摘要:

SummarySeveral covalently closed circular halobacterial megaplasmids (up to more than 500 kb) from different strains ofHalolerax mediterranei, have been resolved by orthogonal‐field alternating gel electro‐phoresis (OFAGE). These molecules seem to be negatively supercoiledin vivo, as deduced from the effect of intercalating agents affecting their topology and, therefore, their electrophoretic mobility. It has also been demonstrated that thetopolsomeraseII Inhibitor novobiocin affects the native topological state of halobacterial megaplasmids impeding their migration in OFAGE under standard conditions for resolution of large supercoiled molecu

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00323.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryA 120 kDa glycoprotein in the larval midgut membrane of the IepidopteranManduca sexta, previously identified as a putative receptor forBacillus thuringiensisCrylA(c) δ‐endotoxin, has been purified by a combination of protoxin affinity Chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin In the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N‐terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3‐fold compared toM. sextabrush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteranM. sextais the metalloprotease aminopepti

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00324.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryPrevious studies demonstrated that the presence of a 7–8 kb plasmid is correlated with expression of the lipopolysaccharide (LPS) O:54 antigen in severalSalmonella entericaserovars. In this study, a 6.7 kb plasmid from a field isolate of S.entericaserovar Borreze was shown to encode enzymes responsible for the synthesis of the O:54 polysaccharide. Curing the plasmid results in simultaneous loss of smooth O‐polysaccharide‐substituted LPS molecules and O:54 serotype. SDS‐PAGE analysis of other 0:54 isolates indicated that the O:54 O‐polysaccharide can be co‐expressed with an additional O‐polysaccharide, likely encoded by chromosomal genes. The structure of the O:54 polysaccharide was determined by a combination of chemical and nuclear magnetic resonance (NMR) methods and was found to be an unusual homopolymer of N‐acetylmannosamine (D‐ManNAc) residues. The polysaccharide contained a disaccharide repeating unit with the structure:→4)‐β‐d‐MANpNAc‐(1→3)‐β‐d‐ManpNAc‐(1 →This structure does not resemble other O‐polysaccharides inS. enterica.To examine the role played by plasmid functions in synthesis of the O:54 polysaccharide, the 6.7 kb plasmid was cloned to produce a hybrid plasmid (pWQ800) in pGEM‐7Zf(+). InEscherichia coliK‐12 Δrfb, pWQ800 directed the synthesis of authentic O:54 polysaccharide. Polymerized O:54 polysaccharide was also produced inS. entericaserovar Typhimuriumrfbandrfcmutants. From these data, we conclude that pWQB00 carries therfbO:54gene cluster and synthesis of the O:54 poiysaccharides does not require host chromosomalrfbfunctions. However, synthesis of the O:54 polysaccharide requires the function of therfeandrffEgenes which are part of the gene cluster encoding enzymes involved in biosynthesis of enterobacterial common antigen. TherffEgene product synthesizes the O:54 precursor, uridine diphospho‐N‐acetylmannosamine. This is the first descript

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00325.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryDNA sequences within the F plasmid transfer origin (oriT) were tested for their ability to initiate or terminate conjugal transfer. Mutant and wild‐typeoriTelements were cloned as direct repetitions flanking therpsLgene on a pBR322‐based plasmid, and the frequency of deletion of this segment during matings sponsored by F’lac(F42) with streptomycin‐resistant recipients was measured. ShortenedoriTelements that lacked adjacent TraM‐binding sites allowed efficient initiation and termination. Some truncatedorirsegments lacking the TraM‐binding sites and the TraY‐binding site,sbyA, initiated transfer inefficiently, but nevertheless promoted efficient termination. Removal of TraM‐, TraY‐, and IHF‐binding sites severely reduced both nicking and termination. Point mutations that previously had been reported to prevent nicking caused reduced levels of both initiation and termination. These results indicate that regions oforiTsupporting initiation are more extensive than those needed for termination, although some regions are required for both. Moreover, termination can be effective for some mutant loci that do not suppor

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00326.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryF plasmidtraYprotein binding to wild‐type or deleted regions containing the TraY‐binding site,sbyA, was studiedin vitro.The principal DNA‐protein complex was formed with DNA segments including thesbyAsite defined by footprinting and (with lesser affinity) with truncated segments that retained the leftward two‐thirds ofsbyA.This located the major sequence determinants for TraY binding between bp 204 and 227 on theoriTmap. For all sequences tested, bound TraY induced bending of approximateiy 50 to 55°, and centred between bp 214 and 221. Thermodynamic and mobility analyses indicated that two TraY protomers bind tosbyA.At higher TraY concentrations, additional TraY bound to the left of thesbyAin a region previously shown to bind IHF (site IHF A). TraY binding to this additional site (sbyC) was inhibited by IHF. Sequence similarities shared bysbyA, sbyB, andSbyCmay include the critical base pairs for TraY

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00327.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe virulence plasmid pXO1 is responsible for toxin production inBacillus anthracis.A DNA fragment from pXO1 was isolated and was shown, by sequence analysis, to contain part of a type 1 DNA topoisomerase gene. Attempts to clone the entire wild‐type gene, designatedtopX, inEscherichia coli, were unsuccessful. In order to obtain the complete gene, it was first insertionally inactivated and then cloned in the mutated form. The deduced amino acid sequence of Topo X1 shows similarities to that of the twoE. colitype 1 DNA topoisomerases. The N‐terminal two‐thirds of the putativeB. anthracisprotein exhibits strongest sequence similarity to topoisomerase III, whereas theC‐terminal portion contains cysteine residues that could form three zinc‐binding domains, as they do in topoisomerase I. The suggested active‐site tyrosine is conserved in all three proteins. The regulation of expression from thetopXpromoter is modified by addition of a gyrase inhibiting antibiotic. The Topo X1 protein is likely to be involved in the stabil

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00328.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryDe‐O‐acylated lipopolysaccharides (LPS) of three polymyxin‐resistantSalmonella typhimurium pmrAmutants and their parent strains were analysed by31P‐NMR (nuclear magnetic resonance) in order to assess, in relation to polymyxin resistance, the types and degree of substitution of phosphates of the LPS and lipid A. in thepmrAmutant LPS phosphate diesters predominated over phosphate monoesters, whereas the latter were more abundant in the parent wild‐type LPS. The increase in the proportion of phosphate diesters was traced to both the core oligosaccharide and the lipld A part. In the latter, the ester‐linked phosphate at position 4’was to a large extent (79–88%) substituted with 4‐amino‐4‐deoxy‐l‐arabinose, whereas in the wild‐type LPS the 4′‐phosphate was mainly present as monoester. In each LPS, regardless of thepmrAmutation, the glycosidically linked phosphate of li

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00329.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe wilt‐inducing phytopathogenPseudomonas solanacearumproduces several extracellular virulence factors, both polysaccharides (EPS I) and proteins (EXPs), which are independently regulated by a LysR‐type transcriptional regulator, PhcA, and a histidine kinase sensor, VsrB. Here we characterize a third locus,vsrA, which is also required for normal production of EPS I, some EXPs and wilt disease. Analysis ofeps::lacZreporters invsrAmutants showed that, likevsrBandphcA, vsrAis required for maximal expression (transcription) ofeps, which contains some of the genes necessary for production of EPS I. UnlikevsrBandphcAmutants, however,epstranscription (and EPS I production) byvsrAmutants varies from 3 to 17% of wild‐type levels, depending on growth conditions. Inactivation ofvsrAalso causes a dramatic reduction in production of three species of EXPs (28kDa, 48kDa, and 66kDa), and an apparent increase in production of a few other EXPs. Unlike most other EPS‐deficientP. solanacearumstrains,vsrAmutants caused almost no disease symptoms when 104cells were stem‐inoculated into tomato plants. This correlated with a greater than 10‐fold reduction in their ability to growin plants. vsrAwas cloned from aP. solanacearumgenomic library by complementation of thevsrAmutant and was further subcloned on a 2.3kb DNA fragment. PhoA fusion analysis and subcellular localization of thevsrAgene product inEscherichia colimaxicells suggest that it is a 53 kDa membrane‐associated protein. Analysis of the nucleotide sequence ofvsrArevealed a 502 residue open reading frame with homology to the histidine kinase domain of sensors in the two‐component regulator family. This discovery shows that EPS I production byP. solanacearumis simultaneously controlled by dual two‐

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00330.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryA sulphated glycosaminoglycan‐dependent mechanism of microbial infection for mammailan cells was characterized for theChlamydia trachomatistrachoma and lymphogranuloma venereum (LGV) biovars. We demonstrated that the trachoma and LGV biovars compete for the same receptor(s) on host cells and that their infectivity was inhibited by heparin or heparan sulphate. Using a specific heparan suiphate lyase (heparitinase) to treat organisms, the Infectivity of both biovars was abolished. Furthermore, exogenous heparan sulphate rescued chlamydial infectivity following treatment with heparitinase and the restored infectivity was neutralized by an anti‐heparan sulphate monoclonal antibody. These data suggest that heparan sulphate‐like‐mediated Interactions betweenC. trachomatisand eukaryotic cells are essential for infe

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00331.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryHelicobacter pyloriis a Gram‐negative bacterium that infects the human gastric mucosa, causes gastritis and contributes to the development of peptic ulcers and gastric cancer. To facilitate molecular genetic analysis of this pathogen, we constructed a ∼20‐fold redundant cosmid library and physical/genetic map of strain NCTC11638. Genomic DNA fragments were cloned into the cosmid vector Lorist6, and clones were ordered by hybridization with several types of probes: (i) ends of cloned DNAs; (ii) chromosomalNotldigest fragments; (iii) cosmids containingNotlsites; and (iv) specific genes. Seven hundred and fifty‐one cosmids were mapped to one of thrM contigs covering>90% of the chromosome, and are represented by a 68‐cosmid miniset. The order of cosmids was confirmed and extents of overlap among them were estimated by restriction analysis.All currently knownH. pylorigenes were mapped, including those for a cytotoxin (vacA), cytotoxin‐associated protein (cagA), urease and regulatory functions (ureAB, ureDandureH), catalase (katA), major and minor flagellins (flaAand flaB), heat‐shock (stress) and chaperone proteins (dnaK, htrA, hspB(groEL)), prokaryotic ferritin (pfr), an adhesin subunit (hpaA), a surface protein (26kDa), and 16S and 23S ribosomal RNAs (two genes each). The orientations of eight genes or clusters were determined, and two repetitive sequences were also found. The gene order and rRNA gene copy number determined here differed from that reported for an unrelated strain, which suggests considerable flexibility inH. pylorigenome

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00332.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY