摘要:

SummarySingle‐strand interruptions in a template DNA are likely to cause collapse of replication forks. We propose a model for the repair of collapsed replication forks inEscherichia coliby the RecBCD recombinational pathway. The model gives reasons for the preferential orientation of Chi sites in theE. colichromosome and accounts for the hyper‐rec phenotype of the strains with increased numbers of single‐strand interruptions in their DNA. On the basis of the model we offer schemes for various repeat‐mediated recombinational events and discuss a mechanism for quasi‐conservative DNA replication explaining the recombinational repair‐associated

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02403.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryThe efficiency of β‐lactam antibiotics, which are among our most useful chemotherapeutic weapons, is continuously challenged by the emergence of resistant bacterial strains. This is most often due to the production of β‐lactamases by the resistant cells. These enzymes inactivate the antibiotics by hydrolysing the β‐lactam amide bond. The elucidation of the structures of some β‐lactamases by X‐ray crystallography has provided precious insights into their catalytic mechanisms and revealed unsuspected similarities with the DD‐transpeptidases, the bacterial enzymes which constitute the lethal targets of β‐lactams. Despite numerous kinetic, structural and site‐directed mutagenesis studies, we have not completely succeeded in explaining the diversity of the specificity profiles of β‐lactamases and their surprising catalytic power. The solutions to these problems represent the cornerstones on which better antibiotics can be designed, hopefu

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02404.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryDespite the isolation of an anti‐sigma factor over 20 years ago, it is only recently that the concept of an anti‐sigma factor emerged as a general mechanism of transcriptional regulation in prokaryotic systems. Anti‐sigma factors bind to sigma factors and inhibit their transcriptional activity. Studies on the mechanism of action of anti‐sigma factors has shed new light on the regulation of gene expression in bacteria, as the anti‐sigma factors add another layer to transcriptional control via negative regulation. Their cellular roles are as diverse as FlgM ofSalmonella typhimurium, which can be exported to sense the structural state of the flagellar organelle, to SpollAB ofBacillus subtilisparticipating in the switch from one cell type to another during the process of sporulation. Additionally, the bacteriophage T4 uses an anti‐sigma factor to sabotage theEscherichia coliE·σ70RNA polymerase in order to direct exclusive transcription of its own genes. Cross‐linking., co‐immuno‐precipitations, and co‐purification indicate that the anti‐sigma factors directly interact with their corresponding sigma factor to negatively regulate transcription. inB. subtilis, anti anti‐sigma factors regulate anti‐sigma factors by preventing an anti‐sigma factor from interacting with its cognate sigma factor, thereby al

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02405.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummarySequence analysis of theBradyrhizobium japonicumhydrogenase promoter regulatory region indicated the presence of a –24/–12 type promoter, which is recognized by RpoN, and a potential integration host factor (IHF)‐binding site.B. japonicum rpoN−/rpoN2double mutants were deficient in hydrogen‐uptake activity. Using plasmid‐bornehup‐lacZfusions, it was shown that therpoNmutants were also deficient in nickel‐dependent transcriptional regulation of hydrogenase. Gel‐shift assays of the hydrogenase promoter regulatory region showed that purified IHF fromEscherichia colibinds to a 210bp fragment. DNase footprint analysis revealed a protected region of 31 bp between bases –44 and –75 from the transcription start site. Western analysis withB. japonicumsoluble extract and antibodies againstE. coliIHF gave two bands equivalent to molecular masses of 12 and 14kDa approximately. When the IHF‐binding area is mutated on a plasmid‐bornehup‐lacZfusion, nickel‐dependent transcriptional regulation of hydrogenase is still observed, but the transcriptional rates are clearly less than in the parenthup‐lacZfusion plasmid. Like the results with nickel, regulation of hydrogenase by other transcriptional regulators (hydrogen and oxygen) still occurs, but at a diminished level in the IHF

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02406.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryTheYAP1andYAP2genes encode yeast transcription factors of the c‐junfamily. We show that yeast mutants deleted for either theYAP1or theYAP2genes are hypersensitive to oxidants, particularly H2O2, and that these genes play a role in regulating the induction of the H2O2adaptive stress response inSaccharomyces cerevisiae.They do not significantly affect the regulation of the superoxide adaptive stress response. The intrinsic resistance of stationary‐phase and respiring yeast cells towards superoxide anions is unaffected by deletion of theYAP1andYAP2genes. However, resistance towards H2O2under these conditions is significantly reduced. We show that expression of the yeastGSH1gene (encoding γ‐glutamylcysteine synthetase) and theSSA1gene (encoding an HSP70 isoform) are induced by oxidants. Unlike theSSA1and thioredoxin (TRX2) genes, expression of theGSH1gene is more strongly induced by superoxide anions than by H2O2. In the absence of added oxidants, transcription of theGSH1gene is reduced in strains carrying theyap1deletion. However, we show that Yap1 is not required for the superoxide anion‐mediated induction ofGSH1gene expression. Furthermore, while the H2O2‐mediated induction ofSSA1expression is shown to beYAP1dependent, the heat‐shock‐mediated induction of theSSA1gene does not requireYAP1.We also present evidence to show that theYAP2gene does not regulate the expression of theTRX2, S

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02407.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryThe toxin coregulated pilus (TCP) is required forVibrio choleraeto colonize the human intestine. The expression of the pilin gene,tcpA, is dependent upon ToxR and upon ToxT. ThetoxTgene was recently mapped within the TCP biogenesis gene cluster and shown to be capable of activating atcpA::TnphoAfusion when cloned inEscherichia coli.In this study, we determined that ToxR/ToxT activation occurs at the level oftcpAtranscription. ToxT expressed inE. colicould activate atcpoperon fusion, while ToxR, ToxR with ToxS, or a ToxR‐PhoA fusion failed to activate thetcpoperon fusion and we could not demonstrate binding of a ToxR extract to thetcpApromoter region in DNA mobility‐shift assays. The start site for the regulated promoter was shown by primer extension to lie 75 bp upstream of the first codon oftcpA.An 800‐basetcpAmessage was identified, by Northern analysis, that correlates by size to the distance between the transcriptional start and a hairpin‐loop sequence betweentcpAandtcpB.The more‐sensitive assay of RNase protection analysis demonstrated that a regulated transcript probably extends through the rest of the downstreamtcpgenes, includingToxTand the adjacent accessory colonization factor (acf) genes. An in‐frametcpAdeletion, but not a polartcpA::TnphoAfusion, could be complemented for pilus surface expression by providingtcpA in trans.This evidence suggests that thetcpgenes, includingtoxT, are organized in an operon directly activated by ToxT in a ToxR‐dependent manner. Most of theToxTexpression under induced conditions requires transcription of thetcpApromoter. Further investigation of howtcp::TnphoAinsertions that are polar onToxTexpression retain regulation showed that a low basal level oftoxTexpression is present intoxRandtcp::TnphoAstrains. Overall, these observations support the ToxR/ToxT cascade of regulation fortcp.Once induced,toxTexpression becomes autoregulatory via thetcppromoter, linkingtcpexpression to that of additional colonization factors, exotoxin production, and genes of unknown function in cholera

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02408.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryHomopolymeric α‐2,8‐linked sialic acid (PSA) has been found as a capsular component of sepsis‐ and meningitis‐causing bacterial pathogens, and on eukaryotic cells as a post‐translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage‐encoded enzyme, the endo‐N‐acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5′ end contains a totally conserved neuraminidase motif. Expressed inEscherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS‐PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage‐induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C‐terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary forin vi

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02409.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryExpression of type IV pili appears to be a requisite determinant of infectivity for the strict human pathogensNeisseria gonorrhoeae and Neisseria meningitidis.The assembly of these colonization factors is a complex process. This report describes a new pilus‐assembly gene,pilG, that immediately precedes the gonococcal (Gc) pilDgene encoding the pre‐pilin leader peptidase. The nucleotide sequence of this region revealed a single complete open reading frame whose derived polypeptide displayed significant identities to the pilus‐assembty protein PilC ofPseudomonas aeruginosaand other polytopic integral cytoplasmic membrane constituents involved in protein export and competence. A unique polypeptide ofMr38kDa corresponding to the gene product was identified. A highly related gene and flanking sequences were cloned from a group E polysaccharide‐producing strain of N. meningitidis (Mc). The results indicate that thepilGgenes and genetic organization at these loci in Gc and Me are extremely conserved. Hybridization studies strongly suggest thatpilG‐related genes exist in commensalNeisseriaspecies and other species known to express type IV pili. Defined genetic lesions were created by using insertional and transposon mutagenesis and moved into the Gc and Me chromosomes by allelic replacement. ChromosomalpilGinsertion mutants were devoid of pili and displayed dramatically reduced competence for transformation. These findings could not be ascribed to pilin‐gene alterations or to polarity exerted onpilDexpression. The results indicated thatPilGexerts its own independent role in neisserial pilus

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02410.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryThe destruction and processing of bacteria by activated macrophages facilitates the presentation of antigens to T cells and thereby promotes the induction of specific immunity. The PhoP‐PhoQ regulatory system that controls the synthesis of manySalmonellaproteins required for virulence and survival within macrophages is one mechanism that this particular intracellular pathogen has evolved to resist destruction. To address whether the phoP locus also influences antigen processing during the interaction ofSalmonella typhimuriumwith macrophages, we tested the effect ofphoPmutations on the processing and presentation of model antigens expressed by the bacteria. Activated macrophages processedphoP−bacteria with greater efficiency than wild‐type bacteria, as measured by the response of antigen‐specific T‐hybridoma cells; Salmonella constitutively expressingPhoPwere processed even less efficiently than wild‐type Salmonella. After heat‐inactivation, however, both wild‐type andphoP−bacteria were efficiently processed. The altered processing and presentation efficiency was not due to differences in the level of antigen expressed by the bacteria or differences in the level of bacterial uptake by the macrophages. In addition,phoP‐regulated gene expression was shown to influence processing of antigen phagocytosed independently of the bacteria. Thus,phoP‐regulated gene products decrease the processing and presentation of S. typhimurium antigens, demonstrating a rote 1or this virulence locus in the inhibition of the induction

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02411.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryThe replication of staphylococcal plasmid pT181 is indirectly controlled at the level of the synthesis of its replication initiator, RepC. As a result, high levels of RepC synthesis per plasmid copy were expected to lead to autocatalytic plasmid replication, which secondarily would affect host physiology. Surprisingly, RepC overexpression was found to lead to a rapid decrease in pT181 copy number and replication rate. These effects depended on the ratio of RepC lo the PT181 replication origin rather than on the absolute amount of RepC in the cell. In a wild‐type host, the increase in RepC/plasmid copy also inhibited chromosome replication and cell division. The changes in host physiology did not play any role in the decrease in pT181 replication caused by RepC overexpression since pT181 replication responded in the same way in a host mutant insensitive to the effects of RepC induction. These results suggest that pT181, the prototype of an entire class of plasmids from Gram‐positive bacteria, responds to overexpression of its replication initiator by a decrease in plasmid replicat

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.tb02412.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY