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1 Institute of Medical Microbiology and Immunology, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark
2 Department of Pathology, Nihon University School of Dentistry, Tokyo, Japan
Correspondence
Knud Poulsen
kp{at}microbiology.au.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers of the iga gene sequences from G. haemolysans strain CCUG 4815 and S. mitis strains SK564 and SK609 determined in this work are AY395568, DQ004563 and DQ004562, respectively.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Kilian et al., 1996). IgA1 is the predominant immunoglobulin subclass in mucosal secretions of the upper respiratory tract and is abundant in serum. Upon cleavage, the resulting Fab fragments retain antigen-binding capacity, whereas Fc-mediated effector functions are abolished. In this way, these bacteria are able to evade the protective functions of this principal mediator of adaptive immunity at mucosal surfaces and may, concomitantly, take advantage of released specific Fab fragments in facilitating adherence and immune disguise (Kilian & Reinholdt, 1987; Kilian et al., 1996; Weiser et al., 2003). The biological significance of these enzymes is indirectly inferred by the facts that nature has developed IgA1-cleaving activity among bacteria colonizing humans along at least five independent evolutionary lineages, and that all three principal bacteria causing meningitis produce an IgA1 protease (Kilian et al., 1996; Kosowska et al., 2002). Metallo-type IgA1 proteases are produced by all members of the species Streptococcus pneumoniae and by some members of the related commensal species Streptococcus sanguinis, Streptococcus oralis, Streptococcus mitis and Streptococcus infantis.
The specificity for human and great ape IgA1 implies that lack of a relevant animal model has precluded definitive proof of the role of the IgA1 proteases in pathogenesis. However, in a mouse model that did not include human IgA1, the pneumococcal IgA1 protease was identified as a virulence factor in colonization as well as in sepsis (Polissi et al., 1998; Chiavolini et al., 2003). This observation suggests that the IgA1 proteases may have features unrelated to cleavage of human IgA1 that contribute to the pathogenesis of infections caused by IgA1-protease-producing bacteria.
Sequence comparisons clearly show that the S. sanguinis, S. pneumoniae and S. oralis IgA1 proteases are closely related (Gilbert et al., 1991; Poulsen et al., 1996, 1998; Wani et al., 1996; reviewed by Kilian & Reinholdt, 2005). However, among S. mitis strains, the IgA1 protease gene iga, in addition to being variably present, shows surprisingly varying degrees of homology to the other streptococcal iga genes, as revealed by Southern blot analysis (Poulsen et al., 1998). No sequence data are available to explain this polymorphism.
Gemella haemolysans is a Gram-positive, facultatively anaerobic coccus frequently found as part of the normal flora of the upper respiratory tract and the oral cavity of humans (Facklam & Elliott, 1995), although it has been implicated in the aetiology of periodontitis (Paster et al., 2001) and is an occasional cause of subacute endocarditis and meningitis (Facklam & Elliott, 1995). G. haemolysans has previously been shown to produce human IgA1-cleaving activity, a feature that is unique to this species within the genus Gemella (Kilian et al., 1983; Lomholt & Kilian, 2000). The activity of the G. haemolysans IgA1 protease is similar to that of Streptococcus species, in that it cleaves at the same Pro–Thr bond in the hinge region of human IgA1 and is inhibited by EDTA, indicating that it is a metalloproteinase (Lomholt & Kilian, 2000). Otherwise, the G. haemolysans IgA1 protease has not been examined and its phylogenetic relationships to other IgA1 proteases are not known. G. haemolysans is not closely related to other bacterial species known to produce IgA1 protease activity, and the evolutionary origin of the IgA1 protease gene in this species is unknown.
To obtain further insight into the phylogeny and function of IgA1 proteases, we sequenced and characterized the iga gene encoding the IgA1 protease in G. haemolysans and in two strains of S. mitis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. We have previously described the iga gene sequence of S. mitis strain SK141 (GenBank accession number Y10285) (Poulsen et al., 1998). However, recent re-evaluation of the taxonomy of the mitis group of Streptococcus reveals that this strain should be assigned to S. oralis (unpublished results). Therefore, we also sequenced the iga gene from two strains that were unambiguously assigned to S. mitis.
All strains were grown on blood agar plates and in Todd–Hewitt broth (Difco) at 37 °C in an atmosphere supplemented with 5 % CO2. Escherichia coli strain TOP10F' (Invitrogen) was grown in 2xyeast tryptone (YT) medium (Sambrook et al., 1989) supplemented with 25 mg tetracycline l–1 and 50 mg ampicillin l–1 when appropriate.
IgA1 protease assay.
For detection of human IgA1-cleaving activity, the sample and PBS, pH 7.4, up to 50 µl, were mixed with 2 µl human myeloma IgA1 (8 mg IgA1 ml–1) and incubated overnight at 37 °C, and characteristic cleavage fragments were detected by reducing SDS-PAGE of 10 µl reaction mixture followed by staining with Coomassie Brilliant Blue. To differentiate between cell-associated and secreted IgA1 protease activity, an overnight culture was centrifuged to separate the fractions and the bacteria were resuspended in an equal volume of PBS, pH 7.4. The protein synthesis was stopped by adding 20 mg chloramphenicol l–1 prior to adding substrate IgA1 and incubation, as described above. Testing serial twofold dilutions of the fractions, combined with visual inspection of the amount of human IgA1 cleaved in the SDS-PAGE analysis, were used to quantify the IgA1 protease activity. For the activity blot experiment, proteins in the supernatant were concentrated 20 times using Centriprep concentrators (Amicon) and separated by 7 % SDS-PAGE under reducing conditions. IgA1 protease activity in the gel upon electrophoresis was detected as described previously (Poulsen et al., 1996). In this assay, human IgA1-cleaving activity in the gel was reflected as loss of staining on the membrane.
PCR and DNA sequencing.
For PCR amplification, we used 
1 ng genomic DNA as template and Ready-To-Go PCR beads (Amersham Biosciences). The temperature profile for the PCR was an initial denaturation at 94 °C for 5 min, followed by 30 cycles at 94 °C for 1 min, 60 °C for 2 min, 72 °C for 2 min, and a final extension at 72 °C for 8 min. When used for DNA sequencing, the resulting amplicons were purified using Wizard Minicolumns (Promega). For sequencing, we used the same primers as for the PCR, as well as primers designed from the previously determined sequence. Sequencing was achieved using the Thermo Sequenase dye terminator cycle sequencing kit (Amersham Bioscience) and the resulting products were analysed with an Applied Biosystems PRISM 377 automated sequencer (PerkinElmer/Applied Biosystems). Based on an alignment of 13 published iga gene sequences from Streptococcus species (Gilbert et al., 1991; Poulsen et al., 1996, 1998; Wani et al., 1996), we designed two degenerate primers, iga-for 5'-GGTAAATCWGGYTWTAACAA-3' and iga-rev 5'-ATGSGTCATYTCATGRGTAT-3', located within conserved regions (corresponding to positions 3949–3968 and 4707–4688, respectively, in the ORF of the S. pneumoniae strain PK81 iga gene, accession number X94909). Inverse PCR was performed as described by Ochman et al. (1988) and Kosowska et al. (2002). Briefly, restriction enzymes selected on the basis of obtained sequences were used to digest the genomic DNA prior to circularization by self-ligation, and outward-pointing oligonucleotide primers, designed from previous sequences, were used for PCR performed as described above. Direct sequencing was performed on both strands of overlapping PCR amplicons (details of the sequencing strategy are available upon request). The presence and sequences of imperfect tandem repeats were confirmed by the size of PCR products using several pairs of primers flanking the repeat region combined with sequencing using repeat-specific primers.
Heterologous expression in E. coli.
The oligonucleotides 5'-ATGGAAGATTTGCAATTATCAGTC-3' and 5'-TTTCCCACCAAATATAGATGTTCT-3' were used in PCR of genomic DNA from G. haemolysans strain CCUG 4815 to amplify a 5541 bp fragment of the ORF encoding the presumed secreted IgA1 protease (see Results and Discussion). Similarly, oligonucleotide 5'-GAAAATAATCTCTTACTGAATTACAATAC-3' combined with 5'-TTTTTTATTCTCAAAAATTGAACTTCTAA-3', and 5'-GGAAAATAATCTTTTACTGAATTATAATACG-3' combined with 5'-TTTTTTATTCTCAAAAATTGAACTTCTAA-3', were used to amplify 5631 and 5754 bp fragments of the iga genes from S. mitis strains SK609 and SK564, respectively. We used the Expand High Fidelity PCR System (Roche Molecular Biochemicals) for the amplification as described by the manufacturer. The PCR products were separated by 1 % agarose gel electrophoresis, visualized by staining with ethidium bromide, and DNA in the band was purified using QIAEXII (Qiagen). Adenosine overhangs were introduced by incubating with Hot Master Mix (Eppendorf) for 8 min at 72 °C. The PCR fragments were cloned using the pBAD-TOPO TA cloning kit and E. coli TOP10F' as host (Invitrogen). The expression vector pBAD-TOPO is intended for cloning PCR products and it adds 14 aa to the N terminus of the recombinant protein and 28 residues, including a V5 epitope and a polyhistidine region, to the C terminus, and expression from the PBAD promoter is controlled by presence of arabinose. A single clone from each of the three transformations with the insert in the right orientation was selected. Expression of the recombinant protein was induced in a fresh broth culture by adding arabinose to a concentration of 0.002 %, as described in the manufacturer's manual. After incubation for 4 h, the bacteria were disrupted by repeated freeze/thawing.
Sequence analyses.
For sequence comparisons and alignments, we used the program PILEUP included in the GCG package (Wisconsin Package Version 10.3; Accelrys).
For searching public sequence databases we used BLAST (available at http://www.ncbi.nlm.nih.gov/blast/). For predicting iga genes, we used the program EasyGene trained on Staphylococcus aureus as host organism (available at http://www.cbs.dtu.dk/services/EasyGene/) (Larsen & Krogh, 2003). For predicting signal peptides, we used the program SignalP (available at http://www.cbs.dtu.dk/services/SignalP) (Nielsen et al., 1997). Transmembrane segments were predicted by the program DAS (available at http://www.sbc.su.se) (Cserzö et al., 1997). Protein domains were predicted by the program Pfam (available at http://pfam.wustl.edu/) (Bateman et al., 2004). For construction of dendrograms based on the neighbour-joining method, we used MEGA3 (available at http://www.megasoftware.net/) (Kumar et al., 2004).
Southern blot analysis.
Whole-cell DNA was purified as described by Hohwy & Kilian (1995). Approximately 10 µg DNA was digested with EcoRI and treated with RNase. DNA fragments were separated by 1 % agarose gel electrophoresis and transferred to and fixed onto a nylon membrane, as described by Sambrook et al. (1989). For detection of iga gene sequences on the blot, we used the Gene Images Alkphos Direct Labelling and Detection System (Amersham Bioscience). The six probes used in the Southern blot experiments were equivalent fragments of the 3' part of the iga genes from G. haemolysans CCUG 4815, S. mitis SK564 and S. pneumoniae TIGR4. The fragments were CCUG 4815-1 and CCUG 4815-2 (positions 4165–4658 and 5577–5906, respectively, in the ORF), SK564-1 and SK564-2 (positions 4354–4844 and 5778–6119, respectively, in the ORF), and TIGR4-1 and TIGR4-2 (positions 4159–4649 and 5577–5915, respectively, in the ORF).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). In the three strains included in the present study, the greater part (>80 %) of the IgA1 protease activity was present in the supernatant of an overnight culture (results not shown). In an activity blot experiment performed upon separation of secreted proteins by reducing SDS-PAGE, the IgA1 protease was present in multiple molecular forms (Fig. 1). In all three strains, the largest protein in the supernatant with human IgA1-cleaving activity had an apparent molecular mass of 220 kDa (Fig. 1). This is very similar to what has previously been observed for other streptococcal IgA1 proteases (Poulsen et al., 1996, 1998). For the S. pneumoniae IgA1 protease, we showed that the smaller molecular forms emerged during concentration and prolonged storage, while fresh cultures only showed the largest forms (unpublished results). Autoproteolysis was presumably the cause of this breakdown of the protein into smaller sizes that retained IgA1 protease activity.
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0.7 kb. Sequencing of the resulting amplicons revealed an ORF in all three bacteria with similarity to the known streptococcal iga genes, suggesting that the amplified fragments represented the homologous iga genes. The sequences of the remaining parts of the presumed iga genes and flanking regions were obtained by ‘walking’ in both directions using inverse PCR. For S. mitis strains SK564 and SK609 and for G. haemolysans strain CCUG 4815, continuous sequences of 7054, 7553 and 7453 nt, respectively, were obtained.
For S. mitis SK564, the sequence revealed an ORF of 6216 nt, starting with ATG, and with the potential of encoding a protein of 2072 aa. The S. mitis SK609 sequence contained an ORF of 6093 nt, also starting with ATG and encoding a protein of 2031 aa. The ORFs of both S. mitis strains showed significant similarity to known streptococcal IgA1 proteases. In both strains, the ORF was preceded by a typical ribosome-binding site (RBS), AAGGAGGA, 3 nt upstream of the proposed start codon. Just upstream of this, –10 and –35 consensus promoter sequences were present. A possible weak transcription terminator with a stem of 6 nt and a loop of 3 nt was present in both sequences, 65 nt downstream of the stop codon. In both sequences the ORF was predicted to represent a real gene by the program EasyGene. In the same orientation and 209 nt upstream of the ORF, both strains harboured an ORF with homology to the C-terminal part of a gene encoding a conserved hypothetical protein, preceding the iga gene in the published genome sequences of S. pneumoniae strains TIGR4 and R6. In S. mitis strain NCTC 12261T, which lacks IgA1 protease activity, the order of genes was as in the pneumococci, except that the iga gene was missing (Fig. 2). In both S. mitis SK564 and SK609, the sequence downstream of the ORF was homologous to that downstream of the iga gene in S. pneumoniae strain PK81 (GenBank accession number X94909), including an ORF with homology to the 5' end of the paralogous zinc metalloproteinase B gene zmpB and iga. A similar gene organization, with the iga gene followed by an IgA1-protease-like gene termed zmpD, has been found in S. pneumoniae strain G54 (Polissi et al., 1998), strain PK81 (Poulsen et al., 1996), and approximately half of clinical isolates of S. pneumoniae (Camilli et al., 2006), whereas this apparent gene duplication is absent in strains TIGR4 and R6. In summary, the gene order in the region around iga was conserved between S. mitis and certain S. pneumoniae strains (Fig. 2).
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). However, the first ATG in the ORF was not preceded by a possible RBS. We propose that the TTG sequence located 51 nt downstream of this ATG codon serves as a start codon in the G. haemolysans iga gene, because of the high degree of similarity with known streptococcal IgA1 proteases at the N terminus, combined with the presence of the putative RBS GAAGGA, 7 nt upstream of the TTG triplet. TTG is often used as a start codon in bacteria (Kozak, 1999). In addition, the program EasyGene predicted this ORF to be a real gene. The ORF has the potential of encoding a protein of 2000 aa. Twenty-six nucleotides downstream, the ORF is followed by a possible transcription terminator, consisting of an inverted repeat structure with a stem of 13 nt and a loop of 13 nt. Putative genes flanking the large ORF, and in the same orientation, were identified at the ends of the sequence obtained. At the upstream end, separated from the proposed iga gene by 263 nt, 621 nt of an ORF encoding a protein with 70 % identity to the C-terminal part of an ABC transporter from Streptococcus agalactiae were present. Five nucleotides downstream of this ORF, an inverted repeat structure, which may constitute a transcription terminator, was identified. At the downstream end, separated from the iga gene by 166 nt, there was an ORF of 397 nt encoding a protein with up to 42 % amino acid identity to the N terminus of a copper homeostasis protein, CutC, found in a variety of prokaryotes and eukaryotes. This ORF was preceded by a typical ribosome-binding sequence GGAGAA, 6 nt upstream of the ATG start codon. Thus, there was no synteny of the area of the genome containing the iga gene in G. haemolysans compared to S. mitis and S. pneumoniae (Fig. 2
).
Heterologous expression of the S. mitis and G. haemolysans iga genes in E. coli
In S. pneumoniae, the iga gene is part of a gene family that includes at least two other paralogous members, zmpB and zmpC. Among these zinc metalloproteinases, only the IgA1 protease cleaves human IgA1 (Oggioni et al., 2003). A higher degree of similarity to iga than zmpB and zmpC suggested that the three sequenced ORFs represented the IgA1 protease in S. mitis SK564, S. mitis SK609 and G. haemolysans CCUG 4815 (see below). In order to verify this, we performed heterologous expression in E. coli. For each of the three bacteria, the part of the ORF encoding the presumed secreted version of the IgA1 protease (see below) was amplified by PCR. The PCR products corresponded to the sequences encoding amino acid positions 154–2072, 154–2031 and 155–2000 in S. mitis SK564, S. mitis SK609 and G. haemolysans CCUG 4815, respectively. The amplicons were cloned into the expression vector pBAD using E. coli TOP10F' as host. The resulting recombinant plasmids with the inserts in the right orientation were termed pBAD-SK564-iga, pBAD-SK609-iga and pBAD-gemella-iga. Expression of the recombinant proteins was induced by adding arabinose. After incubation the bacteria were disrupted by repeated freezing and thawing. Each of the three resulting lysates possessed human IgA1-cleaving activity, demonstrating that the three sequenced ORFs encode the IgA1 protease (Fig. 3).
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). The amino acid sequence HEMTH followed by an E residue 20 aa downstream is typical of bacterial metalloproteinases of the gluzincin type (Vallee & Auld, 1990; Hooper, 1994), and the IgA1 proteases belong to the clan MA(E) family M26 of proteases listed in the MEROPS database (available at http://merops.sanger.ac.uk).
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). Otherwise, all three IgA1 proteases were devoid of cysteine residues. The streptococcal and G. haemolysans signal peptides included a sequence similar to the motif YSIRK-G/S, which is associated with proteins carrying a Gram-positive anchor domain at the C terminus (Tettelin et al., 2001; Bae & Schneewind, 2003).
In the two S. mitis and the G. haemolysans IgA1 proteases, as well as in all other known streptococcal IgA1 proteases, a cell-wall-anchor motif LPNTG (LPYTG in S. mitis strain SK609) (Navarre & Schneewind, 1999; Pallen et al., 2001) is present 53 aa downstream of the proposed signal peptidase site, and followed by two hydrophobic domains with the potential of spanning the cell membrane (Fig. 4). The region between the two putative transmembrane segments is rich in lysines and, according to the positive-inside rule, this region is predicted to face the cytoplasm (von Heijne, 1989). We have previously hypothesized that the streptococcal protease is cleaved at the LPNTG sortase processing site at its N terminus, traverses the cytoplasmic membrane twice, and is released by proteolysis (Poulsen et al., 1998). N-terminal sequence analysis of the S. pneumoniae strain PK81 IgA1 protease showed that it is cleaved at the Ala153–Leu154 peptide bond, just after the last hydrophobic region, presumably by the signal peptidase enzyme (unpublished results). Cleavage of the S. mitis SK564, S. mitis SK609 and G. haemolysans CCUG 4815 IgA1 proteases after the corresponding alanine would result in secreted proteases with molecular masses of 213 415 Da with a pI of 5.01, 209 722 Da with a pI of 5.13, and 205 586 Da with a pI of 5.1, respectively. The proposed processing is in agreement with results from the activity blot experiment, which showed that the largest species of secreted proteins with human IgA1-cleaving activity in each of the three strains had an apparent molecular mass of 220 kDa.
Following the putative cleavage site, the three IgA1 proteases contained 18–23 imperfect repeats, consisting of repeat sequences 16–20 aa in length. Repeat structures are commonly found in surface-exposed and secreted proteins in Gram-positive bacteria, and variation in the number and sequence of repeats may contribute to antigenic variation and thereby to immune evasion (Kehoe, 1994). The repeat structure in the S. sanguinis IgA1 protease has been found to be immunodominant and non-essential for enzyme activity (Gilbert et al., 1993).
In contrast to that of G. haemolysans, all the streptococcal IgA1 proteases harboured a sequence with similarity to the G5 domain, which has been suggested to be a lectin that binds N-acetylglucosamine (Bateman et al., 2005). The sequences contained four of the five conserved glycine residues in the consensus G5 domain (Bateman et al., 2005). This domain was located within the otherwise highly variable repeat structure, suggesting that it has an essential function. In S. pneumoniae, the greater part of the IgA1 protease is non-covalently bound to the cell wall (unpublished results). Further experiments are needed to identify the potential ligand of this domain.
Southern blot analysis
We previously found that the iga gene sequences of S. sanguinis and S. oralis are highly conserved within each of the two species, whereas Southern blot analysis has revealed extensive gene polymorphism among S. pneumoniae and S. mitis strains (Poulsen et al., 1996, 1998). Hakenbeck et al. (2001) have also shown variation in the iga gene among S. pneumoniae strains. Here, we used iga gene probes from G. haemolysans CCUG 4815, S. mitis SK564 and S. pneumoniae TIGR4 in Southern blot analyses to assay for the degree of similarity in iga genes within and between the species G. haemolysans, S. mitis, S. oralis and S. infantis. The two probes from each of the three strains represented equivalent areas flanking the active site (encoding amino acid positions 1388–1552 and 1859–1968 in the G. haemolysans CCUG 4815 IgA1 protease; Fig. 4). An example of the Southern blot analysis is shown in Fig. 5 and the results are compiled in Table 1. The iga genes, including the areas covered by the probes, are supposed to be homologous. Lack of hybridization for some of the probe/genomic DNA combinations under the stringency conditions used suggests lack of significant similarity in the DNA sequences. For example, the 490 bp SK564-1 probe sequence showed only 74 % similarity to the G. haemolysans CCUG 4815 iga gene in a 132 bp segment without longer stretches with identity, and the 611 bp SK564-2 probe had no significant similarity to the gemella iga gene. A potential complication in interpreting the hybridization results is the presence in S. pneumoniae and S. mitis genomes, and presumably also in some of the other species, of paralogues of the iga gene with varying degrees of homology (Kilian & Reinholdt, 2005). However, the specificity of the hybridization is supported by the fact that S. mitis strain SK142 (NCTC 12261), which lacks the iga gene but has two paralogous genes (unfinished genome sequence available at http://www.tigr.org), showed no hybridization.
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), three of which produced IgA1 protease activity. None of the S. infantis strains showed hybridization to any of the probes, indicating that the IgA1 protease-positive strains of this species have a more divergent iga gene sequence. Though highly unlikely, it is a theoretical possibility that the S. infantis iga gene may not be evolutionarily related and therefore not homologous to iga genes of the other streptococci and G. haemolysans.
Phylogenetic considerations
The genera Streptococcus and Gemella both belong to the Gram-positive bacteria with a low G+C content. Phylogeny based on 16S rRNA sequences indicates that Gemella is more closely related to the Bacillus–Staphylococcus complex than to streptococci and lactobacilli. IgA1 protease with significant sequence similarity to that of G. haemolysans described here has been exclusively found among Streptococcus species of the mitis group (Kilian et al., 1996; Kilian & Reinholdt, 2005). A likely explanation of the distribution of this finding is horizontal gene transfer, rather than vertical inheritance from a common ancestor combined with subsequent loss of the gene in some species. Deviation in G+C content of a gene compared to the whole genome is often a valuable marker for identifying genes acquired recently by horizontal transfer. The G+C content of the G. haemolysans iga gene was 34 % which is very similar to the 33–35 % reported for the genome (Reyn, 1986). The streptococci displaying IgA1 protease activity have a genome G+C content of 38–46 % and the content for their iga genes varies from 34 to 41 %. However, because Gemella and Streptococcus have similar low G+C content, it is not possible from these data to draw conclusions on the origin of the iga gene. Several other observations support the hypothesis that the iga gene is transferred from Streptococcus to G. haemolysans. The iga gene is apparently ancient within the mitis group of the genus Streptococcus. This is supported by its presence in several closely related species and by the conserved gene order in the iga gene region of S. pneumoniae and S. mitis, which is different from that in G. haemolysans (Fig. 2). An ancestral streptococcus with an iga gene is also supported by the significant sequence diversification resulting in species-specific clusters of iga gene sequences (Fig. 6). In the genus Gemella, only G. haemolysans displays an iga gene, and the Southern blot analysis indicates that the gene is highly conserved within this species. This argues strongly for the transfer of the iga gene from a mitis group Streptococcus to the ancestor of G. haemolysans after its separation from other Gemella species.
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. This was reflected in the Southern blot hybridizations. The CCUG4815-1 probe located upstream of the active site hybridized strongly to S. mitis SK609 genomic DNA, whereas the CCUG4815-2 probe downstream of the active site only showed weak hybridization to SK609 (Fig. 4, Table 1). This pattern of hybridization with the G. haemolysans iga gene probes was detected in five of eight IgA1 protease-positive strains of S. mitis, suggesting that the SK609 iga gene version is common among members of this species.
Concluding remarks
The sequence of the G. haemolysans IgA1 protease was homologous to streptococcal IgA1 proteases that belong to a family of zinc metalloproteinases found in the mitis group of Streptococcus. The genus Gemella is only distantly related to Streptococcus and we argue that the iga gene in G. haemolysans was acquired from Streptococcus by horizontal gene transfer. Structural features conserved among members of this family of IgA1 proteases may delineate sequences essential for enzymic function, including mechanisms of secretion and substrate specificity. Heterologous expression in E. coli allows for purification and manipulation and therefore further studies of these intriguing proteases.
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ACKNOWLEDGEMENTS |
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Received 22 December 2005;
revised 3 March 2006;
accepted 9 March 2006.
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