Characterization of the gene cluster involved in isoprene metabolism in Rhodococcus sp. strain AD45

The genes involved in isoprene (2-methyl-1,3-butadiene) utilization in Rhodococcus sp. strain AD45 were cloned and characterized. Sequence analysis of an 8.5-kb DNA fragment showed the presence of 10 genes of which 2 encoded enzymes which were previously found to be involved in isoprene degradation: a glutathione S-transferase with activity towards 1,2-epoxy-2-methyl-3-butene (isoI) and a 1-hydroxy-2-glutathionyl-2-methyl-3-butene dehydrogenase (isoH). Furthermore, a gene encoding a second glutathione S-transferase was identified (isoJ). The isoJ gene was overexpressed in Escherichia coli and was found to have activity with 1-chloro-2,4-dinitrobenzene and 3,4-dichloro-1-nitrobenzene but not with 1, 2-epoxy-2-methyl-3-butene. Downstream of isoJ, six genes (isoABCDEF) were found; these genes encoded a putative alkene monooxygenase that showed high similarity to components of the alkene monooxygenase from Xanthobacter sp. strain Py2 and other multicomponent monooxygenases. The deduced amino acid sequence encoded by an additional gene (isoG) showed significant similarity with that of alpha-methylacyl-coenzyme A racemase. The results are in agreement with a catabolic route for isoprene involving epoxidation by a monooxygenase, conjugation to glutathione, and oxidation of the hydroxyl group to a carboxylate. Metabolism may proceed by fatty acid oxidation after removal of glutathione by a still-unknown mechanism.     

Insights into the genetic diversity of initial dioxygenases from PAH-degrading bacteria

Alpha subunit genes of initial polyaromatic hydrocarbon (PAH) dioxygenases were used as targets for the PCR detection of PAH-degrading strains of the genera Pseudomonas, Comamonas and Rhodococcus which were obtained from activated sludge or soil samples. Sequence analysis of PCR products from several Pseudomonas strains showed that alpha subunits (nahAc allele) of this genus are highly conserved. PCR primers for the specific detection of alpha subunit genes of initial PAH dioxygenases from Pseudomonas strains were not suitable for detecting the corresponding genes from the genera Comamonas and Rhodococcus. Southern analysis using a heterologous gene probe derived from the P. putida OUS82 PAH dioxygenase alpha subunit identified segments of the PAH-degradation gene cluster from C. testosteroni strain H. Parts of this gene cluster containing three subunits of the initial PAH dioxygenase were isolated. These three subunits [ferredoxin (pahAb), alpha (pahAc) and beta (pahAd) subunit] were amplified by PCR as one fragment and expressed in Escherichia coli DH5alpha, resulting in an active initial dioxygenase with the ability to transform indole and phenanthrene. The DNA sequence alignment of alpha subunits from C. testosteroni H and various PAH-degrading bacteria permitted the design of new primers and oligonucleotide probes which are useful for the detection of the initial PAH dioxygenases from strains of Pseudomonas, Comamonas and Rhodococcus.     

Cloning, sequencing, and characterization of a gene cluster involved in EDTA degradation from the bacterium BNC1

EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.     

mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.     

Rapid detection and isolation of known and putative alpha-L-arabinofuranosidase genes using degenerate PCR primers

alpha-L-Arabinofuranosidases (AFases; EC 3.2.1.55) are exo-type enzymes, which hydrolyze terminal nonreducing arabinose residues from various polysaccharides such as arabinan and arabinoxylan. Genome-wide BLAST search showed that various bacterial strains possess the putative AFase genes with well-conserved motif sequences at the nucleotide and amino acid sequence levels. In this study, two sets of degenerate PCR primers were designed and tested to detect putative AFase genes, based on their three highly conserved amino acid blocks (PGGNFV, GNEMDG, and DEWNVW). Among 20 Bacillus-associated species, 13 species were revealed to have putative AFase genes in their genome and they share over 67% of amino acid identities with each other. Based on the partial sequence obtained from an isolate, an AFase from Geobacillus sp. was cloned and expressed in E. coli. Enzymatic characterization has verified that the resulting enzyme corresponds to a typical AFase. Accordingly, degenerate PCR primers developed in this work can be used for fast, easy, and specific detection and isolation of putative AFase genes from bacterial cells.     

Cloning and expression of the s-triazine hydrolase gene (trzA) from Rhodococcus corallinus and development of Rhodococcus recombinant strains capable of dealkylating and dechlorinating the herbicide atrazine.

We used degenerate oligodeoxyribonucleotides derived from the N-terminal sequence of the s-triazine hydrolase from Rhodococcus corallinus NRRL B-15444R in an amplification reaction to isolate a DNA segment containing a 57-bp fragment from the trzA gene. By using the nucleotide sequence of this fragment, a nondegenerate oligodeoxyribonucleotide was synthesized and used to screen a genomic library of R. corallinus DNA for fragments containing trzA. A 5.3-kb PstI fragment containing trzA was cloned, and the nucleotide sequence of a 2,450-bp region containing trzA was determined. No trzA expression was detected in Escherichia coli or several other gram-negative bacteria. The trzA gene was subcloned into a Rhodococcus-E. coli shuttle vector, pBS305, and transformed into several Rhodococcus strains. Expression of trzA was demonstrated in all Rhodococcus transformants. Rhodococcus sp. strain TE1, which possesses the catabolic gene (atrA) for the N-dealkylation of the herbicides atrazine and simazine, was able to dechlorinate the dealkylated metabolites of atrazine and simazine when carrying the trzA gene on a plasmid. A plasmid carrying both atrA and trzA was constructed and transformed into three atrA- and trzA-deficient Rhodococcus strains. Both genes were expressed in the transformants. The s-triazine hydrolase activity of the recombinant strains carrying the trzA plasmid were compared with that of the R. corallinus strain from which it was derived.     

The alkene monooxygenase from Xanthobacter strain Py2 is closely related to aromatic monooxygenases and catalyzes aromatic monohydroxylation of benzene, toluene, and phenol

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical. The genes and predicted polypeptides are aamA, encoding the 497-residue oxygenase alpha-subunit (XamoA); aamB, encoding the 88-residue oxygenase gamma-subunit (XamoB); aamC, encoding the 122-residue ferredoxin (XamoC); aamD, encoding the 101-residue coupling or effector protein (XamoD); aamE, encoding the 341-residue oxygenase beta-subunit (XamoE); and aamF, encoding the 327-residue reductase (XamoF). A sequence with >60% concurrence with the consensus sequence of sigma54 (RpoN)-dependent promoters was identified upstream of the aamA gene. Detailed comparison of XamoA with the oxygenase alpha-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase from Rhodococcus rhodochrous B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates. On the basis of the similarity between Xamo and the aromatic monooxygenases, Xanthobacter strain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not. Benzene was oxidized to phenol, which accumulated transiently before being further oxidized. Toluene was oxidized to a mixture of o-, m-, and p-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol, none of which were further oxidized. In growth studies Xanthobacter strain Py2 was found to grow on phenol and catechol but not on benzene or toluene; growth on phenol required a functional alkene monooxygenase. However, there is no evidence of genes encoding steps in the metabolism of catechol in the vicinity of the aam gene cluster. This suggests that the inducer specificity of the alkene monooxygenase may have evolved to benefit from the naturally broad substrate specificity of this class of monooxygenase and the ability of the host strain to grow on catechol.     

Cloning of contiguous genomic fragments from human chromosome 21 harbouring three trefoil peptide genes

A group of small peptides with a typical cysteine-rich domain (termed trefoil motif or P-domain) is abundantly expressed at mucosal surfaces of specific normal and neoplastic tissues. Their association with the maintenance of surface integrity was suggested. The first known human trefoil peptide (pS2) was isolated from breast cancer cells (MCF7). Its oestrogen-inducible gene, and the human homologue to the porcine spasmolytic peptide gene (hSP/SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be localised at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene were described recently. By using suitable oligonucleotide primers and PCR and isolating large (110–250 kb) genomic recombinants cloned in the bacterial artificial chromosome (BAC) system, we present a genomic region from chromosome band 21q22.3 cloned in contiguous sequences and encoding all three members of human P-domain/trefoil peptides proving a physical linkage of all three trefoil peptide genes. Such genomic sequences will provide useful experimental material for analysis of gene regulation, for gene modification experiments and for establishing transgenic cells or animals. Received: 2 January 1996 / Revised: 4 March 1996     

Polymerase chain reaction for identification of aldoxime dehydratase in aldoxime- or nitrile-degrading microorganisms

We developed a molecular screening procedure using Southern hybridization and polymerase chain reaction (PCR) to identify aldoxime dehydratase (Oxd) encoding genes (oxds) among 14 aldoxime- or nitrile-degrading microorganisms. When an oxd gene of Rhodococcus erythropolis N-771 was used as a probe, positive hybridization signals were seen with the chromosomal DNA of eight strains, suggesting that these strains have similar oxd genes to R. erythoropolis N-771. By analyzing the PCR-amplified fragments with degenerate consensus primers, the occurrence of homologous Oxd coexisting with Fe-containing NHase in the active eight strains was demonstrated coinciding with the results of Southern hybridization. Whole length of oxd gene was cloned as an example from one of the positive strains, Pseudomonas sp. K-9, sequenced, and expressed in E. coli. Analysis of the primary structure of the protein (OxdK) encoded by the oxd gene of Pseudomonas sp. K-9 led to identify an Oxd having a new primary structure. Thus, the PCR-based analysis of oxd gene is a useful tool to detect and analyze the "aldoxime-nitrile pathway" in nature, since Oxd is the key enzyme for the pathway.     

Cloning of the genes for degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine from Rhodococcus sp. strain TE1.

The degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine (2-chloro-4-ethyl-amino-6-isopropylamino-1,3,5-triazine) is associated with an indigenous plasmid in Rhodococcus sp. strain TE1. Plasmid DNA libraries of Rhodococcus sp. strain TE1 were constructed in a Rhodococcus-Escherichia coli shuttle vector, pBS305, and transferred into Rhodococcus sp. strain TE3, a derivative of Rhodococcus sp. strain TE1 lacking herbicide degradation activity, to select transformants capable of growing on EPTC as the sole source of carbon (EPTC+). Analysis of plasmids from the EPTC+ transformants indicated that the eptA gene, which codes for the enzyme required for EPTC degradation, residues on a 6.2-kb KpnI fragment. The cloned fragment also harbored the gene required for atrazine N dealkylation (atrA). The plasmid carrying the cloned fragment could be electroporated into a number of other Rhodococcus strains in which both eptA and atrA were fully expressed. No expression of the cloned genes was evident in E. coli strains. Subcloning of the 6.2-kb fragment to distinguish between EPTC- and atrazine-degrading genes was not successful.     

3α-羟类固醇脱氢酶基因的质粒载体构建和表达

目的 实现3α-羟类固醇脱氢酶基因在大肠埃希菌中的高可溶性表达.方法 从土壤中分离睾丸酮丛毛单胞菌,提取其基因组DNA,PCR扩增3α-羟类固醇脱氢酶(3α-HSD)基因,将它克隆到原核表达载体上进行诱导表达.提取细菌总蛋白进行SDS-PAGE分析并测定酶活性.结果 经核苷酸序列测定和酶切鉴定结果表明,成功地构建了重组质粒,IPTG诱导表达后,获得融合蛋白,SDS-PAGE初步测定目的蛋白的相对分子量约为29kDa,与预期理论值一致;酶活性测定结果表明菌体可溶性总蛋白HSD酶比活性为142.81 U/mg,是对照BL21的12.97倍.结论 该研究成功地构建了3α-羟类固醇脱氢酶基因高效原核表达系统,为利用基因工程手段大量制备3α-HSD的工作奠定了基础.     

Cloning and Characterization of Benzoate Catabolic Genes in the Gram-Positive Polychlorinated Biphenyl Degrader Rhodococcus sp. Strain RHA1

Benzoate catabolism is thought to play a key role in aerobic bacterial degradation of biphenyl and polychlorinated biphenyls (PCBs). Benzoate catabolic genes were cloned from a PCB degrader, Rhodococcus sp. strain RHA1, by using PCR amplification and temporal temperature gradient electrophoresis separation. A nucleotide sequence determination revealed that the deduced amino acid sequences encoded by the RHA1 benzoate catabolic genes, benABCDK, exhibit 33 to 65% identity with those of Acinetobacter sp. strain ADP1. The gene organization of the RHA1 benABCDK genes differs from that of ADP1. The RHA1 benABCDK region was localized on the chromosome, in contrast to the biphenyl catabolic genes, which are located on linear plasmids. Escherichia coli cells containing RHA1 benABCD transformed benzoate to catechol via 2-hydro-1,2-dihydroxybenzoate. They transformed neither 2- nor 4-chlorobenzoates but did transform 3-chlorobenzoate. The RHA1 benA gene was inactivated by insertion of a thiostrepton resistance gene. The resultant mutant strain, RBD169, neither grew on benzoate nor transformed benzoate, and it did not transform 3-chlorobenzoate. It did, however, exhibit diminished growth on biphenyl and growth repression in the presence of a high concentration of biphenyl (13 mM). These results indicate that the cloned benABCD genes could play an essential role not only in benzoate catabolism but also in biphenyl catabolism in RHA1. Six rhodococcal benzoate degraders were found to have homologs of RHA1 benABC. In contrast, two rhodococcal strains that cannot transform benzoate were found not to have RHA1 benABC homologs, suggesting that many Rhodococcus strains contain benzoate catabolic genes similar to RHA1 benABC.     

Molecular and culture-based analyses of aerobic carbon monoxide oxidizer diversity

Isolates belonging to six genera not previously known to oxidize CO were obtained from enrichments with aquatic and terrestrial plants. DNA from these and other isolates was used in PCR assays of the gene for the large subunit of carbon monoxide dehydrogenase (coxL). CoxL and putative coxL fragments were amplified from known CO oxidizers (e.g., Oligotropha carboxidovorans and Bradyrhizobium japonicum), from novel CO-oxidizing isolates (e.g., Aminobacter sp. strain COX, Burkholderia sp. strain LUP, Mesorhizobium sp. strain NMB1, Stappia strains M4 and M8, Stenotrophomonas sp. strain LUP, and Xanthobacter sp. strain COX), and from several well-known isolates for which the capacity to oxidize CO is reported here for the first time (e.g., Burkholderia fungorum LB400, Mesorhizobium loti, Stappia stellulata, and Stappia aggregata). PCR products from several taxa, e.g., O. carboxidovorans, B. japonicum, and B. fungorum, yielded sequences with a high degree (>99.6%) of identity to those in GenBank or genome databases. Aligned sequences formed two phylogenetically distinct groups. Group OMP contained sequences from previously known CO oxidizers, including O. carboxidovorans and Pseudomonas thermocarboxydovorans, plus a number of closely related sequences. Group BMS was dominated by putative coxL sequences from genera in the Rhizobiaceae and other alpha-PROTEOBACTERIA: PCR analyses revealed that many CO oxidizers contained two coxL sequences, one from each group. CO oxidation by M. loti, for which whole-genome sequencing has revealed a single BMS-group putative coxL gene, strongly supports the notion that BMS sequences represent functional CO dehydrogenase proteins that are related to but distinct from previously characterized aerobic CO dehydrogenases.     

Identification of two gene clusters involved in cyclohexanone oxidation in Brevibacterium epidermidis strain HCU

Brevibacterium epidermidis HCU can grow on cyclic ketones and alcohols as a sole carbon source. We have previously reported the identification of two cyclohexanone-induced Bayer-Villiger monooxygenase genes by mRNA differential display. Using the related technique of Out-PCR, we have amplified large DNA fragments flanking the two monooxygenase genes. Two large gene clusters were sequenced. Several ORFs in each gene cluster encoded proteins homologous to cyclohexanol and cyclohexanone oxidation enzymes from Acinetobacter. However, the structure of these two gene clusters differs significantly from that of Acinetobacter, where the complete pathway has been described. To assess activity of these genes, they were cloned and expressed in Escherichia coli. In vivo and in vitro assays enabled us to assign functions to the expressed ORFs. These ORFs included a cyclohexanol dehydrogenase, two different epsilon-caprolactone hydrolases and two 6-hydroxyhexanoate dehydrogenases belonging to different enzyme families. Because this environmental isolate is difficult to manipulate, we cannot determine at this time which cluster is involved in the degradation of cyclohexanone under physiological conditions. However, the original differential display experiments and some of the experiments reported here suggest the involvement of both gene clusters in the oxidation of cyclic ketones.     

高效产氢菌B49菌株adh和L-ldh基因克隆及序列分析

设计细菌adh基因和L-ldh基因简并引物,采用降落PCR并结合Cassette PCR方法从高效产氢菌B49中扩增全长基因.共获得两段基因组DNA片段.其中一段序列长1902 bp.包括adh基因开放阅读框 1101 bp,共编码366个氨基酸,编码产物分子量为 39.71 kD,理论等电点为pH5.93.该基因与Clostridium thermocellum ATCC 27405的adh位点序列一致性为73%.另一段序列长2490bp,包括L-ldh基因开放阅读框951 bp,共编码316个氨基酸,编码产物分子量为34.23 kD,理论等电点为pH 6.09.该基因与Bacillus megaterium的L-ldh位点一致性为74%.试验结果表明,采用CodeHop和Genefisher程序化设计的简并引物可信性强,阳性率高,CodeHop设计简并引物效果要好于Genefisher.adh和L-ldh的成功克隆为通过代谢工程手段敲除adh和L-ldh基因提供了科学依据,从而为进一步提高B49产氢能力的代谢工程研究奠定基础.     

AlkB homologues in thermophilic bacteria of the genus Geobacillus

Screening of alkane hydroxylase genes (alkB) was performed in the thermophilic aerobic bacteria of the genus Geobacillus. Total DNA was extracted from the biomass of 11 strains grown on the mixture of saturated C10-C20 hydrocarbons, PCR amplification of fragments of alkB genes was performed with degenerate oligonucleotide primers, PCR products were cloned and sequenced. For the first time in the genome of thermophilic bacteria the presence of a set of alkB gene homologues was revealed. The strains each contain three to six homologues among which only two are universal for all of the strains. Comparative phylogenetic analysis of the nucleotide sequences and the inferred amino acid sequences showed close relatedness of six of the revealed variants of geobacilli sequences to the alkB4, alkB3, and alkB2 genes that had previously been revealed by other authors in Rhodococcus erythropolis strains NRRL B-16531 and Q15. The rest two variants of alkB sequences were unique. Analysis of the GC composition of all the Geobacillus alkB homologues revealed closer proximity to the rhodococcal chromosomal DNA than to the chromosomal DNA of geobacilli. This may be an indication of the introduction of the alkB genes into the Geobacillus genome by interspecies horizontal transfer; and rhodococci or other representatives of the Actinobacteria phylum were probably the donors of these genes. Analysis of the codon usage in fragments of alkB genes confirms the suggestion that the pool of these genes is common to the majority of Gram-positive and certain Gram-negative bacteria. Formation of a set of several alkB homologues in a genome of a particular microorganism may result from free gene exchange within this pool.     

高效产氢菌B49菌株adh和L-ldh基因克隆及序列分析

设计细菌adh基因和L-ldh基因简并引物, 采用降落PCR并结合Cassette PCR方法从高效产氢菌B49中扩增全长基因。共获得两段基因组DNA片段。其中一段序列长1902 bp, 包括adh基因开放阅读框1101 bp, 共编码366个氨基酸, 编码产物分子量为39.71 kD, 理论等电点为pH 5.93。该基因与Clostridium thermocellum ATCC 27405的adh位点序列一致性为73%。另一段序列长2490 bp, 包括L-ldh基因开放阅读框951 bp, 共编码316个氨基酸, 编码产物分子量为34.23 kD, 理论等电点为pH 6.09。该基因与Bacillus megaterium的L-ldh位点一致性为74%。试验结果表明, 采用CodeHop和Genefisher程序化设计的简并引物可信性强, 阳性率高, CodeHop设计简并引物效果要好于Genefisher。adh和L-ldh的成功克隆为通过代谢工程手段敲除adh和L-ldh基因提供了科学依据, 从而为进一步提高B49产氢能力的代谢工程研究奠定基础。