摘要:

SummaryA novel genetic determinant (comA) has been identified and found to be required for the transformation of piliatedNeisseria gonorrhoeae.Mutants incomAof strain MS11 grow normally and are DNA‐uptake proficient but blocked in the translocation of DNA into the cytoplasm. Here we show by site‐specific mutagenesis and genetic complementation that only one of two open reading frames identified incomAis essential for competence: it encodes a protein (ComA) with a predicted size of 74kDa. ThecomAgene maps upstream of theigalocus and is transcribed in the opposite orientation, probably under the control of a putative σ;54type promoter. While DNA probes specific for theN. gonorrhoeae igalocus reveal only a little cross‐reactivity with commensalNeisseriaspecies, the neighbouringcomAgene appears to be present in most of them. ComA fusion proteins were obtained byin vitrotranslation. The synthesized gene products migrated atypically in SDS gels indicating its strong hydrophobicity. Several transmembrane α‐helices were predicted from the amino acid sequence of ComA which, in the context of an observed sequence similarity with other inner membrane proteins, suggests a location for the protein in the inner membrane. Using piliated and non‐piliatedcomAmutants the consequences of transformation deficiency on pilin phase variation were assessed. We show that thecomAdefect affects some but not all types of DNA rearrangements associated withpilEvariation. The results are in agreement with previous observations supporting the notion that multiple recombination pathways contribute to the variabi

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb00942.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryThecarRregion encodes a light‐inducible promoter, a negative regulator of the promoter and atrans‐acting activator that controls the light‐inducibleMyxococcus xanthuscarotenoid biosynthesis regulon. DNA sequence analysis revealed, downstream of the promoter, three translationally coupled genes,carQ, carRandcarS.Sequencing of mutations demonstrated thatcarRencoded the negative regulator and was an integral membrane protein. Mutant construction and sequencing revealed thatcarSwas thetrans‐acting activator and thatcarQwas a positive regulator of the promoter. Neither gene encodes proteins with known sequence‐specific DNA‐binding motifs. The sequence of the light‐inducible promoter region, identified by primer extension analysis, showed similarity to the consensus sequence of theEscherichia colistress response (‘heat‐

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb00943.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryExpression of thepfloperon ofEscherichia coliIs induced 12‐ to 15‐fold by anaerobiosis and transcription is mediated by seven co‐ordinately regulated promoters. The 5′ non‐translated regulatory region of the operon is approximately 450 bp in length and contains two of the seven promoters, termed promoter 6 and promoter 7. Site‐directed mutagenesis was used to aid the identification of DNA sequences important in directing transcription from the two promoters and to examine the effects such mutations had on the regulation of anaerobicpfloperon expression. Introduction of chromosomal mutations either in the FNR‐binding site or ‐10 region of promoter 6 blocked transcription from this promoter, as determined by primer extension. Similarly, mutation of the ‐10 region or the putative FNR half‐site located at ‐50 relative to the transcription start site of promoter 7 severely reduced transcription from that promoter. Prevention of transcription from promoter 6 by the ‐10 box mutation had no influence on promoter 7 transcription. Surprisingly, however, alteration of the FNR‐binding site at promoter 6 did reduce transcription from promoter 7. Thus, acismutation located 280bp downstream on the DNA had a profound effect on promoter 7 transcription. This effect would be commensurate with this mutation disrupting an important interaction between proteins bound at promoter 7 with those bound at promoter 6. Primer extension demonstrated that the promoter 7 mutations had no apparent influence on promoter 6 transcription. By usingpfl–lacZgene fusions it could be shown that the FNR‐binding site and ‐10 region mutations at promoter 6 abolished FNR‐dependent anaerobic regulation ofpfloperon expression. The equivalent mutations at promoter 7 caused a 25% reduction in anaerobic expression. The residual anaerobic expression in such constructs was FNR‐, but no longer ArcA‐dependent. A construct in which the ‐10 region of both promoters 6 and 7 was mutated showed no anaerobic induction ofpfloperon expression. This indicates that transcription from both promoters is required for maxim

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb00944.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryThe pigment‐binding proteins ofRhodobacter capsulatusare encoded by the polycistronicpufandpucoperons. Both operons show higher expression under low oxygen tension than under high oxygen tension in the wild‐type strain. The Tn5 mutant strain AH2 shows only low levels ofpufandpucmRNA under high and low oxygen tension, indicating that it lacks a gene product required for stimulation ofpufandpucgene expression under low oxygen tension. The formation of wild‐type levels of photosynthetic complexes and normal oxygen regulation could be restored by the expressionin transof a 1.7 kb fragment of the R.capsulatuswild‐type chromosome or by addition of 10μg I‐1vitamin B12to the growth medium. An open reading frame of 798 nucleotides containing the Tn5 insertion was identified on the 1.7kb fragment. This open reading frame shows no homology to known genes and has a remarkably high GC cont

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb00945.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryGlobal gene expression is dramatically altered by genomic rearrangements inStreptomyces ambofaciensRP181110. Partial genome mapping of two derivatives of strain RP181110 (strains NSA205 and NSA228) revealed rearrangements located in the unstable region of the genome (deletion in strain NSA228; deletion and amplification in strain NSA205). Computerized comparisons of pulse‐labelled proteins separated by two‐dimensional electrophoresis have revealed numerous differences in gene expression among the three strains during both exponential and stationary phases of growth: 31 proteins were absent in both mutant strains, 16 were absent only in strain NSA228, 17 were absent only in strain NSA205 and 9 were found to be present or over expressed in strain NSA205. Thus, in spite of the scarcity of genetic markers in the unstable region and its dispensability for growth under laboratory conditions, these results suggest that it includes genes which are actively expressed. Spontaneous gene amplifications, which occur frequently in this region of the chromosome, can further activate their express

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb00946.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryThe interaction between the DNA replication terminator, IRI, ofBacillus subtilisand its cognate replication terminator protein (RTP) has been examined by the technique of missing nucleoside interference (MNI). IRI contains two adjacent binding sites (A and B) for RTP dimers. The B site is proximal to the replication fork arrest site. The present results have shown that nucleoside contacts with RTP in the two sites are very different. There are more extensive contacts of nucleosides in both strands of the B site with RTP compared with the A site. The data also strongly suggest that filling by RTP of the B site occurs first and is needed for subsequent co‐operative filling of an overlapping A site. The A site alone binds RTP poorly. The findings are consistent with interaction occurring between RTP dimers bound to adjacent sites of IRI, which would explain why RTP bound to the B site alone cannot cause replication fork arres

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb00947.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryA transposon mutant ofEscherichia coli5K was isolated which reduced 10‐ to 50‐fold the secreted extracellular haemolytic activity of cells carrying the completehlyCABDoperon while leaving unaffected the intracellular haemolytic activity and the levels of intracellular and extracellular haemolysin protein, HlyA. The transposon insertion was identified within therfaPgene (required for attachment of phosphate‐containing substituents to the lipopolysaccharide inner core), and extracellular haemolytic activity was restoredin transby the intactrfaPgene. The toss in cytolytic activity of the secreted HlyA protein was not related to the HlyC‐directed acylation of the protoxin. Activity of the secreted toxin was restored by chaotropic agents and during rate‐zonal centrifugation the mutant‐secreted HlyA migrated as a larger species than the wild type. The results indicate that therfaPmutation affects the aggregation behaviour of the active toxin during or following the signal peptide‐independent secr

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb00948.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryThe effects of a number of mutations incrphave been measured at different cyclic AMP receptor protein (CRP)‐dependent Class II promoters, where the CRP‐binding site is centred around 411/2 base pairs upstream from the transcription start point. The amino acid substitutions HL159 and TA158 result in reduced CRP‐dependent activation, but the reduction varies from one Class II promoter to another. Deletions in theC‐terminus of the RNA polymerase alpha subunit suppress the effects of HL159 and TA158. The role of theC‐terminus of alpha at these promoters is assessed. Other changes at E58, K52 and E96 affect CRP activity specifically at Class II promoters and their role is

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb00949.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummarypSAM2 is an 11 kb integrating element fromStreptomyces ambofaciensthat is capable of replication. It generates single‐stranded DNA during replication, and is therefore the firstStreptomycesintegrating element to be described that may belong to the family of elements, called the ssDNA elements, that replicate by a rolling‐circle mechanism. The direction of replication has been identified. The plus origin (ori) of replication and minus origin (M‐O) have been located.Streptomyces lividansharbouring replicating pSAM2 also contain numerous small covalently closed circular DNA molecules (scm) derived from pSAM2. These scm containoriand extend on both sides of the putative nick site. Sequences at the junction points of these scm are heterogeneous but short direct repeats were always found in the vicinity of these junc

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb00950.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryFunctional chimeras have been generated from the transcriptional activators VnfA and AnfA, which control expression of the alternative nitrogenases inAzotobacter vinelandii.The activation profiles of the native and chimeric proteins have been determined usinglacZfusions toA. vinelandii anfandvnfpromoters inKlebsiella pneumoniae.Repiacing theC‐terminal domain of AnfA with that of VnfA gives a protein with the promoter specificity of VnfA, confirming that theC‐terminal domain contains the determinants of promoter specificity. However, substituting the VnfA sequence from the turn in the helix‐turn‐helix motif to theC‐terminus does not alter the promoter specificity of AnfA. These changes in promoter specificity were reflected in changes in affinity for a VnfA‐binding site, as measured by anin vivorepression assay using alacZfusion to a synthetic promoter. This supports the assumption that promoter recognition is determined by activator binding to enhancer‐like sequences, and shows that the principal determinants of specific DNA‐binding lie outside the‘recognition’helix. This may be a general feature of transcriptional activators dependent on σ;N(σ54). The chimera with the promoter specificity of VnfA retained the dependence on nitrogenase Fe protein characteristic of AnfA, indicating that this property is not related to particular promoter sequences, but is a function of the central orN‐t

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb00951.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY