A novel aromatic-ring-hydroxylating dioxygenase from the diterpenoid-degrading bacterium Pseudomonas abietaniphila BKME-9

Pseudomonas abietaniphila BKME-9 is able to degrade dehydroabietic acid (DhA) via ring hydroxylation by a novel dioxygenase. The ditA1, ditA2, and ditA3 genes, which encode the alpha and beta subunits of the oxygenase and the ferredoxin of the diterpenoid dioxygenase, respectively, were isolated and sequenced. The ferredoxin gene is 9. 2 kb upstream of the oxygenase genes and 872 bp upstream of a putative meta ring cleavage dioxygenase gene, ditC. A Tn5 insertion in the alpha subunit gene, ditA1, resulted in the accumulation by the mutant strain BKME-941 of the pathway intermediate, 7-oxoDhA. Disruption of the ferredoxin gene, ditA3, in wild-type BKME-9 by mutant-allele exchange resulted in a strain (BKME-91) with a phenotype identical to that of the mutant strain BKME-941. Sequence analysis of the putative ferredoxin indicated that it is likely to be a [4Fe-4S]- or [3Fe-4S]-type ferredoxin and not a [2Fe-2S]-type ferredoxin, as found in all previously described ring-hydroxylating dioxygenases. Expression in Escherichia coli of ditA1A2A3, encoding the diterpenoid dioxygenase without its putative reductase component, resulted in a functional enzyme. The diterpenoid dioxygenase attacks 7-oxoDhA, and not DhA, at C-11 and C-12, producing 7-oxo-11, 12-dihydroxy-8,13-abietadien acid, which was identified by 1H nuclear magnetic resonance, UV-visible light, and high-resolution mass spectrometry. The organization of the genes encoding the various components of the diterpenoid dioxygenase, the phylogenetic distinctiveness of both the alpha subunit and the ferredoxin component, and the unusual Fe-S cluster of the ferredoxin all suggest that this enzyme belongs to a new class of aromatic ring-hydroxylating dioxygenases.     

Diversity among aromatic hydrocarbon-degrading bacteria and their meta-cleavagegenes

Sixty-one strains of bacteria capable of growth on 4-methyl benzoic acid (29 isolates) ornaphthalene (32 isolates) as the sole source of carbon and energy were isolated from sedimentsand water samples from the River Tyne, UK. Random amplification of polymorphic DNA fromgenomic DNA extracted from the different strains demonstrated that 14 of the 4-methylbenzoate-degrading isolates were unique and the remainder fell into seven groups containing twoor three isolates that produced identical banding patterns. Thirteen of the naphthalene-degradingisolates were unique and nine groups with two or three identical representatives encompassed allother isolates. Screening of the bacterial strains for the presence of genes homologous to xylE , nahC and bphC by polymerase chain reaction and dot blot hybridizationdemonstrated that most strains harboured xylE - and/or nahC -like genes and only asingle isolate was found that did not harbour any of these genes. None of the isolates harboured bphC -like genes. It was concluded that, while considerable diversity existed in host strainsisolated using a single simple enrichment procedure, the extradiol dioxygenase genes involved inaromatic ring cleavage, present in these strains, were conserved to a considerable degree.     

Diversity of Burkholderia isolates from woodland rhizosphere environments

AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.     

Pseudomonas, the dominant polycyclic aromatic hydrocarbon-degrading bacteria isolated from Antarctic soils and the role of large plasmids in horizontal gene transfer

Twenty-two polycyclic aromatic hydrocarbon (PAH)-degrading bacterial strains were isolated from Antarctic soils with naphthalene or phenanthrene as a sole carbon source, while no degrader was obtained from an unpolluted sampling site. Phylogenetic analysis showed that all belonged to the genus Pseudomonas except one that was identified as the genus of Rahnella. Some of them were closely related to previously reported cold-tolerant species, while some were separated in deeply rooted branches and represent new strains. All these strains showed a high efficiency to degrade naphthalene at 4 degrees C, and some additionally degraded phenanthrene. Using degenerate primers and polymerase chain reaction (PCR) amplification, ndo gene encoding naphthalene dioxygenase (NDO) was detected from all the isolates. Phylogenetic analysis grouped these genes into two clusters which shared 94% similarity to each other, and showed about 97% similarity within a cluster. However, no obvious difference was observed with mesophilic ndo genes; this indicates that the host cell is pivotal in cold adaptation. In addition, the mismatch between 16S rRNA and NDO phylogenetic trees strongly indicates horizontal gene transfer among these isolates and may have happened in situ. Further, Southern hybridization and plasmid curing confirmed that ndo genes were located on a large self-transmissible plasmid, which can be transferred to a mesophilic strains. The transconjugants acquired the ability to utilize naphthalene and phenanthrene. Results of this article imply that Pseudomonas plays an important role in PAH biodegradation in Antarctic soils, and the related genes might be originally transferred from outside Antarctica and spread among indigenous species.     

Occurrence of Two Resin Acid-Degrading Bacteria and a Gene Encoding Resin Acid Biodegradation in Pulp and Paper Mill Effluent Biotreatment Systems Assayed by PCR

Abstract We examined the distribution of two dehydroabietic acid-degrading bacteria, Pseudomonas abietaniphila BKME-9 and Zoogloea resiniphila DhA-35, in biotreatment systems for pulp and paper mill effluents (PPMEs) using PCR assays. These two bacteria were first isolated from two PPME biotreatment systems and can degrade both dehydroabietic acid (DhA) and other abietane resin acids. We also examined the distribution of a catabolic gene, ditA1, encoding the α subunit of an aromatic ring-hydroxylating dioxygenase involved in DhA degradation by BKME-9. PCR primers specific for the 16S rDNA of BKME-9 and of DhA-35 and specific for ditA1 were used. Among 3 laboratory- and 17 full-scale PPME biotreatment systems, 10 contained phylotype BKME-9, 3 contained phylotype DhA-35, and 11 contained ditA1, indicating the wider distribution of phylotype BKME-9 than of phylotype DhA-35. Both phylotype BKME-9 and ditA1 were detected in the biotreatment system from which BKME-9 was originally isolated in 1994, suggesting the persistance of BKME-9 in that biotreatment system. The detection limit of the PCR assay was one cell per PCR reaction, which corresponds to one BKME-9 cell per 6 × 10⁷ total sludge bacteria. A competitive PCR assay indicated that ditA1 ranged from 51 to 250 copies/mg of dry biomass. BKME-9 appears to contribute to the biodegration of resin acids in some PPME biotreatment systems. Using degenerate PCR primers and touchdown PCR, we obtained from our DhA-degrading strain collection six DNA sequences putatively homologous to that of ditA1. Cluster analysis of these DNA sequences suggests that ditA1 encodes a representative of a novel class of dioxygenase enzymes. Received: 12 February 1999; Accepted: 4 May 1999     

三株Pseudomonas sp.的吡啶降解多样性分析

目的:为了探讨3株Pseudomonas sp.对吡啶降解存在多样性.方法:基于16S rRNA和ISR序列分析,对3株分离菌株进行初步鉴定,进而通过Touch -Down PCR,对3株细菌降解吡啶的多样性进行分析.结果:3株细菌XJUHX -1、XJUHX - 12和XJUHX - 16初步鉴定为Pseudomonas,3株实验菌株的部分降解基因的扩增条带有差异.结论:同属的3株 Pseudomonas在吡啶降解上存在多样性.     

Assessment of toluene/biphenyl dioxygenase gene diversity in benzene-polluted soils: links between benzene biodegradation and genes similar to those encoding isopropylbenzene dioxygenases

The PCR-single-strand conformation polymorphism (SSCP) technique was used to assess the diversity and distribution of Rieske nonheme iron oxygenases of the toluene/biphenyl subfamily in soil DNA and bacterial isolates recovered from sites contaminated with benzene, toluene, ethylbenzene, and xylenes (BTEX). The central cores of genes encoding the catalytic alpha subunits were targeted, since they are responsible for the substrate specificities of these enzymes. SSCP functional genotype fingerprinting revealed a substantial diversity of oxygenase genes in three differently BTEX-contaminated soil samples, and sequence analysis indicated that in both the soil DNA and the bacterial isolates, genes for oxygenases related to the isopropylbenzene (cumene) dioxygenase branch of the toluene/biphenyl oxygenase subfamily were predominant among the detectable genotypes. The peptide sequences of the two most abundant alpha subunit sequence types differed by only five amino acids (residues 258, 286, 288, 289, and 321 according to numbering in cumene dioxygenase alpha subunit CumA1 of Pseudomonas fluorescens IP01). However, a strong correlation between sequence type and substrate utilization pattern was observed in isolates harboring these genes. Two of these residues were located at positions contributing, according to the resolved crystal structure of cumene dioxygenase from Pseudomonas fluorescens IP01, to the inner surface of the substrate-binding pocket. Isolates containing an alpha subunit with isoleucine and leucine at positions 288 and 321, respectively, were capable of degrading benzene and toluene, whereas isolates containing two methionine substitutions were found to be incapable of degrading toluene, indicating that the more bulky methionine residues significantly narrowed the available space within the substrate-binding pocket.     

Diversity of the abundant pKLC102/PAGI-2 family of genomic islands in Pseudomonas aeruginosa

The known genomic islands of Pseudomonas aeruginosa clone C strains are integrated into tRNA(Lys) (pKLC102) or tRNA(Gly) (PAGI-2 and PAGI-3) genes and differ from their core genomes by distinctive tetranucleotide usage patterns. pKLC102 and the related island PAPI-1 from P. aeruginosa PA14 were spontaneously mobilized from their host chromosomes at frequencies of 10% and 0.3%, making pKLC102 the most mobile genomic island known with a copy number of 30 episomal circular pKLC102 molecules per cell. The incidence of islands of the pKLC102/PAGI-2 type was investigated in 71 unrelated P. aeruginosa strains from diverse habitats and geographic origins. pKLC102- and PAGI-2-like islands were identified in 50 and 31 strains, respectively, and 15 and 10 subtypes were differentiated by hybridization on pKLC102 and PAGI-2 macroarrays. The diversity of PAGI-2-type islands was mainly caused by one large block of strain-specific genes, whereas the diversity of pKLC102-type islands was primarily generated by subtype-specific combination of gene cassettes. Chromosomal loss of PAGI-2 could be documented in sequential P. aeruginosa isolates from individuals with cystic fibrosis. PAGI-2 was present in most tested Cupriavidus metallidurans and Cupriavidus campinensis isolates from polluted environments, demonstrating the spread of PAGI-2 across habitats and species barriers. The pKLC102/PAGI-2 family is prevalent in numerous beta- and gammaproteobacteria and is characterized by high asymmetry of the cDNA strands. This evolutionarily ancient family of genomic islands retained its oligonucleotide signature during horizontal spread within and among taxa.     

Detection of polycyclic aromatic hydrocarbon degradation genes in different soil bacteria by polymerase chain reaction and DNA hybridization

Twenty different strains of Pseudomonas, Mycobacterium, Gordona, Sphingomonas, Rhodococcus and Xanthomonas which degrade polycyclic aromatic hydrocarbons (PAH) were characterized in respect to genes encoding degradation enzymes for PAH. Genomic DNA from these strains was hybridized with a fragment of ndoB, coding for the large iron sulfur protein (ISP alpha) of the naphthalene dioxygenase from Pseudomonas putida PaW736 (NCIB 9816). A group of seven naphthalene-degrading Pseudomonas strains showed strong hybridization with the ndoB probe, and five Gordona, Mycobacterium, Rhodococcus and Pseudomonas strains able to degrade higher molecular weight PAH showed weaker hybridization signals. Using a polymerase chain reaction (PCR) approach, seven naphthalene-degrading Pseudomonas strains showed a PCR fragment of the expected size with ndoB-specific primers and additionally ten strains of Gordona, Mycobacterium, Pseudomonas, Sphingomonas and Xanthomonas able to degrade higher molecular weight PAH were detected with degenerate primer-pools specific for the ISP alpha [2Fe-2S]-Rieske center of diverse aromatic hydrocarbon dioxygenases. This suggests a molecular relationship between genes coding for PAH catabolism in various PAH-degrading bacterial taxa, which could be used to evaluate the PAH-degradation potential of mixed populations.     

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.     

Assessment of Toluene/Biphenyl Dioxygenase Gene Diversity in Benzene-Polluted Soils: Links between Benzene Biodegradation and Genes Similar to Those Encoding Isopropylbenzene Dioxygenases

The PCR-single-strand conformation polymorphism (SSCP) technique was used to assess the diversity and distribution of Rieske nonheme iron oxygenases of the toluene/biphenyl subfamily in soil DNA and bacterial isolates recovered from sites contaminated with benzene, toluene, ethylbenzene, and xylenes (BTEX). The central cores of genes encoding the catalytic α subunits were targeted, since they are responsible for the substrate specificities of these enzymes. SSCP functional genotype fingerprinting revealed a substantial diversity of oxygenase genes in three differently BTEX-contaminated soil samples, and sequence analysis indicated that in both the soil DNA and the bacterial isolates, genes for oxygenases related to the isopropylbenzene (cumene) dioxygenase branch of the toluene/biphenyl oxygenase subfamily were predominant among the detectable genotypes. The peptide sequences of the two most abundant α subunit sequence types differed by only five amino acids (residues 258, 286, 288, 289, and 321 according to numbering in cumene dioxygenase α subunit CumA1 of Pseudomonas fluorescens IP01). However, a strong correlation between sequence type and substrate utilization pattern was observed in isolates harboring these genes. Two of these residues were located at positions contributing, according to the resolved crystal structure of cumene dioxygenase from Pseudomonas fluorescens IP01, to the inner surface of the substrate-binding pocket. Isolates containing an α subunit with isoleucine and leucine at positions 288 and 321, respectively, were capable of degrading benzene and toluene, whereas isolates containing two methionine substitutions were found to be incapable of degrading toluene, indicating that the more bulky methionine residues significantly narrowed the available space within the substrate-binding pocket.     

Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. nodule bacteria on two Mimosa spp. in Costa Rica

rRNA gene sequencing and PCR assays indicated that 215 isolates of root nodule bacteria from two Mimosa species at three sites in Costa Rica belonged to the genera Burkholderia, Cupriavidus, and Rhizobium. This is the first report of Cupriavidus sp. nodule symbionts for Mimosa populations within their native geographic range in the neotropics. Burkholderia spp. predominated among samples from Mimosa pigra (86% of isolates), while there was a more even distribution of Cupriavidus, Burkholderia, and Rhizobium spp. on Mimosa pudica (38, 37, and 25% of isolates, respectively). All Cupriavidus and Burkholderia genotypes tested formed root nodules and fixed nitrogen on both M. pigra and M. pudica, and sequencing of rRNA genes in strains reisolated from nodules verified identity with inoculant strains. Inoculation tests further indicated that both Cupriavidus and Burkholderia spp. resulted in significantly higher plant growth and nodule nitrogenase activity (as measured by acetylene reduction assays) relative to plant performance with strains of Rhizobium. Given the prevalence of Burkholderia and Cupriavidus spp. on these Mimosa legumes and the widespread distribution of these plants both within and outside the neotropics, it is likely that both beta-proteobacterial genera are more ubiquitous as root nodule symbionts than previously believed.     

Molecular detection and diversity of polycyclic aromatic hydrocarbon-degrading bacteria isolated from geographically diverse sites

Nineteen polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from environmental samples in Kuwait, Indonesia, Thailand, and Japan by enrichment with either naphthalene or phenanthrene as a sole carbon source. Sequence analyses of the 16-S rRNA gene indicated that at least seven genera (Ralstonia, Sphingomonas, Burkholderia, Pseudomonas, Comamonas, Flavobacterium, and Bacillus) were present in this collection. Determination of the ability of the isolates to use PAH and its presumed catabolic intermediates suggests that the isolates showed multiple phenotypes in terms of utilization and degradation pathways. The large subunit of the terminal oxygenase gene (phnAc) from Burkholderia sp. strain RP007 hybridized to 32% (6/19) of the isolates, whilst gene probing using the large subunit of terminal oxygenase gene (pahAc) from Pseudomonas putida strain OUS82 revealed no pahAc-like genes amongst the isolates. Using three degenerated primer sets (pPAH-F/NR700, AJ025/26, and RieskeF/R), targeting a conserved region with the genes encoding the large subunit of terminal oxygenase successfully amplified material from 6 additional PAH-degrading isolates. Sequence analyses showed that the large subunit of terminal oxygenase in 4 isolates was highly homologous to the large subunit of naphthalene dioxygenase gene from Ralstonia sp. strain U2. However, we could not obtain any information on the oxygenase system involved in the naphthalene and/or phenathrene degradation by 7 other strains. These results suggest that PAH-degrading bacteria are diverse, and that there are still many unidentified PAH-degrading bacteria.     

Variations in the gene for vacuolating toxin, vacA, in Japanese isolates of Helicobacter pylori.

Each of 284 strains of Helicobacter pylori which had been isolated in Japan was shown, by use of the polymerase chain reaction (PCR), to be positive for the vacA genes. The amplified vacA genes generated by PCR were classified into six classes (five for the clinical isolates, and one which corresponded to the standard strains). Endoscopic analysis revealed that cases of gastritis were most likely to be associated with class D, while none were associated with class A. The patterns of products of PCR obtained from the Japanese isolates were compared with theoretical patterns derived from sequences of vacA which had been reported previously. The nucleotide sequences of amplified fragments of vacA from representative strains in each class were determined and compared with those of previously reported vacA genes.     

Characterization of Rhizobium loti strains from the Salado River Basin

Thirty indigenous rhizobia strains, isolated from Lotus tenuis in the area of Chascomús and other regions of the Salado River Basin (Argentina), were characterized based on generation time, acid production, carbon utilization, protein profile, and molecular characterization by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by the polymerase chain reaction (PCR). The results indicated that native rhizobia isolates from the Chascomús area are predominantly fast and intermediate-growers. The unclassified rhizobia examined by PCR-RFLP were found to be closely related to the reference strains of validly described Rhizobium species.     

Molecular characterization of dioxygenases from polycyclic aromatic hydrocarbon-degrading Mycobacterium spp

Polycyclic aromatic hydrocarbon (PAH)-degrading genes nidA and nidB that encode the alpha and beta subunits of the aromatic ring-hydroxylating dioxygenase have been cloned and sequenced from Mycobacterium vanbaalenii PYR-1 [Khan et al., Appl. Environ Microbiol. 67 (2001) 3577-3585]. In this study, the presence of nidA and nidB in 12 other Mycobacterium or Rhodococcus strains was investigated. Initially, all strains were screened for their ability to degrade PAHs by a spray plate method, and for the presence of the dioxygenase Rieske center region by polymerase chain reaction (PCR). Only Mycobacterium sp. PAH 2.135 (RJGII-135), M. flavescens PYR-GCK (ATCC 700033), M. gilvum BB1 (DSM 9487) and M. frederiksbergense FAn9T (DSM 44346), all previously known PAH degraders, were positive in both tests. From the three positive strains, complete open reading frames of the nidA and nidB genes were amplified by PCR, using primers designed according to the known nidA and nidB sequences from PYR-1, cloned in the pBAD/Thio-TOPO vector and sequenced. The sequences showed >98% identity with the M. vanbaalenii PYR-1 nidA and nidB genes. Southern DNA-DNA hybridization using nidA and nidB probes from PYR-1 revealed that there is more than one copy of nidA and nidB genes in the strains PYR-1, BB1, PYR-GCK and FAn9T. However, only one copy of each gene was observed in PAH2.135.