摘要:

SummaryThefirAgene is essential for growth ofEscherichia coligrowth and lies in the 4‐minute region of the genome.firAencodes the FirA protein which contains 341 amino acids and has an apparent molecular mass of 36 kDa. Genetic evidence suggests that FirA plays a role in transcription since certainfirAalleles confer temperature sensitivity for growth and RNA synthesis as well as reversing the rifampin resistance of rifampin‐resistantrpoBmutants (‘fir’ effect). FirA co‐immunoprecipitates with RNA polymerase holoenzyme, implying a physical association with the transcriptional machinery, possibly with the beta subunit of RNA polymerase. FirA contains a previously undescribed isoleucine/valine‐rich six‐amino‐acid repeat occurring 14 times within theN‐terminal and 12 more times within theC‐terminal half of the protein. This repeat can be formulated as [HXXXhZ]n with ‘H’ representing a large non‐polar residue (usually isoleucine),‘h’ representing a smaller non‐polar residue, Z’ representing either charged/polar or aromatic residues, XXX representing residues typical of beta turns, and ‘n’ being equal to the repeat number. We refer to this repeat as an isoleucine patch. Proteins encoded by threeE. coliacyltransferases also contain this motif which is roughly positioned in each case, within the amino‐ and carboxyl termini, as in FirA. When the sequences of these proteins are aligned, a region of poor similarity separates the isoleucine patches. The significance of these repeats remains unknown although we speculate that they play an important structural role in the organization and function of FirA (and other proteins containing isoleucine patches), possibly by acting as homo‐ or hetero‐dimerization interfaces. This is supported by the finding that mutations conferring temperature sensitivity for growth or both temperature sensitivity for growth as well as the ‘fir’ effect, map to hypoth

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01532.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryVirtually every organism so far tested has been found to possess an extremely efficient DNA repalr mechanism to ensure that certaln alkylated oxygens do not accumulate in the genome. The repalr is executed by DNA methyltransferases (MTases) which repalr DNA O6‐methylguanine (O6MeG), O4‐methylthymine (O4MeT) and methylphosphotriesters (MePT). The mechanism is rather extravagant because an entire protein molecule is expended for the repalr of just one, or sometimes two, O‐alkyl DNA adduct(s). Cells profit from such an expensive transaction by earning protection agalnst death and mutation by alkylating agents. This review considers the structure, function and biological roles of a number of well‐characterized microbial DNA repalr

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01533.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryShigella flexneri, a Gram‐negative bacillus belonging to the family Enterobacteriaceae, causes bacillary dysentery in humans by invading colonic epithelial cells. Processes by which epithelial cells, which are not professional phagocytes, may limit the spread of the invading microorganisms are poorly understood. This paper shows that IcsA (VirG), a 120kDa bacterial outer membrane protein responsible for intracellular and cell‐to‐cell spread through polymerization of actin, is a major substrate for phosphorylation by cyclic‐dependent protein kinases. Site‐directed mutagenesis of a sequence encoding phosphorylation consensus motif SSRRASS, located at residues 754–760, almost completely abolished the ability of this protein to be phosphorylated by protein kinase A. Such mutants expressed a ‘super Ics’ phenotype, characterized by an increased capacity to spread from cell‐to‐cell during the first three hours of infection in the HeLa cell infection assay. These data suggest that host‐cell phosphorytation of key virulence proteins located on the bacterial surface may represent a significant host defence mechanism during

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01534.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryTheRhizobium leguminosarum nodMgene product shows strong homology to theEscherichia coli gImSgene product that catalyses the formation of glucosamine 6‐P from fructose 6‐P and glutamine. DNA hybridization withnodMindicated that, in addition tonodMon the symbiotic plasmid, another homologous gene was present elsewhere in theR. leguminosarumgenome. A glucosamine‐requiring mutant was isolated and its auxotrophy could be corrected by two different genetic loci. It could grow without glucosamine when thenodMgene on the symbiotic plasmid was induced or if the clonednodMgene was expressed from a vector promoter. Alternatively, it could be complemented by a second fragment ofR. leguminosarumDNA that carries a region homologous toE. coli gImS.Biochemical assays of glucosamine 6‐P formation confirmed that the twoR. leguminosarumgenesnodMandgimShave interchangeable functions. No nodulation of peas or vetch was observed with a doublenodM gImSmutant, and this block occurred at a very early stage since no root‐hair deformation or infection threads were seen. Nodulation and root‐hair deformation did occur with either thenodM orthegImSmutant, showing that the gene products of either of these genes can be involved in the formation of the lipo‐oligosaccharide nodulation signal. However, thegImSmutant formed nodules that had greatly reduced nitrogen fixation. Constitutive expression ofnodMrestored nitrogen fixation to thegImSmutant. Therefore the reduced nitrogen fixation probably occurs becausegImSis absent andnodMis not normally expressed in nodules and, in the absence of glucosamine precursors, normal bacteroid maturati

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01535.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryParasitism of host epithelial cells byTrichomonas vaginalisis a highly specific event. Four trichomonad surface proteins (adhesins) with molecular masses of 65000 daltons (65kDa; AP65), 51 kDa (AP51), 33kDa (AP33), and 23 kDa (AP23) mediate the interaction ofT. vaginaliswith epithelial cells. Fresh isolates, when compared with long‐term‐grown isolates, had greater amounts of adhesins, which corresponded with increased levels of cytoadherence. Anti‐adhesin antibodies reacted by immunobiol only with the respective protein and detected, by indirect immunofluore‐scence, each adhesin on the parasite surface. These antibodies inhibited the binding of live parasites to epithelial cells and protected epithelial cells from contact‐dependent cytotoxicity. The pretreatment of epithelial cells with a preparation of purified adhesins also blocked trichomonal cytoadherence. Moreover, HeLa cells possessed molecules which recognized and bound to adhesins on nitrocellul

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01536.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryPhospholipase C has been increasingly recognized as a significant virulence determinant in the pathogenesis of Gram‐negative and Gram‐positive infections.Pseudomonas aeruginosacarries two, non‐tandem genes encoding phospholipase C (PLC) activity. One PLC (PLC‐H) haemolyses human and sheep erythrocytes while the other is not haemolytic for these kinds of red blood cells. It was previously determined that the synthesis of both PLCs is regulated by inorganic phosphate (Pi), but little else was known regarding the regulation of these potentially important virulence determinants ofP. aeruginosa.In this report, data are presented demonstrating that both PLC genes are regulated at the transcriptional level by Pi and by aP. aeruginosahomologue of the positive regulator of genes in the Pi regulon ofEscherichia coli, i.e. PhoB. In addition to Pi, it is also shown in this report that the synthesis of both PLC‐H and PLC‐N is induced by compounds which are not only derived from the substrate product of both enzymes, i.e. phosphorylcholine, but are also known osmoprotectants in eukaryotic and prokaryotic cells. The osmoprotective derivatives of phosphorylcholine which induce the synthesis of PLC inP. aeruginosainclude choline, glycine betaine, and dimethylglycine, but not sarcosine (monomethylglycine) or glycine. By constructing mutants which are deficient in the production of each separate PLC and in the production of PhoB it was determined that induction of PLC‐H by the osmoprotective compounds is independent of Pi concentration and PhoB, while induction of PLC‐N by these compounds requires Pi‐deficient conditions and PhoB. It was further demonstrated that while an ionic osmolyte, such as NaCl, inhibited production of both enzymes in Pi‐deficient conditions, the presence of osmoprotective compounds, such as choline, restored synthesis of PLC whenP. aeruginosawas grown in 0.5 M NaCl. In contrast to the inhibition of PLC production in 0.5 M NaCl in Pi‐deficient conditions, PLC production in an equivalent osmotic pressure generated by the neutral osmolyte sucrose was not inhibited, and choline markediy induced production of PLC in this ionically neutral, high osmotic environment. These observations are especially relevant to the pathogenic potential of PLC in the dehydrating (hence high osmotic) conditions of the lungs of cys

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01537.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryTheSalmonella typhimuriumLT2 sialidase (neuraminidase, EC 3.2.1.18) structural gene,nanH, has been cloned and sialidase overproduced from multicopy plasmids inEscherichia coli.Sialidase expression was regulated positively by cAMP. In contrast, certain Tn1000insertions located upstream ofnanHcoding sequences reduced sialidase activity. AnanHchromosomal insertion mutation constructed by marker exchange demonstrated a single sialidase gene copy InS. typhimuriumLT2. The complete nucleotide sequence ofnanH, encoding a 41 300 dalton polypeptide, was determined and the derived primary structure was similar to sialidases fromClostridium perfringens, Clostridium sordellii, Bacteroides fragills, andTrypanosoma cruzi.Comparative sequence analysis, including codon usage and secondary structure predictions, indicated that the S.typhimuriumand clostridial sialidases are homologous, strongly suggestive of an interspecies gene transfer event. At least two primary sequence motifs of the bacterial enzymes were detected in influenza A virus sialidases. The predicted secondary structure ol the bacterial enzymes was strikingly similar to viral sialidase. From the population distribution ofnanHdetected within a collection of salmonellae, it was apparent thatS. typhimuriumobtained itsnanHcopy most recently fromSalmonella arizonae. S. typhimuriumLT2 is thus a genetic mosaic that differs from other strains of even the same serotype bynanHplus potentially additional characters linked tonanH.These results have relevance to the evolution and function of sialidases in pathogenic microbes, and to the origin of the sialic acids.

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01538.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryPsiB, an anti‐SOS protein, shown previously to prevent activation of RecA protein, was purified from the crude extract of PsiB overproducing cells. PsiB is probably a tetrameric protein, whose subunit has a sequence‐deduced molecular mass of 15741 daltons. Using an immuno‐assay with anti‐PsiB antibodies, we have monitored PsiB cell concentrations produced by F and R6‐5 piasmids: the latter type produces a detectable level of PsiB protein while the former does not. The discrepancy can be assigned to a Tn10outgoing promoter located upstream ofpsiB.When we inserted a Tn10promoter upstream of FpsiB, the F PsiB protein concentration reached the level of R6‐5 PsiB.We describe here the physiological role that PsiB protein may have in the cell and how it causes an anti‐SOS function. We observed that PsiB protein was transiently expressed by a wild‐type F sex factor during its transmission to anEscherichia coliK‐12 recipient, In an F+× F‐cross, PsiB concentration increased at least 10‐fold in F‐recipient bacteria after 90 minutes and declined thereafter; thepsiBgene may be repressed when F plasm id replicates vegetatively. PsiB protein may be induced zygotically so as to protect F single‐stranded DNA transferred upon conjugation. PsiB protein, when overproduced, may interfere with RecA protein at chromosomal single‐stranded DNA sites generated by discontinuous DNA replication, thus causing

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01539.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThe chromosomal genesgefandrelFfromEscherichia coliand the plasmid‐encoded geneshok, flmA, srnB, andpndAconstitute thegefqene family, which encodes a cell‐killing function. In order to investigate the mechanism of cell killing we have isolated anE. colimutant strain that is resistant to the overexpression of the toxic proteins encoded by thegefgene family. This phenotype requires at least two mutations, one of which has been mapped to 55.2 minutes. This mutation was sequenced and shown to represent a single base substitution in an open reading frame (ORF178) encoding a putative membrane protein having a molecular mass of 20.1 kDa. ORF178 and an upstream frame, ORF190, probably constitute an ope

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01540.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThekilBlocus (which is unclonable in the absence ofkorB) of broad‐host‐range plasmid RK2 (60kb) lies between thetrfAoperon (co‐ordinates 16.4 to 18.2kb), which encodes a protein essential for vegetative replication, and the Tra2 block of conjugative transfer genes (co‐ordinates 20.0 to 27.0 kb). Promoter probe studies indicated thatkilBis transcribed clockwise from a region containing closely spaced divergent promoters, one of which is thetrfApromoter. The repression of both promoters bykorBsuggested thatkilBmay also play a role in stable maintenance of RK2. We have sequenced the region containingkilBand analysed it by deletion and insertion mutagenesis. Loss of the KilB+phenotype does not result in decreased stability of mini RK2 plasmids. However insertion in ORFI (kilBl) of the region analysed results in a Tra‐phenotype in plasmids which are otherwise competent for transfer, demonstrating that this locus is essential for transfer and is probably the first gene of the Tra2 region. From thekilBiDNA sequenceKilBlis predicted to be 34995Da, In line with Mr= 36000 observed by sodium dodecyl sulphate/potyacrylamide gel electrophoresis, and contains a type I ATP‐binding motif. The purified product was used to raise antibody which allowed the level ofKilBIproduced from RK2 to be estimated at approximately 2000 molecules per bacterium. Protein sequence comparisons showed the highest homology score with VirB11, which is essential for the transfer of theAgrobacterium tume‐ faciensTi plasmid DNA from bacteria to plant cells. The sequence similarity of bothKilBIand VirB11 to a family of protein export functions sugge

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01541.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY