The intD mobile genetic element from Dichelobacter nodosus, the causative agent of ovine footrot, is associated with the benign phenotype.

The Gram-negative anaerobic pathogen Dichelobacter nodosus is the principal causative agent of footrot in sheep. The intA, intB and intC elements are mobile genetic elements which integrate into two tRNA genes downstream from csrA (formerly glpA) and pnpA in the D. nodosus chromosome. CsrA homologues act as global repressors of virulence in several bacterial pathogens, as does polynucleotide phosphorylase, the product of pnpA. We have proposed a model in which virulence in D. nodosus is controlled in part by the integration of genetic elements downstream from csrA and pnpA, altering the expression of these putative global regulators of virulence. We describe here a novel integrated genetic element, the intD element, which is 32kb in size and contains an integrase gene, intD, several genes related to genes on other integrated elements of D. nodosus, a type IV secretion system and a putative mobilisation region, suggesting that the intD element has a role in the transfer of other genetic elements. Most of the D. nodosus strains examined which contained the intD gene were benign, with intD integrated next to pnpA, supporting our previous observation that virulent strains of D. nodosus have the intA element next to pnpA.

A bacteroides conjugative transposon, CTnERL, can transfer a portion of itself by conjugation without excising from the chromosome.

CTnERL, a Bacteroides conjugative transposon, transferred DNA by an Hfr-type mechanism during conjugation when it was excision deficient due to an insertion in the integrase gene. Rescue of the conjugative transposon sequences required the recipient to be RecA proficient and to contain an integrated CTnERL. The transfer efficiency was only 10- to 30-fold lower than the normal element transfer efficiency, and the direction of transfer from the oriT gene showed that the integrase end was transferred first and that the transfer genes were transferred last.

Identification of a new ribosomal protection type of tetracycline resistance gene, tet(36), from swine manure pits.

Previously, only one ribosome protection type of a tetracycline resistance gene, tetQ, had been identified in Bacteroides spp. During an investigation of anaerobic bacteria present in swine feces and manure storage pits, a tetracycline-resistant Bacteroides strain was isolated. Subsequent analysis showed that this new Bacteroides strain, Bacteroides sp. strain 139, did not contain tetQ but contained a previously unidentified tetracycline resistance gene. Sequence analysis showed that the tetracycline resistance gene from Bacteroides sp. strain 139 encoded a protein (designated Tet 36) that defines a new class of ribosome protection types of tetracycline resistance. Tet 36 has 60% amino acid identity over 640 aa to TetQ and between 31 and 49% amino acid identity to the nine other ribosome protection types of tetracycline resistance genes. The tet(36) region was not observed to transfer from Bacteroides sp. strain 139 to another Bacteroides sp. under laboratory conditions. Yet tet(36) was found in other genera of bacteria isolated from the same swine manure pits and from swine feces. Phylogenetic analysis of the tet(36)-containing isolates indicated that tet(36) was present not only in the Cytophaga-Flavobacter-Bacteroides group to which Bacteroides sp. strain 139 belongs but also in gram-positive genera and gram-negative proteobacteria, indicating that horizontal transfer of tet(36) is occurring between these divergent phylogenetic groups in the farm environment.

Characterization of genes involved in modulation of conjugal transfer of the Bacteroides conjugative transposon CTnDOT.

In previous studies we identified an 18-kb region of the Bacteroides conjugative transposon CTnDOT that was sufficient for mobilization of coresident plasmids and unlinked integrated elements, as well as self-transfer from Bacteroides to Escherichia coli. When this 18-kb region was cloned on a plasmid (pLYL72), the plasmid transferred itself constitutively in the absence of a coresident conjugative transposon. However, when this plasmid was present in a Bacteroides strain containing a coresident conjugative transposon, conjugal transfer was repressed in the absence of tetracycline and enhanced in the presence of tetracycline. These results suggested that a negative and a positive regulator of conjugal transfer were encoded outside the transfer region of the CTnDOT element. In this work, a minimal and inducible transfer system was constructed and used in transfer and Western blot analyses to identify the differentially regulated genes from CTnDOT responsible for the enhancement and repression of pLYL72 conjugal transfer. Both of these regulatory functions have been localized to a region of the CTnDOT element that is essential for CTn excision. In the presence of tetracycline, the regulatory protein RteC activates the expression of a putative topoisomerase gene, exc, which in turn results in an increase in transfer protein expression and a concomitant 100- to 1,000-fold increase in the frequency of pLYL72 transfer. Our results also suggest that since exc alone cannot result in enhancement of transfer, other factors encoded upstream of exc are also required. Conversely, in the absence of tetracycline, a gene located near the 3' end of exc is responsible for the repression of transfer protein expression and also results in a 100- to 1,000-fold decrease in the frequency of pLYL72 transfer.

Analysis of sequences flanking the vap regions of Dichelobacter nodosus: evidence for multiple integration events, a killer system, and a new genetic element.

Dichelobacter nodosus is the causative agent of ovine footrot. The vap regions of the D. nodosus genome may have arisen by the integration of a genetic element and may have a role in virulence. The virulent D. nodosus strain A198 has multiple copies of the vap regions. In the present study, sequences to the left and right of vap regions 1, 2 and 3 of strain A198 were analysed by Southern blotting and DNa sequencing. The results suggest that vap regions 1 and 2 rose by independent integration events into different tRNA genes. The discovery of a second integrase gene (intB), a gene with similarity to bacteriophage repressor proteins (regA), and a gene similar to an ORF from a conjugative transposon (gepA), suggests that a second genetic element, either a bacteriophage or a conjugative transposon, is integrated next to vap region 3 in the D. nodosus genome. The arrangement of intB and the vap regions in three other virulent strains and one benign strain was determined using using Southern blotting and PCR. One strain, H1215, contained vapE' and not vapE, and thus resembles vap region 3, suggesting that vap region 3 also may have arisen by an independent integration event. In all strains, a copy of intB was found next to the vap regions. The vap regions contain two genes, vapA and toxA, with similarity to the hig genes of the killer plasmid Rts1. Evidence is presented that vapA and toxA have a similar function in D. nodosus.