γ-Resorcylate catabolic-pathway genes in the soil actinomycete Rhodococcus jostii RHA1.

The Rhodococcus jostii RHA1 gene cluster required for γ-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.

Specific gene responses of Rhodococcus jostii RHA1 during growth in soil.

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.

Characterization of nitrate and nitrite utilization system in Rhodococcus jostii RHA1.

A polychlorinated-biphenyl degrader, Rhodococcus jostii RHA1, has the potential to be used in soil for the remediation of environmental contamination. It has been found that RHA1 genes, ro06365 (narK) and ro06366, encoding a nitrate/nitrite transporter and nitrite reductase, respectively, were highly upregulated during the growth in sterile soil. In this study, these genes and ro00862, a paralog of ro06366 were characterized to reveal the nitrate and nitrite utilization systems of RHA1. The transcriptional induction of ro06366 (nirB1) and ro00862 (nirB2) by either nitrate or nitrite was revealed by qRT-PCR. Deletion mutants for each gene exhibited retarded growth on either nitrate or nitrite as a sole nitrogen source. Furthermore, their double mutant, Dnit, grew on and consumed neither nitrate nor nitrite as a sole nitrogen source, suggesting that both nirB1 and nirB2 are involved in the utilization of nitrite and nitrate. A narK mutant, DnarK, exhibited no growth on nitrate and retarded growth on nitrite as the sole nitrogen source. DnarK showed no consumption of nitrate and reduced consumption of nitrite, suggesting that narK is essential for nitrate uptake and is partially involved in nitrite uptake. The induced transcription of nirB1, nirB2, and narK was repressed in the presence of 3 mM ammonium or more. The upregulation of nirB1 and narK in sterilized soil containing ammonium and nitrate suggests that the ammonium concentration of the sterilized soil is equivalent to less than 3 mM. The unique nitrogen metabolism system of RHA1 and its importance for the growth in soil are discussed.