Identification of genes coding for exported proteins of Actinobacillus actinomycetemcomitans.

Escherichia coli. Twenty-four PhoA 1 clones were isolated and sequenced. Membrane localization signals in the form of signal sequences were deduced from most of these sequences. Several of the deduced amino acid sequences were found to be homologous to known exported or membrane-associated proteins. The complete genes corresponding to two of these sequences were isolated from an A. actinomycetemcomitans lambda phage library. One gene was found to be homologous to the outer membrane lipoprotein LolB. The second gene product had homology with a Haemophilus influenzae protein and was localized to the inner membrane of A. actinomycetemcomitans. Actinobacillus actinomycetemcomitans, a gram-negative bacterium, is strongly implicated as a causative agent of juvenile and adult periodontitis (21, 22, 27). Colonization of the oral cavity by A. actinomycetemcomitans and other oral pathogens may result in bleeding, loss of tissue matrix components, and ultimately tooth loss. A. actinomycetemcomitans expresses a number of virulence factors that are important in the disease process (7). Many of these virulence factors are proteins that are exported from the cytoplasm to the bacterial surface. These proteins may initially be directed to the surface of the bacterium by molecular information encoded in the signal sequence (26). The signal sequence is a transient extension of the NH2 terminus of the protein and is removed by enzymes following the translocation of the protein across the inner membrane. A genetic system that identifies genes coding for exported proteins by using a truncated gene for alkaline phosphatase (phoA) that lacks a functional signal sequence has been developed (15). The system takes advantage of the fact that the translocation of alkaline phosphatase (PhoA) across the cytoplasmic membrane is required for enzymatic activity. Gene fusions between the coding region of heterologous signal sequences and phoA result in the expression of PhoA activity. We have utilized this genetic system to identify genes that code for exported proteins of A. actinomycetemcomitans. Construction and screening of an A. actinomycetemcomitans DNA signal sequence library. Cesium chloride-purified chromosomal DNA of A. actinomycetemcomitans SUNY 465 was restricted with Sau3A and separated by agarose gel electrophoresis. DNA fragments corresponding to 300 to 600 bp were excised and purified by electroelution. The vector containing the truncated phoA (pHRM 104), provided by H. R. Masure (St. Jude Children’s Research Hospital, Memphis, Tenn.), was restricted with BamHI and ligated with the 300- to 600-bp Sau3A DNA fragments. Escherichia coli CC118 (phoA mutant) cells were transformed by electroporation with the ligation mixture and incubated in SOC medium (20) for 1 h. The cells were then plated on Luria-Bertani (LB) agar plates containing 500 mg of erythromycin per ml and 80 mg of 5-bromo-4-chloro3-indolylphosphate (XP) per ml. Translocation of alkaline phosphatase across the bacterial inner membrane resulted in the hydrolysis of XP and the development of a blue colony phenotype. Approximately 2,500 individual colonies were screened, and 28 colonies were found to be PhoA 1 . These results indicate that fusion proteins were derived from plasmids containing an A. actinomycetemcomitans DNA sequence harboring a promoter, a translational start site, and a functional signal sequence. The presence of these sequences is based on the absence of an intrinsic promoter and signal sequence upstream from the phoA in the original plasmid (15). In addition, PhoA activity was indicative of the heterologous DNA sequence being in frame with the phoA for the translation of a functional protein.