Search

Coastal salt marshes are highly sensitive wetland ecosystems that can sustain long-term impacts from anthropogenic events such as oil spills. In this study, we examined the microbial communities of a Gulf of Mexico coastal salt marsh during and after the influx of petroleum hydrocarbons following the Deepwater Horizon oil spill. Total hydrocarbon concentrations in salt marsh sediments were highest in June and July 2010 and decreased in September 2010. Coupled PhyloChip and GeoChip microarray analyses demonstrated that the microbial community structure and function of the extant salt marsh hydrocarbon-degrading microbial populations changed significantly during the study. The relative richness and abundance of phyla containing previously described hydrocarbon-degrading bacteria (Proteobacteria, Bacteroidetes, and Actinobacteria) increased in hydrocarbon-contaminated sediments and then decreased once hydrocarbons were below detection. Firmicutes, however, continued to increase in relative richness and abundance after hydrocarbon concentrations were below detection. Functional genes involved in hydrocarbon degradation were enriched in hydrocarbon-contaminated sediments then declined significantly (p,0.05) once hydrocarbon concentrations decreased. A greater decrease in hydrocarbon concentrations among marsh grass sediments compared to inlet sediments (lacking marsh grass) suggests that the marsh rhizosphere microbial communities could also be contributing to hydrocarbon degradation. The results of this study provide a comprehensive view of microbial community structural and functional dynamics within perturbed salt marsh ecosystems.

Reference phylogenies are crucial for providing a taxonomic framework for interpretation of marker gene and metagenomic surveys, which continue to reveal novel species at a remarkable rate. Greengenes is a dedicated full-length 16S rRNA gene database that provides users with a curated taxonomy based on de novo tree inference. We developed a 'taxonomy to tree' approach for transferring group names from an existing taxonomy to a tree topology, and used it to apply the Greengenes, National Center for Biotechnology Information (NCBI) and cyanoDB (Cyanobacteria only) taxonomies to a de novo tree comprising 408,315 sequences. We also incorporated explicit rank information provided by the NCBI taxonomy to group names (by prefixing rank designations) for better user orientation and classification consistency. The resulting merged taxonomy improved the classification of 75% of the sequences by one or more ranks relative to the original NCBI taxonomy with the most pronounced improvements occurring in under-classified environmental sequences. We also assessed candidate phyla (divisions) currently defined by NCBI and present recommendations for consolidation of 34 redundantly named groups. All intermediate results from the pipeline, which includes tree inference, jackknifing and transfer of a donor taxonomy to a recipient tree (tax2tree) are available for download. The improved Greengenes taxonomy should provide important infrastructure for a wide range of megasequencing projects studying ecosystems on scales ranging from our own bodies (the Human Microbiome Project) to the entire planet (the Earth Microbiome Project). The implementation of the software can be obtained from http://sourceforge.net/projects/tax2tree/.

Author Manuscript date: 2013 February 05., Studies of the human microbiome have revealed that even healthy individuals differ remarkably in the microbes that occupy habitats such as the gut, skin and vagina. Much of this diversity remains unexplained, although diet, environment, host genetics and early microbial exposure have all been implicated. Accordingly, to characterize the ecology of human-associated microbial communities, the Human Microbiome Project has analysed the largest cohort and set of distinct, clinically relevant body habitats so far. We found the diversity and abundance of each habitat’s signature microbes to vary widely even among healthy subjects, with strong niche specialization both within and among individuals. The project encountered an estimated 81–99% of the genera, enzyme families and community configurations occupied by the healthy Western microbiome. Metagenomic carriage of metabolic pathways was stable among individuals despite variation in community structure, and ethnic/racial background proved to be one of the strongest associations of both pathways and microbes with clinical metadata. These results thus delineate the range of structural and functional configurations normal in the microbial communities of a healthy population, enabling future characterization of the epidemiology, ecology and translational applications of the human microbiome.

Animals have co-evolved with mutualistic microbial communities, known as the microbiota, which are essential for organ development and function. We hypothesize that modern animal husbandry practices exert an impact on the intestinal microbiota. In this study, we compared the structure of the fecal microbiota between feral and domestic goats using the G2 PhyloChip and assessed the presence of five tetracycline resistance genes [tet(M), tet(S), tet(O), tet(Q) and tet(W)] by PCR. Feces were collected from 10 goats: 5 domestic from a farm in the main island of Puerto Rico and 5 feral from the remote dry island of Mona. There were 42 bacterial phyla from 153 families detected in the goats' feces. A total of 84 PhyloChip-OTUs were different in the fecal microbiota of feral and domestic goat. Both feral and domestic goats carried antibiotic resistance genes tet(O) and tet(W), but domestic goats additionally carried tet(Q). Diet, host genetics and antibiotic exposure are likely determinant factors in shaping the intestinal microbiota and may explain the differences observed between feral and domestic goats fecal microbiota.

Bacteria of the genus Dehalococcoides play an important role in the reductive dechlorination of chlorinated ethenes. A systems-level approach was taken in this study to examine the global transcriptomic and proteomic responses of exponentially growing cells of Dehalococcoides ethenogenes strain 195 to fixed nitrogen limitation (FNL), as dechlorination activity and cell yield both decrease during FNL. As expected, the nitrogen-fixing ( nif ) genes were differentially upregulated in the transcriptome and proteome of strain 195 during FNL. Aside from the nif operon, a putative methylglyoxal synthase-encoding gene (DET1576), the product of which is predicted to catalyze the formation of the toxic electrophile methylglyoxal and is implicated in the uncoupling of anabolism from catabolism in bacteria, was strongly upregulated in the transcriptome and could potentially play a role in the observed growth inhibition during FNL. Carbon catabolism genes were generally downregulated in response to FNL, and a number of transporters were differentially regulated in response to nitrogen limitation, with some playing apparent roles in nitrogen acquisition, while others were associated with general stress responses. A number of genes related to the functions of nucleotide synthesis, replication, transcription, translation, and posttranslational modifications were also differentially expressed. One gene coding for a putative reductive dehalogenase (DET1545) and a number of genes coding for oxidoreductases, which have implications in energy generation and redox reactions, were also differentially regulated. Interestingly, most of the genes within the multiple integrated elements were not differentially expressed. Overall, this study elucidates the molecular responses of strain 195 to FNL and identifies differentially expressed genes that are potential biomarkers to evaluate environmental cellular nitrogen status.

A high-density phylogenetic microarray (PhyloChip) was applied to track bacterial and archaeal populations through different phases of remediation at Ft. Lewis, WA, a trichloroethene (TCE)-contaminated groundwater site. Biostimulation with whey, and bioaugmentation with a Dehalococcoides-containing enrichment culture were strategies implemented to enhance dechlorination. As a measure of species richness, over 1300 operational taxonomic units (OTUs) were detected in DNA from groundwater samples extracted during different stages of treatment and in the bioaugmentation culture. In order to determine active members within the community, 16S rRNA from samples were analyzed by microarray and ~600 OTUs identified. A cDNA clone library of the expressed 16S rRNA corroborated the observed diversity and activity of some of the phyla. Principle component analysis of the treatment plot samples revealed that the microbial populations were constantly changing during the course of the study. Dynamic analysis of the archaeal population showed significant increases in methanogens at the later stages of treatment that correlated with increases in methane concentrations of over two orders of magnitude. Overall, the PhyloChip analyses in this study have provided insights into the microbial ecology and population dynamics at the TCE-contaminated field site useful for understanding the in situ reductive dechlorination processes.

Culture-dependent and -independent approaches were employed to identify the bacterial community structure from olive-mill wastewater produced from three olive-fruit varieties. The 233 bacterial isolates recovered were phylogenetically related to 38 members of Firmicutes, Actinobacteria, α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, and Bacteroidetes. Employing a novel microarray-based approach (PhyloChip) a high bacterial diversity was revealed consisting of 18 different phyla with representatives from 99 different families. The bacterial diversity in olive-mill wastewater from the three olive tree varieties was dominated by α-, β-, γ-, δ-, e-Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, Cyanobacteria, and Actinobacteria. This in-depth analysis of the indigenous microbiota indicated a cultivar-specific bacterial profile. Interestingly, the common bacterial taxa present in all three varieties examined were restricted indicating that the bacterial communities present in the olive-mill wastewater are greatly influenced by the olive-fruit variety.

Dehalococcoides ethenogenes strain 195 (DE195) was grown in a sustainable syntrophic association with Desulfovibrio vulgaris Hildenborough (DVH) as a co-culture, as well as with DVH and the hydrogenotrophic methanogen Methanobacterium congolense (MC) as a tri-culture using lactate as the sole energy and carbon source. In the co- and tri-cultures, maximum dechlorination rates of DE195 were enhanced by approximately three times (11.0±0.01 μmol per day for the co-culture and 10.1±0.3 μmol per day for the tri-culture) compared with DE195 grown alone (3.8±0.1 μmol per day). Cell yield of DE195 was enhanced in the co-culture (9.0±0.5 × 10(7) cells per μmol Cl(-) released, compared with 6.8±0.9 × 10(7) cells per μmol Cl(-) released for the pure culture), whereas no further enhancement was observed in the tri-culture (7.3±1.8 × 10(7) cells per μmol Cl(-) released). The transcriptome of DE195 grown in the co-culture was analyzed using a whole-genome microarray targeting DE195, which detected 102 significantly up- or down-regulated genes compared with DE195 grown in isolation, whereas no significant transcriptomic difference was observed between co- and tri-cultures. Proteomic analysis showed that 120 proteins were differentially expressed in the co-culture compared with DE195 grown in isolation. Physiological, transcriptomic and proteomic results indicate that the robust growth of DE195 in co- and tri-cultures is because of the advantages associated with the capabilities of DVH to ferment lactate to provide H(2) and acetate for growth, along with potential benefits from proton translocation, cobalamin-salvaging and amino acid biosynthesis, whereas MC in the tri-culture provided no significant additional benefits beyond those of DVH.

This study demonstrates the prevalence, phylogenetic diversity, and physiology of nitrate-reducing microorganisms capable of utilizing reduced humic acids (HA) as electron donors in agricultural soils. Most probable number (MPN) enumeration of agricultural soils revealed large populations (104 to 106 cells g−1 soil) of microorganisms capable of reducing nitrate while oxidizing the reduced HA analog 2,6-anthrahydroquinone disulfonate (AH2DS) to its corresponding quinone. Nitrate-dependent HA-oxidizing organisms isolated from agricultural soils were phylogenetically diverse and included members of the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Advective up-flow columns inoculated with corn plot soil and amended with reduced HA and nitrate supported both HA oxidation and enhanced nitrate reduction relative to no-donor or oxidized HA controls. The additional electron donating capacity of reduced HA could reasonably be attributed to the oxidation of reduced functional groups. Subsequent 16S rRNA gene-based high-density oligonucleotide microarray (PhyloChip) indicated that reduced HA columns supported the development of a bacterial community enriched with members of the Acidobacteria, Firmicutes, and Betaproteobacteria relative to the no-donor control and initial inoculum. This study identifies a previously unrecognized role for HA in stimulating denitrification processes in saturated soil systems. Furthermore, this study indicates that reduced humic acids impact soil geochemistry and the indigenous bacterial community composition., IMPORTANCE This study identifies a new metabolic capacity in soil microbial communities that may be responsible for the mediation of significant nitrogen losses from soil systems. Nitrate-dependent humic acid (HA)-oxidizing organisms isolated from agricultural soils were phylogenetically diverse and included members of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Advective up-flow columns inoculated with corn plot soil and amended with reduced HA and nitrate supported both HA oxidation and enhanced nitrate reduction relative to no-donor or oxidized HA controls. The additional electron donating capacity of reduced HA could reasonably be attributed to the oxidation of reduced functional groups.

Interest in coral microbial ecology has been increasing steadily over the last decade, yet standardized methods of sample collection still have not been defined. Two methods were compared for their ability to sample coral-associated microbial communities: tissue punches and foam swabs, the latter being less invasive and preferred by reef managers. Four colonies of star coral, Montastraea annularis, were sampled in the Dry Tortugas National Park (two healthy and two with white plague disease). The PhyloChip™ G3 microarray was used to assess microbial community structure of amplified 16S rRNA gene sequences. Samples clustered based on methodology rather than coral colony. Punch samples from healthy and diseased corals were distinct. All swab samples clustered closely together with the seawater control and did not group according to the health state of the corals. Although more microbial taxa were detected by the swab method, there is a much larger overlap between the water control and swab samples than punch samples, suggesting some of the additional diversity is due to contamination from water absorbed by the swab. While swabs are useful for noninvasive studies of the coral surface mucus layer, these results show that they are not optimal for studies of coral disease.

One of the major factors associated with global change is the ever-increasing concentration of atmospheric CO(2). Although the stimulating effects of elevated CO(2) (eCO(2)) on plant growth and primary productivity have been established, its impacts on the diversity and function of soil microbial communities are poorly understood. In this study, phylogenetic microarrays (PhyloChip) were used to comprehensively survey the richness, composition and structure of soil microbial communities in a grassland experiment subjected to two CO(2) conditions (ambient, 368 p.p.m., versus elevated, 560 p.p.m.) for 10 years. The richness based on the detected number of operational taxonomic units (OTUs) significantly decreased under eCO(2). PhyloChip detected 2269 OTUs derived from 45 phyla (including two from Archaea), 55 classes, 99 orders, 164 families and 190 subfamilies. Also, the signal intensity of five phyla (Crenarchaeota, Chloroflexi, OP10, OP9/JS1, Verrucomicrobia) significantly decreased at eCO(2), and such significant effects of eCO(2) on microbial composition were also observed at the class or lower taxonomic levels for most abundant phyla, such as Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Acidobacteria, suggesting a shift in microbial community composition at eCO(2). Additionally, statistical analyses showed that the overall taxonomic structure of soil microbial communities was altered at eCO(2). Mantel tests indicated that such changes in species richness, composition and structure of soil microbial communities were closely correlated with soil and plant properties. This study provides insights into our understanding of shifts in the richness, composition and structure of soil microbial communities under eCO(2) and environmental factors shaping the microbial community structure.

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.

A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces.

Comparative genomics of Dehalococcoides strains and an enrichment were performed using a microarray targeting genes from all available sequenced genomes of the Dehalococcoides genus. The microarray was designed with 4305 probe sets to target 98.6% of the open-reading frames from strains 195, CBDB1, BAV1 and VS. The microarrays were validated and applied to query the genomes of two recently isolated Dehalococcoides strains, ANAS1 and ANAS2, and their enrichment source (ANAS) to understand the genome–physiology relationships. Strains ANAS1 and ANAS2 can both couple the reduction of trichloroethene, cis-dichloroethene (DCE) and 1,1-DCE, but not tetrachloroethene and trans-DCE with growth, whereas only strain ANAS2 couples vinyl chloride reduction to growth. Comparative genomic analysis showed that the genomes of both strains are similar to each other and to strain 195, except for genes that are within the previously defined integrated elements or high-plasticity regions. Combined results of the two isolates closely matched the results obtained using genomic DNA of the ANAS enrichment. The genome similarities, together with the distinct chlorinated ethene usage of strains ANAS1, ANAS2 and 195 demonstrate that closely phylogenetically related strains can be physiologically different. This incongruence between physiology and core genome phylogeny seems to be related to the presence of distinct reductive dehalogenase-encoding genes with assigned chlorinated ethene functions (pceA, tceA in strain 195; tceA in strain ANAS1; vcrA in strain ANAS2). Overall, the microarrays are a valuable high-throughput tool for comparative genomics of unsequenced Dehalococcoides-containing samples to provide insights into their gene content and dechlorination functions.

International audience; Bacteria inhabiting crystalline rocks from two terrestrial Icelandic volcanic lava flows of similar age and from the same geographical region, but differing in porosity and mineralogy, were characterised. Microarray (PhyloChip) and clone library analysis of 16S rRNA genes revealed the presence of a diverse assemblage of bacteria in each lava flow. Both methods suggested a more diverse community at the Dómadalshraun site (rhyolitic/andesitic lava flow) than that present at the Hnausahraun site (basaltic lava flow). Proteobacteria dominated the clone library at the Dómadalshraun site, while Acidobacteria was the most abundant phylum in the Hnausahraun site. Although analysis of similarities of denaturing gradient gel electrophoresis profiles suggested a strong correlation of community structure with mineralogy, rock porosity may also play an important role in shaping the bacterial community in crystalline volcanic rocks. Clone sequences were most similar to uncultured microorganisms, mainly from soil environments. Of these, Antarctic soils and temperate rhizosphere soils were prominent, as were clones retrieved from Hawaiian and Andean volcanic soils. The novel diversity of these Icelandic microbial communities was supported by the finding that up to 46% of clones displayed

Microbial communities are each a composite of populations whose presence and relative abundance in water or other environmental samples are a direct manifestation of environmental conditions, including the introduction of microbe-rich fecal material and factors promoting persistence of the microbes therein. As shown by culture-independent methods, different animal-host fecal microbial communities are distinctive, suggesting that their community profiles can be used to differentiate fecal samples and to potentially reveal the presence of host fecal material in environmental waters. Cross-comparisons of microbial communities from different hosts also reveal relative abundances of genetic groups that can be used to distinguish sources. In increasing order of their information richness, several community analysis methods hold promise for MST applications: phospholipid fatty acid (PLFA) analysis, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (TRFLP), cloning/sequencing, and PhyloChip. Specific case studies involving TRFLP and PhyloChip approaches demonstrate the ability of community-based analyses of contaminated waters to confirm a diagnosis of water quality based on host-specific marker(s). The success of community-based MST for comprehensively confirming fecal sources relies extensively upon using appropriate multivariate statistical approaches. While community-based MST is still under evaluation and development as a primary diagnostic tool, results presented herein demonstrate its promise. Coupled with its inherently comprehensive ability to capture an unprecedented amount of microbiological data that is relevant to water quality, the tools for microbial community analysis are increasingly accessible, and community-based approaches have unparalleled potential for translation into rapid, perhaps real-time, monitoring platforms.

Esophageal adenocarcinoma (EA), the type of cancer linked to heartburn due to gastroesophageal reflux diseases (GERD), has increased six fold in the past 30 years. This cannot currently be explained by the usual environmental or by host genetic factors. EA is the end result of a sequence of GERD-related diseases, preceded by reflux esophagitis (RE) and Barrett’s esophagus (BE). Preliminary studies by Pei and colleagues at NYU on elderly male veterans identified two types of microbiotas in the esophagus. Patients who carry the type II microbiota are >15 fold likely to have esophagitis and BE than those harboring the type I microbiota. In a small scale study, we also found that 3 of 3 cases of EA harbored the type II biota. The findings have opened a new approach to understanding the recent surge in the incidence of EA.Our long-term goal is to identify the cause of GERD sequence. The hypothesis to be tested is that changes in the foregut microbiome are associated with EA and its precursors, RE and BE in GERD sequence. We will conduct a case control study to demonstrate the microbiome disease association in every stage of GERD sequence, as well as analyze the trend in changes in the microbiome along disease progression toward EA, by two specific aims. Aim 1 is to conduct a comprehensive population survey of the foregut microbiome and demonstrate its association with GERD sequence. Furthermore, spatial relationship between the esophageal microbiota and upstream (mouth) and downstream (stomach) foregut microbiotas as well as temporal stability of the microbiome-disease association will also be examined. Aim 2 is to define the distal esophageal metagenome and demonstrate its association with GERD sequence. Detailed analyses will include pathway-disease and gene-disease associations. Archaea, fungi and viruses, if identified, also will be correlated with the diseases. A significant association between the foregut microbiome and GERD sequence, if demonstrated, will be the first step for eventually testing whether an abnormal microbiome is required for the development of the sequence of phenotypic changes toward EA. If EA and its precursors represent a microecological disease, treating the cause of GERD might become possible, for example, by normalizing the microbiota through use of antibiotics, probiotics, or prebiotics. Causative therapy of GERD could prevent its progression and reverse the current trend of increasing incidence of EA.

Author(s): Parnell, J Jacob; Rompato, Giovanni; Latta, Leigh C; Pfrender, Michael E; Van Nostrand, Joy D; He, Zhili; Zhou, Jizhong; Andersen, Gary; Champine, Patti; Ganesan, Balasubramanian; Weimer, Bart C | Abstract: BackgroundHorizontal gene transfer (HGT) plays a major role in speciation and evolution of bacteria and archaea by controlling gene distribution within an environment. However, information that links HGT to a natural community using relevant population-genetics parameters and spatial considerations is scarce. The Great Salt Lake (Utah, USA) provides an excellent model for studying HGT in the context of biogeography because it is a contiguous system with dispersal limitations due to a strong selective salinity gradient. We hypothesize that in spite of the barrier to phylogenetic dispersal, functional characteristics--in the form of HGT--expand beyond phylogenetic limitations due to selective pressure.Methodology and resultsTo assay the functional genes and microorganisms throughout the GSL, we used a 16S rRNA oligonucleotide microarray (Phylochip) and a functional gene array (GeoChip) to measure biogeographic patterns of nine microbial communities. We found a significant difference in biogeography based on microarray analyses when comparing Sorensen similarity values for presence/absence of function and phylogeny (Student's t-test; p = 0.005).Conclusion and significanceBiogeographic patterns exhibit behavior associated with horizontal gene transfer in that informational genes (16S rRNA) have a lower similarity than functional genes, and functional similarity is positively correlated with lake-wide selective pressure. Specifically, high concentrations of chromium throughout GSL correspond to an average similarity of chromium resistance genes that is 22% higher than taxonomic similarity. This suggests active HGT may be measured at the population level in microbial communities and these biogeographic patterns may serve as a model to study bacteria adaptation and speciation.

The bacterial diversity and comparative community structure of a clean room used for assembling the Phoenix spacecraft was characterized throughout the spacecraft assembly process by using 16S rRNA gene cloning/sequencing and DNA microarray (PhyloChip) technologies. Samples were collected from several locations of the clean room at three time points: before Phoenix's arrival (PHX-B), during hardware assembly (PHX-D), and after the spacecraft was removed for launch (PHX-A). Bacterial diversity comprised of all major bacterial phyla of PHX-B was found to be statistically different from PHX-D and PHX-A samples. Due to stringent cleaning and decontamination protocols during assembly, PHX-D bacterial diversity was dramatically reduced when compared to PHX-B and PHX-A samples. Comparative community analysis based on PhyloChip results revealed similar overall trends as were seen in clone libraries, but the high-density phylogenetic microarray detected larger diversity in all sampling events. The decrease in community complexity in PHX-D compared to PHX-B, and the subsequent recurrence of these organisms in PHX-A, speaks to the effectiveness of NASA cleaning protocols. However, the persistence of a subset of bacterial signatures throughout all spacecraft assembly phases underscores the need for continued refinement of sterilization technologies and the implementation of safeguards that monitor and inventory microbial contaminants.

PhyloTrac is an integrated desktop application for analysis of PhyloChip microarray data. PhyloTrac combined with PhyloChip provides turnkey and comprehensive identification and analysis of bacterial and archaeal communities in complex environmental samples. PhyloTrac is free for noncommercial organizations and is available for all major operating systems at http://www.phylotrac.org/ .

Bacterial communities in the airways of cystic fibrosis (CF) patients are, as in other ecological niches, influenced by autogenic and allogenic factors. However, our understanding of microbial colonization in younger versus older CF airways and the association with pulmonary function is rudimentary at best. Using a phylogenetic microarray, we examine the airway microbiota in age stratified CF patients ranging from neonates (9 months) to adults (72 years). From a cohort of clinically stable patients, we demonstrate that older CF patients who exhibit poorer pulmonary function possess more uneven, phylogenetically-clustered airway communities, compared to younger patients. Using longitudinal samples collected form a subset of these patients a pattern of initial bacterial community diversification was observed in younger patients compared with a progressive loss of diversity over time in older patients. We describe in detail the distinct bacterial community profiles associated with young and old CF patients with a particular focus on the differences between respective “early” and “late” colonizing organisms. Finally we assess the influence of Cystic Fibrosis Transmembrane Regulator (CFTR) mutation on bacterial abundance and identify genotype-specific communities involving members of the Pseudomonadaceae, Xanthomonadaceae, Moraxellaceae and Enterobacteriaceae amongst others. Data presented here provides insights into the CF airway microbiota, including initial diversification events in younger patients and establishment of specialized communities of pathogens associated with poor pulmonary function in older patient populations.

Microbial fuel cells (MFCs) have applications other than electricity production, including the capacity to power desirable reactions in the cathode chamber. However, current knowledge of the microbial ecology and physiology of biocathodes is minimal, and as a result more research dedicated to understanding the microbial communities active in cathode biofilms is required. Here we characterize the microbiology of denitrifying bacterial communities stimulated by reducing equivalents generated from the anodic oxidation of acetate. We analyzed biofilms isolated from two types of cathodic denitrification systems: (1) a loop format where the effluent from the carbon oxidation step in the anode is subjected to a nitrifying reactor which is fed to the cathode chamber and (2) an alternative non-loop format where anodic and cathodic feed streams are separated. The results of our study indicate the superior performance of the loop reactor in terms of enhanced current production and nitrate removal rates. We hypothesized that phylogenetic or structural features of the microbial communities could explain the increased performance of the loop reactor. We used PhyloChip with 16S rRNA (cDNA) and fluorescent in situ hybridization to characterize the active bacterial communities. Our study results reveal a greater richness, as well as an increased phylogenetic diversity, active in denitrifying biofilms than was previously identified in cathodic systems. Specifically, we identified Proteobacteria, Firmicutes and Chloroflexi members that were dominant in denitrifying cathodes. In addition, our study results indicate that it is the structural component, in terms of bacterial richness and evenness, rather than the phylogenetic affiliation of dominant bacteria, that best corresponds to cathode performance.

The intestinal microbiota of beef cattle are important for animal health, food safety, and methane emissions. This full-length sequencing survey of 11,171 16S rRNA genes reveals animal-to-animal variation in communities that cannot be attributed to breed, gender, diet, age, or weather. Beef communities differ from those of dairy. Core bovine taxa are identified.

Hexavalent Chromium is a widespread contaminant found in soil, sediment, and groundwater. In order to stimulate microbially-mediated reduction of Cr(VI), a poly-lactate compound (HRC) was injected into the Chromium-contaminated aquifer at the Hanford (WA) 100H site in 2004. Cr(VI) concentrations rapidly declined to below the detection limit and remained so for more than three years after injection. Based on the results of the bacterial community composition using high-density DNA 16S rRNA gene microarrays, we observed the community to transition through denitrifying, ironreducing and sulfate-reducing populations. As a result, we specifically focused isolation efforts on three bacterial species that were significant components of the community. Positive enrichments in defined anaerobic media resulted in the isolation of an iron-reducing Geobacter metallireducens-like isolate, a sulfate-reducing Desulfovibrio vukgaris-like strain and a nitrate-reducing Pseudomonas stutzeri-like isolate among several others. All of these isolates were capable of reducing Cr(VI) anoxically and have been submitted for genome sequencing to JGI. To further characterize the microbial, and geochemical mechanisms associated with in situ Cr(VI) reduction at the site, additional HRC was injected in 2008. The goal was to restimulate the indigenous microbial community and to regenerate the reducing conditions necessary for continued Cr(VI) bio-immobilization in the groundwater. Analysismore » of the microbial populations post-injection revealed that they recovered to a similar density as after the first injection in 2004. In this study, we present the results from our investigation into microbially-mediated Cr(VI) reduction at Hanford, and a comparison of the microbial community development following two HRC injections four years apart.« less

Although the cultivable and noncultivable microbial diversity of spacecraft assembly clean rooms has been previously documented using conventional and state-of-the-art molecular techniques, the occurrence of obligate anaerobes within these clean rooms is still uncertain. Therefore, anaerobic bacterial communities of three clean-room facilities were analyzed during assembly of the Mars Science Laboratory rover. Anaerobic bacteria were cultured on several media, and DNA was extracted from suitable anaerobic enrichments and examined with conventional 16S rRNA gene clone library, as well as high-density phylogenetic 16S rRNA gene microarray (PhyloChip) technologies. The culture-dependent analyses predominantly showed the presence of clostridial and propionibacterial strains. The 16S rRNA gene sequences retrieved from clone libraries revealed distinct microbial populations associated with each clean-room facility, clustered exclusively within gram-positive organisms. PhyloChip analysis detected a greater microbial diversity, spanning many phyla of bacteria, and provided a deeper insight into the microbial community structure of the clean-room facilities. This study presents an integrated approach for assessing the anaerobic microbial population within clean-room facilities, using both molecular and cultivation-based analyses. The results reveal that highly diverse anaerobic bacterial populations persist in the clean rooms even after the imposition of rigorous maintenance programs and will pose a challenge to planetary protection implementation activities.

Asthma increased dramatically in the last decades of the 20th century and is representative of chronic diseases that have been linked to altered microbial exposure and immune responses. Here we evaluate the effects of environmental exposures typically associated with asthma protection or risk on the microbial community structure of household dust (dogs, cats, and day care). PCR-denaturing gradient gel analysis (PCR-DGGE) demonstrated that the bacterial community structure in house dust is significantly impacted by the presence of dogs or cats in the home ( P = 0.0190 and 0.0029, respectively) and by whether or not children attend day care ( P = 0.0037). In addition, significant differences in the dust bacterial community were associated with asthma outcomes in young children, including wheezing ( P = 0.0103) and specific IgE ( P = 0.0184). Our findings suggest that specific bacterial populations within the community are associated with either risk or protection from asthma.

A microbial census on deep biosphere (1.34 km depth) microbial communities was performed in two soil samples collected from the Ross and number 6 Winze sites of the former Homestake gold mine, Lead, South Dakota using high-density 16S microarrays (PhyloChip). Soil mineralogical characterization was carried out using X-ray diffraction, X-ray photoelectron, and Mossbauer spectroscopic techniques which demonstrated silicates and iron minerals (phyllosilicates and clays) in both samples. Microarray data revealed extensive bacterial diversity in soils and detected the largest number of taxa in Proteobacteria phylum followed by Firmicutes and Actinobacteria. The archael communities in the deep gold mine environments were less diverse and belonged to phyla Euryarchaeota and Crenarchaeota. Both the samples showed remarkable similarities in microbial communities (1,360 common OTUs) despite distinct geochemical characteristics. Fifty-seven phylotypes could not be classified even at phylum level representing a hitherto unidentified diversity in deep biosphere. PhyloChip data also suggested considerable metabolic diversity by capturing several physiological groups such as sulfur-oxidizer, ammonia-oxidizers, iron-oxidizers, methane-oxidizers, and sulfate-reducers in both samples. High-density microarrays revealed the greatest prokaryotic diversity ever reported from deep subsurface habitat of gold mines.

A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.

Dehalococcoides ethenogenes strain 195 utilizes corrinoid-containing reductive dehalogenases to reduce the environmental pollutants tetrachloroethene and trichloroethene to ethene. Although corrinoids are essential for dehalogenation activity, strain 195 cannot biosynthesize corrinoids de novo. To improve our understanding of corrinoid physiology in this bacterium, whole-genome microarrays were applied to characterize the transcriptome during growth with excess and limiting concentrations of the corrinoid cyanocobalamin. Additional studies examined the effects of exposure to spent medium from a methanogenic chloroethene-dehalogenating enrichment culture (designated ANAS). Both excess cyanocobalamin and ANAS spent medium resulted in the downregulation of two genes (DET0125-0126) that are encoded downstream of a putative cobalamin riboswitch. In contrast, only ANAS spent medium resulted in the downregulation of three duplicated genes (DET0657-0659/DET0691-0693) encoded downstream of a second putative cobalamin riboswitch. These latter genes are predicted to be involved in synthesizing the lower ligand base that is attached to cobyric acid. It is also notable that only excess cyanocobalamin resulted in the downregulation of a predicted cobalamin transport system. Together, these results imply that ANAS spent medium contains corrinoid forms different from cyanocobalamin and that strain 195 adjusts its metabolism according to the corrinoid forms available for uptake.

Antarctic ecosystems are fascinating in their limited trophic complexity, with decomposition and nutrient cycling functions being dominated by microbial activities. Not only are Antarctic habitats exposed to extreme environmental conditions, the Antarctic Peninsula is also experiencing unequalled effects of global warming. Owing to their uniqueness and the potential impact of global warming on these pristine systems, there is considerable interest in determining the structure and function of microbial communities in the Antarctic. We therefore utilized a recently designed 16S rRNA gene microarray, the PhyloChip, which targets 8741 bacterial and archaeal taxa, to interrogate microbial communities inhabiting densely vegetated and bare fell-field soils along a latitudinal gradient ranging from 51°S (Falkland Islands) to 72°S (Coal Nunatak). Results indicated a clear decrease in diversity with increasing latitude, with the two southernmost sites harboring the most distinct Bacterial and Archaeal communities. The microarray approach proved more sensitive in detecting the breadth of microbial diversity than polymerase chain reaction-based bacterial 16S rRNA gene libraries of modest size (190 clones per library). Furthermore, the relative signal intensities summed for phyla and families on the PhyloChip were significantly correlated with the relative occurrence of these taxa in clone libraries. PhyloChip data were also compared with functional gene microarray data obtained earlier, highlighting numerous significant relationships and providing evidence for a strong link between community composition and functional gene distribution in Antarctic soils. Integration of these PhyloChip data with other complementary methods provides an unprecedented understanding of the microbial diversity and community structure of terrestrial Antarctic habitats. © 2009 International Society for Microbial Ecology All rights reserved.

The bacterial diversity associated with citrus leaf midribs was characterized for citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rRNA gene microarrays and 16S rRNA gene clone library sequencing to determine the microbial community composition for symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria in 15 phyla were present in the citrus leaf midribs, while 20 orders in 8 phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs than in asymptomatic midribs. “ Candidatus Liberibacter asiaticus” was detected at a very low level in asymptomatic plants but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis results were further verified by sequencing 16S rRNA gene clone libraries, which indicated the dominance of “ Candidatus Liberibacter asiaticus” in symptomatic leaves. These data implicate “ Candidatus Liberibacter asiaticus” as the pathogen responsible for HLB disease.

Increasing evidence confirms the crucial role bacteria and archaea play within the coral holobiont, that is, the coral host and its associated microbial community. The bacterial component constitutes a community of high diversity, which appears to change in structure in response to disease events. In this study, we highlight the limitation of 16S rRNA gene (16S rDNA) clone library sequencing as the sole method to comprehensively describe coral-associated communities. This limitation was addressed by combining a high-density 16S rRNA gene microarray with, clone library sequencing as a novel approach to study bacterial communities in healthy versus diseased corals. We determined an increase in diversity as well as a significant shift in community structure in Montastraea faveolata colonies displaying phenotypic signs of White Plague Disease type II (WPD-II). An accumulation of species that belong to families that include known coral pathogens (Alteromonadaceae, Vibrionaceae), bacteria previously isolated from diseased, stressed or injured marine invertebrates (for example, Rhodobacteraceae), and other species (for example, Campylobacteraceae) was observed. Some of these species were also present in healthy tissue samples, but the putative primary pathogen, Aurantimonas corallicida, was not detected in any sample by either method. Although an ecological succession of bacteria during disease progression after causation by a primary agent represents a possible explanation for our observations, we also discuss the possibility that a disease of yet to be determined etiology may have affected M. faveolata colonies and resulted in (or be a result of) an increase in opportunistic pathogens.

The most prevalent microorganisms, viruses, bacteria, and fungi, are introduced into the atmosphere from many anthropogenic sources such as agricultural, industrial and urban activities, termed microbial air pollution (MAP), and natural sources. These include soil, vegetation, and ocean surfaces that have been disturbed by atmospheric turbulence. The airborne concentrations range from nil to great numbers and change as functions of time of day, season, location, and upwind sources. While airborne, they may settle out immediately or be transported great distances. Further, most viable airborne cells can be rendered nonviable due to temperature effects, dehydration or rehydration, UV radiation, and/or air pollution effects. Mathematical microbial survival models that simulate these effects have been developed.

DNA from low-biodiversity fracture water collected at 2.8-kilometer depth in a South African gold mine was sequenced and assembled into a single, complete genome. This bacterium, Candidatus Desulforudis audaxviator , composes >99.9% of the microorganisms inhabiting the fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate-reducing, chemoautotrophic thermophile that can fix its own nitrogen and carbon by using machinery shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent life-style well suited to long-term isolation from the photosphere deep within Earth's crust and offers an example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome.

Significant effort is currently focused on microbial fuel cells (MFCs) as a source of renewable energy. Most studies concentrate on operation at mesophilic temperatures. However, anaerobic digestion studies have reported on the superiority of thermophilic operation and demonstrated a net energy gain in terms of methane yield. As such, our studies focused on MFC operation and microbiology at 55 degrees C. Over a 100-day operation, these MFCs were stable and achieved a power density of 37 mW m(-2) with a coulombic efficiency of 89%. To infer activity and taxonomic identity of dominant members of the electricity-producing community, we performed phylogenetic microarray and clone library analysis with small subunit ribosomal RNA (16S rRNA) and ribosomal RNA gene (16S rDNA). The results illustrated the dominance (80% of clone library sequences) of the Firmicutes in electricity production. Similarly, rRNA sequences from Firmicutes accounted for 50% of those taxa that increased in relative abundance from current-producing MFCs, implying their functional role in current production. We complemented these analyses by isolating the first organisms from a thermophilic MFC. One of the isolates, a Firmicutes Thermincola sp. strain JR, not only produced more current than known organisms (0.42 mA) in an H-cell system but also represented the first demonstration of direct anode reduction by a member of this phylum. Our research illustrates the importance of using a variety of molecular and culture-based methods to reliably characterize bacterial communities. Consequently, we revealed a previously unidentified functional role for Gram-positive bacteria in MFC current generation.

“ Dehalococcoides ” bacteria can reductively dehalogenate a wide range of halogenated organic pollutants. In this study, DNA microarrays were used to monitor dynamic changes in the transcriptome as “ Dehalococcoides ethenogenes” strain 195 transitioned from exponential growth into stationary phase. In total, 415 nonredundant genes were identified as differentially expressed. As expected, genes involved with translation and energy metabolism were down-regulated while genes involved with general stress response, transcription, and signal transduction were up-regulated. Unexpected, however, was the 8- to 10-fold up-regulation of four putative reductive dehalogenases (RDases) (DET0173, DET0180, DET1535, and DET1545). Another unexpected finding was the up-regulation of a large number of genes located within integrated elements, including a putative prophage and a multicopy transposon. Finally, genes encoding the dominant hydrogenase-RDase respiratory chain of this strain (Hup and TceA) were expressed at stable levels throughout the experiment, providing molecular evidence that strain 195 can uncouple dechlorination from net growth.

We present TaqMan-minor groove binding (MGB) assays for an SNP that separates the Yersinia pestis strain CO92 from all other strains and for another SNP that separates North American strains from all other global strains.

We engineered a strain of the bacterium Caulobacter crescentus to fluoresce in the presence of micromolar levels of uranium at ambient temperatures when it is exposed to a hand-held UV lamp. Previous microarray experiments revealed that several Caulobacter genes are significantly upregulated in response to uranium but not in response to other heavy metals. We designated one of these genes urcA (for u ranium r esponse in c aulobacter). We constructed a reporter that utilizes the urcA promoter to produce a UV-excitable green fluorescent protein in the presence of the uranyl cation, a soluble form of uranium. This reporter is specific for uranium and has little cross specificity for nitrate (Caulobacter reporter strain can provide on-demand usability in the field; it requires minimal sample processing and no equipment other than a hand-held UV lamp, and it may be sprayed directly on soil, groundwater, or industrial surfaces.

Background Accurate taxonomy is best maintained if species are arranged as hierarchical groups in phylogenetic trees. This is especially important as trees grow larger as a consequence of a rapidly expanding sequence database. Hierarchical group names are typically manually assigned in trees, an approach that becomes unfeasible for very large topologies. Results We have developed an automated iterative procedure for delineating stable (monophyletic) hierarchical groups to large (or small) trees and naming those groups according to a set of sequentially applied rules. In addition, we have created an associated ungrouping tool for removing existing groups that do not meet user-defined criteria (such as monophyly). The procedure is implemented in a program called GRUNT (GRouping, Ungrouping, Naming Tool) and has been applied to the current release of the Greengenes (Hugenholtz) 16S rRNA gene taxonomy comprising more than 130,000 taxa. Conclusion GRUNT will facilitate researchers requiring comprehensive hierarchical grouping of large tree topologies in, for example, database curation, microarray design and pangenome assignments. The application is available at the greengenes website [1].

By using a high-throughput screening method, a mutant of a uropathogenic Escherichia coli strain affected in the rapA gene was isolated. The mutant formed normal-architecture biofilms but showed decreased penicillin G resistance, although the mutation did not affect planktonic cell resistance. Transcriptome analysis showed that 22 genes were down-regulated in the mutant biofilm. One of these genes was yhcQ , which encodes a putative multidrug resistance pump. Mutants with mutations in this gene also formed biofilms with decreased resistance, although the effect was less pronounced than that of the rapA mutation. Thus, an additional mechanism(s) controlled by a rapA -regulated gene(s) was involved in wild-type biofilm resistance. The search for this mechanism was guided by the fact that another down-regulated gene in rapA biofilms, yeeZ , is suspected to be involved in extra cell wall-related functions. A comparison of the biofilm matrix of the wild-type and rapA strains revealed decreased polysaccharide quantities and coverage in the mutant biofilms. Furthermore, the (fluorescent) functional penicillin G homologue Bocillin FL penetrated the mutant biofilms more readily. The results strongly suggest a dual mechanism for the wild-type biofilm penicillin G resistance, retarded penetration, and effective efflux. The results of studies with an E. coli K-12 strain pointed to the same conclusion. Since efflux and penetration can be general resistance mechanisms, tests were conducted with other antibiotics. The rapA biofilm was also more sensitive to norfloxacin, chloramphenicol, and gentamicin.

Management of airway infections caused by Pseudomonas aeruginosa is a serious clinical challenge, but little is known about the microbial ecology of airway infections in intubated patients. We analyzed bacterial diversity in endotracheal aspirates obtained from intubated patients colonized by P. aeruginosa by using 16S rRNA clone libraries and microarrays (PhyloChip) to determine changes in bacterial community compositions during antibiotic treatment. Bacterial 16S rRNA genes were absent from aspirates obtained from patients briefly intubated for elective surgery but were detected by PCR in samples from all patients intubated for longer periods. Sequencing of 16S rRNA clone libraries demonstrated the presence of many orally, nasally, and gastrointestinally associated bacteria, including known pathogens, in the lungs of patients colonized with P. aeruginosa . PhyloChip analysis detected the same organisms and many additional bacterial groups present at low abundance that were not detected in clone libraries. For each patient, both culture-independent methods showed that bacterial diversity decreased following the administration of antibiotics, and communities became dominated by a pulmonary pathogen. P. aeruginosa became the dominant species in six of seven patients studied, despite treatment of five of these six with antibiotics to which it was sensitive in vitro. Our data demonstrate that the loss of bacterial diversity under antibiotic selection is highly associated with the development of pneumonia in ventilated patients colonized with P. aeruginosa. Interestingly, PhyloChip analysis demonstrated reciprocal changes in abundance between P. aeruginosa and the class Bacilli , suggesting that these groups may compete for a similar ecological niche and suggesting possible mechanisms through which the loss of microbial diversity may directly contribute to pathogen selection and persistence.

Geochemical, microbiological, and molecular analyses of alkaline saline groundwater at 2.8 kilometers depth in Archaean metabasalt revealed a microbial biome dominated by a single phylotype affiliated with thermophilic sulfate reducers belonging to Firmicutes . These sulfate reducers were sustained by geologically produced sulfate and hydrogen at concentrations sufficient to maintain activities for millions of years with no apparent reliance on photosynthetically derived substrates.

The bacterium Caulobacter crescentus and related stalk bacterial species are known for their distinctive ability to live in low-nutrient environments, a characteristic of most heavy metal-contaminated sites. Caulobacter crescentus is a model organism for studying cell cycle regulation with well-developed genetics. We have identified the pathways responding to heavy-metal toxicity in C. crescentus to provide insights for the possible application of Caulobacter to environmental restoration. We exposed C. crescentus cells to four heavy metals (chromium, cadmium, selenium, and uranium) and analyzed genome-wide transcriptional activities postexposure using an Affymetrix GeneChip microarray. C. crescentus showed surprisingly high tolerance to uranium, a possible mechanism for which may be the formation of extracellular calcium-uranium-phosphate precipitates. The principal response to these metals was protection against oxidative stress (up-regulation of manganese-dependent superoxide dismutase sodA). Glutathione S -transferase, thioredoxin, glutaredoxins, and DNA repair enzymes responded most strongly to cadmium and chromate. The cadmium and chromium stress response also focused on reducing the intracellular metal concentration, with multiple efflux pumps employed to remove cadmium, while a sulfate transporter was down-regulated to reduce nonspecific uptake of chromium. Membrane proteins were also up-regulated in response to most of the metals tested. A two-component signal transduction system involved in the uranium response was identified. Several differentially regulated transcripts from regions previously not known to encode proteins were identified, demonstrating the advantage of evaluating the transcriptome by using whole-genome microarrays.

A microarray has been designed using 62,358 probes matched to both prokaryotic and eukaryotic small-subunit ribosomal RNA genes. The array categorized environmental DNA to specific phylogenetic clusters in under 9 h. To a background of DNA generated from natural outdoor aerosols, known quantities of rRNA gene copies from distinct organisms were added producing corresponding hybridization intensity scores that correlated well with their concentrations ( r = 0.917). Reproducible differences in microbial community composition were observed by altering the genomic DNA extraction method. Notably, gentle extractions produced peak intensities for Mycoplasmatales and Burkholderiales , whereas a vigorous disruption produced peak intensities for Vibrionales , Clostridiales , and Bacillales.

Motivation: Prokaryotic organisms have been identified utilizing the sequence variation of the 16S rRNA gene. Variations steer the design of DNA probes for the detection of taxonomic groups or specific organisms. The long-term goal of our project is to create probe arrays capable of identifying 16S rDNA sequences in unknown samples. This necessitated the authentication, categorization and alignment of the >75 000 publicly available‘ 16S’ sequences. Preferably, the entire process should be computationally administrated so the aligned collection could periodically absorb 16S rDNA sequences from the public records. A complete multiple sequence alignment would provide a foundation for computational probe selection and facilitates microbial taxonomy and phylogeny. Results: Here we report the alignment and similarity clustering of 62 662 16S rDNA sequences and an approach for designing effective probes for each cluster. A novel alignment compression algorithm, NAST (Nearest Alignment Space Termination), was designed to produce the uniform multiple sequence alignment referred to as the prokMSA. From the prokMSA, 9020 Operational Taxonomic Units (OTUs) were found based on transitive sequence similarities. An automated approach to probe design was straightforward using the prokMSA clustered into OTUs. As a test case, multiple probes were computationally picked for each of the 27 OTUs that were identified within the Staphylococcus Group. The probes were incorporated into a customized microarray and were able to correctly categorize Staphylococcus aureus and Bacillus anthracis into their correct OTUs. Although a successful probe picking strategy is outlined, the main focus of creating the prokMSA was to provide a comprehensive, categorized, updateable 16S rDNA collection useful as a foundation for any probe selection algorithm. Availability: http://greengenes.llnl.gov/16S/ Contact: GLAndersen@lbl.gov Supplementary Information: Complete prokMSA in aligned fasta format: http://greengenes.llnl.gov/16S_cgi/download/update_28Dec02/prokMSA_aligned.fasta.Z Sequences within each Operational Taxonomic Unit: http://greengenes.llnl.gov/16S_cgi/download/update_15Mar02/SeqDescByOTU.txt DNA probe sequences: http://greengenes.llnl.gov/16S_cgi/download/update_15Mar02/15Mar02_StaphGrpPrbs.xls * To whom correspondence should be addressed.

Comparative bacterial genomics shows that even different isolates of the same bacterial species can vary significantly in gene content. An effective means to survey differences across whole genomes would be highly advantageous for understanding this variation. Here we show that suppression subtractive hybridization (SSH) provides high, representative coverage of regions that differ between similar genomes. Using Helicobacter pylori strains 26695 and J99 as a model, SSH identified approximately 95% of the unique open reading frames in each strain, showing that the approach is effective. Furthermore, combining data from parallel SSH experiments using different restriction enzymes significantly increased coverage compared to using a single enzyme. These results suggest a powerful approach for assessing genome differences among closely related strains when one member of the group has been completely sequenced.

Yersinia pestis, the causative agent of bubonic plague, emerged recently (20000 years ago) as a clone of Yersinia pseudotuberculosis. There is scant evidence of genome diversity in Y. pestis, although it is possible to differentiate three biovars (antiqua, mediaevalis or orientalis) based on two biochemical tests. There are a few examples of restriction fragment length polymorphisms (RFLPs) within Y. pestis; however, their genetic basis is poorly understood. In this study, six difference regions (DFRs) were identified in Y. pestis, by using subtractive hybridization, which ranged from 4.6 to 19 kb in size. Four of the DFRs are flanked by insertion sequences, and their sequences show similarity to bacterial genes encoding proteins for flagellar synthesis, ABC transport, insect toxicity and bacteriophage functions. The presence or absence of these DFRs (termed the DFR profile) was demonstrated in 78 geographically diverse strains of Y. pestis. Significant genome plasticity was observed among these strains and suggests the acquisition and deletion of these DNA regions during the recent evolution of Y. pestis. Y. pestis biovar orientalis possesses DFR profiles that are different from antiqua and mediaevalis biovars, reflecting the recent origins of this biovar. Whereas some DFR profiles are specific for antiqua and mediaevalis, some DFR profiles are shared by both biovars. Furthermore, the progenitor of Y. pestis, Y. pseudotuberculosis (an enteric pathogen), possesses its own DFR profile. The DFR profiles detailed here demonstrate genome plasticity within Y. pestis, and they imply evolutionary relationships among the three biovars of Y. pestis, as well as between Y. pestis and Y. pseudotuberculosis.

Aims: A high-volume aerosol collector was developed to efficiently capture airborne bacteria in order to assess levels of diversity in the air. Methods and Results: Particulate matter was collected on a device designed to filter 1·4 × 106 litres of air in a 24 h period on a 1-lm pore size polyester membrane. Methods were optimized for extraction of genomic DNA from the air filter concentrate. Preparation times of 90 s with 0·5-0·05 mm diameter zirconia/silica beads yielded the highest concentration genomic DNA that was able to support PCR. A 24-h air sample was taken in Salt Lake City, Utah and the microbial composition was determined by the amplification and sequence analysis of 16S ribosomal DNA fragments. Conclusions: Sequence analysis revealed a large diversity in the type of microbial species present including clones matching the sequence of Clostridium botulinum. The primary components of the aerosol sample included many different spore-forming bacteria as well as more fragile members of the Proteobacteria division. Significance and Impact of the Study: The high-volume air collection and genomic DNA recovery system allows for the rapid detection of both cultivable as well as culture-resistant organisms in the environment.