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Infect. Immun. doi:10.1128/IAI.01687-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A screen for leukotoxin mutants in Aggregatibacter actinomycetemcomitans: genes of the phosphotranserase system (PTS) are required for leukotoxin biosynthesis

Department of Oral Biology, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103

^* To whom correspondence should be addressed. Email: kachlasc{at}umdnj.edu.

	Abstract

Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans is a pathogen that causes localized aggressive periodontitis (LAP) and extra-oral infections including infective endocarditis. Recently, we reported that A. actinomycetemcomitans is -hemolytic on certain growth media due to the production of leukotoxin (LtxA). Based on this observation and our ability to generate random transposon insertions in A. actinomycetemcomitans, we developed and carried out a rapid screen for leukotoxin mutants. Using PCR, we mapped several of the mutations to genes that are known or predicted to be required for LtxA production, including ltxA, ltxB, ltxD, and tdeA. In addition, we identified an insertion in a gene previously not recognized to be involved in LtxA biosynthesis, ptsH. ptsH encodes the protein HPr, a phosphocarrier protein that is part of the sugar phosphotransferase system (PTS). HPr results in the phosphorylation of other proteins and ultimately in the activation of adenylate cyclase and cAMP production. The ptsH mutant showed only partial hemolysis on blood agar and did not produce LtxA. The phenotype was complemented by supplying wild type ptsH in trans and real-time PCR (RT-PCR) analysis showed that the ptsH mutant produced approximately 10-fold less ltxA mRNA compared to the wild type strain. The levels of cAMP in the ptsH mutant were significantly lower than the wild type strain and LtxA production could be restored by adding exogenous cAMP to the culture.