摘要:

SummaryRolling circle‐replicating plasmids constitute a group of small, promiscuous multicopy replicons spread among eubacteria. Until recently, rolling circle replication seemed to be limited to small plasmids from Gram‐positive hosts and to single‐stranded bacteriophages from Gram‐negative bacteria. However, characterization of two small plasmids from Gram‐negative hosts has shown that this replication mechanism is general among eubacteria. This review focuses on a family of highly related promiscuous plasmids that replicate by the rolling circle mechanism, and that have been isolated from various Gram‐positive bacteria and from the Gram‐negative bacteriumHelicobacter.They all share homologies at the leading‐strand origins and at the initiator of replication proteins. The plasmids of this family have directly repeated sequences at their plus origin of replication, which is located 5′ from the start point of the mRNA for the initiation of replication protein. Replication is controlled by an antisense RNA and by a transcriptional repressor protein. The features and regulatory circuits of replication of this plasmid family seem to be unique among rolling circle‐replicating plasmids. Members of this family replicate autonomously in Gram‐positi

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01625.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryCatabolite gene activator protein (CAP)‐dependent promoters can be grouped into three classes, based on the requirement for transcription activation and the position of the DNA site for CAP. Class I CAP‐dependent promoters require only CAP for transcription activation and have the DNA site for CAP located upstream of the DNA site for RNA polymerase. Amino acids 156 to 162 of the promoter‐proximal subunit of CAP are essential for transcription activation at Class I CAP‐dependent promoters, but are not essential for DNA binding, and are not essential for DNA bending. In the structure of the CAP‐DNA complex, these amino acids are located in a surface loop and form a cluster on the surface of the CAP‐DNA complex. Amino acids 261, 265, and 270 of the α subunit of RNA polymerase are essential for response to transcription activation by CAP at Class I CAP‐dependent promoters. Several lines of evidence indicate that transcription activation at Class I CAP‐dependent promoters requires a direct protein‐protein contact between amino acids 156 to 162 of the promoter‐proximal subunit of CAP and a molecule of RNA polymerase bound adjacent to CAP on the same face of the DNA helix. It is a strong possibility that this direct protein–protein contact involves amino acids 261 and 265 of the α su

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01626.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryElectrophoretic karyotyping of the two most widely studied strains ofPhanerochaete chrysosporium, BKMF‐1767 and ME‐446, has been determined using transverse alternating field etectrophoresis. The genomic DNA of BKMF‐1767 was resolved into 10 chromosomes ranging in size from 1.8–5.0 Mb, amounting to a total genome size of about 29 Mb. The genomic DNA of strain ME‐446, on the other hand, was resolved into 11 chromosomes, amounting to a total genome size of about 32Mb. Lignin peroxidase genes have been localized to five chromosomes in strain BKMF‐1767 and to four chromosomes in st

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01627.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryThe major virulence factor of the important human pathogenStreptococcus pyogenesis the M protein, which prevents phagocytosis of the bacterium. In different strains of streptococci, there are over 80 serologically different M proteins and there are additional M‐like proteins, some of which bind immunoglobulins. Although the sequence of the M molecules differs among differentS. pyogenesstrains, all M proteins, and some of the immunogiobulin‐binding molecules, have at least two copies of the C repeat region. We describe construction of a deletion mutation inS. pyogenes, which has only one C repeat copy, and show that the mutant strain is still resistant to phagocytosis. The mutation was constructedin vitroand used to replace the residentemmallele in anS. pyogenesstrain. To facilitate homologous recombination into the streptococcal chromosome, we adapted a shuttle vector which is temperature sensitive for replication in Gram‐positive bacteria but not in Gram‐negative hosts. This new method for delivery of a homologous DNA fragment to theS. pyogeneschromosome is efficient and reproducible and should be of gene

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01628.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryThe chromosomal region ofBacillus subtiliscomprising the entiresrfAoperon,sfpand about four kilo‐bases in between have been completely sequenced and functionally characterized. ThesrfAgene codes for three large subunits of surfactin synthetase, 402, 401 and 144 kDa, respectively, arranged in a series of seven amino acid activating domains which, as shown in the accompanying communication, recognize and bind the seven amino acids of the surfactin peptide. ThesrfAamino acid activating domains share homologies with similar domains of other peptide synthetases; in particular, regions can be identified which are more homologous in domains activating the same amino acid. A fourth gene insrfAencodes a polypeptide homologous togrsT.Four genes are positioned betweensrfAandsfp, the disruption of which does not affect surfactin biosynthesi

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01629.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummarysrfAis a locus required for the production of the lipopeptide antibiotic surfactin. This locus is also necessary for efficient sporulation and competence development. Mutations in the 5′ portion of thesrfAoperon affect all three of these processes, whereas mutations in the 3′ portion ofsrfAonly affect sporulation and surfactin production. Analysis of the proteins encoded by thesrfAlocus revealed seven large domains which are likely to be responsible for the activation and binding of the seven amino acids of surfactin. Identification of the amino acid that is activated by thesrfAdomains was determined by amino acid‐dependent pyrophosphate exchange reactions on partially purified cell extracts of strains carrying differentsrfAmutations. These results indicate colin‐earity between the order of the domains in thesrfAlocus and the amino acid sequence of surfactin. The minimal genetic element ofsrfArequired for the establishment of competence was shown to be the 5′ region of the second open reading ofsrfA, which encodes the valine activation domain. This portion ofsrfA, when cloned on a plasmid, complemented the competence deficiency of asrfAdeletion mutan

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01630.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryWe showed previously that a mutant strain of group BStreptococcus(GBS) defective in capsule production was avirulent. This study describes the derivation of an unencapsulated mutant from a highly encapsulated wild‐type strain of type III GBS, COH1, by transposon mutagenesis with Tn916ΔE. The mutant, COH1‐13, was sensitive to phagocytic killing by human leukocytesin vitroand was relatively avirulent in a neonatal rat sepsis model compared with the wild‐type strain. No capsular polysaccharide was evident in the cytoplasm or on the cell surface of the mutant strain. The Tn916ΔE insertion site in COH1‐13 was mapped to the same chromosomal location as the Tn916insertion site in the unencapsulated type III mutant COH31‐15 reported previously. Nucleotide sequencing of DNA flanking the insertion site in COH1‐13 revealed an open reading frame, designatedcpsD, with significant homology to therfbPgene ofSalmonella typhimurium. RfbPencodes a galactosyl transferase enzyme that catalyses the transfer of galactose to undecaprenol phosphate, the initial step inO‐polysaccharide synthesis. A particulate fraction of a lysate of wild‐type strain GBS COH1 mediated the transfer of galactose from UDP‐galactose to an endogenous acceptor. The galactose–acceptor complex partitioned into organic solvents, suggesting it is lipid in nature or membrane‐associated. Galactosyl transferase activity was significantly reduced in the unencapsulated mutant strain COH1‐13. These results, together with the similarity in deduced amino acid sequence betweencpsDandrfbPsuggest thatcpsDencodes a galactosyl transferase essential for assembly of the GBS type II

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01631.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryOne characteristic of pigmented (Pgm+) cells ofYersinia pestisis the adsorption of sufficient quantities of exogenous haemin during growth at 26°C to form dark‐brown colonies. Carriage of the cloned haemin‐storage (hms) locus in pHMS1 restores this phenotype to spontaneous Pgm−chromosomal deletion mutants ofY. pestis.We have mapped the location of the structural genes for four proteins encoded on pHMS1 using minicell,in vitrotranscription/translation, and complementation analysis. ThehmsHandhmsFgenes encode 90kDa and 72 kDa protein precursors processed to surface‐exposed, outer membrane proteins of 86kDa and 70kDa, respectively. Beta‐galactosidase positive MudII1734 insertions inhmsRsuggest that it encodes a protein that is also essential for haemin storage. Finally, the structural gene for a 41 kDa protein lies distal to thehmsHgene but, unlikehmsH, hmsF, andhmsR, its expression is not essential for the Hms+phenotype i

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01632.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryThe heat stable enterotoxins (ST) of enterotoxigenicEscherichia coli(ETEC) cause diarrhoea by binding specific intestinal receptors. Precise histochemical localization of ST receptors could provide more information about the pathophysiology of secretory diarrhoea and the role of ST receptors in normal biology. To accomplish this, we quantitatively coupled biotin to theN‐terminus of ST1b using biotin‐X‐X‐N‐hydroxysuccinimide ester. The derivatized toxin (BST) has an apparentKdof 11.7±10 nM for rat brush border receptors. We used BST in an affinity panning cell‐capture system, to validate its ability to discriminate between receptor‐positive and receptor‐negative cells. Cell lines expressing ST receptors (human colon carcinoma T84, and COS cells transfected with guanylyl cyclase‐C (GC‐C) ST receptor cDNA) were captured to streptavidin and anti‐biotin‐coated plates with high efficiency and specificity. This system provides a novel approach to screening cells for the presence of unique ST‐binding proteins. BST was then used with streptavidin‐gold to demonstrate the cellular topography of ST receptors at the light microscopic level. Villus enterocytes were intensely stained, but only a faint signal was observed in upper crypts of rat small intestine. Thus, a gradient of increasing receptor density was seen as upper crypt cells matured into villus enterocytes. Higher magnification revealed that ST receptors are concentrated at the apical aspect of villus enterocytes. Recently, guanylin, a putative endogenous ligand for ST receptors, has been localized to Paneth cells, at the base of intestinal crypts. Thus, ST receptors, are concentrated in villus entercoytes, while guanylin appears to be produced at the base of the crypts. This topographical arrangement suggests that there are autocrine or paracrine pathways by which ST receptors inte

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01633.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryMutations in the structural gene (hns) for theEscherichia colinucleoid‐associated DNA‐binding protein H‐NS cause highly pleiotropic effects on gene expression, site‐specific recombination, transposition of phage Mu, the stability of the genetic material and the topological state of the DNA. We have investigated the regulation ofhnsexpression and found thathnstranscription is subjected to stationary phase induction and negative autoregulation. A set ofhns–lacZprotein and operon fusions was constructedin vitroand integrated in single copy into theattBsite of the bacterial genome. Quantification of β‐galactosidase activity along the bacterial growth curve showed thathnsexpression increases approximately 10‐fold in stationary phase compared with exponentially growing cells. Immunological detection of the H‐NS protein in growing and stationary phase cells supported the genetic data and showed that H‐NS synthesis varies with growth phase. In addition, primer extension experiments demonstrated that the amount ofhnsmRNA is elevated in stationary phase cultures and thathnstranscription is directed by a unique promoter functioning in both log and stationary phase. Disruption of thehnsgene by an insertion mutation led to the derepression (approximately fourfold) of the expression of anhns–lacZoperon fusion integrated at theattBsite, showing thathnstranscription is subjected to negative regulation by its own gene product. Autoregulation ofhnsexpression is particularly pronounced in log phase. Both stationary phase control and autoregulation ofhnstranscription are associated with a 130 bp fragment that contains thehnspromoter. In order to study the interaction of H‐NS with its own regulatory region, we developed an efficient overproduction procedure and a simple purification scheme for H‐NS. DNA gel retardation assays showed that the H‐NS protein can preferentially interact with a restriction fragment carrying thehnspromoter. This restriction fragment showed features of curved DNA as judged by two‐dimensional polyacrylamide gel electrophores

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01634.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY