Identification and characterization of conjugative transposons CTn86 and CTn9343 in Bacteroides fragilis strains

The related genetic elements flanking the Bacteroides fragilis pathogenicity island (PAI) in enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 and also present in pattern III nontoxigenic B. fragilis (NTBF) NCTC 9343 were defined as putative conjugative transposons (CTns), designated CTn86 and CTn9343, respectively (A. A. Franco, J. Bacteriol. 181:6623-6633, 2004). CTn86 and CTn9343 have the same basic structures except that their encoded transposases have low similarity and CTn9343 lacks the B. fragilis PAI and contains an extra 7-kb region not present in CTn86. In this study, using DNA hybridization and PCR analysis, we characterized the genetic element flanking the PAI in a collection of ETBF strains and the related genetic elements in a collection of NTBF pattern III strains. We found that in all 123 ETBF strains, the PAI is contained in a genetic element similar to CTn86. Of 73 pattern III strains, 26 (36%) present a genetic element similar to CTn9343, 38 (52%) present a genetic element similar to CTn9343 but lack the 7-kb region that is also absent in CTn86 (CTn9343-like element), and 9 (12%) present a genetic element similar to CTn86 but lacking the PAI (CTn86-like element). In addition to containing CTn86, ETBF strains can also contain CTn9343, CTn9343-like, or CTn86-like elements. CTn86, CTn9343, CTn86-like, and CTn9343-like elements were found exclusively in B. fragilis strains and predominantly in division I, cepA-positive strains.     

Identification and Characterization of Conjugative Transposons CTn86 and CTn9343 in Bacteroides fragilis Strains

The related genetic elements flanking the Bacteroides fragilis pathogenicity island (PAI) in enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 and also present in pattern III nontoxigenic B. fragilis (NTBF) NCTC 9343 were defined as putative conjugative transposons (CTns), designated CTn86 and CTn9343, respectively (A. A. Franco, J. Bacteriol. 181:6623-6633, 2004). CTn86 and CTn9343 have the same basic structures except that their encoded transposases have low similarity and CTn9343 lacks the B. fragilis PAI and contains an extra 7-kb region not present in CTn86. In this study, using DNA hybridization and PCR analysis, we characterized the genetic element flanking the PAI in a collection of ETBF strains and the related genetic elements in a collection of NTBF pattern III strains. We found that in all 123 ETBF strains, the PAI is contained in a genetic element similar to CTn86. Of 73 pattern III strains, 26 (36%) present a genetic element similar to CTn9343, 38 (52%) present a genetic element similar to CTn9343 but lack the 7-kb region that is also absent in CTn86 (CTn9343-like element), and 9 (12%) present a genetic element similar to CTn86 but lacking the PAI (CTn86-like element). In addition to containing CTn86, ETBF strains can also contain CTn9343, CTn9343-like, or CTn86-like elements. CTn86, CTn9343, CTn86-like, and CTn9343-like elements were found exclusively in B. fragilis strains and predominantly in division I, cepA-positive strains.     

Molecular Evolution of the Pathogenicity Island of Enterotoxigenic Bacteroides fragilis Strains

Enterotoxigenic Bacteroides fragilis (ETBF) strains, which produce a 20-kDa zinc metalloprotease toxin (BFT), have been associated with diarrheal disease in animals and young children. Studying a collection of ETBF and nontoxigenic B. fragilis (NTBF) strains, we found that bft and a second metalloprotease gene (mpII) are contained in an approximately 6-kb pathogenicity island (termed B. fragilis pathogenicity island or BfPAI) which is present exclusively in all 113 ETBF strains tested (pattern I). Of 191 NTBF strains, 100 (52%) lack both the BfPAI and at least a 12-kb region flanking BfPAI (pattern II), and 82 of 191 NTBF strains (43%) lack the BfPAI but contain the flanking region (pattern III). The nucleotide sequence flanking the left end of the BfPAI revealed a region with the same organization as the mobilization region of the 5-nitroimidazole resistance plasmid pIP417 and the clindamycin resistance plasmid pBFTM10, that is, two mobilization genes (bfmA and bfmB) organized in one operon and a putative origin of transfer (oriT) located in a small, compact region. The region flanking the right end of the BfPAI contains a gene (bfmC) whose predicted protein shares significant identity to the TraD mobilization proteins encoded by plasmids F and R100 from Escherichia coli. Nucleotide sequence analysis of one NTBF pattern III strain (strain I-1345) revealed that bfmB and bfmC are adjacent to each other and separated by a 16-bp GC-rich sequence. Comparison of this sequence with the appropriate sequence of ETBF strain 86-5443-2-2 showed that in this ETBF strain the 16-bp sequence is replaced by the BfPAI. This result defined the BfPAI as being 6,036 bp in length and its precise integration site as being between the bfmB and bfmC stop codons. The G+C content of the BfPAI (35%) and the flanking DNA (47 to 50%) differ greatly from that reported for the B. fragilis chromosome (42%), suggesting that the BfPAI and its flanking region are two distinct genetic elements originating from very different organisms. ETBF strains may have evolved by horizontal transfer of these two genetic elements into a pattern II NTBF strain.     

Transfer region of a bacteroides conjugative transposon, CTnDOT

Bacteroides species harbor large self-transmissible integrated elements called conjugative transposons (CTns). In this paper, we report the first complete sequence analysis of the transfer region of a Bacteroides CTn. The transfer region contained 17 genes (designated orfA-orfQ). Only 2 of the genes shared sequence similarity with genes in the databases and only 1 of these genes was associated with self-transmissible elements.     

Unexpected effect of a Bacteroides conjugative transposon, CTnDOT, on chromosomal gene expression in its bacterial host

Foreign DNA elements such as plasmids and conjugative transposons are constantly entering new bacterial hosts. A possible outcome of such events that has not been considered previously is that regulatory genes carried on some of them might affect the expression of chromosomal genes of the new host. To assess this possibility, we investigated the effect of the Bacteroides conjugative transposon CTnDOT on expression of chromosomal genes in Bacteroides thetaiotaomicron 5482 (BT4001). Most of the upregulated genes were genes of unknown function, but a number of them were associated with a region of the chromosome that contained a putative conjugative transposon, which had been tentatively designated as CTn4-bt. Upregulation of CTn4-bt genes and other chromosomal genes affected by CTnDOT was controlled by two regulatory genes on CTnDOT, rteA and rteB, which encode a two-component regulatory system. Transfer of CTn4-bt was also mediated by rteA and rteB. Three other putative CTns, CTn1-bt, CTn2-bt and CTn3-bt, were mobilized by CTnERL, a CTn closely related to CTnDOT, but genes from CTnERL other than rteA and rteB were also required. Unexpectedly, homologous recombination was required for CTn1-bt, CTn2-bt, CTn3-bt and CTn4-bt to integrate in the recipient. Our results show that regulatory genes on an incoming mobile element can have multiple effects on its new host, including the activation of previously non-transmissible elements.     

A multicomponent system is required for tetracycline-induced excision of Tn4555

Bacteroides spp. are the predominant organisms in the intestinal tract, and they also are important opportunistic pathogens. Antibiotic therapy of Bacteroides infections often is complicated by the prevalence of drug-resistant organisms which acquire resistance genes from a variety of mobile genetic elements including conjugative transposons (CTns) and mobilizable transposons (MTns). Tn4555 is an MTn that encodes beta-lactam resistance, and it is efficiently mobilized by the Bacteroides CTns via a tetracycline (TET)-inducible mechanism. In this study a model system with CTn341 and a Tn4555 minielement was used to examine Tn4555 excision from the chromosome. Using PCR and mobilization assays it was established that excision was stimulated by TET in the presence of CTn341. In order to determine which Tn4555 genes were required for excision, int, tnpA, tnpC, xis, and mobA mutants were examined. The results indicated that int plus two additional genes, tnpC and xis, were required for optimal excision. In addition, there was no requirement for the mobA gene, as had been shown for another MTn, NBU1. The Xis protein sequence is related to a family of plasmid excisionases, but the TnpC gene product did not match anything in the sequence databases. Evidence also was obtained that suggested that Xis is involved in the control of TET-induced excision and in control of mobilization by CTn341. Overall, these results indicate that excision of MTns is a complex process that requires multiple gene products.     

Interstrain Variation of the Polysaccharide B Biosynthesis Locus of Bacteroides fragilis: Characterization of the Region from Strain 638R

The sequence and analysis of the capsular polysaccharide biosynthesis locus, PS B2, of Bacteroides fragilis 638R are described, and the sequence is compared with that of the PS B1 biosynthesis locus of B. fragilis NCTC 9343. Two genes of the region, wcgD and wcgC, are shown by complementation to encode a UDP-N-acetylglucosamine 2-epimerase and a UDP-N-acetylmannosamine dehydrogenase, respectively.     

Integration and excision of a Bacteroides conjugative transposon, CTnDOT

Bacteroides conjugative transposons (CTns) are thought to transfer by first excising themselves from the chromosome to form a nonreplicating circle, which is then transferred by conjugation to a recipient. Earlier studies showed that transfer of most Bacteroides CTns is stimulated by tetracycline, but it was not known which step in transfer is regulated. We have cloned and sequenced both ends of the Bacteroides CTn, CTnDOT, and have used this information to examine excision and integration events. A segment of DNA that contains the joined ends of CTnDOT and an adjacent open reading frame (ORF), intDOT, was necessary and sufficient for integration into the Bacteroides chromosome. Integration of this miniature form of the CTn was not regulated by tetracycline. Excision of CTnDOT and formation of the circular intermediate were detected by PCR, using primers designed from the end sequences. Sequence analysis of the PCR products revealed that excision and integration involve a 5-bp coupling sequence-type mechanism possibly similar to that used by CTn Tn916, a CTn found originally in enterococci. PCR analysis also demonstrated that excision is a tetracycline-regulated step in transfer. The integrated minielement containing intDOT and the ends of CTnDOT did not excise, nor did a larger minielement that also contained an ORF located immediately downstream of intDOT designated orf2. Thus, excision involves other genes besides intDOT and orf2. Both intDOT and orf2 were disrupted by single-crossover insertions. Analysis of the disruption mutants showed that intDOT was essential for excision but orf2 was not. Despite its proximity to the integrase gene, orf2 appears not to be essential for excision.     

Characterization of the Bacteroides fragilis pathogenicity island in human blood culture isolates

Bacteroides fragilis is an important anaerobic pathogen accounting for up to 10% of bacteremias in adult patients. Enterotoxin producing B. fragilis (ETBF) strains have been identified as enteric pathogens of children and adults. In order to further characterize the B. fragilis pathogenicity island (BfPAI) and using PCR assays for bft- and mpII-metalloprotease genes, we determined the frequency of B. fragilis strains with pattern I (containing the BfPAI and its flanking region), pattern II (lacking both the BfPAI and the flanking region), and pattern III (lacking the BfPAI but containing the flanking region) in 63 blood culture isolates. The results were compared to 197 B. fragilis isolates from different clinical sources. We found 19% of blood culture isolates were pattern I (ETBF), 43% were pattern II (NTBF) and 38% were pattern III (NTBF). Comparatively, B. fragilis isolates from other clinical sources were 10% pattern I, 47% pattern II and 43% pattern III. This suggests that the pathogenicity island and the flanking elements may be general virulence factors of B. fragilis.     

Investigation of antigenic expression of Bacteroides fragilis by immunogold labelling and immunoblotting with a monoclonal antibody

Abstract Electron microscopy and immunogold labelling with monoclonal antibody (McAb) Bfl identified an antigen expressed on some in vitro and in vivo grown Bacteroides fragilis NCTC9343 cells.
Immunoprecipitation with this McAb was used to enrich for B. fragilis NCTC9343 cells expressing the Bfl antigen. The McAb Bfl bound to an epitope close to the surface of the outer membrane, but the fibrous capsular network radiating from the bacterial surface was not labelled. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting identified 3 high- M _r bands which resisted heating and protease digestion but were partially sensitive to sodium periodate treatment.     

UDP-glucuronic acid decarboxylases of Bacteroides fragilis and their prevalence in bacteria

Xylose is rarely described as a component of bacterial glycans. UDP-xylose is the nucleotide-activated form necessary for incorporation of xylose into glycans and is synthesized by the decarboxylation of UDP-glucuronic acid (UDP-GlcA). Enzymes with UDP-GlcA decarboxylase activity include those that lead to the formation of UDP-xylose as the end product (Uxs type) and those synthesizing UDP-xylose as an intermediate (ArnA and RsU4kpxs types). In this report, we identify and confirm the activities of two Uxs-type UDP-GlcA decarboxylases of Bacteroides fragilis, designated BfUxs1 and BfUxs2. Bfuxs1 is located in a conserved region of the B. fragilis genome, whereas Bfuxs2 is in the heterogeneous capsular polysaccharide F (PSF) biosynthesis locus. Deletion of either gene separately does not result in the loss of a detectable phenotype, but deletion of both genes abrogates PSF synthesis, strongly suggesting that they are functional paralogs and that the B. fragilis NCTC 9343 PSF repeat unit contains xylose. UDP-GlcA decarboxylases are often annotated incorrectly as NAD-dependent epimerases/dehydratases; therefore, their prevalence in bacteria is underappreciated. Using available structural and mutational data, we devised a sequence pattern to detect bacterial genes encoding UDP-GlcA decarboxylase activity. We identified 826 predicted UDP-GlcA decarboxylase enzymes in diverse bacterial species, with the ArnA and RsU4kpxs types confined largely to proteobacterial species. These data suggest that xylose, or a monosaccharide requiring a UDP-xylose intermediate, is more prevalent in bacterial glycans than previously appreciated. Genes encoding BfUxs1-like enzymes are highly conserved in Bacteroides species, indicating that these abundant intestinal microbes may synthesize a conserved xylose-containing glycan.     

Characterization of restriction endonuclease BfrBI from Bacteroides fragilis strains BE3 and AIP 10006

A new type II restriction endonuclease, named BfrBI, was detected in two strains of Bacteroides fragilis, BE3 and AIP 10006 (NCTC 9343T). The enzyme BfrBI, an isoschizomer of NsiI and AvaIII, recognized the hexanucleotide sequence [5'-ATG decreases CAT-3'], with a cleavage site generating blunt ends.     

A new Bacteroides conjugative transposon that carries an ermB gene

The erythromycin resistance gene ermB has been found in a variety of gram-positive bacteria. This gene has also been found in Bacteroides species but only in six recently isolated strains; thus, the gene seems to have entered this genus only recently. One of the six Bacteroides ermB-containing isolates, WH207, could transfer ermB to Bacteroides thetaiotaomicron strain BT4001 by conjugation. WH207 was identified as a Bacteroides uniformis strain based on the sequence of its 16S rRNA gene. Results of pulsed-field gel electrophoresis experiments demonstrated that the transferring element was normally integrated into the Bacteroides chromosome. The element was estimated from pulsed-field gel data to be about 100 kb in size. Since the element appeared to be a conjugative transposon (CTn), it was designated CTnBST. CTnBST was able to mobilize coresident plasmids and the circular form of the mobilizable transposon NBU1 to Bacteroides and Escherichia coli recipients. A 13-kb segment that contained ermB was cloned and sequenced. Most of the open reading frames in this region had little similarity at the amino acid sequence level to any proteins in the sequence databases, but a 1,723-bp DNA segment that included a 950-bp segment downstream of ermB had a DNA sequence that was virtually identical to that of a segment of DNA found previously in a Clostridium perfringens strain. This finding, together with the finding that ermB is located on a CTn, supports the hypothesis that CTnBST could have entered Bacteroides from some other genus, possibly from gram-positive bacteria. Moreover, this finding supports the hypothesis that many transmissible antibiotic resistance genes in Bacteroides are carried on CTns.     

Bioinformatic evidence and characterization of novel putative large conjugative transposons residing in genomes of genera <Emphasis Type="Italic">Bacteroides</Emphasis> and <Emphasis Type="Italic">Prevotella</Emphasis>

Bioinformatic evidence of the presence of a large conjugative transposon in ruminal bacterium Prevotella bryantii B₁4^T is presented. The described transposon appears to be related to another large conjugative transposon CTnBST, described in Bacteroides uniformis WH207 and to the conjugative transposon CTn3-Bf, which was observed in the genome of Bacteroides fragilis strain YCH46. All three transposons share tra gene regions with high amino acid identity and clearly conserved gene order. Additionally, a second conserved region consisting of hypothetical genes was discovered in all three transposons and named the GG region. This region served as a specific sequence signature and made possible the discovery of several other apparently related hypothetical conjugative transposons in bacteria from the genus Bacteroides. A cluster of genes involved in sugar utilization and metabolism was discovered within the hypothetical CTnB₁4, to a certain extent resembling the polysaccharide utilization loci which were described recently in some Bacteroides strains. This is the first firm report on the presence of a large mobile genetic element in any strain from the genus Prevotella.     

Analysis of a Bacteroides conjugative transposon using a novel "targeted capture" model system

Large conjugative transposons (CTn's) are widespread among Bacteroides spp. and they are responsible for the high rates of Bacteroides tetracycline resistance, which is mediated by the tetQ gene. These elements are self-transmissible and conjugation can be induced up to 1000-fold by the addition of tetracycline to cultures prior to mating. In addition to self-transfer, the Bacteroides CTn's, such as CTn341, are able to mobilize unlinked genetic elements such as plasmids and mobilizable transposons in a tetracycline-inducible manner. To study the molecular properties of these unique elements, a vector was designed to capture CTn's for analysis in heterologous hosts. This plasmid, pFD670, consisted of the low-copy vector pWSK29, the RK2 oriT, an ermF gene, and a tetQ gene fragment containing the N-terminus and promoter. The vector was transferred into Bacteroides recipients containing CTn341 where it integrated into the tetQ gene by homologous recombination. This integrated construct then was transferred back into an Escherichia coli host where it replicated as a plasmid, pFD699, about 56 kb in size. Further analysis showed that pFD699 could be transferred into Bacteroides hosts where it displayed the same tetracycline-inducible properties as the native CTn341. The captured element appeared to utilize a circular intermediate in both transfer and transposition, and integration into the chromosome seemed to be random. Hybridization studies with a range of Bacteroides CTn's encoding tetracycline resistance revealed a great deal of homology between most of the CTn's but there was much variation seen in the restriction patterns of these elements, suggesting great diversity among this group.     

Prevalence of enterotoxigenic Bacteroides fragilis strains (ETBF) in the gut of children with clinical diagnosis of antibiotic associated diarrhoea (AAD)

Prevalence of enterotoxin producing Bacteroides fragilis (ETBF) strains in faecal samples of children with clinical diagnosis antibiotic associated diarrhoea (AAD) was investigated. Out of faecal samples collected from sixty children, thirty C. difficile strains were isolated. Enterotoxigenic B. fragilis (ETBF) strains were cultured from four children what made 6.7% of investigated faecal samples. Out of two samples toxinogenic C. difficile strains [tox A(+) tox B(+)] were cultured together with enterotoxinogenic B. fragilis. From sample of one child C. difficile A negative/B positive strains [tox A(-) tox B(+)] was found together with B. fragilis (ETBF). From faecal sample of one child enterotoxinogenic B. fragilis only was isolated. It was shown that in the gut of children with clinical diagnosis of (AAD) enterotoxinogenic B. fragilis (ETBF) can be present. B. fragilis (ETBF) can be observed in concomitance with toxinogenic C. difficile.     

Possible origins of CTnBST, a conjugative transposon found recently in a human colonic Bacteroides strain

A previous survey of Bacteroides isolates suggested that the ermB gene entered Bacteroides spp. recently. Previously, ermB had been found almost exclusively in gram-positive bacteria. In one Bacteroides strain, ermB was located on 100-kb conjugative transposon (CTn) CTnBST. To assess the possible origin of this CTn, we obtained the full DNA sequence of CTnBST and used this information to investigate its possible origins. Over one-half of CTnBST had high sequence identity to a putative CTn found in the genome of Bacteroides fragilis YCH46. This included the ends of the CTn and genes involved in integration, transfer, and excision. However, the region around the ermB gene contained genes that appeared to originate from gram-positive organisms. In particular, a 7-kb segment containing the ermB gene was 100% identical to an ermB region found in the genome of the gram-positive bacterium Arcanobacterium pyogenes. A screen of Bacteroides isolates whose DNA cross-hybridized with a CTnBST probe revealed that several isolates did not carry the 7-kb region, implying that the acquisition of this region may be more recent than the acquisition of the entire CTnBST element by Bacteroides spp. We have also identified other Bacteroides isolates that carry a slightly modified 7-kb region but have no other traces of CTnBST. Thus, it is possible that this 7-kb region could itself be part of a mobile element that has inserted in a Bacteroides CTn. Our results show that CTnBST is a hybrid element which has acquired a portion of its coding region from gram-positive bacteria but which may originally have come from Bacteroides spp. or some related species.     

Nature, type of linkage, quantity, and absolute configuration of (3-hydroxy) fatty acids in lipopolysaccharides from Bacteroides fragilis NCTC 9343 and related strains.

The main fatty acids present in lipopolysaccharides from Bacteroides fragilis NCTC 9343 were identified as 13-methyl-tetradecanoic, D-3-hydroxypentadecanoic, D-3-hydroxyhexadecanoic, D-3-hydroxy-15-methyl-hexadecanoic, and D-3-hydroxyheptadecanoic acids. Of these, 13-methyl-tetradecanoic acid is exclusively ester bound, and 3-hydroxy-15-methyl-hexadecanoic acid is exclusively involved in amide linkage. The other 3-hydroxy fatty acids are both ester and amide bound. All 3-hydroxy fatty acids possess the D configuration, and the 3-hydroxyl group of ester-linked 3-hydroxy fatty acids is not substituted. Lipopolysaccharides of related Bacteroides species (B. thetaiotaomicron, B. ovatus, B. distasonis, and B. vulgatus) showed a fatty acid spectrum with both similar and distinct features compared to that of B. fragilis lipopolysaccharides.