Search

Natural gas seeps release significant amounts of methane and other gases including ethane and propane contributing to global climate change. In this study, bacterial actively consuming short-chain alkanes were identified by cultivation, whole-genome sequencing, and stable-isotope probing (SIP)-metagenomics using 13C-propane and 13C-ethane from two different natural gas seeps, Pipe Creek and Andreiasu Everlasting Fire. Nearly 100 metagenome-assembled genomes (MAGs) (completeness 70–99%) were recovered from both sites. Among these, 16 MAGs had genes encoding the soluble di-iron monooxygenase (SDIMO). The MAGs were affiliated to Actinobacteria (two MAGs), Alphaproteobacteria (ten MAGs), and Gammaproteobacteria (four MAGs). Additionally, three gaseous-alkane degraders were isolated in pure culture, all of which could grow on ethane, propane, and butane and possessed SDIMO-related genes. Two Rhodoblastusstrains (PC2 and PC3) were from Pipe Creek and a Mycolicibacteriumstrain (ANDR5) from Andreiasu. Strains PC2 and PC3 encoded putative butane monooxygenases (MOs) and strain ANDR5 contained a propane MO. Mycolicibacteriumstrain ANDR5 and MAG19a, highly abundant in incubations with 13C-ethane, share an amino acid identity (AAI) of 99.3%. We show using a combination of enrichment and isolation, and cultivation-independent techniques, that these natural gas seeps contain a diverse community of active bacteria oxidising gaseous-alkanes, which play an important role in biogeochemical cycling of natural gas.

Natural gas seeps release significant amounts of methane and other gases including ethane and propane contributing to global climate change. In this study, bacterial actively consuming short-chain alkanes were identified by cultivation, whole-genome sequencing, and stable-isotope probing (SIP)-metagenomics using 13C-propane and 13C-ethane from two different natural gas seeps, Pipe Creek and Andreiasu Everlasting Fire. Nearly 100 metagenome-assembled genomes (MAGs) (completeness 70–99%) were recovered from both sites. Among these, 16 MAGs had genes encoding the soluble di-iron monooxygenase (SDIMO). The MAGs were affiliated to Actinobacteria (two MAGs), Alphaproteobacteria (ten MAGs), and Gammaproteobacteria (four MAGs). Additionally, three gaseous-alkane degraders were isolated in pure culture, all of which could grow on ethane, propane, and butane and possessed SDIMO-related genes. Two Rhodoblastusstrains (PC2 and PC3) were from Pipe Creek and a Mycolicibacteriumstrain (ANDR5) from Andreiasu. Strains PC2 and PC3 encoded putative butane monooxygenases (MOs) and strain ANDR5 contained a propane MO. Mycolicibacteriumstrain ANDR5 and MAG19a, highly abundant in incubations with 13C-ethane, share an amino acid identity (AAI) of 99.3%. We show using a combination of enrichment and isolation, and cultivation-independent techniques, that these natural gas seeps contain a diverse community of active bacteria oxidising gaseous-alkanes, which play an important role in biogeochemical cycling of natural gas.

Coastal saltmarsh sediments represent an important source of natural methane emissions, much of which originates from quaternary and methylated amines, such as choline and trimethylamine. In this study, we combine DNA stable isotope probing with high throughput sequencing of 16S rRNA genes and 13C2-choline enriched metagenomes, followed by metagenome data assembly, to identify the key microbes responsible for methanogenesis from choline. Microcosm incubation with 13C2-choline leads to the formation of trimethylamine and subsequent methane production, suggesting that choline-dependent methanogenesis is a two-step process involving trimethylamine as the key intermediate. Amplicon sequencing analysis identifies Deltaproteobacteriaof the genera Pelobacteras the major choline utilizers. Methanogenic Archaea of the genera Methanococcoidesbecome enriched in choline-amended microcosms, indicating their role in methane formation from trimethylamine. The binning of metagenomic DNA results in the identification of bins classified as Pelobacterand Methanococcoides. Analyses of these bins reveal that Pelobacterhave the genetic potential to degrade choline to trimethylamine using the choline-trimethylamine lyase pathway, whereas Methanococcoidesare capable of methanogenesis using the pyrrolysine-containing trimethylamine methyltransferase pathway. Together, our data provide a new insight on the diversity of choline utilizing organisms in coastal sediments and support a syntrophic relationship between Bacteria and Archaea as the dominant route for methanogenesis from choline in this environment.

Isoprene (2-methyl-1,3-butadiene), the most abundantly produced biogenic volatile organic compound (BVOC) on Earth, is highly reactive and can have diverse and often detrimental atmospheric effects, which impact on climate and health. Most isoprene is produced by terrestrial plants, but (micro)algal production is important in aquatic environments, and the relative bacterial contribution remains unknown. Soils are a sink for isoprene, and bacteria that can use isoprene as a carbon and energy source have been cultivated and also identified using cultivation-independent methods from soils, leaves and coastal/marine environments. Bacteria belonging to the Actinobacteria are most frequently isolated and identified, and Proteobacteria have also been shown to degrade isoprene. In the freshwater-sediment isolate, Rhodococcusstrain AD45, initial oxidation of isoprene to 1,2-epoxy-isoprene is catalyzed by a multicomponent isoprene monooxygenase encoded by the genes isoABCDEF. The resultant epoxide is converted to a glutathione conjugate by a glutathione S-transferase encoded by isoI, and further degraded by enzymes encoded by isoGHJ. Genome sequence analysis of actinobacterial isolates belonging to the genera Rhodococcus, Mycobacteriumand Gordoniahas revealed that isoABCDEFand isoGHIJare linked in an operon, either on a plasmid or the chromosome. In Rhodococcusstrain AD45 both isoprene and epoxy-isoprene induce a high level of transcription of 22 contiguous genes, including isoABCDEFand isoGHIJ. Sequence analysis of the isoAgene, encoding the large subunit of the oxygenase component of isoprene monooxygenase, from isolates has facilitated the development of PCR primers that are proving valuable in investigating the ecology of uncultivated isoprene-degrading bacteria.

Dimethylsulfide (DMS) is an environmentally important trace gas with roles in sulfur cycling, signalling to higher organisms and in atmospheric chemistry. DMS is believed to be predominantly produced in marine environments via microbial degradation of the osmolyte dimethylsulfoniopropionate (DMSP). However, significant amounts of DMS are also generated from terrestrial environments, for example, peat bogs can emit ~6 μmol DMS m−2per day, likely via the methylation of methanethiol (MeSH). A methyltransferase enzyme termed ‘MddA’, which catalyses the methylation of MeSH, generating DMS, in a wide range of bacteria and some cyanobacteria, may mediate this process, as the mddA gene is abundant in terrestrial metagenomes. This is the first study investigating the functionality of MeSH-dependent DMS production (Mdd) in a wide range of aerobic environments. All soils and marine sediment samples tested produced DMS when incubated with MeSH. Cultivation-dependent and cultivation-independent methods were used to assess microbial community changes in response to MeSH addition in a grassland soil where 35.9% of the bacteria were predicted to contain mddA. Bacteria of the genus Methylotenera were enriched in the presence of MeSH. Furthermore, many novel Mdd+bacterial strains were isolated. Despite the abundance of mddA in the grassland soil, the Mdd pathway may not be a significant source of DMS in this environment as MeSH addition was required to detect DMS at only very low conversion rates.

Dimethylsulfide (DMS) is an environmentally important trace gas with roles in sulfur cycling, signalling to higher organisms and in atmospheric chemistry. DMS is believed to be predominantly produced in marine environments via microbial degradation of the osmolyte dimethylsulfoniopropionate (DMSP). However, significant amounts of DMS are also generated from terrestrial environments, for example, peat bogs can emit ~6?µmol DMS?m-2per day, likely via the methylation of methanethiol (MeSH). A methyltransferase enzyme termed ‘MddA’, which catalyses the methylation of MeSH, generating DMS, in a wide range of bacteria and some cyanobacteria, may mediate this process, as the mddAgene is abundant in terrestrial metagenomes. This is the first study investigating the functionality of MeSH-dependent DMS production (Mdd) in a wide range of aerobic environments. All soils and marine sediment samples tested produced DMS when incubated with MeSH. Cultivation-dependent and cultivation-independent methods were used to assess microbial community changes in response to MeSH addition in a grassland soil where 35.9% of the bacteria were predicted to contain mddA. Bacteria of the genus Methylotenerawere enriched in the presence of MeSH. Furthermore, many novel Mdd+bacterial strains were isolated. Despite the abundance of mddAin the grassland soil, the Mdd pathway may not be a significant source of DMS in this environment as MeSH addition was required to detect DMS at only very low conversion rates.

Howard Dalton was an outstanding microbiologist who, after his remarkably productive DPhil work in the Nitrogen Fixation Laboratory at the University of Sussex, and a short period in the USA, spent his research career at the University of Warwick. He devoted himself to the elucidation of the process of methane oxidation by bacteria that use this relatively inert gas as their sole source of carbon and energy. He discovered two completely novel multicomponent monooxygenase enzymes responsible for the initial oxidation of methane to methanol. He then continued to elucidate their functions, mechanisms, regulation and structures. Their wide substrate specificity led to his interest in using these and related enzymes for biocatalysis, biological transformations and bioremediation. While remaining at Warwick University he also acted as a highly appreciated Chief Scientific Advisor to the UK Government at the Department for the Environment and Rural Affairs (Defra). Howard was a highly effective scientist, a down-to-earth, self-effacing man, outgoing and witty, an inspirational colleague who above all else made science fun.

Stable-isotope probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without the prerequisite of cultivation. Stable-isotope-labeled carbon (13C) or nitrogen (15N) sources are assimilated into microbial biomass of environmental samples. Separation and molecular analysis of labeled nucleic acids (DNA or RNA) reveals phylogenetic and functional information about the microorganisms responsible for the metabolism of a particular substrate. Here, we highlight general guidelines for incubating environmental samples with labeled substrate and provide a detailed protocol for separating labeled DNA from unlabeled community DNA. The protocol includes a modification of existing published methods, which maximizes the recovery of labeled DNA from CsCl gradients. The separation of DNA and retrieval of unlabeled and labeled fractions can be performed in 4–5 days, with much of the time being committed to the ultracentrifugation step.

Oligonucleotide probes targeting the 16S rRNA of distinct phylogenetic groups of methanotrophs were designed for the in situ detection of these organisms. A probe, MG-64, detected specifically type I methanotrophs, while probes MA-221 and MA-621, detected type II methanotrophs in whole cell hybridisations. A probe Mc1029 was also designed which targeted only organisms from the Methylococcusgenus after whole cell hybridisations. All probes were labelled with the fluorochrome Cy3 and optimum conditions for hybridisation were determined. Non-specific target sites of the type I (MG-64) and type II (MA-621) probes to non-methanotrophic organisms are highlighted. The probes are however used in studying enrichment cultures and environments where selective pressure favours the growth of methanotrophs over other organisms. The application of these probes was demonstrated in the detection of type I methanotrophs with the MG-64 probe in an enrichment culture from an estuarine sample demonstrating methane oxidation. The detection of type I methanotrophs was confirmed by a 16S rDNA molecular analysis of the estuarine enrichment culture which demonstrated that the most abundant bacterial clone type in the 16S rDNA library was most closely related to Methylobactersp. strain BB5.1, a type I methanotroph also isolated from an estuarine environment.

Abstract: Several bacteria from soil and rainwater samples were enriched and isolated with propanesulfonate or butanesulfonate as sole carbon and energy source. Most of the strains isolated utilized nonsubstituted alkanesulfonates with a chain length of C3–C6 and the substituted sulfonates taurine and isethionate as carbon and energy source. A gram-positive isolate, P40, and a gram-negative isolate, P53, were characterized in more detail. Phylogenetic analysis grouped strain P40 within group IV of the genus Rhodococcus and showed a close relationship with Rhodococcus opacus. After phylogenetic and physiological analyses, strain P53 was identified as Comamonas acidovorans. Both bacteria also utilized a wide range of sulfonates as sulfur source. Strain P40, but not strain P53, released sulfite into the medium during dissimilation of sulfonated compounds. Cell-free extracts of strain P53 exhibited high sulfite oxidase activity [2.34 U (mg protein)–1] when assayed with ferricyanide, but not with cytochrome c. Experiments with whole-cell suspensions of both strains showed that the ability to dissimilate 1-propanesulfonate was specifically induced during growth on this substrate and was not present in cells grown on propanol, isethionate or taurine. Whole-cell suspensions of both strains accumulated acetone when oxidizing the non-growth substrate 2-propanesulfonate. Strain P40 cells also accumulated sulfite under these conditions. Stoichiometric measurements with 2-propanesulfonate as substrate in oxygen electrode experiments indicate that the nonsubstituted alkanesulfonates were degraded by a monooxygenase. When strain P53 grew with nonsubstituted alkanesulfonates as carbon and energy source, cells expressed high amounts of yellow pigments, supporting the proposition that an oxygenase containing iron sulfur centres or flavins was involved in their degradation.

NAD+-dependent propan-1-ol and propan-2-ol dehydrogenase activities were detected in cell-free extracts of Rhodococcus rhodochrous PNKb1 grown on propane and potential intermediates of propane oxidation. However, it was unclear whether this activity was mediated by one or more enzymes. The isolation of mutants unable to utilize propan-1-ol (alcA-) or propan-2-ol (alcB-) as sole carbon and energy sources demonstrated that these substrates are metabolized by different alcohol dehydrogenases. These mutants were also unable to utilize propane as a growth substrate indicating that both alcohols are intermediates of propane metabolism. Therefore, propane is metabolized by terminal and sub-terminal oxidation pathways. Westernblot analysis demonstrated that a previously purified NAD+-dependent propan-2-ol dehydrogenase (Ashraf and Murrell 1990) was only synthesized after growth on propane and sub-terminal oxidation intermediates (but not acetone), and not propan-1-ol or terminal oxidation intermediates. Therefore, our evidence suggest that another dehydrogenase is involved in the metabolism of propan-1-ol and this agrees with the isolation of the alcA- and alcB- phenotypes. The previously characterized NAD+-dependent propan-2-ol dehydrogenase from R. rhodochrous PNKb1 is highly conserved amongst members of the propane-utilizing Rhodococcus-Nocardia complex.

Two new methane-oxidizing bacteria have been isolated from seawater samples from Plymouth Sound. These marine methanotrophs have an obligate requirement for NaCl and exhibit many properties of typical Type I methanotrophs previously isolated from freshwater environments. However, they are different from all other methanotrophs thus far described in that they failed to grow on all solid media tested. The nitrogen metabolism of both strains was investigated. They were not N2-fixers nor would they use ammonia as nitrogen source. They appeared to utilize the glutamate dehydrogenase pathway for the assimilation of ammonia under all growth conditions tested.

NAD+-linked primary and secondary alcohol dehydrogenase activity was detected in cell-free extracts of propane-grown Rhodococcus rhodochrous PNKb1. One enzyme was purified to homogeneity using a two-step procedure involving DEAE-cellulose and NAD-agarose chromatography and this exhibited both primary and secondary NAD+-linked alcohol dehydrogenase activity. The Mr of the enzyme was approximately 86,000 with subunits of Mr 42,000. The enzyme exhibited broad substrate specificity, oxidizing a range of short-chain primary and secondary alcohols (C2-C8) and representative cyclic and aromatic alcohols. The pH optimum was 10. At pH 6.5, in the presence of NADH, the enzyme catalysed the reduction of ketones to alcohols. The Km values for propan-1-ol, propan-2-ol and NAD were 12 mM, 18 mM and 0.057 mM respectively. The enzyme was inhibited by metal-complexing agents and iodoacetate. The properties of this enzyme were compared with similar enzymes in the current literature, and were found to be significantly different from those thus far described. It is likely that this enzyme plays a major role in the assimilation of propane by R. rhodochrous PNKb1.

Methane monooxygenase (MMO) is the enzyme responsible for the conversion of methane to methanol in methanotrophic bacteria. In addition, this enzyme complex oxidizes a wide range of aliphatic and aromatic compounds in a number of potentially useful biotransformations. In this study, we have used biochemical data obtained from purification and characterization of the soluble MMO from Methylococcus capsulatus (Bath), to identify structural genes encoding this enzyme by oligonucleotide probing. The genes encoding the ß and ? subunits of MMO were found to be chromosomally located and were linked in this organism. We report here on the analysis of a recombinant plasmid containing 12 kilobases of Methylococcus DNA and provide the first evidence for the localization and linkage of genes encoding the methane monooxygenase enzyme complex. DNA sequence analysis suggests that the primary structures of the ß and ? subunit of MMO are completely novel and the complete sequence of these genes is presented.

Zygosaccharomyces rouxii catalysed the reduction of ethyl 4-chloroacetoacetate (ethyl 4-chloro-3-oxobutanoate) to the corresponding (S)-hydroxy ester (ethyl (S)-4-chloro-3-hydroxybutanoate) in high enantiomeric excess. The productivity of non-immobilised cells was compared to cells immobilised on a range of organic and inorganic supports. Cells immobilised in calcium alginate displayed a catalytic activity significantly higher than that of non-immobilised cells. A time dependent fall in the enantiomeric purity of the product was observed with the use of this matrix. This phenomenon was not seen in the reduction catalysed by non-immobilised cells.

Six yeasts were studied for their ability to reduce ethyl 4-chloroacetoacetate (ethyl 4-chloro-3-oxobutanoate) stereoselectively. Five species reduced the substrate to ethyl (S)-4-chloro-3-hydroxybutanoate of high (92-99%) optical purity. With glucose-grown cells, substrate reduction could only be demonstrated when growth was oxygen-limited, whereas xylose-grown Pichia capsulata could be grown under conditions of oxygen excess without losing its reducing ability. Zygosaccha-romyces rouxii exhibited high enantioselectivity (≥98% ee (S)-enantiomer) under all conditions tested, whilst in P. capsulata, a novel switch was observed from producing mainly the (R)-enantiomer using glucose as co-substrate to producing mainly the (R)-enantiomer using 2-propanol as co-substrate. This switch was correlated with a change in reduction predominantly from an NADPH-dependent dehydrogenase system to an NADH-dependent system. In the production of ethyl (R)-4-chloro-3-hydroxybutanoate with P. capsulata, the enantioselectivity was also found to depend upon growth conditions. With glucose-grown cells, higher enantioselectivity was observed using cells harvested in stationary phase (93-94% ee) compared with cells harvested in exponential phase (43-60% ee). Growing P. capsulata with xylose rather than glucose as the major source of carbon for growth resulted in an eight-fold increase in the specific rate of ethyl (R)-4-chloro-3-hydroxybutanoate production using 2-propanol as co-substrate, although enantioselectivity was slightly reduced (65-81% ee) compared with the maximum achieved with glucose-grown cells. The effect of growth on xylose could also be correlated with enhanced activity of an NADH-dependent (R)-selective dehydrogenase system.

Trichloroethylene (TCE) oxidation was examined in 9 different methanotrophs grown under conditions favoring expression of the membrane associated methane monooxygenase. Depending on the strain, TCE oxidation rates varied from 1 to 677 pmol/min/mg cell protein. Levels of TCE in the reaction mixture were reduced to below 40 nmolar in some strains. Cells incubated in the presence of acetylene, a selective methane monooxygenase inhibitor, did not oxidize TCE.

Methanotrophs are a fascinating group of bacteria that have the unique ability to grow on methane as their sole carbon and energy source. They appear to be widespread in nature and have been isolated from a number of different environments, including soils, sediments, freshwater, marine sediments, seawater, acid peat bogs, hot springs and cold environments such as the Antarctic. There are now eight recognised genera of methanotrophs. Methanotrophs have attracted a great deal of interest over the past 30 years since they have considerable potential for the production of bulk chemicals, fine chemicals and in bioremediation processes, such as the degradation of the groundwater pollutant trichloroethylene. More recently, they are being extensively studied in a wide variety of environments since methanotrophs play a critical role in the global methane cycle. Polymerase chain reaction-based methods have been used to study the ecology and diversity of methanotrophs. We review here molecular ecology methods that are already available or which are currently being developed for methanotrophs. These are based on 16S ribosomal RNA technology and specific amplification of ‘functional genes’, such as those encoding unique enzymes in the metabolism of these organisms including methane monooxygenase and methanol dehydrogenase.

The potential for methane oxidation was measured, and methanotroph gene sequences studied, in a peat core from the Moorhouse Nature Reserve, UK. Methane oxidation potential was observed in all depths of the peat core (down to 30 cm), and was inhibited by addition of acetylene, indicating the involvement of methane-oxidising bacteria. A peak of activity was shown in the 10–12 cm horizon, below which activity decreased with depth. Above this horizon, methane oxidation was relatively high and showed little change with depth. 16S rDNA libraries from several sections of the peat core were screened with methanotroph 16S rDNA probes designed to detect the genera Methylomonas, Methylococcus, Methylobacterand Methylosinus. Two clones, MHP14 and MHP17, hybridised strongly with the Methylosinusprobe and upon complete sequencing and phylogenetic analysis were shown to group closely to the Methylosinus/Methylocystisgenera of methanotrophs. However, the clones do form a distinct branch of their own, supported by BOOTSTRAP values, and may represent a novel group of acidophilic methanotrophs which have yet to be cultured.

Total base sequences of the 16S rRNA genes ofThiobacillus halophilus andThiobacillus aquaesulis show that these bacteria fall into the gamma- and beta-subdivisions, respectively of the Proteobacteria. The closest relative ofT. halophilus isThiobacillus hydrothermalis (with 98.7% similarity), and the closest relative ofT. aquaesulis isThiobacillus thioparus (93.2% similarity). Physiological properties and mol% G+C content of their DNA serve to confirm that these four organisms are all distinct species. It is reiterated that the species currently assigned to the genusThiobacillus are clearly so diverse that they need reclassification into several genera. The type species,T. thioparus, is unequivocally placed in the beta-subdivision of the Proteobacteria, thus requiring that the use of the genus nameThiobacillus be restricted to the chemolithoautotrophic species falling into that group.T. aquaesulis andT. thioparus may thus be regarded as true species ofThiobacillus. The relatively large number of obligately chemolithoautotrophicThiobacillus species falling in the gamma-subdivision of the Proteobacteria need further study in order to assess the case for reclassification into one or more new or different genera.

Abstract: The isolation and properties of a novel species of pink-pigmented methylotroph, Methylobacterium thiocyanatum, are described. This organism satisfied all the morphological, biochemical, and growth-substrate criteria to be placed in the genus Methylobacterium. Sequencing of the gene encoding its 16S rRNA confirmed its position in this genus, with its closest phylogenetic relatives being M. rhodesianum, M. zatmanii and M. extorquens, from which it differed in its ability to grow on several diagnostic substrates. Methanol-grown organisms contained high activities of hydroxypyruvate reductase [3 μmol NADH oxidized min–1 (mg crude extract protein)–1], showing that the serine pathway was used for methylotrophic growth. M. thiocyanatum was able to use thiocyanate or cyanate as the sole source of nitrogen for growth, and thiocyanate as the sole source of sulfur in the absence of other sulfur compounds. It tolerated high concentrations (at least 50 mM) of thiocyanate or cyanate when these were supplied as nitrogen sources. Growing cultures degraded thiocyanate to produce thiosulfate as a major sulfur end product, apparently with the intermediate formation of volatile sulfur compounds (probably hydrogen sulfide and carbonyl sulfide). Enzymatic hydrolysis of thiocyanate by cell-free extracts was not demonstrated. Cyanate was metabolized by means of a cyanase enzyme that was expressed at approximately sevenfold greater activity during growth on thiocyanate [V max 634 24 nmol NH3 formed min–1 (mg protein)–1] than on cyanate [89 9 nmol NH3 min–1 (mg protein)–1]. Kinetic study of the cyanase in cell-free extracts showed the enzyme (1) to exhibit high affinity for cyanate (K m 0.07 mM), (2) to require bicarbonate for activity, (3) to be subject to substrate inhibition by cyanate and competitive inhibition by thiocyanate (K i 0.65 mM), (4) to be unaffected by 1 mM ammonium chloride, (5) to be strongly inhibited by selenocyanate, and (6) to be slightly inhibited by 5 mM thiosulfate, but unaffected by 0.25 mM sulfide or 1 mM thiosulfate. Polypeptides that might be a cyanase subunit (mol.wt. 17.9 kDa), a cyanate (and/or thiocyanate) permease (mol.wt. 25.1 and 27.2 kDa), and a putative thiocyanate hydrolase (mol.wt. 39.3 kDa) were identified by SDS-PAGE. Correlation of the growth rate of cultures with thiocyanate concentration (both stimulatory and inhibitory) and the kinetics of cyanase activity might indicate that growth on thiocyanate involved the intermediate formation of cyanate, hence requiring cyanase activity. The very high activity of cyanase observed during growth on thiocyanate could be in compensation for the inhibitory effect of thiocyanate on cyanase. Alternatively, thiocyanate may be a nonsubstrate inducer of cyanase, while thiocyanate degradation itself proceeds by a carbonyl sulfide pathway not involving cyanate. A formal description of the new species (DSM 11490) is given.

Abstract: Two novel genera of restricted facultative methylotrophs are described; both Methylosulfonomonas and Marinosulfonomonas are unique in being able to grow on methanesulfonic acid as their sole source of carbon and energy. Five identical strains of Methylosulfonomonas were isolated from diverse soil samples in England and were shown to differ in their morphology, physiology, DNA base composition, molecular genetics, and 16S rDNA sequences from the two marine strains of Marinosulfonomonas, which were isolated from British coastal waters. The marine strains were almost indistinguishable from each other and are considered to be strains of one species. Type species of each genus have been identified and named Methylosulfonomonas methylovora (strain M2) and Marinosulfonomonas methylotropha (strain PSCH4). Phylogenetic analysis using 16S rDNA sequencing places both genera in the α-Proteobacteria. Methylosulfonomonas is a discrete lineage within the α-2 subgroup and is not related closely to any other known bacterial genus. The Marinosulfonomonas strains form a monophyletic cluster in the α-3 subgroup of the Proteobacteria with Roseobacter spp. and some other partially characterized marine bacteria, but they are distinct from these at the genus level. This work shows that the isolation of bacteria with a unique biochemical character, the ability to grow on methanesulfonic acid as energy and carbon substrate, has resulted in the identification of two novel genera of methylotrophs that are unrelated to any other extant methylotroph genera.

Abstract: Three distinct strains (KL1, KS1, and KS2) of facultatively chemolitho-autotrophic bacteria able to use carbon disulfide or carbonyl sulfide as sole energy substrates were identified as novel strains of Paracoccus denitrificans. Evidence for their identity as biovars of P. denitrificans and as close relatives of Paracoccus versutus is based on their DNA composition, total sequencing of the genes for their 16S rRNA, muropeptide profiles, amino acid composition of peptidoglycan, kinetics of murein degradation by lysozyme, possession of large plasmids (91–98 kb) and megaplasmids (> 450 kb), and plasmid transfer between the strains and with P. denitrificans and P. versutus. No functions have been identified for the 91- to 98-kb plasmids of strains KL1 and KS2, but curing strain KL1 of its plasmid did not affect growth on carbon disulfide, thiosulfate or succinate. Emendation of the formal description of Paracoccus denitrificans is presented. Autotrophic growth on carbon disulfide and thiosulfate was confirmed by 14CO2 fixation. Evidence is presented for initiation of carbon disulfide oxidation by an NADH-dependent oxygenase. Cell-free extracts catalyzed (1) NADH-stimulated uptake of oxygen in the presence of carbon disulfide, and (2) carbon-disulfide-stimulated oxidation of NADH. The activity was not sedimented at 50,000 �g. Intermediates in aerobic carbon disulfide metabolism were shown by GC and GC/MS to include carbonyl sulfide and hydrogen sulfide, but anaerobic production of COS and H2S from carbon disulfide did not occur. SDS-PAGE of cell-free extracts showed polypeptides that were unique to growth on carbon disulfide, common to carbon disulfide and carbonyl sulfide, or found after growth on carbon disulfide, carbonyl sulfide or thiosulfate. The possible identity of these as proteins involved in sulfur compound metabolism is discussed.

Abstract: Acetylene reduction was observed with ferrous-iron-oxidizing Thiobacillus ferrooxidans, as expected from previous studies with this bacterium. Acetylene reduction was also found during the growth of T. ferrooxidans on tetrathionate. Only Leptospirillum ferrooxidans, one of several other phylogenetically diverse, ferrous-iron- and/or sulfur-oxidizing acidophilic microorganisms, also reduced acetylene. A reduction of the oxygen concentration in the culture atmosphere was necessary to alleviate inhibition of nitrogenase activity. DNA sequences homologous to nif structural genes were found in both organisms. Diazotrophic growth of L. ferrooxidans was inferred from an increase in iron oxidation in ammonium-free medium when the oxygen concentration was limited and from apparent inhibition by acetylene under these conditions.

Prymnesium parvumis a toxin-producing microalga, which causes harmful algal blooms globally, frequently leading to massive fish kills that have adverse ecological and economic implications for natural waterways and aquaculture alike. The dramatic effects observed on fish are thought to be due to algal polyether toxins, known as the prymnesins, but their lack of environmental detection has resulted in an uncertainty about the true ichthyotoxic agents. Using qPCR, we found elevated levels of P. parvumand its lytic virus, PpDNAV-BW1, in a fish-killing bloom on the Norfolk Broads, United Kingdom, in March 2015. We also detected, for the first time, the B-type prymnesin toxins in Broads waterway samples and gill tissue isolated from a dead fish taken from the study site. Furthermore, Norfolk Broads P. parvumisolates unambiguously produced B-type toxins in laboratory-grown cultures. A 2 year longitudinal study of the Broads study site showed P. parvumblooms to be correlated with increased temperature and that PpDNAV plays a significant role in P. parvumbloom demise. Finally, we used a field trial to show that treatment with low doses of hydrogen peroxide represents an effective strategy to mitigate blooms of P. parvumin enclosed water bodies.

N2-fixation was investigated in the chemoautotrophic hydrogen bacterium Xanthobacter H4-14. N2-fixing batch cultures of this organism could only be grown at pO2 values of around 0.02 bar, and in continuous culture dissolved oxygen tensions above 16 µM were found to inhibit N2-fixation. Xanthobacter H4-14 utilized a variety of amino acids, nitrate and ammonia as nitrogen sources. Cell-free extracts from steady-state continuous cultures of ammonia grown, nitrate grown and N2-fixing Xanthobacter were assayed for the presence of ammonia assimilation enzymes. No alanine dehydrogenase or glutamate dehydrogenase activity was detected. Ammonia was assimilated exclusively via the glutamine synthetase/glutamate synthase pathway, irrespective of the extracellular concentration of ammonia.

Enrichment and isolation of methyl chloride utilising bacteria from a variety of pristine terrestrial, freshwater, estuarine and marine environments resulted in the detection of six new methyl chloride utilising Hyphomicrobium strains, strain CMC related to Aminobacter spp. and to two previously isolated methyl halide utilising bacteria CC495 and IMB-1, and a Gram-positive isolate SAC-4 phylogenetically related to Nocardioides spp. All the pristine environments sampled for enrichment resulted in the successful isolation of methyl chloride utilising organisms.

The production of green fluorescent protein (GFP) in Methylobacterium extorquens was studied by creating four different constructs using pJB3KmD, pRK310 and pVK101 vectors, as well as pLac and soluble methane monooxygenase (sMMO) promoters. Plasmids were introduced into the cells by electroporation. Expression of GFP by selected clones was evaluated by growing cells in complex or defined media. The use of pRK310 as an expression vector containing the lacZ promoter resulted in a 100-fold increase of GFP production when compared to cells containing the pLac-GFP-pJB3KmD construct. Higher production of GFP was observed also in cells containing pLac-GFP-pRK310 and pmmoX-GFP-pVK101 constructs. While the transcriptional regulation of the smmo gene in Methylosinus trichosporium OB3b is known to be copper-dependent, expression of GFP by M. extorquens clones harboring pmmoX-promoters was not strongly controlled by the presence of copper in the medium. The production of GFP was generally constant throughout the growth of M. extorquens carrying the pLac-GFP-pRK310 construct. GFP yields varied between 850 and 1000 μg of GFP g biomass−1. However, the yield of GFP in cells carrying pmmoX-GFP-pVK101 was somewhat reduced after the mid-exponential phase of growth.

A novel thermophilic methane-oxidising bacterium was isolated from underground hot springs in Hungary. Strain HB grew on methane at up to 72°C, the highest recorded growth temperature for a methanotroph. 16S rDNA phylogenetic analysis showed that strain HB was the first known representative of a novel, deep-branching group of the γ-Proteobacteria quite distinct from extant methanotrophs. The nucleotide sequence of pmoA, encoding particulate methane monooxygenase, was determined for this novel thermophile. Sequence comparison showed that the methane monooxygenase of this strain was most closely related to that of Methylocaldum and Methylococcus species. Particulate methane monooxygenase gene fragments having a high degree of identity to that of pmoA from strain HB were amplified by PCR from DNA isolated from thermophilic methane-oxidising enrichments inoculated with hot spring samples (55–70°C) from Japan, suggesting that this novel genus, for which we informally suggest the name ‘Methylothermus’, is widespread in thermophilic environments.

The hifH gene from Klebsiella pneumoniae, which codes for the Fe protein component of nitrogenase, was used as a probe to detect nifH homologues in total cellular DNA from 13 obligate methane oxidizing bacteria. All but one of those strains that had previously been shown capable of fixing dinitrogen contained sequences homologous to nifH. DNAs from three of the six non-diazotrophic strains were also found to possess such homology.

Southern hybridization techniques were used to examine the DNA homologies between the three nitrogenase structural genes nifH, nifD and nifK of Klebsiella pneumoniae and DNA from the obligate methane oxidizing bacterium Methylococcus capsulatus (Bath). The high degree of homology between methanotroph DNA sequences and the KlebsiellanifH and nifD genes was used to isolate and clone the corresponding MethylococcusnifH and nifD genes. Subsequent restriction analysis revealed that all three nif structural genes were contiguous in the Methylococcu genome in the order nifH, nifD and nifK, as found for the majority of other diazotrophs.

The particulate methane monooxygenase gene pmoA, encoding the 27 kDa polypeptide of the membrane-bound particulate methane monooxygenase, was amplified by PCR from DNA isolated from a blanket peat bog and from enrichment cultures established, from the same environment, using methane as sole carbon and energy source. The resulting 525 bp PCR products were cloned and a representative number of clones were sequenced. Phylogenetic analysis of the derived amino acid sequences of the pmoA clones retrieved directly from environmental DNA samples revealed that they form a distinct cluster within representative PmoA sequences from type II methanotrophs and may originate from a novel group of acidophilic methanotrophs. The study also demonstrated the utility of the pmoA gene as a phylogenetic marker for identifying methanotroph-specific DNA sequences in the environment.

Genes encoding paniculate methane monooxygenase and ammonia monooxygenase share high sequence identity. Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa polypeptides, which are believed to contain the active sites, of particulate methane monooxygenase and ammonia monooxygenase. A 525-bp internal DNA fragment of the genes encoding these polypeptides (pmoA and amoA) from a variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced. Representatives of each of the phylogenetic groups of both methanotrophs (α- and γ-Proteobacteria) and ammonia-oxidizing nitrifying bacteria (β-and y-Proteobacteria) were included. Analysis of the predicted amino acid sequences of these genes revealed strong conservation of both primary and secondary structure. Nitrosococcus oceanus AmoA showed higher identity to PmoA sequences from other members of the γ-Proteobacteria than to AmoA sequences. These results suggest that the particulate methane monooxygenase and ammonia monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria.