Novel forms of ring-hydroxylating dioxygenases are widespread in pristine and contaminated soils

Ring-hydroxylating dioxygenases (RHDs) are of central importance to bacterial recycling of aromatic hydrocarbons, including anthropogenic pollutants. The database of presently characterized RHDs is biased towards those from organisms readily isolated on anthropogenic substrates. To investigate the extent to which RHDs from extant organisms reflect the natural diversity of these enzymes, we developed a polymerase chain reaction (PCR) method for retrieval of RHD gene fragments from environmental samples. Gene libraries from two contaminated and two pristine soil samples were constructed. None of the inferred peptides from clones examined were identical to previously described RHDs; however, all showed significant sequence homology and contained key catalytic residues. On the basis of sequence identity, the environmental clones clustered into six distinct groups, only one of which included known RHDs. One of the new sequence groupings was particularly widespread, being recovered from all soil samples tested. Comparison of inferred peptide sequences of the environmental clones and known RHDs showed the former to have greater sequence variation at sites thought to influence accessibility of the active site than that seen between currently known RHDs. We conclude that presently characterized RHDs do not adequately represent the diversity of function found in in situ forms.     

Diversity and catalytic potential of PAH-specific ring-hydroxylating dioxygenases from a hydrocarbon-contaminated soil

Ring-hydroxylating dioxygenases (RHDs) catalyze the initial oxidation step of a range of aromatic hydrocarbons including polycyclic aromatic hydrocarbons (PAHs). As such, they play a key role in the bacterial degradation of these pollutants in soil. Several polymerase chain reaction (PCR)-based methods have been implemented to assess the diversity of RHDs in soil, allowing limited sequence-based predictions on RHD function. In the present study, we developed a method for the isolation of PAH-specific RHD gene sequences of Gram-negative bacteria, and for analysis of their catalytic function. The genomic DNA of soil PAH degraders was labeled in situ by stable isotope probing, then used to PCR amplify sequences specifying the catalytic domain of RHDs. Sequences obtained fell into five clusters phylogenetically linked to RHDs from either Sphingomonadales or Burkholderiales. However, two clusters comprised sequences distantly related to known RHDs. Some of these sequences were cloned in-frame in place of the corresponding region of the phnAIa gene from Sphingomonas CHY-1 to generate hybrid genes, which were expressed in Escherichia. coli as chimerical enzyme complexes. Some of the RHD chimeras were found to be competent in the oxidation of two- and three-ring PAHs, but other appeared unstable. Our data are interpreted in structural terms based on 3D modeling of the catalytic subunit of hybrid RHDs. The strategy described herein might be useful for exploring the catalytic potential of the soil metagenome and recruit RHDs with new activities from uncultured soil bacteria.     

Characterization of hydrocarbon-degrading microbial populations in contaminated and pristine Alpine soils

Biodegradation of petroleum hydrocarbons in cold environments, including Alpine soils, is a result of indigenous cold-adapted microorganisms able to degrade these contaminants. In the present study, the prevalence of seven genotypes involved in the degradation of n-alkanes (Pseudomonas putida GPo1 alkB; Acinetobacter spp. alkM; Rhodococcus spp. alkB1, and Rhodococcus spp. alkB2), aromatic hydrocarbons (P. putida xylE), and polycyclic aromatic hydrocarbons (P. putida ndoB and Mycobacterium sp. strain PYR-1 nidA) was determined in 12 oil-contaminated (428 to 30,644 mg of total petroleum hydrocarbons [TPH]/kg of soil) and 8 pristine Alpine soils from Tyrol (Austria) by PCR hybridization analyses of total soil community DNA, using oligonucleotide primers and DNA probes specific for each genotype. The soils investigated were also analyzed for various physical, chemical, and microbiological parameters, and statistical correlations between all parameters were determined. Genotypes containing genes from gram-negative bacteria (P. putida alkB, xylE, and ndoB and Acinetobacter alkM) were detected to a significantly higher percentage in the contaminated (50 to 75%) than in the pristine (0 to 12.5%) soils, indicating that these organisms had been enriched in soils following contamination. There was a highly significant positive correlation (P < 0.001) between the level of contamination and the number of genotypes containing genes from P. putida and Acinetobacter sp. but no significant correlation between the TPH content and the number of genotypes containing genes from gram-positive bacteria (Rhodococcus alkB1 and alkB2 and Mycobacterium nidA). These genotypes were detected at a high frequency in both contaminated (41.7 to 75%) and pristine (37.5 to 50%) soils, indicating that they are already present in substantial numbers before a contamination event. No correlation was found between the prevalence of hydrocarbon-degradative genotypes and biological activities (respiration, fluorescein diacetate hydrolysis, lipase activity) or numbers of culturable hydrocarbon-degrading soil microorganisms; there also was no correlation between the numbers of hydrocarbon degraders and the contamination level. The measured biological activities showed significant positive correlation with each other, with the organic matter content, and partially with the TPH content and a significant negative correlation with the soil dry-mass content (P < 0.05 to 0.001).     

Bioremediation of oil by marine microbial mats

Cyanobacterial mats developing in oil-contaminated sabkhas along the African coasts of the Gulf of Suez and in the pristine Solar Lake, Sinai, were collected for laboratory studies. Samples of both mats showed efficient degradation of crude oil in the light, followed by development of an intense bloom of Phormidium spp. and Oscillatoria spp. Isolated cyanobacterial strains, however, did not degrade crude oil in axenic cultures. Strains of sulfate-reducing bacteria and aerobic heterotrophs were capable of degrading model compounds of aliphatic and aromatic hydrocarbons. Results indicate that degradation of oil was done primarily by aerobic heterotrophic bacteria. The oxygenic photosynthesis of oil-insensitive cyanobacteria supplied the molecular oxygen for the efficient aerobic metabolism of organisms, such as Marinobacter sp. The diurnal shifts in environmental conditions at the mat surface, from highly oxic conditions in the light to anaerobic sulfide-rich habitat in the dark, may allow the combined aerobic and anaerobic degradation of crude oil at the mat surface. Hence, coastal cyanobacterial mats may be used for the degradation of coastline oil spills. Oxygen microelectrodes detected a significant inhibition of photosynthetic activity subsequent to oil addition. This prevailed for a few hours and then rapidly recovered. In addition, shifts in bacterial community structure following exposure to oil were determined by denaturing gradient gel electrophoresis of PCR-amplified fractions of 16S rRNA from eubacteria, cyanobacteria and sulfate-reducing bacteria. Since the mats used for the present study were obtained from oil-contaminated environments, they were believed to be preequilibrated for petroleum remediation. The mesocosm system at Eilat provided a unique opportunity to study petroleum degradation by mats formed under different salinities (up to 21%). These mats, dominated by cyanobacteria, can serve as close analogues to the sabkhas contaminated during the Gulf War in Kuwait and Saudi Arabia. Electronic Publication     

Roles of ring-hydroxylating dioxygenases in styrene and benzene catabolism in Rhodococcus jostii RHA1

Proteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene in Rhodococcus jostii RHA1, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the ΔbphAa and etbAa1::cmrA etbAa2::aphII mutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion of pcaL of the β-ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded via meta- and ortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latter's initial transformation. The stress response may augment RHA1's ability to degrade PCBs and other pollutants that induce similar uncoupling.     

Multiplicity of genes for aromatic ring-hydroxylating dioxygenases in Mycobacterium isolate KMS and their regulation

Mycobacterium sp. strain KMS has bioremediation potential for polycyclic aromatic hydrocarbons (PAHs), such as pyrene, and smaller ring aromatics, such as benzoate. Degradation of these aromatics involves oxidation catalyzed by aromatic ring-hydroxylating dioxygenases. Multiple genes encoding dioxygenases exist in KMS: ten genes encode large-subunits with homology to phenylpropionate dioxygenase genes, sixteen pairs of adjacent genes encode alpha- and beta-subunits of dioxygenase and two genes encode beta-subunits. These genes include orthologs of nid genes essential for degradation of multi-ring PAHs in M. vanbaalenii isolate PYR-1. The multiplicity of genes in part is explained by block duplication that results in two or three copies of certain genes on the chromosome, a linear plasmid, and a circular plasmid within the KMS genome. Quantitative real-time PCR showed that four dioxygenase beta-subunit nid genes from operons with almost identical promoter sequences otherwise unique in the genome were induced by pyrene to similar extents. No induction occurred with benzoate. Unlike isolate PYR-1, isolate KMS has an operon specifying benzoate catabolism and the expression of the alpha-subunit dioxygenase gene was activated by benzoate but not pyrene. These studies showed that isolate KMS had a genome well adapted to utilization of different aromatic compounds.     

Diversity and distribution in hypersaline microbial mats of bacteria related to Chloroflexus spp

Filamentous bacteria containing bacteriochlorophylls c and a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to Chloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of "green nonsulfur bacteria." PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to Chloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.     

Detection of known and novel genes encoding aromatic ring-hydroxylating dioxygenases in soils and in aromatic hydrocarbon-degrading bacteria

The 14-3-3 family of proteins function as small adaptors that facilitate a diverse array of cellular processes by mediating specific protein interactions. One such process is the DNA damage checkpoint, where these proteins prevent inappropriate activation of cyclin-dependent kinases. The filamentous fungus Aspergillus nidulans possesses a highly conserved 14-3-3 homologue (artA) that may function in an analogous manner to prevent septum formation. However, instead of blocking septation, over-expression of artA causes a severe delay in the polarization of conidiospores. This observation suggests that these proteins play an important role in hyphal morphogenesis.     

Molecular analysis of microbial community structures in pristine and contaminated aquifers: field and laboratory microcosm experiments

This study used phylogenetic probes in hybridization analysis to (i) determine in situ microbial community structures in regions of a shallow sand aquifer that were oxygen depleted and fuel contaminated (FC) or aerobic and noncontaminated (NC) and (ii) examine alterations in microbial community structures resulting from exposure to toluene and/or electron acceptor supplementation (nitrate). The latter objective was addressed by using the NC and FC aquifer materials for anaerobic microcosm studies in which phylogenetic probe analysis was complemented by microbial activity assays. Domain probe analysis of the aquifer samples showed that the communities were predominantly Bacteria; Eucarya and Archaea were not detectable. At the phylum and subclass levels, the FC and NC aquifer material had similar relative abundance distributions of 43 to 65% beta- and gamma-Proteobacteria (B+G), 31 to 35% alpha-Proteobacteria (ALF), 15 to 18% sulfate-reducing bacteria, and 5 to 10% high G+C gram positive bacteria. Compared to that of the NC region, the community structure of the FC material differed mainly in an increased abundance of B+G relative to that of ALF. The microcosm communities were like those of the field samples in that they were predominantly Bacteria (83 to 101%) and lacked detectable Archaea but differed in that a small fraction (2 to 8%) of Eucarya was detected regardless of the treatment applied. The latter result was hypothesized to reflect enrichment of anaerobic protozoa. Addition of nitrate and/or toluene stimulated microbial activity in the microcosms, but only supplementation of toluene alone significantly altered community structures. For the NC material, the dominant subclass shifted from B+G to ALF, while in the FC microcosms 55 to 65% of the Bacteria community was no longer identifiable by the phylum or subclass probes used. The latter result suggested that toluene exposure fostered the proliferation of phylotype(s) that were otherwise minor constituents of the FC aquifer community. These studies demonstrated that alterations in aquifer microbial communities resulting from specific anthropogenic perturbances can be inferred from microcosm studies integrating chemical and phylogenetic probe analysis and in the case of hydrocarbon contamination may facilitate the identification of organisms important for in situ biodegradation processes. Further work integrating and coordinating microcosm and field experiments is needed to explore how differences in scale, substrate complexity, and other hydrogeological conditions may affect patterns observed in these systems.     

Diversity of phototrophic bacteria in microbial mats from Arctic hot springs (Greenland)

We investigated the genotypic diversity of oxygenic and anoxygenic phototrophic microorganisms in microbial mat samples collected from three hot spring localities on the east coast of Greenland. These hot springs harbour unique Arctic microbial ecosystems that have never been studied in detail before. Specific oligonucleotide primers for cyanobacteria, purple sulfur bacteria, green sulfur bacteria and Choroflexus/Roseiflexus-like green non-sulfur bacteria were used for the selective amplification of 16S rRNA gene fragments. Amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced. In addition, several cyanobacteria were isolated from the mat samples, and classified morphologically and by 16S rRNA-based methods. The cyanobacterial 16S rRNA sequences obtained from DGGE represented a diverse, polyphyletic collection of cyanobacteria. The microbial mat communities were dominated by heterocystous and non-heterocystous filamentous cyanobacteria. Our results indicate that the cyanobacterial community composition in the samples were different for each sampling site. Different layers of the same heterogeneous mat often contained distinct and different communities of cyanobacteria. We observed a relationship between the cyanobacterial community composition and the in situ temperatures of different mat parts. The Greenland mats exhibited a low diversity of anoxygenic phototrophs as compared with other hot spring mats which is possibly related to the photochemical conditions within the mats resulting from the Arctic light regime.     

Diversity of 746 heterotrophic bacteria isolated from microbial mats from ten Antarctic lakes

Microbial mats, growing in Antarctic lakes constitute unique and very diverse habitats. In these mats microorganisms are confronted with extreme life conditions. We isolated 746 bacterial strains from mats collected from ten lakes in the Dry Valleys (lakes Hoare and Fryxell), the Vestfold Hills (lakes Ace, Druzhby, Grace, Highway, Pendant, Organic and Watts) and the Larsemann Hills (lake Reid), using heterotrophic growth conditions. These strains were investigated by fatty acid analysis, and by numerical analysis, 41 clusters, containing 2 to 77 strains, could be delineated, whereas 31 strains formed single branches. Several fatty acid groups consisted of strains from different lakes from the same region, or from different regions. The 16S rRNA genes from 40 strains, representing 35 different fatty acid groups were sequenced. The strains belonged to the alpha, beta and gamma subclasses of the Proteobacteria, the high and low percent G+C Gram-positives, and to the Cytophaga-Flavobacterium-Bacteroides branch. For strains representing 16 fatty acid clusters, validly named nearest phylogenetic neighbours showed pairwise sequence similarities of less than 97%. This indicates that the clusters they represent, belong to taxa that have not been sequenced yet or as yet unnamed new taxa, related to Alteromonas, Bacillus, Clavibacter, Cyclobacterium, Flavobacterium, Marinobacter, Mesorhizobium, Microbacterium, Pseudomonas, Saligentibacter, Sphingomonas and Sulfitobacter.     

The relationship between the microbial activity of the autochthonous microorganisms of pristine and contaminated soils and their potential for the degradation of mineral oil hydrocarbons

The microbial activity of pristine and contaminated soils was investigated by measuring the following parameters: glucose induced respiration, dimethylsulphoxide reduction and the hydrolysis of fluorescein diacetate. The viable counts were determined by the plate count method. The ability of the autochthonous microorganisms of the investigated soils to degrade diesel fuel was determined in a closed system on the basis of the oxygen consumption and by direct measurements of the hydrocarbon concentrations. As expected, compost showed the highest microbial activity with regard to all three parameters, followed by the grassland and the arable soil samples which were also found to have high activity. However, soils that had been exposed to mineral oil for a long period of time showed significantly lower values. Microorganisms from contaminated sites had a high degradation potential; few pristine soils reached similar turnover rates. The investigations showed that the level of the degradation of diesel fuel in pristine soils correlated with their microbial activity, but this correlation was not found in the investigated contaminated soils.     

Rapid microbial production of filamentous sulfur mats at hydrothermal vents

During recent oceanographic cruises to Pacific hydrothermal vent sites (9 degrees N and the Guaymas Basin), the rapid microbial formation of filamentous sulfur mats by a new chemoautotrophic, hydrogen sulfide-oxidizing bacterium was documented in both in situ and shipboard experiments. Observations suggest that formation of these sulfur mats may be a factor in the initial colonization of hydrothermal surfaces by macrofaunal Alvinella worms. This novel metabolic capability, previously shown to be carried out by a coastal strain in H2S continuous-flow reactors, may be an important, heretofore unconsidered, source of microbial organic matter production at deep-sea hydrothermal vents.     

Comparative genomic analysis of pyrene-degrading <Emphasis Type="Italic">Mycobacterium</Emphasis> species: Genomic islands and ring-hydroxylating dioxygenases involved in pyrene degradation

The genome sequences of two pyrene-degrading bacterial strains of Mycobacterium spp. PYR10 and PYR15, isolated from the estuarine wetland of the Han river, South Korea, were determined using the PacBio RS II sequencing platform. The complete genome of strain PYR15 was 6,037,017 bp in length with a GC content of 66.5%, and contained 5,933 protein-coding genes. The genome of strain PYR10 was 5,999,427 bp in length with a GC content of 67.7%, and contained 5,767 protein-coding genes. Based on the average nucleotide identity values, these strains were designated as M. gilvum PYR10 and M. pallens PYR15. A genomic comparison of these pyrene-degrading Mycobacterium strains with pyrene-non-degrading strains revealed that the genomes of pyrene-degrading strains possessed similar repertoires of ringhydroxylating dioxygenases (RHDs), including the pyrenehydroxylating dioxygenases encoded by nidA and nidA3, which could be readily distinguished from those of pyrenenon-degraders. Furthermore, genomic islands, containing catabolic gene clusters, were shared only among the pyrenedegrading Mycobacterium strains and these gene clusters contained RHD genes, including nidAB and nidA3B3. Our genome data should facilitate further studies on the evolution of the polycyclic aromatic hydrocarbon-degradation pathways in the genus Mycobacterium.     

Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading Mycobacterium sp. strain 6PY1. [(14)C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity. As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis. Pyrene-induced proteins were tentatively identified by peptide sequence analysis. Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from Nocardioides sp. strain KP7. The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned. Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria. When overproduced in Escherichia coli, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene. The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells. The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with M(r)s of about 52,000 and 20,000. Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells. In contrast, Pdo2 could be detected only in PAH-grown cells. These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth.     

中原石油污染土壤原位微生物生态修复技术的应用

利用优化原位土著微生物菌群辅以物理和化学相结合的生态修复技术, 进行了河南中原油田石油残留污染土壤的野外修复应用研究。修复结果显示, 土壤中残留石油含量平均在2 898.25 mg/kg时, 经过99 d微生物生态修复技术的实施, 土壤中石油含量降解可达99%以上, 为油田区土壤石油残留污染的修复提供了技术方法和推广应用的可行性研究。     

Phototrophs in high iron microbial mats: microstructure of mats in iron-depositing hot springs

Chocolate Pots Hot Springs in Yellowstone National Park are high in ferrous iron, silica and bicarbonate. The springs are contributing to the active development of an iron formation. The microstructure of photosynthetic microbial mats in these springs was studied with conventional optical microscopy, confocal laser scanning microscopy and transmission electron microscopy. The dominant mats at the highest temperatures (48-54 degrees C) were composed of Synechococcus and Chloroflexus or Pseudanabaena and Mastigocladus. At lower temperatures (36-45 degrees C), a narrow Oscillatoria dominated olive green cyanobacterial mats covering most of the iron deposit. Vertically oriented cyanobacterial filaments were abundant in the top 0.5 mm of the mats. Mineral deposits accumulated beneath this surface layer. The filamentous microstructure and gliding motility may contribute to binding the iron minerals. These activities and heavy mineral encrustation of cyanobacteria may contribute to the growth of the iron deposit. Chocolate Pots Hot Springs provide a model for studying the potential role of photosynthetic prokaryotes in the origin of Precambrian iron formations.