INTRODUCTION

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Actinobacillus actinomycetemcomitans is a gram-negative bacterium associated with several human diseases (8, 36, 47). The most predominant of these is known as localized juvenile periodontitis, a severe disease of adolescents that is characterized by bone and tissue destruction and ultimately loss of teeth if untreated. A. actinomycetemcomitans is also a member of a clinically important group of bacteria implicated in infective endocarditis (39). These bacteria are referred to as the HACEK group (Haemophilus aphrophilus, A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) (9). Vegetative growths and inflammation of the heart valves caused by the HACEK bacteria result in serious complications owing to the formation of bacterial masses on the valves.

A. actinomycetemcomitans expresses several potential virulence factors (8), but only the RTX-type leukotoxin has been studied in detail at the molecular and genetic levels (14, 24). Other possible factors include a cytolethal distending toxin, iron and hemin binding proteins, a trypsin-like protease, an OmpA family member, capsular polysaccharide biosynthetic proteins, catalase, and a GroEL-like protein (5, 12, 13, 23, 26, 28, 35, 41, 43, 46). However, their potential roles in pathogenesis are unknown. A. actinomycetemcomitans is also able to invade epithelial cells, and the bacteria can be transferred between cells (8, 27).

A striking and characteristic property of fresh clinical isolates of A. actinomycetemcomitans is their ability to form tenacious biofilms on solid surfaces, including glass, plastics, and hydroxyapatite (6, 21, 30). This property is very likely required for pathogenesis by allowing for colonization of teeth in an environment of continuous salivary flow. Clinical isolates form rough-appearing colonies, autoaggregate, and express bundles of fimbria-like structures that may be important for adherence and colonization (18, 30, 34).

Genetic analysis of rough, adherent strains has proven to be difficult. The distinctive adherence property of clinical strains is easily lost, as nonadherent, smooth-colony variants readily emerge during subculture (7, 45). In addition, while DNA can be introduced into A. actinomycetemcomitans by transformation (37) or conjugation (11), the efficiency of DNA transfer into rough, adherent strains is too low for standard transposon mutagenesis protocols involving suicide vectors. Recently we reported the development of transposon IS903kan, which carries a cryptic kanamycin resistance gene that can be activated upon insertion of the transposon into an expressed gene, and we have demonstrated its utility in the direct selection of random insertions in genetically recalcitrant bacteria, such as A. actinomycetemcomitans (38). Here we report the use of IS903kan to obtain adherence-defective mutants of A. actinomycetemcomitans CU1000, a well-characterized rough clinical isolate (7, 20, 30). Genetic and nucleotide sequence analyses of these mutants have allowed us to identify a locus of seven novel genes that are required for tenacious adherence, autoaggregation, and the production of bundled fibers. Examination of genome sequences of Bacteria and Archaea revealed that surprisingly diverse microorganisms are predicted to carry tad-related genes. We discuss the likely function of these genes and the significance of their widespread occurrence.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacterial strains and growth conditions. A. actinomycetemcomitans CU1000N is a spontaneous nalidixic acid-resistant (Nal^r) derivative of the rough clinical isolate CU1000 (6). CU1060N is a Nal^r derivative of CU1060 (6), a spontaneous smooth-colony variant of CU1000. A. actinomycetemcomitans strains were stored and cultured as previously described (6). Bacteria were streaked from 70°C frozen stocks onto agar plates containing A. actinomycetemcomitans growth medium (AAGM) (11) and incubated in a sealed chamber enriched with 5% CO₂ at 37°C for several days. Individual colonies were inoculated into AAGM broth (11) in sealed, screw-cap tubes and incubated at 37°C. For light microscopy, bacteria were grown in broth for 1 day (CU1060N) or 2 days (CU1000N). For CU1000N, the cells were scraped from the walls and resuspended before preparation of wet mounts. To assay adherence of cells onto the surface of culture tubes, 2-day cultures were subjected to vortex mixing and the tube contents were discarded. An equal volume of 5-µg/ml ethidium bromide solution was added to the tubes, left for 10 min at room temperature, and then poured off, and the tubes were washed with water. The tubes were photographed in UV light.

IS903kan mutagenesis of A. actinomycetemcomitans. Plasmid pVJT128, which carries a chloramphenicol resistance (Cm^r) gene and the cryptic IS903kan transposon, was mobilized into rough strain CU1000N by conjugation with an Escherichia coli donor strain (SK338) that contains pVJT128 and an oriT-defective derivative of RK2, pRK21761 (38). CU1000N(pVJT128) transconjugants were selected on AAGM medium containing nalidixic acid (20 µg/ml) and chloramphenicol (4 µg/ml) and screened for sensitivity to kanamycin to confirm that pRK21761 had not transferred and that transposition of IS903kan had not occurred. One such CU1000N(pVJT128) transconjugant was grown in AAGM broth containing chloramphenicol and then plated onto medium containing 4 µg of chloramphenicol per ml and 2 mM IPTG (isopropyl--D-thiogalactopyranoside) (to induce transposition of IS903kan), and colonies were grown for 3 days. Several pools of colonies were each plated onto solid medium containing 20 or 40 µg of kanamycin per ml. Because the kanamycin-resistant (Km^r) colonies were small on this medium, the colonies from each pool were mixed and replated onto nonselective medium, where colony morphology was easily distinguished. To ensure that the mutants were independent isolates, only one smooth-colony mutant was kept from each original pool. Mutants were grown in broth in the absence of chloramphenicol to allow loss of the resident pVJT128 plasmid, as described previously (38).

DNA procedures. Preparation of plasmid DNA from E. coli was done by the alkaline lysis protocol (1). Agarose gel electrophoresis has been described previously (33). DNA manipulations with restriction endonucleases and T4 DNA ligase were done according to the manufacturers' recommendations. Amplification of DNA by PCR was done with Taq DNA polymerase (32). All cloned PCR products were confirmed by nucleotide sequencing. Transformation of E. coli was done by the method of Cohen et al. (4). Inverse PCR with IS903kan insertion mutants was done as previously described (38). Approximately 10 to 20 µg of genomic DNA was digested with EcoRI (which does not cleave IS903kan), followed by ethanol precipitation, dilution, and ligation to circularize genomic fragments. PCR amplification was carried out for 30 cycles using primers directed outward from the ends of IS903kan. Amplified products were cloned into pCR2.1 (Invitrogen, Carlsbad, Calif.) or pT7blue-3 (Novagen, Madison, Wis.) for sequencing. Nucleotide sequence determination was done by the Columbia University DNA Sequencing Facility using a Perkin-Elmer Applied Biosystems Automated DNA sequencer 373A.

Genetic complementation analysis. The desired open reading frame (ORF) was amplified by PCR, cloned into pCR2.1, sequenced, and then subcloned into the IncQ expression vector pJAK16 (a gift of J. Kornacki), a derivative of the tacp expression vector pMMB67 (10). The pJAK16 derivatives were mobilized by conjugation from E. coli donors to the appropriate A. actinomycetemcomitans strains (38). Because the tacp promoter is leaky even in the presence of functional LacI repressor, complementation did not require induction by IPTG.

Electron microscopy. Colonies were resuspended in 0.1 M Tris-HCl (pH 7.5), and a drop of the suspension was placed on a Formvar- and carbon-coated copper grid (EM Sciences, Fort Washington, Pa.) and left for approximately 10 min. Grids were immersed in a drop of 1% uranyl acetate and removed immediately; the excess stain was wicked away. Grids were viewed in a JEOL 1200EX transmission electron microscope at 80.0 kV.

Sequence analysis and similarity searching. Nucleotide sequences potentially coding for proteins similar to the tad gene products were identified using the BLAST alignment program to search both GenBank and the Unfinished Genomes Database available on the National Center for Biotechnology Information web page (http://www.ncbi.nlm.nih.gov/BLAST/unfinishedgenome.html). Default settings of the basic BLAST program were used. All reported sequences significantly exceeded the default criteria and were among the best hits when compared to the corresponding Tad polypeptides. Contiguity and sequential order of tad-related ORFs were established using the ORF finder program to characterize the region around each tad-like sequence. The putative products of all neighboring ORFs were compared to sequences in GenBank using the pBlast function. Preliminary sequence data for Methanococcus jannaschii, Archeoglobus fulgidus, and Chlorobium tepidum were obtained from The Institute for Genomic Research website at http://www.tigr.org. Bordetella pertussis and Yersinia pestis sequences were produced by the respective Sanger Centre sequencing groups (http://www.sanger.ac.uk/), Pyrococcus horikoshii sequences were produced by NITE (http://www.nite.go.jp), the Pyrococcus abyssi sequence was produced by Genoscope (http://www.genoscope.cns.fr), the Pyrococcus furiosus sequence was produced by the Utah Genome Center (http://www.genome.utah.edu), the Pseudomonas aeruginosa sequence was produced by the Pseudomonas Genome Project (http://www.pseudomonas.com), the Methanobacterium thermoautotrophicum sequence was produced by the Genome Therapeutics Center (http://www.genomecorp.com), and the Pasteurella multocida sequence was produced by the University of Minnesota P. multocida Genome Project (http://www.cbc.umn.edu/ResearchProjects/AGAC/Pm/index.html). A compilation of Intein sequences is found in InBase, the New England BioLabs Intein Database (http://www.neb.com/inteins). Accession numbers for tadA- and tadB-related sequences, respectively, in other organisms are as follows: M. jannaschii, Q58191 and Q58189; P. abyssi, CAB50295 and CAB50294; P. horikoshii, BAA29741 and BAA29744; A. fulgidus, AAB90582 and AAB90583; and M. thermoautotrophicum, AAB86174 and AAB85476. The GenBank accession number for plasmid pCL1 from Chlorobium limicola is U77780, that for KlbA (TrbB) is C44020, and that for VirB11 is AAA88655.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Isolation of adherence-defective mutants by transposon mutagenesis. A. actinomycetemcomitans strain CU1000 was originally isolated from a 13-year-old, African-American female with localized juvenile periodontitis (7). To facilitate the study and genetic manipulation of CU1000, we isolated a spontaneous nalidixic acid-resistant variant, CU1000N. Like its parent, CU1000N exhibits the characteristic rough, opaque colony morphology typical of fresh clinical isolates (Fig. 1a). In broth, CU1000N adheres tightly to the walls of the culture vessels (Fig. 1d). The biofilm is resistant to vortex mixing or vigorous agitation, and the bacteria must be physically scraped from the walls. The rough colony phenotype can be maintained indefinitely during subcultures on solid medium. Rough A. actinomycetemcomitans strains give rise to nonadherent, planktonic variants, whose colonies on solid medium are smooth, translucent, and larger than those of rough strains (7, 20, 30, 45) (Fig. 1e and h). One such derivative of CU1000 is CU1060 (7), and its Nal^r derivative is CU1060N. Cells of the smooth, nonadherent variants, including CU1060N, do not display the striking bundles of parallel, fimbria-like fibers normally present in abundance on cells of rough strains (Fig. 1c and 2). It has been suggested that these fibrils mediate adherence (30, 34). Cells of the rough strains and smooth variants also differ dramatically in their ability to autoaggregate (Fig. 1b and f).

View larger version (54K): [in this window] [in a new window]   FIG. 1.   Phenotypes of isogenic rough- and smooth-colony strains of A. actinomycetemcomitans. Top panels, rough-colony strain CU1000N; bottom panels: smooth-colony variant CU1060N. (a and e) Images from stereomicroscopy of 3-day-old colonies. Bars, 1.0 mm. (b and f) Images from light microscopy of bacterial cells using differential interference contrast optics. (c and g) Images from transmission electron microscopy of negatively stained cells. Bars, 0.2 µm. (d and h) Adherence of cells to the culture tube surface, as visualized by fluorescence of ethidium bromide-stained cells in UV light.

View larger version (120K): [in this window] [in a new window]   FIG. 2.   Electron micrograph of fibril bundles produced by the rough strain CU1000N. (a) Fibril bundle showing individual strands separating from the bundle at several places. Bar, 50 nm. (b) Parallel array of six or seven fibrils passing over the edge of a bacterial cell. The spherical structures at the bacterial cell surface are likely to be membranous vesicles, which are commonly seen in preparations of rough strains of A. actinomycetemcomitans (30). Bar, 20 nm.

903kan

903kan

View larger version (107K): [in this window] [in a new window]   FIG. 3.   Phenotypes and genetic complementation of a tadC mutant. Top panels, mutant strain Aa1359 (tadC::IS903kan). Middle panels, strain Aa1359 containing the pJAK16 vector plasmid. Bottom panels, Aa1359 containing pJAK16 with the cloned tadC ORF from CU1000N. The methods used are identical to those described for Fig. 1. Results for complementation of the other mutants were essentially the same (Table 1). Bars, 1.0 mm (a, d, and g), 0.2 µm (b and e), and 0.5 µm (h).

Identification of seven tad genes. To map the sites of IS903kan insertion for several Tad mutants, we did inverse PCR on genomic DNA with primers directed outward from the ends of the transposon (38). Using these sequences and partial nucleotide sequences from the ongoing A. actinomycetemcomitans Genome Sequencing Project to design additional PCR primers, we found that all of the insertions mapped to a continuous 7-kb region of the A. actinomycetemcomitans genome. We determined the sequence for both strands of this region for the rough strain CU1000N. The sequence revealed a cluster of seven closely spaced ORFs oriented in the same direction (Fig. 4). At least one IS903kan insertion mutant was isolated for each ORF, and all mutants were defective in adherence (Table 1) and autoaggregation (data not shown). Genetic complementation analysis with the appropriate ORFs on plasmids showed that the IS903kan insertions are not polar on expression of downstream genes (Table 1). The complemented Tad mutants produced rough-appearing colonies on solid medium, although not to the same degree as those of the wild-type rough strain (Fig. 3a, d, and g), presumably due to nonstoichiometric levels of expression from the plasmid-borne genes. However, cells of the complemented mutants clearly regained the tight adherence phenotype of the rough parental strain (Fig. 3c, f, and i) and the ability to autoaggregate (data not shown). All mutants with insertions in different ORFs were examined by electron microscopy, and all were found to lack fibrils (Fig. 3b), unless the complementing plasmid was present (Table 1, Fig. 3h). Controls with the vector (Fig. 3d, e, and f) or plasmids with noncognate ORFs (Table 1) showed no evidence of complementation for any of the phenotypes. These results confirmed that all seven ORFs are required for tight adherence, and we therefore designated them genes tadA, tadB, tadC, tadD, tadE, tadF, and tadG.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Arrangement of tad genes in A. actinomycetemcomitans and other microorganisms. The top line shows the tadABCDEFG genes of A. actinomycetemcomitans (Aa) strain CU1000N. The locations of the IS903kan insertions in specific mutant strains are indicated by downward arrows. Below are tad-related genes from P. multocida (Pm), Y. pestis (Yp), P. aeruginosa (Pa), C. tepidum (Ct), and P. horikoshii (Ph). The tad sequences for organisms other than A. actinomycetemcomitans were obtained from individual genome sequencing projects as described in Materials and Methods. The scale bar at the bottom is in kilobase pairs.

Tad proteins may constitute a fibril secretion system. We searched GenBank to identify other proteins that are related to the tad gene products. The predicted polypeptide products of the tadB, tadC, tadD, tadE, tadF, and tadG genes showed no significant similarities to any other proteins of known function in the database. However, PSORT (29) and TMPred (17) predicted the presence of six (TadB), five (TadC), and one (TadD, TadE, TadF, and TadG) membrane-spanning regions. Thus, these proteins are likely to be localized to the membrane.

tad genes are widespread. We also compared each Tad protein individually to the Unfinished Microbial Genomes Database, and we were surprised to find significant similarities for each of the Tad proteins with predicted proteins of unknown function in Y. pestis, the causative agent of bubonic plague, and P. multocida, a pathogen that infects humans and animals. The coding regions for the Tad-like proteins in both organisms show all seven ORFs in an arrangement identical to that of the tadABCDEFG region of A. actinomycetemcomitans. Not only do the predicted P. multocida and Y. pestis proteins show significant percent identities with the predicted Tad proteins, but the sizes and isoelectric points (pIs) of the corresponding proteins are strikingly similar (Table 2). Recently, a complete tad region coding for putative polypeptides with high similarities to the seven A. actinomycetemcomitans tad gene products has been identified in the genome of Haemophilus ducreyi, the causative agent of chancroid (E. J. Hansen, personal communication).