Evolutionary patterns and heterogeneity of Dengue Virus serotypes in Pakistan.

Comprehensive and systematic examination of Dengue virus (DENV) evolution is essential in the context of Pakistan as the virus presents a significant public health challenge with the ability to adapt and evolve. To shed light on intricate evolutionary patterns of all four DENV serotypes, we analyzed complete genome sequences (n=43) and envelope (E) gene sequences (n=44) of all four DENV serotypes collected in Pakistan from 1994 to 2023 providing a holistic view of their genetic evolution. Our findings revealed that all four serotypes of DENV co-circulate in Pakistan with a close evolutionary relationship between DENV-1 and DENV-3. Genetically distinct serotypes DENV-2 and DENV-4 indicate that DENV-4 stands out as the most genetically different, while DENV-2 exhibits greater complexity due to the presence of multiple genotypes and the possibility of temporal fluctuations in genotype prevalence. Selective pressure analysis in Envelope (E) gene revealed heterogeneity among sequences (n=44) highlighting 46 codons in the genome experiencing selective pressure, characterized by a bias towards balancing selection indicating genetic stability of the virus. Furthermore, our study suggested an intriguing evolutionary shift of DENV-4 towards the DENV-2 clade, potentially influenced by antibodies with cross-reactivity to multiple serotypes providing a critical insight into the complex factors shaping DENV evolution and contributing to the emergence of new serotype.

Methylotrophic bacteria from rice paddy soils: mineral-nitrogen-utilizing isolates richness in bulk soil and rhizosphere.

Methanol, the second most abundant volatile organic compound, primarily released from plants, is a major culprit disturbing atmospheric chemistry. Interestingly, ubiquitously found methanol-utilizing bacteria, play a vital role in mitigating atmospheric methanol effects. Despite being extensively characterized, the effect of nitrogen sources on the richness of methanol-utilizers in the bulk soil and rhizosphere is largely unknown. Therefore, the current study was planned to isolate, characterize and explore the richness of cultivable methylotrophs from the bulk soil and rhizosphere of a paddy field using media with varying nitrogen sources. Our data revealed that more genera of methylotrophs, including Methylobacterium, Ancylobacter, Achromobacter, Xanthobacter, Moraxella, and Klebsiella were enriched with the nitrate-based medium compared to only two genera, Hyphomicrobium and Methylobacterium, enriched with the ammonium-based medium. The richness of methylotrophic bacteria also differed substantially in the bulk soil as compared to the rhizosphere. Growth characterization revealed that majority of the newly isolated methanol-utilizing strains in this study exhibited better growth at 37 °C instead of 30 or 45 °C. Moreover, Hyphomicrobium sp. FSA2 was the only strain capable of utilizing methanol even at elevated temperature 45 °C, showing its adaptability to a wide range of temperatures. Differential carbon substrate utilization profiling revealed the facultative nature of all isolated methanol-utilizer strains with Xanthobacter sp. TS3, being an important methanol-utilizer capable of degrading toxic compounds such as acetone and ethylene glycol. Overall, our study suggests the role of nutrients and plant-microbial interaction in shaping the composition of methanol-utilizers in terrestrial environment.

Genomic characterization and comparative genomic analysis of HS-associated Pasteurella multocida serotype B:2 strains from Pakistan.

BACKGROUND: Haemorrhagic septicaemia (HS) is a highly fatal and predominant disease in livestock, particularly cattle and buffalo in the tropical regions of the world. Pasteurella multocida (P. multocida), serotypes B:2 and E:2, are reported to be the main causes of HS wherein serotype B:2 is more common in Asian countries including Pakistan and costs heavy financial losses every year. As yet, very little molecular and genomic information related to the HS-associated serotypes of P. multocida isolated from Pakistan is available. Therefore, this study aimed to explore the characteristics of novel bovine isolates of P. multocida serotype B:2 at the genomic level and perform comparative genomic analysis of various P. multocida strains from Pakistan to better understand the genetic basis of pathogenesis and virulence. RESULTS: To understand the genomic variability and pathogenomics, we characterized three HS-associated P. multocida serotype B:2 strains isolated from the Faisalabad (PM1), Peshawar (PM2) and Okara (PM3) districts of Punjab, Pakistan. Together with the other nine publicly available Pakistani-origin P. multocida strains and a reference strain Pm70, a comparative genomic analysis was performed. The sequenced strains were characterized as serotype B and belong to ST-122. The strains contain no plasmids; however, each strain contains at least two complete prophages. The pan-genome analysis revealed a higher number of core genes indicating a close resemblance to the studied genomes and very few genes (1%) of the core genome serve as a part of virulence, disease, and defense mechanisms. We further identified that studied P. multocida B:2 strains harbor common antibiotic resistance genes, specifically PBP3 and EF-Tu. Remarkably, the distribution of virulence factors revealed that OmpH and plpE were not present in any P. multocida B:2 strains while the presence of these antigens was reported uniformly in all serotypes of P. multocida. CONCLUSION: This study's findings indicate the absence of OmpH and PlpE in the analyzed P. multocida B:2 strains, which are known surface antigens and provide protective immunity against P. multocida infection. The availability of additional genomic data on P. multocida B:2 strains from Pakistan will facilitate the development of localized therapeutic agents and rapid diagnostic tools specifically targeting HS-associated P. multocida B:2 strains.

Inhibition of nitrous oxide reduction in forest soil microcosms by different forms of methanobactin.

Copper plays a critical role in controlling greenhouse gas emissions as it is a key component of the particulate methane monooxygenase and nitrous oxide reductase. Some methanotrophs excrete methanobactin (MB) that has an extremely high copper affinity. As a result, MB may limit the ability of other microbes to gather copper, thereby decreasing their activity as well as impacting microbial community composition. Here, we show using forest soil microcosms that multiple forms of MB; MB from Methylosinus trichosporium OB3b (MB-OB3b) and MB from Methylocystis sp. strain SB2 (MB-SB2) increased nitrous oxide (N2 O) production as well caused significant shifts in microbial community composition. Such effects, however, were mediated by the amount of copper in the soils, with low-copper soil microcosms showing the strongest response to MB. Furthermore, MB-SB2 had a stronger effect, likely due to its higher affinity for copper. The presence of either form of MB also inhibited nitrite reduction and generally increased the presence of genes encoding for the iron-containing nitrite reductase (nirS) over the copper-dependent nitrite reductase (nirK). These data indicate the methanotrophic-mediated production of MB can significantly impact multiple steps of denitrification, as well as have broad effects on microbial community composition of forest soils.

Facultative methanotrophs - diversity, genetics, molecular ecology and biotechnological potential: a mini-review.

Methane-oxidizing bacteria (methanotrophs) play a vital role in reducing atmospheric methane emissions, and hence mitigating their potent global warming effects. A significant proportion of the methane released is thermogenic natural gas, containing associated short-chain alkanes as well as methane. It was one hundred years following the description of methanotrophs that facultative strains were discovered and validly described. These can use some multi-carbon compounds in addition to methane, often small organic acids, such as acetate, or ethanol, although Methylocella strains can also use short-chain alkanes, presumably deriving a competitive advantage from this metabolic versatility. Here, we review the diversity and molecular ecology of facultative methanotrophs. We discuss the genetic potential of the known strains and outline the consequent benefits they may obtain. Finally, we review the biotechnological promise of these fascinating microbes.

Carbon source regulation of gene expression in Methylosinus trichosporium OB3b.

Gene expression in methanotrophs has been shown to be affected by the availability of a variety of metals, most notably copper regulating expression of alternative forms of methane monooxygenase. Here, we show that growth substrate also affects expression of genes encoding for enzymes responsible for the oxidation of methane to formaldehyde and the assimilation of carbon. Specifically, in Methylosinus trichosporium OB3b, expression of genes involved in the conversion of methane to methanol (pmoA and mmoX) and methanol to formaldehyde (mxaF, xoxF1, and xoxF2) as well as in carbon assimilation (fae1, fae2, metF, and sga) decreased when this strain was grown on methanol vs. methane, indicating that methanotrophs manipulate gene expression in response to growth substrate as well as the availability of copper. Interestingly, growth of M. trichosporium OB3b on methane vs. methanol was similar despite such large changes in gene expression. Finally, methanol-grown cultures of M. trichosporium OB3b also exhibited the "copper-switch." That is, expression of pmoA increased and mmoX decreased in the presence of copper, indicating that copper still controlled the expression of alternative forms of methane monooxygenase in M. trichosporium OB3b even though methane was not provided. Such findings indicate that methanotrophs can sense and respond to multiple environmental parameters simultaneously.

A TonB-Dependent Transporter Is Responsible for Methanobactin Uptake by Methylosinus trichosporium OB3b.

Methanobactin, a small modified polypeptide synthesized by methanotrophs for copper uptake, has been found to be chromosomally encoded. The gene encoding the polypeptide precursor of methanobactin, mbnA, is part of a gene cluster that also includes several genes encoding proteins of unknown function (but speculated to be involved in methanobactin formation) as well as mbnT, which encodes a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine if mbnT is truly responsible for methanobactin uptake, a knockout was constructed in Methylosinus trichosporium OB3b using marker exchange mutagenesis. The resulting M. trichosporium mbnT::Gm(r) mutant was found to be able to produce methanobactin but was unable to internalize it. Further, if this mutant was grown in the presence of copper and exogenous methanobactin, copper uptake was significantly reduced. Expression of mmoX and pmoA, encoding polypeptides of the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), respectively, also changed significantly when methanobactin was added, which indicates that the mutant was unable to collect copper under these conditions. Copper uptake and gene expression, however, were not affected in wild-type M. trichosporium OB3b, indicating that the TonB-dependent transporter encoded by mbnT is responsible for methanobactin uptake and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When the mbnT::Gm(r) mutant was grown under a range of copper concentrations in the absence of methanobactin, however, the phenotype of the mutant was indistinguishable from that of wild-type M. trichosporium OB3b, indicating that this methanotroph has multiple mechanisms for copper uptake.

Marker Exchange Mutagenesis of mxaF, Encoding the Large Subunit of the Mxa Methanol Dehydrogenase, in Methylosinus trichosporium OB3b.

Methanotrophs have remarkable redundancy in multiple steps of the central pathway of methane oxidation to carbon dioxide. For example, it has been known for over 30 years that two forms of methane monooxygenase, responsible for oxidizing methane to methanol, exist in methanotrophs, i.e., soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), and that expression of these two forms is controlled by the availability of copper. Specifically, sMMO expression occurs in the absence of copper, while pMMO expression increases with increasing copper concentrations. More recently, it was discovered that multiple forms of methanol dehydrogenase (MeDH), Mxa MeDH and Xox MeDH, also exist in methanotrophs and that the expression of these alternative forms is regulated by the availability of cerium. That is, expression of Xox MeDH increases in the presence of cerium, while Mxa MeDH expression decreases in the presence of cerium. As it had been earlier concluded that pMMO and Mxa MeDH form a supercomplex in which electrons from Mxa MeDH are back donated to pMMO to drive the initial oxidation of methane, we speculated that Mxa MeDH could be rendered inactive through marker-exchange mutagenesis but growth on methane could still be possible if cerium was added to increase the expression of Xox MeDH under sMMO-expressing conditions. Here we report that mxaF, encoding the large subunit of Mxa MeDH, could indeed be knocked out in Methylosinus trichosporium OB3b, yet growth on methane was still possible, so long as cerium was added. Interestingly, growth of this mutant occurred in both the presence and the absence of copper, suggesting that Xox MeDH can replace Mxa MeDH regardless of the form of MMO expressed.

Cerium regulates expression of alternative methanol dehydrogenases in Methylosinus trichosporium OB3b.

Methanotrophs have multiple methane monooxygenases that are well known to be regulated by copper, i.e., a "copper switch." At low copper/biomass ratios the soluble methane monooxygenase (sMMO) is expressed while expression and activity of the particulate methane monooxygenase (pMMO) increases with increasing availability of copper. In many methanotrophs there are also multiple methanol dehydrogenases (MeDHs), one based on Mxa and another based on Xox. Mxa-MeDH is known to have calcium in its active site, while Xox-MeDHs have been shown to have rare earth elements in their active site. We show here that the expression levels of Mxa-MeDH and Xox-MeDH in Methylosinus trichosporium OB3b significantly decreased and increased, respectively, when grown in the presence of cerium but the absence of copper compared to the absence of both metals. Expression of sMMO and pMMO was not affected. In the presence of copper, the effect of cerium on gene expression was less significant, i.e., expression of Mxa-MeDH in the presence of copper and cerium was slightly lower than in the presence of copper alone, but Xox-MeDH was again found to increase significantly. As expected, the addition of copper caused sMMO and pMMO expression levels to significantly decrease and increase, respectively, but the simultaneous addition of cerium had no discernible effect on MMO expression. As a result, it appears Mxa-MeDH can be uncoupled from methane oxidation by sMMO in M. trichosporium OB3b but not from pMMO.

Methanobactin from Methylocystis sp. strain SB2 affects gene expression and methane monooxygenase activity in Methylosinus trichosporium OB3b.

Methanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 µM CuCl2, expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl2, the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl2. mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl2. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin "piracy" may be commonplace.

Fluorescence-based bacterial bioreporter for specific detection of methyl halide emissions in the environment.

Methyl halides are volatile one-carbon compounds responsible for substantial depletion of stratospheric ozone. Among them, chloromethane (CH3Cl) is the most abundant halogenated hydrocarbon in the atmosphere. Global budgets of methyl halides in the environment are still poorly understood due to uncertainties in their natural sources, mainly from vegetation, and their sinks, which include chloromethane-degrading bacteria. A bacterial bioreporter for the detection of methyl halides was developed on the basis of detailed knowledge of the physiology and genetics of Methylobacterium extorquens CM4, an aerobic alphaproteobacterium which utilizes chloromethane as the sole source of carbon and energy. A plasmid construct with the promoter region of the chloromethane dehalogenase gene cmuA fused to a promotorless yellow fluorescent protein gene cassette resulted in specific methyl halide-dependent fluorescence when introduced into M. extorquens CM4. The bacterial whole-cell bioreporter allowed detection of methyl halides at femtomolar levels and quantification at concentrations above 10 pM (approximately 240 ppt). As shown for the model chloromethane-producing plant Arabidopsis thaliana in particular, the bioreporter may provide an attractive alternative to analytical chemical methods to screen for natural sources of methyl halide emissions.

Detection of BK and JC polyomaviruses in sewage water of the urban areas of Lahore, Pakistan.

The surveillance of sewage water has become an extremely essential tool to trace the circulation of viruses in a population and to predict the outbreak of viral diseases. Sewage monitoring is important for those viruses which cause subclinical infections since it is difficult to determine their prevalence. Polyomaviruses are ubiquitously present, circular double-stranded DNA viruses that can infect humans as well. Among all human polyomaviruses, BK polyomavirus and JC polyomavirus associated with the development of aggressive diseases in immunocompromised individuals, are highly prevalent. This study aimed to investigate the presence and the quantitative prevalence of these two disease-associated human polyomaviruses in sewage water collected from six drains of Lahore, Pakistan. The viruses present in the environmental samples were concentrated by PEG method before isolating viral nucleic acids. Conventional PCR amplifications were performed for molecular detection of BK polyomavirus and JC polyomavirus targeting their large tumor antigen genetic region. The presence of BK polyomavirus and JC polyomavirus was confirmed in the DNA extracted from concentrated sewage samples of each drain by performing both qualitative and quantitative PCR. Our data shows that the viral load ranged from 1278 to 178368 copies per µg of environmental DNA for BK polyomavirus and 5173 to 79129 copies per µg of environmental DNA for JC polyomavirus. In conclusion, here we report first time the detection of BK polyomavirus and JC polyomavirus in sewage water collected from six main drains in urban areas of Lahore, Pakistan showing the high prevalence of these viruses in the Pakistani population. This assay could be used as a proxy to determine the prevalence of these viruses in the Pakistani population.

Complete genome sequences of six strains of the genus Methylobacterium.

The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches and environmental constraints.

Identification of active gaseous-alkane degraders at natural gas seeps.

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 Rhodoblastus strains (PC2 and PC3) were from Pipe Creek and a Mycolicibacterium strain (ANDR5) from Andreiasu. Strains PC2 and PC3 encoded putative butane monooxygenases (MOs) and strain ANDR5 contained a propane MO. Mycolicibacterium strain 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.

Antimicrobial activity of bacteria associated with the rhizosphere and phyllosphere of Avena fatua and Brachiaria reptans.

Environmental pollution especially heavy metal-contaminated soils adversely affects the microbial communities associated with the rhizosphere and phyllosphere of plants growing in these areas. In the current study, we identified and characterized the rhizospheric and phyllospheric bacterial strains from Avena fatua and Brachiaria reptans with the potential for antimicrobial activity and heavy metal resistance. A total of 18 bacterial strains from the rhizosphere and phyllosphere of A. fatua and 19 bacterial strains from the rhizosphere and phyllosphere of B. reptans were identified based on 16S rRNA sequence analysis. Bacterial genera, including Bacillus, Staphylococcus, Pseudomonas, and Enterobacter were dominant in the rhizosphere and phyllosphere of A. fatua and Bacillus, Marinobacter, Pseudomonas, Enterobacter, and Kocuria, were the dominating bacterial genera from the rhizosphere and phyllosphere of B. reptans. Most of the bacterial strains were resistant to heavy metals (Cd, Pb, and Cr) and showed antimicrobial activity against different pathogenic bacterial strains. The whole-genome sequence analysis of Pseudomonas putida BR-PH17, a strain isolated from the phyllosphere of B. reptans, was performed by using the Illumina sequencing approach. The BR-PH17 genome contained a chromosome with a size of 5774330 bp and a plasmid DNA with 80360 bp. In this genome, about 5368 predicted protein-coding sequences with 5539 total genes, 22 rRNAs, and 75 tRNA genes were identified. Functional analysis of chromosomal and plasmid DNA revealed a variety of enzymes and proteins involved in antibiotic resistance and biodegradation of complex organic pollutants. These results indicated that bacterial strains identified in this study could be utilized for bioremediation of heavy metal-contaminated soils and as a novel source of antimicrobial drugs.

Novel coronavirus disease (COVID-19) pandemic: A recent mini review.

The COVID-19, caused by a novel coronavirus, was declared as a global pandemic by WHO more than five months ago, and we are still experiencing a state of global emergency. More than 74.30 million confirmed cases of the COVID-19 have been reported globally so far, with an average fatality rate of almost 3.0%. Seven different types of coronaviruses had been detected from humans; three of them have resulted in severe outbreaks, i.e., MERS-CoV, SARS-CoV, and SARS-CoV-2. Phylogenetic analysis of the genomes suggests that the possible occurrence of recombination between SARS-like-CoVs from pangolin and bat might have led to the origin of SARS-CoV-2 and the COVID-19 outbreak. Coronaviruses are positive-sense, single-stranded RNA viruses and harbour a genome (30 kb) consisting of two terminal untranslated regions and twelve putative functional open reading frames (ORFs), encoding for non-structural and structural proteins. There are sixteen putative non-structural proteins, including proteases, RNA-dependent RNA polymerase, helicase, other proteins involved in the transcription and replication of SARS-CoV-2, and four structural proteins, including spike protein (S), envelope (E), membrane (M), and nucleocapsid (N). SARS-CoV-2 infection, with a heavy viral load in the body, destroys the human lungs through cytokine storm, especially in elderly persons and people with immunosuppressed disorders. A number of drugs have been repurposed and employed, but still, no specific antiviral medicine has been approved by the FDA to treat this disease. This review provides a current status of the COVID-19, epidemiology, an overview of phylogeny, mode of action, diagnosis, and possible treatment methods and vaccines.

Human BK and JC polyomaviruses: Molecular insights and prevalence in Asia.

Polyomaviridae family consists of small circular dsDNA viruses. Out of the 14 human polyomaviruses described so far, BKPyV and JCPyV have been studied extensively since their discovery in 1971. Reportedly, both BKPyV and JCPyV are widely distributed across the globe with the frequency of 80-90 % in different populations. The primary infection of these viruses is usually asymptomatic and latent which is activated as a consequence of immunosuppression. Activated BKPyV and JCPyV viruses lead to the development of BK Virus Associated Nephropathy and Progressive Multifocal Leukoencephalopathy, respectively. Immense progress has been made during the last few decades regarding the molecular understanding of polyomaviruses. Epidemiology of polyomaviruses has also been studied extensively. However, most of the epidemiological studies have focused on European and American populations. Therefore, limited data is available regarding the geographical distribution of these potentially oncogenic viruses in Asian countries. In this article, we have presented a compendium of latest advances in the molecular understanding of polyomaviruses and their pathobiology. We also present a comprehensive review of published literature regarding the epidemiology and prevalence of BKPyV and JCPyV in Asian regions. For this purpose, a thorough search of available online resources was performed. As a result, we retrieved 24 studies for BKPyV and 22 studies for JCPyV, that describe their prevalence in Asia. These studies unanimously report high occurrence of both BKPyV and JCPyV in Asian populations. The available data from these studies was categorized into two groups: on the basis of prevalence (low, medium and high) and disease development (healthy and diseased). Altogether, Korean population hasbeen evidenced to possess highest frequency of BKPyV (66.7 %), while JCPyV was found to be most prevalent in Taiwan (88 %). Due to high and ubiquitous distribution of these viruses, frequent studies are required to develop a better understanding regarding the epidemiology and pathobiology of these viruses in Asia.

Novel facultative Methylocella strains are active methane consumers at terrestrial natural gas seeps.

BACKGROUND: Natural gas seeps contribute to global climate change by releasing substantial amounts of the potent greenhouse gas methane and other climate-active gases including ethane and propane to the atmosphere. However, methanotrophs, bacteria capable of utilising methane as the sole source of carbon and energy, play a significant role in reducing the emissions of methane from many environments. Methylocella-like facultative methanotrophs are a unique group of bacteria that grow on other components of natural gas (i.e. ethane and propane) in addition to methane but a little is known about the distribution and activity of Methylocella in the environment. The purposes of this study were to identify bacteria involved in cycling methane emitted from natural gas seeps and, most importantly, to investigate if Methylocella-like facultative methanotrophs were active utilisers of natural gas at seep sites. RESULTS: The community structure of active methane-consuming bacteria in samples from natural gas seeps from Andreiasu Everlasting Fire (Romania) and Pipe Creek (NY, USA) was investigated by DNA stable isotope probing (DNA-SIP) using 13C-labelled methane. The 16S rRNA gene sequences retrieved from DNA-SIP experiments revealed that of various active methanotrophs, Methylocella was the only active methanotrophic genus common to both natural gas seep environments. We also isolated novel facultative methanotrophs, Methylocella sp. PC1 and PC4 from Pipe Creek, able to utilise methane, ethane, propane and various non-gaseous multicarbon compounds. Functional and comparative genomics of these new isolates revealed genomic and physiological divergence from already known methanotrophs, in particular, the absence of mxa genes encoding calcium-containing methanol dehydrogenase. Methylocella sp. PC1 and PC4 had only the soluble methane monooxygenase (sMMO) and lanthanide-dependent methanol dehydrogenase (XoxF). These are the first Alphaproteobacteria methanotrophs discovered with this reduced functional redundancy for C-1 metabolism (i.e. sMMO only and XoxF only). CONCLUSIONS: Here, we provide evidence, using culture-dependent and culture-independent methods, that Methylocella are abundant and active at terrestrial natural gas seeps, suggesting that they play a significant role in the biogeochemical cycling of these gaseous alkanes. This might also be significant for the design of biotechnological strategies for controlling natural gas emissions, which are increasing globally due to unconventional exploitation of oil and gas.

Methanethiol and Dimethylsulfide Cycling in Stiffkey Saltmarsh.

Methanethiol (MeSH) and dimethylsulfide (DMS) are volatile organic sulfur compounds (VOSCs) with important roles in sulfur cycling, signaling and atmospheric chemistry. DMS can be produced from MeSH through a reaction mediated by the methyltransferase MddA. The mddA gene is present in terrestrial and marine metagenomes, being most abundant in soil environments. The substrate for MddA, MeSH, can also be oxidized by bacteria with the MeSH oxidase (MTO) enzyme, encoded by the mtoX gene, found in marine, freshwater and soil metagenomes. Methanethiol-dependent DMS production (Mdd) pathways have been shown to function in soil and marine sediments, but have not been characterized in detail in the latter environments. In addition, few molecular studies have been conducted on MeSH consumption in the environment. Here, we performed process measurements to confirm that Mdd-dependent and Mdd-independent MeSH consumption pathways are active in tested surface saltmarsh sediment when MeSH is available. We noted that appreciable natural Mdd-independent MeSH and DMS consumption processes masked Mdd activity. 16S rRNA gene amplicon sequencing and metagenomics data showed that Methylophaga, a bacterial genus known to catabolise DMS and MeSH, was enriched by the presence of MeSH. Moreover, some MeSH and/or DMS-degrading bacteria isolated from this marine environment lacked known DMS and/or MeSH cycling genes and can be used as model organisms to potentially identify novel genes in these pathways. Thus, we are likely vastly underestimating the abundance of MeSH and DMS degraders in these marine sediment environments. The future discovery and characterization of novel enzymes involved in MeSH and/or DMS cycling is essential to better assess the role and contribution of microbes to global organosulfur cycling.

Complete Genome Sequence of the Aerobic Facultative Methanotroph Methylocella tundrae Strain T4.

Methylocella tundrae T4T is a facultative aerobic methanotroph which was isolated from an acidic tundra wetland and possesses only a soluble methane monooxygenase. The complete genome, which includes two megaplasmids, was sequenced using a combination of Illumina and Nanopore technologies. One of the megaplasmids carries a propane monooxygenase gene cluster.