Substrate-specific clades of active marine methylotrophs associated with a phytoplankton bloom in a temperate coastal environment

Marine microorganisms that consume one-carbon (C1) compounds are poorly described, despite their impact on global climate via an influence on aquatic and atmospheric chemistry. This study investigated marine bacterial communities involved in the metabolism of C1 compounds. These communities were of relevance to surface seawater and atmospheric chemistry in the context of a bloom that was dominated by phytoplankton known to produce dimethylsulfoniopropionate. In addition to using 16S rRNA gene fingerprinting and clone libraries to characterize samples taken from a bloom transect in July 2006, seawater samples from the phytoplankton bloom were incubated with 13C-labeled methanol, monomethylamine, dimethylamine, methyl bromide, and dimethyl sulfide to identify microbial populations involved in the turnover of C1 compounds, using DNA stable isotope probing. The [13C]DNA samples from a single time point were characterized and compared using denaturing gradient gel electrophoresis (DGGE), fingerprint cluster analysis, and 16S rRNA gene clone library analysis. Bacterial community DGGE fingerprints from 13C-labeled DNA were distinct from those obtained with the DNA of the nonlabeled community DNA and suggested some overlap in substrate utilization between active methylotroph populations growing on different C1 substrates. Active methylotrophs were affiliated with Methylophaga spp. and several clades of undescribed Gammaproteobacteria that utilized methanol, methylamines (both monomethylamine and dimethylamine), and dimethyl sulfide. rRNA gene sequences corresponding to populations assimilating 13C-labeled methyl bromide and other substrates were associated with members of the Alphaproteobacteria (e.g., the family Rhodobacteraceae), the Cytophaga-Flexibacter-Bacteroides group, and unknown taxa. This study expands the known diversity of marine methylotrophs in surface seawater and provides a comprehensive data set for focused cultivation and metagenomic analyses in the future

Temporal and spatial stability of ammonia-oxidizing archaea and bacteria in aquarium biofilters

Nitrifying biofilters are used in aquaria and aquaculture systems to prevent accumulation of ammonia by promoting rapid conversion to nitrate via nitrite. Ammonia-oxidizing archaea (AOA), as opposed to ammonia-oxidizing bacteria (AOB), were recently identified as the dominant ammonia oxidizers in most freshwater aquaria. This study investigated biofilms from fixed-bed aquarium biofilters to assess the temporal and spatial dynamics of AOA and AOB abundance and diversity. Over a period of four months, ammonia-oxidizing microorganisms from six freshwater and one marine aquarium were investigated at 4-5 time points. Nitrogen balances for three freshwater aquaria showed that active nitrification by aquarium biofilters accounted for >= 81-86% of total nitrogen conversion in the aquaria. Quantitative PCR (qPCR) for bacterial and thaumarchaeal ammonia monooxygenase (amoA) genes demonstrated that AOA were numerically dominant over AOB in all six freshwater aquaria tested, and contributed all detectable amoA genes in three aquarium biofilters. In the marine aquarium, however, AOB outnumbered AOA by three to five orders of magnitude based on amoA gene abundances. A comparison of AOA abundance in three carrier materials (fine sponge, rough sponge and sintered glass or ceramic rings) of two three-media freshwater biofilters revealed preferential growth of AOA on fine sponge. Denaturing gel gradient electrophoresis (DGGE) of thaumarchaeal 16S rRNA genes indicated that community composition within a given biofilter was stable across media types. In addition, DGGE of all aquarium biofilters revealed low AOA diversity, with few bands, which were stable over time. Nonmetric multidimensional scaling (NMDS) based on denaturing gradient gel electrophoresis (DGGE) fingerprints of thaumarchaeal 16S rRNA genes placed freshwater and marine aquaria communities in separate clusters. These results indicate that AOA are the dominant ammonia-oxidizing microorganisms in freshwater aquarium biofilters, and that AOA community composition within a given aquarium is stable over time and across biofilter support material types

Developmental succession of the microbiome of Culex mosquitoes

Background: The native microflora associated with mosquitoes have important roles in mosquito development and vector competence. Sequencing of bacterial V3 region from 16S rRNA genes across the developmental stages of Culex mosquitoes (early and late larval instars, pupae and adults) was used to test the hypothesis that bacteria found in the larval stage of Culex are transstadially transmitted to the adult stage, and to compare the microbiomes of field-collected versus laboratory-reared mosquitoes.Results: Beta diversity analysis revealed that bacterial community structure differed among three life stages (larvae, pupae and adults) of Culex tarsalis. Although only similar to 2 % of the total number of bacterial OTUs were found in all stages, sequences from these OTUs accounted for nearly 82 % of the total bacterial sequences recovered from all stages. Thorsellia (Gammaproteobacteria) was the most abundant bacterial taxon found across all developmental stages of field-collected Culex mosquitoes, but was rare in mosquitoes from laboratory-reared colonies. The proportion of Thorsellia sequences in the microbiomes of mosquito life stages varied ontogenetically with the greatest proportions recovered from the pupae of C. tarsalis and the lowest from newly emerged adults. The microbiome of field-collected late instar larvae was not influenced significantly by differences in the microbiota of the habitat due to habitat age or biopesticide treatments. The microbiome diversity was the greatest in the early instar larvae and the lowest in laboratory-reared mosquitoes.Conclusions: Bacterial communities in early instar C. tarsalis larvae were significantly more diverse when compared to late instar larvae, pupae and newly emerged adults. Some of the bacterial OTUs found in the early instar larvae were also found across developmental stages. Thorsellia dominated the bacterial communities in field-collected immature stages but occurred at much lower relative abundance in adults. Differences in microbiota observed in larval habitats did not influence bacterial community profiles of late instar larvae or adults. However, bacterial communities in laboratory-reared C. tarsalis larvae differed significantly from the field. Determining the role of Thorsellia in mosquitoes and its distribution across different species of mosquitoes warrants further investigation

mockrobiota: a Public Resource for Microbiome Bioinformatics Benchmarking.

Mock communities are an important tool for validating, optimizing, and comparing bioinformatics methods for microbial community analysis. We present mockrobiota, a public resource for sharing, validating, and documenting mock community data resources, available at http://caporaso-lab.github.io/mockrobiota/. The materials contained in mockrobiota include data set and sample metadata, expected composition data (taxonomy or gene annotations or reference sequences for mock community members), and links to raw data (e.g., raw sequence data) for each mock community data set. mockrobiota does not supply physical sample materials directly, but the data set metadata included for each mock community indicate whether physical sample materials are available. At the time of this writing, mockrobiota contains 11 mock community data sets with known species compositions, including bacterial, archaeal, and eukaryotic mock communities, analyzed by high-throughput marker gene sequencing. IMPORTANCE The availability of standard and public mock community data will facilitate ongoing method optimizations, comparisons across studies that share source data, and greater transparency and access and eliminate redundancy. These are also valuable resources for bioinformatics teaching and training. This dynamic resource is intended to expand and evolve to meet the changing needs of the omics community

Targeted metagenomics of active microbial populations with stable-isotope probing

The ability to explore microbial diversity and function has been enhanced by novel experimental and computational tools. The incorporation of stable isotopes into microbial biomass enables the recovery of labeled nucleic acids from active microorganisms, despite their initial abundance and culturability. Combining stable-isotope probing (SIP) with metagenomics provides access to genomes from microorganisms involved in metabolic processes of interest. Studies using metagenomic analysis on DNA obtained from DNA-SIP incubations can be ideal for the recovery of novel enzymes for biotechnology applications, including biodegradation, biotransformation, and biosynthesis. This chapter introduces metagenomic and DNA-SIP methodologies, highlights biotechnology-focused studies that combine these approaches, and provides perspectives on future uses of these methods as analysis tools for applied and environmental microbiology

Microbiota variations in Culex nigripalpus disease vector mosquito of West Nile virus and Saint Louis Encephalitis from different geographic origins

Although mosquito microbiota are known to influence reproduction, nutrition, disease transmission, and pesticide resistance, the relationship between host-associated microbial community composition and geographical location is poorly understood. To begin addressing this knowledge gap, we characterized microbiota associated with adult females of Culex nigripalpus mosquito vectors of Saint Louis Encephalitis and West Nile viruses sampled from three locations in Florida (Vero Beach, Palmetto Inland, and Palmetto Coast). High-throughput sequencing of PCR-amplified 16S rRNA genes demonstrated significant differences among microbial communities of mosquitoes sampled from the three locations. Mosquitoes from Vero Beach (east coast Florida) were dominated by uncultivated Asaia sp. (Alphaproteobacteria), whereas microbiota associated with mosquitoes collected from two mosquito populations at Palmetto (west coast Florida) sites were dominated by uncultured Spironema culicis (Spirochaetes), Salinisphaera hydrothermalis (Gammaproteobacteria), Spiroplasma (Mollicutes), uncultured Enterobacteriaceae, Candidatus Megaira (Alphaproteobacteria; Rickettsiae), and Zymobacter (Gammaproteobacteria). The variation in taxonomic profiles of Cx. nigripalpus gut microbial communities, especially with respect to dominating taxa, is a potentially critical factor in understanding disease transmission and mosquito susceptibility to insecticides among different mosquito populations

Anadromous Arctic Char Microbiomes: Bioprospecting in the High Arctic

Northern populations of Arctic char (Salvelinus alpinus) can be anadromous, migrating annually from the ocean to freshwater lakes and rivers in order to escape sub-zero temperatures. Such seasonal behavior demands that these fish and their associated microbiomes adapt to changes in salinity, temperature, and other environmental challenges. We characterized the microbial community composition of anadromous S. alpinus, netted by Inuit fishermen at freshwater and seawater fishing sites in the high Arctic, both under ice and in open water. Bacterial profiles were generated by DNA extraction and high-throughput sequencing of PCR-amplified 16S ribosomal RNA genes. Results showed that microbial communities on the skin and intestine of Arctic char were statistically different when sampled from freshwater or saline water sites. This association was tested using hierarchical Ward's linkage clustering, showing eight distinct clusters in each of the skin and intestinal microbiomes, with the clusters reflecting sampling location between fresh and saline environments, confirming a salinity-linked turnover. This analysis also provided evidence for a core composition of skin and intestinal bacteria, with the phyla Proteobacteria, Firmicutes, and Cyanobacteria presenting as major phyla within the skin-associated microbiomes. The intestine-associated microbiome was characterized by unidentified genera from families Fusobacteriaceae, Comamonadaceae, Pseudomonadaceae, and Vibrionaceae. The salinity-linked turnover was further tested through ordinations that showed samples grouping based on environment for both skin- and intestine-associated microbiomes. This finding implies that core microbiomes between fresh and saline conditions could be used to assist in regulating optimal fish health in aquaculture practices. Furthermore, identified taxa from known psychrophiles and with nitrogen cycling properties suggest that there is additional potential for biotechnological applications for fish farm and waste management practices