Christine Florizone

Genetic and Genomic Insights into the Role of Benzoate-Catabolic Pathway Redundancy in Burkholderia xenovorans LB400

ABSTRACT 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 (boxC) and the megaplasmid (boxM). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the ben-cat pathway (the ΔbenABCD::kan mutant), the boxC pathway (the ΔboxABC::kan mutant), and both pathways (the ΔbenABCDΔ boxABC::kan mutant). All three mutants oxidized benzoate in resting-cell assays. However, the ΔbenABCD::kan and ΔbenABCD ΔboxABC::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 ΔbenABCD::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 LB400s 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.

Catabolism of Benzoate and Phthalate in Rhodococcus sp. Strain RHA1: Redundancies and Convergence

Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in Rhodococcus sp. strain RHA1, a polychlorinated biphenyl-degrading actinomycete. Sequence analyses identified genes involved in the catabolism of benzoate (ben) and phthalate (pad), the uptake of phthalate (pat), and two branches of the beta-ketoadipate pathway (catRABC and pcaJIHGBLFR). The regulatory and structural ben genes are separated by genes encoding a cytochrome P450. The pad and pat genes are contained on a catabolic island that is duplicated on plasmids pRHL1 and pRHL2 and includes predicted terephthalate catabolic genes (tpa). Proteomic analyses demonstrated that the beta-ketoadipate pathway is functionally convergent. Specifically, the pad and pat gene products were only detected in phthalate-grown cells. Similarly, the ben and cat gene products were only detected in benzoate-grown cells. However, pca-encoded enzymes were present under both growth conditions. Activity assays for key enzymes confirmed these results. Disruption of pcaL, which encodes a fusion enzyme, abolished growth on phthalate. In contrast, after a lag phase, growth of the mutant on benzoate was similar to that of the wild type. Proteomic analyses revealed 20 proteins in the mutant that were not detected in wild-type cells during growth on benzoate, including a CatD homolog that apparently compensated for loss of PcaL. Analysis of completed bacterial genomes indicates that the convergent beta-ketoadipate pathway and some aspects of its genetic organization are characteristic of rhodococci and related actinomycetes. In contrast, the high redundancy of catabolic pathways and enzymes appears to be unique to RHA1 and may increase its potential to adapt to new carbon sources.

Growth Substrate- and Phase-Specific Expression of Biphenyl, Benzoate, and C1 Metabolic Pathways in Burkholderia xenovorans LB400

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 approximately 16-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.

Roles of Ring-Hydroxylating Dioxygenases in Styrene and Benzene Catabolism in Rhodococcus jostii RHA1

Proteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene in Rhodococcus jostii RHA1, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the delta bphAa and etbAa1::cmrA etbAa2::aphII mutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion of pcaL of the beta-ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded via meta- and ortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latters initial transformation. The stress response may augment RHA1s ability to degrade PCBs and other pollutants that induce similar uncoupling.

Functional Characterization of pGKT2, a 182-Kilobase Plasmid Containing the xplAB Genes, Which Are Involved in the Degradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine by Gordonia sp. Strain KTR9

ABSTRACT Several microorganisms have been isolated that can transform hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a cyclic nitramine explosive. To better characterize the microbial genes that facilitate this transformation, we sequenced and annotated a 182-kb plasmid, pGKT2, from the RDX-degrading strain Gordonia sp. KTR9. This plasmid carries xplA, encoding a protein sharing up to 99% amino acid sequence identity with characterized RDX-degrading cytochromes P450. Other genes that cluster with xplA are predicted to encode a glutamine synthase-XplB fusion protein, a second cytochrome P450, Cyp151C, and XplR, a GntR-type regulator. Rhodococcus jostii RHA1 expressing xplA from KTR9 degraded RDX but did not utilize RDX as a nitrogen source. Moreover, an Escherichia coli strain producing XplA degraded RDX but a strain producing Cyp151C did not. KTR9 strains cured of pGKT2 did not transform RDX. Physiological studies examining the effects of exogenous nitrogen sources on RDX degradation in strain KTR9 revealed that ammonium, nitrite, and nitrate each inhibited RDX degradation by up to 79%. Quantitative real-time PCR analysis of glnA-xplB, xplA, and xplR showed that transcript levels were 3.7-fold higher during growth on RDX than during growth on ammonium and that this upregulation was repressed in the presence of various inorganic nitrogen sources. Overall, the results indicate that RDX degradation by KTR9 is integrated with central nitrogen metabolism and that the uptake of RDX by bacterial cells does not require a dedicated transporter.

Proteomic Analysis of Survival of Rhodococcus jostii RHA1 during Carbon Starvation

ABSTRACT Rhodococcus jostii RHA1, a catabolically diverse soil actinomycete, is highly resistant to long-term nutrient starvation. After 2 years of carbon starvation, 10% of the bacterial culture remained viable. To study the molecular basis of such resistance, we monitored the abundance of about 1,600 cytosolic proteins during a 2-week period of carbon source (benzoate) starvation. Hierarchical cluster analysis elucidated 17 major protein clusters and showed that most changes occurred during transition to stationary phase. We identified 196 proteins. A decrease in benzoate catabolic enzymes correlated with benzoate depletion, as did induction of catabolism of alternative substrates, both endogenous (lipids, carbohydrates, and proteins) and exogenous. Thus, we detected a transient 5-fold abundance increase for phthalate, phthalate ester, biphenyl, and ethyl benzene catabolic enzymes, which coincided with at least 4-fold increases in phthalate and biphenyl catabolic activities. Stationary-phase cells demonstrated an ∼250-fold increase in carbon monoxide dehydrogenase (CODH) concurrent with a 130-fold increase in CODH activity, suggesting a switch to CO or CO2 utilization. We observed two phases of stress response: an initial response occurred during the transition to stationary phase, and a second response occurred after the cells had attained stationary phase. Although SigG synthesis was induced during starvation, a ΔsigG deletion mutant showed only minor changes in cell survival. Stationary-phase cells underwent reductive cell division. The extreme capacity of RHA1 to survive starvation does not appear to involve novel mechanisms; rather, it seems to be due to the coordinated combination of earlier-described mechanisms.

Specificity Fingerprinting of Retaining β-1,4-Glycanases in the Cellulomonas fimi Secretome Using Two Fluorescent Mechanism-Based Probes

Functional proteomics methods are crucial for activity‐ and mechanism‐based investigation of enzymes in biological systems at a post‐translational stage. Glycosidases have central roles in cellular metabolism and its regulation, and their dysfunction can have detrimental effects. These enzymes also play key roles in biomass conversion. A functional profiling methodology was developed for direct, fluorescence‐based, in‐gel analysis of retaining β‐glycosidases. Two spectrally nonoverlapping fluorescent, mechanism‐based probes containing different recognition elements for retaining cellulases and xylanases were prepared. The specificity‐based covalent labelling of retaining glycanases by the two probes was demonstrated in model enzyme mixtures. Using the two probes and mass spectrometry, the secretomes of the biomass‐converting bacterium Cellulomonas fimi, under induction by different polyglycan growth substrates, were analysed to obtain a specificity profile of the C. fimi retaining β‐glycanases. This is a facile strategy for the analysis of glycosidases produced by biomass‐degrading organisms.

Distinct Roles for Two CYP226 Family Cytochromes P450 in Abietane Diterpenoid Catabolism by Burkholderia xenovorans LB400

The 80-kb dit cluster of Burkholderia xenovorans LB400 encodes the catabolism of abietane diterpenoids. This cluster includes ditQ and ditU, predicted to encode cytochromes P450 (P450s) belonging to the poorly characterized CYP226A subfamily. Using proteomics, we identified 16 dit-encoded proteins that were significantly more abundant in LB400 cells grown on dehydroabietic acid (DhA) or abietic acid (AbA) than in succinate-grown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s; DitU was detected only in the AbA-grown cells, whereas DitQ was expressed both during growth on DhA and during growth on AbA. Analyses of insertion mutants showed that ditQ was required for growth on DhA, ditU was required for growth on AbA, and neither gene was required for growth on the central intermediate, 7-oxo-DhA. In cell suspension assays, patterns of substrate removal and metabolite accumulation confirmed the role of DitU in AbA transformation and the role of DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (dissociation constant, 0.98 +/- 0.01 microM) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate the distinct roles of the P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway.

Genomic analysis of the phenylacetyl-CoA pathway in Burkholderia xenovorans LB400

The phenylacetyl-CoA (Paa) catabolic pathway and genome-wide gene expression responses to phenylacetate catabolism were studied in the polychlorinated biphenyl (PCB)-degrading strain Burkholderia xenovorans LB400. Microarray and RT-qPCR analyses identified three non-contiguous chromosomal clusters of genes that are predicted to encode a complete Paa pathway that were induced up to 40-fold during growth of LB400 on phenylacetate: paaGHIJKR, paaANEBDF, and paaC. Comparison of the available genome sequences revealed that this organization is unique to Burkholderiaceae. Parallel proteomic studies identified 7 of the 14 predicted Paa proteins, most of which were detected only in phenylacetate-grown cells, but not in benzoate- or succinate-grown cells. Finally, the transcriptomic and proteomic analyses revealed the induction of at least 7 predicted catabolic pathways of aromatic compounds and some aromatic plant products (phenols, mandelate, biphenyl, C1 compounds, mevalonate, opine, and isoquinoline), as well as an oxidative stress response and a large group of transporters. Most of these genes were not induced during growth on benzoate or biphenyl, suggesting that phenylacetate or a metabolite may act as a signal that triggers multiple physiological processes. Identifying the components of the Paa pathway is important since the pathway appears to contribute to virulence of Burkholderia pathogens.