Bioavailability of clay-adsorbed dioxin to Sphingomonas wittichii RW1 and its associated genome-wide shifts in gene expression.

Polychlorinated dibenzo-p-dioxins and dibenzofurans are a group of chemically-related pollutants categorically known as dioxins. Some of their chlorinated congeners are among the most hazardous pollutants that persist in the environment. This persistence is due in part to the limited number of bacteria capable of metabolizing these compounds, but also to their limited bioavailability in soil. We used Sphingomonas wittichii strain RW1 (RW1), one of the few strains able to grow on dioxin, to characterize its ability to respond to and degrade clay-bound dioxin. We found that RW1 grew on and completely degraded dibenzo-p-dioxin (DD) intercalated into the smectite clay saponite (SAP). To characterize the effects of DD sorption on RW1 gene expression, we compared transcriptomes of RW1 grown with either free crystalline DD or DD intercalated clay, i.e. sandwiched between the clay interlayers (DDSAP). Free crystalline DD appeared to cause greater expression of toxicity and stress related functions. Genes coding for heat shock proteins, chaperones, as well as genes involved in DNA repair, and efflux were up-regulated during growth on crystalline dioxin compared to growth on intercalated dioxin. In contrast, growth on intercalated dioxin up-regulated genes that might be important in recognition and uptake mechanisms, as well as surface interaction/attachment/biofilm formation such as extracellular solute-binding protein and LuxR. These differences in gene expression may reflect the underlying adaptive mechanisms by which RW1 cells sense and deploy pathways to access dioxin intercalated into clay. These data show that intercalated DD remains bioavailable to the degrading bacterium with implications for bioremediation alternatives.

Sphingomonas wittichii Strain RW1 Genome-Wide Gene Expression Shifts in Response to Dioxins and Clay.

Sphingomonas wittichii strain RW1 (RW1) is one of the few strains that can grow on dibenzo-p-dioxin (DD). We conducted a transcriptomic study of RW1 using RNA-Seq to outline transcriptional responses to DD, dibenzofuran (DF), and the smectite clay mineral saponite with succinate as carbon source. The ability to grow on DD is rare compared to growth on the chemically similar DF even though the same initial dioxygenase may be involved in oxidation of both substrates. Therefore, we hypothesized the reason for this lies beyond catabolic pathways and may concern genes involved in processes for cell-substrate interactions such as substrate recognition, transport, and detoxification. Compared to succinate (SUC) as control carbon source, DF caused over 240 protein-coding genes to be differentially expressed, whereas more than 300 were differentially expressed with DD. Stress response genes were up-regulated in response to both DD and DF. This effect was stronger with DD than DF, suggesting a higher toxicity of DD compared to DF. Both DD and DF caused changes in expression of genes involved in active cross-membrane transport such as TonB-dependent receptor proteins, but the patterns of change differed between the two substrates. Multiple transcription factor genes also displayed expression patterns distinct to DD and DF growth. DD and DF induced the catechol ortho- and the salicylate/gentisate pathways, respectively. Both DD and DF induced the shared down-stream aliphatic intermediate compound pathway. Clay caused category-wide down-regulation of genes for cell motility and chemotaxis, particularly those involved in the synthesis, assembly and functioning of flagella. This is an environmentally important finding because clay is a major component of soil microbes' microenvironment influencing local chemistry and may serve as a geosorbent for toxic pollutants. Similar to clay, DD and DF also affected expression of genes involved in motility and chemotaxis.

Environmentally relevant parameters affecting PCB degradation: carbon source- and growth phase-mitigated effects of the expression of the biphenyl pathway and associated genes in Burkholderia xenovorans LB400.

The principal means for microbial degradation of polychlorinated biphenyls (PCBs) is through the biphenyl pathway. Although molecular aspects of the regulation of the biphenyl pathway have been studied, information on environmental facets such as the effect of alternative carbon sources on (polychlorinated) biphenyl degradation is limited. Here we explore the effect of environmental conditions (e.g., carbon source and growth phase) on the variation in PCB degradation profiles of Burkholderia xenovorans LB400. Genome-wide expression patterns reveal 25 genes commonly up-regulated during PCB degradation and growth on biphenyl to be upregulated in the transition to stationary phase (relative to growth on succinate) including two putative detoxification pathways. Quantitative reverse transcription PCR (Q-RT-PCR) analysis of the upper biphenyl pathway (bphA, bphD, and bphR1), and detoxification genes in response to environmental conditions suggest associated regulation of the biphenyl pathway and chloroacetaldehyde dehydrogenase. The response of genes in the upper biphenyl pathway to carbon source competition and growth phase reveals inhibition of the biphenyl pathway by PCBs. Although PCBs are not degraded during growth on succinate with PCBs, expression data indicate that the biphenyl pathway is induced, suggesting that post-transcriptional regulation or active transport of biphenyl maybe limiting PCB degradation. Identification of the involvement of peripheral pathways in degradation of PCBs is crucial to understanding PCB degradation in an environmental context as bacteria capable of biodegradation experience a range of carbon sources and growth phases.

DNA-stable isotope probing integrated with metagenomics for retrieval of biphenyl dioxygenase genes from polychlorinated biphenyl-contaminated river sediment.

Stable isotope probing with [(13)C]biphenyl was used to explore the genetic properties of indigenous bacteria able to grow on biphenyl in PCB-contaminated River Raisin sediment. A bacterial 16S rRNA gene clone library generated from [(13)C]DNA after a 14-day incubation with [(13)C]biphenyl revealed the dominant organisms to be members of the genera Achromobacter and Pseudomonas. A library built from PCR amplification of genes for aromatic-ring-hydroxylating dioxygenases from the [(13)C]DNA fraction revealed two sequence groups similar to bphA (encoding biphenyl dioxygenase) of Comamonas testosteroni strain B-356 and of Rhodococcus sp. RHA1. A library of 1,568 cosmid clones was produced from the [(13)C]DNA fraction. A 31.8-kb cosmid clone, detected by aromatic dioxygenase primers, contained genes of biphenyl dioxygenase subunits bphAE, while the rest of the clone's sequence was similar to that of an unknown member of the Gammaproteobacteria. A discrepancy in G+C content near the bphAE genes implies their recent acquisition, possibly by horizontal transfer. The biphenyl dioxygenase from the cosmid clone oxidized biphenyl and unsubstituted and para-only-substituted rings of polychlorinated biphenyl (PCB) congeners. A DNA-stable isotope probing-based cosmid library enabled the retrieval of functional genes from an uncultivated organism capable of PCB metabolism and suggest dispersed dioxygenase gene organization in nature.

Coping with polychlorinated biphenyl (PCB) toxicity: Physiological and genome-wide responses of Burkholderia xenovorans LB400 to PCB-mediated stress.

The biodegradation of polychlorinated biphenyls (PCBs) relies on the ability of aerobic microorganisms such as Burkholderia xenovorans sp. LB400 to tolerate two potential modes of toxicity presented by PCB degradation: passive toxicity, as hydrophobic PCBs potentially disrupt membrane and protein function, and degradation-dependent toxicity from intermediates of incomplete degradation. We monitored the physiological characteristics and genome-wide expression patterns of LB400 in response to 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 LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility.

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.

Degradation of aroclor 1242 dechlorination products in sediments by Burkholderia xenovorans LB400(ohb) and Rhodococcus sp. strain RHA1(fcb).

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.

Validation of a more sensitive method for using spotted oligonucleotide DNA microarrays for functional genomics studies on bacterial communities.

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.

Use of both 16S rRNA and engineered functional genes with real-time PCR to quantify an engineered, PCB-degrading Rhodococcus in soil.

A real-time PCR (RTm-PCR) assay using fluorescently labeled oligonucleotides (TaqMan probes) was used to detect and quantify the recombinant Rhodococcus sp. strain RHA1(fcb) in soil. One primer and probe set targeted a hypervariable region of the 16S rRNA gene unique to strain RHA1(fcb) and its phylogenetic relatives, and the other set targeted the recombinant 4-chlorobenzoate (4-CBA) degradation operon (fcb) and was strain-specific. The method had a 6-log dynamic range of detection (10(2)-10(7) cells ml(-1)) for both probes when DNA from pure cultures was used. Although the method was less sensitive in soil, the estimated number of cells in soil by real-time PCR corresponded to the measured number of RHA1(fcb) cells determined by colony-forming units.