摘要:

SummaryThe effects of defined mutations In the lipopolysaccharide (LPS) and the outer membrane protein OmpA of the recipient cell on mating‐pair formation in liquid media by the transfer systems of the F‐Iike plasmids pOX38 (F), ColB2 and R100‐1 were investigated. Transfer of all three plasmids was affected differently by mutations in therfa(LPS) locus of the recipient cell, the F plasmid being most sensitive to mutations that affectedrfaPgene expression which is responslbie for the addition of pyrophosphorylethanolamine (PPEA) to heptose I of the inner core of the LPS. CoIB2 transfer was more strongly affected by mutations in the heptose II‐heptose III region of the LPS(rfaF)whereas R100‐1 was not strongly affected by any of therfamutations tested.ompAbut notrfamutations further decreased the mating efficiency of an F plasmid carrying a mutation in the mating‐pair stabilization protein TraN. An F derivative with a chloramphenicol acetyltransferase (CAT) cassette interrupting thetraApilin gene was constructed and pilin genes from F‐like plasmids (F, ColB2, R100‐1) were used to complement this mutation. Unexpectediy, the results suggested that the differences in the pilin sequences were not responsible for recognizing specific groups in the LPS, OmpA or the TraT surface exclusion protein. Other corroborating evidence is presented suggesting the presence of an adhesin at

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00486.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThetragene ofStreptomyces lividansplasmid plJ101 is required for both plasmid DNA transfer and plJ101‐induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy oftramediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here namedclt.Insertional mutation or deletion ofcltfrom plJ101 reduced plasmid transfer mediated by either plasmid‐borne or chromosomally locatedtraby at least three orders of magnitude, abolished the transfer‐associated pocking phenomenon, and interfered with the ability oftra+plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer inS. lividansinvolves acis‐acting function dispensable for chromosomal gene transfer and imply that either theS. lividanschromosome encodes its ownclt‐like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different m

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00487.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe toxicity to mosquito larvae of the parasporal body produced byBacillus thuringiensissubsp.israelensisand the PG‐14 isolate ofB. thuringiensissubsp.morrisoniis at least 20‐fold greater than any of the four mosquitocidal proteins of which It is composed (CytA, CrylVA, B, and D). This high toxicity is postulated to be due to synergistic interactions among parasporal proteins. However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae. In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CrylVD toxins were cloned and expressed in acrystalliferousB. thuringiensissubsp.israelensiscells using the shuttle vector pHT3101. CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bio‐assays against first instars ofAedes aegypti.The LC50for the CytA inclusion was 60 ng ml−1, whereas the LC50for the CryIVD was 85ng ml−1In comparison, the LC50s for different combinations of CytA and CrylVD inclusions ranged from 12–15 ng ml−1, 4–5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins. These results suggest that the high toxicity of the wild‐type parasporal bodies ofB. thuringiensissubspp.israelensisandmorrisoniIs due to synergism among three or four of the

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00488.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryA new locus required for type 4 pilus biogenesis byPseudomonas aeruginosahas been identified. ApilEmutant, designated MJ‐6, was broadly resistant to pili‐specific phages and unable to translocate across solid surfaces by the pilus‐dependent mechanism of twitching motility (Twt−). Immunoblot analysis demonstrated that MJ‐6 was devoid of pili (Pil−) but was unaffected in the production of unassembled pilin pools. Genetic studies aimed at localizing thepilEmutation on theP. aeruginosaPAO chromosome demonstrated a strong co‐linkage between MJ‐6 phage resistance and theproBmarker located at 71 min. Cloning of thepilEgene was facilitated by the isolation and identification of a proB+‐containing plasmid from a PAO1 cosmid library. Upon introduction of the PA01proB+cosmid clone into MJ‐6, sensitivity to pili‐specific phage, twitching motility and pilus production were restored. The nucleotide sequence of a 1 kbEcoRV‐Clalfragment containing thepilEregion revealed a single complete open reading frame with characteristicP. aeruginosacodon bias. PilE, a protein with a molecular weight of 15278, showed significant sequence identity to the pilin precursors ofP. aeruginosaand to other type 4 prepilin proteins. The region of highest homology was localized to theN‐terminal 40 amino acid residues. The putative PilEN‐terminus contained a seven‐residue basic leader sequence followed by a consensus cleavage site for prepilin pep‐tidase and a largely hydrophobic region which contained tyrosine residues (Tyr‐24 and Tyr‐27) previously implicated in maintaining pilin subunit‐subunit interactions. The requirement of PilE in pilus biogenesis was confirmed by demonstrating that chromosomalpilEinsertion mutants were pi

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00489.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryDeletion mutants of R100‐1 were constructed by classical methods to remove various segments of thetraMopen reading frame, pTraM‐binding sites and thetraMpromoters. Complementation tests showed thattraMwas efficiently complemented only when thefrans‐actingfragment contained both the completetraMgene and the adjacenttraJpromoter and leader sequences. The conclusion is thattraMandtraJconstitute a complex operon. A deletion mutant lacking all of thefraJgene, and one containing a frameshiftingtraMdeletion, retained the ability to transfer at a low level, thereby showing that neither pTraM nor pTraJ is absolutely essential for tra

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00490.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThreonine is found at the third position of the second α‐helix in the helix‐turn‐helix motifs of most bacterial DNA‐binding proteins. To investigate the role of this conserved residue inEscherichia coliTrp repressor function, plasmids encoding mutant Trp repressers with each of the 19 amino acid changes of Thr‐81 were made by site‐directed mutagenesis. All 19 changes decrease the activity of Trp holorepressor, indicating that the Thr‐81 side‐chain is critical for TrpR function. Three mutant repressors, Ser‐81, Lys‐81 and Arg‐81, retain partial DNA‐binding activity and inhibit transcription from the wild‐typetrppromoter/operator complex; challenge‐phage assays show that Ser‐81 and Lys‐81 holorepressors have altered DNA‐binding specificities. The side‐chain of Thr‐81 may make direct contacts with base pairs 4 and 3 of thetrpoperator, consistent with the nuclear magnetic resonance solution structure

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00491.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe distribution, characterization and function of thetcpAgene was investigated inVibrio choleraeO1 strains of the El Tor biotype and in a newly emergent non‐O1 strain classified as serogroup O139. TheV. cholerae tcpAgene from the classical biotype strain O395 was used as a probe to identify a clone carrying thetcpAgene from the El Tor biotype strain E7946. The sequence of the E7946tcpAgene revealed that the mature El Tor TcpA pilin has the same number of residues as, and is 82% identical to, TcpA of classical biotype strain O395. The majority of differences in primary structure are either conservative or clustered in a manner such that compensatory changes retain regional amino acid size, polarity and charge. In a functional analysis, the cloned gene was used to construct an El Tor mutant strain containing an insertion intcpA.This strain exhibited a colonization defect in the infant mouse cholera model similar in magnitude to that previously described for classical biotypetcpAmutants, thus establishing an equivalent role for TCP in intestinal colonization by El Tor biotype strains. ThetcpAanalysis was further extended to both a prototype El Tor strain from the Peru epidemic and to the first non‐O1 strain known to cause epidemic cholera, an O139V. choleraeisolate from the current widespread Asian epidemic. These strains were shown to carrytcpAwith a sequence identical to E7946. These results provide further evidence that the newly emergent non‐O1 serogroup O139 strain represents a derivative of an El Tor biotype strain and, despite its different LPS structure, shares common TCP‐associated antigens. Therefore, there appear to be only two related sequences associated with TCP pilin required for colonization by all strains responsible for epidemic cholera, one primary sequence associated with classical strains and one for El Tor strains and the recent O139 derivative. A diagnostic correlation between the presence oftcpAand the V. cholerae to colonize and cause clinical is now extended to strains of both O1 and non‐O1

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00492.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe stationary‐phase‐specific sigma factor σS(RpoS/KatF) is required forEscherichia colito induce expression of fibronectin‐binding curli organelles upon reaching stationary phase. We show that thecsgAgene which encodes the curlin subunit protein belongs to a dicistronic operon,csgBA.The transcriptional start site ofcsgBAwas determined and an AT‐rich upstream activating sequence (UAS) required for transcriptional activation was identified. ThepcsgBApromoter is not specific for σSsince the same promoter sequence can be used by Eσ70in vivoin a strain lacking nucleoid‐associated protein H‐NS and σS. Transcription remained growth‐phase induced and dependent upon the UAS in such a double mutant. Furthermore, we demonstrate that an additional operon,hdeAB, which is also dependent upon σSfor transcription, can be transcribed by Eσ70in vivoin the absence of H‐NS by utilizing thephdeABpromoter. Two other genes known to be under the control of σSfor expression,bolAandkatE, remained transcriptionally silent in the absence of H‐NS. It is suggested that a subset ofE. colipromoters can be recognized by both EσSand Eσ70in vivobut H‐NS interacting with these sequences prevents formation of succesful transcription‐

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00493.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe structure, genomic organization and transcription of the gene encoding histone H2B in the protozoan parasiteTrypanosoma cruzihave been studied. This gene consists of a 746‐nucleotlde unit, tandemly repeated at least 18 times in each of two clusters. DNA probes corresponding to histones H2B and H3 hybridized to different chromosomes revealing that the genes coding for these two histones are not physically linked in the genome ofT. cruzi.The primary transcription product of the H2B gene is processed by trans‐splicing and polyadenylation. Inhibition of DNA synthesis with aphidicolin resulted in the reduction of histone H2B mRNA to undetectable levels in about two hours, suggesting that its abundance is regulated throughout the cell cycle as it occurs in other eukaryotes. in addition, a concomitant inhibition of translation by cycloheximide reverted this effect indicating thatde novoprotein synthesis is required for RNA instability. Histone mRNA abundance was dependent on the life‐cycle stage ofT. cruzi: abundant in amastigotes and epimastigotes, the dividing forms in the host cell and the insect vector, respectively, white undetected in trypomastigotes, the parasite's non‐dividing lif

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00494.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryExpression from both theEscherichia coli nirandnrfpromoters is dependent on anaerobic induction by FNR but is further regulated by NarL and NarP in response to the presence of nitrite and nitrate in the growth medium. Thenirpromoter is activated by NarL in response to nitrate and nitrite and activated by NarP in response to nitrate but not nitrite. The effects of point mutations suggest that NarL and NarP both bind to the same target, which is a pair of heptamer sequences organized as an inverted repeat, centred 691/2 bp upstream of the transcript startpoint. Thenrfpromoter can be activated by either NarP or NarL in response to nitrite but is repressed by NarL in response to nitrate. Mutational analysis of thenrfpromoter has been exploited to corroborate the location of the ‐10 hexamer and the FNR‐binding site, and to find the sites essential for nitrite‐dependent activation and nitrate‐dependent repression. Optimal activation by NarP or NarL in response to nitrite requires an inverted pair of heptamer sequences, similar to that found at thenirpromoter, but centred 741/2 bp upstream from the transcript start. NarL‐dependent repression by nitrate is due to two heptamer sequences that flank the FNR‐binding sequence. We conclude that NarL and NarP bind to the same heptamer sequences, but that the affinities for the two factors vary from s

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00495.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY