摘要:

SummaryMany genes ofEscherichia colihave been shown to be sensitive to DNA superhelicity. The superhelicity of the chromosome is itself also supercoiling‐dependent. We have developed a general strategy for investigating how a particular gene responds to changes in DNA topology. This approach is used to study theE. coliligase gene. The thermosensitivity of theE. coliligts251 mutation can be phenotypically suppressed by mutations which map close to, or in, thegyrBgene and which affect the degree of DNA supercoillng. The level of suppression correlates with the degree of DNA relaxation observed, suggesting that the gene encoding theE. coliDNA ligase is activated by relaxation of the chromosomal DN

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00171.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryThe gene encoding plastocyanin(petE1)fromAnabaenasp. PCC 7937 was isolated using two sets of mixed oligonucleotide hybridization probes derived from conserved regions in the protein. Plastocyanin is encoded as a preprotein of 139 amino acids. The amino‐terminal extension of 34 residues has all the characteristics of a signal peptide and is probably involved in translocation of preplastocyanin over the thylakoid membrane. The level of thepetE1mRNA, a single transcript of about 740 bases, was found to be severely reduced under conditions of Cu2+deficiency. ThepetE1gene was transferred to the genome ofSynechococcussp. PCC 7942, which did not appear to contain a structural gene for plastocyanin itself. The integrated gene becomes expressed at the transcriptional level, regardless of the amount of Cu2+availabl

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00172.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryThe phytopathogenic enterobacteriumErwinia chrysanthemisecretes a number of enzymes involved in plant‐tissue degradation, notably the five isoenzymes of pectate lyase. We have cloned a region involved in pectate lyase and cellulase secretion by complementation of non‐secretoryoutJmutants ofE. chrysanthemistrain 3937 using the RP4::miniMu plasmid pULB110. The cloned region maps near theade‐22marker on theE. chrysanthemi3937 chromosome. An R‐prime containing a chromosomal DNA insert of about 30 kb was first obtained; subcloning into pBR325 permitted the isolation of a 4 kb Cial/Sspl fragment able to complementoutJmutations inE. chrysanthemi.The isolation ofphoAfusions in this fragment allowed us to determine the direction of transcription of the encoding region, which extends over about 2.5 kb, and demonstrate that this region encodes exported protein(s). When theTnphoAinsertions were transferred back intoE. chrysanthemichromosome, the recombined strains no longer secreted pectate lyases or cellulases. Identification of the products encoded by the Cial/Sspl fragment demonstrated thatoufJencodes an 83 kD polypeptide which is processed to an 81 kD polypeptide by cleavage of a signal sequence. The cloned DNA fragment did not endowEscherichia coliwith the ability to secrete pectate

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00173.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryThe gene cluster (rfbregion) which determines the synthesis of O101 lipopolysaccharide (LPS) O‐antigen was cloned from theEscherichia coliO101:K99:F41 reference strain B41 to give plasmid pPM1301. The smallest subclones represented by pPM1305 and pPM 1330expressed O‐antigen inE. coliK‐12 similar to (but not identical to) B41, as judged by immunogold electron microscopy and silver staining of LPS separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). At least six proteins were detected by minicell analysis of proteins encoded by pPM1305, which suggests that O‐antigen synthesis is genetically complex.Restriction and deletion analysis demonstrated that a minimum of 8.9 kb and a maximum of 11.8 kb are required for O101 O‐antigen biosynthesis inE. coliK‐12. Examination of LPS banding patterns of other O101 isolates by SDS‐PAGE suggested heterogeneity of LPS structure. Southern DNA hybridization analysis using radiolabelled subclones of pPM1305 demonstrated that there was a close relationship among the 0

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00174.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryThe genes determining the biosynthesis of the colonization factor CS5 have been cloned fromEscherichia coli0115:H40:PCF8775 isolated during an outbreak of diarrhoea among aboriginal children in Central Australia. Electron microscopy has shown purified CS5 to be of semi‐rigid fimbrial type. NH2‐terminal analysis has shown the CS5 determinant to be distinct from other fimbriae, although there is some conservation of certain residues. Expression in minicells of the cloned fimbrial genes encoded on pPM1312 has shown that proteins of 70 and 46.5 kD which co‐purify with the 23kD major fimbrial subunit protein are also co‐expressed along with proteins of 45, 31, 17 and 14kD. The major CS5 subunit is synthesized in precursor form (approximately 26 kD). A synthetic oligonucleotide to the NH2‐terminal amino acid coding sequence of the purified protein has been used in Southern hybridization analyses to define the region on pPM1312 encoding the structural gene for the major pil

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00175.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryThe major outer membrane protein (MOMP) is the prime candidate for the development of a chlamydial vaccine. Antibodies to the subspecies‐specific epitope neutralize chlamydial infection. Monoclonal antibodies (MAbs) to this epitope were prepared either by immunization with whole chlamydiae or with a 16 amino acid synthetic peptide. The critical binding site on the subspecies epitope for these MAbs was determined to single amino acid resolution using several hundred solid‐phase peptides. A frame shift of just one amino acid in critical binding site completely prevented antibody binding to viable chlamydiae. A single MAb to whole organisms was capable of spanning both the surface‐exposed, conformation‐dependent, subspecies epitope and a buried, conformation‐independent species epitope some 10 Å distant. Immunization with peptide generated an MAb with reduced binding constraints which permitted the antibody to bind with broadened species‐specificity at the subspecies binding site. The results show for the first time the importance of both critical binding site and conformation at the subspecies epitope. We suggest that the conformational flexibility of short, epitopic peptide vaccines may in some cases be advantageous, giving rise to extended specificity not attained with the nat

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00176.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryProtein G (also designated Fc receptor type III) is the IgG‐binding protein of group C and G streptococci. Protein G has also been shown to bind human serum albumin but at a site that is structurally separated from the IgG‐binding region. From the known gene sequence of protein G, two synthetic oligonucleotides were constructed for use as probes in DNA‐hybridization experiments to study the structure and distribution of the albumin‐ and IgG‐binding regions in bacterial strains belonging to different species. Thus, one of the probes corresponded to repeats within the IgG‐binding region (I) and the other corresponded to repeats in the albumin‐binding encoding region (II). Probe I showed strong hybridization to DNA isolated from 31 human group C and G strains, whereas hybridization to probe II was variable. With the three restriction endonucleases used, three restriction patterns were found in Southern blot experiments. No fundamental difference could be detected in hybridization experiments, either between strains of group C and G streptococci, or between isolates of different clinical origin. No hybridization to DNA from other bacterial spec

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00177.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryInteraction of the basement‐membrane binding O75X adhesin of uropathogenicEscherichia coliwith various extracellular matrix proteins was studied. The adhesin showed strong binding to type IV collagen immobilized on microtitre plates, whereas other collagens, laminin and fibronectin, were only weakly recognized. Similarly, specific binding of [125I]‐labelled type IV collagen to 075X‐positive bacteria was shown. Interaction of the two proteins was also demonstrated by affinity chromatography of the O75X adhesin on immobilized type IV collagen. The adhesin bound strongly to the immobilized N‐terminal 7S domain of type IV collagen, and the binding of [125I]‐labelled type IV collagen to O75X‐positive bacteria was inhibited by the soluble 7S domain. Binding of O75X to type IV collagen and to its 7S domain was specifically inhibited by chloramphenicol but was not affected by periodate or endoglycosidase‐H treatment of the glycoproteins. Our results show that the 7S domain of type IV collagen is the basement membrane receptor for the O75X adhesin and suggest an interaction based on protein‐protein recognition. Inhibition of the interaction by chloramphenicol favours the supposition that a modified tyrosine is involved in t

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00178.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryThe chromophoric protein phycocyanin is the major protein in the phycobilisome rod of the cyanobacteriumSynechococcus6301 (formerly designatedAna‐cystis nidulanssp. UTEX 625). The gene clusters coding for the β‐ and α‐subunits of phycocyanin are duplicated on the chromosome ofSynechococcus6301 and separated by an intergenic region 2.5 kb long. The structure of the phycocyanin operons of the phycobilisome mutant strains AN112 and AN135 was compared to that of wild‐typeSynechococcus6301. Both mutants have an altered phycobilisome structure resulting in the appearance of rods of a shorter overall length. Genetic mapping indicated that the mutant strain AN112 completely lacked the intergenic region and carried only one set of phycocyanin subunit genes. No gross structural difference in the genetic organization of the corresponding region of mutant strain AN135 was detected. Northern blot analysis and primer extension experiments were used to monitor the transcriptional pattern of the phycocyanin rod operon. It was found that AN112 had lost the 3.7 kb minor mRNA, which covers the intergenic region, and only produced two major 1.3 and 1.4 kb mRNA species. These transcripts were identified as fusion products of the 5′ end of the transcriptional unit originating from the promoter region of the left‐hand phycocyanin gene cluster and the 3′ end of the transcriptional unit covering the right‐hand phycocyanin gene cluster. The mutant strain AN 135 had a transcriptional pattern very similar to that of the wild type. The level of transcription of the major transcripts covering the phycocyanin gene clusters was similar in the wild‐type and mutant strains. The results indicate that genes coding for two of the linker proteins, namely the 30 and 33 kD poly‐peptides, are located in the intergenic region between the duplicated phycocyanin gene clusters inSynecho‐coccus6301. The results also show that loss of the right‐hand phycocyanin promoter does not drastically imp

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00179.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

SummaryA series of expression plasmids containing either the complete insert from plasmid pUCDBKI (Peopleset al., 1987) or sub‐fragments thereof were constructed in atacpromoter vector. Analysis of protein lysates of induced cultures of these clones identified the gene encoding NADPH‐specific acetoacetyl‐CoA reductase in the 2.3 kb of sequence located downstream from the β‐ketothiolase gene in plasmid pUCDBK1. The complete nucleotide sequence (2.1 kb) of this region was determined. An open reading frame was located 88 bp downstream from the stop codon of the thiotase gene encoding a potential polypeptide ofM, 25000, which is in good agreement with that observed for the overexpressed protein on SDS‐PAGE. N‐terminal protein sequence data obtained by Edman degradation of the purifiedMr= 25000 polypeptide were used to identify the correct start of the NADPH‐specific acetoacetyl‐CoA reductase gene. Hence inZ. ramigera, the genes encoding β‐ketothiolase(phbA)and NADPH‐specific acetoacetyl‐CoA reductase(phbB)are organized asphbA‐phbB.S1‐nuclease analysis ofZ. ramigeraRNA identified a transcription start site 85 bp upstream from thephbAstructural gene l

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1989.tb00180.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY