RESUMO
A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformable Neisseria mutants. By random insertion of a selectable marker into the conjugative Neisseria plasmid ptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned in Escherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of an E. coli replicon that does not support autonomous replication in Neisseria, e.g. ColE1, p15A, or ori(fd), fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformable Neisseria strain involves a three-step process: (i) insertion of the desired gene into a +Hermes vector; (ii) transformation of Hermes into a Neisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the final Neisseria recipient. Several applications for the genetic manipulation of pathogenic Neisseriae are described.
Assuntos
Genes Bacterianos , Vetores Genéticos , Neisseria gonorrhoeae/genética , Transformação Bacteriana , Sequência de Bases , Clonagem Molecular , Conjugação Genética , Elementos de DNA Transponíveis , Escherichia coli , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Oligodesoxirribonucleotídeos , Fenótipo , Plasmídeos , Recombinases Rec A/biossíntese , Recombinases Rec A/genética , Mapeamento por RestriçãoRESUMO
Opacity proteins (Opa) of Neisseria gonorrhoeae, a family of variant outer membrane proteins implicated in pathogenesis, are subject to phase variation. In strain MS11, 11 different opa gene alleles have been identified, the expression of which can be turned on and off independently. Using a reverse genetic approach, we demonstrate that a single Opa protein variant of strain MS11, Opa50, enables gonococci to invade epithelial cells. The remaining variant Opa proteins show no, or very little, specificity for epithelial cells but instead confer interaction with human polymorphonuclear neutrophils (PMNs). Thus, depending on the opa allele expressed, gonococci are capable of invading epithelial cells or of interacting with human leukocytes. The respective properties of Opa proteins are maintained independent of the gonococcal strain; thus, the specificity for epithelial cells or leukocytes is intrinsic to Opa proteins. Significant homology exists in the surface exposed variable regions of two invasion supporting Opa proteins from independent strains. Efficient epithelial cell invasion is favoured by high level Opa production, however, a 10-fold reduction still allows significant invasion by gonococci. In contrast, recombinant Escherichia coli expressing Opa proteins adhered or invaded poorly under similar experimental conditions, thus indicating that additional factors besides Opa are required in the Opa-mediated interaction with human cells.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Leucócitos/microbiologia , Neisseria gonorrhoeae/fisiologia , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Células Cultivadas , Clonagem Molecular , DNA Bacteriano , Epitélio/microbiologia , Escherichia coli , Expressão Gênica , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Células Tumorais CultivadasRESUMO
Variants of Neisseria gonorrhoeae MS11 show distinct colony morphologies because of the expression of a class of surface components called opacity (Opa, PII) proteins. Southern analyses combined with molecular cloning of genomic DNA from a single variant of MS11 has identified 11 opa genes contained in separate loci. These opa genes code for distinct opacity proteins which are distinguishable at their variable domains. The opa gene analyses were also extended to divergent variants of MS11. These studies have shown that, during in vitro and in vivo culture, 10 of the 11 opa genes did not undergo significant change in their primary sequence. However, in these variants, one gene (opaE) underwent non-reciprocal inter-opa recombinations to generate newer Opa variants. Phylogenic analysis of the opa gene sequences suggests that the opa gene family have evolved by a combination of gene duplication, gene replacement and partial inter-opa recombination events.
Assuntos
Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Genes Bacterianos/genética , Família Multigênica/genética , Neisseria gonorrhoeae/genética , Sequência de Aminoácidos , Variação Antigênica/genética , Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Sequência de Bases , Evolução Biológica , Southern Blotting , Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica/fisiologia , Variação Genética/genética , Dados de Sequência Molecular , Recombinação Genética/genéticaRESUMO
The coding part of the cDNA of cardiac fatty acid-binding protein (cFABP) from bovine heart was cloned into the vector pKK233-2. After induction with isopropyl-beta-D-thiogalactopyranoside cFABP was found in a soluble form in the cytosol of plasmid transformed E. coli amounting up to 5.7% of the soluble protein. cFABP was detected after SDS-polyacrylamide gelelectrophoresis and/or isoelectric focusing and Western blot by immuno-staining and was determined quantitatively by a solid phase enzyme-linked immuno sorbent assay. The cFABP produced by bacteria binds oleic acid with high affinity as shown by comigration of protein and ligand in both gelfiltration and isoelectric focusing. cFABP was purified from bacterial lysates to near homogeneity and resolved into four isoproteins.
