The emerging role for bacteria in lignin degradation and bio-product formation

With advances in biotechnology, bioremediation has become one of the most rapidly developing fields of environmental restoration, utilizing microorganisms to reduce the concentration and toxicity of various chemical pollutants, such as petroleum hydrocarbons, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, phthalate esters, nitroaromatic compounds, industrial solvents, pesticides and metals. A number of bioremediation strategies have been developed to treat contaminated wastes and sites. Selecting the most appropriate strategy to treat a specific site can be guided by considering three basic principles: the amenability of the pollutant to biological transformation to less toxic products (biochemistry), the accessibility of the contaminant to microorganisms (bioavailability) and the opportunity for optimization of biological activity (bioactivity). Recent advances in the molecular genetics of biodegradation and studies on enzyme-tailoring and DNA-shuffling are discussed in this paper.

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Biotechnology and bioremediation: successes and limitations.

Biodegradation potential of the genus Rhodococcus.

Biodegradation and Rhodococcus – masters of catabolic versatility

The microbial degradation of polychlorinated biphenyls (PCBs) has been extensively studied in recent years The genetic organization of biphenyl catabolic genes has been elucidated in various groups of microorganisms, their structures have been analyzed with respect to their evolutionary relationships, and new information on mobile elements has become available Key enzymes, specifically biphenyl 2,3-dioxygenases, have been intensively characterized, structure/sequence relationships have been determined and enzymes optimized for PCB transformation However, due to the complex metabolic network responsible for PCB degradation, optimizing degradation by single bacterial species is necessarily limited As PCBs are usually not mineralized by biphenyl-degrading organisms, and cometabolism can result in the formation of toxic metabolites, the degradation of chlorobenzoates has received special attention A broad set of bacterial strategies to degrade chlorobenzoates has recently been elucidated, including new pathways for the degradation of chlorocatechols as central intermediates of various chloroaromatic catabolic pathways To optimize PCB degradation in the environment beyond these metabolic limitations, enhancing degradation in the rhizosphere has been suggested, in addition to the application of surfactants to overcome bioavailability barriers However, further research is necessary to understand the complex interactions between soil/sediment, pollutant, surfactant and microorganisms in different environments

Aerobic degradation of polychlorinated biphenyls

Rhodococcus sp. strain RHA1 is a gram-positive polychlorinated biphenyl (PCB) degrader which can degrade 10 ppm of PCB48 (equivalent to Aroclor1248), including tri-, tetra-, and pentachlorobiphenyls, in a few days. We isolated the 7.6-kb EcoRI-BamHI fragment carrying the biphenyl catabolic genes of RHA1 and determined their nucleotide sequence. On the basis of deduced amino acid sequence homology, we identified six bph genes, bphA1A2A3A4, bphB, and bphC, that are responsible for the initial three steps of biphenyl degradation. The order of bph genes in RHA1 is bphA1A2A3A4-bphC-bphB. This gene order differs from that of other PCB degraders reported previously. The amino acid sequences deduced from the RHA1 bph genes have a higher degree of homology with the tod genes from Pseudomonas putida F1 (49 to 79%) than with the bph genes of Pseudomonas sp. strains KF707 and KKS102 (30 to 65%). In Escherichia coli, bphA gene activity was not observed even when expression vectors were used. The activities of bphB and bphC, however, were confirmed by observing the transformation of biphenyl to a meta-cleavage compound with the aid of benzene dioxygenase activity that complemented the bphA gene activity (S. Irie, S. Doi, T. Yorifuji, M. Takagi, and K. Yano, J. Bacteriol. 169:5174-5179, 1987). The expected products of the cloned bph genes, except bphA3, were observed in E. coli in an in vitro transcription-translation system. Insertion mutations of bphA1 and bphC of Rhodococcus sp. strain RHA1 were constructed by gene replacement with cloned gene fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of biphenyl catabolic genes of gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1.

The two 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD) hydrolase genes, etbD1 and etbD2, were cloned from a strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, and their nucleotide sequences were determined. The etbD2 gene was located in the vicinity of bphA gene homologs and encoded an enzyme whose amino-terminal sequence was very similar to the amino-terminal sequence of the HOHD hydrolase which was purified from RHA1. Using the etbD2 gene fragment as a probe, we cloned the etbD1 gene encoding the purified HOHD hydrolase by colony hybridization. Both genes encode a product having 274 amino acid residues and containing the nucleophile motif conserved in alpha/beta hydrolase fold enzymes. The deduced amino acid sequences were quite similar to the amino acid sequences of the products of the single-ring aromatic hydrolase genes, such as dmpD, cumD, todF, and xylF, and not very similar to the amino acid sequences of the products of bphD genes from PCB degraders, including RHA1. The two HOHD hydrolase genes and the RHA1 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HPDA) hydrolase gene, bphD, were expressed in Escherichia coli, and their relative enzymatic activities were examined. The product of bphD was very specific to HPDA, and the products of etbD1 and etbD2 were specific to HOHD. All of the gene products exhibited poor activities against the meta-cleavage product of catechol. These results agreed with the results obtained for BphD and EtbD1 hydrolases purified from RHA1. The three hydrolase genes exhibited similar induction patterns both in an RNA slot blot hybridization analysis and in a reporter gene assay when a promoter probe vector was used. They were induced by biphenyl, ethylbenzene, benzene, toluene, and ortho-xylene. Strain RCD1, an RHA1 mutant strain lacking both the bphD gene and the etbD2 gene, grew well on ethylbenzene. This result suggested that the etbD1 gene product is involved in the meta-cleavage metabolic pathway of ethylbenzene.

Two Nearly Identical Aromatic Compound Hydrolase Genes in a Strong Polychlorinated Biphenyl Degrader, 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 (PbphA1) was identified and was shown to promote transcription induction by biphenyl and ethylbenzene. An 8.8-kb HindIII fragment that promotes transcription induction of PbphA1 in Rhodococcus erythropolis IAM1399 was isolated from the region downstream of bphB by using a reporter plasmid containing PbphA1. 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 PbphA1 and that both bphS and bphT are required for the elevated basal activation of and transcriptional induction by biphenyl of PbphA1. 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 PbphA1 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.

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

Crystal structure of 2-hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid (HPDA) hydrolase (BphD enzyme) from the Rhodococcus sp. strain RHA1 of the PCB degradation pathway.

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.

https://www.tandfonline.com/doi/pdf/10.1271/bbb.68.1249

Akihiro Yamada

Papers

Characterization of biphenyl catabolic genes of gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1.

Two Nearly Identical Aromatic Compound Hydrolase Genes in a Strong Polychlorinated Biphenyl Degrader, Rhodococcus sp. Strain RHA1

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

Crystal structure of 2-hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid (HPDA) hydrolase (BphD enzyme) from the Rhodococcus sp. strain RHA1 of the PCB degradation pathway.

Biphenyl-inducible Promoters in a Polychlorinated Biphenyl-degrading Bacterium, Rhodococcus sp. RHA1