Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven ''central aromatic'' and twenty ''peripheral aromatic'' pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.genomics ͉ niche adaptation ͉ evolution ͉ biodegradation ͉ redundancy
We designed and successfully implemented the use of in situ-synthesized 45-mer oligonucleotide DNA microarrays (XeoChips) for genome-wide expression profiling of Burkholderia xenovorans LB400, which is among the best aerobic polychlorinated biphenyl degraders known so far. We conducted differential gene expression profiling during exponential growth on succinate, benzoate, and biphenyl as sole carbon sources and investigated the transcriptome of early-stationary-phase cells grown on biphenyl. Based on these experiments, we outlined metabolic pathways and summarized other cellular functions in the organism relevant for biphenyl and benzoate degradation. All genes previously identified as being directly involved in biphenyl degradation were up-regulated when cells were grown on biphenyl compared to expression in succinate-grown cells. For benzoate degradation, however, genes for an aerobic coenzyme A activation pathway were upregulated in biphenyl-grown cells, while the pathway for benzoate degradation via hydroxylation was upregulated in benzoate-grown cells. The early-stationary-phase biphenyl-grown cells showed similar expression of biphenyl pathway genes, but a surprising up-regulation of C 1 metabolic pathway genes was observed. The microarray results were validated by quantitative reverse transcription PCR with a subset of genes of interest. The XeoChips showed a chip-to-chip variation of 13.9%, compared to the 21.6% variation for spotted oligonucleotide microarrays, which is less variation than that typically reported for PCR product microarrays.
Recent microarray experiments suggested that Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB)-degrading bacterium, utilizes up to three apparently redundant benzoate pathways and a C 1 metabolic pathway during biphenyl and benzoate metabolism. To better characterize the roles of these pathways, we performed quantitative proteome profiling of cells grown on succinate, benzoate, or biphenyl and harvested during either mid-logarithmic growth or the transition between the logarithmic and stationary growth phases. The Bph enzymes, catabolizing biphenyl, were ϳ16-fold more abundant in biphenyl-versus succinate-grown cells. Moreover, the upper and lower bph pathways were independently regulated. Expression of each benzoate pathway depended on growth substrate and phase. Proteins specifying catabolism via benzoate dihydroxylation and catechol ortho-cleavage (ben-cat pathway) were approximately an order of magnitude more abundant in benzoate-versus biphenyl-grown cells at the same growth phase. The chromosomal copy of the benzoyl-coenzyme A (CoA) (box C ) pathway was also expressed during growth on biphenyl: Box C proteins were approximately twice as abundant as Ben and Cat proteins under these conditions. By contrast, proteins of the megaplasmid copy of the benzoyl-CoA (box M ) pathway were only detected in transition-phase benzoate-grown cells. Other proteins detected at increased levels in benzoate-and biphenyl-grown cells included general stress response proteins potentially induced by reactive oxygen species formed during aerobic aromatic catabolism. Finally, C 1 metabolic enzymes were present in biphenyl-grown cells during transition phase. This study provides insights into the physiological roles and integration of apparently redundant catabolic pathways in large-genome bacteria and establishes a basis for investigating the PCB-degrading abilities of this strain.
Transcriptomic and proteomic analyses of Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB) degrader, have implicated growth substrate-and phase-dependent expression of three benzoate-catabolizing pathways: a catechol ortho cleavage (ben-cat) pathway and two benzoyl-coenzyme A pathways, encoded by gene clusters on the large chromosome (box C ) and the megaplasmid (box M ). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the ben-cat pathway (the ⌬benABCD::kan mutant), the box C pathway (the ⌬boxAB C ::kan mutant), and both pathways (the ⌬benABCD⌬ boxAB C ::kan mutant). All three mutants oxidized benzoate in resting-cell assays. However, the ⌬benABCD::kan and ⌬ben-ABCD ⌬boxAB C ::kan mutants grew at reduced rates on benzoate and displayed increased lag phases. By contrast, growth on succinate, on 4-hydroxybenzoate, and on biphenyl was unaffected. Microarray and proteomic analyses revealed that cells of the ⌬benABCD::kan mutant growing on benzoate expressed both box pathways. Overall, these results indicate that all three pathways catabolize benzoate. Deletion of benABCD abolished the ability of LB400 to grow using 3-chlorobenzoate. None of the benzoate pathways could degrade 2-or 4-chlorobenzoate, indicating that the pathway redundancy does not directly contribute to LB400's PCB-degrading capacities. Finally, an extensive sigmaE-regulated oxidative stress response not present in wild-type LB400 grown on benzoate was detected in these deletion mutants, supporting our earlier suggestion that the box pathways are preferentially active under reduced oxygen tension. Our data further substantiate the expansive network of tightly interconnected and complexly regulated aromatic degradation pathways in LB400.
Spotted oligonucleotide microarrays potentially offer a wide scope of applications for microbial ecology, especially as they improve the flexibility of design and the specificity of detection compared to PCR product based microarrays. Sensitivity, however, was expected to be problematic, as studies with the more sensitive PCR-based cDNA microarrays indicate that only genes from populations contributing to more than 5% of the community DNA can be detected. We evaluated several parameters to increase sensitivity and then tested applicability for bacterial functional genomics. The optimal parameters were the use of 5'-C6-amino-modified 70-mers printed on CMT-GAPS II substrates at a 40 micro M concentration combined with the use of Tyramide Signal Amplification labelling. This protocol allowed detection of single copy genes belonging to an organism contributing to 1% or more of the total community. To demonstrate its application, we detected the specific aromatic oxygenase genes in a soil community degrading polychlorinated biphenyls (PCBs). This increase in sensitivity is important if oligonucleotide microarrays are to be used for simultaneous monitoring of a range of functions performed by different microorganisms in the environment.
Commercially used polychlorinated biphenyls (PCBs), which are mixtures of more than 60 individual chlorinated biphenyl congeners, are among the most persistent anthropogenic chemical pollutants that threaten natural ecosystems and human health (1). Numerous biphenyl-degrading microorganisms have been isolated and studied, especially for the range of PCB congeners that they degrade. Research has been primarily focused on the biodegradative pathways and the biphenyl dioxygenases responsible for initial PCB oxidation by isolated bacteria (14,27). Knowledge, however, is limited concerning the indigenous microbial populations that metabolize PCBs in the environment. Stable isotope probing (SIP) coupled with metagenomics is one approach to more directly explore which organisms and genetic information may be involved in PCB degradation in PCB-contaminated sites.SIP was developed to separate and concentrate the nucleic acids or fatty acids of microbial populations that metabolize and, hence, assimilate the isotopically labeled substrates into new cell material (4, 5, 28). Recently, the active PCB degraders in a biofilm community on PCB droplets were revealed as Burkholderia species by using DNA-SIP (32). In another DNA-SIP study, 75 different genera that acquired carbon from [ 13 C]biphenyl were found in the PCB-contaminated root zone of a pine tree (22). In addition, that heavy [13 C]DNA fraction revealed new dioxygenase sequences and possible PCB degradation pathways from GeoChip (16) results and from PCRamplified sequences obtained by using primers targeting aromatic-ring-hydroxylating dioxygenase (ARHD) genes (22).A major hurdle in using DNA-SIP for metagenomic analyses (9) is the very small amount of heavy DNA that is produced and, hence, recovered, making library construction difficult. Two studies have shown the feasibility of DNA-SIP for metagenomic analyses for C-1 compound-utilizing communities, but they first increased the amount of the heavy DNA fraction by multiple-displacement amplification (6,10) or enriched the community by growth in sediment slurries. (18).In this study, we used [ 13 C]biphenyl to probe for potential PCB-degrading populations in a PCB-contaminated river sediment and to recover genes potentially involved in the critical first step of PCB degradation, the dioxygenase attack. We found a 31.8-kb cosmid clone that contained a biphenyl dioxygenase sequence (bphAE) and demonstrated its activity on PCBs. MATERIALS AND METHODSSample description and SIP microcosms. Sediment historically contaminated with Aroclor 1248 at concentrations of 0.2 to 4.6 mg kg Ϫ1 was collected in October 2000 from River Raisin at Monroe, MI. The sediment samples were stored at 4°C under river water until use.Five replicate microcosms, each containing 5 g of sediment amended with 10 mg of uniformly labeled [ 13 C]biphenyl (99 atom % 13 C) (Sigma-Aldrich) and 10 ml of K1 minimal medium (34) were placed in 160-ml serum bottles. The sample bottles were sealed with Teflon stoppers and aluminum crimp caps and
the presence of Aroclor 1242 (500 ppm) under low expression of the structural biphenyl pathway (succinate and benzoate growth) and under induction by biphenyl. We found no inhibition of growth or change in fatty acid profile due to PCBs under nondegrading conditions. Moreover, we observed no differential gene expression due to PCBs themselves. However, PCBs did have a slight effect on the biosurface area of LB400 cells and caused slight membrane separation. Upon activation of the biphenyl pathway, we found growth inhibition from PCBs beginning after exponential-phase growth suggestive of the accumulation of toxic compounds. Genome-wide expression profiling revealed 47 differentially expressed genes (0.56% of all genes) under these conditions. The biphenyl and catechol pathways were induced as expected, but the quinoprotein methanol metabolic pathway and a putative chloroacetaldehyde dehydrogenase were also highly expressed. As the latter protein is essential to conversion of toxic metabolites in dichloroethane degradation, it may play a similar role in the degradation of chlorinated aliphatic compounds resulting from PCB degradation.
Burkholderia xenovorans strain LB400, which possesses the biphenyl pathway, was engineered to contain the oxygenolytic ortho dehalogenation (ohb) operon, allowing it to grow on 2-chlorobenzoate and to completely mineralize 2-chlorobiphenyl. A two-stage anaerobic/aerobic biotreatment process for Aroclor 1242-contaminated sediment was simulated, and the degradation activities and genetic stabilities of LB400(ohb) and the previously constructed strain RHA1(fcb), capable of growth on 4-chlorobenzoate, were monitored during the aerobic phase. The population dynamics of both strains were also followed by selective plating and real-time PCR, with comparable results; populations of both recombinants increased in the contaminated sediment. Inoculation at different cell densities (10 4 or 10 6 cells g ؊1 sediment) did not affect the extent of polychlorinated biphenyl (PCB) biodegradation. After 30 days, PCB removal rates for high and low inoculation densities were 57% and 54%, respectively, during the aerobic phase.
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