Project description:Genes encoding vanillin dehydrogenase (vdh) and vanillate O-demethylase (vanAB) were identified in Rhodococcus jostii RHA1 using gene disruption and enzyme activities. During growth on vanillin or vanillate, vanA was highly upregulated while vdh was not. This study contributes to our understanding of lignin degradation by RHA1 and other actinomycetes.
Project description:Oxysterols from steroid autooxidation have numerous harmful effects, but their biodegradation is poorly understood. Microarrays were used to study mineralization of the most common oxysterol, 7-ketocholesterol (7KC), by Rhodococcus jostii RHA1. Growth on 7KC versus growth on cholesterol resulted in 363 differentially expressed genes, including upregulation of two large gene clusters putatively encoding steroid catabolism. Despite this difference, 7KC degradation required key genes involved in cholesterol degradation, indicating a common catabolic route.
Project description:The Rhodococcus jostii RHA1 gene cluster required for ?-resorcylate (GRA) catabolism was characterized. The cluster includes tsdA, tsdB, tsdC, tsdD, tsdR, tsdT, and tsdX, which encode GRA decarboxylase, resorcinol 4-hydroxylase, hydroxyquinol 1,2-dioxygenase, maleylacetate reductase, an IclR-type regulator, a major facilitator superfamily transporter, and a putative hydrolase, respectively. The tsdA gene conferred GRA decarboxylase activity on Escherichia coli. Purified TsdB oxidized NADH in the presence of resorcinol, suggesting that tsdB encodes a unique NADH-specific single-component resorcinol 4-hydroxylase. Mutations in either tsdA or tsdB resulted in growth deficiency on GRA. The tsdC and tsdD genes conferred hydroxyquinol 1,2-dioxygenase and maleylacetate reductase activities, respectively, on E. coli. Inactivation of tsdT significantly retarded the growth of RHA1 on GRA. The growth retardation was partially suppressed under acidic conditions, suggesting the involvement of tsdT in GRA uptake. Reverse transcription-PCR analysis revealed that the tsd genes constitute three transcriptional units, the tsdBADC and tsdTX operons and tsdR. Transcription of the tsdBADC and tsdTX operons was induced during growth on GRA. Inactivation of tsdR derepressed transcription of the tsdBADC and tsdTX operons in the absence of GRA, suggesting that tsd gene transcription is negatively regulated by the tsdR-encoded regulator. Binding of TsdR to the tsdR-tsdB and tsdT-tsdR intergenic regions was inhibited by the addition of GRA, indicating that GRA interacts with TsdR as an effector molecule.
Project description: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 CO(2) 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.
Project description:Transcriptome analysis of Rhodococcus jostii RHA1 during growth in sterilized soil was performed. A total of 165 soil-specific genes were identified by subtracting genes upregulated in late growth phases and on solid medium from 264 genes commonly upregulated during growth on biphenyl or pyruvate in sterilized soil. Classification of the 165 genes into functional categories indicated that this soil-specific group is rich in genes for the metabolism of fatty acids, amino acids, carbohydrates, and nitrogen and relatively poor in those for cellular processes and signaling. The ro06365-ro06369 gene cluster, in which ro06365 to ro06368 were highly upregulated in transcriptome analysis, was characterized further. ro06365 and ro06366 show similarity to a nitrite/nitrate transporter and a nitrite reductase, respectively, suggesting their involvement in nitrogen metabolism. A strain with an ro06366 deletion, D6366, showed growth retardation when we used nitrate as the sole nitrogen source and no growth when we used nitrite. A strain with a deletion of ro06365 to ro06368, DNop, utilized neither nitrite nor nitrate and recovered growth using nitrite and nitrate by introduction of the deleted genes. Both of the mutants showed growth retardation in sterilized soil, and the growth retardation of DNop was more significant than that of D6366. When these mutants were cultivated in medium containing the same proportions of ammonium, nitrate, and nitrite ions as those in the sterilized soil, they showed growth retardation similar to that in the soil. These results suggest that the ro06365-ro06369 gene cluster has a significant role in nitrogen utilization in sterilized soil.
Project description:Rhodococcus jostii RHA1 was engineered to utilise the cellulose component of lignocellulose, as well as the lignin fraction, by introduction of cellulase genes. The genome of R. jostii RHA1 was found to contain two β-glucosidase genes, RHA1_ro01034 and RHA1_ro02947, which support growth on cellobiose as growth substrate. Five Gram-positive endocellulase genes and one exocellulase gene were cloned into expression vector pTipQC2, and expressed in R. jostii RHA1. Endoglucanase activity was detected, with highest activity using Cellulomonas fimi cenA, and this recombinant strain grew on minimal media containing 0.5% carboxymethylcellulose (CMC). The R. jostii RHA1 genome was also found to contain a 3-dehydroshikimate dehydratase gene RHA1_ro01367, which supports growth on quinic acid as growth substrate, and conversion to protocatechuic acid. Therefore, this bacterium shows promise for further engineering to utilise cellulose for conversion to protocatechuic acid-derived bioproducts.
Project description:Rhodococcus jostii RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen Mycobacterium tuberculosis , are discussed. Bioinformatics and data processing: All database searches were performed using SEQUEST algorithm, embedded in BioworksTM (Rev. 3.3.1, Thermo Fisher Scientific Inc., Waltham, MA), with a RHA1 database containing 9145 sequences. Only tryptic peptides with up to two missed cleavages were accepted. No fixed modifications were considered. Oxidation of methionine was permitted as variable modification. The mass tolerance for precursor ions was set to 10 ppm; the mass tolerance for fragment ions was set to 1 amu. For protein identification a threshold for deltaCn (0.08) and for XCorr values was defined, depending on the peptide charge (>2.5 (+2); > 3.5 (+3)). A protein was considered identified if at least two different peptides met these criteria. To assess the false discovery rate (FDR) of protein identification, a database with reversed protein sequences was searched retaining the search parameters and filter criteria. The FDR was calculated by dividing the absolute number of hits from the reversed database through the sum of hits from both database searches (reversed database and original database). Using the stringent criteria described above, no reversed database hit was found; therefore, the FDR was 0% in all measurements. This low error rate can be attributed to the additional demand for two different peptide matches per protein, which eliminated all otherwise observed protein hits against the decoy database.
Project description:Here we report the first transcriptomic analysis of a Gram-positive bacterium to desiccation. Filtered RHA1 cells incubated at either low relative humidity (20%), as an air drying treatment, or high relative humidity (100%), as a control, were transcriptionally profiled over a comprehensive time series. Keywords: stress response time course
Project description:Rhodococcus jostii RHA1 is a soil-residing actinomycete with many favorable metabolic capabilities that make it an ideal candidate for the bioremediation of contaminated soils. Arguably the most basic requirement for life is water, yet some nonsporulating bacteria, like RHA1, can survive lengthy droughts. Here we report the first transcriptomic analysis of a gram-positive bacterium during desiccation. Filtered RHA1 cells incubated at either low relative humidity (20%), as an air-drying treatment, or high relative humidity (100%), as a control, were transcriptionally profiled over a comprehensive time series. Also, the morphology of RHA1 cells was characterized by cryofixation scanning electron microscopy during each treatment. Desiccation resulted in a transcriptional response of approximately 8 times more differentially regulated genes than in the control (819 versus 106 genes, respectively). Genes that were differentially expressed during only the desiccation treatment primarily had expression profiles that were maximally up-regulated upon complete drying of the cells. The microarray expression ratios for some of the highly up-regulated genes were verified by reverse transcriptase quantitative PCR. These genes included dps1, encoding an oxidative stress protection protein which has not previously been directly associated with desiccation, and the two genes encoding sigma factors SigF1 and SigF3, possibly involved in the regulatory response to desiccation. RHA1 cells also induced the biosynthetic pathway for the compatible solute ectoine. These desiccation-specific responses represent the best candidates for important mechanisms of desiccation resistance in RHA1.