摘要:

SummaryWhether infection by bacteriophage P2 results in lysogenization of the host or vegetative growth of the phage depends upon a race between transcription from the repressor promoter Pc and the early promoter Pe; transcription from these promoters is mutually exclusive, since the Pcrepressor Cox is formed from the Petranscript and the Perepressor C from the Pctranscript. The involvement of integration host factor (IHF) in the lysogenization ofEscherichia coliK12 by P2 was tested by comparing wild‐type and IHF‐deficient (him Aandhim D)mutants. No lysogenic clones were formed following infection of the mutant bacteria. A switch plasmid that contains Pc‐C‐car andPe‐cox‐kanwas used to test the choice for expression of Pc versus Pe. In the wild‐type K12 bacteria, 20% of the clones expressed Pe transcription and 80% Pc transcription, whereas all transformed IHF‐defective clones expressed transcription from Peonly. The effects of IHF on thein vivoexpression of the Peand Pcpromoters were only marginal. The IHF protein was found to bind upstream of the Pepromoter, where a potentialihfsequ

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb02009.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryThe regulatory region of two divergently oriented transcriptional units involved in the formation of the gas‐evolving hydrogenase (isoenzyme 3) ofEscherichia coliwas investigated. DNA sequence analysis revealed the existence of a 210bp non‐coding region containing two sequences showing homology to ‐24/‐12 NtrA‐dependent promoters. These sequences were arranged in a divergent orientation entirety consistent with their being involved in transcribing the divergent operons. Through S1 protection experiments it could be shown that transcription of both promoters was NtrA‐dependent and that it was regulated in an identical manner: oxygen repressed expression, as did anaerobic growth in the presence of nitrate; transcription was induced in cells grown anaerobically in the absence of exogenous electron acceptors and formate was found to be obligately required for this anaerobic induction. Lying at an approximately equal distance between both promoters was a short stretch of DNA which showed similarity to the sequence previously identified (Birkmann and Böck, 1989a) as being necessary for formate induction of

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb02010.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryFNR, the activator of anaerobic respiratory genes ofEscherichia coli, has previously only been isolated as a protein ofMr, 29 000, which lacks nineN‐terminal amino acid residues. The underlying proteolytic events have been studied with the aim of isolating intact FNR and determining whether cleavage is the result of a physiologically significant intracellular processing mechanism or proteolytic degradation during isolation.The FNR protein was present in aerobically and anaerobically grown bacteria as the intact protein (Mr, 30 000). Proteolysis only occurred during and shortly after disruption of the bacteria. The production of FNR (Mr, 29 000) must therefore be regarded as an isolation artefact. The proteolysis was caused by a protease which is located outside the cytoplasmic membrane or activated upon disruption of the membrane. Protease inhibitors directed against serine, cysteine or metalloproteases failed to prevent cleavage of FNR. InE. colistrain CAG627, proteolysis was greatly reduced making it possible to isolate FNR ofMr, 30 000. TheN‐terminal sequence of FNR (Mr, 30 000) was identical to that predicted from thefnrgene starting with the initiating methionine residue and including a four‐cysteine cluster (16)Cys–X3–Cys–X2–C

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb02011.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryA model for the domain structure of σ54‐dependent transcriptional activators, based on sequence data, has been tested by examining the function of truncated and chimaeric proteins. Removal of theN‐terminal domain of NtrC abolishes transcriptional activation, indicating that this domain is positively required for activator function. Over‐expression of this domain as a separate peptide appears to titrate out the phosphorylating activity of NtrB. Removal of theN‐terminal domain of NifA reduces activation 3–4‐fold. The residual activity is particularly sensitive to inhibition by NifL, suggesting that the role of theN‐terminal domain is to block the action of NifL in derepressing conditions. TheC‐terminal domain of NtrC showed repressor activity when expressed as a separate peptide. This domain is necessary for activator function even when NtrC binding sites are deleted from promoters. A point mutation in the ATP‐binding motif of the NtrC central domain, Ser 169 to Ala, also abolished activator function. Exchanging theN‐terminal domains ofKlebsiella pneumoniaeNtrC, NifA andEscherichia co/rOmpR, did not produce any hybrid activity, suggesting thatN‐terminal domains in the native proteins specifically recognize th

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb02012.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryEscherichia colicontaining a cloned gene encoding theBordetella pertussisserotype 2 fimbrial subunit failed to produce detectable levels of the gene product in whole‐cell extracts. To engineer plasmids capable of directing the expression inE. coliof high levels of this product, both as a pre‐protein and as a methionylated mature form the upstream signals of the fimbrial subunit gene were replaced by the lambda PLand PRpromoters and theE. coli atpEtranslational initiation region. These constructs did not result in the expression of fimbrial subunit at detectable levels in severalE. colistrains including DH5. However, they did inE. coliCAG629, which is Ion protease and heat shock protein deficient. Both pre‐protein and methionylated mature protein had molecular weights of 25.0 kD, which indicated that correct processing of the leader sequence had occurred and thus that it was transposed across the inner membrane. Electron microscopic investigation of the cell surface ofE. colicells expressing either form of the fimbrial gene failed to detect the presence of filamentous structures. The methionytated mature form of the recombinant fimbrial subunit was purified to apparent homogeneity. After dialysis in appropriate conditions it was seen to autoassemble into protein polymers. Antibodies raised against polymerized recombinant subunit reacted weakly with wholeB. pertussisserotype 2 fimbriae in immunodot blot assays. However, such antibodies reacted in Western blots equally well with the recombinant and wild‐type form of the fimbrial subunit. These results suggest that the assem

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb02013.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryThe nucieotide sequence of the traT gene present in the virulence‐associated pfasmid ofSalmonella typhimuriumwas determined. The predicted TraT protein encoded by this gene was found to consist of 243 amino acids and to resemble the known TraT proteins of the plasmids of the F incompatibility group. Thus it contains a signal sequence of 20 amino acids, an amino‐terminal lipid attachment site, and two strongly hydrophobic regions close to each other in the mature protein. A mutation leading to increased permeability of the outer membrane to hydrophobic agents, previousfy focalrzed to thetraTgene, was shown to change a glycine residue to arginine within one of these hydrophobic regions. The same principle was found to apply to TraT of R6‐5: the introduction, by site‐directed mutagenesis, of either positively or negatively charged amino acids or the helix‐disrupting proline in the corresponding hydrophobic region led to increased hydrophobic permeability of the outer

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb02014.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryThree different techniques, protease accessibility, ceil fractionation andin situimmunocytochemistry, were used to study the location of the lipoprotein pullulanase produced byEscherichia coliK12 carrying the cloned pullulanase structural gene (pulA)fromKlebsiella pneumoniae, with or without theK. pneumoniaegenes required to transport pullulanase to the cell surface (secretion‐competent and secretion‐incompetent, respectively). Pullulanase produced by secretion‐competent strains could be slowly but quantitatively released into the medium by growing the cells in medium containing pronase. The released pullulanase lacked theN‐terminal fatty‐acylated cystelne residue (and probably also a shortN‐terminal segment of the pullulanase polypeptide), confirming that theN‐terminus is the sole membrane anchor in the protein. Pullulanase produced by secretion‐incompetent strains was not affected by proteases, confirming that it is not exposed on the cell surface. Pullulanase cofractionated with both outer and inner membrane vesicles upon isopycnic sucrose gradient centrifugation, irrespective of the secretion competence of the strain. Examination by electron‐microscopy of vesicles labelled with antipullulanase serum and protein A‐gold confirmed that pullulanase was associated with both types of vesicles. When thin‐sectioned cells were examined by the same technique, pullulanase was found to be located mainly on the cell surface of the secretion‐competent cells and mainly in the proximity of the inner membrane in the secretion‐incompetent cells. Thus, while the results from three independent techniques (substrate accessibility, protease accessibility andin situimmunocytochemistry) show that pullulanase is transported to the cell surface of secretion‐competent cells, this could not be confirmed by cell‐fractionation techniques. Possible explanations for t

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb02015.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryThe determined nucleotide sequence of theKlebsielia pneumoniaeUNF5023 genepulAcomprises a single open reading frame coding for a 1090‐residue precursor of the secreted protein pullulanase. The predicted sequence of this protein is highly homologous to that of pullulanase ofKiebsiella aerogenesstrain W70. However, the UNF5023 pullulanase lacks a collagen‐like sequence present at theN‐terminus of the mature W70 enzyme and differs further from the W70 pullulanase around residue 300 and at theC‐terminus. Pullulanases with or without the collagen‐like sequence could not be separated by gel electrophoresis under denaturing or non‐denaturing conditions, and were unaffected by collagenase. A large central domain which is highly conserved in both UNF5023 and W70 polypeptides contains eight short sequences that are also found in amylases and iso‐amylases. Linker mutations in the region of the UNF5023pulAgene coding for this domain abolished catalytic activity without affecting transport of the polypeptide across the outer membrane. Hybrid proteins comprising at least the amino‐terminal 656 residues of pre‐pullulanase fused to alkaline phosphatase were partially localized to the cell surface, as judged by their accessibility to anti‐pullulanase serum in immuno‐fluorescence tests. On the basis of these results, we tentatively propose that secretion signals required for recognition and translocation across the outer membrane via the pullulanase‐specific extension of the secretion pathway are located near the N‐terminus of th

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb02016.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryThe relationship between phenotypic variation and nucleotide sequence variation of the gene encodingVibrio parahaemolyticusthermostable direct haemolysin (tdhgene) was examined. Strains showing a typical haemolysin‐positive phenotype carried two chromosomal gene copies (designatedtdh1andtdh2)while fdh‐gene‐positive strains showing a weakly positive or negative haemolysin phenotype possessed only a single chromosomal gene copy. Both gene copies from a typical haemolysin‐positive strain were cloned and sequenced and possessed 97.2% homo‐logy. Comparison of the amino acid sequence predicted from the nucleotide sequence with the protein sequence determined by Edman degradation as well as construction of atdh1‐deficient yet haemolytic strain ofV. parahaemolyticussuggest that thetdh2locus is primarily responsible for the haemolytic phenotype. Two othertdhgene copies were cloned from a phenotypically negative strain which was unusual in that it contained one gene copy on a plasmid (designatedtdh4)in addition to a single copy on the chromosome (tdhS).Bothtdh3andtdh4were expressed inEscherichia coliand TDHs with haemolytic activity were produced. These gene copies were sequenced and shared 96.7% homology with thetdh1gene. TheV. parahaemolyticusstrain carryingtdh3andtdh4gene copies did not produce detectable amounts oftdh‐specific RNA transcript. It seems, therefore, that differences in the transcriptional control are primarily responsible for the differences seen in haemoly

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb02017.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

SummaryContrary to what would be expected from data in the literature, mutations in thefsoCgene of the F71(fso)P‐fimbrial gene cluster do not completely block fimbrial biogenesis.fsoCmutants still express small amounts of fimbriae of normal length, which carry the non‐adhesive minor subunit protein, FsoE, but lack the adhesin, FsoG. The FsoC protein operates at the same stage in fimbrial biogenesis as the FsoF and FsoG proteins. The data suggest that FsoC, FsoF and FsoG interact to form an initiation complex for fimbrial biogene

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1990.tb02018.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY