Behavioral response of dissimilatory perchlorate-reducing bacteria to different electron acceptors

The response behavior of three dissimilatory perchlorate-reducing bacteria to different electron acceptors (nitrate, chlorate, and perchlorate) was investigated with two different assays. The observed response was species-specific, dependent on the prior growth conditions, and was inhibited by oxygen. We observed attraction toward nitrate when Dechloromonas aromatica strain RCB and Azospira suillum strain PS were grown with nitrate. When D. aromatica and Dechloromonas agitata strain CKB were grown with perchlorate, both responded to nitrate, chlorate, and perchlorate. When A. suillum was grown with perchlorate, the organism responded to chlorate and perchlorate but not nitrate. A gene replacement mutant in the perchlorate reductase subunit (pcrA) of D. aromatica resulted in a loss of the attraction response toward perchlorate but had no impact on the nitrate response. Washed-cell suspension studies revealed that the perchlorate grown cells of D. aromatica reduced both perchlorate and nitrate, while A. suillum cells reduced perchlorate only. Based on these observations, energy taxis was proposed as the underlying mechanism for the responses to (per)chlorate by D. aromatica. To the best of our knowledge, this study represents the first investigation of the response behavior of perchlorate-reducing bacteria to environmental stimuli. It clearly demonstrates attraction toward chlorine oxyanions and the unique ability of these organisms to distinguish structurally analogous compounds, nitrate, chlorate, and perchlorate and respond accordingly.     

Direct enrichment of perchlorate-reducing microbial community for efficient electroactive perchlorate reduction in biocathodes

Biological reduction of perchlorate (ClO₄ ^?) has emerged as a promising solution for the removal of perchlorate in contaminated water and soils. In this work, we demonstrate a simple process to enrich perchlorate-reducing microbial communities separately using acetate as electron donor and the municipal aerobic membrane bioreactor sludge as inoculum. Inoculation of cathodes in microbial fuel cells (MFCs) with these enrichments, and further electrochemical enrichment at constant resistance operation of the MFCs, led to perchlorate-reducing biocathodes with peak reduction rates of 0.095 mM/day (2 mg/m²/day). Analysis of the microbial diversity of perchlorate-reducing biocathodes using PCR-DGGE revealed unique community profiles when compared to the denitrifying biocathode communities. More importantly, the total time taken for enrichment of the electroactive communities was reduced from several months reported previously in literature to less than a month in this work.     

Interplay between Bacillus subtilis RecD2 and the RecG or RuvAB helicase in recombinational repair

Bacillus subtilis AddAB, RecS, RecQ, PcrA, HelD, DinG, RecG, RuvAB, PriA and RecD2 are genuine recombinational repair enzymes, but the biological role of RecD2 is poorly defined. A ΔrecD2 mutation sensitizes cells to DNA-damaging agents that stall or collapse replication forks. We found that this ΔrecD2 mutation impaired growth, and that a mutation in the pcrA gene (pcrA596) relieved this phenotype. The ΔrecD2 mutation was not epistatic to ΔaddAB, ΔrecQ, ΔrecS, ΔhelD, pcrA596 and ΔdinG, but epistatic to recA. Specific RecD2 degradation caused unviability in the absence of RecG or RuvAB, but not on cells lacking RecU. These findings show that there is notable interplay between RecD2 and RecG or RuvAB at arrested replication forks, rather than involvement in processing Holliday junctions during canonical double strand break repair. We propose that there is a trade-off for efficient genome duplication, and that recombinational DNA helicases directly or indirectly provide the cell with the means to tolerate chromosome segregation failures.     

The effect of multiple primer–template mismatches on quantitative PCR accuracy and development of a multi-primer set assay for accurate quantification of pcrA gene sequence variants

Quantitative PCR (qPCR) is a critical tool for quantifying the abundance of specific organisms and the level or expression of target genes in medically and environmentally relevant systems. However, often the power of this tool has been limited because primer–template mismatches, due to sequence variations of targeted genes, can lead to inaccuracies in measured gene quantities, detection failures, and spurious conclusions. Currently available primer design guidelines for qPCR were developed for pure culture applications, and available primer design strategies for mixed cultures were developed for detection rather than accurate quantification. Furthermore, past studies examining the impact of mismatches have focused only on single mismatches while instances of multiple mismatches are common. There are currently no appropriate solutions to overcome the challenges posed by sequence variations. Here, we report results that provide a comprehensive, quantitative understanding of the impact of multiple primer–template mismatches on qPCR accuracy and demonstrate a multi-primer set approach to accurately quantify a model gene pcrA (encoding perchlorate reductase) that has substantial sequence variation. Results showed that for multiple mismatches (up to 3 mismatches) in primer regions where mismatches were previously considered tolerable (middle and 5′ end), quantification accuracies could be as low as ~ 0.1%. Furthermore, tests were run using a published pcrA primer set with mixtures of genomic DNA from strains known to harbor the target gene, and for some mixtures quantification accuracy was as low as ~ 0.8% or was non-detect. To overcome these limitations, a multiple primer set assay including minimal degeneracies was developed for pcrA genes. This assay resulted in nearly 100% accurate detection for all mixed microbial communities tested. The multi-primer set approach demonstrated herein can be broadly applied to other genes with known sequences.     

At least four regulatory genes control sulphur metabolite repression in Aspergillus nidulans

The Staphylococcus aureus chromosomal gene pcrA, identified by mutations, such as pcrA3, that affect plasmid pT181 replication, has been cloned and sequenced. The pcrA gene encodes a protein with significant similarity (40% identity) to two Escherichia coli helicases: the helicase II encoded by the uvrD gene and the Rep helicase. The pcrA3 mutation was found to be a C to T transition leading to a threonine to isoleucine substitution at amino acid residue 61 of the protein. The pcrA gene seems to belong to an operon containing at least one other gene, tentatively named pcrB, upstream from pcrA. The PcrA protein was shown to be essential for cell viability and overproduction has deleterious effects on the host and plasmid replication.     

Characterization and quantification of class 1 integrons and associated gene cassettes in sewage treatment plants

Three hydrogen-based membrane biofilm reactors (MBfR) biologically reduced nitrate and perchlorate in a synthetic ion-exchange (IX) brine. Inocula from different natural saline environments were employed to initiate the three MBfRs. Under high-salinity (3%) conditions, bioreduction of nitrate and perchlorate occurred simultaneously, and the three MBfRs from the different inocula exhibited similar removal fluxes for nitrate and perchlorate. Clone libraries were generated from samples of the biofilms in the three MBfRs and compared to those of their inocula. When H₂ was the sole exogenous electron donor under high-salinity conditions, MBfR-driven community shifts were observed with a similar pattern regardless of inoculum. The following 16S rRNA gene phylogenetic analysis showed the presence of novel perchlorate-reducing bacteria in the salt-tolerant mBfR communities. These findings suggest that autohydrogenotrophic and high-salinity conditions provided strong selective pressure for novel perchlorate-reducing populations in the mBfRs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Protective Role of the PG1036-PG1037-PG1038 Operon in Oxidative Stress in Porphyromonas gingivalis W83

As an anaerobe, Porphyromonas gingivalis is significantly affected by the harsh inflammatory environment of the periodontal pocket during initial colonization and active periodontal disease. We reported previously that the repair of oxidative stress-induced DNA damage involving 8-oxo-7,8-dihydroguanine (8-oxoG) may occur by an undescribed mechanism in P. gingivalis. DNA affinity fractionation identified PG1037, a conserved hypothetical protein, among other proteins, that were bound to the 8-oxoG lesion. PG1037 is part of the uvrA-PG1037-pcrA operon in P. gingivalis which is known to be upregulated under H₂O₂ induced stress. A PCR-based linear transformation method was used to inactivate the uvrA and pcrA genes by allelic exchange mutagenesis. Several attempts to inactivate PG1037 were unsuccessful. Similar to the wild-type when plated on Brucella blood agar, the uvrA and pcrA-defective mutants were black-pigmented and beta-hemolytic. These isogenic mutants also had reduced gingipain activities and were more sensitive to H₂O₂ and UV irradiation compared to the parent strain. Additionally, glycosylase assays revealed that 8-oxoG repair activities were similar in both wild-type and mutant P. gingivalis strains. Several proteins, some of which are known to have oxidoreducatse activity, were shown to interact with PG1037. The purified recombinant PG1037 protein could protect DNA from H₂O₂-induced damage. Collectively, these findings suggest that the uvrA-PG1037-pcrA operon may play an important role in hydrogen peroxide stress-induced resistance in P. gingivalis.     

Bacterial communities in water and sediment shaped by paper mill pollution and indicated bacterial taxa in sediment in Daling River

Effluents from paper mills are highly toxic and are a major source of aquatic pollution. In this study, we collected water and sediment samples to examine the microbial communities using denaturing gradient gel electrophoresis and identified bacterial taxa greatly affected by paper mill pollution using next-generation sequencing data. Our results indicated that bacterial communities in downstream sediments were similar to those in paper mill discharge sites, indicating obvious effects of pollution, while bacterial communities in downstream water samples showed similar profiles to those in upstream sites, both being quite different from the bacterial communities in paper mill discharge sites. This was possibly because of the short contact period. In addition, bacterial communities in the estuary were quite different from those in other water and sediment samples, which was owing to the special habitat type. Considering the storage of paper mill pollutants in sediment and the significant effect on shifts in bacterial communities, we selected Clostridia and Epsilonproteobacteria at the class level and Fusibacter and Desulfobulbus at the genus level as bacterial indicators of paper mill pollution. To monitor the remediation of polluted aquatic environments, we propose Sphingobacteria, Alphaproteobacteria, Actinobacteria, Subdivision3, Planctomycetacia and Verrucomicrobiae at the class level and Bacillus, Steroidobacter, Nocardioides, Terrimonas, Pirellula and Methylibium at the genus level.     

Comparison of bacterial communities of biofilms formed on different membrane surfaces

Bacterial biofilm communities formed on different membrane surfaces were investigated based on 16S rRNA gene sequence analysis. The biofilm communities were distinct from those of mixed-liquor and consisted mainly of Beta- and Gammaproteobacteria. Sequences of Xathomonas and Aquabacterium were mostly retrieved from the biofilm samples rather than from the mixed liquor. Furthermore, statistical analyses demonstrated the importance of a physico-chemical property of membrane, surface roughness, in structuring the bacterial biofilm communities.     

Description of the novel perchlorate-reducing bacteria Dechlorobacter hydrogenophilus gen. nov., sp. nov. and Propionivibrio militaris, sp. nov.

Novel dissimilatory perchlorate-reducing bacteria (DPRB) were isolated from enrichments conducted under conditions different from those of all previously described DPRB. Strain LT-1^T was enriched using medium buffered at pH 6.6 with 2-(N-morpholino)ethanesulfonic acid (MES) and had only 95% 16S rRNA gene identity with its closest relative, Azonexus caeni. Strain MP^T was enriched in the cathodic chamber of a perchlorate-reducing bioelectrical reactor (BER) and together with an additional strain, CR (99% 16S rRNA gene identity), had 97% 16S rRNA gene identity with Propionivibrio limicola. The use of perchlorate and other electron acceptors distinguished strains MP^T and CR from P. limicola physiologically. Strain LT-1^T had differences in electron donor utilization and optimum growth temperatures from A. caeni. Strains LT-1^T and MP^T are the first DPRB to be described in the Betaproteobacteria outside of the Dechloromonas and Azospira genera. On the basis of phylogenetic and physiological features, strain LT-1^T represents a novel genus in the Rhodocyclaceae; strain MP^T represents a novel species within the genus Propionivibrio. The names Dechlorobacter hydrogenophilus gen. nov., sp. nov and Propionivibrio militaris sp. nov. are proposed.     

Floristic groups and plant communities of southeastern Santa Fe,Argentina

This paper is a survey of the vegetation of the southeastern departments in the Province of Santa Fe (Argentina). The vegetation was analyzed following Braun-Blanquet's approach modified by Mueller-Dombois & Ellenberg (1974). The most relevant species of the region were placed in 25 groups according to their requirements or general behaviour. Most of the communities are herbaceous, and apart from the woody and some other miscellaneous ones they were grouped into three ecologically and floristically defined sets. The first set, the Stipa grasslands and related communities, which are characterized by the more or less abundant presence of Stipa hyalina, Stipa neesiana and Stipa papposa, comprises five different communities. The second, the halophilous communities, comprises five communities, the two Spartina ssp. grasslands, the halophilous prairies of Distichlis spicata, the short sedge Scirpus americanus communities and the ‘pela-dales’. The third, the hygrophilous communities, comprises nine communities which are not so well defined as the ones in the other sets. Besides, two further communities have been included, the Paspalum quadrifarium and the Melica macra tall grasslands.     

Yeast Communities of Diverse Drosophila Species: Comparison of Two Symbiont Groups in the Same Hosts

The combination of ecological diversity with genetic and experimental tractability makes Drosophila a powerful model for the study of animal-associated microbial communities. Despite the known importance of yeasts in Drosophila physiology, behavior, and fitness, most recent work has focused on Drosophila-bacterial interactions. In order to get a more complete understanding of the Drosophila microbiome, we characterized the yeast communities associated with different Drosophila species collected around the world. We focused on the phylum Ascomycota because it constitutes the vast majority of the Drosophila-associated yeasts. Our sampling strategy allowed us to compare the distribution and structure of the yeast and bacterial communities in the same host populations. We show that yeast communities are dominated by a small number of abundant taxa, that the same yeast lineages are associated with different host species and populations, and that host diet has a greater effect than host species on yeast community composition. These patterns closely parallel those observed in Drosophila bacterial communities. However, we do not detect a significant correlation between the yeast and bacterial communities of the same host populations. Comparative analysis of different symbiont groups provides a more comprehensive picture of host-microbe interactions. Future work on the role of symbiont communities in animal physiology, ecological adaptation, and evolution would benefit from a similarly holistic approach.     

Analysis of bacterial communities in sponges and coral inhabiting Red Sea,using barcoded 454 pyrosequencing

Microbial communities are linked with marine sponge are diverse in their structure and function. Our understanding of the sponge-associated microbial diversity is limited especially from Red Sea in Saudi Arabia where few species of sponges have been studied. Here we used pyrosequencing to study two marine sponges and coral species sampled from Obhur region from Red sea in Jeddah. A total of 168 operational taxonomic units (OTUs) were identified from Haliclona caerulea, Stylissa carteri and Rhytisma fulvum. Taxonomic identification of tag sequences of 16S ribosomal RNA revealed 6 different bacterial phyla and 9 different classes. A proportion of unclassified reads were was also observed in sponges and coral sample. We found diverse bacterial communities associated with two sponges and a coral sample. Diversity and richness estimates based on OUTs revealed that sponge H. caerulea had significantly high bacterial diversity. The identified OTUs showed unique clustering in three sponge samples as revealed by Principal coordinate analysis (PCoA). Proteobacteria (88–95%) was dominant phyla alonwith Bacteroidetes, Planctomycetes, Cyanobacteria, Firmicutes and Nitrospirae. Seventeen different genera were identified where genus Pseudoalteromonas was dominant in all three samples. This is first study to assess bacterial communities of sponge and coral sample that have never been studied before to unravel their microbial communities using 454-pyrosequencing method.     

Bacterial Community Response to Petroleum Hydrocarbon Amendments in Freshwater,Marine, and Hypersaline Water-Containing Microcosms

Hydrocarbon-degrading bacterial communities from freshwater, marine, and hypersaline Brazilian aquatic ecosystems (with water salinities corresponding to 0.2%, 4%, and 5%, respectively) were enriched with different hydrocarbons (heptadecane, naphthalene, or crude oil). Changes within the different microcosms of bacterial communities were analyzed using cultivation approaches and molecular methods (DNA and RNA extraction, followed by genetic fingerprinting and analyses of clone libraries based on the 16S rRNA-coding gene). A redundancy analysis (RDA) of the genetic fingerprint data and a principal component analysis (PCA) of the clone libraries revealed hydrocarbon-enriched bacterial communities specific for each ecosystem studied. However, within the same ecosystem, different bacterial communities were selected according to the petroleum hydrocarbon used. In general, the results demonstrated that Acinetobacter and Cloacibacterium were the dominant genera in freshwater microcosms; the Oceanospirillales order and the Marinobacter, Pseudomonas, and Cycloclasticus genera predominated in marine microcosms; and the Oceanospirillales order and the Marinobacter genus were selected in the different hydrocarbon-containing microcosms in hypersaline water. Determination of total petroleum hydrocarbons (TPHs) in all microcosms after 32 days of incubation showed a decrease in the hydrocarbon concentration compared to that for the controls. A total of 50 (41.3%) isolates from the different hydrocarbon-contaminated microcosms were associated with the dominant operational taxonomic units (OTUs) obtained from the clone libraries, and their growth in the hydrocarbon contaminating the microcosm from which they were isolated as the sole carbon source was observed. These data provide insight into the general response of bacterial communities from freshwater, marine, and hypersaline aquatic ecosystems to petroleum hydrocarbon contamination.     

Spatial scales of bacterial community diversity at cold seeps (Eastern Mediterranean Sea)

Cold seeps are highly productive, fragmented marine ecosystems that form at the seafloor around hydrocarbon emission pathways. The products of microbial utilization of methane and other hydrocarbons fuel rich chemosynthetic communities at these sites, with much higher respiration rates compared with the surrounding deep-sea floor. Yet little is known as to the richness, composition and spatial scaling of bacterial communities of cold seeps compared with non-seep communities. Here we assessed the bacterial diversity across nine different cold seeps in the Eastern Mediterranean deep-sea and surrounding seafloor areas. Community similarity analyses were carried out based on automated ribosomal intergenic spacer analysis (ARISA) fingerprinting and high-throughput 454 tag sequencing and were combined with in situ and ex situ geochemical analyses across spatial scales of a few tens of meters to hundreds of kilometers. Seep communities were dominated by Deltaproteobacteria, Epsilonproteobacteria and Gammaproteobacteria and shared, on average, 36% of bacterial types (ARISA OTUs (operational taxonomic units)) with communities from nearby non-seep deep-sea sediments. Bacterial communities of seeps were significantly different from those of non-seep sediments. Within cold seep regions on spatial scales of only tens to hundreds of meters, the bacterial communities differed considerably, sharing <50% of types at the ARISA OTU level. Their variations reflected differences in porewater sulfide concentrations from anaerobic degradation of hydrocarbons. This study shows that cold seep ecosystems contribute substantially to the microbial diversity of the deep-sea.     

Natural Communities of Achromatium oxaliferum Comprise Genetically, Morphologically, and Ecologically Distinct Subpopulations

The diversity and ecology of natural communities of the uncultivated bacterium Achromatium oxaliferum were studied by use of culture-independent approaches. 16S rRNA gene sequences were PCR amplified from DNA extracted from highly purified preparations of cells that were morphologically identified as A. oxaliferum present in freshwater sediments from three locations in northern England (Rydal Water, Jenny Dam, Hell Kettles). Cloning and sequence analysis of the PCR-amplified 16S rRNA genes revealed that multiple related but divergent sequences were routinely obtained from the A. oxaliferum communities present in all the sediments examined. Whole-cell in situ hybridization with combinations of fluorescence-labelled oligonucleotide probes revealed that the divergent sequences recovered from purified A. oxaliferum cells corresponded to genetically distinct Achromatium subpopulations. Analysis of the cell size distribution of the genetically distinct subpopulations demonstrated that each was also morphologically distinct. Furthermore, there was a high degree of endemism in the Achromatium sequences recovered from different sediments; identical sequences were never recovered from different sampling locations. In addition to ecological differences that were apparent between Achromatium communities from different freshwater sediments, the distribution of different subpopulations of Achromatium in relation to sediment redox profiles indicated that the genetically and morphologically distinct organisms that coexisted in a single sediment were also ecologically distinct and were adapted to different redox conditions. This result suggests that Achromatium populations have undergone adaptive radiation and that the divergent Achromatium species occupy different niches in the sediments which they inhabit.     

Phyllosphere Bacterial Community of Floating Macrophytes in Paddy Soil Environments as Revealed by Illumina High-Throughput Sequencing

The phyllosphere of floating macrophytes in paddy soil ecosystems, a unique habitat, may support large microbial communities but remains largely unknown. We took Wolffia australiana as a representative floating plant and investigated its phyllosphere bacterial community and the underlying driving forces of community modulation in paddy soil ecosystems using Illumina HiSeq 2000 platform-based 16S rRNA gene sequence analysis. The results showed that the phyllosphere of W. australiana harbored considerably rich communities of bacteria, with Proteobacteria and Bacteroidetes as the predominant phyla. The core microbiome in the phyllosphere contained genera such as Acidovorax, Asticcacaulis, Methylibium, and Methylophilus. Complexity of the phyllosphere bacterial communities in terms of class number and α-diversity was reduced compared to those in corresponding water and soil. Furthermore, the bacterial communities exhibited structures significantly different from those in water and soil. These findings and the following redundancy analysis (RDA) suggest that species sorting played an important role in the recruitment of bacterial species in the phyllosphere. The compositional structures of the phyllosphere bacterial communities were modulated predominantly by water physicochemical properties, while the initial soil bacterial communities had limited impact. Taken together, the findings from this study reveal the diversity and uniqueness of the phyllosphere bacterial communities associated with the floating macrophytes in paddy soil environments.     

Free-Living and Particle-Associated Bacterioplankton in Large Rivers of the Mississippi River Basin Demonstrate Biogeographic Patterns

The different drainage basins of large rivers such as the Mississippi River represent interesting systems in which to study patterns in freshwater microbial biogeography. Spatial variability in bacterioplankton communities in six major rivers (the Upper Mississippi, Missouri, Illinois, Ohio, Tennessee, and Arkansas) of the Mississippi River Basin was characterized using Ion Torrent 16S rRNA amplicon sequencing. When all systems were combined, particle-associated (>3 μm) bacterial assemblages were found to be different from free-living bacterioplankton in terms of overall community structure, partly because of differences in the proportional abundance of sequences affiliated with major bacterial lineages (Alphaproteobacteria, Cyanobacteria, and Planctomycetes). Both particle-associated and free-living communities ordinated by river system, a pattern that was apparent even after rare sequences or those affiliated with Cyanobacteria were removed from the analyses. Ordination of samples by river system correlated with environmental characteristics of each river, such as nutrient status and turbidity. Communities in the Upper Mississippi and the Missouri and in the Ohio and the Tennessee, pairs of rivers that join each other, contained similar taxa in terms of presence-absence data but differed in the proportional abundance of major lineages. The most common sequence types detected in particle-associated communities were picocyanobacteria in the Synechococcus/Prochlorococcus/Cyanobium (Syn/Pro) clade, while free-living communities also contained a high proportion of LD12 (SAR11/Pelagibacter)-like Alphaproteobacteria. This research shows that while different tributaries of large river systems such as the Mississippi River harbor distinct bacterioplankton communities, there is also microhabitat variation such as that between free-living and particle-associated assemblages.