Multiple Polychlorinated Biphenyl Transformation Systems in the Gram-Positive Bacterium Rhodococcus sp. Strain RHA1

The cloned bphA gene of the polychlorinated biphenyl (PCB) degrader Rhodococcus sp. strain RHA1 was expressed in Rhodococcus erythropolis IAM1399 cells, resulting in the transformation of di-, tri-, and tetrachlorobiphenyls. Disruption of the bphA1 gene in RHA1 resulted in a lack of growth on biphenyl and a loss of PCB transformation activity. However, the bphA1 insertion mutant of RHA1, designated RDA1, retained the ability to transform PCB congeners when grown on ethylbenzene as its carbon source. It also transformed 4-chlorobiphenyl to 4-chlorobenzoate, although it was suspected to be deficient in bphB and bphC gene activities as well as bphA. This suggested that an alternative PCB degradation system distinct from the one encoded by the cloned bph genes was present.     

Characterization of Pseudomonas putida mutants unable to catabolize benzoate: cloning and characterization of Pseudomonas genes involved in benzoate catabolism and isolation of a chromosomal DNA fragment able to substitute for xylS in activation of the TOL lower-pathway promoter.

Mutants of Pseudomonas putida mt-2 that are unable to convert benzoate to catechol were isolated and grouped into two classes: those that did not initiate attack on benzoate and those that accumulated 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (benzoate diol). The latter mutants, represents by strain PP0201, were shown to lack benzoate diol dehydrogenase (benD) activity. Mutants from the former class were presumed either to carry lesions in one or more subunit structural genes of benzoate dioxygenase (benABC) or the regulatory gene (benR) or to contain multiple mutations. Previous work in this laboratory suggested that benR can substitute for the TOL plasmid-encoded xylS regulatory gene, which promotes gene expression from the OP2 region of the lower or meta pathway operon. Accordingly, structural and regulatory gene mutations were distinguished by the ability of benzoate-grown mutant strains to induce expression from OP2 without xylS by using the TOL plasmid xylE gene (encoding catechol 2,3-dioxygenase) as a reporter. A cloned 12-kb BamHI chromosomal DNA fragment from the P. aeruginosa PAO1 chromosome complemented all of the mutations, as shown by restoration of growth on benzoate minimal medium. Subcloning and deletion analyses allowed identification of DNA fragments carrying benD, benABC, and the region possessing xylS substitution activity, benR. Expression of these genes was examined in a strain devoid of benzoate-utilizing ability, Pseudomonas fluorescens PFO15. The disappearance of benzoate and the production of catechol were determined by chromatographic analysis of supernatants from cultures grown with casamino acids. When P. fluorescens PFO15 was transformed with plasmids containing only benABCD, no loss of benzoate was observed. When either benR or xylS was cloned into plasmids compatible with those plasmids containing only the benABCD regions, benzoate was removed from the medium and catechol was produced. Regulation of expression of the chromosomal structural genes by benR and xylS was quantified by benzoate diol dehydrogenase enzyme assays. The results obtained when xylS was substituted for benR strongly suggest an isofunctional regulatory mechanism between the TOL plasmid lower-pathway genes (via the OP2 promoter) and chromosomal benABC. Southern hybridizations demonstrated that DNA encoding the benzoate dioxygenase structural genes showed homology to DNA encoding toluate dioxygenase from the TOL plasmid pWW0, but benR did not show homology to xylS. Evolutionary relationships between the regulatory systems of chromosomal and plasmid-encoded genes for the catabolism of benzoate and related compounds are suggested.     

Catabolism of benzoate and phthalate in Rhodococcus sp. strain RHA1: redundancies and convergence

Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in Rhodococcus sp. strain RHA1, a polychlorinated biphenyl-degrading actinomycete. Sequence analyses identified genes involved in the catabolism of benzoate (ben) and phthalate (pad), the uptake of phthalate (pat), and two branches of the beta-ketoadipate pathway (catRABC and pcaJIHGBLFR). The regulatory and structural ben genes are separated by genes encoding a cytochrome P450. The pad and pat genes are contained on a catabolic island that is duplicated on plasmids pRHL1 and pRHL2 and includes predicted terephthalate catabolic genes (tpa). Proteomic analyses demonstrated that the beta-ketoadipate pathway is functionally convergent. Specifically, the pad and pat gene products were only detected in phthalate-grown cells. Similarly, the ben and cat gene products were only detected in benzoate-grown cells. However, pca-encoded enzymes were present under both growth conditions. Activity assays for key enzymes confirmed these results. Disruption of pcaL, which encodes a fusion enzyme, abolished growth on phthalate. In contrast, after a lag phase, growth of the mutant on benzoate was similar to that of the wild type. Proteomic analyses revealed 20 proteins in the mutant that were not detected in wild-type cells during growth on benzoate, including a CatD homolog that apparently compensated for loss of PcaL. Analysis of completed bacterial genomes indicates that the convergent beta-ketoadipate pathway and some aspects of its genetic organization are characteristic of rhodococci and related actinomycetes. In contrast, the high redundancy of catabolic pathways and enzymes appears to be unique to RHA1 and may increase its potential to adapt to new carbon sources.     

Characterization and functional analysis of a novel gene cluster involved in biphenyl degradation in Rhodococcus sp. strain R04

AIMS: Isolation of the genes relative to PCB biodegradation and identification of the bph gene function in Rhodococcus sp. R04. METHODS AND RESULTS: A 8.7-kb fragment carrying the biphenyl catabolic genes bphABCD was isolated from the gene library in Rhodococcus sp. R04. Based on the deduced amino acid sequence homology, seven bph genes, bphA1A2A3A4, bphB, bphC and bphD, were thought to be responsible for the initial four steps of biphenyl degradation. In Escherichia coli, BphA exhibited poor activity for biphenyl transformation, and BphB, BphC and BphD were found to be catalytically active towards 2,3-dihydro-2,3-dihydroxybiphenyl, 2,3-dihydroxybiphenyl and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate, respectively (activities of 50, 8.1 and 2.4 micromol l(-1) min(-1) mg(-1)). SDS-PAGE analysis indicated that the sizes of bphA1A2A3A4, bphB, bphC and bphD gene products were 49, 19, 14, 47, 32, 30 and 31 kDa, respectively. After disruption of bph genes, the bphA1 mutants lost the ability to grow on biphenyl, the bphB and bphD mutants were able to transform a little of biphenyl, but hardly grew on biphenyl. CONCLUSION: The cloned bph genes indeed play an important role in the biphenyl catabolism in this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This bph gene organization in Rhodococcus sp. R04 differs from that of other biphenyl degraders reported previously, indicating it is a novel type of bph gene cluster. Analysis of the phylogenetic tree suggested that BphA1 and BphA2 in Rhodococcus sp. R04 had a different evolutionary relationship with those in the other PCB degraders.     

Sphingobium fuliginis HC3: A Novel and Robust Isolated Biphenyl- and Polychlorinated Biphenyls-Degrading Bacterium without Dead-End Intermediates Accumulation

Biphenyl and polychlorinated biphenyls (PCBs) are typical environmental pollutants. However, these pollutants are hard to be totally mineralized by environmental microorganisms. One reason for this is the accumulation of dead-end intermediates during biphenyl and PCBs biodegradation, especially benzoate and chlorobenzoates (CBAs). Until now, only a few microorganisms have been reported to have the ability to completely mineralize biphenyl and PCBs. In this research, a novel bacterium HC3, which could degrade biphenyl and PCBs without dead-end intermediates accumulation, was isolated from PCBs-contaminated soil and identified as Sphingobium fuliginis. Benzoate and 3-chlorobenzoate (3-CBA) transformed from biphenyl and 3-chlorobiphenyl (3-CB) could be rapidly degraded by HC3. This strain has strong degradation ability of biphenyl, lower chlorinated (mono-, di- and tri-) PCBs as well as mono-CBAs, and the biphenyl/PCBs catabolic genes of HC3 are cloned on its plasmid. It could degrade 80.7% of 100 mg L ⁻¹ biphenyl within 24 h and its biphenyl degradation ability could be enhanced by adding readily available carbon sources such as tryptone and yeast extract. As far as we know, HC3 is the first reported that can degrade biphenyl and 3-CB without accumulation of benzoate and 3-CBA in the genus Sphingobium, which indicates the bacterium has the potential to totally mineralize biphenyl/PCBs and might be a good candidate for restoring biphenyl/PCBs-polluted environments.     

Polychlorinated biphenyl/biphenyl degrading gene clusters in Rhodococcus sp. K37, HA99, and TA431 are different from well-known bph gene clusters of Rhodococci

Four kinds of polychlorinated biphenyl (PCB)-degrading Rhodococcus sp. (TA421, TA431, HA99, and K37) have been isolated from termite ecosystem and under alkaline condition. The bph gene cluster involved in the degradation of PCB/biphenyl has been analyzed in strain TA421. This gene cluster was highly homologous to bph gene clusters in R. globerulus P6 and Rhodococcus sp. RHA1. In this study, we cloned and analyzed the bph gene cluster essential to PCB/biphenyl degradation from R. rhodochrous K37. The order of the genes and the sequence were different in K37 than in P6, RHA1, and TA421. The bphC8(K37) gene was more homologous to the meta-cleavage enzyme involved in phenanthrene metabolism than bphC genes involved in biphenyl metabolism. Two other Rhodococcus strains (HA99 and TA431) had PCB/biphenyl degradation gene clusters similar to that in K37. These findings suggest that these bph gene clusters evolved separately from the well-known bph gene clusters of PCB/biphenyl degraders.     

Proteomic analysis of survival of Rhodococcus jostii RHA1 during carbon starvation

Rhodococcus jostii RHA1, a catabolically diverse soil actinomycete, is highly resistant to long-term nutrient starvation. After 2 years of carbon starvation, 10% of the bacterial culture remained viable. To study the molecular basis of such resistance, we monitored the abundance of about 1,600 cytosolic proteins during a 2-week period of carbon source (benzoate) starvation. Hierarchical cluster analysis elucidated 17 major protein clusters and showed that most changes occurred during transition to stationary phase. We identified 196 proteins. A decrease in benzoate catabolic enzymes correlated with benzoate depletion, as did induction of catabolism of alternative substrates, both endogenous (lipids, carbohydrates, and proteins) and exogenous. Thus, we detected a transient 5-fold abundance increase for phthalate, phthalate ester, biphenyl, and ethyl benzene catabolic enzymes, which coincided with at least 4-fold increases in phthalate and biphenyl catabolic activities. Stationary-phase cells demonstrated an ～250-fold increase in carbon monoxide dehydrogenase (CODH) concurrent with a 130-fold increase in CODH activity, suggesting a switch to CO or CO(2) utilization. We observed two phases of stress response: an initial response occurred during the transition to stationary phase, and a second response occurred after the cells had attained stationary phase. Although SigG synthesis was induced during starvation, a ΔsigG deletion mutant showed only minor changes in cell survival. Stationary-phase cells underwent reductive cell division. The extreme capacity of RHA1 to survive starvation does not appear to involve novel mechanisms; rather, it seems to be due to the coordinated combination of earlier-described mechanisms.     

基因工程菌Pseudomonas sp.B4的构建及其产邻苯二酚发酵条件的初步研究

利用PCR技术以Pseudomonas sp. B3-1基因组DNA为模板,扩增出2.9kb编码苯甲酸双加氧酶基因簇benABC。将该基因簇连接于pLAFRJ载体,电转化至E．coli DH5α,再通过三亲本结合法导入野生菌株Pseudomonas sp. B3-1中,得到了一株邻苯二酚产量提高的基因工程菌,命名为Pseudomonas sp.B4。发酵条件优化表明,当苯甲酸钠浓度为6.0 g/L,聚蛋白胨浓度为2.0 g/L,温度为32℃以及pH值为6.0时,工程菌在200rpm旋转摇床发酵36小时后,邻苯二酚产量达到0.7 mg/ml,比优化前提高了20％。     

Identification of a catabolic transposon, Tn4371, carrying biphenyl and 4-chlorobiphenyl degradation genes in Alcaligenes eutrophus A5.

Alcaligenes eutrophus A5 catabolizes biphenyl to CO2 via benzoate and 4-chlorobiphenyl to 4-chlorobenzoate. In curing and conjugation experiments, the A5 endogenous 51-kb IncP1 plasmid pSS50 was found to be dispensable for biphenyl and 4-chlorobiphenyl catabolism. Transfer of the biphenyl- and 4-chlorobiphenyl-degrading phenotype by means of pSS50 was observed at a frequency of 10(-5) per transferred plasmid in matings of A5 with other A. eutrophus strains. Transconjugants harbor enlarged pSS50 derivatives which contain additional genetic information governing the oxidation of biphenyl and 4-chlorobiphenyl to benzoate and 4-chlorobenzoate and originating from the chromosome of strain A5. The following observations indicate that the catabolic genes reside on a 59-kb large transposon (Tn4371) for which a restriction map is presented. (i) Tn4371 transposes between different replicons and at different locations of the same replicon. (ii) Transposition was observed in a Rec- strain of A. eutrophus. (iii) Tn4371 transposes as a single, contiguous piece of DNA. Although an RP4::Tn4371 plasmid was stably maintained in different hosts, the plasmid conferred growth on biphenyl only when present in strains of A. eutrophus and in an Acinetobacter sp. strain.     

Adventures in Rhodococcus - from steroids to explosives

Rhodococcus is a genus of mycolic-acid-containing actinomycetes that utilize a remarkable variety of organic compounds as growth substrates. This degradation helps maintain the global carbon cycle and has increasing applications ranging from the biodegradation of pollutants to the biocatalytic production of drugs and hormones. We have been using Rhodococcus jostii RHA1 as a model organism to understand the catabolic versatility of Rhodococcus and related bacteria. Our approach is exemplified by the discovery of a cluster of genes specifying the catabolism of cholesterol. This degradation proceeds via β-oxidative degradation of the side chain and O2-dependent cleavage of steroid ring A in a process similar to bacterial degradation of aromatic compounds. The pathway is widespread in Actinobacteria and is critical to the pathogenesis of Mycobacterium tuberculosis, arguably the world's most successful pathogen. The close similarity of some of these enzymes with biphenyl- and polychlorinated-biphenyl-degrading enzymes that we have characterized is facilitating inhibitor design. Our studies in RHA1 have also provided important insights into a number of novel metalloenzymes and their biosynthesis, such as acetonitrile hydratase (ANHase), a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. Molecular genetic and biochemical studies have identified AnhE as a dimeric metallochaperone that delivers cobalt to ANHase, enabling its maturation in vivo. Other metalloenzymes we are characterizing include N-acetylmuramic acid hydroxylase, which catalyzes an unusual hydroxylation of the rhodococcal and mycobacterial peptidoglycan, and 2 RHA1 dye-decolorizing peroxidases. Using molecular genetic and biochemical approaches, we have demonstrated that one of these enzymes is involved in the degradation of lignin. Overall, our studies are providing fundamental insights into a range of catabolic processes that have a wide variety of applications.     

Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp. strain B13.

DNA fragments containing the xylD and xylL genes of TOL plasmid pWW0 -161 of Pseudomonas putida, which code for the catabolic enzymes toluate 1,2-dioxygenase and dihydrodihydroxybenzoic acid dehydrogenase, respectively, and the nahG gene of the NAH plasmid NAH7 , which codes for salicylate hydroxylase, were cloned in pBR322 vector plasmid. Deletion and insertion mutagenesis were used to localize these genes with respect to crucial endonuclease cleavage sites. The pBR322-based plasmids were ligated to the broad host range cloning vector pKT231 , or derivatives of it, and the hybrid plasmids were introduced into Pseudomonas sp. B13( WR1 ), a bacterium able to degrade 3-chlorobenzoate but not 4-chlorobenzoate, 3,5- dichlorobenzoate , salicylate, or chlorosalicylates . The cloned xylD gene expanded the catabolic range of WR1 to include 4-chlorobenzoate, whereas the cloned xylD - xylL genes enabled the isolation of derivatives of WR1 that degraded 3-chlorobenzoate, 4-chlorobenzoate, and 3,5- dichlorobenzoate . The cloned nahG gene extended the catabolic range of WR1 to include salicylate and 3-, 4-, and 5- chlorosalicylate .     

Molecular cloning and expression of the 3-chlorobenzoate-degrading genes from Pseudomonas sp. strain B13.

The genes specifying the utilization of 3-chlorobenzoate by Pseudomonas sp. strain B13 WR1 have been cloned by using a broad-host-range cosmid cloning system. Analysis of the catabolic products of the enzymatic reactions encoded by two hybrid cosmids, pMW65 and pMW90, by thin-layer and high-performance liquid chromatography demonstrated that both encoded the genes for the complete catabolism of 3-chlorobenzoate. Physical analysis of one of the cosmid derivatives, pMW65, by restriction endonuclease mapping and subcloning demonstrated that the pathway genes are encoded on a fragment no larger than 11 kilobases.     

Multiplicity of aromatic ring hydroxylation dioxygenase genes in a strong PCB degrader, Rhodococcus sp. strain RHA1 demonstrated by denaturing gradient gel electrophoresis

To address the multiplicity of aromatic ring hydroxylation dioxygenases, we used PCR amplification and denaturing gradient gel electrophoresis (DGGE). The amplified DNA fragments separated into five bands, A to E. Southern hybridization analysis of RHA1 total DNA using the probes for each band showed that band C originated from a couple of homologous genes. The nucleotide sequences of the bands showed that bands A, C, and E would be parts of new dioxygenase genes in RHA1. That of band B agreed with the bphA1 gene, which was characterized previously. That of band D did not correspond to any known gene sequences. The regions including the entire open reading frames (ORFs) were cloned and sequenced. The nucleotide sequences of ORFs suggested that the genes of bands A, C, and E may respectively encode benzoate, biphenyl, and polyhydrocarbon dioxygenases. Northern hybridization indicated the induction of the gene of band A by benzoate and biphenyl, and that of the gene of band C by biphenyl and ethylbenzene, supporting the above notions. The gene of band E was not induced by any of these substrates. Thus the combination of DGGE and Southern hybridization enable us to address the multiplicity of the ring hydroxylation dioxygenase genes and to isolate some of them.     

Association of Phosphatidylcholine with Soybean 11S Globulin

To address the multiplicity of aromatic ring hydroxylation dioxygenases, we used PCR amplification and denaturing gradient gel electrophoresis (DGGE). The amplified DNA fragments separated into five bands, A to E. Southern hybridization analysis of RHA1 total DNA using the probes for each band showed that band C originated from a couple of homologous genes. The nucleotide sequences of the bands showed that bands A, C, and E would be parts of new dioxygenase genes in RHA1. That of band B agreed with the bphA1 gene, which was characterized previously. That of band D did not correspond to any known gene sequences. The regions including the entire open reading frames (ORFs) were cloned and sequenced. The nucleotide sequences of ORFs suggested that the genes of bands A,C, and E may respectively encode benzoate, biphenyl, and polyhydrocarbon dioxygenases. Northern hybridization indicated the induction of the gene of band A by benzoate and biphenyl, and that of the gene of band C by biphenyl and ethylbenzene, supporting the above notions. The gene of band E was not induced by any of these substrates. Thus the combination of DGGE and Southern hybridization enable us to address the multiplicity of the ring hydroxylation dioxygenase genes and to isolate some of them.     

Three of the seven bphC genes of Rhodococcus erythropolis TA421, isolated from a termite ecosystem, are located on an indigenous plasmid associated with biphenyl degradation.

Rhodococcus erythropolis TA421, a polychlorinated biphenyl and biphenyl degrader isolated from a termite ecosystem, has seven bphC genes expressing 2,3-dihydroxybiphenyl dioxygenase activity. R. erythropolis TA421 harbored a large and probably linear plasmid on which three (bphC2, bphC3, and bphC4) of the seven bphC genes were located. A non-biphenyl-degrading mutant, designated strain TA422, was obtained spontaneously from R. erythropolis TA421. TA422 lacked the plasmid, suggesting that the three bphC genes were involved in the degradation of biphenyl. Southern blot analyses showed that R. erythropolis TA421 and Rhodococcus globerulus P6 have a similar set of bphC genes and that the genes for biphenyl catabolism are located on plasmids of different sizes. These results indicated that the genes encoding the biphenyl catabolic pathway in Rhodococcus strains are borne on plasmids.     

Expression in Escherichia coli of biphenyl 2,3-dioxygenase genes from a Gram-positive polychlorinated biphenyl degrader, Rhodococcus jostii RHA1

Rhodococcus jostii RHA1 is a polychlorinated biphenyl degrader. Multi-component biphenyl 2,3-dioxygenase (BphA) genes of RHA1 encode large and small subunits of oxygenase component and ferredoxin and reductase components. They did not express enzyme activity in Escherichia coli. To obtain BphA activity in E. coli, hybrid BphA gene derivatives were constructed by replacing ferredoxin and/or reductase component genes of RHA1 with those of Pseudomonas pseudoalcaligenes KF707. The results obtained indicate a lack of catalytic activity of the RHA1 ferredoxin component gene, bphAc in E. coli. To determine the cause of inability of RHA1 bphAc to express in E. coli, the bphAc gene was introduced into Rosetta (DE3) pLacI, which has extra tRNA genes for rare codons in E. coli. The resulting strain abundantly produced the bphAc product, and showed activity. These results suggest that codon usage bias is involved in inability of RHA1 bphAc to express its catalytic activity in E. coli.