摘要:

A 30 kbp chromosomal region containing the S‐layer gene (slpA) fromThermus thermophilusHB8 was cloned from a λ phage gene library. DNA sequence analysis of the region upstream to theslpAgene revealed the presence of an open reading frame (ORF) which coded for a 604‐amino‐acid protein highly homologous to the glucosamine−6‐P synthases (EC 2.6.1.16) of both prokaryotic and eukaryotic origin. The identification of this ORF as the glucosamine−6‐P synthase gene fromT. thermophilus(glmSth) has been carried out using three different strategies: (i) complementation of anEscherichia coliglmSmutant; (ii)in vivoinsertional inactivation of the gene; and (iii)in vitrosynthesis of glucosamine−6‐P at 60°C by a cytoplasmic extract of an overproducingE. colistrain. TheglmSthgene is transcribed diver‐gently fromslpAin a 2.0 kb mRNA which probably also includes a tryptophan tRNA gene (trpTth) identified at its 3′ extreme. As the products of both theglmSthand theslpAgenes are main components of the cell envelope ofT. thermophilus, their unusual clustering in the chromosome could be related to the existence of specific mechanisms for their

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17010001.x

出版商:Blackwell Scientific Publications    

年代:1995

数据来源: WILEY

摘要:

A functional DNA replication terminator ofBacillus subtiliscontains two overlapping binding sites, A and B, for the replication terminator protein (RTP). A degenerate 17‐mer oligonucleotide corresponding to the consensus B site has been used to detect four new terminators in theB. subtilischromosome, in addition to the previously identified and closely spaced IRI and IRII. All the new terminators lie in the terminus region of the chromosome, on both sides of IRI and IRII, with their positions spanning<1O% of its length. Their DNA sequences are characterized by clearly identifiable A‐ and B‐binding sites. They bind RTP in a manner indistinguishable from IRI, although precise affinities have not been compared. Each new terminator is functional in causing fork arrest when present in a plasmid replicating inB. subtilis. Three of the four were tested for polarity in fork‐arrest activity and exhibited the polarity expected. The total of six terminators now identified inB. subtilishave been namedTerI‐TerVI.TerIandTerIIcorrespond to the previously identified IRI and IRII, respectively. The chromosomal orientations of all but one of the terminators (TerIV) have been established and they conform to an arrangement similar to that inEscherichia coliin which two opposed groups of polar terminators provide a replication‐fork trap ensuring that the approaching forks meet within a restricted region of the chromosome. The development of a strikingly similar arrangement of terminators in the two organisms, despite the lack of any detectable similarity in their respective DNA terminators and terminator proteins, emphasizes the importance of the replication‐fork trap

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17010013.x

出版商:Blackwell Scientific Publications    

年代:1995

数据来源: WILEY

摘要:

Each cysteine residue in the MerT and MerP polypeptides of bacterial transposon Tn501was replaced by serine, and the mercury‐resistance phenotypes of the mutants were determined inEscherichia coli. Cys−24 and Cys−25 in the first transmembrane region of MerT were essential for transport of mercuric ions through the cytoplasmic membrane, and mutations Cys−76‐Ser, Cys−82‐Ser or Gly−38‐Asp in MerT or Cys−36‐Ser in MerP all reduced transport and resistance. Deletion of themerPgene slightly reduced mercuric ion resistance and transport, whereas a Cys−33‐Ser mutation in MerP appears to block transport of mercuric ions by MerT. The effects of deletingmerPon mutations inmerTwere tested. The 116‐amino‐acid MerT protein is sufficient for mercuric ion transport ac

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17010025.x

出版商:Blackwell Scientific Publications    

年代:1995

数据来源: WILEY

摘要:

A gene (sigF) encoding a new sigma factor was isolated fromStreptomyces aureofaciensusing a degenerate oligonucleotide probe designed from the GLI(KDNE)A motif lying within the well‐conserved region 2.2 of the eubacterial σ70family. Homologues were present in otherStreptomycesspp., and that of the genetically well‐studiedStreptomyces coelicolorA3(2) was also cloned. The nucleotide sequences of the twosigFgenes were determined and shown to encode primary translation products of 287 (S. coelicolor) and 295 (S. aureofaciens) amino acid residues, both showing greatest similarity to σBofBacillus subtilis. However, while σBis involved in stationary‐phase gene expression and in the general stress response inB. subtilis, σFaffects morphological differentiation inStreptomyces, Disruption ofsigFdid not affect vegetative growth but did cause awhimutant phenotype. Microscopic examination showed that thesigFmutant produced spores that were smaller and deformed compared with those of the wild type, that the spore walls were thinner and sensitive to detergents and that insigFmutant spores the chromosome failed to condense. σFis proposed to control the late stages of spore development inSt

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17010037.x

出版商:Blackwell Scientific Publications    

年代:1995

数据来源: WILEY

摘要:

Yersinia enterocoliticacells, when cultured at 30°C or below, are flagellated and motile. Cells cultured at 37°C or above lack flagella and are non‐motile. To identify flagellin genes that are a target of this temperature‐dependent regulation, a library ofY. enterocoliticagenomic inserts in a phage λ vector was probed with theSalmonella typhimuriumfliC(flagellin) gene. A DNA fragment subcloned from a recombinant phage which hybridizes with the probe complements a non‐motileS. typhimuriumfliC−fljB−(flagellin‐minus) mutant. DNA sequence analysis shows thatY. enterocoliticacontains three tandem flagellin genes, designatedfleA,fleBandfleC. All three genes are co‐ordinately transcribed at low, but not high, temperature fromfliA‐dependent (σF) promoters. Flagellin transcription arrests rapidly after upshift to 37°C (host temperature). In contrast, flagellin transcription resumes only after several generations when cells cultured at 37°C ar

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17010049.x

出版商:Blackwell Scientific Publications    

年代:1995

数据来源: WILEY

摘要:

Burkholderia cepacia(basonymPseudomonas cepacia), the type speciesof the new genusBurkholderia, is of interest, not only because of its broad catabolic capacity and its ability to antagonize soil‐borne plant pathogens, but also because of its causative role in infections in man, which are particularly evident in patients with cystic fibrosis. A physical map of the 8.1 Mb genome of theB. cepaciatype‐strain ATCC 25416 was constructed by applying two‐dimensional pulsed‐field gel electrophoresis techniques. Placed onto the macrorestriction map were 38Spel, 11Swal, 11Pacl, 11Pmeland six l‐Ceul sites, resulting in an average resolution of 1O5 kbp. Random single‐hit linearization by irradiation and restriction mapping uncovered the presence of four circular replicons of 3.65 Mb, 3.17 Mb, 1.07 Mb and 200 kbp in size. The largest replicon harbours fourrrnoperons while the other two Megabase‐size replicons each contain a singlerrnoperon, suggesting that the genome has three chromosomes and a large plasmid. Within the beta subdivision of proteobacteria, the existence of multiple replicons is not confined toB. cepacia. The phylogenetically related speciesBurkholderia glumae,Burkholderia pickettii,Burkholderia solanacearum,Alcaligenes eutrophusand the so far unassignedPseudomonas glatheiwere also found to harbour more than one Megabase

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17010057.x

出版商:Blackwell Scientific Publications    

年代:1995

数据来源: WILEY

摘要:

Cytoadherence to the vaginal epithelium is a critical step in infection by the eukaryotic flagellateTrichomonas vaginalis. Four trichomonad surface proteins (AP65, AP51, AP33 and AP23) mediate cytoadherence. The cDNA encoding the AP65 adhesin was isolated from a phagemid cDNA expression library by screening with antiserum and monoclonal antibody (mAb) raised against the purified trichomonad AP65 protein. Two clones, F11.2 and F11.5, coded for immuno‐crossreactive recombinant proteins that possessed functional properties equal to theT. vaginalisAP65 adhesin. Analysis of full‐length sequences corresponding to the F11.2 and F11.5 cDNAs revealed that both contained 1701‐base open reading frames (ORFs) that encoded proteins of 63 281 daltons and 63087 daltons, respectively. Comparison of the full‐length sequences showed 87% identity at the nucleotide level and 91% identity at the protein level. Restriction‐enzyme mapping and Southern analysis reaffirmed the distinctness of the F11.2 and F11.5 cDNAs, indicating that two different AP65 genes (now calledap65−1andap65−2) are present in theT. vaginalisgenome in at least two copies each. Northern analysis detected high levels of transcript of ∼1.8 kb for bothap65−1andap65‐2genes in trichomonads grown only in high‐iron medium, confirming the transcriptional regulation of adhesin synthesis by iron. Homology searches revealed significant similarity (38% amino acid identity and 54% nucleotide identity) to malic enzymes. However, purified malic enzyme and mAb to AP65 crossreactive with malic enzyme neither inhibited cytoadherence ofT. vaginalisto host cells nor prevented binding of the trichomonad AP65 to HeLa ce

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17010069.x

出版商:Blackwell Scientific Publications    

年代:1995

数据来源: WILEY

摘要:

We report the presence of long stretches of tandem repeats in the genome of the halophilic ArchaeaHaloferax mediterraneiandHaloferax volcaniiA 30 bp sequence with dyad symmetry (including 5 bp inverted repeats) was repeated in tandem, interspersed with 33–39 bp unique sequences. This structure extends for long stretches — 1.4kb at one location inH. mediterraneichromosome and about 3kb in theH. volcaniichromosome. The tandem repeats (designated TREPs) show a similar distribution in both organisms, appearing once or twice in theH. volcaniiandH. mediterraneichromosomes, and once in the largest, probably essential megaplasmid of each organism but not in the smaller replicons. Sequencing of the structures in bothH. volcaniireplicons revealed an extremely high sequence conservation in both replicons within the species, as well as in the different organisms. Homologous sequences have also been found in other more distantly related halophilic members of the Archaea. Transformation ofH. volcaniiwith a recombinant plasmid containing a 1.1 kb fragment of the TREPs produced significant alterations in the host cells, particularly in terms of cell viability. The introduction of extra copies of TREPs within the vector significantly alters the distribution of the genome among the daughter cells, as observed by DAPI staining. Although the precise biological role cannot be completely ascertained, all the data conform with the tandem repeats being involved in replicon partitioning in halobacte

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17010085.x

出版商:Blackwell Scientific Publications    

年代:1995

数据来源: WILEY

摘要:

We have cloned and characterized a homologue of the previously isolatedGPD1gene, encodingsn‐glycerol 3‐phosphate dehydrogenase (NAD+) inSaccharomyces cerevisiae. This second gene, calledGPD2, encodes a protein of 384 amino acids that shares 69% sequence identity withGPD1. LikeGPD1it has an amino‐terminal extension of unknown function.GPD2is located on chromosome VII and cross‐hybridizes withGPD1at chromosome IV as well as with an unknown homologue at chromosome XV. Disruption of theGPD2gene did not reveal any observable phenotypic effects, whereas overexpression resulted in a slight, but significant, increase of GPD enzyme activity in wild‐type cells. Analysis of gene transcription by a CAT‐reporter gene fused to theGPDpromoters revealed decreased transcriptional activity of theGPD2promoter in cells grown on non‐fermentable as opposed to fermentable carbon sources, and no induction in cells exposed to high osmolarity or heat shock. Similar analysis ofGPD1demonstrated an 8–17‐fold higher basal level of transcription compared toGPD2. Furthermore, such analysis revealed that theGPD1promoter was it induced by increased osmolarity essentially independent of the type of stress solute used, the level ofGPD1transcription being increased about sevenfold in cells gro

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17010095.x

出版商:Blackwell Scientific Publications    

年代:1995

数据来源: WILEY

摘要:

The uropathogenicEscherichia colistrain 536 (O6:K15:H31) carries two unstable DNA regions on its chromosome which were termed pathogenicity islands (Pais). Both pathogenicity islands, Pai I and Pai II, are incorporated into tRNA specific loci: Pai I is located in the tRNA gene for selenocysteine (selC), and Pai II is integrated in the leucine‐specific tRNA locusleuX. Mutant strain 536−21 has lost the two pathogenicity islands together with the intact tRNA genes. While 536 is a virulent strain, 536−21 has lost a number of properties, including in vivo virulence. In previous publications we reported that the genes coding for two haemolysins (hlyI,hlyII) and P‐related fimbria (prf) are located on the Pais. In this paper, we demonstrate that the expression of other gene products influencing metabolic properties in addition toin vivovirulence are strongly dependent on the intact tRNA lociselCandleuX. In order to determine the influence of the two tRNAs on the expression of these properties, the genesseICandleuXwere cloned from the genome of strain 536 and then introduced into the mutant 536−21. Our results clearly show that the selenocysteine‐specific tRNA (tRNASec) directly influences the ability of the bacteria to grow under anaerobic conditions, because selenocysteine is part of the enzyme formate dehydrogenase (FDH) which is involved in mixed acid fermentation. The rare leucine‐specific tRNA5Leu, encoded byleuX, influences a number of properties including type 1 fimbria production, flagellation and motility, production of enterobactin and serum resistance, and is also necessary for fullin vivovirulence. While the tRNASecis directly involved in the production of FDHs, theleuXspecific tRNA5Leuappears to influence the expression of various factors through specific transcriptional or translational contr

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_17010109.x

出版商:Blackwell Scientific Publications    

年代:1995

数据来源: WILEY