Spectroscopic Characterisation of the Naphthalene Dioxygenase from Rhodococcus sp. Strain NCIMB12038.

Polycyclic aromatic hydrocarbons (PAHs), such as naphthalene, are potential health risks due to their carcinogenic and mutagenic effects. Bacteria from the genus Rhodococcus are able to metabolise a wide variety of pollutants such as alkanes, aromatic compounds and halogenated hydrocarbons. A naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038 has been characterised for the first time, using electron paramagnetic resonance (EPR) spectroscopy and UV-Vis spectrophotometry. In the native state, the EPR spectrum of naphthalene 1,2-dioxygenase (NDO) is formed of the mononuclear high spin Fe(III) state contribution and the oxidised Rieske cluster is not visible as EPR-silent. In the presence of the reducing agent dithionite a signal derived from the reduction of the [2Fe-2S] unit is visible. The oxidation of the reduced NDO in the presence of O2-saturated naphthalene increased the intensity of the mononuclear contribution. A study of the "peroxide shunt", an alternative mechanism for the oxidation of substrate in the presence of H2O2, showed catalysis via the oxidation of mononuclear centre while the Rieske-type cluster is not involved in the process. Therefore, the ability of these enzymes to degrade recalcitrant aromatic compounds makes them suitable for bioremediative applications and synthetic purposes.

Analysis of viral and bacterial communities in groundwater associated with contaminated land.

This work aimed at the comprehensive analysis of total microbial communities inhabiting a typical hydrocarbon-polluted site, where chemical characteristics of the groundwater were readily available. To achieve this, a joint metagenomic characterization of bacteria and viruses surrounding a contaminant plume was performed over a one-year period. The results presented demonstrated that both potential hydrocarbon degraders and their bacteriophages were dominant around the plume, and that the viral and bacterial diversities found at the site were probably influenced by the pH of the groundwater. Niche-specific and dispersed associations between phages and bacteria were identified. The niche phage-host associations were found at the edge of the site and at the core of the plume where pH was the highest (9.52). The identified host populations included several classes of bacteria (e.g. Clostridia and Proteobacteria). Thirty-six viral generalists were also discovered, with BGW-G9 having the broadest host range across 23 taxa, including Pseudomonas, Polycyclovorans, Methylocaldum and Candidatus Magnetobacterium species. The phages with broad host ranges are presumed to have significant effects on prokaryotic production and horizontal gene transfer, and therefore impact the biodegradation processes conducted by various bacteria of the environment studied. This study for the first time characterized the phages and their bacterial hosts associated with a contaminant plume.

Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA genes in the viral metagenome of the marine sponge Hymeniacidon perlevis.

Marine sponges have never been directly examined with respect to the presence of viruses or their potential involvement in horizontal gene transfer. Here we demonstrate for the first time, to our knowledge, the presence of viruses in the marine sponge Hymeniacidon perlevis. Moreover, bacterial 16S rDNA was detected in DNA isolated from these viruses, indicating that phage-derived transduction appears to occur in H. perlevis. Phylogenetic analysis revealed that bacterial 16S rDNA isolated from sponge-derived viral and total DNA differed significantly, indicating that not all species are equally involved in transduction.

Extent and variation of phage-borne bacterial 16S rRNA gene sequences in wastewater environments.

Phage metagenomes isolated from wastewater over a 12-month period were analyzed. The results suggested that various strains of Proteobacteria, Bacteroidetes, and other phyla are likely to participate in transduction. The patterns of 16S rRNA sequences found in phage metagenomes did not follow changes in the total bacterial community.

Analysis of transduction in wastewater bacterial populations by targeting the phage-derived 16S rRNA gene sequences.

Bacterial 16S rRNA genes transduced by bacteriophages were identified and analyzed in order to estimate the extent of the bacteriophage-mediated horizontal gene transfer in the wastewater environment. For this purpose, phage and bacterial DNA was isolated from the oxidation tank of a municipal wastewater treatment plant. Phylogenetic analysis of the 16S rRNA gene sequences cloned from a phage metagenome revealed that bacteriophages transduce genetic material in several major groups of bacteria. The groups identified were as follows: Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Actinomycetales and Firmicutes. Analysis of the 16S rRNA gene sequences in the total bacterial DNA from the same sample revealed that several bacterial groups found in the oxidation tank were not present in the phage metagenome (e.g. Deltaproteobacteria, Nitrospira, Planctomycetes and many Actinobacteria genera). These results suggest that transduction in a wastewater environment occurs in several bacterial groups; however, not all species are equally involved into this process. The data also showed that a number of distinctive bacterial strains participate in transduction-mediated gene transfer within identified bacterial groupings. Denaturing gradient gel electrophoresis analysis confirmed that profiles of the transduced 16S rRNA gene sequences and those present in the whole microbial community show significant differences.

Genomes of "phiKMV-like viruses" of Pseudomonas aeruginosa contain localized single-strand interruptions.

The "phiKMV-like viruses" comprise an important genus of T7 related phages infecting Pseudomonas aeruginosa. The genomes of these bacteriophages have localized single-strand interruptions (nicks), a distinguishing genomic trait previously thought to be unique for T5 related coliphages. Analysis of this feature in the newly sequenced phage phikF77 shows all four nicks to be localized on the non-coding DNA strand. They are present with high frequencies within the phage population and are introduced into the phage DNA at late stages of the lytic cycle. The general consensus sequence in the nicks (5'-CGACxxxxxCCTAoh pCTCCGG-3') was shown to be common among all phiKMV-related phages.

Resolving genetic functions within microbial populations: in situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell raman-fluorescence in situ hybridization.

Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope ((13)C)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) recovered the key phylogenetic and functional RNAs. This was followed by single-cell physiological analyses of these populations to determine and quantify in situ functions within an aerobic naphthalene-degrading groundwater microbial community. Using these culture-independent approaches, we identified three prokaryote species capable of naphthalene biodegradation within the groundwater system: two taxa were isolated in the laboratory (Pseudomonas fluorescens and Pseudomonas putida), whereas the third eluded culture (an Acidovorax sp.). Using parallel population and single-cell stable isotope technologies, we were able to identify an unculturable Acidovorax sp. which played the key role in naphthalene biodegradation in situ, rather than the culturable naphthalene-biodegrading Pseudomonas sp. isolated from the same groundwater. The Pseudomonas isolates actively degraded naphthalene only at naphthalene concentrations higher than 30 muM. This study demonstrated that unculturable microorganisms could play important roles in biodegradation in the ecosystem. It also showed that the combined RNA SIP-Raman-fluorescence in situ hybridization approach may be a significant tool in resolving ecology, functionality, and niche specialization within the unculturable fraction of organisms residing in the natural environment.

Structure and increased thermostability of Rhodococcus sp. naphthalene 1,2-dioxygenase.

Rieske nonheme iron oxygenases form a large class of aromatic ring-hydroxylating dioxygenases found in microorganisms. These enzymes enable microorganisms to tolerate and even exclusively utilize aromatic compounds for growth, making them good candidates for use in synthesis of chiral intermediates and bioremediation. Studies of the chemical stability and thermostability of these enzymes thus become important. We report here the structure of free and substrate (indole)-bound forms of naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038. The structure of the Rhodococcus enzyme reveals that, despite a approximately 30% sequence identity between these naphthalene dioxygenases, their overall structures superpose very well with a root mean square deviation of less than 1.6 A. The differences in the active site of the two enzymes are pronounced near the entrance; however, indole binds to the Rhodococcus enzyme in the same orientation as in the Pseudomonas enzyme. Circular dichroism spectroscopy experiments show that the Rhodococcus enzyme has higher thermostability than the naphthalene dioxygenase from Pseudomonas species. The Pseudomonas enzyme has an apparent melting temperature of 55 degrees C while the Rhodococcus enzyme does not completely unfold even at 95 degrees C. Both enzymes, however, show similar unfolding behavior in urea, and the Rhodococcus enzyme is only slightly more tolerant to unfolding by guanidine hydrochloride. Structure analysis suggests that the higher thermostability of the Rhodococcus enzyme may be attributed to a larger buried surface area and extra salt bridge networks between the alpha and beta subunits in the Rhodococcus enzyme.

Aminobacter ciceronei sp. nov. and Aminobacter lissarensis sp. nov., isolated from various terrestrial environments.

The bacterial strains IMB-1(T) and CC495(T), which are capable of growth on methyl chloride (CH(3)Cl, chloromethane) and methyl bromide (CH(3)Br, bromomethane), were isolated from agricultural soil in California fumigated with CH(3)Br, and woodland soil in Northern Ireland, respectively. Two pesticide-/herbicide-degrading bacteria, strains ER2 and C147, were isolated from agricultural soil in Canada. Strain ER2 degrades N-methyl carbamate insecticides, and strain C147 degrades triazine herbicides widely used in agriculture. On the basis of their morphological, physiological and genotypic characteristics, these four strains are considered to represent two novel species of the genus Aminobacter, for which the names Aminobacter ciceronei sp. nov. (type strain IMB-1(T)=ATCC 202197(T)=CIP 108660(T)=CCUG 50580(T); strains ER2 and C147) and Aminobacter lissarensis sp. nov. (type strain CC495(T)=NCIMB 13798(T)=CIP 108661(T)=CCUG 50579(T)) are proposed.

Analysis of genes involved in methyl halide degradation in Aminobacter lissarensis CC495.

Aminobacter lissarensis CC495 is an aerobic facultative methylotroph capable of growth on glucose, glycerol, pyruvate and methylamine as well as the methyl halides methyl chloride and methyl bromide. Previously, cells grown on methyl chloride have been shown to express two polypeptides with apparent molecular masses of 67 and 29 kDa. The 67 kDa protein was purified and identified as a halomethane:bisulfide/halide ion methyltransferase. This study describes a single gene cluster in A. lissarensis CC495 containing the methyl halide utilisation genes cmuB, cmuA, cmuC, orf 188, paaE and hutI. The genes correspond to the same order and have a high similarity to a gene cluster found in Aminobacter ciceronei IMB-1 and Hyphomicrobium chloromethanicum strain CM2 indicating that genes encoding methyl halide degradation are highly conserved in these strains.

Biodegradation and Rhodococcus--masters of catabolic versatility.

The genus Rhodococcus is a very diverse group of bacteria that possesses the ability to degrade a large number of organic compounds, including some of the most difficult compounds with regard to recalcitrance and toxicity. They achieve this through their capacity to acquire a remarkable range of diverse catabolic genes and their robust cellular physiology. Rhodococcus appear to have adopted a strategy of hyper-recombination associated with a large genome. Notably, they harbour large linear plasmids that contribute to their catabolic diversity by acting as 'mass storage' for a large number of catabolic genes. In addition, there is increasing evidence that multiple pathways and gene homologues are present that further increase the catabolic versatility and efficiency of Rhodococcus.

Web-type evolution of rhodococcus gene clusters associated with utilization of naphthalene.

Clusters of genes which include determinants for the catalytic subunits of naphthalene dioxygenase (narAa and narAb) were analyzed in naphthalene-degrading Rhodococcus strains. We demonstrated (i) that in the region analyzed homologous gene clusters are separated from each other by nonhomologous DNA, (ii) that there are various degrees of homology between related genes, and (iii) that nar genes are located on plasmids in strains NCIMB12038 and P400 and on a chromosome in P200. These observations suggest that genetic exchange and reshuffling of genetic modules, as well as vertical descent of the genetic information, were the main routes in the evolution of naphthalene degradation in Rhodococcus. These conclusions were supported by studies of transcription patterns in the region analyzed. It was found that the nar region is not organized into a single operon but there are several transcription units which differ in the strains investigated. The narA and narB genes were found to be transcribed as a single unit in all strains analyzed, and their transcription was induced by naphthalene. The putative aldolase gene (narC) was found on the same transcript only in strains P200 and P400. In NCIMB12038 transcription of two more gene clusters was induced by growth on naphthalene. Transcription start sites for narA and narB were found to be different in all of the strains studied. Putative regulatory genes (narR1 and narR2) were transcribed as a single mRNA in naphthalene-induced cells. At the same time, a number of the genes known to be essential for naphthalene catabolism in gram-negative bacteria were not found in the region analyzed.

Multiple inverted DNA repeats of Bacteroides fragilis that control polysaccharide antigenic variation are similar to the hin region inverted repeats of Salmonella typhimurium.

The important opportunistic pathogen Bacteroides fragilis is a strictly anaerobic Gram-negative bacterium and a member of the normal resident human gastrointestinal microbiota. Our earlier studies indicated that there is considerable within-strain variation in polysaccharide expression, as detected by mAb labelling. Analysis of the genome sequence has revealed multiple invertible DNA regions, designated fragilis invertible (fin) regions, seven of which are upstream of polysaccharide biosynthesis loci and are approximately 226 bp in size. Using orientation-specific PCR primers and sequence analysis with populations enriched for one antigenic type, two of these invertible regions were assigned to heteropolymeric polysaccharides with different sizes of repeating units, as determined by PAGE pattern. The implication of these findings is that inversion of the fin regions switches biosynthesis of these polysaccharides off and on. The invertible regions are bound by inverted repeats of 30 or 32 bp with striking similarity to the Salmonella typhimurium H flagellar antigen inversion cross-over (hix) recombination sites of the invertible hin region. It has been demonstrated that a plasmid-encoded Hin invertase homologue (FinB), present in B. fragilis NCTC 9343, binds specifically to the invertible regions and the recombination sites have been designated as fragilis inversion cross-over (fix) sites.

Crystallization and preliminary X-ray diffraction analysis of naphthalene dioxygenase from Rhodococcus sp. strain NCIMB 12038.

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by Rhodococcus sp. strain NCIMB 12038. The terminal oxygenase component (naphthalene 1,2-dioxygenase) that catalyzes this reaction belongs to the aromatic ring hydroxylating dioxygenase family and has been crystallized. These enzymes utilize a mononuclear non-heme iron centre to catalyze the addition of dioxygen to their respective substrates. In this reaction, two electrons, two protons and a dioxygen molecule are consumed. The Rhodococcus enzyme has only 33 and 29% sequence identity to the corresponding alpha- and beta-subunits of the NDO system of Pseudomonas putida NCIMB 9816-4, for which the tertiary structure has been reported. In order to determine the three-dimensional structure of the Rhodococcus NDO, diffraction-quality crystals have been prepared by the hanging-drop method. The crystals belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 144, c = 185.6 A, alpha = beta = gamma = 90 degrees, and diffract to 2.3 A resolution.

Tandem enzyme-catalysed oxidations of alkyl phenyl sulfides and alkyl benzenes: enantiocomplementary routes to chiral phenols.

Dioxygenase-catalysed trioxygenation of alkyl phenyl sulfides and alkyl benzenes yields enantiopure cis-dihydrodiol sulfoxides and triols respectively; naphthalene cis-dihydrodiol dehydrogenase-catalysed aromatisation of these diastereoisomers gives enantiopure catechols of either configuration.

Analysis of bacteria contaminating ultrapure water in industrial systems.

Bacterial populations inhabiting ultrapure water (UPW) systems were investigated. The analyzed UPW systems included pilot scale, bench scale, and full size UPW plants employed in the semiconductor and other industries. Bacteria present in the polishing loop of the UPW systems were enumerated by both plate counts and epifluorescence microscopy. Assessment of bacterial presence in UPW by epifluorescence microscopy (cyanotolyl tetrazolium chloride [CTC] and DAPI [4',6'-diamidino-2-phenylindole] staining) showed significantly higher numbers (10 to 100 times more bacterial cells were detected) than that determined by plate counts. A considerable proportion of the bacteria present in UPW (50 to 90%) were cells that did not give a positive signal with CTC stain. Bacteria isolated from the UPW systems were mostly gram negative, and several groups seem to be indigenous for all of the UPW production systems studied. These included Ralstonia pickettii, Bradyrhizobium sp., Pseudomonas saccharophilia, and Stenotrophomonas strains. These bacteria constituted a significant part of the total number of isolated strains (>or=20%). Two sets of primers specific to R. pickettii and Bradyrhizobium sp. were designed and successfully used for the detection of the corresponding bacteria in the concentrated UPW samples. Unexpectedly, nifH gene sequences were found in Bradyrhizobium sp. and some P. saccharophilia strains isolated from UPW. The widespread use of nitrogen gas in UPW plants may be associated with the presence of nitrogen-fixing genes in these bacteria.

Characterization of IS2112, a new insertion sequence from Rhodococcus, and its relationship with mobile elements belonging to the IS110 family.

A new insertion sequence (IS2112) was identified in the genome of the 1-haloalkane-utilizing bacterium Rhodococcus rhodochrous NCIMB 13064. The insertion element is 1415 bp long, does not contain terminal inverted repeats, and is not flanked by directly repeated sequences. IS2112 belongs to the IS110 family of transposable elements, and forms a separate subfamily, along with IS116. Two copies of IS2112 were found in R. rhodochrous NCIMB 13064 and one, two or three copies of a similar sequence were detected in five other 1-haloalkane-degrading Rhodococcus strains. There were no sequences homologous to IS2112 found in the 1-haloalkane-degrading 'Pseudomonas pavonaceae' 170 and Rhodococcus sp. HA1 or in several Rhodococcus strains which do not utilize haloalkanes. IS2112 was originally found in plasmid pRTL1 of R. rhodochrous NCIMB 13064, which harbours genes encoding utilization of 1-haloalkanes, and was located 5 kbp upstream of the haloalkane dehalogenase gene (dhaA). Although the second copy of IS2112 in strain NCIMB 13064 was also present on the pRTL1 plasmid, these sequences do not apparently comprise a single composite transposon encoding haloalkane utilization. An analysis of derivatives of NCIMB 13064 revealed that IS2112 was involved in genome rearrangements. IS2112 appeared to change its location as a result of transposition and as a result of other rearrangements of the NCIMB 13064 genome.

Cloning of new Rhodococcus extradiol dioxygenase genes and study of their distribution in different Rhodococcus strains.

Four extradiol dioxygenase genes which encode enzymes active against catechol and substituted catechols were cloned from two different Rhodococcus strains, and their nucleotide sequences were determined. A catechol 2,3-dioxygenase gene (edoC) was shown to be identical to the previously described ipbC gene from the isopropylbenzene operon of Rhodococcus erythropolis. Amino acid sequences deduced from the three other genes (edoA, edoB and edoD) were shown to have various degrees of homology to different extradiol dioxygenases. The EdoA and EdoB dioxygenases were classified as belonging to the third family of type I oxygenases and represented two new subfamilies, whereas the EdoD dioxygenase was a type II enzyme. Analysis of six Rhodococcus strains revealed a wide distribution of the above dioxygenase genes. Rhodococcus sp. 11 was shown to harbour all four of the analysed dioxygenase genes. Nucleotide sequences homologous to the edoB gene were present in all of the strains, including R. erythropolis NCIMB 13065, which did not utilize any of the aromatic compounds analysed. The latter finding points to the existence of a silent pathway(s) for degradation of aromatic compounds in this Rhodococcus strain.

The plasmid-located haloalkane dehalogenase gene from Rhodococcus rhodochrous NCIMB 13064.

The haloalkane dehalogenase (dhaA) gene from Rhodococcus rhodochrous NCIMB 13064 was cloned and sequenced. Its comparison with the previously studied dhlA gene from Xanthobacter autotrophicus GJ10 did not show homology. However, the amino acid sequences of the products of these genes showed approximately 30% identity and several of the catalytic amino acid residues were conserved in the NCIMB 13,064 dehalogenase. A high level of dhaA expression was demonstrated in Escherichia coli cells and this gene was shown to encode a dehalogenase with the activity against chloroalkanes of chain length C3-C10. Also, some dehalogenase activity against 1,2-dichloroethane encoded by the cloned dhaA gene was detected. The analysis of NCIMB 13,064 derivatives lacking dehalogenase activity showed that the dhaA gene was located on the 100 kbp pRTL1 plasmid. It was also found that reversible rearrangements of DNA in the dhaA region may be responsible for the control of expression of haloalkane dehalogenase in R. rhodochrous NCIMB 13064. A number of repeated and inverted sequences which may cause genetic instability at the locus were found in the haloalkane dehalogenase gene region.