摘要:

SummaryPertussis toxin, a major virulence factor of Bordetella pertussis, is an oligomeric protein composed of five different subunits that are exported individually to the periplasmic space by the signal peptide–dependent pathway. After assembly, the protein is exported from the periplasm to the extracellular compartment. We show that pertussis toxin secretion across the outer membrane requires the gene product of at least one gene (ptlC) that is located downstream from the pertussis toxin operon. The amino acid sequence of PtlC shows a high degree of homology to VirB4, a protein encoded by thevirBoperon, which contains 11 open reading frames that are involved in the transfer of T‐DNA fromAgrobaderium tumefaciensto the plant cells. This is a novel mechanism of protein export in Gram‐negative bac

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01587.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryCheA is a dimeric autophosphorylating protein kinase that plays a critical role in the signal transduction network controlling chemotaxis InEscherichia coli. The autophosphorylation reaction was analysed using mutant proteins defective in kinase and regulatory functions. Proteins in which the site of autophosphorylation was mutated (CheA48HQ) or missing (CheAs) were found to phosphorylate the kinase‐defective mutant, CheA470GK. The kinetics of this reaction support the hypothesis that autophosphorylation is the result of trans‐phosphorylation within a dimer. The carboxy‐terminal portion of CheA was previously shown to be dispensable for autophosphorylation, but required for regulation in response to environmental signals transmitted through a transducer and CheW. Mixing of CheA48HQ or CheA470GK with a truncated protein lacking this regulatory domain demonstrated that regulated autophosphoryltion requires the presence of both carboxy‐terminal portions in a CheA dimer. These results indicate that the dimeric form of CheA plays an integral role in signal transduction in bacterial che

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01588.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryThe chemical mutagen ethylmethanesulphonate (EMS) has been used to generate mutants ofErwinia carotovorasubspeciescarotovorawhich are defective in the secretion of pectinases (Pel) and cellulases (Cel) but unaltered for protease (Prt) secretion. Such mutants, called Out−still synthesize Pel and Cel but these enzymes accumulate within the periplasm. Cosmid clones carrying wild‐typeE. carotovrassp.carotovoraDNA, identified by their ability to restore the Out+phenotype when transferred to some Out−mutants, were classified into six complementation groups using cosmids and cosmid derivatives. Analysis of the nucleotide sequence of a 12.7 kb DNA fragment, encompassing complementing cosmid inserts, revealed a coding capacity for 13 potential open reading frames (ORFs), and these were designatedoutC‐outO. Some of the out gene products were visualized using a T7 gene 10 expression system. The predicted Out proteins are highly similar to components of extracellular enzyme secretion systems from a diverse range of eubacteria includingErwinia chrysanthemi, Klebsiella oxytoca, Aeromonas hydrophila, Pseudomonas aeruginosa and Xanthomonas campestris.Lower levels of similarity exist betweenEccOut proteins and components of macromolecular trafficking systems fromBacillus subtilis, Haemophilus influenzae, Agrobacterium tumefaciens, Yersinia pestisand a protein involved in the morphogenesis of filamentous bacteriophages such

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01589.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryUpstream of themoxFJGIRgenes ofParacoccus denitrificansa regulatory region involved in methanol oxidation was identified. The nucleotide sequence of this region was determined and revealed three genes,moxZ, moxYandmoxX, which are transcribed opposite tomoxFand which encode proteins of 16.4, 48.2 and 24.5kDa, respectively. Computer alignment analysis revealed that the gene products of moxyandmoxXhave homology with the protein histidine kinases and the response regulators, respectively, forming the two‐component regulatory systems. No significant homology of themoxZgene product with any known protein, sequenced thus far, was found. The MoxZ, MoxY and MoxX proteins were identified inEscherichia coliin a heterologous expression system. Mutants with an insertion of a kanamycin‐resistance marker inmoxZ, moxYandmoxXwere isolated. These mutant strains were unable to grow on methanol while growth on methylamine was not affected. In themoxZmutant both subunits of methanol dehydrogenase and cytochrome c5511were not synthesized, methanol dehydrogenase activity was absent, and hardly any expression of amoxZ‐lacZtranscriptional fusion was found. Complementation of the mutation was observed after addition of the three genesmoxZ, Y and X,in trans.This indicates that the two‐component regulatory system is involved in activation of themoxFpromoter. A mutant with an unmarked deletion inmoxZwas isolated. This mutant showed reduced growth on methanol relative to the wild type. Expression of themoxF‐lacZtranscriptional fusion gene and methanol dehydrogenase activity in this strain were also lower than those found in the wild type. Therefore, besides the two proteins of the two‐component regulatory pair, a third protein, MoxZ, appears to be involved in regulation of methanol dehydrogenas

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01590.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryThe nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from theRhizobium leguminosarumbv. viciae hydrogenase gene cluster has been determined. Six genes, arranged as a single operon, were identified, and designatedhypA, B, F, C, DandEbased on the sequence similarities of all of them, excepthypF, to genes from the hydrogenase pleiotropic operon (hyp) fromEscherichia coli.The gene products fromhypBFCDEwere identified byin vivoexpression analysis inE. coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence. Transposon Tn5 insertions intohypB, hypF, hypDandhypEresulted inR. leguminosarummutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit. The gene products HypA, HypB, HypF and HypD contained CX2C motifs characteristic of metal‐binding proteins. In addition, HypB bore a long histidine‐rich stretch of amino acids near the N‐terminus, suggesting a possible role in nickel binding for this protein. The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX2CX81CX2C) with a capacity to form zinc finger‐like structures in theN‐terminal third of the protein. A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01591.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryWithin the capsule gene complex (cps) ofNeisseria meningitidistwo functional regions B and C are involved in surface translocation of the cytoplasmically synthesized capsular polysaccharide, which is a homopolymer of α‐2,8 polyneuraminic acid. The region‐C gene products share characteristics with transporter proteins of the ABC (ATP‐binding cassette) superfamily of active transporters. For analysis of the role of region B in surface translocation of the capsular polysaccharide we purified the polysaccharides of region B‐ and region C‐defectiveEscherichia coliclones by affinity chromatography. The molecular weights of the polysaccharides were determined by gel filtration and the polysaccharides were analysed for phospholipid substitution by polyacrylamide gel electrophoresis and immunoblotting. The results indicate that the full‐size capsular polysaccharide with a phospholipid anchor is synthesized intracellularly and that lipid modification is a strong requirement for translocation of the poly saccharide to the cell surface. Proteins encoded by region B are involved in phospholipid substitution of the capsular polysaccharide. Nucleotide sequence analysis of region B revealed two open reading frames, which encode proteins with molecular masses of 45.1

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01592.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryA novobiocin producer,Streptomyces sphaeroides, contains two genes, designatedgyrBsandgyrBR, that encode novobiocin‐sensitive and ‐resistant DNA gyrase B proteins, respectively. The cloning ofgyrBRwas reported earlier; here, we describe the cloning ofgyrBS. Both genes have been sequenced (the deduced products ofgyrBsandgyrBRhaveMrvalues of 87.6K and 86.5 K, respectively) and their transcripts have been mapped. Downstream ofgyrBS, and co‐transcribed with it, is the solegyrAgene (encoding DNA gyrase A protein). By constructing hybridgyrBSgenes, using fragments ofgyrBSandgyrBR, a specific portion of theN‐terminal domain of the gyrase B protein (corresponding to amino acid residues 134–256 ofEscherichia coligyrase B) has been implicated in the binding of n

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01593.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryDNA supercoiling is known to influence the pattern of gene expression in prokaryotes. Thus the mechanism of transcription initiation and the topological state of the template are intimately related. Usingin vitroreconstituted transcription assays, composed of purified RNA polymerase and promoters in their natural topological state, we have conducted a detailed study of transcription initiation from T7 early promoters including the following steps: the formation of ternary complexes, acquisition of rifampicin resistance, release of sigma factor and the capacity for RNA chain elongation in complexes. We determined the order of these events and the length of the transcripts when each step occurred during initiation of transcription on supercoiled templates. The length of the transcripts varied in a promoter‐specific manner. Analysis of abortive products formed during the initiation showed that stronger promoters go to the elongation mode at transcript lengths shorter than that required for weaker promoter

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01594.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryThe human sexually transmittted parasiteTrichomonas vaginalisis a representative of one of the three earliest evolving eukaryotic lineages. We investigated whetherT. vaginalishas DNA sequences and peptides related to cell division control molecules universal among yeasts and higher eukaryotes. AT. vaginalisceil division control (CDC2/28) homologue was amplified by the polymerase chain reaction and sequenced. The absolute similarity with other CDC2/28 genes was 47%, with conservative replacement similarity of 67%. Western blots demonstrated a singleT. vaginalispeptide reactive with antiserum to the PSTAIRE peptide, an expressed component of CDC2/28 genes in higher eukaryotes. Although eukaryotic,T. vaginalishas properties similar to those of bacteria and is the earlist evolving eukaryote reported to possess CDC2/28 DNA and peptide homologues. These observations suggest that the molecular origins of cell division control in eukaroytes preceded mitochondria, 28S ribosomes and regulated glycolysis.

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01595.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryIsoniazid‐resistant isolates ofMycobacterium tuberculosiswere transformed with a plasmid vector carrying the functional catalase‐peroxidase (katG) gene. Expression ofkatGrestored full drug susceptibility in isolates initially resistant to concentrations ranging from 3.2 to>50μgml−1. Transformation with the correspondingkatGgene fromEscherichia coliresulted in low‐level expression of catalase and peroxidase activities and conferred partial isoniazid sen

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01596.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY