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Vibrio parahaemolyticus is the most common cause of seafood-borne illness reported in the United States. The draft genomes of 132 North American clinical and oyster V. parahaemolyticus isolates were sequenced to investigate their phylogenetic and biogeographic relationships. The majority of oyster isolate sequence types (STs) were from a single harvest location; however, four were identified from multiple locations. There was population structure along the Gulf and Atlantic Coasts of North America, with what seemed to be a hub of genetic variability along the Gulf Coast, with some of the same STs occurring along the Atlantic Coast and one shared between the coastal waters of the Gulf and those of Washington State. Phylogenetic analyses found nine well-supported clades. Two clades were composed of isolates from both clinical and oyster sources. Four were composed of isolates entirely from clinical sources, and three were entirely from oyster sources. Each single-source clade consisted of one ST. Some human isolates lack tdh, trh, and some type III secretion system (T3SS) genes, which are established virulence genes of V. parahaemolyticus. Thus, these genes are not essential for pathogenicity. However, isolates in the monophyletic groups from clinical sources were enriched in several categories of genes compared to those from monophyletic groups of oyster isolates. These functional categories include cell signaling, transport, and metabolism. The identification of genes in these functional categories provides a basis for future in-depth pathogenicity investigations of V. parahaemolyticus. IMPORTANCEVibrio parahaemolyticus is the most common cause of seafood-borne illness reported in the United States and is frequently associated with shellfish consumption. This study contributes to our knowledge of the biogeography and functional genomics of this species around North America. STs shared between the Gulf Coast and the Atlantic seaboard as well as Pacific waters suggest possible transport via oceanic currents or large shipping vessels. STs frequently isolated from humans but rarely, if ever, isolated from the environment are likely more competitive in the human gut than other STs. This could be due to additional functional capabilities in areas such as cell signaling, transport, and metabolism, which may give these isolates an advantage in novel nutrient-replete environments such as the human gut.

Community profiling of the oral microbiome requires the recovery of quality sequences in order to accurately describe microbial community structure and composition. Our objective was to assess the effects of specimen collection method, storage medium, and storage conditions on the relative abundance of taxa in saliva and plaque identified using 16S rRNA genes. We also assessed short-term changes in taxon composition and relative abundance and compared the salivary and dental plaque communities in children and adults. Over a 2-week period, four successive saliva and dental plaque specimens were collected from four adults with no dental decay (108 samples), and two successive specimens were collected from six children with four or more erupted teeth (48 samples). There were minimal differences in community composition at the phylum and operational taxonomic unit levels between dental plaque collection using a scaler and collection using a CytoSoft brush. Plaque samples stored in OMNIgene medium showed higher within-sample Shannon diversity, were compositionally different, and were more similar to each other than plaque stored in liquid dental transport medium. Saliva samples stored in OMNIgene recovered similar communities for at least a week following storage at room temperature. However, the microbial communities recovered from plaque and saliva stored in OMNIgene were significantly different in composition from their counterparts stored in liquid dental transport medium. Dental plaque communities collected from the same tooth type over four successive visits from the same adult did not significantly differ in structure or composition. IMPORTANCE Large-scale epidemiologic studies require collection over time and space, often with multiple teams collecting, storing, and processing data. Therefore, it is essential to understand how sensitive study results are to modest changes in collection and storage protocols that may occur with variation in personnel, resources available at a study site, and shipping requirements. The research presented in this paper measures the effects of multiple storage parameters and collection methodologies on the measured ecology of the oral microbiome from healthy adults and children. These results will potentially enable investigators to conduct oral microbiome studies at maximal efficiency by guiding informed administrative decisions pertaining to the necessary field or clinical work.

Magnetotactic bacteria (MTB) comprise a phylogenetically diverse group of prokaryotes capable of orienting and navigating along magnetic field lines. Under oxic conditions, MTB in natural environments in the Northern Hemisphere generally display north-seeking (NS) polarity, swimming parallel to the Earth's magnetic field lines, while those in the Southern Hemisphere generally swim antiparallel to magnetic field lines (south-seeking [SS] polarity). Here, we report a population of an uncultured, monotrichously flagellated, and vibrioid MTB collected from a brackish lagoon in Brazil in the Southern Hemisphere that consistently exhibits NS polarity. Cells of this organism were mainly located below the oxic-anoxic interface (OAI), suggesting it is capable of some type of anaerobic metabolism. Magnetosome crystalline habit and composition were consistent with elongated prismatic magnetite (Fe 3 O 4 ) particles. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that this organism belongs to a distinct clade of the Gammaproteobacteria class. The presence of NS MTB in the Southern Hemisphere and the previously reported finding of SS MTB in the Northern Hemisphere reinforce the idea that magnetotaxis is more complex than we currently understand and may be modulated by factors other than O 2 concentration and redox gradients in sediments and water columns. IMPORTANCE Magnetotaxis is a navigational mechanism used by magnetotactic bacteria to move along geomagnetic field lines and find an optimal position in chemically stratified sediments. For that, magnetotactic bacteria swim parallel to the geomagnetic field lines under oxic conditions in the Northern Hemisphere, whereas those in the Southern Hemisphere swim antiparallel to magnetic field lines. A population of uncultured vibrioid magnetotactic bacteria was discovered in a brackish lagoon in the Southern Hemisphere that consistently swim northward, i.e., the opposite of the overwhelming majority of other Southern Hemisphere magnetotactic bacteria. This finding supports the idea that magnetotaxis is more complex than previously thought.

Crown gall disease of grapevine is caused by virulent Agrobacterium strains and establishes a suitable habitat for agrobacteria and, potentially, other bacteria. The microbial community associated with grapevine plants has not been investigated with respect to this disease, which frequently results in monetary losses. This study compares the endophytic microbiota of organs from grapevine plants with or without crown gall disease and the surrounding vineyard soil over the growing seasons of 1 year. Amplicon-based community profiling revealed that the dominating factor causing differences between the grapevine microbiota is the sample site, not the crown gall disease. The soil showed the highest microbial diversity, which decreased with the distance from the soil over the root and the graft union of the trunk to the cane. Only the graft union microbiota was significantly affected by crown gall disease. The bacterial community of graft unions without a crown gall hosted transient microbiota, with the three most abundant bacterial species changing from season to season. In contrast, graft unions with a crown gall had a higher species richness, which in every season was dominated by the same three bacteria ( Pseudomonas sp., Enterobacteriaceae sp., and Agrobacterium vitis ). For in vitro -cultivated grapevine plantlets, A. vitis infection alone was sufficient to cause crown gall disease. Our data show that microbiota in crown galls is more stable over time than microbiota in healthy graft unions and that the microbial community is not essential for crown gall disease outbreak. IMPORTANCE The characterization of bacterial populations in animal and human diseases using high-throughput deep-sequencing technologies, such as 16S amplicon sequencing, will ideally result in the identification of disease-specific microbiota. We analyzed the microbiota of the crown gall disease of grapevine, which is caused by infection with the bacterial pathogen Agrobacterium vitis. All other Agrobacterium species were found to be avirulent, even though they lived together with A. vitis in the same crown gall tumor. As has been reported for human cancer, the crown gall tumor also hosted opportunistic bacteria that are adapted to the tumor microenvironment. Characterization of the microbiota in various diseases using amplicon sequencing may help in early diagnosis, to serve as a preventative measure of disease in the future.

Escherichia coli has been proposed to have two habitats—the intestines of mammals/birds and the nonhost environment. Our goal was to assess whether certain strains of E. coli have evolved toward adaptation and survival in wastewater. Raw sewage samples from different treatment plants were subjected to chlorine stress, and ∼59% of the surviving E. coli strains were found to contain a genetic insertion element (IS 30 ) located within the uspC-flhDC intergenic region. The positional location of the IS 30 element was not observed across a library of 845 E. coli isolates collected from various animal hosts or within GenBank or whole-genome reference databases for human and animal E. coli isolates ( n = 1,177). Phylogenetics clustered the IS 30 element-containing wastewater E. coli isolates into a distinct clade, and biomarker analysis revealed that these wastewater isolates contained a single nucleotide polymorphism (SNP) biomarker pattern that was specific for wastewater. These isolates belonged to phylogroup A, possessed generalized stress response (RpoS) activity, and carried the locus of heat resistance, features likely relevant to nonhost environmental survival. Isolates were screened for 28 virulence genes but carried only the fimH marker. Our data suggest that wastewater contains a naturalized resident population of E. coli . We developed an endpoint PCR targeting the IS 30 element within the uspC-flhDC intergenic region, and all raw sewage samples ( n = 21) were positive for this marker. Conversely, the prevalence of this marker in E. coli -positive surface and groundwater samples was low (≤5%). This simple PCR assay may represent a convenient microbial source-tracking tool for identification of water samples affected by municipal wastewater. IMPORTANCE The results of this study demonstrate that some strains of E. coli appear to have evolved to become naturalized populations in the wastewater environment and possess a number of stress-related genetic elements likely important for survival in this nonhost environment. The presence of non-host-adapted strains in wastewater challenges our understanding of using E. coli as a microbial indicator of wastewater treatment performance, suggesting that the E. coli strains present in human and animal feces may be very different from those found in treated wastewater.

Arbuscular mycorrhizal (AM) fungi form mutualisms with plant roots that increase plant growth and shape plant communities. Each AM fungal cell contains a large amount of genetic diversity, but it is unclear if this diversity varies across evolutionary lineages. We found that sequence variation in the nuclear large-subunit (LSU) rRNA gene from 29 isolates representing 21 AM fungal species generally assorted into genus- and species-level clades, with the exception of species of the genera Claroideoglomus and Entrophospora . However, there were significant differences in the levels of sequence variation across the phylogeny and between genera, indicating that it is an evolutionarily constrained trait in AM fungi. These consistent patterns of sequence variation across both phylogenetic and taxonomic groups pose challenges to interpreting operational taxonomic units (OTUs) as approximations of species-level groups of AM fungi. We demonstrate that the OTUs produced by five sequence clustering methods using 97% or equivalent sequence similarity thresholds failed to match the expected species of AM fungi, although OTUs from AbundantOTU, CD-HIT-OTU, and CROP corresponded better to species than did OTUs from mothur or UPARSE. This lack of OTU-to-species correspondence resulted both from sequences of one species being split into multiple OTUs and from sequences of multiple species being lumped into the same OTU. The OTU richness therefore will not reliably correspond to the AM fungal species richness in environmental samples. Conservatively, this error can overestimate species richness by 4-fold or underestimate richness by one-half, and the direction of this error will depend on the genera represented in the sample. IMPORTANCE Arbuscular mycorrhizal (AM) fungi form important mutualisms with the roots of most plant species. Individual AM fungi are genetically diverse, but it is unclear whether the level of this diversity differs among evolutionary lineages. We found that the amount of sequence variation in an rRNA gene that is commonly used to identify AM fungal species varied significantly between evolutionary groups that correspond to different genera, with the exception of two genera that are genetically indistinguishable from each other. When we clustered groups of similar sequences into operational taxonomic units (OTUs) using five different clustering methods, these patterns of sequence variation caused the number of OTUs to either over- or underestimate the actual number of AM fungal species, depending on the genus. Our results indicate that OTU-based inferences about AM fungal species composition from environmental sequences can be improved if they take these taxonomically structured patterns of sequence variation into account.

Listeria monocytogenes is a ubiquitous bacterium that may cause the foodborne illness listeriosis. Only a small amount of data about the population genetic structure of strains isolated from food is available. This study aimed to provide an accurate view of the L. monocytogenes food strain population in France. From 1999 to 2014, 1,894 L. monocytogenes strains were isolated from food at the French National Reference Laboratory for L. monocytogenes and classified according to the five risk food matrices defined by the European Food Safety Authority (EFSA). A total of 396 strains were selected on the basis of different pulsed-field gel electrophoresis (PFGE) clusters, serotypes, and strain origins and typed by multilocus sequence typing (MLST), and the MLST results were supplemented with MLST data available from Institut Pasteur, representing human and additional food strains from France. The distribution of sequence types (STs) was compared between food and clinical strains on a panel of 675 strains. High congruence between PFGE and MLST was found. Out of 73 PFGE clusters, the two most prevalent corresponded to ST9 and ST121. Using original statistical analysis, we demonstrated that (i) there was not a clear association between ST9 and ST121 and the food matrices, (ii) serotype IIc, ST8, and ST4 were associated with meat products, and (iii) ST13 was associated with dairy products. Of the two major STs, ST121 was the ST that included the fewest clinical strains, which might indicate lower virulence. This observation may be directly relevant for refining risk analysis models for the better management of food safety. IMPORTANCE This study showed a very useful backward compatibility between PFGE and MLST for surveillance. The results enabled better understanding of the population structure of L. monocytogenes strains isolated from food and management of the health risks associated with L. monocytogenes food strains. Moreover, this work provided an accurate view of L. monocytogenes strain populations associated with specific food matrices. We clearly showed that some STs were associated with food matrices, such as meat, meat products, and dairy products. We opened the way to source attribution modeling in order to quantify the relative importance of the main food matrices.

Given that epiphytic microbes are often found in large population sizes on plants, we tested the hypothesis that plants are quantitatively important local sources of airborne microorganisms. The abundance of microbial communities, determined by quantifying bacterial 16S RNA genes and the fungal internal transcribed spacer (ITS) region, in air collected directly above vegetation was 2- to 10-fold higher than that in air collected simultaneously in an adjacent nonvegetated area 50 m upwind. Nonmetric multidimensional scaling revealed that the composition of airborne bacteria in upwind air samples grouped separately from that of downwind air samples, while communities on plants and downwind air could not be distinguished. In contrast, fungal taxa in air samples were more similar to each other than to the fungal epiphytes. A source-tracking algorithm revealed that up to 50% of airborne bacteria in downwind air samples were presumably of local plant origin. The difference in the proportional abundances of a given operational taxonomic unit (OTU) between downwind and upwind air when regressed against the proportional representation of this OTU on the plant yielded a positive slope for both bacteria and fungi, indicating that those taxa that were most abundant on plants proportionally contributed more to downwind air. Epiphytic fungi were less of a determinant of the microbiological distinctiveness of downwind air and upwind air than epiphytic bacteria. Emigration of epiphytic bacteria and, to a lesser extent, fungi, from plants can thus influence the microbial composition of nearby air, a finding that has important implications for surrounding ecosystems, including the built environment into which outdoor air can penetrate. IMPORTANCE This paper addresses the poorly understood role of bacterial and fungal epiphytes, the inhabitants of the aboveground plant parts, in the composition of airborne microbes in outdoor air. It is widely held that epiphytes contribute to atmospheric microbial assemblages, but much of what we know is limited to qualitative assessments. Elucidating the sources of microbes in outdoor air can inform basic biological processes seen in airborne communities (e.g., dispersal and biogeographical patterns). Furthermore, given the considerable contribution of outdoor air to microbial communities found within indoor environments, the understanding of plants as sources of airborne microbes in outdoor air might contribute to our understanding of indoor air quality. With an experimental design developed to minimize the likelihood of other-than-local plant sources contributing to the composition of airborne microbes, we provide direct evidence that plants are quantitatively important local sources of airborne microorganisms, with implications for the surrounding ecosystems.

Stress-induced abdominal dysfunction is an attractive target for probiotics. To investigate the effects of the probiotic Lactobacillus casei strain Shirota on abdominal dysfunction, a double-blind, placebo-controlled trial was conducted with healthy medical students undertaking an authorized nationwide examination for academic advancement. For 8 weeks, until the day before the examination, 23 and 24 subjects consumed an L. casei strain Shirota-fermented milk and a placebo milk daily, respectively. In addition to assessments of abdominal symptoms, psychophysical state, and salivary stress markers, gene expression changes in peripheral blood leukocytes and composition of the gut microbiota were analyzed using DNA microarray analysis and 16S rRNA gene amplicon sequence analysis, respectively, before and after the intervention. Stress-induced increases in a visual analog scale measuring feelings of stress, the total score of abdominal dysfunction, and the number of genes with changes in expression of more than 2-fold in leukocytes were significantly suppressed in the L. casei strain Shirota group compared with those in the placebo group. A significant increase in salivary cortisol levels before the examination was observed only in the placebo group. The administration of L. casei strain Shirota, but not placebo, significantly reduced gastrointestinal symptoms. Moreover, 16S rRNA gene amplicon sequencing demonstrated that the L. casei strain Shirota group had significantly higher numbers of species, a marker of the alpha-diversity index, in their gut microbiota and a significantly lower percentage of Bacteroidaceae than the placebo group. Our findings indicate that the daily consumption of probiotics, such as L. casei strain Shirota, preserves the diversity of the gut microbiota and may relieve stress-associated responses of abdominal dysfunction in healthy subjects exposed to stressful situations. IMPORTANCE A novel clinical trial was conducted with healthy medical students under examination stress conditions. It was demonstrated that the daily consumption of lactic acid bacteria provided health benefits to prevent the onset of stress-associated abdominal symptoms and a good change of gut microbiota in healthy medical students.

Chthonomonas calidirosea T49 T is a low-abundance, carbohydrate-scavenging, and thermophilic soil bacterium with a seemingly disorganized genome. We hypothesized that the C. calidirosea genome would be highly responsive to local selection pressure, resulting in the divergence of its genomic content, genome organization, and carbohydrate utilization phenotype across environments. We tested this hypothesis by sequencing the genomes of four C. calidirosea isolates obtained from four separate geothermal fields in the Taupō Volcanic Zone, New Zealand. For each isolation site, we measured physicochemical attributes and defined the associated microbial community by 16S rRNA gene sequencing. Despite their ecological and geographical isolation, the genome sequences showed low divergence (maximum, 1.17%). Isolate-specific variations included single-nucleotide polymorphisms (SNPs), restriction-modification systems, and mobile elements but few major deletions and no major rearrangements. The 50-fold variation in C. calidirosea relative abundance among the four sites correlated with site environmental characteristics but not with differences in genomic content. Conversely, the carbohydrate utilization profiles of the C. calidirosea isolates corresponded to the inferred isolate phylogenies, which only partially paralleled the geographical relationships among the sample sites. Genomic sequence conservation does not entirely parallel geographic distance, suggesting that stochastic dispersal and localized extinction, which allow for rapid population homogenization with little restriction by geographical barriers, are possible mechanisms of C. calidirosea distribution. This dispersal and extinction mechanism is likely not limited to C. calidirosea but may shape the populations and genomes of many other low-abundance free-living taxa. IMPORTANCE This study compares the genomic sequence variations and metabolisms of four strains of Chthonomonas calidirosea , a rare thermophilic bacterium from the phylum Armatimonadetes . It additionally compares the microbial communities and chemistry of each of the geographically distinct sites from which the four C. calidirosea strains were isolated. C. calidirosea was previously reported to possess a highly disorganized genome, but it was unclear whether this reflected rapid evolution. Here, we show that each isolation site has a distinct chemistry and microbial community, but despite this, the C. calidirosea genome is highly conserved across all isolation sites. Furthermore, genomic sequence differences only partially paralleled geographic distance, suggesting that C. calidirosea genotypes are not primarily determined by adaptive evolution. Instead, the presence of C. calidirosea may be driven by stochastic dispersal and localized extinction. This ecological mechanism may apply to many other low-abundance taxa.

The use of a multilocus sequence typing (MLST) technique has identified the intraspecific genetic diversity of U.S. Flavobacterium psychrophilum , an important pathogen of salmonids worldwide. Prior to this analysis, little U.S. F. psychrophilum genetic information was known; this is of importance when considering targeted control strategies, including vaccine development. Herein, MLST was used to investigate the genetic diversity of 96 F. psychrophilum isolates recovered from rainbow trout ( Oncorhynchus mykiss ), coho salmon ( Oncorhynchus kisutch ), and Chinook salmon ( Oncorhynchus tshawytscha ) that originated from nine U.S. states. The isolates fell into 34 distinct sequence types (STs) that clustered in 5 clonal complexes (CCs) ( n = 63) or were singletons ( n = 33). The distribution of STs varied spatially, by host species, and in association with mortality events. Several STs (i.e., ST9, ST10, ST30, and ST78) were found in multiple states, whereas the remaining STs were localized to single states. With the exception of ST256, which was recovered from rainbow trout and Chinook salmon, all STs were found to infect a single host species. Isolates that were collected during bacterial cold water disease outbreaks most frequently belonged to CC-ST10 (e.g., ST10 and ST78). Collectively, the results of this study clearly demonstrate the genetic diversity of F. psychrophilum within the United States and identify STs of clinical significance. Although the majority of STs described herein were novel, some (e.g., ST9, ST10, ST13, ST30, and ST31) were previously recovered on other continents, which demonstrates the transcontinental distribution of F. psychrophilum genotypes. IMPORTANCE Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS) and is an important bacterial pathogen of wild and farmed salmonids worldwide. These infections are responsible for large economic losses globally, yet the genetic diversity of this pathogen remains to be fully investigated. Previous studies have identified the genetic diversity of this pathogen in other main aquaculture regions; however, little effort has been focused on the United States. In this context, this study aims to examine the genetic diversity of F. psychrophilum from the United States, as this region remains important in salmonid aquaculture.

Sponges harbor a remarkable diversity of microbial symbionts in which signal molecules can accumulate and enable cell-cell communication, such as quorum sensing (QS). Bacteria capable of QS were isolated from marine sponges; however, an extremely small fraction of the sponge microbiome is amenable to cultivation. We took advantage of community genome assembly and binning to investigate the uncultured majority of sponge symbionts. We identified a complete N -acyl-homoserine lactone (AHL)-QS system (designated TswIR) and seven partial luxI homologues in the microbiome of Theonella swinhoei . The TswIR system was novel and shown to be associated with an alphaproteobacterium of the order Rhodobacterales , here termed Rhodobacterales bacterium TS309. The tswI gene, when expressed in Escherichia coli , produced three AHLs, two of which were also identified in a T. swinhoei sponge extract. The taxonomic affiliation of the 16S rRNA of Rhodobacterales bacterium TS309 to a sponge-coral specific clade, its enrichment in sponge versus seawater and marine sediment samples, and the presence of sponge-specific features, such as ankyrin-like domains and tetratricopeptide repeats, indicate a likely symbiotic nature of this bacterium.

Faecalibacterium prausnitzii depletion in intestinal diseases has been extensively reported, but little is known about intraspecies variability. This work aims to determine if subjects with gastrointestinal disease host mucosa-associated F. prausnitzii populations different from those hosted by healthy individuals. A new species-specific PCR-denaturing gradient gel electrophoresis (PCR-DGGE) method targeting the 16S rRNA gene was developed to fingerprint F. prausnitzii populations in biopsy specimens from 31 healthy control (H) subjects and 36 Crohn's disease (CD), 23 ulcerative colitis (UC), 6 irritable bowel syndrome (IBS), and 22 colorectal cancer (CRC) patients. The richness of F. prausnitzii subtypes was lower in inflammatory bowel disease (IBD) patients than in H subjects. The most prevalent operational taxonomic units (OTUs) consisted of four phylotypes (OTUs with a 99% 16S rRNA gene sequence similarity [OTU99]), which were shared by all groups of patients. Their distribution and the presence of some disease-specific F. prausnitzii phylotypes allowed us to differentiate the populations in IBD and CRC patients from that in H subjects. At the level of a minimum similarity of 97% (OTU97), two phylogroups accounted for 98% of the sequences. Phylogroup I was found in 87% of H subjects but in under 50% of IBD patients ( P = 0.003). In contrast, phylogroup II was detected in >75% of IBD patients and in only 52% of H subjects ( P = 0.005). This study reveals that even though the main members of the F. prausnitzii population are present in both H subjects and individuals with gut diseases, richness is reduced in the latter and an altered phylotype distribution exists between diseases. This approach may serve as a basis for addressing the suitability of F. prausnitzii phylotypes to be quantified as a putative biomarker of disease and depicting the importance of the loss of these subtypes in disease pathogenesis.

Most DNA-based microbial source tracking (MST) approaches target host-associated organisms within the order Bacteroidales , but the gut microbiota of humans and other animals contain organisms from an array of other taxonomic groups that might provide indicators of fecal pollution sources. To discern between human and nonhuman fecal sources, we compared the V6 regions of the 16S rRNA genes detected in fecal samples from six animal hosts to those found in sewage (as a proxy for humans). We focused on 10 abundant genera and used oligotyping, which can detect subtle differences between rRNA gene sequences from ecologically distinct organisms. Our analysis showed clear patterns of differential oligotype distributions between sewage and animal samples. Over 100 oligotypes of human origin occurred preferentially in sewage samples, and 99 human oligotypes were sewage specific. Sequences represented by the sewage-specific oligotypes can be used individually for development of PCR-based assays or together with the oligotypes preferentially associated with sewage to implement a signature-based approach. Analysis of sewage from Spain and Brazil showed that the sewage-specific oligotypes identified in U.S. sewage have the potential to be used as global alternative indicators of human fecal pollution. Environmental samples with evidence of prior human fecal contamination had consistent ratios of sewage signature oligotypes that corresponded to the trends observed for sewage. Our methodology represents a promising approach to identifying new bacterial taxa for MST applications and further highlights the potential of the family Lachnospiraceae to provide human-specific markers. In addition to source tracking applications, the patterns of the fine-scale population structure within fecal taxa suggest a fundamental relationship between bacteria and their hosts.

Symbiosis between insects and bacteria result in a variety of arrangements, genomic modifications, and metabolic interconnections. Here, we present genomic, phylogenetic, and morphological characteristics of a symbiotic system associated with Melophagus ovinus , a member of the blood-feeding family Hippoboscidae. The system comprises four unrelated bacteria representing different stages in symbiosis evolution, from typical obligate mutualists inhabiting bacteriomes to freely associated commensals and parasites. Interestingly, the whole system provides a remarkable analogy to the association between Glossina and its symbiotic bacteria. In both, the symbiotic systems are composed of an obligate symbiont and two facultative intracellular associates, Sodalis and Wolbachia . In addition, extracellular Bartonella resides in the gut of Melophagus . However, the phylogenetic origins of the two obligate mutualist symbionts differ. In Glossina , the mutualistic Wigglesworthia appears to be a relatively isolated symbiotic lineage, whereas in Melophagus , the obligate symbiont originated within the widely distributed Arsenophonus cluster. Although phylogenetically distant, the two obligate symbionts display several remarkably similar traits (e.g., transmission via the host's “milk glands” or similar pattern of genome reduction). To obtain better insight into the biology and possible role of the M. ovinus obligate symbiont, “ Candidatus Arsenophonus melophagi,” we performed several comparisons of its gene content based on assignments of the Cluster of Orthologous Genes (COG). Using this criterion, we show that within a set of 44 primary and secondary symbionts, “ Ca . Arsenophonus melophagi” is most similar to Wigglesworthia . On the other hand, these two bacteria also display interesting differences, such as absence of flagellar genes in Arsenophonus and their presence in Wigglesworthia . This finding implies that a flagellum is not essential for bacterial transmission via milk glands.

Bacterial endosymbionts have been identified as potentially useful biological control agents for a range of invertebrate vectors of disease. Previous studies of Culicoides (Diptera: Ceratopogonidae) species using conventional PCR assays have provided evidence of Wolbachia (1/33) and Cardinium (8/33) infections. Here, we screened 20 species of Culicoides for Wolbachia and Cardinium , utilizing a combination of conventional PCR and more sensitive quantitative PCR (qPCR) assays. Low levels of Cardinium DNA were detected in females of all but one of the Culicoides species screened, and low levels of Wolbachia were detected in females of 9 of the 20 Culicoides species. Sequence analysis based on partial 16S rRNA gene and gyrB sequences identified “ Candidatus Cardinium hertigii” from group C, which has previously been identified in Culicoides from Japan, Israel, and the United Kingdom. Wolbachia strains detected in this study showed 98 to 99% sequence identity to Wolbachia previously detected from Culicoides based on the 16S rRNA gene, whereas a strain with a novel wsp sequence was identified in Culicoides narrabeenensis. Cardinium isolates grouped to geographical regions independent of the host Culicoides species, suggesting possible geographical barriers to Cardinium movement. Screening also identified Asaia bacteria in Culicoides . These findings point to a diversity of low-level endosymbiont infections in Culicoides , providing candidates for further characterization and highlighting the widespread occurrence of these endosymbionts in this insect group.

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of bloody diarrhea and extraintestinal sequelae in humans, most importantly hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Besides the bacteriophage-encoded Shiga toxin gene ( stx ), EHEC harbors the locus of enterocyte effacement (LEE), which confers the ability to cause attaching and effacing lesions. Currently, the vast majority of EHEC infections are caused by strains belonging to five O serogroups (the “big five”), which, in addition to O157, the most important, comprise O26, O103, O111, and O145. We hypothesize that these four non-O157 EHEC serotypes differ in their phylogenies. To test this hypothesis, we used multilocus sequence typing (MLST) to analyze a large collection of 250 isolates of these four O serogroups, which were isolated from diseased as well as healthy humans and cattle between 1952 and 2009. The majority of the EHEC isolates of O serogroups O26 and O111 clustered into one sequence type complex, STC29. Isolates of O103 clustered mainly in STC20, and most isolates of O145 were found within STC32. In addition to these EHEC strains, STC29 also included stx -negative E. coli strains, termed atypical enteropathogenic E. coli (aEPEC), yet another intestinal pathogenic E. coli group. The finding that aEPEC and EHEC isolates of non-O157 O serogroups share the same phylogeny suggests an ongoing microevolutionary scenario in which the phage-encoded Shiga toxin gene stx is transferred between aEPEC and EHEC. As a consequence, aEPEC strains of STC29 can be regarded as post- or pre-EHEC isolates. Therefore, STC29 incorporates phylogenetic information useful for unraveling the evolution of EHEC.

Anaerobic oxidation of methane (AOM) coupled to nitrite reduction is a novel AOM process that is mediated by denitrifying methanotrophs. To date, enrichments of these denitrifying methanotrophs have been confined to freshwater systems; however, the recent findings of 16S rRNA and pmoA gene sequences in marine sediments suggest a possible occurrence of AOM coupled to nitrite reduction in marine systems. In this research, a marine denitrifying methanotrophic culture was obtained after 20 months of enrichment. Activity testing and quantitative PCR (qPCR) analysis were then conducted and showed that the methane oxidation activity and the number of NC10 bacteria increased correlatively during the enrichment period. 16S rRNA gene sequencing indicated that only bacteria in group A of the NC10 phylum were enriched and responsible for the resulting methane oxidation activity, although a diverse community of NC10 bacteria was harbored in the inoculum. Fluorescence in situ hybridization showed that NC10 bacteria were dominant in the enrichment culture after 20 months. The effect of salinity on the marine denitrifying methanotrophic culture was investigated, and the apparent optimal salinity was 20.5‰, which suggested that halophilic bacterial AOM coupled to nitrite reduction was obtained. Moreover, the apparent substrate affinity coefficients of the halophilic denitrifying methanotrophs were determined to be 9.8 ± 2.2 μM for methane and 8.7 ± 1.5 μM for nitrite.

Two glucoamylase-like genes, TVN1315 and Ta0286 , from the archaea Thermoplasma volcanium and T. acidophilum , respectively, were expressed in Escherichia coli . The gene products, TVN1315 and Ta0286, were identified as archaeal trehalases. These trehalases belong to the CAZy database family GH15, although they have putative (α/α) 6 barrel catalytic domain structures similar to those of GH37 and GH65 family trehalases from other organisms. These newly identified trehalases function within a narrow range of acidic pH values (pH 3.2 to 4.0) and at high temperatures (50 to 60°C), and these enzymes display K m values for trehalose higher than those observed for typical trehalases. These enzymes were inhibited by validamycin A; however, the inhibition constants ( K i ) were higher than those of other trehalases. Three TVN1315 mutants, corresponding to E408Q, E571Q, and E408Q/E571Q mutations, showed reduced activity, suggesting that these two glutamic acid residues are involved in trehalase catalysis in a manner similar to that of glucoamylase. To date, TVN1315 and Ta0286 are the first archaeal trehalases to be identified, and this is the first report of the heterologous expression of GH15 family trehalases. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 family enzymes as well as glycoside hydrolase family enzymes; additionally, these enzymes provide insight into archaeal trehalose metabolism.

Riemerella anatipestifer is a well-described pathogen of waterfowl and other avian species that can cause septicemic and exudative diseases. In this study, we sequenced the complete genome of R. anatipestifer strain Yb2 and analyzed it against the published genomic sequences of R. anatipestifer strains DSM15868, RA-GD, RA-CH-1, and RA-CH-2. The Yb2 genome contains one circular chromosome of 2,184,066 bp with a 35.73% GC content and no plasmid. The genome has 2,021 open reading frames that occupy 90.88% of the genome. A comparative genomic analysis revealed that genome organization is highly conserved among R. anatipestifer strains, except for four inversions of a sequence segment in Yb2. A phylogenetic analysis found that the closest neighbor of Yb2 is RA-GD. Furthermore, we constructed a library of 3,175 mutants by random transposon mutagenesis, and 100 mutants exhibiting more than 100-fold-attenuated virulence were obtained by animal screening experiments. Southern blot analysis and genetic characterization of the mutants led to the identification of 49 virulence genes. Of these, 25 encode cytoplasmic proteins, 6 encode cytoplasmic membrane proteins, 4 encode outer membrane proteins, and the subcellular localization of the remaining 14 gene products is unknown. The functional classification of orthologous-group clusters revealed that 16 genes are associated with metabolism, 6 are associated with cellular processing and signaling, and 4 are associated with information storage and processing. The functions of the other 23 genes are poorly characterized or unknown. This genome-wide study identified genes important to the virulence of R. anatipestifer .

Vanilla beans were analyzed using biochemical methods, which revealed that glucovanillin disperses from the inner part to the outer part of the vanilla bean during the curing process and is simultaneously hydrolyzed by β- d -glucosidase. Enzymatic hydrolysis was found to occur on the surface of the vanilla beans. Transcripts of the β- d -glucosidase gene ( bgl ) of colonizing microorganisms were detected. The results directly indicate that colonizing microorganisms are involved in glucovanillin hydrolysis. Phylogenetic analysis based on 16S rRNA gene sequences showed that the colonizing microorganisms mainly belonged to the Bacillus genus. bgl was detected in all the isolates and presented clustering similar to that of the isolate taxonomy. Furthermore, inoculation of green fluorescent protein-tagged isolates showed that the Bacillus isolates can colonize vanilla beans. Glucovanillin was metabolized as the sole source of carbon in a culture of the isolates within 24 h. These isolates presented unique glucovanillin degradation capabilities. Vanillin was the major volatile compound in the culture. Other compounds, such as α-cubebene, β-pinene, and guaiacol, were detected in some isolate cultures. Colonizing Bacillus isolates were found to hydrolyze glucovanillin in culture, indirectly demonstrating the involvement of colonizing Bacillus isolates in glucovanillin hydrolysis during the vanilla curing process. Based on these results, we conclude that colonizing Bacillus isolates produce β- d -glucosidase, which mediates glucovanillin hydrolysis and influences flavor formation.

Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that may be responsible for severe human infections. Only a limited number of serotypes, including O26:H11, are involved in the majority of serious cases and outbreaks. The main virulence factors, Shiga toxins (Stx), are encoded by bacteriophages. Seventy-four STEC O26:H11 strains of various origins (including human, dairy, and cattle) were characterized for their stx subtypes and Stx phage chromosomal insertion sites. The majority of food and cattle strains possessed the stx 1a subtype, while human strains carried mainly stx 1a or stx 2a . The wrbA and yehV genes were the main Stx phage insertion sites in STEC O26:H11, followed distantly by yecE and sbcB . Interestingly, the occurrence of Stx phages inserted in the yecE gene was low in dairy strains. In most of the 29 stx -negative E. coli O26:H11 strains also studied here, these bacterial insertion sites were vacant. Multilocus sequence typing of 20 stx -positive or stx -negative E. coli O26:H11 strains showed that they were distributed into two phylogenetic groups defined by sequence type 21 (ST21) and ST29. Finally, an EspK-carrying phage was found inserted in the ssrA gene in the majority of the STEC O26:H11 strains but in only a minority of the stx -negative E. coli O26:H11 strains. The differences in the stx subtypes and Stx phage insertion sites observed in STEC O26:H11 according to their origin might reflect that strains circulating in cattle and foods are clonally distinct from those isolated from human patients.

Large sulfur-oxidizing bacteria in the family Beggiatoaceae are important players in the global sulfur cycle. This group contains members of the well-known genera Beggiatoa , Thioploca , and Thiomargarita but also recently identified and relatively unknown candidate taxa, including “ Candidatus Thiopilula” spp. and “ Ca . Thiophysa” spp. We discovered a population of “ Ca . Thiopilula” spp. colonizing cold seeps near Barbados at a ∼4.7-km water depth. The Barbados population consists of spherical cells that are morphologically similar to Thiomargarita spp., with elemental sulfur inclusions and a central vacuole, but have much smaller cell diameters (5 to 40 μm). Metatranscriptomic analysis revealed that when exposed to anoxic sulfidic conditions, Barbados “ Ca . Thiopilula” organisms expressed genes for the oxidation of elemental sulfur and the reduction of nitrogenous compounds, consistent with their vacuolated morphology and intracellular sulfur storage capability. Metatranscriptomic analysis further revealed that anaerobic methane-oxidizing and sulfate-reducing organisms were active in the sediment, which likely provided reduced sulfur substrates for “ Ca . Thiopilula” and other sulfur-oxidizing microorganisms in the community. The novel observations of “ Ca . Thiopilula” and associated organisms reported here expand our knowledge of the globally distributed and ecologically successful Beggiatoaceae group and thus offer insight into the composition and ecology of deep cold seep microbial communities.

Red soils, which are widely distributed in tropical and subtropical regions of southern China, are characterized by low organic carbon, high content of iron oxides, and acidity and, hence, are likely to be ideal habitats for acidophilic actinomycetes. However, the diversity and biosynthetic potential of actinomycetes in such habitats are underexplored. Here, a total of 600 actinomycete strains were isolated from red soils collected in Jiangxi Province in southeast China. 16S rRNA gene sequence analysis revealed a high diversity of the isolates, which were distributed into 26 genera, 10 families, and 7 orders within the class Actinobacteria ; these taxa contained at least 49 phylotypes that are likely to represent new species within 15 genera. The isolates showed good physiological potentials for biosynthesis and biocontrol. Chemical screening of 107 semirandomly selected isolates spanning 20 genera revealed the presence of at least 193 secondary metabolites from 52 isolates, of which 125 compounds from 39 isolates of 12 genera were putatively novel. Macrolides, polyethers, diketopiperazines, and siderophores accounted for most of the known compounds. The structures of six novel compounds were elucidated, two of which had a unique skeleton and represented characteristic secondary metabolites of a putative novel Streptomyces phylotype. These results demonstrate that red soils are rich reservoirs for diverse culturable actinomycetes, notably members of the families Streptomycetaceae , Pseudonocardiaceae , and Streptosporangiaceae , with the capacity to synthesize novel bioactive compounds.

With an aim to develop a highly efficient method for the recovery of rare earth elements (REEs) by using microorganisms, we attempted to isolate dysprosium (Dy)-accumulating microorganisms that grow under acidic conditions from environmental samples containing high concentrations of heavy metals. One acidophilic strain, T9, which was isolated from an abandoned mine, decreased the concentration of Dy in medium that contained 100 mg/liter Dy to 53 mg/liter Dy after 3 days of cultivation at pH 2.5. The Dy content in the cell pellet of the T9 strain was 910 μg/mg of dry cells. The T9 strain also accumulated other REEs. Based on the results of 28S-D1/D2 rRNA gene sequencing and morphological characterization, we designated this fungal strain Penidiella sp. T9. Bioaccumulation of Dy was observed on the cell surface of the T9 strain by elemental mapping using scanning electron microscopy-energy dispersive X-ray spectroscopy. Our results indicate that Penidiella sp. T9 has the potential to recover REEs such as Dy from mine drainage and industrial liquid waste under acidic conditions.

The traditional view of the dependency of subsurface environments on surface-derived allochthonous carbon inputs is challenged by increasing evidence for the role of lithoautotrophy in aquifer carbon flow. We linked information on autotrophy (Calvin-Benson-Bassham cycle) with that from total microbial community analysis in groundwater at two superimposed—upper and lower—limestone groundwater reservoirs (aquifers). Quantitative PCR revealed that up to 17% of the microbial population had the genetic potential to fix CO 2 via the Calvin cycle, with abundances of cbbM and cbbL genes, encoding RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) forms I and II, ranging from 1.14 × 10 3 to 6 × 10 6 genes liter −1 over a 2-year period. The structure of the active microbial communities based on 16S rRNA transcripts differed between the two aquifers, with a larger fraction of heterotrophic, facultative anaerobic, soil-related groups in the oxygen-deficient upper aquifer. Most identified CO 2 -assimilating phylogenetic groups appeared to be involved in the oxidation of sulfur or nitrogen compounds and harbored both RubisCO forms I and II, allowing efficient CO 2 fixation in environments with strong oxygen and CO 2 fluctuations. The genera Sulfuricella and Nitrosomonas were represented by read fractions of up to 78 and 33%, respectively, within the cbbM and cbbL transcript pool and accounted for 5.6 and 3.8% of 16S rRNA sequence reads, respectively, in the lower aquifer. Our results indicate that a large fraction of bacteria in pristine limestone aquifers has the genetic potential for autotrophic CO 2 fixation, with energy most likely provided by the oxidation of reduced sulfur and nitrogen compounds.

Botrytis cinerea is a pathogenic fungus causing gray mold on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea . The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A −1 frameshifting region (designated the KNOT element) containing a shifty heptamer, a heptanucleotide spacer, and an H-type pseudoknot was predicted in the junction region of ORF1 and ORF2. The −1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein (RFP)-green fluorescent protein (GFP) by the plasmid containing the construct dsRed- KNOT- eGFP in Escherichia coli . BcRV1 belongs to a taxonomically unassigned double-stranded RNA (dsRNA) mycovirus group. It is closely related to grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in strain BerBc-1 was found capable of being transmitted vertically through macroconidia and horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was found to be positively correlated with hypovirulence in B. cinerea , with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1.

Many stinkbugs (Insecta: Hemiptera: Heteroptera) are associated with bacterial symbionts in a posterior region of the midgut. In these stinkbugs, adult females excrete symbiont-containing materials from the anus for transmission of the beneficial symbionts to their offspring. For ensuring the vertical symbiont transmission, a variety of female-specific elaborate traits at the cellular, morphological, developmental, and behavioral levels have been reported from diverse stinkbugs of the families Plataspidae, Urostylididae, Parastrachiidae, etc. Meanwhile, such elaborate female-specific traits for vertical symbiont transmission have been poorly characterized for the largest and economically important stinkbug family Pentatomidae. Here, we investigated the midgut symbiotic system of a pentatomid stinkbug, Plautia splendens . A specific gammaproteobacterial symbiont was consistently present extracellularly in the cavity of numerous crypts arranged in four rows on the midgut fourth section. The symbiont was smeared on the egg surface upon oviposition by adult females, orally acquired by newborn nymphs, and thereby transmitted vertically to the next generation and important for growth and survival of the host insects. We found that, specifically in adult females, several rows of crypts at the posterior end region of the symbiotic midgut were morphologically differentiated and conspicuously enlarged, often discharging the symbiotic bacteria from the crypt cavity to the main tract of the symbiotic midgut. The female-specific enlarged end crypts were also found in other pentatomid stinkbugs Plautia stali and Carbula crassiventris . These results suggest that the enlarged end crypts represent a female-specific specialized morphological trait for vertical symbiont transmission commonly found among stinkbugs of the family Pentatomidae.

Bacillus cereus is an opportunistic human pathogen responsible for food poisoning and other, nongastrointestinal infections. Due to the emergence of multidrug-resistant B. cereus strains, the demand for alternative therapeutic options is increasing. To address these problems, we isolated and characterized a Siphoviridae virulent phage, PBC1, and its lytic enzymes. PBC1 showed a very narrow host range, infecting only 1 of 22 B. cereus strains. Phylogenetic analysis based on the major capsid protein revealed that PBC1 is more closely related to the Bacillus clarkii phage BCJA1c and phages of lactic acid bacteria than to the phages infecting B. cereus . Whole-genome comparison showed that the late-gene region, including the terminase gene, structural genes, and holin gene of PBC1, is similar to that from B. cereus temperate phage 250, whereas their endolysins are different. Compared to the extreme host specificity of PBC1, its endolysin, LysPBC1, showed a much broader lytic spectrum, albeit limited to the genus Bacillus . The catalytic domain of LysPBC1 when expressed alone also showed Bacillus -specific lytic activity, which was lower against the B. cereus group but higher against the Bacillus subtilis group than the full-length protein. Taken together, these results suggest that the virulent phage PBC1 is a useful component of a phage cocktail to control B. cereus , even with its exceptionally narrow host range, as it can kill a strain of B. cereus that is not killed by other phages, and that LysPBC1 is an alternative biocontrol agent against B. cereus .

Pseudomonas fluorescensis commonly considered a saprophytic rhizobacterium devoid of pathogenic potential. Nevertheless, the recurrent isolation of strains from clinical human cases could indicate the emergence of novel strains originating from the rhizosphere reservoir, which could be particularly resistant to the immune system and clinical treatment. The importance of type three secretion systems (T3SSs) in the relatedPseudomonas aeruginosanosocomial species and the occurrence of this secretion system in plant-associatedP. fluorescensraise the question of whether clinical isolates may also harbor T3SSs. In this study, isolates associated with clinical infections and identified in hospitals as belonging toP. fluorescenswere compared with fluorescent pseudomonads harboring T3SSs isolated from plants. Bacterial isolates were tested for (i) their genetic relationships based on their 16S rRNA phylogeny, (ii) the presence of T3SS genes by PCR, and (iii) their infectious potential on animals and plants under environmental or physiological temperature conditions. Two groups of bacteria were delineated among the clinical isolates. The first group encompassed thermotolerant (41°C) isolates from patients suffering from blood infections; these isolates were finally found to not belong toP. fluorescensbut were closely related and harbored highly conserved T3SS genes belonging to the Ysc-T3SS family, like the T3SSs fromP. aeruginosa. The second group encompassed isolates from patients suffering from cystic fibrosis; these isolates belonged toP. fluorescensand harbored T3SS genes belonging to the Hrp1-T3SS family found commonly in plant-associatedP. fluorescens.

An aseptic meningitis outbreak occurred in Luoding City of Guangdong, China, in 2012, and echovirus type 30 (ECHO30) was identified as the major causative pathogen. Environmental surveillance indicated that ECHO30 was detected in the sewage of a neighboring city, Guangzhou, from 2010 to 2012 and also in Luoding City sewage samples (6/43, 14%) collected after the outbreak. In order to track the potential origin of the outbreak viral strains, we sequenced the VP1 genes of 29 viral strains from clinical patients and environmental samples. Sequence alignments and phylogenetic analyses based on VP1 gene sequences revealed that virus strains isolated from the sewage of Guangzhou and Luoding cities matched well the clinical strains from the outbreak, with high nucleotide sequence similarity (98.5% to 100%) and similar cluster distribution. Five ECHO30 clinical strains were clustered with the Guangdong environmental strains but diverged from strains from other regions, suggesting that this subcluster of viruses most likely originated from the circulating virus in Guangdong rather than having been more recently imported from other regions. These findings underscore the importance of long-term, continuous environmental surveillance and genetic analysis to monitor circulating enteroviruses.

Lake Vanda is a perennially ice-covered and stratified lake in the McMurdo Dry Valleys, Antarctica. The lake develops a distinct chemocline at about a 50-m depth, where the waters transition from cool, oxic, and fresh to warm, sulfidic, and hypersaline. The bottom water brine is unique, as the highly chaotropic salts CaCl 2 and MgCl 2 predominate, and CaCl 2 levels are the highest of those in any known microbial habitat. Enrichment techniques were used to isolate 15 strains of heterotrophic bacteria from the Lake Vanda brine. Despite direct supplementation of the brine samples with different organic substrates in primary enrichments, the same organism, a relative of the halophilic bacterium Halomonas ( Gammaproteobacteria ), was isolated from all depths sampled. The Lake Vanda (VAN) strains were obligate aerobes and showed broad pH, salinity, and temperature ranges for growth, consistent with the physicochemical properties of the brine. VAN strains were halophilic and quite CaCl 2 tolerant but did not require CaCl 2 for growth. The fact that only VAN strain-like organisms appeared in our enrichments hints that the highly chaotropic nature of the Lake Vanda brine may place unusual physiological constraints on the bacterial community that inhabits it.

In ecological studies, microbial diversity is nowadays mostly assessed via the detection of phylogenetic marker genes, such as 16S rRNA. However, PCR amplification of these marker genes produces a significant amount of artificial sequences, often referred to as chimeras. Different algorithms have been developed to remove these chimeras, but efforts to combine different methodologies are limited. Therefore, two machine learning classifiers (reference-based and de novo CATCh) were developed by integrating the output of existing chimera detection tools into a new, more powerful method. When comparing our classifiers with existing tools in either the reference-based or de novo mode, a higher performance of our ensemble method was observed on a wide range of sequencing data, including simulated, 454 pyrosequencing, and Illumina MiSeq data sets. Since our algorithm combines the advantages of different individual chimera detection tools, our approach produces more robust results when challenged with chimeric sequences having a low parent divergence, short length of the chimeric range, and various numbers of parents. Additionally, it could be shown that integrating CATCh in the preprocessing pipeline has a beneficial effect on the quality of the clustering in operational taxonomic units.

Microbiologically influenced corrosion (MIC) of metallic materials imposes a heavy economic burden. The mechanism of MIC of metallic iron (Fe 0 ) under anaerobic conditions is usually explained as the consumption of cathodic hydrogen by hydrogenotrophic microorganisms that accelerates anodic Fe 0 oxidation. In this study, we describe Fe 0 corrosion induced by a nonhydrogenotrophic nitrate-reducing bacterium called MIC1-1, which was isolated from a crude-oil sample collected at an oil well in Akita, Japan. This strain requires specific electron donor-acceptor combinations and an organic carbon source to grow. For example, the strain grew anaerobically on nitrate as a sole electron acceptor with pyruvate as a carbon source and Fe 0 as the sole electron donor. In addition, ferrous ion and l -cysteine served as electron donors, whereas molecular hydrogen did not. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MIC1-1 was a member of the genus Prolixibacter in the order Bacteroidales . Thus, Prolixibacter sp. strain MIC1-1 is the first Fe 0 -corroding representative belonging to the phylum Bacteroidetes . Under anaerobic conditions, Prolixibacter sp. MIC1-1 corroded Fe 0 concomitantly with nitrate reduction, and the amount of iron dissolved by the strain was six times higher than that in an aseptic control. Scanning electron microscopy analyses revealed that microscopic crystals of FePO 4 developed on the surface of the Fe 0 foils, and a layer of FeCO 3 covered the FePO 4 crystals. We propose that cells of Prolixibacter sp. MIC1-1 accept electrons directly from Fe 0 to reduce nitrate.

Large spatial scales and long-term shifts of bacterial community composition (BCC) in the open ocean can often be reliably predicted based on the dynamics of physical-chemical variables. The power of abiotic factors in shaping BCC on shorter time scales in shallow estuarine mixing zones is less clear. We examined the diurnal variation in BCC at different water depths in the spring and fall of 2011 at a station in the Gray's Reef National Marine Sanctuary (GRNMS). This site is located in the transition zone between the estuarine plume and continental shelf waters of the South Atlantic Bight. A total of 234,516 pyrotag sequences of bacterial 16S rRNA genes were recovered; they were taxonomically affiliated with >200 families of 23 bacterial phyla. Nonmetric multidimensional scaling analysis revealed significant differences in BCC between spring and fall samples, likely due to seasonality in the concentrations of dissolved organic carbon and nitrate plus nitrite. Within each diurnal sampling, BCC differed significantly by depth only in the spring and differed significantly between day and night only in the fall. The former variation largely tracked changes in light availability, while the latter was most correlated with concentrations of polyamines and chlorophyll a . Our results suggest that at the GRNMS, a coastal mixing zone, diurnal variation in BCC is attributable to the mixing of local and imported bacterioplankton rather than to bacterial growth in response to environmental changes. Our results also indicate that, like members of the Roseobacter clade, SAR11 bacteria may play an important role in processing dissolved organic material in coastal oceans.

Methanogenic archaea play a key role in biogas-producing anaerobic digestion and yet remain poorly taxonomically characterized. This is in part due to the limitations of low-throughput Sanger sequencing of a single (16S rRNA) gene, which in the past may have undersampled methanogen diversity. In this study, archaeal communities from three sludge digesters in Hong Kong and one wastewater digester in China were examined using high-throughput pyrosequencing of the methyl coenzyme M reductase ( mcrA ) and 16S rRNA genes. Methanobacteriales , Methanomicrobiales , and Methanosarcinales were detected in each digester, indicating that both hydrogenotrophic and acetoclastic methanogenesis was occurring. Two sludge digesters had similar community structures, likely due to their similar design and feedstock. Taxonomic classification of the mcrA genes suggested that these digesters were dominated by acetoclastic methanogens, particularly Methanosarcinales , while the other digesters were dominated by hydrogenotrophic Methanomicrobiales . The proposed euryarchaeotal order Methanomassiliicoccales and the uncultured WSA2 group were detected with the 16S rRNA gene, and potential mcrA genes for these groups were identified. 16S rRNA gene sequencing also recovered several crenarchaeotal groups potentially involved in the initial anaerobic digestion processes. Overall, the two genes produced different taxonomic profiles for the digesters, while greater methanogen richness was detected using the mcrA gene, supporting the use of this functional gene as a complement to the 16S rRNA gene to better assess methanogen diversity. A significant positive correlation was detected between methane production and the abundance of mcrA transcripts in digesters treating sludge and wastewater samples, supporting the mcrA gene as a biomarker for methane yield.