MOLECULAR ANALYSIS OF A POTENTIAL TRANSPORT OPERON IN AA

点击翻译按钮获取中文摘要

Actinobacillus actinomcetemcomitans (Aa) is a primary pathogen associated with a variety of oral and non-oral infections. Most pathogenic microorganisms release into the host environment virulence factors that enhance their pathogenicity. The molecular basis for this protein export has been characterized in some of these bacteria. For example, the Bordetella pertussis pertussis toxin liberation (ptl) operon and the Agrobacterium tumefaciens virulence (virB) operon are two well characterized, homologous transport operons. However, a mechanism by which Aa transports substances from the intracellular to the extracellular environment has not been elucidated. We have identified seven open reading frames (ORFs) encoding putative proteins homologous with those predicted by the ptl and virB transport operons on the Aa plasmid, pVT745, isolated from Aa strain VT745 (originally designated JP2). Southern blot analyses showed that many of these pVT745 sequences also are present in the chromosome of Aa strain VT747. The DNA sequence of one of one of these chromosomally-encoded genes from strain VT747 shared 95% homology at the DNA level with the corresponding pVT745-encoded gene. The amino acid sequences of the predicted proteins of these two genes showed 58% and 56% homology respectively, with the predicted PtlC protein of B. Pertussis. And 47% and 50% homology, respectively, with the predicted VirB4 protein of A. tumefaciens. It is hypothesized that these Aa plasmid and chromosomal genes are parts of operons similar to the ptl or virB transport operons, and that the gene products may be responsible for macromolecular transport in Aa. The following Specific Aims are proposed to address this hypothesis: 1. To use recombinant DNA technology to insertionally inactivate (a) the chromosomally-encoded ptlC/virB4 gene homolog in Aa strain VT747 and (b) the corresponding plasmid-encoded gene in strain VT745 to demonstrate their importance in transport in Aa. 2. To (a) identify additional genes present on the chromosome of Aa VT747 that share homology with the potential transport genes on the plasmid, pVT745, and (b) to determine whether the identified chromosomal and plasmid genes are transcribed, either individually or as part of an operon. 3. To clone and sequence the chromosomally-encoded Aa genes identified in Specific Aim 2. Identification of genes involved in the secretion of macromolecules from Aa may lead to the development of strategies aimed at diminishing the virulence potential of this pathogenic microorganism.