Distribution and Diversity of Archaeal and Bacterial Ammonia Oxidizers in Salt Marsh Sediments

Diversity and abundance of ammonia-oxidizing Betaproteobacteria (β-AOB) and archaea (AOA) were investigated in a New England salt marsh at sites dominated by short or tall Spartina alterniflora (SAS and SAT sites, respectively) or Spartina patens (SP site). AOA amoA gene richness was higher than β-AOB amoA richness at SAT and SP, but AOA and β-AOB richness were similar at SAS. β-AOB amoA clone libraries were composed exclusively of Nitrosospira-like amoA genes. AOA amoA genes at SAT and SP were equally distributed between the water column/sediment and soil/sediment clades, while AOA amoA sequences at SAS were primarily affiliated with the water column/sediment clade. At all three site types, AOA were always more abundant than β-AOB based on quantitative PCR of amoA genes. At some sites, we detected 10⁹ AOA amoA gene copies g of sediment⁻¹. Ratios of AOA to β-AOB varied over 2 orders of magnitude among sites and sampling dates. Nevertheless, abundances of AOA and β-AOB amoA genes were highly correlated. Abundance of 16S rRNA genes affiliated with Nitrosopumilus maritimus, Crenarchaeota group I.1b, and pSL12 were positively correlated with AOA amoA abundance, but ratios of amoA to 16S rRNA genes varied among sites. We also observed a significant effect of pH on AOA abundance and a significant salinity effect on both AOA and β-ΑΟΒ abundance. Our results expand the distribution of AOA to salt marshes, and the high numbers of AOA at some sites suggest that salt marsh sediments serve as an important habitat for AOA.Nitrification, the sequential oxidation of ammonia to nitrite and nitrate, is a critical step in the nitrogen cycle and is mediated by a suite of phylogenetically and physiologically distinct microorganisms. The recent discovery of ammonia oxidation among Archaea (17, 38) has led to a dramatic shift in the current model of nitrification and to new questions of niche differentiation between putative ammonia-oxidizing Archaea (AOA) and the more-well-studied ammonia-oxidizing Betaproteobacteria (β-AOB). Based on surveys of 16S rRNA genes and archaeal amoA genes, it is evident that AOA occupy a wide range of niches (10), suggesting a physiologically diverse group of Archaea. Additionally, in studies where AOA and β-AOB were both targeted, AOA were typically more abundant than their bacterial counterparts (19, 21, 42). However, there are reports of β-AOB outnumbering AOA in estuarine systems (6, 33), suggesting a possible shift in competitive dominance under certain conditions.Patterns of β-AOB diversity in estuaries have been well characterized and appear to be regulated by similar mechanisms within geographically disparate systems (4, 11, 32). However, AOA distribution and their role in nitrification relative to β-AOB remain to be determined. A few studies have begun to address this question in different estuaries, but no unifying patterns or mechanisms have emerged. Although β-AOB have been well studied along estuarine salinity gradients (1, 3, 4, 7, 11, 13, 22, 33, 39) and recent studies have begun to address AOA in estuaries (1, 6, 22, 32, 33), few have investigated β-AOB in salt marshes (9), and none has included AOA.In this study, we investigated the distribution and abundance of AOA and β-AOB based on the distribution and abundance of amoA genes in salt marsh sediments dominated by different types of vegetation. Although we equate the presence of archaeal amoA genes with the genetic potential to oxidize ammonia, we acknowledge the possibility that all Archaea that have amoA genes may not all represent functional ammonia oxidizers. Vegetation patterns of New England salt marshes are strongly correlated with marsh elevation and are controlled by a combination of interspecific competition and tolerance to physico-chemical stress (28). The dominant grasses of New England salt marshes are Spartina alterniflora and Spartina patens, which typically grow as pure stands. S. alterniflora is found in two phenotypically distinct but genetically identical forms, a tall and a short growth form (34). The tall S. alterniflora grows to heights of 1 to 2 m and is typically found at the edges of the marsh and along creek banks (SAT sites), while the short-form S. alterniflora may reach heights of only 30 cm and is found in sites (SAS sites) slightly higher on the marsh where soil drainage is limited and conditions are more reduced compared to SAT sites (14). Conversely, S. patens, due to its lower tolerance of salt and more reduced conditions, is found in sites (SP sites) highest on the marsh, in areas that receive less flooding (5). Because the marsh is subjected to daily tidal fluctuations, most sites experience periods of anoxia, the degree of which depends on the marsh elevation. We hypothesized that ammonia-oxidizing communities in areas dominated by different marsh grasses would reflect the different edaphic conditions associated with each type of grass, due to differences in vertical zonation in the marsh.     

Responses of Ammonia-Oxidizing Bacterial and Archaeal Populations to Organic Nitrogen Amendments in Low-Nutrient Groundwater

To evaluate the potential for organic nitrogen addition to stimulate the in situ growth of ammonia oxidizers during a field scale bioremediation trial, samples collected from the Eastern Snake River Plain Aquifer in Idaho before, during, and after the addition of molasses and urea were subjected to PCR analysis of ammonia monooxygenase subunit A (amoA) genes. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) were present in all of the samples tested, with AOA amoA genes outnumbering AOB amoA genes in all of the samples. Following urea addition, nitrate levels rose and bacterial amoA copy numbers increased dramatically, suggesting that urea hydrolysis stimulated nitrification. Bacterial amoA diversity was limited to two Nitrosomonas phylotypes, whereas archaeal amoA analyses revealed 20 distinct operational taxonomic units, including several that were markedly different from all previously reported sequences. Results from this study demonstrate the likelihood of stimulating ammonia-oxidizing communities during field scale manipulation of groundwater conditions to promote urea hydrolysis.Subsurface calcite precipitation driven by microbial urea hydrolysis has been proposed as a means of remediating trace metal or radionuclide contaminants (e.g., strontium-90) that can be coprecipitated and retained in the solid phase (11, 12, 42). Urea hydrolysis generates carbonate alkalinity and raises pH, both of which promote calcite precipitation. However, another product of urea hydrolysis is ammonium, as shown in the following equation: In low-nutrient groundwater, the ammonium resulting from urea hydrolysis can have a number of fates, including uptake by nitrogen-limited microorganisms or transformation to nitrite by ammonia-oxidizing microorganisms. Microbial oxidation of ammonia is a net acid-yielding process. The resultant acidity from this reaction could inhibit calcite precipitation or promote destabilization of preexisting calcite, potentially liberating contaminants from the solid phase. In addition, the further transformation of nitrite by nitrite-oxidizing bacteria leads to the formation of nitrate, a regulated contaminant of drinking water.The first step of bacterial ammonia oxidation, the conversion of ammonia to hydroxylamine, is catalyzed by the membrane-bound enzyme ammonia monooxygenase. The gene coding for the catalytic α subunit of this enzyme, amoA, has proven to be an effective molecular marker for ammonia-oxidizing bacteria (AOB) (20, 34). All of the currently known chemoautotrophic AOB are associated with the Nitrosomonas and Nitrosospira genera within the Betaproteobacteria or the genus Nitrosococcus within the Gammaproteobacteria (15, 32). Although ammonia oxidation was long believed to be carried out exclusively by members of the domain Bacteria, considerable evidence now suggests that recently discovered ammonia-oxidizing archaea (AOA) (18) are key players in this critical step of the microbial nitrogen cycle (8).The archaeal amoA gene has been found in a wide range of environments (9; reviewed in references 8 and 31), and its expression has been documented in enrichment cultures (35) and soil microcosms (40), as well as in marine and terrestrial environments (21, 23). Reported quantitative PCR (qPCR) analyses of amoA in marine and terrestrial environments suggest that AOA typically outnumber AOB by orders of magnitude (23, 26, 44), and AOA abundance has also recently been shown to be highly correlated with water column ¹⁵NH₄⁺ oxidation rates (1). However, some recent studies have reported that AOB are more abundant under certain conditions (6, 27, 35, 43, 45).In an effort to better understand the fate of ammonium generated from urea hydrolysis, we monitored the abundance and diversity of bacterial and archaeal amoA genes during a field experiment designed to test stimulation of urea hydrolysis in groundwater. Dilute molasses and urea were sequentially introduced into a well in the Eastern Snake River Plain Aquifer (ESRPA) in Idaho (13). Previous laboratory experiments indicated that molasses, an inexpensive and commonly used bioremediation amendment (14), was effective in increasing overall microbial populations, as well as total ureolytic activity (13, 39). The ESRPA is a deep basalt aquifer and is considered oligotrophic (4, 22, 29); however, previous work has demonstrated the presence of ureolytic microbes in this environment (11, 13). Erwin et al. also reported evidence of AOB during the analysis of methane monooxygenase clone libraries from ESRPA samples (7), but in general, the structure and function of ammonia-oxidizing microbial communities (and especially AOA) in deep aquifers like the ESRPA have been relatively unexplored.     

Vertical Distribution of Ammonia-Oxidizing Crenarchaeota and Methanogens in the Epipelagic Waters of Lake Kivu (Rwanda-Democratic Republic of the Congo)

Four stratified basins in Lake Kivu (Rwanda-Democratic Republic of the Congo) were sampled in March 2007 to investigate the abundance, distribution, and potential biogeochemical role of planktonic archaea. We used fluorescence in situ hybridization with catalyzed-reported deposition microscopic counts (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) fingerprinting, and quantitative PCR (qPCR) of signature genes for ammonia-oxidizing archaea (16S rRNA for marine Crenarchaeota group 1.1a [MCG1] and ammonia monooxygenase subunit A [amoA]). Abundance of archaea ranged from 1 to 4.5% of total DAPI (4′,6-diamidino-2-phenylindole) counts with maximal concentrations at the oxic-anoxic transition zone (∼50-m depth). Phylogenetic analysis of the archaeal planktonic community revealed a higher level of richness of crenarchaeal 16S rRNA gene sequences (21 of the 28 operational taxonomic units [OTUs] identified [75%]) over euryarchaeotal ones (7 OTUs). Sequences affiliated with the kingdom Euryarchaeota were mainly recovered from the anoxic water compartment and mostly grouped into methanogenic lineages (Methanosarcinales and Methanocellales). In turn, crenarchaeal phylotypes were recovered throughout the sampled epipelagic waters (0- to 100-m depth), with clear phylogenetic segregation along the transition from oxic to anoxic water masses. Thus, whereas in the anoxic hypolimnion crenarchaeotal OTUs were mainly assigned to the miscellaneous crenarchaeotic group, the OTUs from the oxic-anoxic transition and above belonged to Crenarchaeota groups 1.1a and 1.1b, two lineages containing most of the ammonia-oxidizing representatives known so far. The concomitant vertical distribution of both nitrite and nitrate maxima and the copy numbers of both MCG1 16S rRNA and amoA genes suggest the potential implication of Crenarchaeota in nitrification processes occurring in the epilimnetic waters of the lake.Lake Kivu is a meromictic lake located in the volcanic region between Rwanda and the Democratic Republic of the Congo and is the smallest of the African Great Rift Lakes. The monimolimnion of the lake contains a large amount of dissolved CO₂ and methane (300 km³ and 60 km³, respectively) as a result of geological and biological activity (24, 73, 85). This massive accumulation converts Lake Kivu into one of the largest methane reservoirs in the world and into a unique ecosystem for geomicrobiologists interested in the methane cycle and in risk assessment and management (34, 71, 72, 85). Comprehensive studies on the diversity and activity of planktonic populations of both large and small eukaryotes and their trophic interplay operating in the epilimnetic waters of the lake are available (33, 39, 49). Recent surveys have also provided a deeper insight into the seasonal variations of photosynthetic and heterotrophic picoplankton (67, 68), although very few data exist on the composition, diversity, and spatial distribution of bacterial and archaeal communities. In this regard, the studies conducted so far of the bacterial/archaeal ecology in Lake Kivu have been mostly focused on the implications on the methane cycle (34, 73), but none have addressed the presence and distribution of additional archaeal populations in the lake.During the last few years, microbial ecology studies carried out in a wide variety of habitats have provided compelling evidence of the ubiquity and abundance of mesophilic archaea (4, 10, 13, 19). Moreover, the discovery of genes encoding enzymes related to nitrification and denitrification in archaeal metagenomes from soil and marine waters (29, 86, 88) and the isolation of the first autotrophic archaeal nitrifier (40) demonstrated that some archaeal groups actively participate in the carbon and nitrogen cycles (56, 64, 69). In relation to aquatic environments, genetic markers of ammonia-oxidizing archaea (AOA) of the marine Crenarchaeota group 1.1a (MCG1) have consistently been found in water masses of several oceanic regions (6, 14, 17, 26, 28, 30, 37, 42, 51, 52, 89), estuaries (5, 9, 26, 53), coastal aquifers (26, 66), and stratified marine basins (15, 41, 44). Although less information is available for freshwater habitats, recent studies carried out in oligotrophic high-mountain and arctic lakes showed an important contribution of AOA in both the planktonic and the neustonic microbial assemblages (4, 61, 89).The oligotrophic nature of Lake Kivu and the presence of a well-defined redoxcline may provide an optimal niche for the development of autotrophic AOA populations. Unfortunately, no studies of the involvement of microbial planktonic populations in cycling nitrogen in the lake exist, and only data on the distribution of dissolved inorganic nitrogen species in relation to phytoplankton ecology (67, 68) and nutrient loading are available (54, 58). Our goals here were to ascertain whether or not archaeal populations other than methane-related lineages were relevant components of the planktonic microbial community and to determine whether the redox gradient imposed by the oxic-anoxic interphase acts as a threshold for their vertical distribution in epipelagic waters (0- to 100-m depth). To further explore the presence and potential activity of nitrifying archaeal populations in Lake Kivu, samples were analyzed for the abundance and vertical distribution of signature genes for these microorganisms, i.e., the 16S rRNA of MCG1 and the ammonia monooxygenase subunit A (amoA) gene by quantitative PCR.     

Effect of Lake Trophic Status and Rooted Macrophytes on Community Composition and Abundance of Ammonia-Oxidizing Prokaryotes in Freshwater Sediments

Communities of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in freshwater sediments and those in association with the root system of the macrophyte species Littorella uniflora, Juncus bulbosus, and Myriophyllum alterniflorum were compared for seven oligotrophic to mesotrophic softwater lakes and acidic heathland pools. Archaeal and bacterial ammonia monooxygenase alpha-subunit (amoA) gene diversity increased from oligotrophic to mesotrophic sites; the number of detected operational taxonomic units was positively correlated to ammonia availability and pH and negatively correlated to sediment C/N ratios. AOA communities could be grouped according to lake trophic status and pH; plant species-specific communities were not detected, and no grouping was apparent for AOB communities. Relative abundance, determined by quantitative PCR targeting amoA, was always low for AOB (<0.05% of all prokaryotes) and slightly higher for AOA in unvegetated sediment and AOA in association with M. alterniflorum (0.01 to 2%), while AOA accounted for up to 5% in the rhizospheres of L. uniflora and J. bulbosus. These results indicate that (i) AOA are at least as numerous as AOB in freshwater sediments, (ii) aquatic macrophytes with substantial release of oxygen and organic carbon into their rhizospheres, like L. uniflora and J. bulbosus, increase AOA abundance; and (iii) AOA community composition is generally determined by lake trophy, not by plant species-specific interactions.Oxygen release from the roots of macrophyte species such as Littorella uniflora (L.) Asch. (shore weed), Lobelia dortmanna L. (water lobelia), and Glyceria maxima (Hartm.) Holmb. (reed sweet grass) stimulates nitrification and coupled nitrification-denitrification in the rhizosphere compared to that in unvegetated sediment (2, 36, 40). These interactions are of high ecological relevance especially in oligotrophic systems, since enhanced nitrogen loss due to rhizosphere-associated denitrification can retard natural eutrophication and succession of plant communities (1). While the microbial communities involved in coupled nitrification-denitrification have been well studied in rice paddy soils (7, 11), less information is available for natural freshwater sediments, especially those from oligotrophic lakes (2, 26).The first key step of coupled nitrification-denitrification, the oxidation of ammonia to nitrite, is catalyzed by two groups of prokaryotes—the ammonia-oxidizing bacteria (AOB) (24) and the only recently recognized ammonia-oxidizing archaea (AOA) (22). For both groups, the gene encoding the alpha-subunit of ammonia monooxygenase (amoA) has been widely used as a functional marker to analyze their community compositions (15, 25); recent studies demonstrated the ubiquity of AOA and their predominance over AOB in a broad range of environments (32, 38). AOA, but not AOB, were also strongly enriched in the rhizosphere of the freshwater macrophyte Littorella uniflora in a mesotrophic Danish lake, suggesting that AOA were primarily responsible for increased rates of nitrification in the rhizosphere of this plant species (19). Moreover, ammonia oxidizer communities differed between rhizosphere and unvegetated sediment, indicating a plant-specific effect on AOA and AOB community composition. The objectives of this study were therefore to test whether (i) AOA generally predominate over AOB in freshwater sediments and especially in macrophyte rhizospheres and (ii) macrophytes have species-specific effects on abundance and community composition of AOA and AOB in rhizosphere sediments and on root surfaces.To address these questions, two shallow heathland pools and five lakes in Denmark and Germany, ranging from low-pH and dystrophic sites to neutral-pH and oligotrophic and mesotrophic sites, were chosen, and three macrophyte species—Littorella uniflora, Juncus bulbosus L. (bulbous rush), and Myriophyllum alterniflorum DC. (alternate water milfoil)—were selected as model systems. These plant species differ in nitrogen nutrition, extent of radial oxygen loss, and lifestyle, presumably resulting in differential, plant species-specific effects on rhizosphere- and root-associated AOA and AOB communities. L. uniflora prefers nitrate as the nitrogen source, while J. bulbosus prefers ammonium (41, 45); oxygen release is high to moderate from the roots of L. uniflora and J. bulbosus (9, 12) but is minor from the roots of M. alterniflorum (M. Herrmann, P. Stief, and A. Schramm, unpublished results); L. uniflora and J. bulbosus remain photosynthetically active throughout the year, while only the below-ground parts of M. alterniflorum are retained during winter.Rhizosphere sediments and roots from each plant species were sampled from three different sites per species, and unvegetated sediment was obtained from all seven sites. The comparison of samples from these different sites and compartments (rhizosphere, root surface, unvegetated sediment) allowed an evaluation of the importance of plant species relative to that of environmental conditions related to lake trophic status and pH on ammonia oxidizer communities.     

High Abundance of Ammonia-Oxidizing Archaea in Coastal Waters,Determined Using a Modified DNA Extraction Method

Molecular characterizations of environmental microbial populations based on recovery and analysis of DNA generally assume efficient or unbiased extraction of DNA from different sample matrices and microbial groups. Appropriate controls to verify this basic assumption are rarely included. Here three different DNA extractions, performed with two commercial kits (FastDNA and UltraClean) and a standard phenol-chloroform method, and two alternative filtration methods (Sterivex and 25-mm-diameter polycarbonate filters) were evaluated, using the addition of Nitrosopumilus maritimus cells to track the recovery of DNA from marine Archaea. After the comparison, a simplified phenol-chloroform extraction method was developed and shown to be significantly superior, in terms of both the recovery and the purity of DNA, to other protocols now generally applied to environmental studies. The simplified and optimized method was used to quantify ammonia-oxidizing Archaea at different depth intervals in a fjord (Hood Canal) by quantitative PCR. The numbers of Archaea increased with depth, often constituting as much as 20% of the total bacterial community.Efficient DNA extraction from environmental samples is fundamental to many culture-independent characterizations (10). Thus, there was an early and concerted effort to establish appropriate methods of DNA extraction from different types of environmental samples (14, 19, 25, 30, 34, 43, 47). DNA extraction efficiency is particularly important for quantitative PCR (qPCR), because poor DNA extraction efficiency results in the underestimation of gene copy numbers in the samples examined (6, 42).Most methodological developments addressed DNA extraction from soil and sediment samples, with fewer comparative studies of the efficiency of collection and extraction from water samples (4, 13, 40). In part, a methodological focus on soils reflected the simplicity of filtration to collect aquatic populations and the generally good recovery of DNA from the Gram-negative bacteria making up a significant fraction of aquatic communities. However, small Archaea are now known to constitute a substantial fraction of the prokaryotic populations in marine and terrestrial systems (2, 7, 9, 20, 26, 31, 33, 45). Since the archaeal cell wall and membrane structures are distinct from those of bacteria, there is no assurance that commonly used extraction methods are adequate. With increasing reliance on commercially available bead-beating-type DNA extraction kits, these methods are now often used for different water samples (1, 5-7, 14, 19, 36). Although most protocols incorporate mechanical disruption to ensure more-uniform extraction than is possible by using methods that rely entirely on enzymatic digestion and/or chemical disruption (4, 13, 40), the suitability of these protocols for the concerted analysis of archaeal and bacterial populations has not been fully evaluated.In the studies reported here, the recently isolated marine archaeon Nitrosopumilus maritimus strain SCM1 (22) was therefore used as a reference standard for evaluation of the commonly employed DNA extraction methods by using qPCR. This archaeon was then used as a reference for the development of a simple, rapid, and efficient method of extracting DNA from both archaeal and bacterial cells. The modified protocol was subsequently employed to characterize the vertical distribution of ammonia-oxidizing Archaea in a fjord (Hood Canal) in Puget Sound (Washington State), revealing a high fractional representation of Archaea relative to Bacteria not observed previously in coastal waters.     

Diversity,Abundance, and Spatial Distribution of Sediment Ammonia-Oxidizing Betaproteobacteria in Response to Environmental Gradients and Coastal Eutrophication in Jiaozhou Bay,China

Ongoing anthropogenic eutrophication of Jiaozhou Bay offers an opportunity to study the influence of human activity on bacterial communities that drive biogeochemical cycling. Nitrification in coastal waters appears to be a sensitive indicator of environmental change, suggesting that function and structure of the microbial nitrifying community may be associated closely with environmental conditions. In the current study, the amoA gene was used to unravel the relationship between sediment aerobic obligate ammonia-oxidizing Betaproteobacteria (Beta-AOB) and their environment in Jiaozhou Bay. Protein sequences deduced from amoA gene sequences grouped within four distinct clusters in the Nitrosomonas lineage, including a putative new cluster. In addition, AmoA sequences belonging to three newly defined clusters in the Nitrosospira lineage were also identified. Multivariate statistical analyses indicated that the studied Beta-AOB community structures correlated with environmental parameters, of which nitrite-N and sediment sand content had significant impact on the composition, structure, and distribution of the Beta-AOB community. Both amoA clone library and quantitative PCR (qPCR) analyses indicated that continental input from the nearby wastewater treatment plants and polluted rivers may have significant impact on the composition and abundance of the sediment Beta-AOB assemblages in Jiaozhou Bay. Our work is the first report of a direct link between a sedimentological parameter and the composition and distribution of the sediment Beta-AOB and indicates the potential for using the Beta-AOB community composition in general and individual isolates or environmental clones in the Nitrosomonas oligotropha lineage in particular as bioindicators and biotracers of pollution or freshwater or wastewater input in coastal environments.Nitrification, the oxidation of ammonia to nitrate via nitrite, plays a critical role in the biogeochemical cycling of nitrogen and the formation of the large deep-sea nitrate reservoir (37, 46, 51). Because the N cycle may affect the global C cycle, shifts in N transformation processes may also affect the climate (30, 40). Nitrification is an important bioremediation process in human-perturbed estuarine and coastal ecosystems, where it may serve as a detoxification process for excess ammonia (14). If coupled to classical denitrification or anaerobic ammonium oxidation (anammox), these processes may remove most of the anthropogenic N pollution (51, 81). Bacterial nitrifiers may also cooxidize a variety of xenobiotic compounds (3, 49). On the other hand, nitrification may lead to enhanced production of the potent greenhouse gases nitric oxide (NO) and nitrous oxide (N₂O) (15). Because the input of excess ammonia stimulates the growth of ammonia-oxidizing microorganisms, research in coastal environments and ecosystems increasingly includes the study of microbial communities involved in nitrification.Marine nitrification is performed by chemolithoautotrophic proteobacteria and the newly discovered ammonia-oxidizing archaea (AOA) (47); nevertheless, reliable information on the individual contributions of each cohort to the process is still lacking (72) and the contributions likely vary in different environments (33, 51, 93). Beta- and gammaproteobacterial aerobic obligate ammonia-oxidizing bacteria (AOB) are known to catalyze the oxidization of ammonia to nitrite, the first and rate-limiting step of nitrification (3). Because of their monophyletic nature, diversity, and important environmental functionality, the betaproteobacterial AOB (Beta-AOB) have served as a model system in the study of fundamental questions in microbial ecology, including microbial community structure, distribution, activity, and environmental response (9, 49, 92).The growth of AOB is slow, and present isolates represent only a fraction of their natural diversity. Culture-independent molecular methods provide a more convenient and accurate approach for community analyses (76, 92). All AOB genomes contain at least one cluster of amoCAB genes encoding functional ammonia monooxygenase (AMO), which catalyzes the oxidation of ammonia to hydroxylamine (4). Because AmoA- and 16S rRNA-based phylogenies are congruent (73), the amoA gene has been extensively used as a molecular marker to explore and characterize the structure and diversity of AOB communities in a variety of estuarine and coastal environments (9, 10, 11, 13, 27, 32, 36, 43, 68, 88). Some of these studies indicated that local environmental factors such as salinity, pH, ammonium, and O₂ concentrations might be drivers for the formation of distinct AOB assemblages, in which individual lineages may have evolved differential ecophysiological adaptivity (4, 73, 85). Furthermore, differences in AmoA sequences may correlate with differences in isotopic discrimination during ammonia oxidation, implicating function-specific ammonia monooxygenases (15). Therefore, the AmoA sequences may provide information about the structure and composition of the AOB communities and their ecological function and response to environmental complexity and variability. Despite long-standing efforts, a complete understanding of these relationships is still lacking (9), especially in complex environments such as anthropogenic activity-impacted coastal areas.China consumes more than 20 million tons of N fertilizer each year, leading to a significant increase of coastal N pollution (38). Thus, the China coast is an important location for intense N biogeochemical cycling. Jiaozhou Bay is a large semienclosed water body of the temperate Yellow Sea in China. Eutrophication has become its most serious environmental problem, along with red tides, species loss, and contamination with toxic chemicals and harmful microbes (21, 23, 24, 28, 82, 91). In similar environments with a high input of nitrogenous compounds, surface sediment is a major site for nitrification due to a relatively high AOB abundance and activity (79).Although the China coast is important in N cycling and in related environmental and climatic issues, surprisingly very little is known, especially about the microbial processes and functions involved. On a global scale, it is currently not well understood how the AOB community structure, abundance, and distribution respond to coastal eutrophication, though partial knowledge is emerging (36, 49, 89). Recent studies indicated that spatial distribution and structure of the sediment AOA community could be influenced by a variety of environmental factors, of which continental input may play important roles in estuary and continental margin systems (22, 26). The sediment AOA community may serve as useful biotracers and bioindicators of specific environmental disturbance. Likewise, the sediment AOB community may also serve as biotracers or bioindicators of continental influence, such as eutrophication in coastal environments. In this study, the bacterial functional marker gene amoA was employed to test this hypothesis in the eutrophied Jiaozhou Bay.     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

Phylogenetic Analysis Indicates Evolutionary Diversity and Environmental Segregation of Marine Podovirus DNA Polymerase Gene Sequences

The distribution of viral genotypes in the ocean and their evolutionary relatedness remain poorly constrained. This paper presents data on the genetic diversity and evolutionary relationships of 1.2-kb DNA polymerase (pol) gene fragments from podoviruses. A newly designed set of PCR primers was used to amplify DNA directly from coastal sediment and water samples collected from inlets adjacent to the Strait of Georgia, British Columbia, Canada, and from the northeastern Gulf of Mexico. Restriction fragment length polymorphism analysis of 160 cloned PCR products revealed 29 distinct operational taxonomic units (OTUs), with OTUs within a site typically being more similar than those among sites. Phylogenetic analysis of the DNA pol gene fragments demonstrated high similarity between some environmental sequences and sequences from the marine podoviruses roseophage SIO1 and cyanophage P60, while others were not closely related to sequences from cultured phages. Interrogation of the CAMERA database for sequences from metagenomics data demonstrated that the amplified sequences were representative of the diversity of podovirus pol sequences found in marine samples. Our results indicate high genetic diversity within marine podovirus communities within a small geographic region and demonstrate that the diversity of environmental polymerase gene sequences for podoviruses is far more extensive than previously recognized.Marine viruses are the most abundant (41) and diverse (2, 6) biological entities in the ocean. They affect community composition by causing the lysis of specific subsets of the microbial community (22, 28, 46, 47) and, by killing numerically dominant host taxa, may influence species evenness and richness (24, 28, 43, 50). Despite the abundance of bacteriophages in marine systems and their important roles in marine microbial composition, little is known about the distribution and diversity of specific groups of marine viruses. However, most marine bacteriophage isolates are tailed phages (3) belonging to the order Caudovirales (27), which comprises the families Myoviridae, Podoviridae, and Siphoviridae.Podoviruses are classified into several groups (e.g., T7-like, P22-like, and phi-29-like) based on genome size, genome arrangement, and shared genes and can be readily isolated from seawater (11, 16, 42, 45). Genomic analysis of roseophage SIO1 (33), cyanophage P60 (7), vibriophage VpV262 (21), and cyanophage PSSP7 (40) suggests that many of the isolates are T7-like. Despite the apparently wide distribution of podoviruses in the sea, and their potential importance as agents of microbial mortality, there has been little effort to explore their diversity.Sequence analysis of representative genes is one approach that has been used to examine the genetic diversity of specific groups of marine viruses. For example, homologues for structural genes (g20 and g23) found in T4-like phages are found in some marine myoviruses (18, 20) and have been used to examine the distribution, diversity, and evolutionary relationships among marine myoviruses (12, 14, 17, 37, 38, 49). Other studies have used DNA polymerase (pol) to examine the diversity of viruses infecting eukaryotic phytoplankton (8, 38) and have shown that phylogenies constructed with this gene are congruent with established viral taxonomy (9, 36, 37).Although it is not universally present, family A DNA pol is a good target for examining the diversity of podoviruses (4). Our study presents a newly designed set of PCR primers that amplify a longer fragment of the DNA polymerase from a much larger suite of podoviruses and shows that the diversity within marine podoviruses as revealed by DNA pol sequences is far greater than previously realized.     

In Situ Production of Crenarchaeol in Two California Hot Springs

Crenarchaeol, a membrane-spanning glycerol dialkyl glycerol tetraether (GDGT) containing a cyclohexane moiety in addition to four cyclopentane moieties, was originally hypothesized to be synthesized exclusively by the mesophilic Crenarchaeota. Recent studies reporting the occurrence of crenarchaeol in hot springs and as a membrane constituent of the recently isolated thermophilic crenarchaeote “Candidatus Nitrosocaldus yellowstonii,” however, have raised questions regarding its taxonomic distribution and function. To determine whether crenarchaeol in hot springs is indeed synthesized by members of the Archaea in situ or is of allochthonous origin, we quantified crenarchaeol present in the form of both intact polar lipids (IPLs) and core lipids in sediments of two California hot springs and in nearby soils. IPL-derived crenarchaeol (IPL-crenarchaeol) was found in both hot springs and soils, suggesting in situ production of this GDGT over a wide temperature range (12°C to 89°C). Quantification of archaeal amoA gene abundance by quantitative PCR showed a good correspondence with IPL-crenarchaeol, suggesting that it was indeed derived from living cells and that crenarchaeol-synthesizing members of the Archaea in our samples may also be ammonia oxidizers.Numerous groups of the Archaea synthesize isoprenoid glycerol dialkyl glycerol tetraethers (GDGTs) as a major component of their core membrane lipids, which can contain up to eight cyclopentane moieties (e.g., see reference 7) (Fig. ​(Fig.1).1). An increase in the number of cyclopentane moieties results in denser packing of membrane lipids, allowing for the maintenance of both cellular membrane integrity at high temperatures and stable proton gradients under low-pH conditions (8). This biophysical characteristic is hypothesized to be among those traits essential for the survival and persistence of the Archaea in the “extreme” environments in which they are commonly found (42). GDGTs are synthesized by a large number of cultivated members of the Archaea (see overviews in references 20 and 34), and in nature, they are abundant in hot springs (24, 25, 34, 46), for example, where members of the Archaea are known to thrive at high temperatures and over a wide pH range (3, 21).Open in a separate windowFIG. 1.Structures of GDGTs referred to in the text. “IS,” C₄₆ internal standard.Crenarchaeol is unique among the GDGTs in that it contains a cyclohexane moiety in addition to four cyclopentane moieties (Fig. ​(Fig.1).1). It was first reported in large abundances from Holocene and ancient sediments collected from various marine settings as supporting evidence for the widespread distribution of low-temperature relatives of the hyperthermophilic Archaea (31). It was later proposed that crenarchaeol was synthesized exclusively by marine group I Crenarchaeota (36), a hypothesis further supported by core lipid analysis of the mesophilic marine group I.1a crenarchaeotes “Cenarchaeum symbiosum” (38) and “Candidatus Nitrosopumilus maritimus” SCM1 (30), which showed that both of these organisms synthesize crenarchaeol at moderate temperatures. In addition to this, the apparent absence of crenarchaeol in cultures of (hyper)thermophilic members of the Archaea (see overviews in references 20 and 34) and molecular modeling (8, 37) led to the hypothesis that crenarchaeol decreases lipid density, effectively allowing archaeal membranes composed of membrane-spanning GDGTs to function at mesophilic temperatures (37). Hence, crenarchaeol synthesis was thought to be instrumental in the evolution and radiation of mesophilic Crenarchaeota from thermophilic habitats (17).Recent studies, however, have reported the occurrence of crenarchaeol in hot springs with temperatures of up to 86.5°C (24, 25, 34, 46). That work has been debated to some extent, as there exists the potential for the allochtonous input of fossilized lipid material from weathering of nearby soils where mesophilic Crenarchaeota may thrive: Schouten et al. (34) previously found large relative amounts of specific soil bacterium biomarkers in tandem with crenarchaeol in Yellowstone hot springs. In contrast, Reigstad et al. (28) reported the occurrence of crenarchaeol in the absence of soil-specific biomarkers in Icelandic hot springs. Furthermore, the recently isolated thermophilic crenarchaeote “Candidatus Nitrosocaldus yellowstonii” was shown to synthesize crenarchaeol at a growth temperature of 72°C (6).Core lipids (CLs) that occur in biological membranes generally contain polar head groups such as sugars and phosphates, which are rapidly cleaved upon cell senescence (10, 44). The loss of head groups from intact polar lipids (IPLs) leaves relatively recalcitrant CLs to accumulate in the environment over time as fossil biomarkers. Therefore, depending on the extraction and/or analytical protocols, CLs present in environmental lipid extracts may be derived from both living cells and fossil biomass, including a mixture of both CL-derived GDGTs (CL-GDGTs) and IPL-derived GDGTs (IPL-GDGTs). Most studies of the presence of crenarchaeol in hot springs reported to date have analyzed directly extracted CL-crenarchaeol or CL-crenarchaeol released by the acid hydrolysis of Bligh-Dyer IPL lipid extracts, i.e., without prior separation of CL-GDGTs from IPL-GDGTs (24, 25, 28, 34, 46). In these cases, the reported GDGT distributions represent an integrated signal of both “living” and fossilized material, rendering it impossible to distinguish what proportion (if any) of the observed crenarchaeol was derived from local living archaeal communities. Thus, the in situ production of crenarchaeol in hot springs and its importance relative to that of the in situ production of other archaeal GDGTs remain uncertain.Here we have used a recently described chromatographic method (22, 26) to separately quantify the potential contributions of both in situ-produced and fossilized crenarchaeol (as well as other archaeal GDGTs) in two Californian hot springs and their surrounding soils. In addition, we have quantified the amounts of archaeal amoA and archaeal 16S rRNA gene copies from one site to make quantitative comparisons between gene abundance and IPL-GDGT concentrations.     

The Single-Stranded DNA Genome of Novel Archaeal Virus Halorubrum Pleomorphic Virus 1 Is Enclosed in the Envelope Decorated with Glycoprotein Spikes

Only a few archaeal viruses have been subjected to detailed structural analyses. Major obstacles have been the extreme conditions such as high salinity or temperature needed for the propagation of these viruses. In addition, unusual morphotypes of many archaeal viruses have made it difficult to obtain further information on virion architectures. We used controlled virion dissociation to reveal the structural organization of Halorubrum pleomorphic virus 1 (HRPV-1) infecting an extremely halophilic archaeal host. The single-stranded DNA genome is enclosed in a pleomorphic membrane vesicle without detected nucleoproteins. VP4, the larger major structural protein of HRPV-1, forms glycosylated spikes on the virion surface and VP3, the smaller major structural protein, resides on the inner surface of the membrane vesicle. Together, these proteins organize the structure of the membrane vesicle. Quantitative lipid comparison of HRPV-1 and its host Halorubrum sp. revealed that HRPV-1 acquires lipids nonselectively from the host cell membrane, which is typical of pleomorphic enveloped viruses.In recent years there has been growing interest in viruses infecting hosts in the domain Archaea (43). Archaeal viruses were discovered 35 years ago (52), and today about 50 such viruses are known (43). They represent highly diverse virion morphotypes in contrast to the vast majority (96%) of head-tail virions among the over 5,000 described bacterial viruses (1). Although archaea are widespread in both moderate and extreme environments (13), viruses have been isolated only for halophiles and anaerobic methanogenes of the kingdom Euryarchaeota and hyperthermophiles of the kingdom Crenarchaeota (43).In addition to soil and marine environments, high viral abundance has also been detected in hypersaline habitats such as salterns (i.e., a multipond system where seawater is evaporated for the production of salt) (19, 37, 50). Archaea are dominant organisms at extreme salinities (36), and about 20 haloarchaeal viruses have been isolated to date (43). The majority of these are head-tail viruses, whereas electron microscopic (EM) studies of highly saline environments indicate that the two other described morphotypes, spindle-shaped and round particles, are the most abundant ones (19, 37, 43). Thus far, the morphological diversity of the isolated haloarchaeal viruses is restricted compared to viruses infecting hyperthermophilic archaea, which are classified into seven viral families (43).All of the previously described archaeal viruses have a double-stranded DNA (dsDNA) genome (44). However, a newly characterized haloarchaeal virus, Halorubrum pleomorphic virus 1 (HRPV-1), has a single-stranded DNA (ssDNA) genome (39). HRPV-1 and its host Halorubrum sp. were isolated from an Italian (Trapani, Sicily) solar saltern. Most of the studied haloarchaeal viruses lyse their host cells, but persistent infections are also typical (40, 44). HRPV-1 is a nonlytic virus that persists in the host cells. In liquid propagation, nonsynchronous infection cycles of HRPV-1 lead to continuous virus production until the growth of the host ceases, resulting in high virus titers in the growth medium (39).The pleomorphic virion of HRPV-1 represents a novel archaeal virus morphotype constituted of lipids and two major structural proteins VP3 (11 kDa) and VP4 (65 kDa). The genome of HRPV-1 is a circular ssDNA molecule (7,048 nucleotides [nt]) containing nine putative open reading frames (ORFs). Three of them are confirmed to encode structural proteins VP3, VP4, and VP8, which is a putative ATPase (39). The ORFs of the HRPV-1 genome show significant similarity, at the amino acid level, to the minimal replicon of plasmid pHK2 of Haloferax sp. (20, 39). Furthermore, an ∼4-kb region, encoding VP4- and VP8-like proteins, is found in the genomes of two haloarchaea, Haloarcula marismortui and Natronomonas pharaonis, and in the linear dsDNA genome (16 kb) of spindle-shaped haloarchaeal virus His2 (39). The possible relationship between ssDNA virus HRPV-1 and dsDNA virus His2 challenges the classification of viruses, which is based on the genome type among other criteria (15, 39).HRPV-1 is proposed to represent a new lineage of pleomorphic enveloped viruses (39). A putative representative of this lineage among bacterial viruses might be L172 of Acholeplasma laidlawii (14). The enveloped virion of L172 is pleomorphic, and the virus has a circular ssDNA genome (14 kb). In addition, the structural protein pattern of L172 with two major structural proteins, of 15 and 53 kDa, resembles that of HRPV-1.The structural approach has made it possible to reveal relationships between viruses where no sequence similarity can be detected. It has been realized that several icosahedral viruses infecting hosts in different domains of life share common virion architectures and folds of their major capsid proteins. These findings have consequences for the concept of the origin of viruses. A viral lineage hypothesis predicts that viruses within the same lineage may have a common ancestor that existed before the separation of the cellular domains of life (3, 5, 8, 26). Currently, limited information is available on the detailed structures of viruses infecting archaea. For example, the virion structures of nontailed icosahedral Sulfolobus turreted icosahedral virus (STIV) and SH1 have been determined (21, 23, 46). However, most archaeal viruses represent unusual, sometimes nonregular, morphotypes (43), which makes it difficult to apply structural methods that are based on averaging techniques.A biochemical approach, i.e., controlled virion dissociation, gives information on the localization and interaction of virion components. In the present study, controlled dissociation was used to address the virion architecture of HRPV-1. A comparative lipid analysis of HRPV-1 and its host was also carried out. Our results show that the unique virion type is composed of a flexible membrane decorated with the glycosylated spikes of VP4 and internal membrane protein VP3. The circular ssDNA genome resides inside the viral membrane vesicle without detected association to any nucleoproteins.     

Haloferax volcanii Flagella Are Required for Motility but Are Not Involved in PibD-Dependent Surface Adhesion

Although the genome of Haloferax volcanii contains genes (flgA1-flgA2) that encode flagellins and others that encode proteins involved in flagellar assembly, previous reports have concluded that H. volcanii is nonmotile. Contrary to these reports, we have now identified conditions under which H. volcanii is motile. Moreover, we have determined that an H. volcanii deletion mutant lacking flagellin genes is not motile. However, unlike flagella characterized in other prokaryotes, including other archaea, the H. volcanii flagella do not appear to play a significant role in surface adhesion. While flagella often play similar functional roles in bacteria and archaea, the processes involved in the biosynthesis of archaeal flagella do not resemble those involved in assembling bacterial flagella but, instead, are similar to those involved in producing bacterial type IV pili. Consistent with this observation, we have determined that, in addition to disrupting preflagellin processing, deleting pibD, which encodes the preflagellin peptidase, prevents the maturation of other H. volcanii type IV pilin-like proteins. Moreover, in addition to abolishing swimming motility, and unlike the flgA1-flgA2 deletion, deleting pibD eliminates the ability of H. volcanii to adhere to a glass surface, indicating that a nonflagellar type IV pilus-like structure plays a critical role in H. volcanii surface adhesion.To escape toxic conditions or to acquire new sources of nutrients, prokaryotes often depend on some form of motility. Swimming motility, a common means by which many bacteria move from one place to another, usually depends on flagellar rotation to propel cells through liquid medium (24, 26, 34). These motility structures are also critical for the effective attachment of bacteria to surfaces.As in bacteria, rotating flagella are responsible for swimming motility in archaea, and recent studies suggest that archaea, like bacteria, also require flagella for efficient surface attachment (37, 58). However, in contrast to bacterial flagellar subunits, which are translocated via a specialized type III secretion apparatus, archaeal flagellin secretion and flagellum assembly resemble the processes used to translocate and assemble the subunits of bacterial type IV pili (34, 38, 54).Type IV pili are typically composed of major pilins, the primary structural components of the pilus, and several minor pilin-like proteins that play important roles in pilus assembly or function (15, 17, 46). Pilin precursor proteins are transported across the cytoplasmic membrane via the Sec translocation pathway (7, 20). Most Sec substrates contain either a class I or a class II signal peptide that is cleaved at a recognition site that lies subsequent to the hydrophobic portion of the signal peptide (18, 43). However, the precursors of type IV pilins contain class III signal peptides, which are processed at recognition sites that precede the hydrophobic domain by a prepilin-specific peptidase (SPase III) (38, 43, 45). Similarly, archaeal flagellin precursors contain a class III signal peptide that is processed by a prepilin-specific peptidase homolog (FlaK/PibD) (3, 8, 10, 11). Moreover, flagellar assembly involves homologs of components involved in the biosynthesis of bacterial type IV pili, including FlaI, an ATPase homologous to PilB, and FlaJ, a multispanning membrane protein that may provide a platform for flagellar assembly, similar to the proposed role for PilC in pilus assembly (38, 44, 53, 54). These genes, as well as a number of others that encode proteins often required for either flagellar assembly or function (flaCDEFG and flaH), are frequently coregulated with the flg genes (11, 26, 44, 54).Interestingly, most sequenced archaeal genomes also contain diverse sets of genes that encode type IV pilin-like proteins with little or no homology to archaeal flagellins (3, 39, 52). While often coregulated with pilB and pilC homologs, these genes are never found in clusters containing the motility-specific flaCDEFG and flaH homologs; however, the proteins they encode do contain class III signal peptides (52). Several of these proteins have been shown to be processed by an SPase III (4, 52). Moreover, in Sulfolobus solfataricus and Methanococcus maripaludis, some of these archaeal type IV pilin-like proteins were confirmed to form surface filaments that are distinct from the flagella (21, 22, 56). These findings strongly suggest that the genes encode subunits of pilus-like surface structures that are involved in functions other than swimming motility.In bacteria, type IV pili are multifunctional filamentous protein complexes that, in addition to facilitating twitching motility, mediate adherence to abiotic surfaces and make close intercellular associations possible (15, 17, 46). For instance, mating between Escherichia coli in liquid medium has been shown to require type IV pili (often referred to as thin sex pili), which bring cells into close proximity (29, 30, 57). Recent work has shown that the S. solfataricus pilus, Ups, is required not only for efficient adhesion to surfaces of these crenarchaeal cells but also for UV-induced aggregation (21, 22, 58). Frols et al. postulate that autoaggregation is required for DNA exchange under these highly mutagenic conditions (22). Halobacterium salinarum has also been shown to form Ca²⁺-induced aggregates (27, 28). Furthermore, conjugation has been observed in H. volcanii, which likely requires that cells be held in close proximity for a sustained period to allow time for the cells to construct the cytoplasmic bridges that facilitate DNA transfer between them (35).To determine the roles played by haloarchaeal flagella and other putative type IV pilus-like structures in swimming and surface motility, surface adhesion, autoaggregation, and conjugation, we constructed and characterized two mutant strains of H. volcanii, one lacking the genes that encode the flagellins and the other lacking pibD. Our analyses indicate that although this archaeon was previously thought to be nonmotile (14, 36), wild-type (wt) H. volcanii can swim in a flagellum-dependent manner. Consistent with the involvement of PibD in processing flagellins, the peptidase mutant is nonmotile. Unlike nonhalophilic archaea, however, the flagellum mutant can adhere to glass as effectively as the wild type. Conversely, the ΔpibD strain fails to adhere to glass surfaces, strongly suggesting that in H. volcanii surface adhesion involves nonflagellar, type IV pilus-like structures.     

Diversity of Dissimilatory Sulfite Reductase Genes (dsrAB) in a Salt Marsh Impacted by Long-Term Acid Mine Drainage

Sulfate-reducing bacteria (SRB) play a major role in the coupled biogeochemical cycling of sulfur and chalcophilic metal(loid)s. By implication, they can exert a strong influence on the speciation and mobility of multiple metal(loid) contaminants. In this study, we combined DsrAB gene sequencing and sulfur isotopic profiling to identify the phylogeny and distribution of SRB and to assess their metabolic activity in salt marsh sediments exposed to acid mine drainage (AMD) for over 100 years. Recovered dsrAB sequences from three sites sampled along an AMD flow path indicated the dominance of a single Desulfovibrio species. Other major sequence clades were related most closely to Desulfosarcina, Desulfococcus, Desulfobulbus, and Desulfosporosinus species. The presence of metal sulfides with low δ³⁴S values relative to δ³⁴S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of ∼2), although possibly within less acidic microenvironments. Interestingly, δ³⁴S values for pore water sulfate were lower than those for sulfate delivered during tidal inundation of marsh sediments. 16S rRNA gene sequence data from sediments and sulfur isotope data confirmed that sulfur-oxidizing bacteria drove the reoxidation of biogenic sulfide coupled to oxygen or nitrate reduction over a timescale of hours. Collectively, these findings imply a highly dynamic microbially mediated cycling of sulfate and sulfide, and thus the speciation and mobility of chalcophilic contaminant metal(loid)s, in AMD-impacted marsh sediments.Salt marshes exhibit high primary production rates (1, 101) and form biogeochemical “transition zones” for nutrient production, transport, and cycling between terrestrial and coastal marine environments (41, 66, 100). These zones also serve to reduce the flux of potentially toxic metals in contaminated groundwater to estuaries (12, 99, 106). Both functions depend strongly on microbial activity, especially that of sulfate-reducing bacteria (SRB) (42, 62, 67). SRB recycle much of the sedimentary organic carbon pool in marsh sediments (42-44) and indirectly inhibit production of the greenhouse gas methane (37, 71). They can restrict the mobility of dissolved contaminant metals by inducing precipitation of poorly soluble metal sulfides, and studies have examined their use in constructed wetlands to bioremediate acid mine drainage (AMD) and other metalliferous waste streams (11, 35, 40, 46, 50, 76, 90, 94, 104). However, the high acidity and metal concentrations inherent to AMD can inhibit SRB growth (15, 88, 98), and preferential growth of iron- and sulfur-oxidizing bacteria over SRB has been observed in some treatment wetlands (39).For natural salt marshes, 16S ribosomal nucleic acid- and phospholipid fatty acid (PLFA)-based analyses have shown that SRB commonly comprise a significant fraction of the microbial community (13, 24, 31, 34, 51, 58). Studies of salt marsh dissimilatory sulfite reductase genes (dsrAB), a highly conserved functional phylogenetic marker of prokaryotic sulfate reducers (49, 57, 102, 103, 107), have revealed both novel and deeply branching clades (3). Studies of mining-impacted sites at pH 2.0 to 7.8 (5, 7, 39, 70, 72, 77, 84), of soils and geothermal settings at a pH of ∼4 (55, 68), of metal-contaminated estuaries at pH 6.8 to 7.2 (65), and of hypersaline lakes at pH 7.5 (56) further outline the distribution and tolerance of specific groups and species of SRB under geochemically stringent conditions. Other findings point toward the existence of deltaproteobacteria in environments at a pH of ∼1 (10), although it is unknown if these include SRB. SRB diversity in salt marshes under long-term contamination by AMD has not been well investigated. Such studies may provide useful information for bioremediation projects in estuarine environments, as well as general insights into relationships between SRB physiology and the geochemistry of AMD.We studied the diversity of SRB, based on phylogenetic analysis of recovered DsrAB gene sequences (∼1.9 kb), in natural salt marsh sediments of the San Francisco Bay impacted by AMD for over 100 years. Sulfur isotope ratio and concentration measurements of pore water sulfate and metal sulfide minerals provided information about the spatial and temporal extent of active bacterial sulfate reduction (BSR) in sediment cores taken from specific sites along an AMD flow path. Collectively, the results revealed a tidal marsh system characterized by rapidly cycling bacterial sulfate reduction and sulfide reoxidation associated with oscillating tidal inundation and groundwater infiltration.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Extraintestinal Escherichia coli Carrying Virulence Genes in Coastal Marine Sediments

Despite the recognized potential of long-term survival or even growth of fecal indicators bacteria (FIB) in marine sediments, this compartment is largely ignored by health protection authorities. We conducted a large-scale study over approximately 50 km of the Marche coasts (Adriatic Sea) at depths ranging from 2 to 5 m. Total and fecal coliforms (FC) were counted by culture-based methods. Escherichia coli was also quantified using fluorescence in situ hybridization targeting specific 16S rRNA sequences, which yielded significantly higher abundances than culture-based methods, suggesting the potential importance of viable but nonculturable E. coli cells. Fecal coliforms displayed high abundances at most sites and showed a prevalence of E. coli. FC isolates (n = 113) were identified by API 20E, additional biochemical tests, and internal transcribed spacer-PCR. E. coli strains, representing 96% of isolates, were then characterized for genomic relatedness and phylogenetic group (A, B1, B2, and D) of origin by randomly amplified polymorphic DNA and multiplex-PCR. The results indicated that E. coli displayed a wide genotypic diversity, also among isolates from the same station, and that 44 of the 109 E. coli isolates belonged to groups B2 and D. Further characterization of B2 and D isolates for the presence of 11 virulence factor genes (pap, sfa/foc, afa, eaeA, ibeA, traT, hlyA, stx₁, stx₂, aer, and fyuA) showed that 90% of B2 and 65% of D isolates were positive for at least one of these. Most of the variance of both E. coli abundance and assemblage composition (>62%) was explained by a combination of physical-chemical and trophic variables. These findings indicate that coastal sediments could represent a potential reservoir for commensal and pathogenic E. coli and that E. coli distribution in marine coastal sediments largely depends upon the physical and trophic status of the sediment. We conclude that future sampling designs aimed at monitoring the microbiological quality of marine coastal areas should not further neglect the analysis of the sediment and that monitoring of these environments can be improved by including molecular methods as a complement of culture-based techniques.Marine environments contaminated by fecal material, derived from human or animal waste, may contain a large variety of pathogenic microorganisms. Health protection and monitoring programs analyze the contamination of aquatic ecosystems (20) but, due to technical and practical difficulties, the search of fecal indicator bacteria (FIB) is routinely preferred to the systematic search of all potential pathogens to assess the sanitary risk of a water body (17). Recreational seawaters are, for instance, classified on the basis of the concentration of Escherichia coli and Enterococcus spp. (21, 33, 40), assumed to be indicators of fecal contamination and of the presence of other pathogenic enteric bacteria. Exposure to waters contaminated with E. coli and Enterococcus spp. have been associated with an increased risk of contracting gastrointestinal and respiratory illnesses (10, 24, 31, 62, 64). Although most E. coli strains are harmless, some strains can cause a variety of intestinal and extraintestinal diseases (11, 57, 58, 62) such as diarrhea, urinary tract infections, bacteremia, sepsis, and meningitis (57). Phylogenetic analyses have shown that E. coli includes four main phylogroups (A, B1, B2, and D) and that most virulent extraintestinal strains belong to the groups B2 and D (11, 23, 46).The microbiological quality of marine waters is typically based exclusively on the water column, whereas sediments have received attention only recently (7, 14, 27, 45). Fecal coliforms (FC) and enterococci have been reported from marine sediments (5, 19, 41), and it has been also proposed that FIB accumulated in the sediments have the potential to contaminate the overlying waters by resuspension of sediment particles (35). There is evidence that FIB and pathogenic bacteria can survive longer in aquatic sediments than in the overlying water column (12, 34). However, the available knowledge on the environmental factors influencing the ecology of pathogenic bacteria in marine sediments is still extremely scant, and there are only few detailed studies on the pathogenic potential, genetic diversity, or population structure of FIB in sediments (1, 63).The development of molecular methods has permitted a range of new approaches to monitor the safety of recreational waters (2). Among the available molecular methods, the fluorescence in situ hybridization (FISH) based on probes specific to 16S or 23S rRNA can be utilized to detect and enumerate specific prokaryotic taxa (16, 59). Since the number of ribosomes varies, generally between 10³ and 10⁵ per cell, depending on the species and physiological state, FISH has also been used to provide evidence of an active metabolic state of the detected cells (2, 8). FISH can thus represent a good complement to culture-based methods, and provides reliable quantitative data in a short time (within 4 h). With regard to FIB, the use of FISH to detect total coliforms (TC) has proven to be difficult, due to their high phylogenetic heterogeneity (55). Conversely, the use of species-specific probes for the detection of single species, such as E. coli, is routinely used (22, 47, 53); however, it has been never tested on marine sediments.The objective of the present study was to investigate the microbiological quality of coastal marine sediments along a large area of the Adriatic Sea (Central Mediterranean Sea) and to evaluate the presence and distribution of specific bacterial genotypes associated with different marine areas. More specifically, it was our aim to evaluate whether marine sediments may be a potential reservoir of active pathogenic E. coli and thus represent a risk for human health. To do this, we analyzed (i) the abundance and distribution of TC and FC; (ii) the abundance and distribution of E. coli strains, along with their genetic relatedness; and (iii) the presence of extraintestinal pathogenic E. coli carrying virulence gene factors. To determine bacterial abundance, culture-dependent (the membrane filtration [MF] technique) and culture-independent (the FISH technique) approaches were used. Finally, to identify the factors potentially responsible for the accumulation and survival of E. coli in the benthic environment, we investigated the environmental variables possibly related to the distribution of FIB.