摘要:

SummaryBacillus subtiliscell extracts, prepared at different times during growth, contained several proteins that were apparently guanylylatedin vitrowith [α‐32P]‐GTP. Four of the proteins were partially purified and theN‐terminal amino acid sequences (13 to 20 residues) were determined. One sequence had 84% identity toBacillus stearothermophliustriosephosphate isomerase, two were 100% identical to the predicted sequences of theB. subtilis ptsIandptsHgenes while no identity was found for the fourth sequence. This apparent guanylylation occurred with proteins involved in glucose metabolism, although the significance is u

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb00882.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThe uropathogenic Gram‐negative bacteriumProteus mirabilisexhibits a form of multicellular behaviour termed swarming, which involves cyclical differentiation of typical vegetative cells into filamentous, multi‐nucleate, hyperflagellate swarm cells capable of rapid and co‐ordinated population migration across surfaces. We observed that differentiation into swarm cells was accompanied by substantial increases in the activities of intracellular urease and extracellular haemolysjn and metalloprotease, which are believed to be central to the pathogenicity ofP. mirabills.In addition, the ability ofP. mirabilisto invade human urothelial cellsin vitrowas primarily a characteristic of differentiated swarm cells, not vegetative cells. These virulence factor activities fell back as the cells underwent cyclical reversion to the vegetative form (consolidation), in parallel with the diagnostic modulation of flagellin levels on the cell surface. Control cellular alkaline phosphatase activities did not increase during differentiation or consolidation. Non‐flagellated, non‐motile transposon insertion mutants were unable to invade urothelial cells and they generated only low‐level activities of haemolysin, urease and protease (0–10% of wild type). Motile mutants unable to differentiate into swarm cells were comparably reduced in their haemolytic, ureolytic and invasive phenotypes and generated threefold less protease activity. Mutants that were able to form swarm cells but exhibited various aberrant patterns of swarming migration produced wild‐type activities of haemotysin, urease and protease, but their ability to enter urothelial cells was three‐ to 10‐fold lower. Analysis of haemolysin(hpmA)transcripts showed that during swarm cell differentiation there was a c. 50‐fold increase in the level of the predicted major 5.2 kb and minor 6.9 kb mRNAs transcribed from thehpmoperon, and assay of mRNA complimentary to urease(ureC)and flagellin(fliC)gene sequences confirmed that modulation of virulence factor activity during the swarming cycle resulted from differential expression of virulence genes in parallel withfliCgene expression. Hybridization of stage‐specific mRNA with 30 random, non‐overlapping chromosomal gene probes provided no evidence for universal changes in the expression of theP. mirabiisgenome, suggesting that differential virulence gene expression m

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb00883.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryTheBacillus cereus cnpgene coding for the thermolysin‐like neutral protease (TNP) has been cloned, sequenced, and expressed inBacillus subtilis.The protease is first produced as a pre‐pro‐protein (Mr= 61000); the pro‐peptide is approximately two‐thirds of the size of the mature protein. The pro‐sequence has been compared with those of six other TNPs, and significant homologies have been found. Additionally, the TNP pro‐sequences are shown to be homologous to the pro‐sequence ofPseudomonas aeruginosaelastase. A mutant has been constructed fromcnp, in which 23 amino acids upstream from the pro‐protein processing site have been deleted. This region has no homologous analogue in any of the other TNP pro‐sequences. The detection results in a delay of six to eight hours in detection of active protease in the growth medium, as well as a 75% decrease in maximum protease production.N‐terminal analysis of the mutant mature protein demonstrates that the processing site is unaltered by the pro‐sequence deletion. The deletion must, therefore, modulate the kinetics of processing and/or secre

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb00884.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryShigella flexnericauses bacillary dysentery by invading epithelial cells of the colonic mucosa. We have characterized theicsBgene which is located on the virulence plasmid pWR100. After inactivation oficsB, the mutant strain remained invasive, but formed abnormally small plaques on HeLa cell monolayers, colonized only the peripheral cells of Caco‐2 islets, and was unable to provoke a keratoconjunctivitis in guinea‐pigs. Examination of infected HeLa cells showed that theicsBmutant was able to lyse the phagocytic vacuole and to form protrusions at the surface of infected cells, but, unlike the wild type, remained trapped in protrusions surrounded by two membranes. These results indicate that IcsB is involved in the lysis of the protrusions, a step necessary for intercellular spr

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb00885.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryCoumarins are inhibitors of the ATP hydrolsis and DNA supercoiling reactions catalysed by DNA gyrase. Their target is the B subunit of gyrase (GyrB), encoded by thegyrBgene. The exact mode and site of action of the drugs is unknown. We have identified four mutations conferring coumarin resistance toEscherichia coli: Arg‐136 to Cys, His or Ser and Gly‐164 to Val.In vitro, the ATPase and supercoiling activities of the mutant GyrB proteins are reduced relative to the wild‐type enzyme and show resistance to the coumarin antibiotics. Significant differences in the susceptibility of mutant GyrB proteins to inhibition by either chlorobiocin and novobiocin or coumermycin have been found, suggesting wider contacts between coumermycin and GyrB. We discuss the significance of Arg‐136 and Gly‐164 in relation to the notion that coumarin drugs act as competitive inhibitors of the ATPase

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb00886.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThe alterNatlve sigma factor σ54is required for transcription of nitrogen fixation genes inKlebsiella pneutnoniaeand other diazotrophs. Thenifgenes, and other Eσ54‐dependent genes whose products are necessary for a wide range of processes, are positively regulated. A unifying model that is well supported by studies onnifand other nitrogen‐regulated(ntr)genes includes the central tenet that σ54confers upon core RNA polymerase the ability to recognize and bind specific promoter sequences, but not the ability to isomerize to the open complex without assistance from the appropriate activator protein. Direct physical evidence for formation of an activator‐independent complex between Eσ54and the NifA‐dependentK. pneumoniae nifHandnifUpromoters has, to date, been lacking. Using purified components we have now demonstrated formation of the closed complex at these promoters, indicating that it is an intermediate along the pathway to open complex formation. The closed complex was not detected when conserved features of the promoter were altered by mutation, nor was its stability increased when integration host factor protein was bound adjacent to the Eσ54recognit

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb00887.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThis paper reports the characterization of a new locus,vagC/vagD, on the virulence plasmid ofSalmonella dublin.Strain G19, harbouring aTnAinsertion invagC, exhibited reduced virulence althoughvagCwas outside the 8 kb essential virulence region. G19 was also unable to grow on minimal‐medium containing various sole carbon/energy sources, unlike the wild‐type and plasmid‐cured strains. Sequencing of the locus revealed the presence of two ORFs (vagCandvagD) which overlapped by one nucleotide. The VagC polypeptide (12 kDa) was observed using minicell expression. Results indicated thatvagDwas responsible for the pheno‐typic differences observed between the wild type and G19, and thatvagCmodulated the activity ofvagD.Furthermore, microscopic analysis of G19 cells harvested from minimal‐medium plates showed that a high proportion of cells were elongated, which suggested thatvagCandvagDmight be involved in coordination of plasmid replication with cell division. We propose thatvagD, under certain environmental conditions, acts to prevent cell division until plasmid replication is complete, thus aiding plasmid maintenance.vagCandvagDare absent from the related virulence plasmid ofSalmonella ty

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb00888.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryInterposon mutagenesis of a region upstream of thepetABC(fbcFBC)operon, encoding the ubiquinol: cytochrome c2oxidoreductase (bc1complex) of the photosynthetic bacteriumRhodobacter capsulatusrevealed the presence of two genes,petPandpetR.DNA nucleotide sequence determination of this region indicated thatpetPandpetRare transcribed In the same direction as thepetABC(fbcFBC)operon, and are translationally coupled. A silent insertion located In the interoperonal region separatingpetPRand thepetABC(fbcFBC)genes indicated that these clusters have separate promoters. The deduced amino acid sequence of the putativepetRgene product is homologous to various bacterial response regulators, especially to those of the OmpR subgroup. Moreover, it was found that PetR‐mutants are unable to grow on rich or minimal media by either photosynthesis or respiration, demonstrating that these gene products are essential for growth ofR. capsulatu

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb00889.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThetusgene encodes a DNA‐binding protein (Tus) that inhibits replication forks at specific block‐sites within the terminus region of theEscherichia colichromosome. One of these block‐sites,TerB, is adjacent to thetusgene. Using primer extension and a promoter fusion to characterizein vivoexpression, we have demonstrated that thetustranscription start site is withinTerB, and that Tus protein autoregulates expression at this weak promoter We have also demonstrated that a minority oftustranscripts are initiated fromanupstream region that contains two additional open reading frames. This readthrough transcription intotusis reduced in the presence of Tus pr

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb00890.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryInvasive pulmonary aspergillosis, usually caused byAspergillus fumigatus, is a life‐threatening condition of immunosuppressed patients. We have created a mutant strain of this fungus that lacks an extracellular alkaline protease (AFAlp). This was accomplished by transformation ofA. fumigatuswith a plasmid containing a selectable marker for hygromycin B resistance, and a 504 bp segment of the AFAlp gene, obtained by polymerase‐chain‐reaction‐based amplification ofA. fumigatusgenomic DNA. Approximately 25% of transformants resulted from disruption of the AFAlp gene. SDS‐polyacrylamide gel etectrophoresis of proteins from the culture filtrate of a strain carrying the AFAlp gene disruption showed that it lacked a major protein of 33 kDa. Furthermore, in contrast to the culture filtrate from wild‐type cells, the mutant had undetectable activity on azocollagen and elastin‐Congo red, over a broad pH range. This shows that AFAlp accounts for most, if not all, of the extracellular elastinolytic activity ofA. fumigatus, and that the mutant strain will be useful in assessing the role of AFAlp in

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb00891.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY