摘要:

SummaryTheEscherichia colioperons daa andsfaencode F1845 and S pill, respectively. In this paper we show that the expression of these operons is under phase variation control at a transcriptional level. The transcription of both operons is dependent on the global regulator leucine‐responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam). Lrp is required for methylation protection of two GATC sites located within conserved DNA sequences in the regulatory regions of these operons. These GATC sites are differentially methylated, establishing a methylation pattern which is characteristic of either the phase ON or phase OFF state. We also show that Lrp binds to thedaaandsfaregulatory regions and that this binding is modulated by the methylation of the GATC sites. These results indicate that the phase variation of thedaaandsfaoperons is regulated by a mechanism involving differential binding of Lrp owing to methylation of GATC sites in the regulatory region, which is similar to the mechanism that controls phase variation of the pap opero

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00340.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryShigella flexnerikills macrophages through apoptosis, involving the induction of host cell DNA fragmentation and characteristic morphological changes. Shigella can only cause damage if it escapes from the phagolysosome into the cytoplasm. TheS. flexnericytotoxic genes have been localized to theipaoperon of shigella's virulence plasmid.ipaB, C and D deletion mutants are not invasive and therefore not cytotoxic. In order to distinguish genes involved in the escape from the phagolysosome as distinct from cytotoxicity, we constructedShigellastrains that secrete low amounts ofEscherichia colihaemolysin (hlylow). These strains can escape into the cytoplasm of the macrophage even in the absence of the invasion plasmid as verified by electron microscopy and resistance to chloroquine. Macrophages were infected with differentipamutants expressing hlylow. Both δipaC hlylowand δipaD hlylowwere cytotoxic whilst δipaB hlylowand a hlylowstrain cured of shigella's pathogenicity plasmid were not. Furthermore, both δipC ahlylowand δipaD hlylowkilled through apoptosis as shown by both changes in ultrastructural morphology and fragmentation of the host ceil DNA. These results demonstrate thatipaBis essential forS. flexnerito induce apoptosis in macroph

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00341.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryDiaminopimelic acid (DAP) is a major component of the peptidoglycan layer of the mycobacterial cell wall. The mycobacterial cell wall has been implicated as a potential virulence factor and is highly immunogenic. The pathway for biosynthesis of DAP may serve as a target in the design of antimycobacterial agents and construction ofin vivoselection systems. Despite its significance, this biosynthetic pathway is poorly understood in mycobacteria. In order to develop a better understanding of mycobacterial DAP biosynthesis, the aspartate semialdehyde dehydrogenase (asd) genes ofMycobacterium smegmatis, bacille Calmette‐Guerin (BCG),Mycobacterium avium, Mycobacterium leprae, andMycobacterium tuberculosiswere isolated. TheM. smegmatis asdgene was isolated by complementation inEscherichia coli.This gene was then used to isolate theasdgenes from other mycobacteria. Theasd‐complemeningfragments from BCG andM. smegmatiswere sequenced. An open reading frame upstream of the mycobacterial asd gene was identified as the mycobacterial aspartokinase gene (ask). Primer extension analysis revealed that the only transcriptional start in this region is found 5’of theaskgene. This observation indicates that the mycobacterialaskandasdgenes are in an o

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00342.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryStudy of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression during macrophage infection. A derivative of Jn903, Tn903dlllacZ, is shown to transpose with high efficiency inL. pneumophila.Tn903dlllacZencodes resistance to kanamycin (KmR) and carries a 5’truncatedlacZgene that can form translational fusions to L. pneumophila genes upon transposition. The cls‐acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. KmRLacZ+insertion mutants ofL. pneumophilawere isolated and shown by DNA hybridization to carry a single Tn903dlllacZinserted within their chromosomes at various locations. One particular KmRLacZ+mutant, AB1156, does not produce the brown pigment (Pig) characteristic ofLegionellaspecies. Tn903dlllacZis responsible for this phenotype since reintroduction of the transposonlinked mutation into a wild‐type background results in a Pig phenotype. L.pneumophilapigment production is normally observed in stationary‐phase growth of cells in culture, and β‐galactosidase activity measured from thepig::lacZfusion increased during the logarithmic‐phase growth and peaked at the onset of stationary phase. Interestingly,pig::lacZexpression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L.pneumophilato grow within or kill host

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00343.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryCells from the early exponential growth phase of cultures of the methanogenic archaeonMethanococcus vannieliihave been shown to containc.180 transcripts of themcrBDCGA (mcr)operon,c.100 transcripts of theMvaL1,L10,L12 (Mva) operon,c.8 transcripts of theargGgene andc.1 transcript of thesecYgene. These values decreased toc.50mcrtranscripts,c.30Mvatranscripts,c.3argGtranscripts and<1 secY transcript per cell as the cultures entered the stationary phase of growth. Addition of bromoethanesulphonate (BES) or removal of H2inhibited growth and RNA synthesisin vivoand, at 37°C in the presence of BES, the half‐lives of themcr, Mva, argGandsecYtranscripts were found to be 15 min, 30 min, 57 min and 7 min, respectively. Addition of puromycin, pseudomonic acid or virginiamycin also inhibited growth but did not inhibit transcription. In the presence of puromycin the half‐lives of themcrandMvatranscripts increased c. 4.6‐fold and c. 3.5‐fold, respectively, and there was a net accumulation of theMvatranscript. Addition of pseudomonic acid or virginiamycin also increased the half‐life of theMvatranscript and also resulted in the accumulation of a second, shorterMvatranscript but did not increase the half‐life of themcrtranscript. Transcription of themcroperon was not stimulated by partial inhibition of me

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00344.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryBinding of the Gram‐positive pathogenic bacteriumStreptococcus pyogenes(group A streptococcus) to respiratory epithelium is mediated by the fibronectinbinding adhesin, protein F. Most strains of streptococci regulate the expression of protein F in response to oxygen levels and redox potential; however, JRS4 constitutively binds high levels of fibronectin under all environmental conditions. In this study, we have examined the regulation of protein F expression in JRS4 using a shuttle mutagenesis strategy novel to S.pyogenes.Cloned DNA representing the chromosomal loci adjacent to the gene which encodes protein F (prtF) was subjected to transposon mutagenesis inEscherichia coliusing a derivative of transposon mγδ that was modified to contain a streptococcal antibiotic‐resistance gene. Mutagenized DNA was then returned to the streptococcal chromosome by allelic replacement. Analysis of the resulting fibronectinbinding phenotypes revealed that insertions in a region upstream ofprtFabolished the constitutive phenotype. However, these mutants now demonstrated regulation in response to both oxygen levels and redox potential. Because these insertions define a locus responsible for the constitutive phenotype, it has been designatedrofA(regulatorofF). Chromosomal interruption studies using integrationat plasmids together with complementation data from a previous study (VanHeyningenetal., 1993) suggested thatrofAacts as a positive trans‐acting regulator ofprtF.Construction of prtF‐lacZ fusions indicated that transcription ofprtFis constitutive in JRS4 but is regulated inrofAmutants. Analysis of the DNA sequence defined by therofAinsertions revealed a 1495bp open reading frame, whose predicted product (RofA) possessed both a putative helix‐turn‐helix motif and limited homology to two other transcriptional activators (Mry, PrgR) of Gram‐positive surface proteins. Sequences homologous torofAwere found in regulated strains of 5.pyogenes, which suggests thatrofAmay act as an activator ofprtFin response to an unidentified environmental signal. We speculate that the allele reported here contains a mutation that renders it consti

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00345.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryWe isolated aRhizobium leguminosarummutant strain altered in theglnBgene. This event, which has never been described in the Rhizobiaceae, is rare in comparison to mutants isolated in the contiguous gene,glnA.TheglnBmutation removes theglnBApromoter but invivodoes not preventglnAexpression from its own promoter, which is not nitrogen regulated. TheglnBmutant strain does not grow on nitrate as a sole nitrogen source and it is Nod+, Fix+. Two –24/–12 promoters, for theglnllandglnBAgenes, are constitutively expressed in theglnBmutant, while two –35/–10‐like promoters forglnAandntrBCare unaffected. We propose that theglnBgene product, the P11protein, plays a negative role in the ability of NtrC to activate transcription from its target promoters and a positive role in the mechanism of nitrate ut

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00346.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe transformation‐deficient strain E26, Isolated as a pHV60 insertion mutant, was used to isolatecomK, a novel transcription unit required for genetic competence inBacillus subtilis.Mutational analysis and sequence determination showed thatcomKcontained one open reading frame (ORF), which could encode a protein of 192 amino acid residues with a predicted molecular weight of 22 500. An integrated copy ofcomKnot only complemented the competence deficiency of acomKdeletion mutant, but also that of strains E26 and FB93. Expression ofcomKoccurred exclusively in glucose‐based minimal medium during the transition to stationary growth phase. Furthermore, the expression of late competence genes appeared to be dependent on the gene product ofcomK, the expression of which in turn depended on the presence of a functionalcomL(orsrfA) transcription unit. These epistatic interactions indicate thatcomKis a competence locus occupying an intermediate position in the competence signal transduction network. Primer extension analysis showed thatcomKhas one major transcription start site, preceded by a sequence resembling the consensus promoter used by the σAform of RNA polyme

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00347.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryPili are one of the adhesins ofPseudomonas aeruginosathat mediate adherence to epithelial cell‐surface receptors. The pili ofP. aeruginosastrains PAK and PAO were examined and found to bind gangliotetraosyl ceramide (asialo‐GM1) and, to a lesser extend, ll3N‐acetylneuraminosylgangliotetraosyl ceramide (GM1) in solid‐phase binding assays. Asialo‐GM1, but not GM1, inhibited both PAK and PAK pili binding to immobilized asialo‐GM1on the microtitre plate. PAO pili competitively inhibited PAK pili binding to asialo‐GM1, suggesting the presence of a structurally similar receptor‐binding domain in both pilus types. The interaction between asialo‐GM1and pili occurs at the pilus tip as asialo‐GM1coated colloidal gold only decorates the tip of purified pili. Three sets of evidence suggest that the C‐terminal disulphide‐bonded region of thePseudomonaspilin is exposed at the tip of the pilus: (i) immunocytochemical studies indicate thatP. aeruginosapili have a basal‐tip structural differentiation where the monoclonal antibody (mAb) PK3B recognizes an antigenic epitope displayed only on the basal ends of pili (produced by shearing) while the mAb PK99H, whose antigenic epitope resides in residues 134–140 (Wonget al., 1992), binds only to the tip of PAK pili; (ii) synthetic peptides, PAK(128–144)ox‐OH and PAO(128–144)ox‐OH, which correspond to theC‐terminal disulphide‐bonded region ofPseudomonaspilin are able to bind to asialo‐GM1and inhibit the binding of pili to the glycolipid; (iii) PK99H was shown to block PAK pilus binding to asialo‐GM1Monoclonal antibody PK3B had no effect on PAK pili binding to asialo‐GM1Thus, the adherence of thePseudomonaspilus to glycosphingolipid receptors is a tip‐associated phenomenon Involving a tip‐exposed

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00348.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryPseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. The pilus adhesin ofP. aeruginosastrains PAK and PAO has been shown to bind to the glycolipid asialo‐GM1(Leeet al., 1994 —accompanying article). PAK and PAO pili were examined for their abilities to bind to the synthetic βGalNAc(1–4)βGal (a minimal structural carbohydrate receptor sequence of asialo‐GM1and asialo‐GM2proposed by Krivanet al., 1988a) using solid‐phase binding assays. Both pill specifically bound to βGalNAc(1–4)βGal. The binding of βGal‐NAc(1–4)βGal‐Biotin to the Immobilized PAK and PAO pili was inhibited by corresponding free pili. The receptor binding domain of the PAK pilus resides in theC‐terminal disulphide‐looped region (residues 128–144) of the pilin structural subunit (Irvinet al., 1989). Biotinylated synthetic peptides corresponding theC‐terminal residues 128–144 ofP. aeruginosaPAK and PAO pilin molecules were shown to bind to the βGalNAc(1–4)βGal‐(bovine serum albumin (BSA)). The binding of biotinylated peptides to βGalNAc‐(1–4)βGal‐BSA was inhibited by PAK pili, Ac‐KCTSDQDEOFIPKGCSK‐OH (AcPAK(128–144)ox‐OH) and Ac‐ACKSTQDPMFTPKGCDN‐OH (AcPAO(128–144)ox‐OH) peptides. (In these peptides Ac denotes Nα‐acetylation of theN‐terminus, ‐OH means a peptide with a free a‐carboxyl group at theC‐terminus and the‘ox’denotes the oxidation of the sulphhydryl groups of Cys–129 and Cys–142.) Both acetylated peptides were also able to inhibit the binding of βGalNAc(1–4)βGal‐biotin to the corresponding BSA‐Peptide(128–144)ox‐OH conjugates. The βGlcNAc(1–3)βGal(1–4)βGlc‐biotin conjugate was unable to specifically bind to either Immobilized PAK and PAO pili or the respectiveC‐termlnal peptides. The data above demonstrated that theP. aeruginosapili recognize asialo‐GM1receptor analogue and that βGalNAc(1–4)βGal disaccharlde is sufficien

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00349.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY