Keisuke Miyauchi

The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse

Rhodococcus sp. RHA1 (RHA1) is a potent polychlorinated biphenyl-degrading soil actinomycete that catabolizes a wide range of compounds and represents a genus of considerable industrial interest. RHA1 has one of the largest bacterial genomes sequenced to date, comprising 9,702,737 bp (67% G+C) arranged in a linear chromosome and three linear plasmids. A targeted insertion methodology was developed to determine the telomeric sequences. RHA1s 9,145 predicted protein-encoding genes are exceptionally rich in oxygenases (203) and ligases (192). Many of the oxygenases occur in the numerous pathways predicted to degrade aromatic compounds (30) or steroids (4). RHA1 also contains 24 nonribosomal peptide synthase genes, six of which exceed 25 kbp, and seven polyketide synthase genes, providing evidence that rhodococci harbor an extensive secondary metabolism. Among sequenced genomes, RHA1 is most similar to those of nocardial and mycobacterial strains. The genome contains few recent gene duplications. Moreover, three different analyses indicate that RHA1 has acquired fewer genes by recent horizontal transfer than most bacteria characterized to date and far fewer than Burkholderia xenovorans LB400, whose genome size and catabolic versatility rival those of RHA1. RHA1 and LB400 thus appear to demonstrate that ecologically similar bacteria can evolve large genomes by different means. Overall, RHA1 appears to have evolved to simultaneously catabolize a diverse range of plant-derived compounds in an O2-rich environment. In addition to establishing RHA1 as an important model for studying actinomycete physiology, this study provides critical insights that facilitate the exploitation of these industrially important microorganisms.

Complete analysis of genes and enzymes for γ-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26

γ-Hexachlorocyclohexane (γ-HCH; also called BHC or lindane) is one of the highly chlorinated pesticides which can cause serious environmental problems. Sphingomonas paucimobilis UT26 degrades γ-HCH under aerobic conditions. The unique degradation pathway of γ-HCH in UT26 is revealed. In the upstream pathway, γ-HCH is transformed to 2,5-dichlorohydroquinone (2,5-DCHQ) by two different dehalogenases (LinA and LinB) and one dehydrogenase (LinC) which are expressed constitutively. In the downstream pathway, 2,5-DCHQ is reductively dehalogenated, and then ring-cleaved by enzymes (LinD and LinE, respectively) whose expressions are regulated. We have cloned and sequenced five structural genes (linA, linB, linC, linD, and linE) directly involved in this degradation pathway. The linD and linE genes form an operon, and its expression is positively regulated by the LysR-type transcriptional regulator (LinR). The genes linA, linB, and linC are constitutively expressed, and are present separately from each other in the UT26 genome. Cell fractionation analysis, Western blotting, and immuno electron microscopy revealed that LinA and LinB are localized in the periplasmic space of UT26.

Novel 2,4-Dichlorophenoxyacetic Acid Degradation Genes from Oligotrophic Bradyrhizobium sp. Strain HW13 Isolated from a Pristine Environment

The tfd genes of Ralstonia eutropha JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria. A new family of 2,4-D degradation genes, cadRABKC, was cloned and characterized from Bradyrhizobium sp. strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals. The cadR gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence. The cadABC genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of Burkholderia cepacia AC1100 and with a putative ferredoxin, ThcC, of Rhodococcus erythropolis NI86/21, respectively. They are thoroughly different from the 2,4-D dioxygenase gene, tfdA, of R. eutropha JMP134. The cadK gene was presumed to encode a 2,4-D transport protein from its deduced amino acid sequence that showed 60% identity with the 2,4-D transporter, TfdK, of strain JMP134. Sinorhizobium meliloti Rm1021 cells containing cadRABKC transformed several phenoxyacetic acids, including 2,4-D and 2,4,5-T, to corresponding phenol derivatives. Frameshift mutations indicated that each of the cadRABC genes was essential for 2,4-D conversion in strain Rm1021 but that cadK was not. Five 2,4-D degraders, including Bradyrhizobium and Sphingomonas strains, were found to have cadA gene homologs, suggesting that these 2,4-D degraders share 2,4-D degradation genes similar to those of strain HW13 cadABC.

PcpA, which is involved in the degradation of pentachlorophenol in Sphingomonas chlorophenolica ATCC39723, is a novel type of ring‐cleavage dioxygenase

The pentachlorophenol (PCP) mineralizing bacterium Sphingomonas chlorophenolica ATCC39723 degrades PCP via 2,6‐dichlorohydroquinone (2,6‐DCHQ). The pathway converting PCP to 2,6‐DCHQ has been established previously; however, the pathway beyond 2,6‐DCHQ is not clear, although it has been suggested that a PcpA plays a role in 2,6‐DCHQ conversion. In this study, PcpA expressed in Escherichia coli was purified to homogeneity and shown to have novel ring‐cleavage dioxygenase activity in conjunction with hydroquinone derivatives, and converting 2,6‐DCHQ to 2‐chloromaleylacetate.

Characterization of Transcriptional Regulatory Genes for Biphenyl Degradation in Rhodococcus sp. Strain RHA1

Transcription of the bphA1A2A3A4C1B genes, which are responsible for the conversion of biphenyl and polychlorinated biphenyl to the meta-cleavage products in Rhodococcus sp. strain RHA1, was examined. The bphA1 promoter (P(bphA1)) was identified and was shown to promote transcription induction by biphenyl and ethylbenzene. An 8.8-kb HindIII fragment that promotes transcription induction of P(bphA1) in Rhodococcus erythropolis IAM1399 was isolated from the region downstream of bphB by using a reporter plasmid containing P(bphA1). Analysis of the nucleotide sequence of this fragment revealed a set of putative two-component regulatory system genes, which were designated bphS and bphT. Deletion analysis of the 8.8-kb HindIII fragment indicated that bphT is responsible for the basal activation of P(bphA1) and that both bphS and bphT are required for the elevated basal activation of and transcriptional induction by biphenyl of P(bphA1). These results support the notion that bphS and bphT encode a sensor kinase and a response regulator, respectively, of a two-component regulatory system. The bphS and bphT genes promote transcriptional induction by a variety of aromatic compounds, including biphenyl, benzene, alkylbenzenes, and chlorinated benzenes. A promoter activity assay and reverse transcription (RT)-PCR analysis revealed a weak constitutive promoter in the adjacent region upstream of bphS. RT-PCR analysis indicated that there is induced transcription of bphA1 through bphT, in which P(bphA1) is thought to take part. An insertionally inactivated bphS mutant, SDR1, did not grow on biphenyl. Growth was restored by introduction of an intact bphS gene into SDR1. These results indicate that at least bphS is indispensably responsible for the growth of RHA1 on biphenyl.

Identification and Characterization of Genes Involved in the Downstream Degradation Pathway of γ-Hexachlorocyclohexane in Sphingomonas paucimobilis UT26

Sphingomonas paucimobilis UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH) as a sole source of carbon and energy. In our previous study, we cloned and characterized genes that are involved in the conversion of gamma-HCH to maleylacetate (MA) via chlorohydroquinone (CHQ) in UT26. In this study, we identified and characterized an MA reductase gene, designated linF, that is essential for the utilization of gamma-HCH in UT26. A gene named linEb, whose deduced product showed significant identity to LinE (53%), was located close to linF. LinE is a novel type of ring cleavage dioxygenase that catalyzes the conversion of CHQ to MA. LinEb expressed in Escherichia coli transformed CHQ and 2,6-dichlorohydroquinone to MA and 2-chloromaleylacetate, respectively. Our previous and present results indicate that UT26 (i) has two gene clusters for degradation of chlorinated aromatic compounds via hydroquinone-type intermediates and (ii) uses at least parts of both clusters for gamma-HCH utilization.

Characterization of the 4-carboxy-4-hydroxy-2-oxoadipate aldolase gene and operon structure of the protocatechuate 4,5-cleavage pathway genes in Sphingomonas paucimobilis SYK-6.

The protocatechuate (PCA) 4,5-cleavage pathway is the essential metabolic route for degradation of low-molecular-weight products derived from lignin by Sphingomonas paucimobilis SYK-6. In the 10.5-kb EcoRI fragment carrying the genes for PCA 4,5-dioxygenase (ligAB), 2-pyrone-4,6-dicarboxylate hydrolase (ligI), 4-oxalomesaconate hydratase (ligJ), and a part of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC), we found the ligK gene, which encodes 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase. The ligK gene was located 1,183 bp upstream of ligI and transcribed in the same direction as ligI. We also found the ligR gene encoding a LysR-type transcriptional activator, which was located 174 bp upstream of ligK. The ligK gene consists of a 684-bp open reading frame encoding a polypeptide with a molecular mass of 24,131 Da. The deduced amino acid sequence of ligK showed 57 to 88% identity with those of the corresponding genes recently reported in Sphingomonas sp. strain LB126, Comamonas testosteroni BR6020, Arthrobacter keyseri 12B, and Pseudomonas ochraceae NGJ1. The ligK gene was expressed in Escherichia coli, and the gene product (LigK) was purified to near homogeneity. Electrospray-ionization mass spectrometry indicated that LigK catalyzes not only the conversion of CHA to pyruvate and oxaloacetate but also that of oxaloacetate to pyruvate and CO(2). LigK is a hexamer, and its isoelectric point is 5.1. The K(m) for CHA and oxaloacetate are 11.2 and 136 micro M, respectively. Inactivation of ligK in S. paucimobilis SYK-6 resulted in the growth deficiency of vanillate and syringate, indicating that ligK encodes the essential CHA aldolase for catabolism of these compounds. Reverse transcription-PCR analysis revealed that the PCA 4,5-cleavage pathway genes of S. paucimobilis SYK-6 consisted of four transcriptional units, including the ligK-orf1-ligI-lsdA cluster, the ligJAB cluster, and the monocistronic ligR and ligC genes.

Multiple-Subunit Genes of the Aromatic-Ring-Hydroxylating Dioxygenase Play an Active Role in Biphenyl and Polychlorinated Biphenyl Degradation in Rhodococcus sp. Strain RHA1

ABSTRACT A gram-positive strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, can degrade PCBs by cometabolism with biphenyl or ethylbenzene. In RHA1, three sets of aromatic-ring-hydroxylating dioxygenase genes are induced by biphenyl. The large and small subunits of their terminal dioxygenase components are encoded by bphA1 and bphA2, etbA1 and etbA2, and ebdA1 and ebdA2, respectively, and the deduced amino acid sequences of etbA1 and etbA2 are identical to those of ebdA1 and ebdA2, respectively. In this study, we examined the involvement of the respective subunit genes in biphenyl/PCB degradation by RHA1. Reverse transcription-PCR and two-dimensional polyacrylamide gel electrophoresis analyses indicated the induction of RNA and protein products of etbA1 and ebdA1 by biphenyl. Single- and double-disruption mutants of etbA1, ebdA1, and bphA1 were constructed by insertional inactivation. The 4-chlorobiphenyl (4-CB) degradation activities of all the mutants were lower than that of RHA1. The results indicated that all of these genes are involved in biphenyl/PCB degradation. Furthermore, we constructed disruption mutants of ebdA3 and bphA3, encoding ferredoxin, and etbA4, encoding ferredoxin reductase components. The 4-CB degradation activities of these mutants were also lower than that of RHA1, suggesting that all of these genes play a role in biphenyl/PCB degradation. The substrate preferences of etbA1A2/ebdA1A2- and bphA1A2-encoded dioxygenases for PCB congeners were examined using the corresponding mutants. The results indicated that these dioxygenase isozymes have different substrate preferences and that the etbA1A2/ebdA1A2-encoded isozyme is more active on highly chlorinated congeners than the bphA1A2-encoded one.

Biphenyl-inducible promoters in a polychlorinated biphenyl-degrading bacterium, Rhodococcus sp. RHA1.

Five transcriptional promoters of biphenyl-degradation genes in Rhodococcus sp. RHA1 were characterized. We newly identified the etbA4 promoter region, which was located adjacent upstream from a ferredoxin reductase gene, etbA4 and a dihydrodiol dehydrogenase gene, bphB2. The etbA4 promoter activity was determined in RHA1 using a promoter probe vector with a luxAB luciferase reporter gene, and was induced by a variety of aromatic compounds as well as the bphA1, ebdA1, etbA1, and etbD1 promoters. All these promoters were induced by aromatic compounds in a closely related heterologous host, R. erythropolis IAM1399 in the presence of RHA1 bphST genes, suggesting that these five promoters are under the control of bphST-coding two-component regulatory system. Sequence comparison of the bphA1 promoter with the ebdA1 and etbA1 promoters, whose transcription starts were determined by primer extension analysis, revealed a consensus sequence centering 42-bp upstream from the transcription start. This consensus was also conserved in the etbA4 and etbD1 promoters, and deletions of the bphA1 promoter affecting the consensus impaired inducible promoter activity. These results suggest that this consensus plays a role in transcription induction and/or the promotion of biphenyl degradation genes in RHA1.

Characterization of Two Biphenyl Dioxygenases for Biphenyl/PCB Degradation in A PCB Degrader, Rhodococcus sp. Strain RHA1

Rhodococcus sp. RHA1 induces two biphenyl dioxygenases, the BphA and EtbA/EbdA dioxygenases, during growth on biphenyl. Their subunit genes were expressed in R. erythropolis IAM1399 to investigate the involvement of each subunit gene in their activity and their substrate preferences. The recombinant expressing ebdA1A2A3etbA4 and that expressing bphA1A2A3A4 exhibited 4-chlorobiphenyl (4-CB) transformation activity, suggesting that these gene sets are responsible for the EtbA/EbdA and BphA dioxygenases respectively. When bphA4 and etbA4 were swapped to construct the recombinants expressing ebdA1A2A3bphA4 and bphA1A2A3etbA4 respectively, compatibility between BphA4 and EtbA4 was suggested by their 4-CB transformation activities. When bphA3 and ebdA3 were swapped, incompatibility between BphA3 and EbdA3 was suggested. BphA and EtbA/EbdA dioxygenases exhibited the highest transformation activity toward biphenyl and naphthalene respectively, and also attacked dibenzofuran and dibenzo-p-dioxin. The wide substrate preference of EtbA/EbdA dioxygenase suggested that it plays a more important role in polychlorinated biphenyl (PCB) degradation than does BphA dioxygenase.