguished only by the fact that the secretion intermediate is less susceptible to proteinase K and trypsin, and is partially inaccessible to substrate or in an inactive conformation in sphaeroplasts. These and other results indicate that the secretion intermediate can acquire considerable higher-ordered structure, including disulphide bridges, before it is transported to the cell surface; this seems to rule out the possibility that it is threaded through this membrane as a locally unfolded polypeptide.

Notizen: The initiator protein RepA1 of the IncFII replicon RepFIC derived from the enterotoxin plasmid EntP307 has been cloned under the control of the λ Pl promoter. This has enabled us to overproduce this protein and study its properties. Here we show that RepA1 is a soluble basic protein with an experimentally determined molecular weight of 40000. Deletion analysis indicates that the overproduced protein originates from the open reading frame which we previously designated as coding for RepA1. We have also shown that the replication function of the replicon RepFIC depends on the intact RepA1 coding frame.

Notizen: Contrary to previous reports, lipopolysaccharides from Pseudomonas cepacia contain a 3-deoxyoct-2-ulosonic acid (probably a single residue). The lipopolysaccharides contain only two phosphate residues, one of which apparently forms a phosphodiester bridge between 4-amino-4-deoxyarabinose and a glucosamine residue in lipid A. The second, unlocated phosphate residue occurs mainly as a monoester in some lipopolysaccharides, and mainly as a diester in others. All lipopolysaccharides lack pyrophosphate residues. The results support the view that the resistance of P. cepacia to cationic antibiotics stems from ineffective binding to the outer membrane, as a consequence of the low number of phosphate and carboxylate groups in the lipopolysaccharide, and the presence of the protonated aminodeoxypentose.

Notizen: Gas vesicles are subcellular inclusions found in a large number of aquatic prokaryotes. The gvpA gene, which frequently occurs as a multigene family, encodes the major gas vesicle structural protein. In several cyanobacteria, another gene, gvpC, encodes a different protein which might be a dispensable element for gas vesicle formation. We report here the molecular characterization of a gvpA gene in Pseudanabaena sp. PCC 6901. In this planktonic cyanobacterium, it is the only gvp gene which could be detected, and electrophoretic analysis of isolated gas vesicles revealed the presence of a single protein. A monocistronic mRNA species corresponds to the transcription of the gvpA gene and the abundance of the gvpA mRNA is inversely correlated with photosynthetic photon flux density, indicating that a light-dependent transcriptional regulation is likely to be involved in the control of gas vacuolation in this strain.

Notizen: The penicillin-binding protein 4 (PBP4), from Escherichia coli, a DD-carboxypeptidase/DD-endopeptidase, was purified in an enzymatically active form to homogeneity by affinity chromatography on 6-aminopeni-cillanic acid/Sepharose and heparin/Sepharose. Polyclonal antibodies raised against the pure protein were used to identify and isolate PBP4 overproducing clones from an E. coli expression library, which was established on the basis of a temperature-inducible runaway replication plasmid. Three positive clones were isolated, one of which carried the intact structural gene dacB that codes for PBP4, on a 1.9kb Smal-Eco RI fragment, whereas the other two carried truncated forms of this gene. The direction of transcription was determined. The PBP4 overproducing strain, when grown in rich medium, tolerated 160-fold overexpression. After disrupting cells by sonication, the majority (80%) of the overproduced PBP4 was detected in the 100000 x g supernatant. Southern blotting analysis using the cloned dacB gene as a probe revealed that, in contrast to that described by Takeda et al. (1981), the plasmid pLC18–38 of the Clarke-Carbon collection does not code for PBP4. The overall composition of murein, synthesized in vitro or in vivo by the PBP4 overproducing strain, as determined by high-performance liquid chromatography analysis, suggests that PBP4 is not involved in transpeptidation but exclusively catalyses a DD-carboxypeptidase and DD-endopeptidase reaction.

Notizen: Synthesis of bacterial flagella and the accompanying array of chemotaxis receptors and transducers represents a major commitment of energy and resources for a growing bacterial cell and is subject to numerous levels of regulation. Genes for flagellar and chemotaxis proteins are expressed in a complex transcriptional cascade. This regulatory hierarchy acts to ensure that the highly expressed filament structural protein, flagellin, is synthesized only after a prerequisite set of structural proteins has been expressed and properly assembled. Recent evidence suggests that many bacteria utilize an alternative sigma (σ) subunit, similar in specificity to the Bacillus subtilisσ28 protein, to direct transcription of flagellin, chemotaxis and motility genes. In Caulobacter crescentus and Campylobacter spp., both a σ54-like factor and a σ28-like factor participate in the transcription of flagellar and chemotaxis genes. Conversely, a σ28-like factor controls non-motility functions in at least one non-flagellated organism.

Notizen: The eda gene that encodes 2-keto-3-deoxy-6-phos-phogluconate aldolase of the Entner-Doudoroff pathway was cloned from Zymomonas mobilis by genetic complementation of an Escherichia coli mutant. The gene is present in a single copy on the Z mobilis genome and is not tightly linked to the edd gene. Nucleotide sequence analysis of the eda region revealed that the structural gene is 627 bp long and capable of encoding a protein of 208 amino acids with a deduced molecular weight of 21 505. The eda gene is monocistronic and is transcribed from a single promoter. The transcriptional initiation site was determined and an improved consensus promoter sequence for Z. mobilis was derived. High-level expression of the eda gene can be attributed to very efficient translational initiation caused by the high quality of the ribosome-binding site and stability of the mRNA, which has a decay rate of 7.6 min. A comparison of highly expressed Z mobilis genes indicated that the relative quality of the ribosome-binding sites of these genes might play an important role in determining the level of enzyme synthesis. This possibility is discussed with regard to the role of gene expression in coordinating the enzyme levels of the Entner-Doudoroff glycolytic pathway.

Notizen: The bacteriophage λ gene product is one of the first genes expressed during phage development. N protein allows the expression of other phage genes by altering the transcription elongation process so as to prevent transcription termination. We have found that N levels may be modulated soon after induction or infection. Using N-lacZ fusions, we determined that cells containing RNaselll have at least a fourfold greater expression than cells defective for RNaselll. This effect is exerted at the post-transcriptional level. RNaselll processes an RNA stem structure in the N- leader RNA. Removal of the stem structure by deletion increases λ expression and prevents further stimulation by RNaselll. The base of this stable stem is adjacent to the λ ribosome binding site. We present a model for control of N synthesis in which this stable stem inhibits ribosome access to the N mRNA.

Notizen: Bacteriophage P1 res and mod genes encode the restriction and modification polypeptides of the Type III restriction enzyme EcoP1. Northern blot analysis using res- and mod-specific probes revealed the presence of two separate transcripts in strains harbouring the Eco P1 restriction and modification genes. Furthermore, by constructing a series of fusions with a promoterless lacZ gene, we show that both the res and mod genes are transcribed from separate promoters. A more detailed investigation of the mod promoter region revealed two promoters located some 70 and 140 bp upstream from the translational start codon. In addition, another pair of promoters and a further separate promoter are located more than 500 bp upstream from this start codon. Two short open reading frames are located between these distal and proximal promoter clusters.Transcription of the res gene is initiated from within the mod open reading frame from two adjacent promoters. In addition a functional promoter is located on the antisense strand close to the res promoter region. The relationship between the transcription units of the res and mod genes is discussed.

Notizen: Primers suitable for the amplification of the gene encoding the class 1 outer membrane protein of Neisseria meningitidis by the polymerase chain reaction (PCR) were designed from published DNA sequences and used to study the gene in eight meningococcal strains of different serogroup, serotype and subtype. At high annealing stringency one product, shown to correspond to the class 1 protein gene, was amplified from each strain. For three strains an additional smaller product, provisionally identified as the gene encoding the class 3 outer membrane protein, was amplified at lower annealing stringencies. Nucleotide sequence analysis of the PCR products corresponding to the class 1 proteins established the differences in the primary structure of the proteins between each of the subtypes and other outer-membrane proteins from Neisseria spp. These differences impose constraints on possible structural models of these proteins. Most amino acid sequence variation occurred in two domains of between 8 and 17 amino acids; there was an additional region which varied mainly between classes of outer membrane protein and there were nine conserved regions. Using appropriate primers it was possible to distinguish between class 1 outer membrane protein genes from strains of different subtypes by the PCR.

Notizen: The gene encoding a 23 kilodalton protein antigen has been cloned from Mycobacterium tuberculosis by screening of a recombinant DNA library with monoclonal antibodies. The product of the gene has been identified as the superoxide dismutase (SOD) of M. tuberculosis on the basis of sequence comparison and by expression of the recombinant protein in a functionally active form. The derived amino acid sequence of M. tuberculosis SOD reveals a close similarity to manganese-containing SODs from other organisms, in spite of the fact that previous studies using the purified enzyme have identified iron as the preferred metal ion ligand. SOD is present in the extracellular fluid of logarithmic-phase cultures of M. tuberculosis, but the structural gene is not preceded by a signal peptide sequence. Insertion of the M. tuberculosis SOD gene into a novel shuttle vector demonstrated the presence of a promoter which functions efficiently in mycobacteria but is ineffective in Escherichia coli

Notizen: The 7E gene is expressed late in normal development of Dictyostelium discoideum after pseudoplasmodium formation. After disaggregation of the developing cells, transcription of this gene depends entirely on exogenous 3′5′ cyclic AMP (cAMP). The 5′ flanking region of the 7E gene contains two TATA box-oligo (dT) promoter motifs but analysis of 7E gene expression by primer extension shows only a single primary transcript with transcription initiating immediately after the most proximal promoter motif during development or in disaggregated cells in the presence of exogenous cAMP. Four C-rich sequence lie within 350 bp upstream of the cap site, analogus to the upstream elements implicated in the cAMP regulation of several other Dictyostelium genes expressed in development.

Notizen: Each of the two mutants isolated from a fliC (=hag, flagellin-deficient) Escherichia coli strain made motile by a plasmid carrying the fliC gene of Salmonella muenchen by selection for motility in the presence of anti-d (Salmonella flagellar antigen) serum had both lost and gained one or more subfactors of the wild-type antigen. In one more mutant codon 246 was GAC (alanine) instead of GCC (asparagine); the other had a deletion of 105 base pairs, explicable by a 10bp direct repeat, starting at bases 782 and 887. The in vitro removal of a 48bp EcoRV(631)/EcoRV(679) fragment produced plasmid pLS408, which was found to lack a subfactor of wild-type antigen d but able to confer motility on flagellin-negative Salmonella sp.(and used for insertion of epitope-specifying oligonucleotides at its EcoRV site). Immunoblotting with absorbed and unabsorbed sera from rabbits immunized with E. coli with wild-type or mutated antigen d showed that the fusion proteins specified by λ gt11 with the N-terminal part of gene lacZ joined to a restriction fragment coding for residues 145–391 of flagellin gave the same patter of parent-specific and mutant-specific reactions as the flagellate bacteria. Four out of five similarly selected mutants had the same 105bp deletion as the first-isolated mutant; the fifth had a 72bp deletion made possible by a 7-base pair direct repeat, starting at positions 649 and 721. All these changes in serological character without loss of function affected segment IV, specifying residues 182 to 308 of the total of 505, where there is little homology between different flagellar antigen alleles.

Notizen: The Phanerochaete chryososporium trpC gene has been isolated by complementation of an Escherichia coli trpC mutant. The full extent of the fungal gene, determined by sequence analysis, was found to be 2414bp. This includes a single intron of 50bp, the presence of which was confirmed by RNA-primed polymerase chain reaction analysis. This feature makes the P. chrysosporium gene unique when compared to equivalent genes from other filamentous fungi. The P. chrysosporium trpC gene encodes a single protein containing three enzyme activities involved in tryptophan biosynthesis arranged in the order: NH2–GAT–IGPS–PRAI–COOH. This order is conserved in all filamentous fungi so far examined and, indeed, is the gene order within the E. coli trp operon.

Notizen: DNA adenine methylation controls DNA replication of plasmids containing the prototypic REPI replicon by affecting protein recognition and by altering the helical stability of the origin. Denaturing gradient gel electrophoresis shows that adenine methylated origin DNA is more easily melted than unmethylated. However, because an added DNA adenine methylation (dam) site at the origin, whether in or out of phase with other helically aligned dam sites, actually prevents replication, we conclude that destabilization of the helix is not the exclusive function of adenine methyfation in REPI replication. We find that the conformation and degree of methylation at the origin, features which are important for protein recognition, are essential for replication. In fact, RepI, a protein required for replication initiation at REPI replicons, contains a region homologous with a domain in proteins which specifically recognize and bind 5′-GATC-3′. We propose that the dam sites in the origin play a dual role: one is destabilization of the helix, and the other is protein recognition.

Notizen: The multi-functional PriA protein of Escherichia coli (formerly replication factor Y or protein n′) serves to guide the ordered assembly of the primosome, a mobile multiprotein replication priming/helicase complex. Primosome assembly is essential for bacteriophage ØX174 complementary DNA strand synthesis and ColE1-type plasmid replication reconstituted in vitro with purified proteins. The biochemical activities of the primosome suggest that it can fulfil the primase/helicase requirement on the lagging-strand DNA template during cellular DNA replication. However, reconstruction in vitro of DNA replication of small plasmids containing the E. coli origin of DNA replication (oriC) does not require the complete complement of primosomal proteins. Thus, the extent to which PriA-catalysed primosome assembly participates in chromosomal replication has remained unclear. The recent isolation of the genes encoding PriA, PriB (protein n), PriC (protein n″), and DnaT (protein i) has provided the necessary tools for addressing this issue. The phenotype of mutations in these genes, and other results described in this review, suggest that assembly of the primosome catalysed by PriA does in fact contribute at some stage to normal cellular DNA replication. A model for primososme-catalysed reactivation of a dysfunctional replication fork is discussed.

Notizen: The cytoplasmic L-A dsRNA virus of Saccharomyces cerevisiae consists of a 4.5kb dsRNA and the two gene products it encodes: the capsid (cap) and at least one copy of the capsid–polymerase (cap–pol) fusion protein. Virion cap–pol catalyses transcription of the plus (sense)-strand; this is extruded from the virus and serves as messenger for synthesis of cap and cap–pol. Nascent cap–pol binds to a specific domain in the plus strand to initiate encapsidation and then catalyses minus-strand synthesis to complete the replication cycle. Products of at least three host genes are required for replication, and virus copy number is kept at tolerable levels by the SKI antivirus system. S. cerevisiae killer viruses are satellite dsRNAs that use a similar encapsidation domain to parasitize the L-A replication machinery. They encode precursors of secreted polypeptide toxins and immunity (specific resistance) determinants and are self-selecting. Three unique killer types, K1, K2 and K28, are currently recognized. They are distinguished by an absence of cross-immunity and by toxin properties and lethal mechanisms; while K1 and K2 toxins bind to cell-wall glucan and disrupt membrane functions, K28 toxin binds to mannoprotein and causes inhibition of DNA synthesis.

Notizen: A Rickettsia rickettsii outer surface membrane protein (rOmp B), of an apparent molecular mass of 120 kilodaltons, is a major surface antigen of the Rickettsiae that displays genus, species, and sub-species specific antigenic determinants. The 5′ portion of this gene was found to be unstable in plasmids, but was stably cloned in a lambda vector. The nucleotide sequence of the 5′ terminus has been determined, thus completing the DNA sequence of the entire gene. Genetic analysis revealed an unusually large open reading frame with the capacity to encode a product much larger than the mature protein. A 32 kilodalton peptide from purified rickettsiae was isolated and the amino terminus was sequenced, which revealed that the peptide is encoded by the 3′ portion of this large open reading frame. This suggests a role for post-translational processing of rOmp B from a large precursor molecule.

Notizen: Two Corynebacterium glutamicum mutants defective in lysine uptake were identified by analysing mutants resistant to S-(2-amtnoethyl)-cysteine (AEC). A 5.6kb genomic DNA fragment restoring AEC sensitivity and lysine uptake was isolated. A 4.2kb subfragment was sequenced and three open reading frames were identified. Subcloning and gene disruption experiments showed that only the first open reading frame, termed lysl, is involved in lysine uptake. Lysl consists of 501 amino acids with a Mr of 53600. The hydrophobicity profile suggests that the lysl gene product is an integral membrane protein with 13 transmembrane segments. The amino acid sequence of lysl displays strong homology to that of the arcD gene product of Pseudomonas aeruginosa, which is proposed to act as an arginine–ornithine antiporter. Investigation of the influence of the lysl gene on lysine secretion suggests the existence of a separate lysine efflux system in C. glutamicum.

Notizen: We identified and sequenced the regulatory syrM and nodD3 genes of Rhizobium meliloti 41. Both genes were shown to contribute to optimal nodulation of alfalfa. In R. meliloti strains carrying syrM and nodD3 on plasmid, the nod genes are expressed constitutively, resulting in host-range extension to siratro. This is due to the presence of multiple syrM copies, suggesting that SyrM participates directly in nod gene activation. NodD3 activates nod genes in conjunction with flavonoids and enhances syrM expression, which is controlled also by its own product, NodD2, and two putative trans-acting factors. nodD3 is regulated by SyrM, NodD1, NodD3, the repressor NoIR, and two putative factors.

Notizen: The expression of the chloramphenicol-inducible chloramphenicol-acetyltransferase gene (cat), encoded on Staphylococcus aureus plasmid pUB112, is regulated via a translational attenuation mechanism. Ribosomes, which are arrested by chloramphenicol during synthesis of a short leader peptide, activate catm RNA translation by opening a 5′-located stem-loop structure, thus setting free the cat ribosome-binding site. We have determined the 5′ and 3′ ends of cat mRNA and analysed its stability in Bacillus subtilis. In the absence of the antibiotic, the half-life of cat mRNA is shorter than 0.5 min; it is enhanced to about 8 min by sub-inhibitory concentrations of the drug. No decay intermediates of cat mRNA could be detected, indicating a very fast degradation after an initial rate-limiting step. ochre nonsense mutations in the 5′ region of the cat structural gene, which eliminate cat mRNA translation, did not affect its chloramphenicol-induced stabilization. Mutations in the leader-peptide coding region, which abolish ribosome stalling and, therefore, cat gene induction, also eliminate cat mRNA stabilization. We conclude that cat mRNA is stabilized on induction by a chloram-phenicol-arrested ribosome, which physically protects a nuclease-sensitive target site in the 5′ region of cat mRNA against exo- or endonucleolytic initiation of degradation. This protection is analogous to ermA and ermC mRNA and seems to reflect a general mechanism for stabilization of mRNA derived from inducible antibiotic resistance genes in B. subtilis.

Notizen: The cpeBA operon of the Group III chromatically adapting cyanobacterium Pseudanabaena species PCC 7409 was cloned, sequenced and characterized. The cpeBA genes are transcribed in green-light-grown cells as an abundant 1400-nucleotide mRNA which initiates 69 nucleotides upstream from the cpeB translation start. Extensive sequence identity, extending 70 nucleotides 5′ to the transcription start, occurs among cpeBA promoters of Group II and III chromatic adapters. Cell extracts of green-light-grown Calothrix species PCC 7601 contain an activity which specifically binds a restriction fragment containing the Pseudanabanea species PCC 7409 cpeBA promoter. Green-light-dependent cpeBA transcription in Group II and III chromatically adapting cyanobacteria is suggested to be similarly controlled by a transcriptional activator.

Notizen: Structural features of three analysed bacterial DNA replication origin classes (six enteric origins, three pseudomonad origins, and the Bacillus subtilis origin region) are compared in order to deduce characteristics common to all bacterial origins and characteristics that distinguish the three origin classes. The two Pseudomonas aeruginosa origins are shown to map within 10 kb of each other, and correlations are drawn with four potential origin regions in B. subtilis. The enteric origin class is further distinguished from the other two classes by its genetic organization, the presence of GATC sites, and the role of Dam methylation in enteric initiation. The pseudomonad origin class has the most features that are common to all of these bacterial origins, and hence may be the paradigm bacterial origin class.

Notizen: The importance of parasite-directed phosphatases in such diseases as smallpox and the bubonic plague emphasizes the need to understand the molecular events associated with normal function of protein tyrosine phosphatase in eukaryotic cells.

Notizen: Pf1 is a filamentous, single-stranded DNA virus that has Pseudomas aeruginosa (strain K) as host. It is the longest of the filamentous bacterial viruses, and the DNA within it has the most extended conformation known. Pfl virus cannot infect Escherichia coli (strain MM294) cells, but when these cells are transfected with the double-stranded replicative form of Pt1 DNA (RF DNA, 7.35 kb), they export low levels of infectious particles that create plaques on lawns of P. aeruginosa. Several different structural species, at least two of which are infectious, are exported. One of them, called Epf1, has virtually the same structure as Pf1, but the amount of Epf1 exported by E. coli is 104 lower than the amount of Pf1 exported by P. aeruginosa. The results imply that host factors affect not only the efficiency of virus assembly and export, but also the actual structures of the species exported.

Notizen: A regulatory gene, cfxR, involved in the carbon dioxide assimilation of Alcaligenes eutrophus H16 has been characterized through the analysis of mutants. The function of cfxR is required for the expression of two cfx operons that comprise structural genes encoding Calvin cycle enzymes. CfxR (34.8 kDa) corresponds with an open reading frame of 954 bp, with a translational initiation codon 167 bp upstream of the chromosomal cfx operon. The cfx operon and cfxR are transcribed divergently. The N-terminal sequence of CfxR is very similar to those of bacterial regulatory proteins belonging to the LysR family. Heterologous expression of cfxR in Escherichia coli was achieved using the pT7-7 system. Mobility shift experiments demonstrated that CfxR is a DNA-binding protein with a target site upstream of both the chromosomal and the plasmid-encoded cfx operons.

Notizen: Comparison of the amino acid sequences of the C-terminal domain of three DNAase type E colicins has identified six candidate specificity determinants for the interaction of these E colicins with their homologous immunity proteins. We have changed these candidate specificity determinants of colicin E9, using site-directed mutagenesis, to the corresponding amino-acids of colicin E8. A‘mutant’colicin E9, in which four of the six candidate specificity determininants have been changed, demonstrated colicin activity against Escherichia coli indicator strains which carried either the E8imm or the E9imm genes, indicative of a‘novel’E colicin. After changing all six of the candidate specificity determinants, the resulting colicin E9‘mutant’exhibited a phenotype very similar to that of colicin E8.

Notizen: The CorA Mg2+ transport system of Salmonella typhimurium mediates both influx and efflux of Mg2+. Mutations at the corA locus (83.5min) confer resistance to Co2+. Using transposon mutagenesis, three additional Co2+ resistance loci (corB, corC, and corD) were found and mapped to 55, 15, and 3min, respectively, on the S. typhimurium chromosome. No mutations corresponding to the reported corB locus at 95min in Escherichia coli were obtained. The corB, corC, and corD mutations confer levels of Co2+ resistance intermediate between those of the wild-type and corA mutations.Isogenic strains were constructed containing combinations of transposon insertion mutations in each of the three Co2+ resistance loci to assess their influence on the CorA Mg2+ transport system. The Vmax and Km values for 28Mg2+ or for 57Co2+ and 63Ni2+ influx, analogues of Mg2+ transported by the CorA system, were changed less than twofold compared with the wild-type values, regardless of the mutation(s) present. However, while efflux of 28Mg2+ through the CorA system was decreased threefold in strains carrying one or two mutant alleles among corB, corC, or corD, efflux was completely abolished in either a corA or a corBCD strain. Thus, although the corA gene product is necessary and sufficent to mediate Mg2+ influx, Mg2+ efflux requires the presence of a wild-type allele of at least one of the corB, corC or corD loci.

Notizen: The insecticidal crystal proteins of Bacillus thuringiensis show a high degree of specificity. In vitro binding studies with several crystal proteins demonstrated a correlation between toxicity and binding to receptors of larval midgut epithelial cells. In order to study the domain-function relationships of the toxic fragment, hybrid crystal proteins based on CrylA(b) and CrylC were constructed. Two out of 11 hybrid proteins constructed exhibited insecticidal activity. Both displayed an insectidial spectrum similar to that of the parental crystal protein from which the C-terminal part of the toxic fragment originated. In addition, in vitro binding studies directly demonstrated the involvement of the C-terminal part of the toxic fragment in receptor binding. These results demonstrate that the C-terminal part of the toxic fragment determines specific receptor binding, which in turn determines, to a large extent, the insect specificity.

Notizen: The sequence of the repressor locus, c, of the Streptomyces temperate phage, φC31, was shown previously to contain an open reading frame encoding a 74 kDa protein. Further analysis of the transcriptional and translational products of the c gene shows a more complex pattern of expression. A nest of three in-frame N-terminally different, C-terminally identical proteins of 74, 54 and 42 kDa were found to be expressed from a corresponding nest of transcripts. The repressor proteins were produced in Escherichia coli and the 42 kDa protein was purified, verified by N-terminal sequencing, and used to raise antibody. The antibody cross-reacted in Western blots with the 74, 54 and 42 kDa proteins expressed in E. coli and Streptomyces lividans and from Streptomyces coelicolorφC31 lysogens. Analysis of transcription of the c gene by S1 mapping and primer extension showed that the nest of transcripts encoding the repressor protein were induced after heat treatment of the cts locus (Sinclair and Bibb, 1989; this paper). Correspondingly, all three of the repressor proteins were induced. In addition to a promoter, cp1, which lies upstream of the 74 kDa open reading frame, the c locus contained at least one internal promoter, cp2, which transcribes DNA encoding the 54 and 42 kDa proteins. Transcripts initiating from cp3 were observed in RNA preparations from S. lividans containing the c gene deleted for cp1 and cp2, but gene fusions using DNA which should contain any putative promoting activity from this region transcriptionally fused to the xylE gene showed very low levels of expression of catechol 2,3 dioxygenase in S. lividans. The 74 kDa protein was not necessary for super-infection immunity. Data described here and current knowledge of the nature of other‘dual start’genes suggest a model for the regulation of lysis versus lysogeny in φC31.

Notizen: Sensory transduction in the gliding bacterium Myxococcus xanthus is mediated by the frz genes. These genes are homologous to the chemotaxis genes of enteric bacteria and control the rate of cell reversal during gliding. Sensory transduction is hypothesized to involve the recognition of substances present in the medium at the cell surface and the subsequent stimulation of a cytoplasmic methyl-accepting protein, FrzCD. Phosphorylation of FrzE is also involved in the sensory transduction pathway. Despite the similarities between the chemotaxis proteins of enteric bacteria and M. xanthus Frz proteins, fundamental differences exist between these different bacteria in terms of the ability of cells to recognize and respond to substances in their environment. The mechanism of directional switching and the nature of the gliding motor remain obscure. It is hoped that the study of the interaction of the Frz proteins will allow greater understanding of these problems.

Notizen: Summary A general method for the evaluation of macrorestriction fragment patterns is presented and its applicability to the taxonomy of bacteria is demonstrated for 32 Pseudomonas species. Strains were differentiated at the species and subspecies level by genome size and macrorestriction fragment fingerprints of the chromosome that had been separated on pulsed-field gels. The relatedness of bacteria was ascertained from the similarity of Asnl, Dral, Spel, Sspl or Xbal fragment patterns. In general, the dendrograms calculated from the genome fingerprints corresponded with the phylogenetic classification obtained from phenotypic marker or nucleic acid hybridization analysis, but several exceptions were noted. The techniques and algorithms presented herein are generally applicable to the genome analysis of bacteria, lower eukaryotes, and DNA fragments cloned in yeast artificial chromosomes.

Notizen: Skp of Escherichia coli (OmpH of Salmonella typhimurium) is a protein whose precise function has been obscured by its ubiquity in a wide range of sub-cellular fractions such as those containing DNA, ribosomes, and outer membranes. Combining in vitro and in vivo techniques we show that Skp is synthesized as a larger precursor that is processed upon translocation across the plasma membrane. Translocation is dependent on the H+-gradient, ATP, SecA, and SecY. Upon cellular subfractionation (avoiding non-specific electrostatic interactions) Skp partitions with β-lactamase into the fraction of soluble, periplasmic proteins. In the context of the export factor properties of Skp previously demonstrated in vitro it is conceivable that this protein is involved in the later steps of protein translocation across the plasma membrane and/or sorting to the outer membrane.

Notizen: Multiple genes (many of which are designated RAD (for radiation resistance)) are required for cellular responses to DNA damage in the yeast Saccharomyces cerevisiae. In recent years a number of these genes have been cloned and sequenced and in some cases their polypeptide products have been purified and characterized biochemically. These studies are beginning to yield clues about the possible nature of the multiple biochemical pathways for DNA-damage processing in yeast.

Notizen: With the determination of the three-dimensional structure of elastase and the probable identification of the active site and key residues involved in proteolytic activity, our knowledge of the molecular details of this interesting protease is rapidly increasing. Pseudomonas elastase appears to be remarkably similar to the Bacillus metalloproteinase thermolysin. A further significant development has been the discovery of the lasA gene and the fact that Pseudomonas elastase and alkaline proteinase appear to act in concert with the LasA protein to display the notable elastolytic activity exhibited by isolates of this organism. Biochemical and genetic studies indicate that LasA is a second elastase which may be an important virulence factor that has been overlooked in previous studies.

Notizen: Crown gall tumorigenesis by Agrobacterium tumefaciens requires the co-ordinate transcriptional induction of a set of pathogenesis genes. At least three classes of environmental stimuli act synergistically to induce these genes, (i) monocyclic aromatic hydrocarbons such as acetosyringone, coniferyl alcohol, and vanillin, (ii) neutral or acidic monosaccharides such as glucose and glucuronic acid, and (iii) acidic pH. Three proteins are required to sense and respond to these stimuli: (i) VirA, a transmembrane sensory protein and histidine protein kinase. (ii) VirG, a transcriptional activator which is phosphorylated by phosphoryl VirA, and (iii) ChvE, a periplasmic sugar-binding protein. VirA and VirG are members of the so-called two-component family of regulatory proteins. This regulatory system continues to offer new discoveries in the areas of signal transduction. host-microbe interactions, and host range.

Notizen: A 1.5kb cryptic plasmid was isolated from Helicobacter pylori. Low-stringency hybridization analysis using this plasmid as a DNA probe revealed base sequence homology with other plasmids in this species. Nucleotide sequence analysis identified an open reading frame encoding a putative polypeptide of 25 kDa. This protein showed marked amino acid sequence similarity to replication-initiation proteins commonly found in small plasmids endogenous to Gram-positive bacteria which replicate by the ‘rolling-circle’ mechanism. Sequence motifs corresponding to the origins-of-replication consensus sequences were found on this cryptic plasmid. DNA and oligonucleotide probes to these plasmid replication sequences were used in hybridization analysis to identify similar sequences in other H. pylori plasmids. We believe this is the first plasmid isolated from a Gram-negative bacterium to show replication determinants characteristic of the ‘rolling-circle’ group of plasmids from Gram-positive bacteria. The cloned plasmid will be used to develop a shuttle-vector for H. pylori.

Notizen: Erwinia chrysanthemi, a Gram-negative phythopathogenic bacterium, secretes two related extracellular metalloproteases, B and C, which do not have N-terminal signal sequences. The specific pathway by which they are secreted, which has been reconstituted in Escherichia coli, comprises three proteins — PrtD, PrtE and PrtF. Hybrid proteins containing segments of these proteins fused to the C-terminus of protease B were purified and used to immunize rabbits. The antisera thus obtained were used to study the location and membrane topology of the three proteins. PrtD and PrtE were found to cofractionate almost exclusively with the cytoplasmic membrane, whereas PrtF was found to co-fractionate mostly with the outer membrane. Proteinase K accessibility experiments as well as sequence data lead us to propose that PrtF has one or both ends exposed to the periplasm, that PrtE has one transmembrane segment with its amino-terminus facing the cytoplasm and its C-terminal hydrophilic domain exposed to the periplasm, and that PrtD has six transmembrane segments with its N-terminus and its C-terminal hydrophilic domain in the cytoplasm.

Notizen: The expression of the virulence-associated genes in Bordetella species is co-ordinately regulated by the gene products encoded by the bvg locus. In Bordetella pertussis the expression of this locus is regulated by the P1, P2, P3 and P4 promoters which are located in a 350bp DNA fragment also containing the PFHA promoter. Here we report the transcriptional regulation of the bvg locus and the fha gene in Bordetella paraper-tussis and a sequence analysis of the byg-regulated promoters. The Pp1, Pp2, Pp4 and PpFHA promoters are indistinguishable, both in transcription initiation sites and environmental regulation, from the corresponding promoters of B. pertussis, while the Pp3 promoter is not active. Sequence homologies from nine bvg-regulated promoters show a conserved dinucleotide, 5′-TG-3′, at approximately one turn of helix upstream of the -10 5′-A.AaTat-3’region, and a 5′-TTTCC-3’sequence in the -90 region. Since the nucleotide sequence of the inactive Pp3 promoter shows several base substitutions with respect to the found sequence homologies, it is likely that some of these bases play an essential role in promoter activity.

Notizen: A macro-restriction map of the Neisseria gonorrhoeae chromosome was constructed using the enzymes Nhel and Spel. Combinations of one-and two-dimensional electrophoresis of completely or partially digested chromosomal DNA were performed to align the restriction fragments. The chromosome is circular, with an estimated size of 2.33Mb±35kb. A genetic map was derived from the physical map; positions of over 60 defined loci were determined by Southern hybridization.

Notizen: The cytochrome d complex of Escherichia coli is a heterodimer located in the bacterial cytoplasmic membrane, where it functions as a terminal oxidase of the aerobic respiratory chain. The topology of each of the two subunits of the cytochrome d complex was analysed by the genetic method involving alkaline phosphatase gene fusions. These fusions were generated by both an in vivo method using the transposon TnphoA and an in vitro method of construction. A total of 48 unique fusions were isolated and the whole-cell alkaline phosphatase-specific activities were determined. Data from these fusions, in combination with information from other studies, provide the basis for two-dimensional models for each of the two subunits, defining the way in which the subunits fold in the inner membrane of E. coli.

Notizen: Proteins that are able to translocate across biological membranes assume of loosely folded structure. In this review it is suggested that the loosely folded structure, referred to here as‘per-folded conformation', is a particular structure that interacts favourably with components of the export apparatus. Two soluble factors, SecB and GroEL, have been implicated in maintenance of the pre-folded conformation and have been termed‘molecular chaperones'. Results suggest that SecB may be a chaperone that is specialized for binding to exported protein precursors, while GroEL may be a general folding modulator that binds to many intracellular proteins.

Notizen: Genetic competence develops as a global response of Bacillus subtilis to the onset of stationary phase, in glucose-minimal salts-based media. The onset of competence is accompanied by the expression of several late gene products that are required for the binding, processing and uptake of transforming DNA. A number of regulatory genes have been identified that are needed for the appropriate synthesis of the late gene products. The regulatory gene products include a number of known transcription factors, as well as several members of the bacterial two-component regulatory system. Genetic analysis has suggested a scheme for the flow of regulatory information signaling the onset of competence. Most of these regulatory products appear to be involved in the response to nutritional status, while the components responsible for growth stage and cell-type-specific control remain unknown. The general implications of this scheme for post-exponential expression are discussed.

Notizen: The finP gene of plasmid R1 is located between the genestraM and traJ, partially overlapping the first few nucleotides of the latter. It codes for a repressor of the conjugation system. The product of this gene is a small RNA of 72 nucleotides and, because it is transcribed from the opposite DNA strand, it is complementary to the 5′ non-translated sequences, the ribosome-binding site, and the first two codons of traJ mRNA. The finP transcript is present in much higher concentrations in R1 than in R1-19 containing cells, the latter being a derepressed mutant of the former. A synthetic finP gene expressed from a synthetic lambda PL promoter markedly reduced the conjugation frequency of pDB12, a multicopy derivative of R l-19. Mutagenesis of finP showed that only finP loop II mutants have lost the ability to repress conjugation of R1-19 intrans. They are also the only ones which derepress conjugal DNA transfer of R1, probably by competing for the finO product, a molecule needed as corepressor for maximal activity. Mutations interrupting potential open reading frames of finP do not abolish finP repressor activity. Hence finP acts as an antisense RNA blocking the expression of the traJ gene by interacting with traJ mRNA through loop II.

Notizen: The lysC/asd gene cluster of Corynebacterium glutamicum ATCC 13032 was cloned and sequenced. The lysC locus coding for aspartokinase consists of two in-frame overlapping genes, lysCα encoding a protein of 421 amino acids (Mr 44300) and lysCβ encoding a protein of 172 amino acids (Mr 18600). The C. glutamicum aspartokinase was purified and found to contain two proteins of Mr 47000 and Mr 18000.A C. glutamicum mutant expressing a feedback-resistant aspartokinase was shown to be changed in a single base pair of the lysCβ gene, leading to an amino acid exchange in the β-subunit of the aspartokinase. In addition, the identified mutation was found to be responsible for the enhanced expression of the asd gene located downstream of lysC.

Notizen: The cenC gene of Cellulomonas fimi, encoding endoglucanase CenC, has an open reading frame of 1101 codons closely followed by a 9bp inverted repeat. The predicted amino acid sequence of mature CenC, which is 1069 amino acids long, is very unusual in that it has a 150-amino-acid tandem repeat at the N-terminus and an unrelated 100-amino-acid tandem repeat at the C-terminus. CenC belongs to subfamily E1 of the β-1,4-glycanases. High-level expression in Escherichia coli of cenC from a 3.6kbp fragment of C. fimi DNA leads to levels of CenC which exceed 10% of total cell protein. Most of the CenC is in the cytoplasm in an inactive form. About 60% of the active fraction of CenC is in the periplasm. The catalytic properties of the active CenC are indistinguishable from those of native CenC from C. fimi. The Mr of CenC from E. coli and C. fimi is approximately 130 kDa. E. coli and C. fimi also produce an endoglucanase, CenC', of approximate Mr, 120kDa and with the same N-terminal amino acid sequence and catalytic properties as CenC. CenC’appears to be a proteolytic product of CenC. CenC and CenC’can bind to cellulose and to Sephadex. CenC is the most active component of the C. fimi cellulase system isolated to date.

Notizen: We report here the formation of symbiotic plasmids (pSyms), by genetic recombination between rearranged pSyms, which lack symbiotic information, and resistance plasmids carrying parts of different symbiotic plasmids (R's). This recombination was found to occur both between plasmids derived from different Rhizobium phaseoli isolates, and between plasmids derived from strains obtained from the same original isolate.We also present evidence on the formation of a functional symbiotic plasmid by recombination of an R, carrying nif and nod genes from strain CFN42, and an indigenous plasmid present in this strain (pCFN42e), which was thought to be unrelated to its symbiotic plasmid (pCFN42d). These data are discussed with respect to the stability and transfer of Rhizobium symbiotic information.

Notizen: Phenotypic analysis of a temperature-sensitive era mutant strain indicates that Escherichia coli cells depleted of Era undergo many physiological changes. At 43° C., a completely non-permissive temperature, growth is arrested because of loss of the gene and depletion of the Era protein. Depletion of Era at 43° C results in depressed synthesis of heat-shock proteins DnaK, GroEL/ES, D33.4 and C62.5, lack of thermal induction of ppGpp pool levels, and increased capacity for carbon source metabolism through the citric acid cycle. Thus, in addition to inhibition of cell growth and viability, ioss of Era function results in pleiotropic changes including abnormal adaptation to thermal stress.

Notizen: The FixL/Fix J two-component system is a global regulator of nitrogen-fixation genes in Rhizobium meliloti. The transcriptional activator FixJ contains two modules: its N-terminal module is homologous with other two-component regulators; its C-terminal module shows homology with various transcriptional activators, and with the C-terminal region of σ factors, which is involved in the discrimination of the -35 region of bacterial promoters. We show that C-terminal module of Fix J contains the entire transcription activation function, and that the N-terminal module regulates this activity negatively. Oligonucleotide directed mutagenesis of the transcriptional activator module demonstrated the importance of a potential helix-turn-helix structure.

Notizen: The Alcaligenes eutrophus genes for β-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase and poly(β-hydroxybutyric acid) synthase (PHB synthase) which comprise the three-step PHB-biosynthetic pathway, were cloned. Molecular studies revealed that these genes are organized in a single operon. The A. eutrophus PHB-biosynthetic genes are readily expressed in other bacteria, and DNA fragments harbouring the operon can be used as a cartridge to confer to other bacteria the ability to synthesize PHB from acetyl-CoA. The biochemical and physiological capabilities of A. eutrophus for the synthesis of a wide variety of polyhydroxyalkanoates are discussed.