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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 µg of erythromycin per ml and 80 µg of 5-bromo-4-chloro-3-indolylphosphate (XP) per ml.

Sequencing and protein homologs of PhoA⁺ colonies. Plasmids from the PhoA⁺ colonies were isolated using Midi columns (Qiagen, Inc., Chatsworth, Calif.). An oligonucleotide primer (5'-AATATCGCCGTGAGC-3') which hybridizes to the negative strand of phoA was used for sequencing the A. actinomycetemcomitans DNA contained on these plasmids. Plasmids were sequenced with a dideoxy terminator cycle sequencing kit (Applied Biosystems, Inc., Foster City, Calif.) and analyzed with an Applied Biosystems automated DNA sequencer. DNA sequencing was performed in the Vermont Cancer Center DNA Analysis Facility. Unique sequences were determined for 24 of the 28 clones. The amino acid sequences deduced from selected DNA sequences are presented in Table 1. Most of the sequences contain a start methionine followed by amino acids that have features characteristic of prokaryotic signal sequences. A signal sequence typically has three distinct domains: an NH₂-terminal positively charged region (1 to 5 amino acids), a central hydrophobic region (7 to 15 amino acids), and a more polar COOH-terminal domain (3 to 7 amino acids) adjacent to the mature protein. Most of the predicted sequences contain lysine residues followed by a run of amino acids rich in leucine and in other hydrophobic amino acids. Following the polar amino acids, a putative signal peptidase cleavage site can be predicted by the (3,1) rule of von Heijne (25, 26). Some plasmids (pVT1049 and pVT1057) contained sequences that did not fit the domain structure of typical signal sequences. These sequences may represent alternative secretory signals utilized for protein translocation.

Isolation of A. actinomycetemcomitans genes coding for exported proteins. The genes corresponding to the DNA sequences identified on the PhoA⁺ clones were isolated from a  EMBL3 A. actinomycetemcomitans DNA library with an average insert size of 15 kb. The library was generated by restriction of chromosomal DNA with Sau3A followed by separation by agarose gel electrophoresis. DNA fragments corresponding to 14- to 23-kbp fragments were isolated by electroelution, and the eluted fragments were ligated with  EMBL3 arms restricted with BamHI. Phage was packaged with the Packagene in vitro system (Promega Corporation, Madison, Wis.) following the manufacturer's instructions. Phage containing A. actinomycetemcomitans DNA inserts was propagated by infection of E. coli LE392 grown in TYN medium (per liter, 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, NaOH to pH 7.2, 10 mM Tris [pH 7.2]) supplemented with 0.2% maltose and 0.01 M MgSO₄.

View larger version (64K): [in this window] [in a new window]   FIG. 1.   Sequence alignment of LolB protein from A. actinomycetemcomitans. Identical residues are indicated by black shading, while conservative substitutions are indicated by gray shading. Alignment was determined by using Vector NTI software from InforMax, Inc., North Bethesda, Md. ACTAC, A. actinomycetemcomitans; ECOLI, E. coli (11); SALTY, Salmonella typhimurium (16); HAEIN, H. influenzae (8).

View larger version (62K): [in this window] [in a new window]   FIG. 2.   Sequence alignment of Imp22 protein from A. actinomycetemcomitans. Identical residues are indicated by black shading, while conservative substitutions are indicated by gray shading. Alignment was determined by using Vector NTI software from InforMax, Inc., North Bethesda, Md. ACTA, A. actinomycetemcomitans; YFGM_ECOLI, hypothetical 22.2-kDa protein of E. coli (3); Y370_HAEIN, hypothetical protein (HI0370) of H. influenzae (8).

Nucleotide sequence accession numbers. The complete DNA sequences for the genes represented by pVT1051 and pVT1057 can be obtained through GenBank under accession no. AF045460 and AF045461, respectively.