BACKGROUND
bacteroides fragilis is a gram-negative member of the normal human gut microbiota.
the bacteroidetes constitutes one of the major bacterial phyla in the healthy human gut  <cit> .
however, b. fragilis is also an important opportunistic pathogen, and it is the most frequently isolated anaerobic bacterium in clinical specimens, including abdominal abscesses and bloodstream infections  <cit> .
indeed, while b. fragilis accounts for only  <dig> to 13% of the normal human fecal microbiota, it is responsible for  <dig> to 80% of bacteroides infections  <cit> .
only a few virulence factors have been described for b. fragilis, with the best characterized being the polysaccharide  capsule  <cit>  and a secreted metalloprotease, fragilysin  <cit> .
the capsule, which displays antigenic variation, promotes the formation of abscesses  <cit> , and the reduction of pro-inflammatory responses to b. fragilis  <cit> .
the metalloprotease fragilysin, which has been linked to diarrheal disease  <cit> , has activity against the zonula junctions between cells, and could disrupt tissue integrity  <cit> .
b. fragilis also encodes homologues of c <dig> proteases  <cit> .
these are members of the ca clan of papain-like proteases.
other c <dig> proteases include the important virulence factors streptococcal pyrogenic exotoxin b  from streptococcus pyogenes and interpain a from prevotella intermedia.
speb cleaves a variety of host protein, including immunoglobulin, fibronectin and vitronectin; it also activates il-1β and releases kinin from kininogen  <cit> .
interestingly, both speb and interpain a target and inactivate complement factor c <dig>  <cit> .
one further characterized c <dig> protease is the periodontain from the oral pathogen porphyromonas gingivalis, which cleaves α1-proteinase inhibitor promoting degradation of connective tissue components  <cit> .
for both speb and another well characterized family of cysteine proteases  expressed in staphylococci , the protease genes are found juxtaposed to genes encoding specific protease inhibitors, spi  <cit>   and staphostatin  <cit>  , respectively.
the genomes of bacteroides spp., including b. fragilis, may include plasmids  <cit> , and typically include multiple prophage remnants, pathogenicity islands and both conjugative and non-conjugative transposons   <cit> .
this would facilitate acquisition and dissemination of virulence markers.
indeed, the fragilysin is encoded on a pathogenicity island which has been shown to be mobile  <cit> .
this study centers on the identification and characterization of genes encoding homologues of speb, their genetic linkage with putative inhibitors, and the association of these homologous genes with mobile genetic elements.
RESULTS
the b. fragilis genome harbours four paralogous c <dig> protease genes
a phylogenetic study was undertaken to determine the relatedness of c <dig> proteases in other members of the bacteroidetes phylum .
this identified eight-four c <dig> protease candidates, ranging in size from  <dig> to  <dig> amino acids, in organisms that occupy both human and environmental niches.
the larger of these proteins  group together along with speb and interpain a. these larger proteins have additional c-terminal domains, the role of which is yet to be determined  <cit> .
the bfp proteases group with proteins < <dig> amino acid residues in length .
although acceptable bootstrap values were obtained for nodes separating deeper phylogenetic levels, the bootstrap values for the shallower divisions were low.
this reflects the unstable phylogeny obtained.
however, it is noteworthy that all of the candidate protease sequences had a variation on the two active site motifs indicated in fig  <dig>
of particular interest was the identification of speb homologues in b. fragilis.
analysis of the b. fragilis 638r ftp://ftp.sanger.ac.uk/pub/pathogens/bf/, ych <dig>  <cit>  and nctc <dig>  <cit>  genome sequences identified genes encoding a paralogous family of c <dig> cysteine proteases named bfp <dig> , bfp <dig> , bfp <dig> , bfp <dig>  for b. fragilis protease, encoded by genes bfp1- <dig> respectively.
the locus identifiers for the unpublished 638r genome, followed by the predicted molecular mass of the preproprotein in daltons are given in parenthesis.
bfp <dig> and bfp <dig> were present in all three strains whereas bfp <dig> and bfp <dig> were present only in b. fragilis 638r .
a. based on analysis of genome sequence only, locus identifier bf <dig> for bfp <dig>  and bf <dig> bfp <dig>  all other strains confirmed by pcr.
b. locus identifier bf <dig> for bfp <dig> and bf <dig> for bfp <dig>
tmd1-tmd3: total microbiota dna, from faeces of  <dig> healthy adult subjects.
similarity between the predicted bfp protein sequences and zymogen speb ranges from 33- <dig> %, with similarity between the paralogues themselves higher  .
these low values are not surprising, as it has been established that the overall sequence identity and similarity between the ca clan of papain-like proteases is low  <cit> .
however, the core of the the protease domains of the c <dig> proteases speb  and interpain   <cit>  are similar in structure , even with only  <dig> % sequence identity.
critically, the active site residues  are highly conserved .
it is probable that the bfp genes encode active proteases, and thus, may contribute to the pathogenesis of bacteroides infections in a manner analogous to the role of speb in streptococcal pathogenesis  <cit> .
a numbers in italics are percentage similarity, numbers in bold type are percentage identities.
bacterial cysteine protease genes have been found coupled to genes encoding specific inhibitors, therefore, the regions both up and downstream of the four bfp genes were analyzed for candidate inhibitors.
three open reading frames encoding small proteins  within  <dig> base pairs of the proteases were identified.
these were named bfi1a , bfi1b  and bfi <dig>  .
the encoded proteins showed no significant identity to the propeptides of any known protease, nor to spi.
surprisingly, they had identity to the c <dig> cysteine proteases inhibitors, the staphostatins, ranging from  <dig> - <dig> % identity and  <dig> - <dig> % similarity .
this is in line with identity between staphostatin a and staphostatin b with  <dig> % identity and  <dig> % similarity.
despite low levels of sequence identity, analysis of the predicted secondary structure and the conservation and alignment of a critical glycine residue in these sequences  when compared to staphostatins, suggested that these bfi genes encode specific protease inhibitors.
a numbers in italics are percentage similarity, numbers in bold type are percentage identities.
b scpb and sspc are staphostatin a and staphostatin b respectively.
to determine the likely cellular location of bfp and bfi proteins, the respective sequences were analyzed using lippred  <cit> , lipop  <cit> , signalp  <cit>  and psortb  <cit> .
these analyses suggested that bfi1a has a typical sec pathway leader sequence and is likely to be exported to the periplasm.
bfi1b, bfi <dig>  bfp <dig>  bfp <dig> and bfp <dig> have predicted lipoprotein signal sequences and are likely to be tethered to the outer membrane  <cit> .
whilst bfp <dig> has a lipoprotein leader sequence it is not clear which membrane it is likely to associate with.
it should be noted that maturation of c <dig> zymogens would release the active protease from the anchoring acyl-lipid into the extracellular milieu.
b. fragilis c <dig> proteases genes, bfp <dig> and bfp <dig>  are co-transcribed with those for predicted staphostatin-like inhibitors
for both the streptococcal and staphylococcal systems, the proteases and adjacently encoded inhibitors are co-transcribed  <cit> .
to determine if this transcriptional coupling of protease and inhibitor genes was also present in b. fragilis, rna was isolated from broth grown 638r cells, and analysed by reverse transcriptase pcr, using a series of specific primers for the protease and inhibitor genes .
amplicons were detected for all c <dig> protease structural genes suggesting that all the proteases were transcribed in vitro .
amplification of a  <dig>  kb product  using primers bfi1a_f and bfi1b_r supports the hypothesis that bfp <dig> is co-transcribed on a single mrna with bfi1a and bfi1b.
in addition, amplification of a  <dig>  kb product with primers bfp4_f and bfi4_r suggests that bfp <dig> is transcriptionally coupled to bfi <dig> .
a primer used in e = expression studies, l = linkage studies, i = integration/excision studies
bfp <dig> and bfp <dig> are located on genome insertions
as mentioned above, two of the protease genes  were identified only in strain 638r enabling a comparison with the two other sequenced strains of b. fragilis.
using the artemis comparison tool  <cit> , alignment of the b. fragilis nctc <dig> and b. fragilis 638r genome sequences identified two large insertions in strain 638r associated with the chromosomal locations of bfp <dig> and bfp <dig>  in b. fragilis 638r, bfp <dig> was found on a  <dig>  kb insertion, called bfgi <dig> in this study.
annotation of this insertion revealed an architecture similar to the ctnerl-type conjugative transposons   <cit>  .
although the expected integrase, excisionase and transfer regions were present in bfgi <dig>  mobility of this insertion could not be established for broth grown cultures treated with mitomycin c, tetracycline, or uv treatment .
these treatments are commonly used to initiate excision of ctn elements  <cit> .
bfgi <dig> showed homology to a region in porphyromonas gingivalis atcc  <dig> which has previously been characterized as a ctn  <cit> .
however, this region of atcc  <dig> did not encode a c <dig> protease.
a percentage identity/similarity, the number in parenthesis is the number of amino acids used in the calculations.
b the organism encoding the b. fragilis 638r gene homologue.
caccession number of the highest scoring blast hit with an annotated function.
the bfp <dig> gene was located on a  <dig> kb insertion, called bfgi <dig> in this study.
analysis of this region predicted functional modules, e.g.
dna metabolism, dna packaging, prophage head, tail and lysis proteins, consistent with a bacteriophage genomic structure similar to the siphoviridae family of bacteriophages .
these phage are known to infect bacteria that reside in the gut, and are the most frequently identified phage infecting b. fragilis  <cit> .
similarly to other siphoviridae, bfgi <dig> inserts into the 3' end of the trnaarg gene  <cit> .
the attb site overlaps the trnaarg gene, however integration of bfgi <dig> regenerates a functional trnaarg gene.
bfgi <dig> had homology only with a region of a genome for an unidentified bacteroides sp.
, which included a homologue of bfp <dig>
24
a percentage identity/similarity, the number in parenthesis is the number of amino acids used in the calculations.
b the organism, with associated bacteriophage in parenthesis where applicable.
caccession number of the highest scoring blast hit with an annotated function.
the regions flanking the c <dig> loci in a range of bacteroidetes , b. uniformis , b. ovatus , b. intestinalis , parabacteroides distasonis , porphyromonas gingivalis  and prevotella intermedia  were examined for the presence of markers for mobile genetic elements .
the genbank accession code or jcvi taxon numbers are given in parenthesis.
a cassette of tra genes  was found  <dig>  kb away from prtt in porphyromonas gingivalis strain w <dig>  and again in strain atcc  <dig> tra i to q were found   <dig>  kb away from prtt  in that strain.
however, no complete ctn or phage could be found adjacent to these or any other c <dig> protease gene.
the bfgi <dig> element harbouring the bfp <dig> gene is capable of excision
the putative att sequence for the integration of bfgi <dig> was identified by analysis of the sequence at the boundaries of the inserted dna in strain 638r compared with nctc <dig>  a short  <dig> bp direct repeat sequence was identified flanking the bfgi <dig> insertion .
pcr primers bfgi2_attb_f and bfgi2_attb_r  were used in a pcr reaction to detect the excision of the bfgi <dig> prophage from mitomycin c treated b. fragilis 638r cells.
the resulting  <dig> bp pcr product is consistent with excision of bfgi <dig> from the b. fragilis 638r genome , and reconstruction of an intact trnaarg gene .
sequencing of this pcr product indicated the presence of a single copy of the  <dig> bp repeat region, the proposed attb site for bfgi <dig> .
the mitomycin c-treated cells were also analysed for the presence of the bfgi <dig> circular intermediate.
the primers bfgi2_int_f and bfgi2_int_r  were designed directed outwards across the proposed attl and attr sites.
using these primers, amplification of product should only occur if a circularized form of bfgi <dig> is present in the cell.
the size  sequence of the resulting pcr product confirmed the presence of the circular intermediate .
attempts to show plaque formation using nctc <dig> as an indicator strain did not produce any visible plaques.
this could be due to the phenomenon of limited host range for the bacteriophage.
however, given that bfgi <dig> circular intermediate was detected it is tempting to speculate that it is, or is a derivative of an active phage and such phage could be transmitted to a non-lysogenized strain of b. fragilis, bringing with it a copy of a c <dig> protease.
c <dig> protease genes are present in clinical isolates of b. fragilis and in the healthy human faecal microbiota
in addition to the  <dig> genome strains, a panel of  <dig> clinical isolates of b. fragilis from several human infection sites  were tested by allele-specific pcr for the c <dig> protease genes they harbour.
the results indicated that this panel of strains have a complement of bfp genes more similar to nctc <dig> than to 638r .
the distribution of bfp genes in the clinical isolates is not identical, and none of the  <dig> isolates carried all four bfp genes.
the bfp1- <dig> genes were detected in  <dig>   <dig>   <dig> and  <dig> clinical isolates respectively.
the bfp <dig> gene was not be detected in any of these clinical strains, while bfp <dig> was not detected in two strains .
in contrast, bfp <dig> was encoded by all strains.
in b. fragilis strain ych <dig>  there is a ctnerl-type conjugative transposon  <dig> bp distance from the bfi1a-bfp1-bfi1b gene cluster.
however, this conjugative transposon is not present in either of the other two sequenced b. fragilis genomes, 638r and nctc  <dig>  the bfp <dig> gene was only detected in one clinical isolate , with a concomitant detection of the bfgi <dig> insertion.
in all cases a  <dig> bp fragment was successfully amplified using the primer pair bfgi2_attb_f and bfgi2_attb_r , indicating the presence of a free integration site for bfgi <dig> in all strains.
it should be noted that for nctc  <dig> and 638r, there was a lower product yield and although not quantitative this is likely due to the integration of bfgi <dig> in a sub-population of the cells.
a. analysis of genome sequence only.
presence of bfp genes in the healthy human intestinal microbiota was investigated by pcr analysis performed on total dna extracted from faeces from three adult subjects.
the amplification of the appropriately sized dna fragments indicated that all  <dig> bfp genes characterized in this study were present in all three subjects whose samples were tested .
interestingly, this analysis also indicated the presence of an integrated bfgi <dig> prophage in these faecal samples, as well as free attb sites.
discussion
this study has established the presence of homologues of the streptococcal virulence factor speb in a significant gut microorganism, b. fragilis.
the amplification of bfp1- <dig> specific sequences from mrna samples supports the idea that these protease genes are expressed in vivo and in two cases the protease genes  are coupled to genes encoding proteins resembling staphostatins-like inhibitors.
a role in protection of the bacterial cells from ectopic protease has been mooted for these inhibitors  <cit> .
from sequence analysis, the bacteroides inhibitors are likely to localize to the periplasm and cell membranes, which could be an additional mechanism to protect the bacterial cell from proteolytic damage, similar to roles suggested for spi and the staphostatins.
the presence of two bfp protease genes on mobile genetic elements parallels some of the paradigms for the acquisition of virulence determinants by other microorganisms.
for example the panton-valentine leukocidin of staphylococcus aureus  <cit> , spec of s. pyogenes  <cit> , diphtheria toxin of corynebacterium diphtheria  <cit>  and cholera toxin of vibrio cholera  <cit>  as well as the fragilysin of b. fragilis  <cit>  are all encoded by mobile genetic elements.
although the latter case has yet to be conclusively established, the other examples cited, and many others in the literature, illustrate an augmentation of virulence in the recipient organism.
thus, the acquisition of additional copies of a protease with homology to speb by lateral gene transfer may increase the ability of b. fragilis to cause disease.
however, establishing the mechanism of transfer of these protease genes and the role of the encoded proteases in b. fragilis opportunistic infections will require further studies.
CONCLUSIONS
the phylum bacteroidetes constitutes a major proportion of the healthy human intestinal microbiota.
variations in the bacteroidetes proportion are linked to disease, and selected species are significant causes of human infectious disease.
alterations in the composition or function of the bacteroidetes component of the intestinal microbiota might plausibly be involved in diseases involving immune dysregulation, including inflammatory bowel disease, or irritable bowel syndrome.
bacterial proteases are particularly relevant in this context, because they might be involved in the perturbed regulation of host matrix metalloproteases, which is a feature of ibd  <cit> .
thus the linkage of c <dig> proteases genes to mobile genetic elements in b. fragilis, and the demonstrated presence of these coding sequences in the healthy adult gut microbiota, is potentially significant.
experiments to investigate the expression and function of these genes in vivo are in progress.

 bacteroides fragilis is a gram-negative member of the normal human gut microbiota. the bacteroidetes constitutes one of the major bacterial phyla in the healthy human gut. however, b. fragilis is also an important opportunistic pathogen, and it is the most frequently isolated anaerobic bacterium in clinical specimens, including abdominal abscesses and bloodstream infections. indeed, while b. fragilis accounts for only to 13% of the normal human fecal microbiota, it is responsible for to 80% of bacteroides infections. only a few virulence factors have been described for b. fragilis, with the best characterized being the polysaccharide capsule and a secreted metalloprotease, fragilysin. the capsule, which displays antigenic variation, promotes the formation of abscesses, and the reduction of pro-inflammatory responses to b. fragilis. the metalloprotease fragilysin, which has been linked to diarrheal disease, has activity against the zonula junctions between cells, and could disrupt tissue integrity. b. fragilis also encodes homologues of c proteases. these are members of the ca clan of papain-like proteases. other c proteases include the important virulence factors streptococcal pyrogenic exotoxin b from streptococcus pyogenes and interpain a from prevotella intermedia. speb cleaves a variety of host protein, including immunoglobulin, fibronectin and vitronectin; it also activates il-1β and releases kinin from kininogen. interestingly, both speb and interpain a target and inactivate complement factor c. one further characterized c protease is the periodontain from the oral pathogen porphyromonas gingivalis, which cleaves α1-proteinase inhibitor promoting degradation of connective tissue components. for both speb and another well characterized family of cysteine proteases expressed in staphylococci, the protease genes are found juxtaposed to genes encoding specific protease inhibitors, spi and staphostatin, respectively. the genomes of bacteroides spp., including b. fragilis, may include plasmids, and typically include multiple prophage remnants, pathogenicity islands and both conjugative and non-conjugative transposons. this would facilitate acquisition and dissemination of virulence markers. indeed, the fragilysin is encoded on a pathogenicity island which has been shown to be mobile. this study centers on the identification and characterization of genes encoding homologues of speb, their genetic linkage with putative inhibitors, and the association of these homologous genes with mobile genetic elements.  the b. fragilis genome harbours four paralogous c protease genes a phylogenetic study was undertaken to determine the relatedness of c proteases in other members of the bacteroidetes phylum. this identified eight-four c protease candidates, ranging in size from to amino acids, in organisms that occupy both human and environmental niches. the larger of these proteins group together along with speb and interpain a. these larger proteins have additional c-terminal domains, the role of which is yet to be determined. the bfp proteases group with proteins < amino acid residues in length. although acceptable bootstrap values were obtained for nodes separating deeper phylogenetic levels, the bootstrap values for the shallower divisions were low. this reflects the unstable phylogeny obtained. however, it is noteworthy that all of the candidate protease sequences had a variation on the two active site motifs indicated in fig of particular interest was the identification of speb homologues in b. fragilis. analysis of the b. fragilis 638r ftp://ftp.sanger.ac.uk/pub/pathogens/bf/, ych and nctc genome sequences identified genes encoding a paralogous family of c cysteine proteases named bfp, bfp, bfp, bfp for b. fragilis protease, encoded by genes bfp1- respectively. the locus identifiers for the unpublished 638r genome, followed by the predicted molecular mass of the preproprotein in daltons are given in parenthesis. bfp and bfp were present in all three strains whereas bfp and bfp were present only in b. fragilis 638r. a. based on analysis of genome sequence only, locus identifier bf for bfp and bf bfp all other strains confirmed by pcr. b. locus identifier bf for bfp and bf for bfp tmd1-tmd3: total microbiota dna, from faeces of healthy adult subjects. similarity between the predicted bfp protein sequences and zymogen speb ranges from 33- %, with similarity between the paralogues themselves higher. these low values are not surprising, as it has been established that the overall sequence identity and similarity between the ca clan of papain-like proteases is low. however, the core of the the protease domains of the c proteases speb and interpain are similar in structure, even with only % sequence identity. critically, the active site residues are highly conserved. it is probable that the bfp genes encode active proteases, and thus, may contribute to the pathogenesis of bacteroides infections in a manner analogous to the role of speb in streptococcal pathogenesis. a numbers in italics are percentage similarity, numbers in bold type are percentage identities. bacterial cysteine protease genes have been found coupled to genes encoding specific inhibitors, therefore, the regions both up and downstream of the four bfp genes were analyzed for candidate inhibitors. three open reading frames encoding small proteins within base pairs of the proteases were identified. these were named bfi1a, bfi1b and bfi. the encoded proteins showed no significant identity to the propeptides of any known protease, nor to spi. surprisingly, they had identity to the c cysteine proteases inhibitors, the staphostatins, ranging from - % identity and - % similarity. this is in line with identity between staphostatin a and staphostatin b with % identity and % similarity. despite low levels of sequence identity, analysis of the predicted secondary structure and the conservation and alignment of a critical glycine residue in these sequences when compared to staphostatins, suggested that these bfi genes encode specific protease inhibitors. a numbers in italics are percentage similarity, numbers in bold type are percentage identities. b scpb and sspc are staphostatin a and staphostatin b respectively. to determine the likely cellular location of bfp and bfi proteins, the respective sequences were analyzed using lippred, lipop, signalp and psortb. these analyses suggested that bfi1a has a typical sec pathway leader sequence and is likely to be exported to the periplasm. bfi1b, bfi bfp bfp and bfp have predicted lipoprotein signal sequences and are likely to be tethered to the outer membrane. whilst bfp has a lipoprotein leader sequence it is not clear which membrane it is likely to associate with. it should be noted that maturation of c zymogens would release the active protease from the anchoring acyl-lipid into the extracellular milieu. b. fragilis c proteases genes, bfp and bfp are co-transcribed with those for predicted staphostatin-like inhibitors for both the streptococcal and staphylococcal systems, the proteases and adjacently encoded inhibitors are co-transcribed. to determine if this transcriptional coupling of protease and inhibitor genes was also present in b. fragilis, rna was isolated from broth grown 638r cells, and analysed by reverse transcriptase pcr, using a series of specific primers for the protease and inhibitor genes. amplicons were detected for all c protease structural genes suggesting that all the proteases were transcribed in vitro. amplification of a kb product using primers bfi1a_f and bfi1b_r supports the hypothesis that bfp is co-transcribed on a single mrna with bfi1a and bfi1b. in addition, amplification of a kb product with primers bfp4_f and bfi4_r suggests that bfp is transcriptionally coupled to bfi. a primer used in e = expression studies, l = linkage studies, i = integration/excision studies bfp and bfp are located on genome insertions as mentioned above, two of the protease genes were identified only in strain 638r enabling a comparison with the two other sequenced strains of b. fragilis. using the artemis comparison tool, alignment of the b. fragilis nctc and b. fragilis 638r genome sequences identified two large insertions in strain 638r associated with the chromosomal locations of bfp and bfp in b. fragilis 638r, bfp was found on a kb insertion, called bfgi in this study. annotation of this insertion revealed an architecture similar to the ctnerl-type conjugative transposons. although the expected integrase, excisionase and transfer regions were present in bfgi mobility of this insertion could not be established for broth grown cultures treated with mitomycin c, tetracycline, or uv treatment. these treatments are commonly used to initiate excision of ctn elements. bfgi showed homology to a region in porphyromonas gingivalis atcc which has previously been characterized as a ctn. however, this region of atcc did not encode a c protease. a percentage identity/similarity, the number in parenthesis is the number of amino acids used in the calculations. b the organism encoding the b. fragilis 638r gene homologue. caccession number of the highest scoring blast hit with an annotated function. the bfp gene was located on a kb insertion, called bfgi in this study. analysis of this region predicted functional modules, e.g. dna metabolism, dna packaging, prophage head, tail and lysis proteins, consistent with a bacteriophage genomic structure similar to the siphoviridae family of bacteriophages. these phage are known to infect bacteria that reside in the gut, and are the most frequently identified phage infecting b. fragilis. similarly to other siphoviridae, bfgi inserts into the 3' end of the trnaarg gene. the attb site overlaps the trnaarg gene, however integration of bfgi regenerates a functional trnaarg gene. bfgi had homology only with a region of a genome for an unidentified bacteroides sp., which included a homologue of bfp 24 a percentage identity/similarity, the number in parenthesis is the number of amino acids used in the calculations. b the organism, with associated bacteriophage in parenthesis where applicable. caccession number of the highest scoring blast hit with an annotated function. the regions flanking the c loci in a range of bacteroidetes, b. uniformis, b. ovatus, b. intestinalis, parabacteroides distasonis, porphyromonas gingivalis and prevotella intermedia were examined for the presence of markers for mobile genetic elements. the genbank accession code or jcvi taxon numbers are given in parenthesis. a cassette of tra genes was found kb away from prtt in porphyromonas gingivalis strain w and again in strain atcc tra i to q were found kb away from prtt in that strain. however, no complete ctn or phage could be found adjacent to these or any other c protease gene. the bfgi element harbouring the bfp gene is capable of excision the putative att sequence for the integration of bfgi was identified by analysis of the sequence at the boundaries of the inserted dna in strain 638r compared with nctc a short bp direct repeat sequence was identified flanking the bfgi insertion. pcr primers bfgi2_attb_f and bfgi2_attb_r were used in a pcr reaction to detect the excision of the bfgi prophage from mitomycin c treated b. fragilis 638r cells. the resulting bp pcr product is consistent with excision of bfgi from the b. fragilis 638r genome, and reconstruction of an intact trnaarg gene. sequencing of this pcr product indicated the presence of a single copy of the bp repeat region, the proposed attb site for bfgi. the mitomycin c-treated cells were also analysed for the presence of the bfgi circular intermediate. the primers bfgi2_int_f and bfgi2_int_r were designed directed outwards across the proposed attl and attr sites. using these primers, amplification of product should only occur if a circularized form of bfgi is present in the cell. the size sequence of the resulting pcr product confirmed the presence of the circular intermediate. attempts to show plaque formation using nctc as an indicator strain did not produce any visible plaques. this could be due to the phenomenon of limited host range for the bacteriophage. however, given that bfgi circular intermediate was detected it is tempting to speculate that it is, or is a derivative of an active phage and such phage could be transmitted to a non-lysogenized strain of b. fragilis, bringing with it a copy of a c protease. c protease genes are present in clinical isolates of b. fragilis and in the healthy human faecal microbiota in addition to the genome strains, a panel of clinical isolates of b. fragilis from several human infection sites were tested by allele-specific pcr for the c protease genes they harbour. the results indicated that this panel of strains have a complement of bfp genes more similar to nctc than to 638r. the distribution of bfp genes in the clinical isolates is not identical, and none of the isolates carried all four bfp genes. the bfp1- genes were detected in and clinical isolates respectively. the bfp gene was not be detected in any of these clinical strains, while bfp was not detected in two strains. in contrast, bfp was encoded by all strains. in b. fragilis strain ych there is a ctnerl-type conjugative transposon bp distance from the bfi1a-bfp1-bfi1b gene cluster. however, this conjugative transposon is not present in either of the other two sequenced b. fragilis genomes, 638r and nctc the bfp gene was only detected in one clinical isolate, with a concomitant detection of the bfgi insertion. in all cases a bp fragment was successfully amplified using the primer pair bfgi2_attb_f and bfgi2_attb_r, indicating the presence of a free integration site for bfgi in all strains. it should be noted that for nctc and 638r, there was a lower product yield and although not quantitative this is likely due to the integration of bfgi in a sub-population of the cells. a. analysis of genome sequence only. presence of bfp genes in the healthy human intestinal microbiota was investigated by pcr analysis performed on total dna extracted from faeces from three adult subjects. the amplification of the appropriately sized dna fragments indicated that all bfp genes characterized in this study were present in all three subjects whose samples were tested. interestingly, this analysis also indicated the presence of an integrated bfgi prophage in these faecal samples, as well as free attb sites. discussion this study has established the presence of homologues of the streptococcal virulence factor speb in a significant gut microorganism, b. fragilis. the amplification of bfp1- specific sequences from mrna samples supports the idea that these protease genes are expressed in vivo and in two cases the protease genes are coupled to genes encoding proteins resembling staphostatins-like inhibitors. a role in protection of the bacterial cells from ectopic protease has been mooted for these inhibitors. from sequence analysis, the bacteroides inhibitors are likely to localize to the periplasm and cell membranes, which could be an additional mechanism to protect the bacterial cell from proteolytic damage, similar to roles suggested for spi and the staphostatins. the presence of two bfp protease genes on mobile genetic elements parallels some of the paradigms for the acquisition of virulence determinants by other microorganisms. for example the panton-valentine leukocidin of staphylococcus aureus, spec of s. pyogenes, diphtheria toxin of corynebacterium diphtheria and cholera toxin of vibrio cholera as well as the fragilysin of b. fragilis are all encoded by mobile genetic elements. although the latter case has yet to be conclusively established, the other examples cited, and many others in the literature, illustrate an augmentation of virulence in the recipient organism. thus, the acquisition of additional copies of a protease with homology to speb by lateral gene transfer may increase the ability of b. fragilis to cause disease. however, establishing the mechanism of transfer of these protease genes and the role of the encoded proteases in b. fragilis opportunistic infections will require further studies.  the phylum bacteroidetes constitutes a major proportion of the healthy human intestinal microbiota. variations in the bacteroidetes proportion are linked to disease, and selected species are significant causes of human infectious disease. alterations in the composition or function of the bacteroidetes component of the intestinal microbiota might plausibly be involved in diseases involving immune dysregulation, including inflammatory bowel disease, or irritable bowel syndrome. bacterial proteases are particularly relevant in this context, because they might be involved in the perturbed regulation of host matrix metalloproteases, which is a feature of ibd. thus the linkage of c proteases genes to mobile genetic elements in b. fragilis, and the demonstrated presence of these coding sequences in the healthy adult gut microbiota, is potentially significant. experiments to investigate the expression and function of these genes in vivo are in progress. 
bacteroides fragilis is a member of the dominant bacteroidetes phylum of the human intestinal microbiota, and is a significant opportunistic pathogen. the c family of cysteine proteases includes enzymes that contribute to the virulence of bacterial pathogens, such as speb in streptococcus pyogenes. two of these protease genes are unique to b. fragilis 638r and are associated with two large genomic insertions. homologues of the b. fragilis c protease genes were present in a panel of clinical isolates, and in dna extracted from normal human faecal microbiota. four homologues of the streptococcal virulence factor speb were identified in the b. fragilis genome. the presence of homologues of cysteine protease genes in human commensal organisms has not been examined. these four protease genes, two were directly contiguous to open reading frames predicted to encode staphostatin-like inhibitors, with which the protease genes were co-transcribed.
 the c family of cysteine proteases includes enzymes that contribute to the virulence of bacterial pathogens, such as speb in streptococcus pyogenes. the presence of homologues of cysteine protease genes in human commensal organisms has not been examined. bacteroides fragilis is a member of the dominant bacteroidetes phylum of the human intestinal microbiota, and is a significant opportunistic pathogen.  four homologues of the streptococcal virulence factor speb were identified in the b. fragilis genome. these four protease genes, two were directly contiguous to open reading frames predicted to encode staphostatin-like inhibitors, with which the protease genes were co-transcribed. two of these protease genes are unique to b. fragilis 638r and are associated with two large genomic insertions. gene annotation indicated that one of these insertions was a conjugative tn-like element and the other was a prophage-like element, which was shown to be capable of excision. homologues of the b. fragilis c protease genes were present in a panel of clinical isolates, and in dna extracted from normal human faecal microbiota.  this study suggests a mechanism for the evolution and dissemination of an important class of protease in major members of the normal human microbiota. 