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We conducted a global characterization of the microbial communities of shipping ports to serve as a novel system to investigate microbial biogeography. The community structures of port microbes from marine and freshwater habitats house relatively similar phyla, despite spanning large spatial scales. As part of this project, we collected 1,218 surface water samples from 604 locations across eight countries and three continents to catalogue a total of 20 shipping ports distributed across the East and West Coast of the United States, Europe, and Asia to represent the largest study of port-associated microbial communities to date. Here, we demonstrated the utility of machine learning to leverage this robust system to characterize microbial biogeography by identifying trends in biodiversity across broad spatial scales. We found that for geographic locations sharing similar environmental conditions, subpopulations from the dominant phyla of these habitats ( Actinobacteria , Bacteroidetes , Cyanobacteria , and Proteobacteria ) can be used to differentiate 20 geographic locations distributed globally. These results suggest that despite the overwhelming diversity within microbial communities, members of the most abundant and ubiquitous microbial groups in the system can be used to differentiate a geospatial location across global spatial scales. Our study provides insight into how microbes are dispersed spatially and robust methods whereby we can interrogate microbial biogeography. IMPORTANCE Microbes are ubiquitous throughout the world and are highly diverse. Characterizing the extent of variation in the microbial diversity across large geographic spatial scales is a challenge yet can reveal a lot about what biogeography can tell us about microbial populations and their behavior. Machine learning approaches have been used mostly to examine the human microbiome and, to some extent, microbial communities from the environment. Here, we display how supervised machine learning approaches can be useful to understand microbial biodiversity and biogeography using microbes from globally distributed shipping ports. Our findings indicate that the members of globally dominant phyla are important for differentiating locations, which reduces the reliance on rare taxa to probe geography. Further, this study displays how global biogeographic patterning of aquatic microbial communities (and other systems) can be assessed through populations of the highly abundant and ubiquitous taxa that dominant the system., (Copyright © 2020 Ghannam et al.)

As important players in the host defense system, commensal microbes and the microbiota influence multiple aspects of host physiology. Bordetella pertussis infection is highly contagious among humans. However, the roles of the microbiota in B. pertussis pathogenesis are poorly understood. Here, we show that antibiotic-mediated depletion of the microbiota results in increased susceptibility to B. pertussis infection during the early stage. The increased susceptibility was associated with a marked impairment of the systemic IgG, IgG2a, and IgG1 antibody responses to B. pertussis infection after antibiotic treatment. Furthermore, the microbiota impacted the short-lived plasma cell responses as well as the recall responses of memory B cells to B. pertussis infection. Finally, we found that the dysbiosis caused by antibiotic treatment affects CD4 + T cell generation and PD-1 expression on CD4 + T cells and thereby perturbs plasma cell differentiation. Our results have revealed the importance of commensal microbes in modulating host immune responses to B. pertussis infection and support the possibility of controlling the severity of B. pertussis infection in humans by manipulating the microbiota., (Copyright © 2019 Zhang et al.)

Slow sand filtration with extensive pretreatment reduces the microbial growth potential of drinking water to a minimum level at four surface water supplies in The Netherlands. The potential of these slow sand filtrates (SSFs) to promote microbial growth in warm tap water installations was assessed by measuring biofilm formation and growth of Legionella bacteria on glass and chlorinated polyvinylchloride (CPVC) surfaces exposed to SSFs at 37 ± 2°C in a model system for up to six months. The steady-state biofilm concentration ranged from 230 to 3,980 pg ATP cm -2 on glass and 1.4 (±0.3)-times-higher levels on CPVC. These concentrations correlated significantly with the assimilable organic carbon (AOC) concentrations of the warm water (8 to 24 µg acetate-C equivalents [ac-C eq] liter -1 ), which were raised about 2 times by mixing cold and heated (70°C) SSFs. All biofilms supported growth of Legionella pneumophila with maximum concentrations ranging from 6 × 10 2 to 1.5 × 10 5 CFU cm -2 Biofilms after ≤50 days of exposure were predominated by Betaproteobacteriales , mainly Piscinibacter , Caldimonas , Methyloversatilis , and an uncultured Rhodocyclaceae bacterium. These rapidly growing primary colonizers most likely served as prey for the host amoebae of L. pneumophila Alphaproteobacteria , mostly Xanthobacteraceae , e.g., Bradyrhizobium , Pseudorhodoplanes , and other amoeba-resistant bacteria, accounted for 37.5% of the clones retrieved. A conceptual model based on a quadratic relationship between the L. pneumophila colony count and the biofilm concentration under steady-state conditions is used to explain the variations in the Legionella CFU pg -1 ATP ratios in the biofilms. IMPORTANCE Proliferation of L. pneumophila in premise plumbing poses a public health threat. Extended water treatment using physicochemical and biofiltration processes, including slow sand filtration, at four surface water supplies in The Netherlands reduces the microbial growth potential of the treated water to a minimum level, and the distributed drinking water complies with high quality standards. However, heating of the water in warm tap water installations increases the concentration of easily assimilable organic compounds, thereby promoting biofilm formation and growth of L. pneumophila Prevention of biofilm formation in plumbing systems by maintenance of a disinfectant residual during distribution and/or further natural organic matter (NOM) removal is not feasible in the supplies studied. Temperature management in combination with optimized hydraulics and material selection are therefore essential to prevent growth of L. pneumophila in premise plumbing systems. Still, reducing the concentration of biodegradable compounds in drinking water by appropriate water treatment is important for limiting the Legionella growth potential., (Copyright © 2018 American Society for Microbiology.)

Correlating the presence of bacteria and the genes they carry with aspects of plant and animal biology is rapidly outpacing the functional characterization of naturally occurring symbioses. A major barrier to mechanistic studies is the lack of tools for the efficient genetic manipulation of wild and diverse bacterial isolates. To address the need for improved molecular tools, we used a collection of proteobacterial isolates native to the zebrafish intestinal microbiota as a testbed to construct a series of modernized vectors that expedite genetic knock-in and knockout procedures across lineages. The innovations that we introduce enhance the flexibility of conventional genetic techniques, making it easier to manipulate many different bacterial isolates with a single set of tools. We developed alternative strategies for domestication-free conjugation, designed plasmids with customizable features, and streamlined allelic exchange using visual markers of homologous recombination. We demonstrate the potential of these tools through a comparative study of bacterial behavior within the zebrafish intestine. Live imaging of fluorescently tagged isolates revealed a spectrum of distinct population structures that differ in their biogeography and dominant growth mode (i.e., planktonic versus aggregated). Most striking, we observed divergent genotype-phenotype relationships: several isolates that are predicted by genomic analysis and in vitro assays to be capable of flagellar motility do not display this trait within living hosts. Together, the tools generated in this work provide a new resource for the functional characterization of wild and diverse bacterial lineages that will help speed the research pipeline from sequencing-based correlations to mechanistic underpinnings. IMPORTANCE A great challenge in microbiota research is the immense diversity of symbiotic bacteria with the capacity to impact the lives of plants and animals. Moving beyond correlative DNA sequencing-based studies to define the cellular and molecular mechanisms by which symbiotic bacteria influence the biology of their hosts is stalling because genetic manipulation of new and uncharacterized bacterial isolates remains slow and difficult with current genetic tools. Moreover, developing tools de novo is an arduous and time-consuming task and thus represents a significant barrier to progress. To address this problem, we developed a suite of engineering vectors that streamline conventional genetic techniques by improving postconjugation counterselection, modularity, and allelic exchange. Our modernized tools and step-by-step protocols will empower researchers to investigate the inner workings of both established and newly emerging models of bacterial symbiosis., (Copyright © 2018 Wiles et al.)

Several studies have shown that rainfall seasonality, soil heterogeneity, and increased nitrogen (N) deposition may have important effects on tropical forest function. However, the effects of these environmental controls on soil microbial communities in seasonally dry tropical forests are poorly understood. In a seasonally dry tropical forest in the Yucatan Peninsula (Mexico), we investigated the influence of soil heterogeneity (which results in two different soil types, black and red soils), rainfall seasonality (in two successive seasons, wet and dry), and 3 years of repeated N enrichment on soil chemical and microbiological properties, including bacterial gene content and community structure. The soil properties varied with the soil type and the sampling season but did not respond to N enrichment. Greater organic matter content in the black soils was associated with higher microbial biomass, enzyme activities, and abundances of genes related to nitrification ( amoA ) and denitrification ( nirK and nirS ) than were observed in the red soils. Rainfall seasonality was also associated with changes in soil microbial biomass and activity levels and N gene abundances. Actinobacteria , Proteobacteria , Firmicutes , and Acidobacteria were the most abundant phyla. Differences in bacterial community composition were associated with soil type and season and were primarily detected at higher taxonomic resolution, where specific taxa drive the separation of communities between soils. We observed that soil heterogeneity and rainfall seasonality were the main correlates of soil bacterial community structure and function in this tropical forest, likely acting through their effects on soil attributes, especially those related to soil organic matter and moisture content. IMPORTANCE Understanding the response of soil microbial communities to environmental factors is important for predicting the contribution of forest ecosystems to global environmental change. Seasonally dry tropical forests are characterized by receiving less than 1,800 mm of rain per year in alternating wet and dry seasons and by high heterogeneity in plant diversity and soil chemistry. For these reasons, N deposition may affect their soils differently than those in humid tropical forests. This study documents the influence of rainfall seasonality, soil heterogeneity, and N deposition on soil chemical and microbiological properties in a seasonally dry tropical forest. Our findings suggest that soil heterogeneity and rainfall seasonality are likely the main factors controlling soil bacterial community structure and function in this tropical forest. Nitrogen enrichment was likely too low to induce significant short-term effects on soil properties, because this tropical forest is not N limited., (Copyright © 2018 American Society for Microbiology.)

Microaerophilic Fe(II) oxidizers are commonly found in habitats containing elevated Fe(II) and low O 2 concentrations and often produce characteristic Fe mineral structures, so-called twisted stalks or tubular sheaths. Isolates originating from freshwater habitats are all members of the Betaproteobacteria , while isolates from marine habitats belong almost exclusively to the Zetaproteobacteria So far, only a few isolates of marine microaerophilic Fe(II) oxidizers have been described, all of which are obligate microaerophilic Fe(II) oxidizers and have been thought to be restricted to Fe-rich systems. Here, we present two new isolates of marine microaerophilic Fe(II)-oxidizing Zetaproteobacteria that originate from typical coastal marine sediments containing only low Fe concentrations (2 to 11 mg of total Fe/g of sediment [dry weight]; 70 to 100 μM dissolved Fe 2+ in the porewater). The two novel Zetaproteobacteria share characteristic physiological properties of the Zetaproteobacteria group, even though they come from low-Fe environments: the isolates are obligate microaerophilic Fe(II) oxidizers and, like most isolated Zetaproteobacteria , they produce twisted stalks. We found a low organic carbon content in the stalks (∼0.3 wt%), with mostly polysaccharides and saturated aliphatic chains (most likely lipids). The Fe minerals in the stalks were identified as lepidocrocite and possibly ferrihydrite. Immobilization experiments with Ni 2+ showed that the stalks can function as a sink for trace metals. Our findings show that obligate microaerophilic Fe(II) oxidizers belonging to the Zetaproteobacteria group are not restricted to Fe-rich environments but can also be found in low-Fe marine environments, which increases their overall importance for the global biogeochemical Fe cycle. IMPORTANCE So far, only a few isolates of benthic marine microaerophilic Fe(II) oxidizers belonging to the Zetaproteobacteria exist, and most isolates were obtained from habitats containing elevated Fe concentrations. Consequently, it was thought that these microorganisms are important mainly in habitats with high Fe concentrations. The two novel isolates of Zetaproteobacteria that are presented in the present study were isolated from typical coastal marine sediments that do not contain elevated Fe concentrations. This increases the knowledge about possible habitats in which Zetaproteobacteria can exist. Furthermore, we show that the physiology and the typical organo-mineral structures (twisted stalks) that are produced by the isolates do not notably differ from the physiology and the cell-mineral structures of isolates from environments with high Fe concentrations. We also showed that the organo-mineral structures can function as a sink for trace metals., (Copyright © 2017 American Society for Microbiology.)

This study aimed to identify the differences in the oral microbial communities in saliva from patients with and without caries by performing sequencing with the Illumina MiSeq platform, as well as to further assess their relationships with environmental factors (salivary pH and iron concentration). Forty-three volunteers were selected, including 21 subjects with and 22 without caries, from one village in Gansu, China. Based on 966,255 trimmed sequences and clustering at the 97% similarity level, 1,303 species-level operational taxonomic units were generated. The sequencing data for the two groups revealed that (i) particular distribution patterns (synergistic effects or competition) existed in the subjects with and without caries at both the genus and species levels and (ii) both the salivary pH and iron concentration had significant influences on the microbial community structure., Importance: The significant influences of the oral environment observed in this study increase the current understanding of the salivary microbiome in caries. These results will be useful for expanding research directions and for improving disease diagnosis, prognosis, and therapy., (Copyright © 2017 Zhou et al.)

The omnivorous cockroach Periplaneta americana hosts a diverse hindgut microbiota encompassing hundreds of microbial species. In this study, we used 16S rRNA gene sequencing to examine the effect of diet on the composition of the P. americana hindgut microbial community. Results show that the hindgut microbiota of P. americana exhibit a highly stable core microbial community with low variance in compositions between individuals and minimal community change in response to dietary shifts. This core hindgut microbiome is shared between laboratory-hosted and wild-caught individuals, although wild-caught specimens exhibited a higher diversity of low-abundance microbes that were lost following extended cultivation under laboratory conditions. This taxonomic stability strongly contrasts with observations of the gut microbiota of mammals, which have been shown to be highly responsive to dietary change. A comparison of P. americana hindgut samples with human fecal samples indicated that the cockroach hindgut community exhibited higher alpha diversity but a substantially lower beta diversity than the human gut microbiome. This suggests that cockroaches have evolved unique mechanisms for establishing and maintaining a diverse and stable core microbiome., Importance: The gut microbiome plays an important role in the overall health of its host. A healthy gut microbiota typically assists with defense against pathogens and the digestion and absorption of nutrients from food, while dysbiosis of the gut microbiota has been associated with reduced health. In this study, we examined the composition and stability of the gut microbiota from the omnivorous cockroach Periplaneta americana. We found that P. americana hosts a diverse core gut microbiome that remains stable after drastic long-term changes in diet. While other insects, notably ant and bee species, have evolved mechanisms for maintaining a stable association with specific gut microbiota, these insects typically host low-diversity gut microbiomes and consume specialized diets. In contrast, P. americana hosts a gut microbiota that is highly species rich and consumes a diverse solid diet, suggesting that cockroaches have evolved unique mechanisms for developing and maintaining a stable gut microbiota., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Besides being part of anti-Helicobacter pylori treatment regimens, proton pump inhibitors (PPIs) are increasingly being used to treat dyspepsia. However, little is known about the effects of PPIs on the human gastric microbiota, especially those related to H. pylori infection. The goal of this study was to characterize the stomach microbial communities in patients with dyspepsia and to investigate their relationships with PPI use and H. pylori status. Using 16S rRNA gene pyrosequencing, we analyzed the mucosa-associated microbial populations of 24 patients, of whom 12 were treated with the PPI omeprazole and 9 (5 treated and 4 untreated) were positive for H. pylori infection. The Proteobacteria, Firmicutes, Bacteroidetes, Fusobacteria, and Actinobacteria phyla accounted for 98% of all of the sequences, with Helicobacter, Streptococcus, and Prevotella ranking among the 10 most abundant genera. H. pylori infection or PPI treatment did not significantly influence gastric microbial species composition in dyspeptic patients. Principal-coordinate analysis of weighted UniFrac distances in these communities revealed clear but significant separation according to H. pylori status only. However, in PPI-treated patients, Firmicutes, particularly Streptococcaceae, were significantly increased in relative abundance compared to those in untreated patients. Consistently, Streptococcus was also found to significantly increase in relation to PPI treatment, and this increase seemed to occur independently of H. pylori infection. Our results suggest that Streptococcus may be a key indicator of PPI-induced gastric microbial composition changes in dyspeptic patients. Whether the gastric microbiota alteration contributes to dyspepsia needs further investigation., Importance: Although PPIs have become a popular treatment choice, a growing number of dyspeptic patients may be treated unnecessarily. We found that patients treated with omeprazole showed gastric microbial communities that were different from those of untreated patients. These differences regarded the abundances of specific taxa. By understanding the relationships between PPIs and members of the gastric microbiota, it will be possible to envisage new strategies for better managing patients with dyspepsia., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: It has been suggested that iron is one of the most important energy sources for photosynthesis-independent microbial ecosystems in the ocean crust. Iron-metabolizing chemolithoautotrophs play a key role as primary producers, but little is known about their distribution and diversity and their ecological role as submarine iron-metabolizing chemolithotrophs, particularly the iron oxidizers. In this study, we investigated the microbial communities in several iron-dominated flocculent mats found in deep-sea hydrothermal fields in the Mariana Volcanic Arc and Trough and the Okinawa Trough by culture-independent molecular techniques and X-ray mineralogical analyses. The abundance and composition of the 16S rRNA gene phylotypes demonstrated the ubiquity of zetaproteobacterial phylotypes in iron-dominated mat communities affected by hydrothermal fluid input. Electron microscopy with energy-dispersive X-ray microanalysis and X-ray absorption fine structure (XAFS) analysis revealed the chemical and mineralogical signatures of biogenic Fe-(oxy)hydroxide species and the potential contribution of Zetaproteobacteria to the in situ generation. These results suggest that putative iron-oxidizing chemolithoautotrophs play a significant ecological role in producing iron-dominated flocculent mats and that they are important for iron and carbon cycles in deep-sea low-temperature hydrothermal environments., Importance: We report novel aspects of microbiology from iron-dominated flocculent mats in various deep-sea environments. In this study, we examined the relationship between Zetaproteobacteria and iron oxides across several hydrothermally influenced sites in the deep sea. We analyzed iron-dominated mats using culture-independent molecular techniques and X-ray mineralogical analyses. The scanning electron microscopy-energy-dispersive X-ray spectroscopy SEM-EDS analysis and X-ray absorption fine structure (XAFS) analysis revealed chemical and mineralogical signatures of biogenic Fe-(oxy)hydroxide species as well as the potential contribution of the zetaproteobacterial population to the in situ production. These key findings provide important information for understanding the mechanisms of both geomicrobiological iron cycling and the formation of iron-dominated mats in deep-sea hydrothermal fields., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Specific bacterial species are implicated in the pathogenesis of exacerbations of chronic obstructive pulmonary disease (COPD). However, recent studies of clinically stable COPD patients have demonstrated a greater diversity of airway microbiota, whose role in acute exacerbations is unclear. In this study, temporal changes in the airway microbiome before, at the onset of, and after an acute exacerbation were examined in 60 sputum samples collected from subjects enrolled in a longitudinal study of bacterial infection in COPD. Microbiome composition and predicted functions were examined using 16S rRNA-based culture-independent profiling methods. Shifts in the abundance (≥ 2-fold, P < 0.05) of many taxa at exacerbation and after treatment were observed. Microbiota members that were increased at exacerbation were primarily of the Proteobacteria phylum, including nontypical COPD pathogens. Changes in the bacterial composition after treatment for an exacerbation differed significantly among the therapy regimens clinically prescribed (antibiotics only, oral corticosteroids only, or both). Treatment with antibiotics alone primarily decreased the abundance of Proteobacteria, with the prolonged suppression of some microbiota members being observed. In contrast, treatment with corticosteroids alone led to enrichment for Proteobacteria and members of other phyla. Predicted metagenomes of particular microbiota members involved in these compositional shifts indicated exacerbation-associated loss of functions involved in the synthesis of antimicrobial and anti-inflammatory products, alongside enrichment in functions related to pathogen-elicited inflammation. These trends reversed upon clinical recovery. Further larger studies will be necessary to determine whether specific compositional or functional changes detected in the airway microbiome could be useful indicators of exacerbation development or outcome., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Bacterial communities migrate continuously from the drinking water treatment plant through the drinking water distribution system and into our built environment. Understanding bacterial dynamics in the distribution system is critical to ensuring that safe drinking water is being supplied to customers. We present a 15-month survey of bacterial community dynamics in the drinking water system of Ann Arbor, MI. By sampling the water leaving the treatment plant and at nine points in the distribution system, we show that the bacterial community spatial dynamics of distance decay and dispersivity conform to the layout of the drinking water distribution system. However, the patterns in spatial dynamics were weaker than those for the temporal trends, which exhibited seasonal cycling correlating with temperature and source water use patterns and also demonstrated reproducibility on an annual time scale. The temporal trends were driven by two seasonal bacterial clusters consisting of multiple taxa with different networks of association within the larger drinking water bacterial community. Finally, we show that the Ann Arbor data set robustly conforms to previously described interspecific occupancy abundance models that link the relative abundance of a taxon to the frequency of its detection. Relying on these insights, we propose a predictive framework for microbial management in drinking water systems. Further, we recommend that long-term microbial observatories that collect high-resolution, spatially distributed, multiyear time series of community composition and environmental variables be established to enable the development and testing of the predictive framework., Importance: Safe and regulation-compliant drinking water may contain up to millions of microorganisms per liter, representing phylogenetically diverse groups of bacteria, archaea, and eukarya that affect public health, water infrastructure, and the aesthetic quality of water. The ability to predict the dynamics of the drinking water microbiome will ensure that microbial contamination risks can be better managed. Through a spatial-temporal survey of drinking water bacterial communities, we present novel insights into their spatial and temporal community dynamics and recommend steps to link these insights in a predictive framework for microbial management of drinking water systems. Such a predictive framework will not only help to eliminate microbial risks but also help to modify existing water quality monitoring efforts and make them more resource efficient. Further, a predictive framework for microbial management will be critical if we are to fully anticipate the risks and benefits of the beneficial manipulation of the drinking water microbiome., (Copyright © 2014 Pinto et al.)

The methanotrophs in rice field soil are crucial in regulating the emission of methane. Drainage substantially reduces methane emission from rice fields. However, it is poorly understood how drainage affects microbial methane oxidation. Therefore, we analyzed the dynamics of methane oxidation rates, composition (using terminal restriction fragment length polymorphism [T-RFLP]), and abundance (using quantitative PCR [qPCR]) of methanotroph pmoA genes (encoding a subunit of particulate methane monooxygenase) and their transcripts over the season and in response to alternate dry/wet cycles in planted paddy field microcosms. In situ methane oxidation accounted for less than 15% of total methane production but was enhanced by intermittent drainage. The dry/wet alternations resulted in distinct effects on the methanotrophic communities in different soil compartments (bulk soil, rhizosphere soil, surface soil). The methanotrophic communities of the different soil compartments also showed distinct seasonal dynamics. In bulk soil, potential methanotrophic activity and transcription of pmoA were relatively low but were significantly stimulated by drainage. In contrast, however, in the rhizosphere and surface soils, potential methanotrophic activity and pmoA transcription were relatively high but decreased after drainage events and resumed after reflooding. While type II methanotrophs dominated the communities in the bulk soil and rhizosphere soil compartments (and to a lesser extent also in the surface soil), it was the pmoA of type I methanotrophs that was mainly transcribed under flooded conditions. Drainage affected the composition of the methanotrophic community only minimally but strongly affected metabolically active methanotrophs. Our study revealed dramatic dynamics in the abundance, composition, and activity of the various type I and type II methanotrophs on both a seasonal and a spatial scale and showed strong effects of dry/wet alternation cycles, which enhanced the attenuation of methane flux into the atmosphere.

Soils were incubated for 80 days in a continuously labeled (14)CO(2) atmosphere to measure the amount of labeled C incorporated into the microbial biomass. Microbial assimilation of (14)C differed between soils and accounted for 0.12% to 0.59% of soil organic carbon (SOC). Assuming a terrestrial area of 1.4 × 10(8) km(2), this represents a potential global sequestration of 0.6 to 4.9 Pg C year(-1). Estimated global C sequestration rates suggest a "missing sink" for carbon of between 2 and 3 Pg C year(-1). To determine whether (14)CO(2) incorporation was mediated by autotrophic microorganisms, the diversity and abundance of CO(2)-fixing bacteria and algae were investigated using clone library sequencing, terminal restriction fragment length polymorphism (T-RFLP), and quantitative PCR (qPCR) of the ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene (cbbL). Phylogenetic analysis showed that the dominant cbbL-containing bacteria were Azospirillum lipoferum, Rhodopseudomonas palustris, Bradyrhizobium japonicum, Ralstonia eutropha, and cbbL-containing chromophytic algae of the genera Xanthophyta and Bacillariophyta. Multivariate analyses of T-RFLP profiles revealed significant differences in cbbL-containing microbial communities between soils. Differences in cbbL gene diversity were shown to be correlated with differences in SOC content. Bacterial and algal cbbL gene abundances were between 10(6) and 10(8) and 10(3) to 10(5) copies g(-1) soil, respectively. Bacterial cbbL abundance was shown to be positively correlated with RubisCO activity (r = 0.853; P < 0.05), and both cbbL abundance and RubisCO activity were significantly related to the synthesis rates of [(14)C]SOC (r = 0.967 and 0.946, respectively; P < 0.01). These data offer new insights into the importance of microbial autotrophy in terrestrial C cycling.

"Candidatus Methylomirabilis oxyfera" is a newly discovered denitrifying methanotroph that is unrelated to previously known methanotrophs. This bacterium is a member of the NC10 phylum and couples methane oxidation to denitrification through a newly discovered intra-aerobic pathway. In the present study, we report the first ultrastructural study of "Ca. Methylomirabilis oxyfera" using scanning electron microscopy, transmission electron microscopy, and electron tomography in combination with different sample preparation methods. We observed that "Ca. Methylomirabilis oxyfera" cells possess an atypical polygonal shape that is distinct from other bacterial shapes described so far. Also, an additional layer was observed as the outermost sheath, which might represent a (glyco)protein surface layer. Further, intracytoplasmic membranes, which are a common feature among proteobacterial methanotrophs, were never observed under the current growth conditions. Our results indicate that "Ca. Methylomirabilis oxyfera" is ultrastructurally distinct from other bacteria by its atypical cell shape and from the classical proteobacterial methanotrophs by its apparent lack of intracytoplasmic membranes.

Sphagnum peatlands are important ecosystems in the methane cycle. Methane-oxidizing bacteria in these ecosystems serve as a methane filter and limit methane emissions. Yet little is known about the diversity and identity of the methanotrophs present in and on Sphagnum mosses of peatlands, and only a few isolates are known. The methanotrophic community in Sphagnum mosses, originating from a Dutch peat bog, was investigated using a pmoA microarray. A high biodiversity of both gamma- and alphaproteobacterial methanotrophs was found. With Sphagnum mosses as the inoculum, alpha- and gammaproteobacterial acidophilic methanotrophs were isolated using established and newly designed media. The 16S rRNA, pmoA, pxmA, and mmoX gene sequences showed that the alphaproteobacterial isolates belonged to the Methylocystis and Methylosinus genera. The Methylosinus species isolated are the first acid-tolerant members of this genus. Of the acidophilic gammaproteobacterial strains isolated, strain M5 was affiliated with the Methylomonas genus, and the other strain, M200, may represent a novel genus, most closely related to the genera Methylosoma and Methylovulum. So far, no acidophilic or acid-tolerant methanotrophs in the Gammaproteobacteria class are known. All strains showed the typical features of either type I or II methanotrophs and are, to the best of our knowledge, the first isolated (acidophilic or acid-tolerant) methanotrophs from Sphagnum mosses.

Members of the neutrophilic iron-oxidizing candidate class Zetaproteobacteria have predominantly been found at sites of microbially mediated iron oxidation in marine environments around the Pacific Ocean. Eighty-four full-length (>1,400-bp) and 48 partial-length Zetaproteobacteria small-subunit (SSU) rRNA gene sequences from five novel clone libraries, one novel Zetaproteobacteria isolate, and the GenBank database were analyzed to assess the biodiversity of this burgeoning class of the Proteobacteria and to investigate its biogeography between three major sampling regions in the Pacific Ocean: Loihi Seamount, the Southern Mariana Trough, and the Tonga Arc. Sequences were grouped into operational taxonomic units (OTUs) on the basis of a 97% minimum similarity. Of the 28 OTUs detected, 13 were found to be endemic to one of the three main sampling regions and 2 were ubiquitous throughout the Pacific Ocean. Additionally, two deeply rooted OTUs that potentially dominate communities of iron oxidizers originating in the deep subsurface were identified. Spatial autocorrelation analysis and analysis of molecular variance (AMOVA) showed that geographic distance played a significant role in the distribution of Zetaproteobacteria biodiversity, whereas environmental parameters, such as temperature, pH, or total Fe concentration, did not have a significant effect. These results, detected using the coarse resolution of the SSU rRNA gene, indicate that the Zetaproteobacteria have a strong biogeographic signal.

Microbiologically influenced corrosion (MIC) of mild steel in seawater is an expensive and enduring problem. Little attention has been paid to the role of neutrophilic, lithotrophic, iron-oxidizing bacteria (FeOB) in MIC. The goal of this study was to determine if marine FeOB related to Mariprofundus are involved in this process. To examine this, field incubations and laboratory microcosm experiments were conducted. Mild steel samples incubated in nearshore environments were colonized by marine FeOB, as evidenced by the presence of helical iron-encrusted stalks diagnostic of the FeOB Mariprofundus ferrooxydans, a member of the candidate class "Zetaproteobacteria." Furthermore, Mariprofundus-like cells were enriched from MIC biofilms. The presence of Zetaproteobacteria was confirmed using a Zetaproteobacteria-specific small-subunit (SSU) rRNA gene primer set to amplify sequences related to M. ferrooxydans from both enrichments and in situ samples of MIC biofilms. Temporal in situ incubation studies showed a qualitative increase in stalk distribution on mild steel, suggesting progressive colonization by stalk-forming FeOB. We also isolated a novel FeOB, designated Mariprofundus sp. strain GSB2, from an iron oxide mat in a salt marsh. Strain GSB2 enhanced uniform corrosion from mild steel in laboratory microcosm experiments conducted over 4 days. Iron concentrations (including precipitates) in the medium were used as a measure of corrosion. The corrosion in biotic samples (7.4 ± 0.1 mM) was significantly higher than that in abiotic controls (5.0 ± 0.1 mM). These results have important implications for the role of FeOB in corrosion of steel in nearshore and estuarine environments. In addition, this work shows that the global distribution of Zetaproteobacteria is far greater than previously thought.

The human oral cavity contains a number of different habitats, including the teeth, gingival sulcus, tongue, cheeks, hard and soft palates, and tonsils, which are colonized by bacteria. The oral microbiome is comprised of over 600 prevalent taxa at the species level, with distinct subsets predominating at different habitats. The oral microbiome has been extensively characterized by cultivation and culture-independent molecular methods such as 16S rRNA cloning. Unfortunately, the vast majority of unnamed oral taxa are referenced by clone numbers or 16S rRNA GenBank accession numbers, often without taxonomic anchors. The first aim of this research was to collect 16S rRNA gene sequences into a curated phylogeny-based database, the Human Oral Microbiome Database (HOMD), and make it web accessible (www.homd.org). The HOMD includes 619 taxa in 13 phyla, as follows: Actinobacteria, Bacteroidetes, Chlamydiae, Chloroflexi, Euryarchaeota, Firmicutes, Fusobacteria, Proteobacteria, Spirochaetes, SR1, Synergistetes, Tenericutes, and TM7. The second aim was to analyze 36,043 16S rRNA gene clones isolated from studies of the oral microbiota to determine the relative abundance of taxa and identify novel candidate taxa. The analysis identified 1,179 taxa, of which 24% were named, 8% were cultivated but unnamed, and 68% were uncultivated phylotypes. Upon validation, 434 novel, nonsingleton taxa will be added to the HOMD. The number of taxa needed to account for 90%, 95%, or 99% of the clones examined is 259, 413, and 875, respectively. The HOMD is the first curated description of a human-associated microbiome and provides tools for use in understanding the role of the microbiome in health and disease.

Bacteria can use branched-chain amino acids (ILV, i.e., isoleucine, leucine, valine) and fatty acids (FAs) as sole carbon and energy sources converting ILV into acetyl-coenzyme A (CoA), propanoyl-CoA, and propionyl-CoA, respectively. In this work, we used the comparative genomic approach to identify candidate transcriptional factors and DNA motifs that control ILV and FA utilization pathways in proteobacteria. The metabolic regulons were characterized based on the identification and comparison of candidate transcription factor binding sites in groups of phylogenetically related genomes. The reconstructed ILV/FA regulatory network demonstrates considerable variability and involves six transcriptional factors from the MerR, TetR, and GntR families binding to 11 distinct DNA motifs. The ILV degradation genes in gamma- and betaproteobacteria are regulated mainly by a novel regulator from the MerR family (e.g., LiuR in Pseudomonas aeruginosa) (40 species); in addition, the TetR-type regulator LiuQ was identified in some betaproteobacteria (eight species). Besides the core set of ILV utilization genes, the LiuR regulon in some lineages is expanded to include genes from other metabolic pathways, such as the glyoxylate shunt and glutamate synthase in Shewanella species. The FA degradation genes are controlled by four regulators including FadR in gammaproteobacteria (34 species), PsrA in gamma- and betaproteobacteria (45 species), FadP in betaproteobacteria (14 species), and LiuR orthologs in alphaproteobacteria (22 species). The remarkable variability of the regulatory systems associated with the FA degradation pathway is discussed from functional and evolutionary points of view.

Although some bacteria require an atmosphere with high CO(2) levels for their growth, CO(2) is not generally supplied to conventional screening cultures. Here, we isolated 84 bacterial strains exhibiting high-CO(2) dependence. Their phylogenetic affiliations imply that high-CO(2) culture has potential as an effective method to isolate unknown microorganisms.

IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha- and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta- and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids.

The diversity of aerobic anoxygenic phototrophic (AAP) bacteria has been examined in marine habitats, but the types of AAP bacteria in estuarine waters and distribution of ecotypes in any environment are not well known. The goal of this study was to determine the diversity of AAP bacteria in the Delaware estuary and to examine the distribution of select ecotypes using quantitative PCR (qPCR) assays for the pufM gene, which encodes a protein in the light reaction center of AAP bacteria. In PCR libraries from the Delaware River, pufM genes similar to those from Beta- (Rhodoferax-like) or Gammaproteobacteria comprised at least 50% of the clones, but the expressed pufM genes from the river were not dominated by these two groups in August 2002 (less than 31% of clones). In four transects, qPCR data indicated that the gammaproteobacterial type of pufM was abundant only near the mouth of the bay whereas Rhodoferax-like AAP bacteria were restricted to waters with a salinity of <5. In contrast, a Rhodobacter-like pufM gene was ubiquitous, but its distribution along the salinity gradient varied with the season. High fractions (12 to 24%) of all three pufM types were associated with particles. The data suggest that different groups of AAP bacteria are controlled by different environmental factors, which may explain current difficulties in predicting the distribution of total AAP bacteria in aquatic environments.

Molybdenum-independent nitrogenases were first described in the nitrogen-fixing bacterium Azotobacter vinelandii and have since been described in other diazotrophic bacteria. Previously, we reported the isolation of seven diazotrophs with Mo-independent nitrogenases from aquatic environments. In the present study, we extend these results to include diazotrophs isolated from wood chip mulch, soil, "paraffin dirt," and sediments from mangrove swamps. Mo-deficient, N-free media under both aerobic and anaerobic conditions were used for the isolations. A total of 26 isolates were genetically and physiologically characterized. Their phylogenetic placement was determined using 16S rRNA gene sequence analysis. Most of the isolates are members of the gamma subdivision of the class Proteobacteria and appear to be specifically related to fluorescent pseudomonads and azotobacteria. Two other isolates, AN1 and LPF4, are closely related to Enterobacter spp. and Paenibacillus spp., respectively. PCR and/or Southern hybridization were used to detect the presence of nitrogenase genes in the isolates. PCR amplification of vnfG and anfG was used to detect the genetic potential for the expression of the vanadium-containing nitrogenase and the iron-only nitrogenase in the isolates. This study demonstrates that diazotrophs with Mo-independent nitrogenases can be readily isolated from diverse natural environments.

Next to the benthic and pelagic compartments, the epiphyton of submerged macrophytes may offer an additional niche for ammonia-oxidizing bacteria in shallow freshwater lakes. In this study, we explored the potential activities and community compositions of ammonia-oxidizing bacteria of the epiphytic, benthic, and pelagic compartments of seven shallow freshwater lakes which differed in their trophic status, distribution of submerged macrophytes, and restoration history. PCR-denaturing gradient gel electrophoresis analyses demonstrated that the epiphytic compartment was inhabited by species belonging to cluster 3 of the Nitrosospira lineage and to the Nitrosomonas oligotropha lineage. Both the ammonia-oxidizing bacterial community compositions and the potential activities differed significantly between compartments. Interestingly, both the ammonia-oxidizing bacterial community composition and potential activity were influenced by the restoration status of the different lakes investigated.

Forty-one flagellated species representing 11 bacterial phyla were used to investigate the origin of secondary flagellar systems and the structure and formation of flagellar gene operons over the course of bacterial evolution. Secondary (i.e., lateral) flagellar systems, which are harbored by five of the proteobacterial species considered, originated twice, once in the alphaproteobacterial lineage and again in the common ancestor of the Beta- and Gammaproteobacteria. The order and organization of flagellar genes have undergone extensive shuffling and rearrangement among lineages, and based on the phylogenetic distributions of flagellar gene complexes, the flagellar gene operons existed as small, usually two-gene units in the ancestor of Bacteria and have expanded through the recruitment of new genes and fusion of gene units. In contrast to the evolutionary trend towards larger flagellar gene complexes, operon structures have been highly disrupted through gene disassociation and rearrangements in the Epsilon- and Alphaproteobacteria. These results demonstrate that the genetic basis of this ancient and structurally conserved organelle has been subject to many lineage-specific modifications.

We investigated the fine-scale population structure of the "Candidatus Accumulibacter" lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (ppk1) as a genetic marker. We retrieved fragments of "Candidatus Accumulibacter" 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the "Candidatus Accumulibacter" lineage. Sequences from at least five clades of "Candidatus Accumulibacter" were recovered by ppk1-targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using "Candidatus Accumulibacter"-specific 16S rRNA and "Candidatus Accumulibacter" clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total "Candidatus Accumulibacter" lineage and the relative distributions and abundances of the five "Candidatus Accumulibacter" clades. The qPCR-based estimation of the total "Candidatus Accumulibacter" fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1, demonstrating the power of ppk1 as a genetic marker for detection of all currently defined "Candidatus Accumulibacter" clades. The relative distributions of "Candidatus Accumulibacter" clades varied among different EBPR systems and also temporally within a system. Our results suggest that the "Candidatus Accumulibacter" lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.

Dissimilatory nitrate reduction is catalyzed by a membrane-bound and a periplasmic nitrate reductase. We set up a real-time PCR assay to quantify these two enzymes, using the narG and napA genes, encoding the catalytic subunits of the two types of nitrate reductases, as molecular markers. The narG and napA gene copy numbers in DNA extracted from 18 different environments showed high variations, with most numbers ranging from 2 x 10(2) to 6.8 x 10(4) copies per ng of DNA. This study provides evidence that, in soil samples, the number of proteobacteria carrying the napA gene is often as high as that of proteobacteria carrying the narG gene. The high correlation observed between narG and napA gene copy numbers in soils suggests that the ecological roles of the corresponding enzymes might be linked.

The Tipula abdominalis larval hindgut microbial community presumably facilitates digestion of the lignocellulosic diet. The microbial community was investigated through characterization of bacterial isolates and analysis of 16S rRNA gene clone libraries. This initial study revealed novel bacteria and provides a framework for future studies of this symbiosis.

Biofilm formation is controlled by an array of coupled physical, chemical, and biotic processes. Despite the ecological relevance of microbial biofilms, their community formation and succession remain poorly understood. We investigated the effect of flow velocity, as the major physical force in stream ecosystems, on biofilm community succession (as continuous shifts in community composition) in microcosms under laminar, intermediate, and turbulent flow. Flow clearly shaped the development of biofilm architecture and community composition, as revealed by microscopic investigation, denaturing gradient gel electrophoresis (DGGE) analysis, and sequencing. While biofilm growth patterns were undirected under laminar flow, they were clearly directed into ridges and conspicuous streamers under turbulent flow. A total of 51 biofilm DGGE bands were detected; the average number ranged from 13 to 16. Successional trajectories diverged from an initial community that was common in all flow treatments and increasingly converged as biofilms matured. We suggest that this developmental pattern was primarily driven by algae, which, as "ecosystem engineers," modulate their microenvironment to create similar architectures and flow conditions in all treatments and thereby reduce the physical effect of flow on biofilms. Our results thus suggest a shift from a predominantly physical control to coupled biophysical controls on bacterial community succession in stream biofilms.

Drosophila melanogaster is one of the most widely used model systems in biology. However, little is known about its associated bacterial community. As a first step towards understanding these communities, we compared bacterial 16S rRNA gene sequence libraries recovered from 11 natural populations of adult D. melanogaster. Bacteria from these sequence libraries were grouped into 74 distinct taxa, spanning the phyla Proteobacteria, Bacteroidetes, and Firmicutes, which were unevenly spread across host populations. Summed across populations, the distribution of abundance of genera was closely fit by a power law. We observed differences among host population locations both in bacterial community richness and in composition. Despite this significant spatial variation, no relationship was observed between species richness and a variety of abiotic factors, such as temperature and latitude. Overall, bacterial communities associated with adult D. melanogaster hosts are diverse and differ across host populations.

Microbially induced concrete corrosion (MICC) in sewer systems has been a serious problem for a long time. A better understanding of the succession of microbial community members responsible for the production of sulfuric acid is essential for the efficient control of MICC. In this study, the succession of sulfur-oxidizing bacteria (SOB) in the bacterial community on corroding concrete in a sewer system in situ was investigated over 1 year by culture-independent 16S rRNA gene-based molecular techniques. Results revealed that at least six phylotypes of SOB species were involved in the MICC process, and the predominant SOB species shifted in the following order: Thiothrix sp., Thiobacillus plumbophilus, Thiomonas intermedia, Halothiobacillus neapolitanus, Acidiphilium acidophilum, and Acidithiobacillus thiooxidans. A. thiooxidans, a hyperacidophilic SOB, was the most dominant (accounting for 70% of EUB338-mixed probe-hybridized cells) in the heavily corroded concrete after 1 year. This succession of SOB species could be dependent on the pH of the concrete surface as well as on trophic properties (e.g., autotrophic or mixotrophic) and on the ability of the SOB to utilize different sulfur compounds (e.g., H2S, S0, and S2O3(2-)). In addition, diverse heterotrophic bacterial species (e.g., halo-tolerant, neutrophilic, and acidophilic bacteria) were associated with these SOB. The microbial succession of these microorganisms was involved in the colonization of the concrete and the production of sulfuric acid. Furthermore, the vertical distribution of microbial community members revealed that A. thiooxidans was the most dominant throughout the heavily corroded concrete (gypsum) layer and that A. thiooxidans was most abundant at the highest surface (1.5-mm) layer and decreased logarithmically with depth because of oxygen and H2S transport limitations. This suggested that the production of sulfuric acid by A. thiooxidans occurred mainly on the concrete surface and the sulfuric acid produced penetrated through the corroded concrete layer and reacted with the sound concrete below.

Scanning electron microscopy revealed great morphological diversity in biofilms from several largely unexplored subterranean thermal Alpine springs, which contain radium 226 and radon 222. A culture-independent molecular analysis of microbial communities on rocks and in the water of one spring, the "Franz-Josef-Quelle" in Bad Gastein, Austria, was performed. Four hundred fifteen clones were analyzed. One hundred thirty-two sequences were affiliated with 14 bacterial operational taxonomic units (OTUs) and 283 with four archaeal OTUs. Rarefaction analysis indicated a high diversity of bacterial sequences, while archaeal sequences were less diverse. The majority of the cloned archaeal 16S rRNA gene sequences belonged to the soil-freshwater-subsurface (1.1b) crenarchaeotic group; other representatives belonged to the freshwater-wastewater-soil (1.3b) group, except one clone, which was related to a group of uncultivated Euryarchaeota. These findings support recent reports that Crenarchaeota are not restricted to high-temperature environments. Most of the bacterial sequences were related to the Proteobacteria (alpha, beta, gamma, and delta), Bacteroidetes, and Planctomycetes. One OTU was allied with Nitrospina sp. (delta-Proteobacteria) and three others grouped with Nitrospira. Statistical analyses suggested high diversity based on 16S rRNA gene analyses; the rarefaction plot of archaeal clones showed a plateau. Since Crenarchaeota have been implicated recently in the nitrogen cycle, the spring environment was probed for the presence of the ammonia monooxygenase subunit A (amoA) gene. Sequences were obtained which were related to crenarchaeotic amoA genes from marine and soil habitats. The data suggested that nitrification processes are occurring in the subterranean environment and that ammonia may possibly be an energy source for the resident communities.

Phylogenetic sequence analysis of single or multiple genes has dominated the study and census of the genetic diversity among closely related bacteria. It remains unclear, however, how the results based on a few genes in the genome correlate with whole-genome-based relatedness and what genes (if any) best reflect whole-genome-level relatedness and hence should be preferentially used to economize on cost and to improve accuracy. We show here that phylogenies of closely related organisms based on the average nucleotide identity (ANI) of their shared genes correspond accurately to phylogenies based on state-of-the-art analysis of their whole-genome sequences. We use ANI to evaluate the phylogenetic robustness of every gene in the genome and show that almost all core genes, regardless of their functions and positions in the genome, offer robust phylogenetic reconstruction among strains that show 80 to 95% ANI (16S rRNA identity, >98.5%). Lack of elapsed time and, to a lesser extent, horizontal transfer and recombination make the selection of genes more critical for applications that target the intraspecies level, i.e., strains that show >95% ANI according to current standards. A much more accurate phylogeny for the Escherichia coli group was obtained based on just three best-performing genes according to our analysis compared to the concatenated alignment of eight genes that are commonly employed for phylogenetic purposes in this group. Our results are reproducible within the Salmonella, Burkholderia, and Shewanella groups and therefore are expected to have general applicability for microevolution studies, including metagenomic surveys.

Microbial community profiles and species composition associated with two black band-diseased colonies of the coral Siderastrea siderea were studied by 16S rRNA-targeted gene cloning, sequencing, and amplicon-length heterogeneity PCR (LH-PCR). Bacterial communities associated with the surface mucopolysaccharide layer (SML) of apparently healthy tissues of the infected colonies, together with samples of the black band disease (BBD) infections, were analyzed using the same techniques for comparison. Gene sequences, ranging from 424 to 1,537 bp, were retrieved from all positive clones (n = 43 to 48) in each of the four clone libraries generated and used for comparative sequence analysis. In addition to LH-PCR community profiling, all of the clone sequences were aligned with LH-PCR primer sequences, and the theoretical lengths of the amplicons were determined. Results revealed that the community profiles were significantly different between BBD and SML samples. The SML samples were dominated by gamma-proteobacteria (53 to 64%), followed by beta-proteobacteria (18 to 21%) and alpha-proteobacteria (5 to 11%). In contrast, both BBD clone libraries were dominated by alpha-proteobacteria (58 to 87%), followed by verrucomicrobia (2 to 10%) and 0 to 6% each of delta-proteobacteria, bacteroidetes, firmicutes, and cyanobacteria. Alphaproteobacterial sequence types related to the bacteria associated with toxin-producing dinoflagellates were observed in BBD clone libraries but were not found in the SML libraries. Similarly, sequences affiliated with the family Desulfobacteraceae and toxin-producing cyanobacteria, both believed to be involved in BBD pathogenesis, were found only in BBD libraries. These data provide evidence for an association of numerous toxin-producing heterotrophic microorganisms with BBD of corals.

Though a large fraction of primary production and organic matter cycling in the oceans occurs on continental shelves dominated by sandy deposits, the microbial communities associated with permeable shelf sediments remain poorly characterized. Therefore, in this study, we provide the first detailed characterization of microbial diversity in marine sands of the South Atlantic Bight through parallel analyses of small-subunit (SSU) rRNA gene (Bacteria), nosZ (denitrifying bacteria), and amoA (ammonia-oxidizing bacteria) sequences. Communities were analyzed by parallel DNA extractions and clone library construction from both sediment core material and manipulated sediment within column experiments designed for geochemical rate determinations. Rapid organic-matter degradation and coupled nitrification-denitrification were observed in column experiments at flow rates resembling in situ conditions over a range of oxygen concentrations. Numerous SSU rRNA phylotypes were affiliated with the phyla Proteobacteria (classes Alpha-, Delta-, and Gammaproteobacteria), Planctomycetes, Cyanobacteria, Chloroflexi, and Bacteroidetes. Detectable sequence diversity of nosZ and SSU rRNA genes increased in stratified redox-stabilized columns compared to in situ sediments, with the Alphaproteobacteria comprising the most frequently detected group. Alternatively, nitrifier communities showed a relatively low and stable diversity that did not covary with the other gene targets. Our results elucidate predominant phylotypes that are likely to catalyze carbon and nitrogen cycling in marine sands. Although overall diversity increased in response to redox stabilization and stratification in column experiments, the major phylotypes remained the same in all of our libraries, indicating that the columns sufficiently mimic in situ conditions.

The influence of altitude and salinity on bacterioplankton community composition (BCC) in 16 high-mountain lakes located at altitudes of 2,817 to 5,134 m on the Eastern Qinghai-Xizang (Tibetan) Plateau, China, spanning a salinity gradient from 0.02% (freshwater) to 22.3% (hypersaline), was investigated. Three different methods, fluorescent in situ hybridization, denaturing gradient gel electrophoresis (DGGE) with subsequent band sequencing, and reverse line blot hybridization (RLB) with probes targeting 17 freshwater bacterial groups, were used for analysis of BCC. Furthermore, the salt tolerances of 47 strains affiliated with groups detected in or isolated from the Tibetan habitats were investigated. Altitude was not found to influence BCC significantly within the investigated range. Several groups of typical freshwater bacteria, e.g., the ACK-M1 cluster and the Polynucleobacter group, were detected in habitats located above 4,400 m. Salinity was found to be the dominating environmental factor controlling BCC in the investigated lakes, resulting in only small overlaps in the BCCs of freshwater and hypersaline lakes. The relative abundances of different classes of Proteobacteria showed a sharp succession along the salinity gradient. Both DGGE and RLB demonstrated that a few freshwater bacterial groups, e.g., GKS98 and LD2, appeared over wide salinity ranges. Six freshwater isolates affiliated with the GKS98 cluster grew in ecophysiological experiments at maximum salinities of 0.3% to 0.7% (oligosaline), while this group was detected in habitats with salinities up to 6.7% (hypersaline). This observation indicated ecologically significant differences in ecophysiological adaptations among members of this narrow phylogenetic group and suggested ecological significance of microdiversity.

The relative abundance of bacteria in the mucus and crushed tissue of the Mediterranean coral Oculina patagonica was determined by analyses of the 16S rRNA genes of isolated colonies and from a 16S rRNA clone library of extracted DNA. By SYBR gold staining, the numbers of bacteria in mucus and tissue samples were 6.2 x 10(7) and 8.3 x 10(8)/cm2 of coral surface, respectively, 99.8% of which failed to produce colonies on Marine Agar. From analysis of mucus DNA, the most-abundant bacterium was Vibrio splendidus, representing 68% and 50% of the clones from the winter and summer, respectively. After removal of mucus from coral by centrifugation, analyses of DNA from the crushed tissue revealed a large diversity of bacteria, with Vibrio species representing less than 5% of the clones. The most-abundant culturable bacteria were a Pseudomonas sp. (8 to 14%) and two different alpha-proteobacteria (6 to 18%). Out of a total 1,088 16S rRNA genes sequenced, 400 different operational taxonomic units were identified (> 99.5% identity). Of these, 295 were novel (< 99% identical to any sequences in the GenBank database). This study provides a comprehensive database for future examinations of changes in the bacterial community during bleaching events.

Bacterial diversity is central to ecosystem sustainability and soil biological function, for which the role of roots is important. The high-throughput analysis potential of taxonomic microarray should match the breadth of bacterial diversity. Here, the power of this technology was evidenced through methodological verifications and analysis of maize rhizosphere effect based on a 16S rRNA-based microarray developed from the prototype of H. Sanguin et al. (Environ. Microbiol. 8:289-307, 2006). The current probe set was composed of 170 probes (41 new probes in this work) that targeted essentially the Proteobacteria. Cloning and sequencing of 16S rRNA amplicons were carried out on maize rhizosphere and bulk soil DNA. All tested clones that had a perfect match with corresponding probes were positive in the hybridization experiment. The hierarchically nested probes were reliable, but the level of taxonomic identification was variable, depending on the probe set specificity. The comparison of experimental and theoretical hybridizations revealed 0.91% false positives and 0.81% false negatives. The microarray detection threshold was estimated at 0.03% of a given DNA type based on DNA spiking experiments. A comparison of the maize rhizosphere and bulk soil hybridization results showed a significant rhizosphere effect, with a higher predominance of Agrobacterium spp. in the rhizosphere, as well as a lower prevalence of Acidobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes, a new taxon of interest in soil. In addition, well-known taxonomic groups such as Sphingomonas spp., Rhizobiaceae, and Actinobacteria were identified in both microbial habitats with strong hybridization signals. The taxonomic microarray developed in the present study was able to discriminate and characterize bacterial community composition in related biological samples, offering extensive possibilities for systematic exploration of bacterial diversity in ecosystems.

The sensitivity of nitrifying bacteria to acidic conditions is a well-known phenomenon and generally attributed to the lack and/or toxicity of substrates (NH3 and HNO2) with decreasing pHs. In contrast, we observed strong nitrification at a pH around 4 in biofilms grown on chalk particles and investigated the following hypotheses: the presence of less acidic microenvironments and/or the existence of acid-tolerant nitrifiers. Microelectrode measurements (in situ and under various experimental conditions) showed no evidence of a neutral microenvironment, either within the highly active biofilm colonizing the chalk surface or within a control biofilm grown on a nonbuffering (i.e., sintered glass) surface under acidic pH. A 16S rRNA approach (clone libraries and fluorescence in situ hybridizations) did not reveal uncommon nitrifying (potentially acid-tolerant) strains. Instead, we found a strongly acidic microenvironment, evidence for a clear adaptation to the low pH in situ, and the presence of nitrifying populations related to subgroups with low Km s for ammonia (Nitrosopira spp., Nitrosomonas oligotropha, and Nitrospira spp.). Acid-consuming (chalk dissolution) and acid-producing (ammonia oxidation) processes are equilibrated on a low-pH steady state that is controlled by mass transfer limitation through the biofilm. Strong affinity to ammonia and possibly the expression of additional functions, e.g., ammonium transporters, are adaptations that allow nitrifiers to cope with acidic conditions in biofilms and other habitats.

We applied nucleic acid-based molecular methods, combined with estimates of biomass (ATP), pigments, and microelectrode measurements of chemical gradients, to map microbial diversity vertically on a millimeter scale in a hypersaline microbial mat from Guerrero Negro, Baja California Sur, Mexico. To identify the constituents of the mat, small-subunit rRNA genes were amplified by PCR from community genomic DNA extracted from layers, cloned, and sequenced. Bacteria dominated the mat and displayed unexpected and unprecedented diversity. The majority (1,336) of the 1,586 bacterial 16S rRNA sequences generated were unique, representing 752 species (> or =97% rRNA sequence identity) in 42 of the main bacterial phyla, including 15 novel candidate phyla. The diversity of the mat samples differentiated according to the chemical milieu defined by concentrations of O(2) and H(2)S. Bacteria of the phylum Chloroflexi formed the majority of the biomass by percentage of bulk rRNA and of clones in rRNA gene libraries. This result contradicts the general belief that cyanobacteria dominate these communities. Although cyanobacteria constituted a large fraction of the biomass in the upper few millimeters (>80% of the total rRNA and photosynthetic pigments), Chloroflexi sequences were conspicuous throughout the mat. Filamentous Chloroflexi bacteria were identified by fluorescence in situ hybridization within the polysaccharide sheaths of the prominent cyanobacterium Microcoleus chthonoplastes, in addition to free living in the mat. The biological complexity of the mat far exceeds that observed in other polysaccharide-rich microbial ecosystems, such as the human and mouse distal guts, and suggests that positive feedbacks exist between chemical complexity and biological diversity. The sequences determined in this study have been submitted to the GenBank database and assigned accession numbers DQ 329539 to DQ 331020, and DQ 397339 to DQ 397511.

An evolutionary algorithm was applied to study the complex interactions between medium parameters and their effects on the isolation of denitrifying bacteria, both in number and in diversity. Growth media with a pH of 7 and a nitrogen concentration of 3 mM, supplemented with 1 ml of vitamin solution but not with sodium chloride or riboflavin, were the most successful for the isolation of denitrifiers from activated sludge. The use of ethanol or succinate as a carbon source and a molar C/N ratio of 2.5, 20, or 25 were also favorable. After testing of 60 different medium parameter combinations and comparison with each other as well as with the standard medium Trypticase soy agar supplemented with nitrate, three growth media were highly suitable for the cultivation of denitrifying bacteria. All evaluated isolation conditions were used to study the cultivable denitrifier diversity of activated sludge from a municipal wastewater treatment plant. One hundred ninety-nine denitrifiers were isolated, the majority of which belonged to the Betaproteobacteria (50.4%) and the Alphaproteobacteria (36.8%). Representatives of Gammaproteobacteria (5.6%), Epsilonproteobacteria (2%), and Firmicutes (4%) and one isolate of the Bacteroidetes were also found. This study revealed a much more diverse denitrifying community than that previously described in cultivation-dependent research on activated sludge.

Although microbes associated with shallow-water corals have been reported, deepwater coral microbes are poorly characterized. A cultivation-independent analysis of Alaskan seamount octocoral microflora showed that Proteobacteria (classes Alphaproteobacteria and Gammaproteobacteria), Firmicutes, Bacteroidetes, and Acidobacteria dominate and vary in abundance. More sampling is needed to understand the basis and significance of this variation.

Rock varnish from Arizona's Whipple Mountains harbors a microbial community containing about 10(8) microorganisms g(-1) of varnish. Analyses of varnish phospholipid fatty acids and rRNA gene libraries reveal a community comprised of mostly Proteobacteria but also including Actinobacteria, eukaryota, and a few members of the Archaea. Rock varnish represents a significant niche for microbial colonization.

Examination of variation in ecological communities can lead to an understanding of the forces that structure communities, the consequences of change at the ecosystem level, and the relevant scales involved. This study details spatial and seasonal variability in the composition of nitrogen-fixing and cyanobacterial (i.e., oxygenic photosynthetic) functional groups of a benthic, hypersaline microbial mat from Salt Pond, San Salvador Island, Bahamas. This system shows extreme annual variability in the salinity of the overlying water and the extent of water coverage. Analysis of molecular variance and F(ST) tests of genetic differentiation of nifH and cyanobacterial 16S rRNA gene clone libraries allowed for changes at multiple taxonomic levels (i.e., above, below, and at the species level) to inform the conclusions regarding these functional groups. Composition of the nitrogen-fixing community showed significant seasonal changes related to salinity, while cyanobacterial composition showed no consistent seasonal pattern. Both functional groups exhibited significant spatial variation, changing with depth in the mat and horizontally with distance from the shoreline. The patterns of change suggest that cyanobacterial composition was more insensitive to water stress, and consequently, cyanobacteria dominated the nitrogen-fixing community during dry months but gave way to a more diverse community of diazotrophs in wet months. This seasonal pattern may allow the mat community to respond quickly to water-freshening events after prolonged dry conditions (system recovery) and maintain ecosystem function in the face of disturbance during the wet season (system resilience).

The acid waters (pH 2.7 to 3.4) originating from the Carnoulès mine tailings contain high concentrations of dissolved arsenic (80 to 350 mg.liter(-1)), iron (750 to 2,700 mg.liter(-1)), and sulfate (2,000 to 7,500 mg.liter(-1)). During the first 30 m of downflow in Reigous creek issuing from the mine tailings, 20 to 60% of the dissolved arsenic is removed by coprecipitation with Fe(III). The microbial communities along the creek have been characterized using terminal-restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene library analyses. The results indicate a low bacterial diversity in comparison with unpolluted water. Eighty percent of the sequences obtained are related to sequences from uncultured, newly described organisms or recently associated with acid mine drainage. As expected owing to the water chemistry, the sequences recovered are mainly related to bacteria involved in the geochemical Fe and S cycles. Among them, sequences related to uncultured TrefC4 affiliated with Gallionella ferruginea, a neutrophilic Fe-oxidizing bacterium, are dominant. The description of the bacterial community structure and its dynamics lead to a better understanding of the natural remediation processes occurring at this site.

Microbial communities in coastal subsurface sediments are scarcely investigated and have escaped attention so far. But since they are likely to play an important role in biogeochemical cycles, knowledge of their composition and ecological adaptations is important. Microbial communities in tidal sediments were investigated along the geochemical gradients from the surface down to a depth of 5.5 m. Most-probable-number (MPN) series were prepared with a variety of different carbon substrates, each at a low concentration, in combination with different electron acceptors such as iron and manganese oxides. These achieved remarkably high cultivation efficiencies (up to 23% of the total cell counts) along the upper 200 cm. In the deeper sediment layers, MPN counts dropped significantly. Parallel to the liquid enrichment cultures in the MPN series, gradient cultures with embedded sediment subcores were prepared as an additional enrichment approach. In total, 112 pure cultures were isolated; they could be grouped into 53 different operational taxonomic units (OTU). The isolates belonged to the Proteobacteria, "Bacteroidetes," "Fusobacteria," Actinobacteria, and "Firmicutes." Each cultivation approach yielded a specific set of isolates that in general were restricted to this single isolation procedure. Analysis of the enrichment cultures by PCR and denaturing gradient gel electrophoresis revealed an even higher diversity in the primary enrichments that was only partially reflected by the culture collection. The majority of the isolates grew well under anoxic conditions, by fermentation, or by anaerobic respiration with nitrate, sulfate, ferrihydrite, or manganese oxides as electron acceptors.

In a model drinking water distribution system characterized by a low assimilable organic carbon content (<10 microg/liter) and no disinfection, the bacterial community was identified by a phylogenetic analysis of rRNA genes amplified from directly extracted DNA and colonies formed on R2A plates. Biofilms of defined periods of age (14 days to 3 years) and bulk water samples were investigated. Culturable bacteria were associated with Proteobacteria and Bacteriodetes, whereas independently of cultivation, bacteria from 12 phyla were detected in this system. These included Acidobacteria, Nitrospirae, Planctomycetes, and Verrucomicrobia, some of which have never been identified in drinking water previously. A cluster analysis of the population profiles from the individual samples divided biofilms and bulk water samples into separate clusters (P = 0.027). Bacteria associated with Nitrospira moscoviensis were found in all samples and encompassed 39% of the sequenced clones in the bulk water and 25% of the biofilm community. The close association with Nitrospira suggested that a large part of the population had an autotrophic metabolism using nitrite as an electron donor. To test this hypothesis, nitrite was added to biofilm and bulk water samples, and the utilization was monitored during 15 days. A first-order decrease in nitrite concentration was observed for all samples with a rate corresponding to 0.5 x 10(5) to 2 x 10(5) nitrifying cells/ml in the bulk water and 3 x 10(5) cells/cm(2) on the pipe surface. The finding of an abundant nitrite-oxidizing microbial population suggests that nitrite is an important substrate in this system, potentially as a result of the low assimilable organic carbon concentration. This finding implies that microbial communities in water distribution systems may control against elevated nitrite concentrations but also contain large indigenous populations that are capable of assisting the depletion of disinfection agents like chloramines.

Active members of the bacterial community in the sediment of Lake Washington, with special emphasis on C1 utilizers, were identified by employing two complementary culture-independent approaches: reverse transcription of environmental mRNA and 16S rRNA combined with PCR (RT-PCR) and stable-isotope probing (SIP) of DNA with the 13C-labeled C1 substrates methanol, methylamine, formaldehyde, and formate. Analysis of RT-PCR-amplified fragments of 16S rRNA-encoding genes revealed that gammaproteobacterial methanotrophs belonging to Methylobacter and Methylomonas dominate the active methylotroph population, while only one other known methylotrophic lineage, Methylophilaceae, was detected via this approach. Analysis of RT-PCR-amplified functional genes, pmoA and fae, allowed detection of alphaproteobacterial (Methylosinus) and gammaproteobacterial (Methylobacter, Methylomonas, and Methylomicrobium) methanotrophs, methylotrophs of the genus Methylobacterium, and yet-unidentified proteobacteria. SIP experiments allowed detection of a broad variety of groups actively metabolizing C1 compounds. Comparisons between 16S rRNA gene pools amplified from [13C]DNA and from [12C]DNA revealed that the proportion of Methylophilus-related sequences increased in the presence of [13C]methanol, [13C]methylamine, and [13C]formaldehyde; Novosphingobium-related sequences were enriched in the presence of [13C]methanol; Gemmatimonadaceae-related sequences were enriched in the presence of [13C]formaldehyde and [13C]formate; and Xanthomonadaceae-related sequences were enriched in the presence of [13C]formate. Analysis of fae genes amplified from [13C]DNAs isolated from different microcosms revealed specific shifts in populations in response to a specific C1 compound: Methylosinus sequences dominated the [13C]methanol microcosm pool, and beta- and gammaproteobacterial sequences dominated the [13C]methylamine microcosm pool. The [13C]formaldehyde microcosm was dominated by betaproteobacterial sequences and by sequences of a nonaffiliated group, while the [13C]formate microcosm was dominated by alpha- and betaproteobacterial sequences. Overall, these data point toward the presence of a diverse population of active methylotrophs in Lake Washington sediments and toward the existence of yet-uncultivated organisms.

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA- and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.