Horizontal gene transfer (HGT) as a mechanism of disseminating RDX-degrading activity among Actinomycete bacteria.

AIMS: Hexahydro-1,3,5-trinitro-1,3,5,-triazine (RDX) is a cyclic nitramine explosive that is a major component in many high-explosive formulations and has been found as a contaminant of soil and groundwater. The RDX-degrading gene locus xplAB, located on pGKT2 in Gordonia sp. KTR9, is highly conserved among isolates from disparate geographical locations suggesting a horizontal gene transfer (HGT) event. It was our goal to determine whether Gordonia sp. KTR9 is capable of transferring pGKT2 and the associated RDX degradation ability to other bacteria. METHODS AND RESULTS: We demonstrate the successful conjugal transfer of pGKT2 from Gordonia sp. KTR9 to Gordonia polyisoprenivorans, Rhodococcus jostii RHA1 and Nocardia sp. TW2. Through growth and RDX degradation studies, it was demonstrated that pGKT2 conferred to transconjugants the ability to degrade and utilize RDX as a nitrogen source. The inhibitory effect of exogenous inorganic nitrogen sources on RDX degradation in transconjugant strains was found to be strain specific. CONCLUSIONS: Plasmid pGKT2 can be transferred by conjugation, along with the ability to degrade RDX, to related bacteria, providing evidence of at least one mechanism for the dissemination and persistence of xplAB in the environment. SIGNIFICANCE AND IMPACT OF STUDY: These results provide evidence of one mechanism for the environmental dissemination of xplAB and provide a framework for future field relevant bioremediation practices.

Analysis of Borrelia burgdorferi vlsE gene expression and recombination in the tick vector.

Expression and recombination of the antigenic variation vlsE gene of the Lyme disease spirochete Borrelia burgdorferi were analyzed in the tick vector. To assess vlsE expression, Ixodes scapularis nymphs infected with the B. burgdorferi strain B31 were fed on mice for 48 or 96 h or to repletion and then crushed and acetone fixed either immediately thereafter (ticks collected at the two earlier time points) or 4 days after repletion. Unfed nymphs also were examined. At all of the time points investigated, spirochetes were able to bind a rabbit antibody raised against the conserved invariable region 6 of VlsE, as assessed by indirect immunofluorescence, but not preimmune serum from the same rabbit. This same antibody also bound to B31 spirochetes cultivated in vitro. Intensity of fluorescence appeared highest in cultured spirochetes, followed by spirochetes present in unfed ticks. Only a dim fluorescent signal was observed on spirochetes at the 48 and 96 h time points and at day 4 postrepletion. Expression of vlsE in vitro was affected by a rise in pH from 7.0 to 8.0 at 34 degrees C. Hence, vlsE expression appears to be sensitive to environmental cues of the type found in the B. burgdorferi natural history. To assess vlsE recombination, nymphs were capillary fed the B. burgdorferi B31 clonal isolate 5A3. Ticks thus infected were either left to rest for 4 weeks (Group I) or fed to repletion on a mouse (Group II). The contents of each tick from both groups were cultured and 10 B. burgdorferi clones from the spirochetal isolate of each tick were obtained. The vlsE cassettes from several of these clones were amplified by PCR and sequenced. Regardless of whether the isolate was derived from Group I or Group II ticks, no changes were observed in the vlsE sequence. In contrast, vlsE cassettes amplified from B. burgdorferi clones derived from a mouse that was infected with B31-5A3 capillary-fed nymphs showed considerable recombination. It follows that vlsE recombination does not occur in the tick vector.

Transcriptional regulation in spirochetes.

Spirochetes belong to a widely diverse family of bacteria. Several species in this family can cause a variety of illnesses including syphilis and Lyme disease. Despite the fact that the complete genome sequence of two species, Borrelia burgdorferi and Treponema pallidum, have been deciphered, much remains to be understood about spirochetal gene regulation. In this review we focus on the environmental transitions that spirochetes undergo during their life cycles and the mechanisms of transcriptional regulation that might possibly mediate spirochetal adaptations to such changes.

DNA-Binding proteins possibly involved in regulation of the post-logarithmic-phase expression of lipoprotein P35 in Borrelia burgdorferi.

Previously, we have shown that the transcription of p35, a lipoprotein gene of Borrelia burgdorferi, is upregulated or initiated during the post-logarithmic bacterial growth phase in vitro. To identify potential regulatory factors, we examined the formation of DNA-protein complexes by electromobility shift assay, using a 157-bp DNA fragment that spans the p35 promoter region and cell-free extracts of spirochetes harvested from both logarithmic and stationary growth phases. The binding properties of the extracts with the promoter region of the flaB gene, a constitutively expressed, growth-phase-independent gene, were also compared. The results from these experiments demonstrate that B. burgdorferi stationary-phase cell-free extracts have a growth-phase-dependent DNA binding protein that interacts specifically with the p35 promoter region. We show, in addition, that a segment from the 157-bp p35 promoter region which contains both a T-rich stretch and an inverted repeat is able to compete off the stationary-phase-specific complex when the segment is present in molar excess.

Borrelia burgdorferi escape mutants that survive in the presence of antiserum to the OspA vaccine are killed when complement is also present.

As an initial attempt to investigate the possible role of outer surface protein A (OspA) escape mutants of Borrelia burgdorferi in decreasing the efficacy of the OspA vaccine, mutants of the HB19 strain of B. burgdorferi sensu stricto were selected in vitro from an uncloned, low-passage-number isolate. The antiserum used for selection was obtained from rhesus monkeys that had been given a vaccine of the same formulation and dose, and by the same route of administration, as that given to humans in several trials. All of the mutants selected in liquid medium and subsequently cloned twice in solid medium expressed a single abundant protein of 28 to 34 kDa instead of both OspA and OspB. Depending on the mutant, this protein reacted strongly, weakly, or not detectably with the anti-OspA antibody used for selection. Analysis of the ospAB locus of each of four representatives from these three groups of mutants by PCR with oligonucleotide primers that hybridize to flanking regions of the ospAB operon, and of the corresponding phenotype with monoclonal antibodies that bind to the amino or carboxyl terminus of the OspA or OspB polypeptide, indicated that in all cases a deletion within the operon had occurred. Spirochetes from the four mutant strains chosen for further analysis could be killed in antibody-dependent, complement-mediated killing assays with the selecting anti-OspA antibody, despite their resistance to killing with this antibody in the absence of complement. Complement-mediated killing occurred at an antibody concentration higher than that required to kill wild-type spirochetes. If anti-OspA antibody acts only within the tick, where complement is probably ineffective due to tick-derived decomplementing factors, then OspA escape mutants, if infectious, could seriously diminish the efficacy of OspA vaccines. On the other hand, if the killing of B. burgdorferi with anti-OspA antibody also takes place within the human host, then our results indicate that chimeric/deletion escape mutants will be killed as well.

Cell-density-dependent expression of Borrelia burgdorferi lipoproteins in vitro.

Previously, we had identified non-OspA-OspB surface proteins of Borrelia burgdorferi that are targeted by the antibody-dependent complement-mediated killing mechanism. Here we demonstrate by Western blotting that one of these proteins, P35, is upregulated at the onset of stationary phase in vitro. Northern analysis revealed that the upregulation of P35 is at the level of transcription. In addition, the expression of an open reading frame (ORF) located downstream of the p35 gene was found to be regulated in the same fashion as that of P35. This ORF encodes a 7.5-kDa lipoprotein. The transcriptional start sites for both of these genes were determined, to aid in the identification of the putative promoter regions. Additional sequencing of the 5' flanking region of the p35 gene revealed a region of dyad symmetry 52 bp upstream of the transcription start site. Southern analysis demonstrated that the expression of these genes was not due to a cell-density-dependent rearrangement in the genome of B. burgdorferi. These findings provide an in vitro model for studying mechanisms of gene regulation in B. burgdorferi.