摘要:

SummaryOur work on bacterial detergent resistance started with the realization that bacteria growing in a sink full of soap must be resistant to the detergents in that soap. We chose sodium dodecyl sulphate (SDS) as a model detergent and decided to see how much SDS the bacterium growing in the sink could tolerate. The research program thus initiated has shown that bacteria such asEnterobacter cloacaecan grow in up to 25% SDS and that SDS‐shock proteins constitutec.8% of the proteins synthesized by SDS‐grownEscherichia coli.It has also provided explanations why enteric bacteria are oxidase negative, and how pyrroloquinoline quinone (POQ) enters the periplasmic space. Finally, forE. coli, it has provided evidence for an alternate, phosphate‐limited, aquatic life style which places greater emphasis on the Entner‐Doudoroff pathway. Detergent resistance is important both medically and ecologically, e.g. entry of pathogens via bile‐salt‐containing intestinal tracts and biodegradation of detergent‐like pollutants such as those resulting from oil spills. Our current research is focused on SDS‐induced modifications of the cytoplasmic membrane and the presence of SDS i

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02161.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryRegulatory outcome in a bacterial operon depends on the interactions of all the components which influence mRNA production. Levels of mRNA can be altered profoundly by both negative and positive regulatory elements which modulate initiation of transcription. The occupancy of regulatory sites on the DNA by repressors and activators is determined not only by the affinity of these proteins for their cognate site(s) but also by the oligomeric state of the regulatory protein. Thelacoperon inEscherichia coliprovides an excellent prototypic example of the influence of protein assembly on the transcriptional status of the associated structural genes. DNA loop formation is essential for maximal repression of thelacoperon and is contingent upon the presence of multiple operator sites in the DNA and the ability of the repressor to self‐associate to form a bidentate tetramer. The stability of this looped complex is enhanced significantly by DNA supercoiling. Tetramer assembly from dimers apparently occurs via interactions of a‘leucine zipper’motif in the C‐terminal domain of the protein, and the tetramer is essential to formation of looped complexes. Furthermore, analysis of the DNA‐binding characteristics of dimeric mutants has established that the monomer‐dimer association and dimer‐DNA binding (monomer does not bind to DNA) are coupled equilibria. Thus, dimer assembly is essential for generating a DNA‐binding unit, and tetramer assembly is required for formation of the stable looped DNA structure that maximally represses mRNA synthesis. Protein‐protein interactions therefore play a pivotal role in the regulatory activities of thelacrepressor and must be considered when analysing the activities of any oligomeric DN

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02162.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryTwo‐dimensional gel electrophoresis was used to follow low changes in gene expression associated with antibiotic (bialaphos) biosynthesis inStreptomyces hygroscopicus.Cultures were pulse‐labelled with [35S]‐methionine before, during, and after the switch from primary to secondary metabolism in order to compare kinetic profiles of bialaphos (antibiotic) production (bap) genes during this metabolic transition. Separation of gene products on two‐dimensional gels revealed that 27 were dependent onbrpAfor optimal expression and were activated as the culture approached stationary phase. Genes which encoded 10brp4‐dependent proteins were mapped to a 10 kbSstlfragment of the 35 kbbapgene cluster by expressing them inStreptomyces lividansusing the thiostrepton‐inducibletipApromoter.N‐terminal amino acid sequences of twobrpA‐dependent proteins, obtained by direct microsequencing of protein spots excised from two‐dimensional gels, identified them as gene products mapping to the same region and involved in secondary metabolic conversions of thebappathway. The kinetics of synthesis of 16brpA‐dependent gene products were characterized using QUEST computer software. Cluster analysis performed on the kinetics of synthesis of 346 of the most highly expressed gene products of HP5‐29, including 16brpA‐dependent ones, identified 75 families having distinct patterns of expression. ManybrpA‐dependent proteins were clustered together; 10 were found in one kinetic family. These kinetic families also includedbrpA‐independent gene products perhaps subject to similar regulatory mechanisms and thus possibly involved in bialaphos biosynthesis. The activation/derepression ofbapexpression took place as cultures approached stationary phase and was temporally re

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02163.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryWhen the genes coding for the outer membrane (OM) proteins OmpA and OmpF ofEscherichia coliare fused to a signal sequence of a bacillar exoenzyme and expressed inBacillus subtilisthey remain cell‐bound and the signal sequence is not cleaved. To identify the step of arrest in the export of these proteins we studied their accessibility to protease applied to intact protoplasts; they remained resistant indicating fully intracellular localization. Both proteins appeared associated with the cell membranes in sedimentation and flotation centrifugation experiments. However, OmpA and OmpF proteins synthesized inB. subtiliswithout a signal sequence were similarly associated with membranes in centrifugation experiments whereas electron microscopy showed the presence of intracytoplasmic inclusion bodies not obviously attached to the cytoplasmic membrane. We conclude that OmpA and OmpF proteins even when provided with a functional signal sequence do not enter the export pathway inB. subtilis, probably owing to lack of a specific export component inB. subtili

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02164.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThis work describes the isolation and characterization of a full‐length cDNA clone encoding β‐tubulin from the pathogenPneumocystis carinii, P. cariniicontains a single gene encoding β‐tubulin. The complete sequence of this cDNA has been determined and its inferred amino acid sequence compared with the β‐tubulins from other organisms. This analysis augments the data indicating thatP. cariniishould be classified as a fungal organism. Further comparisons between theP. cariniiβ‐tubulin and those of fungal β‐tubulins resistant to benomyl, a β‐tubulin‐binding drug, indicate a difference which may be exploited in the development of a new drug therapy forP. cariniipneumonitis. These results suggest that, theoretically, a drug presently administered for treatment of nematode worm infections may be an effective agent againstP. carinii, without being toxic to the mammalian host. This possibility is current

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02165.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummarySynthesis and secretion of the 110kDa haemolysin toxin ofEscherichia coliand other pathogenic Gram‐negative bacteria are governed by the four genes of thehlyoperon. We have identified, by transposon mutagenesis, anE. colicellular locus,hlyT, required for the synthesis and secretion of haemolysin encodedin transby intacthlyoperons carrying thehlyupstream regulatory region. Mutation of thehlyTlocus specifically reduced the level ofhlyAstructural gene transcript 20‐100‐fold and thus markedly lowered both intracellular and extracellular levels of the HlyA protein. Genetic and structural analysis of thehlyTlocus mapped it at co‐ordinate 3680 kbp (minute 87) on the chromosome adjacent to thefadBAoperon, and identified it specifically as therfaH (sfrB)locus which is required for transcription of the genes encoding synthesis of the sex pilus and also the lipopolysaccharide core for attachment of the O‐antigen ofE. coliandSalmonella.Expression of thehlyoperon in theE. coli hlyTmutant was restoredin transby both thehlyTandrfaHgenes, suggesting that therfaHgene is an important activator of regulon structures that are central to the fertility and virulence of these pathogenic bacteria. DNA sequencing of thehlyTlocus identifies the HlyT/RfaH transcriptional activator as a protein of 162 amino acids (Mr18325) which shows no identity to characterized transcriptio

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02166.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThe nucleotide sequence of thexynAgene ofRuminococcus flavefaciens17 was determined and found to consist of a 2862 bp open reading frame beginning with a TTG start codon. The predicted product, XYLA, consisted of distinct amino‐terminal (A) and carboxy terminal (C) domains (248 amino acids, including a putative signal sequence, and 332 amino acids, respectively) linked by a repetitive sequence (B, 374 amino acids) extraordinarily rich in asparagine (45%) and glutamine (26%) residues. Domains A and C were shown to be capable of expressing xylanase activity independently of each other when suitably truncated derivatives of thexynAcoding region were expressed aslacZfusions. The activities associated with the two domains were shown to differ with respect to the average size of hydrolysis products formed from oat‐spelt xylan, with domain C releasing relatively more xylose and domain A more xylo‐oligosaccharides. The amino acid sequence of domain A of XYLA closely resembled that of theBacillus pumilus xynAenzyme (45% identical residues). On the other hand domain C showed significant similarity (33% to 40% identical residues) to a different group of bacterial xylanases and exoglucanases exemplified by theCaldocellum saccharolyticum xynAandcelBproducts. ThexynAproduct is, therefore, a bifunctional enzyme having two dissimilar catalytic domains capable of acting on

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02167.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryAs with many other fungi, including the budding yeast Saccharomyces cerevisiae, the dimorphic fungus Candida albicans encodes the novel translation factor, elongation factor 3 (EF‐3). Using a rapid affinity chromatography protocol, EF‐3 was purified to homogeneity from C. albicans and shown to have an apparent molecular mass of 128 kDa. A polyclonal antibody raised against C. albicans EF‐3 also showed cross‐reactivity with EF‐3 from S. cerevisiae. Similariy, the S. cerevisiae TEF3 gene (encoding EF‐3) showed cross‐hybridization with genomic DNA from C. albicans in Southern hybridization anaiysis, demonstrating the existence of a single gene closely related to TEF3 in the C. albicans genome. This gene was cloned by using a 0.7 kb polymerase chain reaction‐amplified DNA fragment to screen a C. albicans gene library. DNA sequence analysis of 200 bp of the cloned fragment demonstrated an open reading frame showing 51% predicted amino acid identity between the putative C. albicans EF‐3 gene and its S. cerevisiae counterpart over the encoded 65‐aminoacid stretch. That the cloned C. aibicans sequence did indeed encode EF‐3 was confirmed by demonstrating its ability to rescue an otherwise non‐viable S. cerevisiae tef3:HIS3 null mutant. Thus EF‐3 from C. albicans shows both structural and functional similarlity

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02168.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThe promoter elements responsible for the expression of the regulatorynifgenesrpoN, nifA1andnifA2ofRhodobacter capsulatuswere mapped by exonuclease‐III‐mediated deletions and by primer extension analysis. TherpoNpromoter maps 600 bp upstream ofrpoNand has the characteristic features of a –24/–12 promoter. The upstream activator sequence (UAS) displays two mismatches with the NIFA consensus sequence and is located 37 bp upstream of a perfect –24/–12 promoter element. The spacing and/or the helical phasing of these two promoter elements was found to be important for promoter function. In addition, an UAS half‐site may contribute to optimal promoter function. TherpoNUAS can partially substitute for the UAS of thenifEpromoter. An open reading frame with homology toKlebsiella pneumoniaeNIFU was identified between therpoNpromoter andrpoNand termednifU2since anothernifU‐likegene (nifUl) is located in a conventionalnifUSVWoperon innifregion A. Thus,rpoN, encoding an alternative sigma factor for RNA polymerase, is cotranscribed with anifUanalogous gene from anrpoN‐dependent promoter. Mapping of the promoter elements involved in the expression ofnif Acopy 1 and copy 2 identified a novel promoter type. A conserved distal promoter element is likely to represent the binding site of NTRC inR. capsulatus.The DNA region preceding the mapped 5′ ends of thenif Atranscripts displays much less homology. The distance between the distal and proximal eleme

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02169.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryTranscription ofRhodobacter capsulatusgenes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen.R capsulatus nifA1andnifA2encode identical NIFA proteins that activate transcription ofnifHDKand othernifgenes. In this study, we report thatnifA1‐lacZandnifA2‐lacZfusions are repressed in the presence of NH3and activated to similar levels under nitrogen‐deficient conditions. This nitrogen‐controlled activation was dependent onR. capsulatus ntrC(which encodes a transcriptional activator) but notrpoN(which encodes an RNA polymerase sigma factor). We have used primer extension analyses ofnifA1, nifA2andnifHand deletion analyses ofnifA1andnifA2upstream regions to define likely promoters andcisupstream activation sequences required for nitrogen control of these genes. Primer extension mapping confirmed thatntrCbut notrpoNis required fornifA1andnifA2activation, and thatnifA1andnifA2do not possess typical RPON‐activated

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb02170.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY