Project description:We developed a laboratory-scale model to improve our understanding and capacity to assess the biological risks of genetically engineered bacteria and their genetic elements in the natural environment. Our hypothetical scenario concerns an industrial bioreactor failure resulting in the introduction of genetically engineered bacteria to a downstream municipal wastewater treatment plant (MWWTP). As the first step towards developing a model for this scenario, we sampled microbial communities from the aeration basin of a MWWTP at three seasonal time points. Having established a baseline for community composition, we investigated how the community changed when propagated in the laboratory, including cell culture media conditions that could provide selective pressure in future studies. Specifically, using PhyloChip 16S rRNA gene-targeting microarrays, we compared the compositions of sampled communities to those of inoculates propagated in the laboratory in simulated wastewater conditionally amended with various carbon sources (glucose, chloroacetate, D-threonine) or the ionic liquid 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl). Proteobacteria, Bacteroidetes, and Actinobacteria were predominant in aeration basin and laboratory-cultured populations. Laboratory-cultured populations were enriched in Gammaproteobacteria. Enterobacteriaceae and Aeromonadaceae were enriched by glucose, Pseudomonadaceae by chloroacetate and D-threonine, and Burkholderiaceae by high (50 mM) concentrations of chloroacetate. Microbial populations cultured with chloroacetate and D-threonine were more similar to sampled populations than thoes cultured with glucose or [C2mim]Cl. Although observed relative richness in operational taxonomic units was lower for laboratory cultures than for sampled populations, both flask and reactor systems cultured phylogenetically diverse communities. These results importantly provide a foundation for laboratory models of industrial bioreactor failure scenarios. 46 samples, flask and reactor experiments were conducted in triplicate with two exceptions: [C2mim]Cl_flask and No-Carbon_flask treatments had only one sample (no replicates).

Project description:To gain improved temporal, spatial and phylogenetic resolution of marine microbial communities, in this study we expanded the original prototype genome proxy array (an oligonucleotide microarray targeting marine microbial genome fragments and genomes), evaluated it against metagenomic sequencing, and applied it to time series samples from the Monterey Bay long term ecological research site. The expanded array targeted 268 microbial genotypes (vs. 14 in the original prototype) across much of the known diversity of cultured and uncultured marine microbes. The target abundances measured by the genome proxy array were highly correlated to pyrosequence-based abundances (linear regression R2 = 0.85-0.91, p<0.0001). Fifty-seven samples from ~4-years in Monterey Bay were examined with the array, spanning the photic zone (0m), the base of the surface mixed layer (30m), and the subphotic zone (200m). A significant portion of the expanded genome proxy array’s targets showed signal (95 out of 268 targets present in ≥ 1 sample). The multi-year community survey showed the consistent presence of a core group of common and abundant targeted taxa at each depth in Monterey Bay, higher variability among shallow than deep samples, and episodic occurrences of more transient marine genotypes. The abundance of the most dominant genotypes peaked after strong episodic upwelling events. The genome-proxy array’s ability to track populations of closely-related genotypes indicated population shifts within several abundant target taxa, with specific populations in some cases clustering by depth or oceanographic season. Although 51 cultivated organisms were targeted (representing 19% of the array) the majority of targets detected and of total target signal (85% and ~92%, respectively) were from uncultivated lineages, often those derived from Monterey Bay. The array provided cost-effective (~$15 per array, for construction and use) insights into the natural history of uncultivated lineages in the wild. To gain improved temporal, spatial and phylogenetic resolution of marine microbial communities, in this study we expanded the original prototype genome proxy array (an oligonucleotide microarray targeting marine microbial genome fragments and genomes), evaluated it against metagenomic sequencing, and applied it to time series samples from the Monterey Bay long term ecological research site. The expanded array targeted 268 microbial genotypes (vs. 14 in the original prototype) across much of the known diversity of cultured and uncultured marine microbes. The target abundances measured by the genome proxy array were highly correlated to pyrosequence-based abundances (linear regression R2 = 0.85-0.91, p<0.0001). Fifty-seven samples from ~4-years in Monterey Bay were examined with the array, spanning the photic zone (0m), the base of the surface mixed layer (30m), and the subphotic zone (200m). A significant portion of the expanded genome proxy array’s targets showed signal (95 out of 268 targets present in ≥ 1 sample). The multi-year community survey showed the consistent presence of a core group of common and abundant targeted taxa at each depth in Monterey Bay, higher variability among shallow than deep samples, and episodic occurrences of more transient marine genotypes. The abundance of the most dominant genotypes peaked after strong episodic upwelling events. The genome-proxy array’s ability to track populations of closely-related genotypes indicated population shifts within several abundant target taxa, with specific populations in some cases clustering by depth or oceanographic season. Although 51 cultivated organisms were targeted (representing 19% of the array) the majority of targets detected and of total target signal (85% and ~92%, respectively) were from uncultivated lineages, often those derived from Monterey Bay. The array provided cost-effective (~$15 per array, for construction and use) insights into the natural history of uncultivated lineages in the wild.

Project description:The lesions of enamel caries can be considered as the outcome of dysbiotic changes in the biofilm community of supragingival dental plaque. Demineralization occurs as the cumulative outcome of repeated shifts towards a less diverse microbiota that produces and tolerates a low pH environment in tooth sites that are sheltered from protective factors in host saliva. Although the etiology of caries is multifactorial, frequent consumption of foods rich in fermentable carbohydrates, notably sucrose, appears to be one of the major factors driving the microbiota in the direction of dysbiosis, particularly in the case of otherwise healthy children with normal salivary flow. Streptococcus mutans and closely related species (such as Streptococcus sobrinus) have long been considered to play a primary etiological role in dental caries. S. mutans responds to sucrose by producing large quantities of lactic acid. It is very tolerant of low pH, and produces an insoluble extracellular polysaccharide that may sequester acid at tooth surfaces. The mechanisms behind those putative virulence factors have been intensively studied in monoculture, and recently in simple multi-species consortia. Much less is known of other species that may also contribute to or protect against dysbiosis driven by dietary carbohydrates. Some strains of “non-mutans” streptococci produce and tolerate acid at levels comparable to S. mutans, while others show increased ariginolytic capabilities, which may act to raise pH within the biofilm matrix. S. mutans tends to be a minority species even in caries-active children, and carious lesions likewise can occur in children with no detectable S. mutans. 16S rDNA-based metagenomic comparisons of caries-active and caries-free subjects have detected associations between caries and a variety of oral species, including not only non-mutans streptococci, but also members of other genera, such as Scardovia and Bifidobacterium. Caries associations have not been consistent between studies. Moreover, different taxonomic clusters have been defined as subgroups within the same study. This raises an important point. Although caries-associated communities are typically less diverse than healthy supragingival plaque overall, those dysbiotic communities still display considerable taxonomic diversity between affected individuals. That in turn raises the question of whether it is desirable to define biomarkers of dysbiosis that are less dependent on taxonomy. The Human Microbiome project generated comprehensive metagenomic data for a wide variety of body sites in healthy subjects, including supragingival plaque. Although most of that data was based on 16S rDNA sequencing, shotgun metagenomics was also used to catalog the functional potential of all microbial genes within a smaller subset of subjects. One of the key findings was that healthy sites from different people were broadly similar with respect to their functional profiles, even though there was extensive individual variation in their taxonomic profiles. It is possible that the “conservation of function” concept may also extend to dysbiotic communities. This would explain why microbial communities associated with caries still show considerable taxonomic variation. In that case, differential patterns of community-wide gene and/or protein expression might provide a more accurate indicator of dysbiosis than can be achieved by counting caries-associated species. Metatranscriptomic or metaproteomic approaches can be used to provide information on function. A recent metatranscriptomic comparison of subgingival plaque from healthy and periodontally diseased sites in three subjects has provided data that support the “conservation of function” concept. They observed that taxonomically diverse diseased sites shared conserved gene expression profiles [20]. By the same token, a recent metaproteomic comparison of gut microbiotas from healthy controls to Crohn’s disease patients found that major shifts in protein expression by function did not always correlate with changes in taxon relative abundance [21]. In this metaproteomic study, we found that sucrose–induced changes in protein expression patterns for pathways involving glycolysis, lactate production, aciduricity and ammonia/glutamate metabolism were likewise conserved in taxonomically diverse dysbiotic oral microcosm biofilm communities.

Project description:To gain improved temporal, spatial and phylogenetic resolution of marine microbial communities, in this study we expanded the original prototype genome proxy array (an oligonucleotide microarray targeting marine microbial genome fragments and genomes), evaluated it against metagenomic sequencing, and applied it to time series samples from the Monterey Bay long term ecological research site. The expanded array targeted 268 microbial genotypes (vs. 14 in the original prototype) across much of the known diversity of cultured and uncultured marine microbes. The target abundances measured by the genome proxy array were highly correlated to pyrosequence-based abundances (linear regression R2 = 0.85-0.91, p<0.0001). Fifty-seven samples from ~4-years in Monterey Bay were examined with the array, spanning the photic zone (0m), the base of the surface mixed layer (30m), and the subphotic zone (200m). A significant portion of the expanded genome proxy array’s targets showed signal (95 out of 268 targets present in ≥ 1 sample). The multi-year community survey showed the consistent presence of a core group of common and abundant targeted taxa at each depth in Monterey Bay, higher variability among shallow than deep samples, and episodic occurrences of more transient marine genotypes. The abundance of the most dominant genotypes peaked after strong episodic upwelling events. The genome-proxy array’s ability to track populations of closely-related genotypes indicated population shifts within several abundant target taxa, with specific populations in some cases clustering by depth or oceanographic season. Although 51 cultivated organisms were targeted (representing 19% of the array) the majority of targets detected and of total target signal (85% and ~92%, respectively) were from uncultivated lineages, often those derived from Monterey Bay. The array provided cost-effective (~$15 per array, for construction and use) insights into the natural history of uncultivated lineages in the wild. To gain improved temporal, spatial and phylogenetic resolution of marine microbial communities, in this study we expanded the original prototype genome proxy array (an oligonucleotide microarray targeting marine microbial genome fragments and genomes), evaluated it against metagenomic sequencing, and applied it to time series samples from the Monterey Bay long term ecological research site. The expanded array targeted 268 microbial genotypes (vs. 14 in the original prototype) across much of the known diversity of cultured and uncultured marine microbes. The target abundances measured by the genome proxy array were highly correlated to pyrosequence-based abundances (linear regression R2 = 0.85-0.91, p<0.0001). Fifty-seven samples from ~4-years in Monterey Bay were examined with the array, spanning the photic zone (0m), the base of the surface mixed layer (30m), and the subphotic zone (200m). A significant portion of the expanded genome proxy array’s targets showed signal (95 out of 268 targets present in ≥ 1 sample). The multi-year community survey showed the consistent presence of a core group of common and abundant targeted taxa at each depth in Monterey Bay, higher variability among shallow than deep samples, and episodic occurrences of more transient marine genotypes. The abundance of the most dominant genotypes peaked after strong episodic upwelling events. The genome-proxy array’s ability to track populations of closely-related genotypes indicated population shifts within several abundant target taxa, with specific populations in some cases clustering by depth or oceanographic season. Although 51 cultivated organisms were targeted (representing 19% of the array) the majority of targets detected and of total target signal (85% and ~92%, respectively) were from uncultivated lineages, often those derived from Monterey Bay. The array provided cost-effective (~$15 per array, for construction and use) insights into the natural history of uncultivated lineages in the wild. The expanded genome proxy array was designed as in (Rich et al., 2008). Briefly, each genotype was targeted using suites of ~20 70-mer oligonucleotide probes designed using the program ArrayOligoSelector (Zhu et al., 2003). Probes had approximately the same %GC (40%) and were distributed across the target genome or genome fragment, with no more than one probe per gene and avoiding 16S and 23S rRNA genes. The array included positive and negative control probes designed using the same method, to Halobacterium salinarum NRC-1 and a random genome sequence, respectively. The expanded array had a broader scope than the prototype of Rich et al., 2008 (268 target genotypes, as opposed to the prototype’s 14) and included a co-spot oligo for spot alignment and gridding purposes (using the “alien” oligo sequence of (Urisman et al., 2005). The targets were selected from fully-sequenced marine microbial genomes, publicly-available marine-derived BAC and fosmid clone sequences, and fully-sequenced clones from the lab’s Monterey Bay and Hawaii environmental BAC- and fosmid-based genomic libraries. Targeted genotypes are detailed in Table S1, summarized in Table S2, and presented in a schematic phylogenetic overview in Fig. 1. Previously-unpublished sequences used for array design were submitted to Genbank under accession numbers GU474833-GU474949. This submission presents the analysis of 57 samples, each hybridized to >= 3 arrays. In addition to negative control probes, for each hyrbdization date and run a negative control array was hyrbdized for which the input DNA was TE, taken through the entire aplification, labeling, and hybridization process.

			Action	DRS
	SRR13517411_1.fastq.gz	Fastqsanger.gz
	SRR13517411_2.fastq.gz	Fastqsanger.gz
	SRR13517412_1.fastq.gz	Fastqsanger.gz
	SRR13517412_2.fastq.gz	Fastqsanger.gz
	SRR13517413_1.fastq.gz	Fastqsanger.gz

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