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A review of bacterial methyl halide degradation: Biochemistry, genetics and molecular ecology

Methyl halide‐degrading bacteria are a diverse group of organisms that are found in both terrestrial and marine environments. They potentially play an important role in mitigating ozone depletion resulting from methyl chloride and methyl bromide emissions. The first step in the pathway(s) of methyl halide degradation involves a methyltransferase and, recently, the presence of this pathway has been studied in a number of bacteria. This paper reviews the biochemistry and genetics of methyl halide utilization in the aerobic bacteria Methylobacterium chloromethanicum CM4 T , Hyphomicrobium chloromethanicum CM2 T , Aminobacter strain IMB‐1 and Aminobacter strain CC495. These bacteria are able to use methyl halides as a sole source of carbon and energy, are all members of the α ‐Proteobacteria and were isolated from a variety of polluted and pristine terrestrial environments. An understanding of the genetics of these bacteria identified a unique gene ( cmuA ) involved in the degradation of methyl halides, which codes for a protein (CmuA) with unique methyltransferase and corrinoid functions. This unique functional gene, cmuA , is being used to develop molecular ecology techniques to examine the diversity and distribution of methyl halide‐utilizing bacteria in the environment and hopefully to understand their role in methyl halide degradation in different environments. These techniques will also enable the detection of potentially novel methyl halide‐degrading bacteria.

Environmental Microbiology

Dryland biological soil crust cyanobacteria show unexpected decreases in abundance under long-term elevated CO 2

Biological soil crusts (biocrusts) cover soil surfaces in many drylands globally. The impacts of 10 years of elevated atmospheric CO 2 on the cyanobacteria in biocrusts of an arid shrubland were examined at a large manipulated experiment in Nevada, USA. Cyanobacteria-specific quantitative PCR surveys of cyanobacteria small-subunit (SSU) rRNA genes suggested a reduction in biocrust cyanobacterial biomass in the elevated CO 2 treatment relative to the ambient controls. Additionally, SSU rRNA gene libraries and shotgun metagenomes showed reduced representation of cyanobacteria in the total microbial community. Taxonomic composition of the cyanobacteria was similar under ambient and elevated CO 2 conditions, indicating the decline was manifest across multiple cyanobacterial lineages. Recruitment of cyanobacteria sequences from replicate shotgun metagenomes to cyanobacterial genomes representing major biocrust orders also suggested decreased abundance of cyanobacteria sequences across the majority of genomes tested. Functional assignment of cyanobacteria-related shotgun metagenome sequences indicated that four subsystem categories, three related to oxidative stress, were differentially abundant in relation to the elevated CO 2 treatment. Taken together, these results suggest that elevated CO 2 affected a generalized decrease in cyanobacteria in the biocrusts and may have favoured cyanobacteria with altered gene inventories for coping with oxidative stress.

Nevada

Organic matter quantity and source affects microbial community structure and function following volcanic eruption on Kasatochi Island, Alaska

In August 2008, Kasatochi volcano erupted and buried a small island in pyroclastic deposits and fine ash; since then, microbes, plants and birds have begun to re-colonize the initially sterile surface. Five years post-eruption, bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) copy numbers and extracellular enzyme activity (EEA) potentials were one to two orders of magnitude greater in pyroclastic materials with organic matter (OM) inputs relative to those without, despite minimal accumulation of OM (<&thinsp;0.2%C). When normalized by OM levels, post-eruptive surfaces with OM inputs had the highest &beta;-glucosidase, phosphatase, NAGase and cellobiohydrolase activities, and had microbial population sizes approaching those in reference soils. In contrast, the strongest factor determining bacterial community composition was the dominance of plants versus birds as OM input vectors. Although soil pH ranged from 3.9 to 7.0, and %C ranged 100&times;, differentiation between plant- and bird-associated microbial communities suggested that cell dispersal or nutrient availability are more likely drivers of assembly than pH or OM content. This study exemplifies the complex relationship between microbial cell dispersal, soil geochemistry, and microbial structure and function; and illustrates the potential for soil microbiota to be resilient to disturbance.

Alaska

Concentration and retention of Toxoplasma gondii oocysts by marine snails demonstrate a novel mechanism for transmission of terrestrial zoonotic pathogens in coastal ecosystems

The parasite T oxoplasma gondii is an environmentally persistent pathogen that can cause fatal disease in humans, terrestrial warm-blooded animals and aquatic mammals. Although an association between T .&thinsp;gondii exposure and prey specialization on marine snails was identified in threatened California sea otters, the ability of kelp-dwelling snails to transmit terrestrially derived pathogens has not been previously investigated. The objective of this study was to measure concentration and retention of T .&thinsp;gondii by marine snails in laboratory aquaria, and to test for natural T .&thinsp;gondii contamination in field-collected snails. Following exposure to T .&thinsp;gondii -containing seawater, oocysts were detected by microscopy in snail faeces and tissues for 10 and 3 days respectively. Nested polymerase chain reaction was also applied as a method for confirming putative T .&thinsp;gondii oocysts detected in snail faeces and tissues by microscopy. T oxoplasma gondii was not detected in field-collected snails. Results suggest that turban snails are competent transport hosts for T .&thinsp;gondii . By concentrating oocysts in faecal pellets, snails may facilitate entry of T .&thinsp;gondii into the nearshore marine food web. This novel mechanism also represents a general pathway by which marine transmission of terrestrially derived microorganisms can be mediated via pathogen concentration and retention by benthic invertebrates.

Environmental Microbiology

Functional metagenomic selection of RubisCOs from uncultivated bacteria

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO 2 -dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of the G allionellaceae family. One of the G allionellaceae RubisCOs was purified and shown to possess CO 2 /O 2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO 2 -fixing enzymes not previously characterized.

Environmental Microbiology

The genetic basis of anoxygenic photosynthetic arsenite oxidation

“Photoarsenotrophy”, the use of arsenite as an electron donor for anoxygenic photosynthesis, is thought to be an ancient form of phototrophy along with the photosynthetic oxidation of Fe(II), H 2 S, H 2 , and NO 2 - . Photoarsenotrophy was recently identified from Paoha Island's (Mono Lake, CA) arsenic-rich hot springs. The genomes of several photoarsenotrophs revealed a gene cluster, arxB2AB1CD, where arxA is predicted to encode for the sole arsenite oxidase. The role of arxA in photosynthetic arsenite oxidation was confirmed by disrupting the gene in a representative photoarsenotrophic bacterium, resulting in the loss of light-dependent arsenite oxidation. In situ evidence of active photoarsenotrophic microbes was supported by arxA mRNA detection for the first time, in red-pigmented microbial mats within the hot springs of Paoha Island. This work expands on the genetics for photosynthesis coupled to new electron donors and elaborates on known mechanisms for arsenic metabolism, thereby highlighting the complexities of arsenic biogeochemical cycling.

Environmental Microbiology

Bacterial, fungal, and plant communities exhibit no biomass or compositional response to two years of simulated nitrogen deposition in a semiarid grassland

Nitrogen (N) deposition affects myriad aspects of terrestrial ecosystem structure and function, and microbial communities may be particularly sensitive to anthropogenic N inputs. However, our understanding of N deposition effects on microbial communities is far from complete, especially for drylands where data are comparatively rare. To address the need for an improved understanding of dryland biological responses to N deposition, we conducted a two-year fertilization experiment in a semiarid grassland on the Colorado Plateau in the southwestern United States. We evaluated effects of varied levels of N inputs on archaeal, bacterial, fungal and chlorophyte community composition within three microhabitats: biological soil crusts (biocrusts), soil below biocrusts, and the plant rhizosphere. Surprisingly, N addition did not affect the community composition or diversity of any of these microbial groups; however, microbial community composition varied significantly among sampling microhabitats. Further, while plant richness, diversity, and cover showed no response to N addition, there were strong linkages between plant properties and microbial community structure. Overall, these findings highlight the potential for some dryland communities to have limited biotic ability to retain augmented N inputs, possibly leading to large N losses to the atmosphere and to aquatic systems.

Environmental Microbiology

Sulfolobus islandicus meta-populations in Yellowstone National Park hot springs

Abiotic and biotic forces shape the structure and evolution of microbial populations. We investigated forces that shape the spatial and temporal population structure of Sulfolobus islandicus by comparing geochemical and molecular analysis from seven hot springs in five regions sampled over 3 years in Yellowstone National Park. Through deep amplicon sequencing, we uncovered 148 unique alleles at two loci whose relative frequency provides clear evidence for independent populations in different hot springs. Although geography controls regional geochemical composition and population differentiation, temporal changes in population were not explained by corresponding variation in geochemistry. The data suggest that the influence of extinction, bottleneck events and/or selective sweeps within a spring and low migration between springs shape these populations. We suggest that hydrologic events such as storm events and surface snowmelt runoff destabilize smaller hot spring environments with smaller populations and result in high variation in the S. islandicus population over time. Therefore, physical abiotic features such as hot spring size and position in the landscape are important factors shaping the stability and diversity of the S. islandicus meta-population within Yellowstone National Park.

Wyoming

Low occurrence of multi-antimicrobial and heavy metal resistance in Salmonella enterica from wild birds in the United States

Wild birds are common reservoirs of Salmonella enterica . Wild birds carrying resistant S . enterica may pose a risk to public health as they can spread the resistant bacteria across large spatial scales within a short time. Here, we whole-genome sequenced 375 S . enterica strains from wild birds collected in 41 U.S. states during 1978–2019 to examine bacterial resistance to antibiotics and heavy metals. We found that Typhimurium was the dominant S . enterica serovar, accounting for 68.3% (256/375) of the bird isolates. Furthermore, the proportions of the isolates identified as multi-antimicrobial resistant (multi-AMR: resistant to at least three antimicrobial classes) or multi-heavy metal resistant (multi-HMR: resistant to at least three heavy metals) were both 1.87% (7/375). Interestingly, all the multi-resistant S . enterica ( n = 12) were isolated from water birds or raptors; none of them was isolated from songbirds. Plasmid profiling demonstrated that 75% (9/12) of the multi-resistant strains carried resistance plasmids. Our study indicates that wild birds do not serve as important reservoirs of multi-resistant S . enterica strains. Nonetheless, continuous surveillance for bacterial resistance in wild birds is necessary because the multi-resistant isolates identified in this study also showed close genetic relatedness with those from humans and domestic animals.

Environmental Microbiology

Methanogenic archaea in subsurface coal seams are biogeographically distinct: An analysis of metagenomically-derived mcrA sequences

The production of methane as an end-product of organic matter degradation in the absence of other terminal electron acceptors is common, and has often been studied in environments such as animal guts, soils, and wetlands due to its potency as a greenhouse gas. To date however, the study of the biogeographic distribution of methanogens across coal seam environments has been minimal. Here, we show that coal seams are host to a diverse range of methanogens, which are distinctive to each geological basin. Based on comparisons to close relatives from other methanogenic environments, the dominant methanogenic pathway in these basins is hydrogenotrophic, with acetoclastic being a second major pathway in the Surat Basin. Finally, mcrA and 16S rRNA gene primer biases were predominantly seen to affect the detection of Methanocellales, Methanomicrobiales and Methanosarcinales taxa in this study. Subsurface coal methanogenic community distributions and pathways presented here provide insights into important metabolites and bacterial partners for in situ coal biodegradation.

Environmental Microbiology

Microbial community composition and endolith colonization at an Arctic thermal spring are driven by calcite precipitation

Environmental conditions shape community composition. Arctic thermal springs provide an opportunity to study how environmental gradients can impose strong selective pressures on microbial communities and provide a continuum of niche opportunities. We use microscopic and molecular methods to conduct a survey of microbial community composition at Troll Springs on Svalbard, Norway, in the high Arctic. Microorganisms there exist under a wide range of environmental conditions: in warm water as periphyton, in moist granular materials, and in cold, dry rock as endoliths. Troll Springs has two distinct ecosystems, aquatic and terrestrial, together in close proximity, with different underlying environmental factors shaping each microbial community. Periphyton are entrapped during precipitation of calcium carbonate from the spring's waters, providing microbial populations that serve as precursors for the development of endolithic communities. This process differs from most endolith colonization, in which the rock predates the communities that colonize it. Community composition is modulated as environmental conditions change within the springs. At Troll, the aquatic environments show a small number of dominant operational taxonomic units (OTUs) that are specific to each sample. The terrestrial environments show a more even distribution of OTUs common to multiple samples.

Environmental Microbiology Reports

Early emergence of mcr-1-positive Enterobacteriaceae in gulls from Spain and Portugal

We tested extended‐spectrum β‐lactamase producing bacteria from wild gulls ( Larus spp.) sampled in 2009 for the presence of mcr ‐1. We report the detection of mcr ‐1 and describe genome characteristics of four Escherichia coli and one Klebsiella pneumoniae isolate from Spain and Portugal that also exhibited colistin resistance. Results represent the earliest evidence for colistin‐resistant bacteria in European wildlife.

Environmental Microbiology Reports

Spatial and temporal changes in microbial community structure associated with recharge-influenced chemical gradients in a contaminated aquifer

In a contaminated water‐table aquifer, we related microbial community structure on aquifer sediments to gradients in 24 geochemical and contaminant variables at five depths, under three recharge conditions. Community amplified ribsosomal DNA restriction analysis (ARDRA) using universal 16S rDNA primers and denaturing gradient gel electrophoresis (DGGE) using bacterial 16S rDNA primers indicated: (i) communities in the anoxic, contaminated central zone were similar regardless of recharge; (ii) after recharge, communities at greatest depth were similar to those in uncontaminated zones; and (iii) after extended lack of recharge, communities at upper and lower aquifer margins differed from communities at the same depths on other dates. General aquifer geochemistry was as important as contaminant or terminal electron accepting process (TEAP) chemistry in discriminant analysis of community groups. The Shannon index of diversity ( H ) and the evenness index ( E ), based on DGGE operational taxonomic units (OTUs), were statistically different across community groups and aquifer depths. Archaea or sulphate‐reducing bacteria 16S rRNA abundance was not clearly correlated with TEAP chemistry indicative of methanogenesis or sulphate reduction. Eukarya rRNA abundance varied by depth and date from 0 to 13% of the microbial community. This contaminated aquifer is a dynamic ecosystem, with complex interactions between physical, chemical and biotic components, which should be considered in the interpretation of aquifer geochemistry and in the development of conceptual or predictive models for natural attenuation or remediation.

Environmental Microbiology

Relevance of ammonium oxidation within biological soil crust communities

Thin, vertically structured topsoil communities that become ecologically important in arid regions (biological soil crusts or BSCs) are responsible for much of the nitrogen inputs into pristine arid lands. We studied N2 fixation and ammonium oxidation (AO) at subcentimetre resolution within BSCs from the Colorado Plateau. Pools of dissolved porewater nitrate/ nitrite, ammonium and organic nitrogen in wetted BSCs were high in comparison with those typical of aridosoils. They remained stable during incubations, indicating that input and output processes were of similar magnitude. Areal N2 fixation rates (6.5-48 ??mol C2H2 m-2 h -1) were high, the vertical distribution of N2 fixation peaking close to the surface if populations of heterocystous cyanobacteria were present, but in the subsurface if they were absent. Areal AO rates (19-46 ??mol N m-2 h-1) were commensurate with N2 fixation inputs. When considering oxygen availability, AO activity invariably peaked 2-3 mm deep and was limited by oxygen (not ammonium) supply. Most probable number (MPN)-enumerated ammonia-oxidizing bacteria (6.7-7.9 ?? 103 cells g-1 on average) clearly peaked at 2-3 mm depth. Thus, AO (hence nitrification) is a spatially restricted but important process in the nitrogen cycling of BSC, turning much of the biologically fixed nitrogen into oxidized forms, the fate of which remains to be determined.

Environmental Microbiology

Methylmercury enters an aquatic food web through acidophilic microbial mats in Yellowstone National Park, Wyoming

Microbial mats are a visible and abundant life form inhabiting the extreme environments in Yellowstone National Park (YNP), WY, USA. Little is known of their role in food webs that exist in the Park's geothermal habitats. Eukaryotic green algae associated with a phototrophic green/purple Zygogonium microbial mat community that inhabits low-temperature regions of acidic (pH ∼ 3.0) thermal springs were found to serve as a food source for stratiomyid (Diptera: Stratiomyidae) larvae. Mercury in spring source water was taken up and concentrated by the mat biomass. Monomethylmercury compounds (MeHg + ), while undetectable or near the detection limit (0.025 ng l −1 ) in the source water of the springs, was present at concentrations of 4–7 ng g −1 dry weight of mat biomass. Detection of MeHg + in tracheal tissue of larvae grazing the mat suggests that MeHg + enters this geothermal food web through the phototrophic microbial mat community. The concentration of MeHg + was two to five times higher in larval tissue than mat biomass indicating MeHg + biomagnification occurred between primary producer and primary consumer trophic levels. The Zygogonium mat community and stratiomyid larvae may also play a role in the transfer of MeHg + to species in the food web whose range extends beyond a particular geothermal feature of YNP.

Wyoming

Microclimate and limits to photosynthesis in a diverse community of hypolithic cyanobacteria in northern Australia

Hypolithic microbes, primarily cyanobacteria, inhabit the highly specialized microhabitats under translucent rocks in extreme environments. Here we report findings from hypolithic cyanobacteria found under three types of translucent rocks (quartz, prehnite, agate) in a semiarid region of tropical Australia. We investigated the photosynthetic responses of the cyanobacterial communities to light, temperature and moisture in the laboratory, and we measured the microclimatic variables of temperature and soil moisture under rocks in the field over an annual cycle. We also used molecular techniques to explore the diversity of hypolithic cyanobacteria in this community and their phylogenetic relationships within the context of hypolithic cyanobacteria from other continents. Based on the laboratory experiments, photosynthetic activity required a minimum soil moisture of 15% (by mass). Peak photosynthetic activity occurred between approximately 8°C and 42°C, though some photosynthesis occurred between −1°C and 51°C. Maximum photosynthesis rates also occurred at light levels of approximately 150–550 μmol m −2 s −1 . We used the field microclimatic data in conjunction with these measurements of photosynthetic efficiency to estimate the amount of time the hypolithic cyanobacteria could be photosynthetically active in the field. Based on these data, we estimated that conditions were appropriate for photosynthetic activity for approximately 942 h (∼75 days) during the year. The hypolithic cyanobacteria community under quartz, prehnite and agate rocks was quite diverse both within and between rock types. We identified 115 operational taxonomic units (OTUs), with each rock hosting 8–24 OTUs. A third of the cyanobacteria OTUs from northern Australia grouped with Chroococcidiopsis , a genus that has been identified from hypolithic and endolithic communities from the Gobi, Mojave, Atacama and Antarctic deserts. Several OTUs identified from northern Australia have not been reported to be associated with hypolithic communities previously.

Environmental Microbiology

Human enteric viruses in groundwater indicate offshore transport of human sewage to coral reefs of the Upper Florida Keys

To address the issue of human sewage reaching corals along the main reef of the Florida Keys, samples were collected from surface water, groundwater and coral [surface mucopolysaccharide layers (SML)] along a 10 km transect near Key Largo, FL. Samples were collected semi-annually between July 2003 and September 2005 and processed for faecal indicator bacteria (faecal coliform bacteria, enterococci and Clostridium perfringens) and human-specific enteric viruses (enterovirus RNA and adenovirus DNA) by (RT)-nested polymerase chain reaction. Faecal indicator bacteria concentrations were generally higher nearshore and in the coral SML. Enteric viruses were evenly distributed across the transect stations. Adenoviruses were detected in 37 of 75 samples collected (49.3%) whereas enteroviruses were only found in 8 of 75 samples (10.7%). Both viruses were detected twice as frequently in coral compared with surface water or groundwater. Offshore, viruses were most likely to be found in groundwater, especially during the wet summer season. These data suggest that polluted groundwater may be moving to the outer reef environment in the Florida Keys.

Florida

Microbial community structure of hydrothermal deposits from geochemically different vent fields along the Mid-Atlantic Ridge

To evaluate the effects of local fluid geochemistry on microbial communities associated with active hydrothermal vent deposits, we examined the archaeal and bacterial communities of 12 samples collected from two very different vent fields: the basalt-hosted Lucky Strike (37&deg;17'N, 32&deg;16.3'W, depth 1600-1750m) and the ultramafic-hosted Rainbow (36&deg;13'N, 33&deg;54.1'W, depth 2270-2330m) vent fields along the Mid-Atlantic Ridge (MAR). Using multiplexed barcoded pyrosequencing of the variable region 4 (V4) of the 16S rRNA genes, we show statistically significant differences between the archaeal and bacterial communities associated with the different vent fields. Quantitative polymerase chain reaction (qPCR) assays of the functional gene diagnostic for methanogenesis ( mcr A), as well as geochemical modelling to predict pore fluid chemistries within the deposits, support the pyrosequencing observations. Collectively, these results show that the less reduced, hydrogen-poor fluids at Lucky Strike limit colonization by strict anaerobes such as methanogens, and allow for hyperthermophilic microaerophiles, like Aeropyrum . In contrast, the hydrogen-rich reducing vent fluids at the ultramafic-influenced Rainbow vent field support the prevalence of methanogens and other hydrogen-oxidizing thermophiles at this site. These results demonstrate that biogeographical patterns of hydrothermal vent microorganisms are shaped in part by large scale geological and geochemical processes.

Environmental Microbiology