RESUMO
pIP501 is a 30.2-kilobase (kb) broad-host-range conjugative streptococcal plasmid which encodes chloramphenicol and erythromycin resistance. A smaller conjugation-proficient derivative of pIP501 has been constructed and designated pVA1702. pVA1702 is 25.2 kb, encodes only kanamycin resistance, and confers conjugative ability in an Enterococcus faecalis mating system. The temperature-sensitive transposon delivery vectors pTV1ts and pTV32 carrying Tn917 and Tn917lac, respectively, were used to create insertion mutants of pVA1702 in E. faecalis. These insertions enabled us to localize the determinants conferring conjugation to two separate regions of pVA1702: the 7.5-kb region A and the 8.8-kb region B. A nested set of deletions of the 7.5-kb region was subcloned in Escherichia coli, and the expression of these deletions was examined in an E. coli minicell system. The genes for three conjugal proteins have been located and designated cnjA, cnjB, and cnjC (for conjugation). Two other peptides that are transcribed in a divergent direction from the cnj genes were encoded in the A region, but it is not known whether they are involved in conjugation. These studies indicate that the conjugation genes of pIP501 are arranged in multiple transcriptional units.
Assuntos
Conjugação Genética , Elementos de DNA Transponíveis , Enterococcus faecalis/genética , Escherichia coli/genética , Genes Bacterianos , Plasmídeos , Streptococcus/genética , Deleção Cromossômica , Clonagem Molecular , Mutação , Mapeamento por RestriçãoRESUMO
pIP501 is a member of a group of conjugative plasmids that are self-transmissible to a wide variety of streptococci as well as to other gram-positive bacteria. Several pIP501 restriction fragment deletion derivatives have been isolated and characterized. In this paper we describe one such derivative (pVA1702) which was conjugally proficient but had a limited host range. The loss of host range ability was seen as decreased conjugal transfer from Enterococcus faecalis to Streptococcus sanguis and was coincident with the deletion of a 4.5-kb DNA fragment. Transformation of pVA1702 into S. sanguis also was dramatically reduced as compared to its progenitor, suggesting the 4.5-kb fragment encoded a factor(s) necessary for stable maintenance in this host but not in E. faecalis. These observations suggest that pIP501 employs specific mechanisms enabling its maintenance in certain gram-positive bacteria.
Assuntos
Conjugação Genética , Plasmídeos/genética , Streptococcus sanguis/genética , Deleção Cromossômica , Enterococcus faecalis/genética , Mapeamento por RestriçãoRESUMO
As the study of oral microorganisms intensified almost 2 decades ago, the application of genetic techniques resulted in important contributions to the understanding of this clinically and ecologically important group of bacteria. The isolation and characterization of mutants of cariogenic streptococci helped to focus attention on traits that were important in colonization and virulence. Such classic genetic approaches gave way to molecular genetic techniques, including recombinant DNA methodology in the late 1970s. Gene cloning systems and methods to move DNA into cells have been developed for oral streptococci. Many streptococcal genes thought to be important in colonization and virulence have since been cloned and their nucleotide sequence determined. Mutant strains have been constructed using defective copies of cloned genes in order to create specific genetic lesions on the bacterial chromosome. By testing such mutants in animal models, a picture of the cellular and molecular basis of dental caries is beginning to emerge. These modern genetic methodologies also are being employed to develop novel and efficacious cell-free or whole cell vaccines against this infection. Genetic approaches and analyses are now being used to dissect microorganisms important in periodontal disease as well. Such systems should be able to exploit advances made in genetically manipulating related anaerobes, such as the intestinal Bacteroides. Gene cloning techniques in oral anaerobes, Actinomyces and Actinobacillus, are already beginning to pay dividends in helping understand gene structure and expression. Additional effort is needed to develop facile systems for genetic manipulation of these important groups of microorganisms.