Dual two-component regulatory systems are involved in aromatic compound degradation in a polychlorinated-biphenyl degrader, Rhodococcus jostii RHA1

Abstract

A Gram-positive polychlorinated-biphenyl (PCB) degrader, Rhodococcus jostii RHA1, degrades PCBs by cometabolism with biphenyl. A two-component BphS1T1 system encoded by bphS1 and bphT1 (formerly bphS and bphT) is responsible for the transcription induction of the five gene clusters, bphAaAbAcAdC1B1, etbAa1Ab1CbphD1, etbAa2Ab2AcD2, etbAdbphB2, and etbD1, which constitute multiple enzyme systems for biphenyl/PCB degradation. The bphS2 and bphT2 genes, which encode BphS2 and BphT2, virtually identical to BphS1 (92%) and BphT1 (97%), respectively, were characterized. BphS2T2 induced the activation of the bphAa promoter in a host, Rhodococcus erythropolis IAM1399, in the presence of a variety of aromatics, including benzene, toluene, ethylbenzene, xylenes, isopropylbenzene, and chlorinated benzenes, as effectively as BphS1T1. The substrate spectrum of BphS2T2 was the same as that of BphS1T1, except for biphenyl, which is a substrate only for BphS1T1. BphS2T2 activated transcription from the five promoters of biphenyl/PCB degradation enzyme gene clusters as effectively as BphS1T1. The targeted disruptions of the bphS1, bphS2, bphT1, and bphT2 genes indicated that all these genes are involved in the growth of RHA1 on aromatic compounds. The hybrid system with bphS1 and bphT2 and that with bphS2 and bphT1 were constructed, and both systems conducted induced activation of the bphAa promoter, indicating cross-communication. These results indicated that RHA1 employs not only multiple enzyme systems, but also dual regulatory systems for biphenyl/PCB degradation. Comparison of the sequences, including bphS2T2, with the bphS1T1-containing sequences and the corresponding sequences in other rhodococcal degraders suggests that bphS2T2 might have originated from bphS1T1.

FIG. 1.

Genetic organization of the biphenyl degradation gene clusters in RHA1 under the control of gene products of bphS1 and bphT1 (BphS1T1), which were previously reported as the bphS and bphT genes, respectively. All the gene clusters are located on either the linear plasmid pRHL1 or pRHL2, as illustrated. The BphS1T1-inducible promoters are indicated by black triangles. Double slashes indicate a space separating two independent gene clusters.

FIG. 2.

Deletion analysis of the 14.1-kb HindIII fragment containing bphS2T2. (A) Physical maps of deletion derivatives. The segments represented by solid bars were inserted into pKLAF1, which is a reporter plasmid that does not contain any bphST segment. (B) Luciferase assay of the deletion derivatives. The IAM1399 cells harboring the plasmids were grown in 1/5 LB in the presence or absence of ethylbenzene and were subjected to the luciferase assay. The activity was expressed as RLU measured by a luminometer per milliliter of culture per OD₆₀₀ unit, as described in Materials and Methods. The data are means ± standard deviations from at least three independent experiments.

FIG. 3.

Constitutive transcription of bphS2. (A) Physical map of the 9.2-kb ApaLI-HindIII fragment containing bphS2T2. The thick bar indicates the position of the 323-bp segment amplified in RT-PCR analysis. The 2.1-kb KpnI-EcoRV fragment, indicated by a two-headed arrow, was cloned in a reporter plasmid, pKLA1, for the luciferase assay. (B) RT-PCR analysis of bphS2 transcription. Total RNA of RHA1 grown in LB (lanes 1 and 2) or on biphenyl (lanes 3 and 4) or ethylbenzene (lanes 5 and 6) was used as a template in the presence (+) or absence (−) of reverse transcriptase. The DNA sizes of molecular size markers (lane M) are indicated on the left. The position and the expected size of the amplified fragment are indicated by a horizontal arrowhead on the right. (C) Luciferase assay of the bphS2 promoter region. The reporter plasmids pKLS2F and pKLS2R containing the 2.1-kb bphS2 upstream region in the forward and reverse orientations to luxAB genes, respectively, and a vector control, pKLA1, were independently introduced into RHA1, and the transformants grown in 1/5 LB in the presence or absence of biphenyl or ethylbenzene were subjected to a luciferase assay. The activity was expressed as in Fig. 2. The data are means ± standard deviations from at least three independent experiments.

FIG. 4.

Growth on biphenyl (A) and ethylbenzene (B) of the disruption mutants of bphS1 or bphS2. The bphS1 mutant, SDR1; the bphS2 mutant, SDR2; the bphS1/S2 double mutant, SDR21; SDR21 harboring pKTS2 containing bphS2; and SDR21 containing a vector control, pK4Ts, were grown in W minimal medium containing biphenyl (2 g/liter) or ethylbenzene. Ethylbenzene was supplied as vapor. Growth was monitored by measuring the OD₆₀₀.

FIG. 5.

Growth on biphenyl of the disruption mutants of bphT1 and bphT2. (A) The bphT1 mutant, TDR1; the bphT2 mutant, TDR2; and the bphT1/T2 double mutant, TDR12, were grown in W minimal medium containing biphenyl (1.54 g/liter). (B) TDR12 harboring pKPA1TKE containing bphT1 or pKPA1T2KE containing bphT2 was grown in W minimal medium containing biphenyl (1.54 g/liter). pKPA1 was used as a vector control. Growth was monitored by measuring the OD₆₀₀. Data are means ± standard deviations from at least three independent experiments.

FIG. 6.

Cross-communication between heterologous BphS and BphT. An original bphT was replaced by a heterologous bphT at the common ApaI site to obtain pKLAS1T2 and pKLAS2T1 containing bphS1 plus bphT2 and bphS2 plus bphT1, respectively. Shown is the luciferase activity of IAM1399 harboring pKLAS1T2 or pKLAS2T1. The cells were incubated in 1/5 LB in the presence or absence of biphenyl or ethylbenzene for 5 h and subjected to a luciferase assay. IAM1399 harboring pKLAST1 or pKLAST2 was used as a control. The activity was expressed as in Fig. 2. The data are means ± standard deviations from at least three independent experiments.

FIG. 7.

Comparison of the DNA sequences surrounding the bphST genes in RHA1 and the Rhodococcus strains, including bphST homologs. pROB02 (GenBank accession number AP011117) and pBD2 (GenBank accession number AY223810) are linear plasmids of R. opacus B4 and R. erythropolis BD2, respectively. The gray-shaded sections represent homologous DNA regions with more than 70% identity between two sequences. The exact percentages of identity are indicated by their shades of gray, which are shown in the gray scale bar at the top. The 24-bp direct repeats, including the bphST-dependent promoter consensus of RHA1, are connected by black lines and a gray line between ro10124 and etbD1 of RHA1.

FIG. 8.

Transcriptional regulation mechanisms of BphS1T1 and BphS2T2 two-component systems in RHA1. BphS1T1 and BphS2T2 share most characteristics. Both BphS1 and BphS2 are estimated to phosphorylate BphT1 and BphT2, which are both responsible for the transcriptional activation of bphAap, etbAa1p, etbAa2p, etbAdp, and etbD1p in the presence of a variety of aromatic compounds, including ethylbenzene, benzene, toluene, xylenes, isopropylbenzene, and chlorinated benzenes (Table 1 shows details). Biphenyl is an inducing substrate for BphS1, but not for BphS2.