Specific gene responses of Rhodococcus jostii RHA1 during growth in soil

Abstract

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.

Fig 1

Summary of transcriptome analyses of genes upregulated in sterilized soil. The circles in the Venn diagram represent the number of upregulated genes in each experiment. The intersection of interest drawn out is presented as a circle in the next Venn diagram. The total number of genes upregulated in each experiment is presented in parentheses behind the abbreviated experimental conditions. Abbreviations: BPH, growth on biphenyl in sterilized soil; PYR, growth on pyruvate in sterilized soil; TRA, growth on biphenyl to the transitional phase in liquid minimal salt medium; STA, growth on biphenyl to the stationary phase in liquid minimal salt medium; MEM, growth on biphenyl on a membrane filter placed on a minimal salt medium agar plate.

Fig 2

Heat maps and gene organization of selected gene clusters among the 165 soil-specific genes. The representative gene clusters discussed in the text are shown. The color scale indicates log₂ values of average normalized expression ratios (treatment/control). The expression ratios are color coded as follows: red, upregulated by treatment; green, downregulated by treatment; black, similar levels of expression under both conditions. Abbreviations for the experimental conditions are as indicated in Fig. 1.

Fig 3

RT-PCR analysis of the region containing ro06365 to ro06369. (A) Gene organization of the region including ro06365 to ro06369. The genes ro06365 to ro06369 are represented by black arrows, and their surrounding genes are represented by gray arrows. The horizontal bars with numbers assigned indicate the regions amplified by RT-PCR. (B) Agarose gel electrophoresis of RT-PCR products. The numbers on the top of each plate correspond to those of the amplified regions indicated in panel A. Total RNA isolated from RHA1 cells, which were grown in the presence of ammonium, nitrite, or nitrate ions as the sole nitrogen source, was used as the template for cDNA synthesis in the presence (+) or absence (−) of reverse transcriptase. Region numbers correspond with each of those listed in Table 1.

Fig 4

qRT-PCR analysis of ro06365 and ro06366. (A) The regions of amplification in ro06365 and ro06366. The genes ro06365 and ro06366 are represented by black arrows, and the regions of amplification are indicated by horizontal bars, to which numbers are assigned. (B) Transcriptional induction of ro06365 and ro06366 in the presence of nitrite or nitrate ions. Total RNA was isolated from RHA1 cells grown on biphenyl in the presence of ammonium, nitrite, or nitrate ions. Quantitative RT-PCR was performed for regions 8 and 9 and normalized to 16S rRNA in the same sample. Each induction ratio was estimated by dividing the amount of transcript in the presence of nitrite or nitrate ions by that in the presence of ammonium ions. The data are mean values plus or minus the standard deviations from three independent experiments. Region numbers correspond with each of those listed in Table 1.

Fig 5

The growths of D6366 and DNop in sterilized soil. The growths of D6366 (open squares) and RHA1 (open circles) on biphenyl (A to C) or pyruvate (D to F) in sterilized soil and those of DNop (open triangles) and RHA1 (open circles) on biphenyl (G to I) in sterilized soil were compared. The results of three separate series of experiments are presented. The data are mean values plus or minus the standard deviations from three independent experiments. Each series of experiments represents the mean values from three independent experiments.

Fig 6

Complementation of the DNop mutant by the intact ro06365 to ro06368 genes. RHA1 containing a vector, pK4 (closed circles), DNop containing pK4 (closed triangles), and DNop containing pKNop with the ro06365–ro06368 gene insert (open triangles) were grown on biphenyl in a minimal salt medium containing 10 mM nitrite (A) or nitrate (B) as the sole nitrogen source. The data are mean values plus or minus the standard deviations from three independent experiments.

Fig 7

The growth of RHA1 (open circles), D6366 (open squares), and DNop (open triangles) in minimal salt medium containing 20 mg/liter ammonium and 179 mg/liter nitrate ions. The data are mean values plus or minus the standard deviations from three independent experiments.