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Journal of Bacteriology, September 2008, p. 5972-5980, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.00548-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Membrane Morphology and Leukotoxin Secretion Are Associated with a Novel Membrane Protein of Aggregatibacter actinomycetemcomitans

Claude V. Gallant,¹ Maja Sedic,¹ Erin A. Chicoine,¹ Teresa Ruiz,² and Keith P. Mintz^1*

Department of Microbiology and Molecular Genetics,¹ Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont²

Received 21 April 2008/ Accepted 30 June 2008

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Gram-negative bacteria display either a flat or an irregular outer membrane. The periodontal pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans has an irregular outer membrane. We have identified a gene that is associated with the biogenesis of this morphology. The gene is part of a three-gene operon and codes for a 141-kDa protein designated morphogenesis protein C (MorC), which is conserved in several gram-negative bacteria including Haemophilus influenzae and Pasteurella multocida. Insertional inactivation of this gene resulted in the conversion of an irregularly shaped membrane to a flat membrane. Associated with this morphological change were the autoaggregation of the bacteria during planktonic growth and a concomitant increase in the surface hydrophobicity of the bacterium. The absence of MorC also resulted in the loss of the secretion of leukotoxin but not the ltxA transcription. Our findings suggest that MorC is critical for membrane morphology and leukotoxin secretion in A. actinomycetemcomitans.

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Aggregatibacter (Actinobacillus) actinomycetemcomitans is a small gram-negative, nonmotile, coccobacillus important in the pathogenesis of localized aggressive periodontitis and some cases of adult periodontitis (43, 53). A. actinomycetemcomitans also translocates from the oral cavity into the circulatory system to cause extraoral infections, including infective endocarditis and abscesses in various body sites (34, 35). A. actinomycetemcomitans is a member of the clinically relevant HACEK group of pathogens, consisting of Haemophilus species (H. parainfluenzae, H. aphrophilus, and H. paraphrophilus), A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella species, which has an enhanced capacity to produce endocardial infections (5). Recently, we demonstrated the interaction of A. actinomycetemcomitans with cardiac valves and the role of this organism in a rabbit endocarditis animal model (49).

Isolates of A. actinomycetemcomitans from the oral cavity typically display long bundled fimbriae (17, 38, 42). In broth cultures, these rough strains autoaggregate and adhere to the walls of the growth vessel. During planktonic growth, small cohorts of bacteria lose the ability to express fimbriae and adopt a smooth phenotype (18, 20). These cells (smooth strains) grow homogenously in broth cultures and demonstrate minimal adherence to glass surfaces. Transmission electron microscopy (TEM) studies reveal distinctive membrane morphologies for both rough and smooth variants (6, 28). The bacterial outer membrane of both phenotypes has an irregular or rugose appearance. This membrane morphology is associated with some gram-negative bacteria, e.g., H. influenzae (2) and Moraxella catarrhalis (16), but not with others, e.g., Escherichia coli, Salmonella spp., and Yersinia spp., which have flat outer membranes. The physiological relevance and biogenesis of these membrane convolutions have not been investigated. The outer membrane as a whole is critical for the physiology and protection of gram-negative bacteria from environmental stresses (33).

A. actinomycetemcomitans pathogenicity is attributed to, among other virulence determinants, the secretion of a leukotoxin across the outer membrane (1). Leukotoxin belongs to the repeat-in-toxin (RTX) protein family of host-specific cytolysins and is encoded by a four-gene operon designated ltxC, ltxA, ltxB, and ltxD (25, 26). The genetic organization and protein sequences are homologous to those of the well-characterized -hemolysin of E. coli (25, 50). The A. actinomycetemcomitans toxin specifically targets human and primate granulocytes and lymphocytes (48), thereby aiding the bacterium in the evasion of the host innate immune defense system (21). The toxin has been demonstrated to be beta-hemolytic on solid medium and is also considered to be a hemolysin (3). There is a strong correlation between the presence of a highly leukotoxic strain and the severity of the disease (15).

In this study, we have generated a mutant of A. actinomycetemcomitans that displays a flat outer membrane without any membrane convolutions. The change in the outer membrane was associated with the disruption of a 3.8-kbp open reading frame (ORF) coding for a 141-kDa protein named morphogenesis protein C (MorC). The atypical outer membrane appearance can be converted back to the wild-type phenotype by complementation of the mutant with the full-length gene in trans. Disruption of the gene was also associated with autoaggregation of the bacteria during planktonic growth and with reduced secretion of leukotoxin compared to that of the wild-type strain. Homology searches indicate that this formerly hypothetical protein is conserved in gram-negative bacteria. The data suggest that MorC is linked to the biogenesis of the outer membrane convolutions and membrane function of A. actinomycetemcomitans and may share functional similarities with other bacteria.

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Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 1. The A. actinomycetemcomitans strains used in this study are based on VT1169, a smooth variant derived from the clinical strain SUNY465 (31). All A. actinomycetemcomitans strains were grown under static conditions in Trypticase soy broth supplemented with 0.6% yeast extract (TSBYE) at 37°C in a humidified, 10% CO₂ atmosphere. E. coli strains were grown in Luria-Bertani (LB) medium at 37°C with agitation. Antibiotics were added to a final concentration of 50 µg/ml of spectinomycin and 1 µg/ml of chloramphenicol for A. actinomycetemcomitans and to 20 µg/ml of chloramphenicol for E. coli.

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TABLE 1. Strains and plasmids

A new shuttle plasmid, designated pKM2, was developed for genetic complementation. This plasmid is based on the backbone of the A. actinomycetemcomitans pPK1 plasmid (45). The antibiotic resistance marker spectinomycin adenyltransferase, aad9 (GenBank accession number M69221) from Enterococcus faecalis (27), was replaced with chloramphenicol acetyltransferase (cat) within the multiple cloning site of pBluescript SK (pBS; Stratagene). cat was excised from p34S-Cm2 (10) with SstI and cloned into the SacI site of pBS. The multiple cloning site from pBS containing cat was excised by digestion with BssHII. The 930-bp fragment was treated with Klenow and cloned into the PvuII site of pPK1. Independent colonies were screened for constructs containing the pBS KpnI site 3' of the cat cassette. The DNA was digested with KpnI to remove aad9, and the DNA fragments were separated by gel electrophoresis. The subsequent 3-kbp band was gel purified and self ligated. The plasmid was sequenced to determine the fidelity of the cloning process. All sequencing was performed at the University of Vermont Cancer Center DNA analysis facility.

A lacZ reporter gene construct was generated to study the 165-bp intergenic region of the morC operon. The lacZ gene was PCR amplified from the pBAD/His/lacZ plasmid (Invitrogen, Carlsbad, CA), using primers with engineered SalI and BamHI restriction enzyme sites. The PCR product was purified and incubated with SalI and BamHI. The gel-purified product was ligated with pKM2 (see description above), previously digested with SalI and BamHI, and used to transform E. coli DH5, generating pKM2(lacZ). The 165-bp region upstream of omp67 was amplified by PCR with engineered XhoI and SalI sites, purified, treated with enzymes, and ligated with pKM2(lacZ). The upstream primer used in the PCR was 5'AGTCGACAATGATAGATCCCGTC-3', and the downstream primer was 5'-AGGGATCCTTATTTTTGACACCA-3' (SigmaGenosys, Woodlands, TX). The ligation mixture was used to transform E. coli DH5, and the isolated plasmid was designated pKM2(165-bp lacZ). Sequence fidelity was determined by DNA sequencing as mentioned above. Both constructs [pKM2(lacZ) and pKM2(165-bp lacZ)] were used to transform the A. actinomycetemcomitans wild-type strain following the protocol described by Sreenivasan and Fives-Taylor (45).

Allelic replacement mutagenesis. An allelic replacement mutant of morC was constructed by the conjugation of a nonreplicating broad host range plasmid as previously described (18). Genomic DNA isolated from VT1169 was used as the template in PCR to generate a 2.35-kbp fragment incorporating 1.1 kbp upstream and 1.25 kbp downstream of the start codon of morC. The upstream primer used in the PCR was 5'-CCGGTGAAGACTACTTACTCAGC-3', and the downstream primer was 5'-CTTGTGCCGTCAGTAAATCTCCTTC-3' (SigmaGenosys, Woodlands, TX). The purified DNA fragment was ligated with the T/A cloning plasmid pGEM T-Easy (Promega, Madison, WI) and used to transform E. coli DH10B electrocompetent cells. The isolated plasmid was used as the template for inverse PCR to introduce a unique StuI site for the insertion of spectinomycin adenyltransferase, aad9, from Enterococcus faecalis (27). The primers used in this reaction were 5'-GAAGGCCTTGGTGCTAACCGATGTGC-3' (corresponding to bases 220 to 253 of morC) and 5'-GAAGGCCTTCTGTGAAATCATCCGCC-3' (corresponding to bases 164 to 189 of the complementary strand). This reaction resulted in a 30-bp deletion within morC. The purified product was treated with StuI, self ligated, and used to transform E. coli DH10B. The isolated plasmid was incubated with the StuI enzyme, heat inactivated, and ligated with aad9 previously treated with StuI. The ligation mixture was used to transform E. coli DH5pir cells, and transformants were selected on LB agar plates containing 50 µg/ml spectinomycin. The isolated plasmid was used to transform E. coli SM10pir cells for conjugation. The resulting insertional mutant was confirmed by PCR using primer sequences outside of the target DNA. The complete sequence of morC is available at GenBank (accession number DQ085781).

Complementation of morC. The complete morC sequence was amplified by PCR using the primers described above, with engineered XhoI and XbaI restriction enzyme sites. The PCR product was ligated with the shuttle plasmid pKM2 (see description above) with complementary sites and used to transform E. coli DH10B cells that were plated on LB agar containing 20 µg/ml chloramphenicol. Separately, the 165-bp intergenic region was amplified with engineered KpnI and XhoI sites at the 5' and 3' ends of the sequence, respectively. The 165-bp product was ligated with the morC/pKM2 construct treated with KpnI and XhoI. The resulting plasmid was purified and used to transform the morC mutant, and the transformants were plated on TSBYE agar containing 1 µg/ml of chloramphenicol.

Isolation of RNA and RT-PCR. Total RNA was isolated from 1 ml of mid-logarithmic-growth-phase bacteria. Total RNA (20 µg) was treated with DNase (Invitrogen) and purified by chromatography (RNeasy columns) according to the manufacturer's instructions (Qiagen, Hilden, Germany). Reverse transcription (RT) reactions were performed using 1 to 3 µg of purified RNA and 100 pmol of morC primer a (5'-CCAACGACGAATCAACCAGG 3') and ppx primer b (5'-CGCATCAACGAACACTTTACGG-3'), following the manufacturer's instructions with minor modifications (SuperScript III first-strand synthesis system [Invitrogen]). The modifications included a longer denaturation time for the RNA template (6 min at 80°C) and an annealing temperature of 56°C. The monocistronic nature of the transcript was determined by amplification across the putative intergenic junctions, using the specific target primer junction pair for omp67-morC, omp67^F c (5'-CGCGATAAAGACAACAGCAA-3') and morC^R d (5'-CGTTTTCTGTGGCAAGCATA-3'), and the morC-ppx junction pair morC^F e (5'-GCACCATTACACCGCGCTTA-3') and ppx^R f (5'-CGATGATCATGTGAAAGCTG-3').

β-Galactosidase assays. Transcription of pKM2(lacZ) and pKM2(165bp-lacZ) fusions in the wild-type A. actinomycetemcomitans was monitored by β-galactosidase assays of cells cultured to mid-exponential phase and stationary phase, based on the method described by Miller (29). Assays were performed in triplicate, and experiments were performed a minimum of three times.

TEM of whole-mount bacterial preparations. The morphology of the outer membrane of each strain was visualized using TEM of whole-cell mount preparations (40). Briefly, one colony of each strain of A. actinomycetemcomitans was grown overnight in 10 ml of TSBYE. Cultures were diluted 10-fold and grown to mid-logarithmic phase (A₄₉₅, 0.3). Cells (5 x 10⁹) were collected by centrifugation, washed twice in phosphate-buffered saline (PBS; 10 mM sodium phosphate, 150 mM sodium chloride [pH 7.4]), and resuspended in 100 µl of PBS. A 5-µl aliquot of the suspension was applied to carbon-coated 400 mesh copper grids, rinsed gently with PBS, and negatively stained using 2% phosphotungstic acid. Images were collected on a 14 µm 2,048-by-2,048-pixel charge-coupled device camera (TVIPS, Gauting, Germany) at a nominal magnification of 67,000, using a Tecnai 12 (FEI, Portland, OR) electron microscope operating at 100,000 V.

Detection of leukotoxin. Secreted leukotoxin was concentrated from stationary phase cell medium by acetone precipitation and detected using an anti-LtxA rabbit polyclonal antibody (provided by E. T. Lally, University of Pennsylvania). Briefly, overnight cultures were inoculated in TSBYE medium supplemented with the appropriate antibiotics. The following morning, 10 ml of the overnight culture was transferred to an Erlenmeyer flask containing 100 ml of fresh TSBYE medium (1/10 dilution) and incubated until stationary phase. Cultures were centrifuged at 10,000 x g for 30 min at 4°C, and the bacterial cell pellet was discarded. The supernatants were filtered through a 0.22-µm filter to remove any remaining bacteria. The filtered supernatants containing secreted proteins were precipitated with cold acetone (3:1 ratio of acetone:supernatant) for 5 min at –80°C, followed by centrifugation at 10,000 x g for 30 min at 4°C. The pellets were air dried at 4°C for 2 to 3 days to remove any trace of acetone. The dried pellets were resuspended in a minimal amount of 10 mM HEPES buffer and concentrated fourfold using a Pall Filtron 30,000-molecular-weight-cutoff concentrator (Nanosep, Ann Arbor, MI). Protein concentration was determined by absorbance at 280 nm. Equivalent amounts of secreted protein were resolved on 5 to 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels, and proteins were visualized by staining with Coomassie brilliant blue R-250, or proteins were transferred to nitrocellulose membranes. The membranes were probed using a polyclonal anti-LtxA antibody and detected with a horseradish peroxidase-conjugated secondary antibody. The membrane was incubated with ECL substrate (Amersham Life Sciences, Little Chalfont, Buckinghamshire, United Kingdom) and exposed to photographic film (Xar-5; Eastman Kodak, Rochester, NY) for 5 min.

Hydrophobicity assay. Bacterial hydrophobicity was measured as described by Rosenberg (39). Cells were resuspended to an optical density at A₄₀₀ of 0.4. An aliquot of cell suspension (4 ml) was mixed with 0.4 ml of n-hexadecane by vortexing for 60 s and allowed to separate for 15 min. The A₄₀₀ of the aqueous phase was then measured spectrophotometrically. The difference between the optical density of the aqueous phase before and that after it was mixed with n-hexadecane was used to calculate the relative hydrophobicity as [1 – (A₄₀₀ after mixing)/A₄₀₀ before mixing)] x 100.

Isolation of inner and outer membrane proteins. The bacterial strains were inoculated into TSBYE overnight at 37°C in 10% CO₂. The following morning, each strain was subcultured (1:10 dilution) in 200 ml of TSBYE until mid-logarithmic phase. The bacterial cells were harvested and centrifuged at 8,000 x g for 10 min. The pellet was then washed once in PBS (pH 7.4), centrifuged, and then resuspended in 3 ml of 10 mM HEPES (pH 7.4) with 1 mM protease inhibitor. Bacterial membranes were isolated by disruption of the bacteria using a French pressure cell at 18,000 lb/in², followed by differential centrifugation (32). Total membranes were resuspended in 10 mM HEPES (pH 7.4). Sodium N-lauroyl sarcosine was added to a final concentration of 1% and incubated at room temperature for 30 min. The mixture was centrifuged at 100,000 x g for 30 min. The supernatant was removed and stored on ice. The pellet was washed in 10 mM HEPES (pH 7.4) and centrifuged as described above. The final pellet was resuspended in 10 mM HEPES (pH 7.4). Based on bacterial membrane protein solubility in sarcosinate, inner membrane proteins are defined as the proteins that are soluble in the detergent, and outer membrane proteins are insoluble in the detergent.

LC-MS of bacterial proteins. Mid-logarithmic-phase cells were collected by centrifugation and resuspended in 10 mM HEPES buffer (pH 7.4). Bacterial whole-cell lysates were separated on a 5 to 8% polyacrylamide-SDS gels and stained with Coomassie brilliant blue R-250 and destained. Bands of interest were excised and incubated with three progressive washes of 25 mM ammonium bicarbonate (NH₄HCO₃) in 50% acetonitrile, followed by reduction with 200 µl of 10 mM dithiothreitol in 100 µl of 100 mM NH₄HCO₃ for 1 h at 56°C and alkylation with 1 µl of 55 mM iodoacetamide in 200 µl of 100 mM NH₄HCO₃ for 30 min in the dark. The hydrated gel slice was covered with 30 µl of trypsin solution (10 ng/µl) and incubated overnight at 37°C. A small amount of 10% acetic acid was then added to stop the digestion. The sample was then centrifuged at 2,800 x g, and the supernatant was saved for analysis by electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS). A fused silica microcapillary LC column (12-cm by 75-µm inside diameter) packed with C₁₈ reversed-phase resin (5-µm-particle size; 20-nm-pore size; Magic C18AQ; Michrom Bioresources Inc., Auburn, CA) was used with nanospray ESI. The nanospray ESI was fitted onto a linear quadrupole ion trap mass spectrometer (Thermo Electron, San Jose, CA) that was operated in a collision-induced dissociation mode to obtain both MS and tandem MS (MS/MS) spectra. Samples of tryptic peptides were loaded onto the microcapillary column and separated by applying a gradient of 5 to 80% acetonitrile in 0.1% formic acid at a flow rate of 250 nl/min for 55 min. Mass spectrometry data were acquired in a data-dependent acquisition mode, in which a full MS scan was followed by 10 MS/MS spectra of the most abundant ion.

After an LC-MS run was completed and spectra obtained, the spectra were searched against the oral pathogen sequence databases (www.oralgen.lanl.gov/) using SEQUEST (Bioworks software, version 3.3; Thermo Electron, San Jose, CA). The search parameters permitted a ±1.0-Da peptide MS tolerance and a ±1.0-Da MS/MS tolerance. Oxidation of methionine and carboxymethylation of cysteines were allowed as variable modifications. Up to two missed tryptic cleavages of peptides were considered. The cutoffs for SEQUEST assignment were cross-correlation (Xcorr) scores greater than 1.9, 2.5, and 3.8 for peptide charge states of 1, 2, and 3, respectively, and a delta-correlation (Cn) score of >0.1. Mass spectroscopy was performed at the Vermont Genetics Network proteomics facility located at the University of Vermont.

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The MorC protein is conserved in gram-negative bacteria. A transposon mutant of A. actinomycetemcomitans was initially identified in our laboratory, and the integration site of the transposon was mapped within a predicted 3.8-kbp ORF. A site-directed insertional mutant was generated and used in all subsequent experiments.

The morC gene encodes a 1,292-amino-acid protein with a predicted molecular weight of 140,867. The MorC protein sequence is present in a large number of gram-negative bacteria, and proteins with homologous sequences are present in pathogenic as well as nonpathogenic bacteria. The highest homologies are found in Mannheimia succiniciproducens (71%), Pasteurella multocida (68%), and H. influenzae (67%). The protein is also predicted to be present in the genomes of E. coli (55%), Salmonella enterica (55%), Salmonella enterica serovar Typhimurium (55%), and Yersinia pestis (54%).

The protein sequence (amino acids 1 to 1292) is considered to belong to the COG2911 family of proteins, which are classified as uncharacterized proteins conserved in bacteria. The carboxyl amino acids 1053 to 1292 of MorC are homologous to those of the DUF490 family of unknown proteins. Although the function of this protein in other bacteria is unknown, the C-terminal domain of MorC contains the greatest percentage of identical and conserved amino acids within the protein (Fig. 1). The conserved nature of this gene and operon structure (see below) argues for an important role for this gene in gram-negative bacteria.

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FIG. 1. Amino acid sequence alignment of the carboxyl termini of the Aggregatibacter actinomycetemcomitans MorC protein and its homologs. Identical residues are highlighted in black, and conserved substitutions are shown in gray. MorC, A. actinomycetemcomitans; PM1808, P. multocida; HI0696, H. influenzae Rd KW20; NTHI0820, H. influenzae 86-028NP; DUF490, E. coli.

A comparison of the protein profile of bacterial whole-cell lysates of the wild-type strain and that of the isogenic mutant by SDS-polyacrylamide gel electrophoresis (PAGE) showed little to no difference in the portion of the gel corresponding to the predicted molecular weight of the protein (Fig. 2A). However, in the lane corresponding to the wild-type strain transformed with the complementing plasmid (Fig. 2, lane 3), there was an increase in the staining intensity of a protein with an apparent molecular weight of 135,000. In addition, a protein at 116 kDa was absent from the morC mutant strain but present in the wild-type and complemented strains (Fig. 2A). The existence of the MorC protein in the bacterial proteome was determined by LC-MS analysis. Identical regions of gels, corresponding to approximate molecular masses between 135 and 145 kDa, from bacterial whole-cell lysates of the wild type, the morC mutant, and the complemented strains (Fig. 2A, arrow) were excised and analyzed. The identities of the five most abundant proteins contained in these gel slices are shown in Fig. 2B. Tryptic peptides corresponding to MorC were present in the wild-type and complemented strains but absent from the isogenic mutant. However, the disruption of morC did not affect the synthesis of the other proteins found in the gel slices of all three strains.

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FIG. 2. Identification of MorC in bacterial whole-cell lysates. (A) Proteins from bacterial whole-cell lysates were resolved by electrophoresis on a 5 to 8% polyacrylamide-SDS gel and stained with Coomassie brilliant blue R-250. Lanes 1, wild-type; 2, morC mutant; 3, complemented strain morC mutant/morC. The arrow indicates the region of the gel excised for analysis by LC-MS. (B) The five most abundant proteins found in the gel slices as identified by LC-MS. MorC, morphogenesis protein; YfhM, conserved hypothetical protein; RpoB, DNA-directed RNA polymerase beta-chain; Rne, RNase E; RpoC, DNA-directed RNA polymerase beta'-chain; AceE, pyruvate dehydrogenase E1 component.

Analysis of the primary amino acid sequence of MorC revealed the presence of a putative signal sequence and a signal peptidase processing site between amino acids A38 and L39. The predicted mature protein has a molecular weight of 136,566, which is similar to the apparent molecular weight determined by SDS-PAGE. Secondary structure predictions for carboxyl terminal amino acids (1235 to 1292) suggest that this sequence has a high probability for forming β-barrel structures, which are important structures for the interaction of proteins with the phospholipid bilayer (51). Together, the data suggested that the protein is localized to the membrane. The protein was localized to the membrane fraction based on cell fractionation studies (Fig. 3). Membrane fragments were solubilized in sarcosine to separate the inner and outer membrane proteins. The outer membrane, inner membrane, and cytoplasmic proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue (Fig. 3). The band corresponding to MorC (Fig. 3, arrow), as confirmed by LC-MS, was dominant in the inner membrane protein preparation, whereas the corresponding protein was minimally detected in the outer membrane and the cytoplasmic protein fractions. The data suggest that MorC is localized to the inner membrane.

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FIG. 3. Membrane localization of MorC. Membrane and cytoplasmic fractions were isolated from the complemented morC mutant/morC strain as described in Materials and Methods. Inner and outer membranes were separated by differential solubilization in sodium N-lauroyl sarcosine. Proteins were resolved by electrophoresis on a 5 to 8% polyacrylamide-SDS gel and stained with Coomassie brilliant blue R-250. OM, outer membrane fraction; IM, inner membrane fraction; Cyto, cytoplasm fraction. The arrow indicates the region of the gel corresponding to MorC as determined by LC-MS.

morC is part of a three-gene operon regulated by a 165-bp promoter region. Examination of the neighboring sequences of morC in the A. actinomycetemcomitans chromosome (Oralgen, Los Alamos, NM) suggested that morC may be part of a three-gene operon (Fig. 4A). An ORF coding for a hypothetical 67-kDa outer membrane protein (omp67) lies upstream of morC. Transcription of omp67 is predicted to terminate 27 bp upstream of the putative start codon of morC. Located two base pairs downstream of the predicted termination codon of morC is an ORF carrying an ortholog of an exopolyphosphatase (ppx) gene, which is associated with the recovery of cells following a stress response to starvation. The minimal number of base pairs separating the putative ORFs and the absence of strong transcriptional termination signals suggested that these genes are cotranscribed.

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FIG. 4. (A) Chromosomal organization of morC and surrounding genes. narP, a nitrate/nitrite response regulator; P, 165-bp intergenic region demonstrated to be a promoter; omp67, a 67-kDa hypothetical outer membrane protein; morC, 141-kDa membrane morphogenesis protein; ppx, an exopolyphosphatase. (B) morC is part of a three-gene operon. The operon structure was determined by RT-PCR, as described in Materials and Methods. Primers were designed to amplify sequences corresponding to the 3' end of the upstream gene and the 5' end of the downstream gene spanning the regions from omp67 to morC and from morC to ppx of the cDNA by PCR. Lanes 1, 140-bp target amplicon generated, spanning the junction between omp67 and morC (primers c and d); 2, 180-bp target amplicon generated, spanning the junction between morC and ppx (primers e and f); 3, negative control for the presence of contaminating DNA (no reverse transcriptase added); 4, positive control for the omp67 and morC transcripts (PCR).

The polycistronic mRNA nature of the operon was determined by RT-PCR. Primers were designed to amplify cDNA sequences corresponding to the 3' end of the upstream gene and the 5' end of the downstream gene spanning the region between omp67 and morC and that between morC and ppx by PCR. As expected for an operon, amplicons of the predicted size were generated for the gene sets (Fig. 4B). Disruption of ppx by insertional mutagenesis did not generate the phenotypes displayed by the morC mutant, confirming that the disruption of morC in the mutant had no polar effects on the downstream ppx gene (data not shown).

Contiguous with and 5' to omp67 is a 165-bp intergenic sequence. We proposed that this sequence may act as a promoter for this gene or the entire operon. To test this hypothesis, a plasmid containing the intergenic sequence with the complete morC sequence was constructed. Transformation of the morC mutant with this construct restored the wild-type phenotypes (see below). A plasmid containing the complete morC sequence without the 165-bp sequence did not complement the mutant. The 165-bp promoter region of the morC operon was also cloned into a promoterless lacZ reporter gene to ascertain if this sequence would promote the expression of a heterologous gene. The level of β-galactosidase activity detected in the A. actinomycetemcomitans strain transformed with pKM2(165bp-lacZ) was 400-fold higher than that of the strain transformed with the promoterless construct (data not shown). Taken together, these data confirm that the 165-bp intergenic region upstream of morC encodes a promoter region.

Directly upstream of the promoter sequence is a gene coding for a NarP homolog. NarP is a DNA-binding response regulator that is controlled by its cognate sensor and histidine kinase, NarQ. The NarQ-NarP two-component regulatory system controls anaerobic respiratory gene transcription in response to nitrate and nitrite levels (36, 46, 47).

Phenotypic characteristics of the morC mutant. The morC mutant strain was observed to grow in macroscopic clumps compared with the parent strain, which grows as small aggregates of bacteria (data not shown). These clumps could not be disrupted by vortexing or sonication. Autoaggregation of the mutant bacteria was reverted to the wild-type phenotype by the introduction of a replicating plasmid containing the intact morC gene with the 165-bp promoter sequence (data not shown). The autoaggregation of the mutant bacteria during planktonic growth suggested a change in the hydrophobicity of the bacterium. Therefore, we used the partitioning of the bacteria into an organic solvent as a relative indication of the overall hydrophobicity of the bacterium. Approximately 70% of the mutant bacteria partitioned into the organic solvent compared with 35% of the wild-type bacteria. The relative hydrophobicity of the complemented strain was comparable to that of the parent strain.

The change in hydrophobicity may be related to changes in the surface of the bacterium. TEM was used to visualize the outer membrane of each strain to determine the possible differences in surface features (Fig. 5). These micrographs clearly demonstrate differences in the outer membranes of the bacteria. The irregular or rugose outer membrane associated with the wild-type bacterium was absent from the morC mutant. The mutant strain displayed a membrane devoid of convolutions. Complementation of the mutant bacteria restored the wild-type membrane morphology. In addition, there was an apparent increase in the number of convolutions observed with the complemented strain, suggesting a direct relationship between the number of MorC molecules in the cell and the relative number of membrane convolutions.

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FIG. 5. Transmission electron micrographs of whole mounts of A. actinomycetemcomitans strains stained with 2% phosphotungstic acid (pH 7). Left panel, wild-type; center panel, morC mutant strain; right panel, complemented strain morC mutant/morC. Arrows indicate EmaA surface structures. The smaller spherical objects in the micrographs represent vesicles secreted by this bacterium. Scale bar = 100 nm.

The protein profile of the bacterial whole-cell lysates and membrane preparations of the wild-type are very similar to those of the morC mutant. However, a protein band with an estimated molecular mass of 116 kDa was absent from the lane corresponding to the bacterial whole-cell lysate of the morC mutant (Fig. 2 and 3). The molecular weight of this protein is similar to that of the leukotoxin secreted by A. actinomycetemcomitans. LC-MS analysis of this protein band identified the protein as leukotoxin. This finding was confirmed by using an anti-LtxA polyclonal antibody (Fig. 6A). Immunoblots of the cytoplasm, membrane, and culture medium from the wild-type, the morC mutant, and the complemented strains revealed the absence of the leukotoxin protein (within the detection capabilities of these reagents) from the mutant strain compared with that of an equal amount of protein from the wild-type and complemented strains.

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FIG. 6. (A) Immunoblot analysis of the cytoplasm, membrane, and culture medium of A. actinomycetemcomitans strains using an anti-LtxA antibody. Equal amounts of protein from the cytoplasm, membrane, and culture medium from each strain were separated by 5 to 8% SDS-PAGE, transferred to nitrocellulose, and probed with the polyclonal anti-LtxA antibody. Culture media were concentrated by acetone precipitation before separation by SDS-PAGE. Lanes 1, wild-type; 2, morC mutant; 3, complemented strain morC mutant/morC. (B) Transcriptional analysis of ltxA by RT-PCR. RT-PCR was performed with the RNA isolated from the different strains, using primers for ltxA and glyA. ltxA, 142-bp amplicon. C, negative control (no reverse transcriptase added); glyA, RNA loading control.

RT-PCR analysis of RNA isolated from the different strains was conducted to determine if MorC was acting directly or indirectly as a possible transcriptional regulatory molecule for leukotoxin synthesis. The results, represented in Fig. 6B, indicate that the ltxA mRNA transcript was present in both the wild-type and the morC mutant strains. Hence, the observed absence of leukotoxin is not due to the lack of an ltxA transcript.

The loss of MorC appears to affect, directly or indirectly, the secretion of leukotoxin in the morC mutant strain. To determine if other proteins present in the culture medium were affected by the absence of MorC or if the secretion defect was specific for leukotoxin, secreted proteins from the three strains were examined by SDS-PAGE. Examination of the silver-stained gel of the concentrated culture medium (Fig. 7) showed similar protein profiles for the three strains, the main difference being the absence of leukotoxin in the mutant.

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FIG. 7. Analysis of proteins secreted from A. actinomycetemcomitans. Bacteria were collected by centrifugation, and the culture media were filtered. The proteins were concentrated by cold acetone precipitation. Equal amounts of protein were separated by 5 to 8% SDS-PAGE and silver stained. Lanes 1, wild-type; 2, morC mutant; 3, complemented strain morC mutant/morC.

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Membrane proteins play an important role in the pathobiology of gram-negative bacteria. The number of membrane proteins characterized in A. actinomycetemcomitans represents only a small portion of the total number of proteins revealed by genomic sequencing. Of the 3,000 ORFs identified in the 2.1-Mb genome of A. actinomycetemcomitans, 30% of the genome (900 ORFs) code for hypothetical proteins or proteins of unknown function (derived from the Oralgen database). In this study, we identified a 141-kDa membrane protein, previously annotated as a hypothetical protein with an unknown function that is linked to membrane morphology and function.

The protein is not exclusive to A. actinomycetemcomitans, since homologous proteins are found in numerous gram-negative bacteria. The MorC homolog in the family Pasteurellaceae contains the greatest amino acid sequence identity and maintains the entire operon structure in the chromosome. Interestingly, H. influenzae displays a rugose membrane similar to that of A. actinomycetemcomitans (2), which suggests that MorC may have a similar function in determining the membrane morphology in this bacterium. In other bacteria, morC is found not in an operon but as an individual gene and has a lower percentage of identical or conserved amino acids. The basis for these differences is not implicit but may be related to the function of this protein in the individual species. The amino acid sequence alignment, however, suggests that the carboxyl terminus of the protein is most likely a fundamental domain required for the function of these proteins.

The morC operon includes a gene (omp67) coding for a conserved hypothetical 67-kDa outer membrane protein with an unknown function and a gene homologous to an exopolyphosphatase (ppx). Ppx hydrolyzes accumulated inorganic polyphosphate [poly(P)], which is found in every living cell and includes linear polymers of orthophosphate with chain lengths of up to 1,000 or more (23). Several biological functions have been suggested for cellular poly(P), including as a reservoir for phosphate and energy, a chelator of metal ions, an alkali buffer, a channel for DNA entry and a regulator for gene expression (22). The operon is suggested to be under the transcriptional control of the global regulator FNR.

FNR is an oxygen-sensitive transcription factor which, in the absence of oxygen, activates transcription of the genes important for anaerobic respiration and represses the genes required for aerobic respiration (44). In silico analysis (41) of the intergenic region revealed the presence of DNA consensus sequences for the binding of several transcription factors, including IHF and FNR. FNR belongs to a family of transcriptional regulators that control gene expression in response to the change between aerobic and anaerobic environments (4). The DNA sequence recognized by FNR consists of an inverted repeat with a consensus sequence of TTGATNNNNATCAA (13). Inspection of the 165-bp promoter sequence revealed two possible binding sites for FNR at positions +85 to 98 and +167 to 180, strongly suggesting that FNR plays a role in regulating the morC operon under different environments.

A homolog of NarP was found immediately upstream of the promoter region. NarP is usually associated with the transcriptional regulation of a battery of genes involved in growth adaptation from an aerobic to an anaerobic environment (9). Although the promoter sequence lacks an NarP consensus binding site, NarP is a known regulator of fnr in Pasteurella (37). A. actinomycetemcomitans is an opportunistic pathogen found in the healthy periodontium, although at very low numbers and in greater numbers during periodontal disease (8). A. actinomycetemcomitans can spread to nonoral tissues, which suggests that the bacteria have the capability to switch from anaerobic to aerobic respiration. Therefore, changes in the redox potential of the environment may modulate the expression of the morC operon.

MorC appears to be present in low abundance in the bacterium, as suggested by the difficulty in visualizing the protein by SDS-PAGE of bacterial whole cell lysates and membrane preparations. The protein is associated with the membrane and appears to be a component of the inner membrane. Changes in membrane characteristics have been reported for A. actinomycetemcomitans when the bacteria were grown under different growth conditions (aerobic versus anaerobic) or with different growth media (agar versus broth) (28). In this study, disruption of morC results in a flat membrane in the mutant as opposed to the convoluted membrane found in the wild-type strain. This phenotype is dependent on morC, as determined by genetic complementation (Fig. 5). The role of morC in membrane biogenesis is unclear; however, it is apparent that this protein is important for and may regulate the presence of convolutions at the surface of the bacterium, either directly or through the interaction with other membrane proteins.

The secretion of leukotoxin is mediated by a type I secretion system, and in order for the toxin to reach the extracellular milieu, the toxin must be transported across two membranes separated by a periplasmic space containing the peptidoglycan cell wall (7, 19). Current understanding of the secretion of leukotoxin in A. actinomycetemcomitans is based on its homology with the E. coli HlyA secretion system. LtxB is suggested to be an integral membrane protein that is a traffic ATPase that provides essential energy for ATP hydrolysis (24). The second component, LtxD, by homology to HlyD, is a member of the membrane fusion protein family that interacts with LtxB (11). HlyD exists as a trimer and interacts with the outer membrane protein TolC (TdeA, the A. actinomycetemcomitans homolog) that forms a tunnel for toxin transport (35). Our data suggest that the absence of MorC perturbs the secretion of leukotoxin in this strain of A. actinomycetemcomitans.

The underlying mechanism controlling the defect in LtxA secretion is not apparent. Figure 6B indicates that ltxA is transcribed, suggesting that the secretion defect is posttranscriptional. In addition, we could not detect any accumulation of LtxA in the cytoplasm of the morC mutant strain by immunoblot analysis, suggesting that if the toxin is unable to translocate across the inner membrane, it may be degraded. This hypothesis is supported by the demonstration that mutations in ltxB and ltxD affect the secretion of leukotoxin without affecting the production of the ltxA transcript (14). In these mutants, the level of LtxA in the cytoplasm is dramatically reduced.

The lack of an effect at the level of the ltxA transcription in the morC mutant and the localization of MorC to the membrane suggest that the defect in LtxA secretion is posttranscriptional under the controlled environmental conditions established in this study. The data suggest a structural role for MorC in the membrane, although we cannot exclude a translational control mechanism for regulation of LtxA or other components of the secretion apparatus. We propose that the presence of MorC in the membrane either stabilizes the interaction between LtxB and LtxD or the interaction between LtxD and TdeA for LtxA secretion. Alternatively, MorC may interact with another unknown protein(s) that stabilizes the membrane for the functional interactions required for LtxA secretion.

The secretome of A. actinomycetemcomitans remains relatively unexplored. We have demonstrated that leukotoxin is not present in the culture medium of the morC mutant, within the detectable limits of the antibodies used in this study. However, at this time, we do not know if the absence of leukotoxin is specific to leukotoxin or other proteins found in the culture medium. Our data indicate that the protein profiles of the bacterial growth medium from the wild type and that of the morC mutants are similar (Fig. 7). Many secreted and outer membrane proteins are translocated across the inner membrane via the Sec translocon (12). A. actinomycetemcomitans EmaA monomers oligomerize to form antennae-like surface appendages required for collagen adhesion (40, 52). These monomers are suggested to be translocated via the Sec translocon before they are integrated into the outer membrane (30). In the morC mutant strain, as with the wild-type strain, the EmaA structures are present at the surface of the bacteria (Fig. 5). These data indicate that other secretion pathways are not affected by the absence of MorC from the strains used in this study.

In summary, we have identified a novel gene that plays an important role in determining the morphology of the outer membrane of A. actinomycetemcomitans and impacts the potential pathogenicity of the bacterium by diminishing transport of leukotoxin across the membrane. The prevalence of this gene among bacterial species suggests a conserved role for this gene product. Although the mechanism that controls the changes in the membrane is currently undefined, our data suggest that MorC is involved in membrane biogenesis and function in a nonfimbriated A. actinomycetemcomitans strain.

 
We thank Edward T. Lally (University of Pennsylvania) for the gift of the anti-LtxA polyclonal antibody. We also thank Travis Bellville and Christopher Lenox for technical assistance and Jacqueline Leung for help in the preparation of the manuscript. We also thank Paula Fives-Taylor for continued support.

This work was supported by Public Health Service grants RO1-DE09760 and RO1-DE013824 awarded to K.P.M.

Publication of this article was made possible by the Vermont Genetics Network through grant P20 RR16462 from the INBRE Program of the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH).

 
* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Rm. 118, Stafford Hall, University of Vermont, Burlington, VT 05405. Phone: (802) 656-0712. Fax: (802) 656-8749. E-mail: Keith.Mintz{at}uvm.edu

Published ahead of print on 11 July 2008.

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Journal of Bacteriology, September 2008, p. 5972-5980, Vol. 190, No. 17
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