Engineering the thermal stability of a polyphosphate kinase by ancestral sequence reconstruction to expand the temperature boundary for an industrially applicable ATP regeneration system.

ATP-dependent energy-consuming enzymatic reactions are widely used in cell-free biocatalysis. However, the direct addition of large amounts of expensive ATP can greatly increase cost, and enzymatic production is often difficult to achieve as a result. Although a polyphosphate kinase (PPK)-polyphosphate-based ATP regeneration system has the potential to solve this challenge, the generally poor thermal stability of PPKs limits the widespread use of this method. In this paper, we evaluated the thermal stability of a PPK from Sulfurovum lithotrophicum (SlPPK2). After directed evolution and computation-supported design, we found that SlPPK2 is very recalcitrant and cannot acquire beneficial mutations. Inspired by the usually outstanding stability of ancestral enzymes, we reconstructed the ancestral sequence of the PPK family and used it as a guide to construct three heat-stable variants of SlPPK2, of which the L35F/T144S variant has a half-life of more than 14 h at 60°C. Molecular dynamics simulations were performed on all enzymes to analyze the reasons for the increased thermal stability. The results showed that mutations at these two positions act synergistically from the interior and surface of the protein, leading to a more compact structure. Finally, the robustness of the L35F/T144S variant was verified in the synthesis of nucleotides at high temperature. In practice, the use of this high-temperature ATP regeneration system can effectively avoid byproduct accumulation. Our work extends the temperature boundary of ATP regeneration and has great potential for industrial applications.IMPORTANCEATP regeneration is an important basic applied study in the field of cell-free biocatalysis. Polyphosphate kinase (PPK) is an enzyme tool widely used for energy regeneration during enzymatic reactions. However, the thermal stability of the PPKs reported to date that can efficiently regenerate ATP is usually poor, which greatly limits their application. In this study, the thermal stability of a difficult-to-engineer PPK from Sulfurovum lithotrophicum was improved, guided by an ancestral sequence reconstruction strategy. The optimal variant has a 4.5-fold longer half-life at 60°C than the wild-type enzyme, thus enabling the extension of the temperature boundary for ATP regeneration. The ability of this variant to regenerate ATP was well demonstrated during high-temperature enzymatic production of nucleotides.

Gene-centered metagenome analysis of Vulcano Island soil (Aeolian archipelago, Italy) reveals diverse microbial key players in methane, hydrogen and sulfur cycles.

The Aeolian archipelago is known worldwide for its volcanic activity and hydrothermal emissions, of mainly carbon dioxide and hydrogen sulfide. Hydrogen, methane, and carbon monoxide are minor components of these emissions which together can feed large quantities of bacteria and archaea that do contribute to the removal of these notorious greenhouse gases. Here we analyzed the metagenome of samples taken from the Levante bay on Vulcano Island, Italy. Using a gene-centric approach, the hydrothermal vent community appeared to be dominated by Proteobacteria, and Sulfurimonas was the most abundant genus. Metabolic reconstructions highlight a prominent role of formaldehyde oxidation and the reverse TCA cycle in carbon fixation. [NiFe]-hydrogenases seemed to constitute the preferred strategy to oxidize H2, indicating that besides H2S, H2 could be an essential electron donor in this system. Moreover, the sulfur cycle analysis showed a high abundance and diversity of sulfate reduction genes underpinning the H2S production. This study covers the diversity and metabolic potential of the microbial soil community in Levante bay and adds to our understanding of the biogeochemistry of volcanic ecosystems.

Small RNAs that target G-rich sequences are generated by diverse biogenesis pathways in Epsilonproteobacteria.

Bacterial small RNAs (sRNAs) are widespread post-transcriptional regulators that control bacterial stress responses and virulence. Nevertheless, little is known about how they arise and evolve. Homologs can be difficult to identify beyond the strain level using sequence-based approaches, and similar functionalities can arise by convergent evolution. Here, we found that the virulence-associated CJnc190 sRNA of the foodborne pathogen Campylobacter jejuni resembles the RepG sRNA from the gastric pathogen Helicobacter pylori. However, while both sRNAs bind G-rich sites in their target mRNAs using a C/U-rich loop, they largely differ in their biogenesis. RepG is transcribed from a stand-alone gene and does not require processing, whereas CJnc190 is transcribed from two promoters as precursors that are processed by RNase III and also has a cis-encoded antagonist, CJnc180. By comparing CJnc190 homologs in diverse Campylobacter species, we show that RNase III-dependent processing of CJnc190 appears to be a conserved feature even outside of C. jejuni. We also demonstrate the CJnc180 antisense partner is expressed in C. coli, yet here might be derived from the 3'UTR (untranslated region) of an upstream flagella-related gene. Our analysis of G-tract targeting sRNAs in Epsilonproteobacteria demonstrates that similar sRNAs can have markedly different biogenesis pathways.

Hydrogenimonas cancrithermarum sp. nov., a hydrogen- and thiosulfate-oxidizing mesophilic chemolithoautotroph isolated from diffuse-flow fluids on the East Pacific Rise, and an emended description of the genus Hydrogenimonas.

A novel mesophilic, hydrogen- and thiosulfate-oxidizing bacterium, strain ISO32T, was isolated from diffuse-flow hydrothermal fluids from the Crab Spa vent on the East Pacific Rise. Cells of ISO32T were rods, being motile by means of a single polar flagellum. The isolate grew at a temperature range between 30 and 55 °C (optimum, 43 °C), at a pH range between 5.3 and 7.6 (optimum, pH 5.8) and in the presence of 2.0-4.0 % NaCl (optimum, 2.5 %). The isolate was able to grow chemolithoautotrophically with molecular hydrogen, thiosulfate or elemental sulfur as the sole electron donor. Thiosulfate, elemental sulfur, nitrate and molecular oxygen were each used as a sole electron acceptor. Phylogenetic analysis of 16S rRNA gene sequences placed ISO32T in the genus Hydrogenimonas of the class Epsilonproteobacteria, with Hydrogenimonas thermophila EP1-55-1 %T as its closest relative (95.95 % similarity). On the basis of the phylogenetic, physiological and genomic characteristics, it is proposed that the organism represents a novel species within the genus Hydrogenimonas, Hydrogenimonas cancrithermarum sp. nov. The type strain is ISO32T (=JCM 39185T =KCTC 25252T). Furthermore, the genomic properties of members of the genus Hydrogenimonas are distinguished from those of members of other thermophilic genera in the orders Campylobacterales (Nitratiruptor and Nitrosophilus) and Nautiliales (Caminibacter, Nautilia and Lebetimonas), with larger genome sizes and lower 16S rRNA G+C content values. Comprehensive metabolic comparisons based on genomes revealed that genes responsible for the Pta-AckA pathway were observed exclusively in members of mesophilic genera in the order Campylobacterales and of the genus Hydrogenimonas. Our results indicate that the genus Hydrogenimonas contributes to elucidating the evolutionary history of Epsilonproteobacteria in terms of metabolism and transition from a thermophilic to a mesophilic lifestyle.

RNase R is associated in a functional complex with the RhpA DEAD-box RNA helicase in Helicobacter pylori.

Ribonucleases are central players in post-transcriptional regulation, a major level of gene expression regulation in all cells. Here, we characterized the 3'-5' exoribonuclease RNase R from the bacterial pathogen Helicobacter pylori. The 'prototypical' Escherichia coli RNase R displays both exoribonuclease and helicase activities, but whether this latter RNA unwinding function is a general feature of bacterial RNase R had not been addressed. We observed that H. pylori HpRNase R protein does not carry the domains responsible for helicase activity and accordingly the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The lack of RNase R helicase domains is widespread among the Campylobacterota, which include Helicobacter and Campylobacter genera, and this loss occurred gradually during their evolution. An in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori was discovered. Purified RhpA facilitates the degradation of double stranded RNA by HpRNase R, showing that this complex is functional. HpRNase R has a minor role in 5S rRNA maturation and few targets in H. pylori, all included in the RhpA regulon. We concluded that during evolution, HpRNase R has co-opted the RhpA helicase to compensate for its lack of helicase activity.

Sequestration and efflux largely account for cadmium and copper resistance in the deep-sea Nitratiruptor sp. SB155-2 (phylum Campylobacterota).

In deep-sea hydrothermal vent environments, metal-enriched fluids and sediments abound, making these habitats ideal to study metal resistance in prokaryotes. In this investigation, we employed transcriptomics and shotgun proteomics with scanning transmission electron microscopy and energy-dispersive x-ray spectroscopy (STEM-EDX) to better understand mechanisms of tolerance for cadmium (Cd) and copper (Cu) at stress-inducing concentrations in Nitratiruptor sp. SB155-2 (phylum Campylobacterota). Transcriptomic profiles were remarkably different in the presence of these two metals, displaying 385 (19%) and 629 (31%) differentially transcribed genes (DTG) in the presence of Cd(II) and Cu(II), respectively, while only 7% of differentially transcribed (DT) genes were shared, with genes for non-specific metal transporters and genes involved in oxidative stress-response predominating. Transcriptomic and proteomic analyses confirmed that metal-specific DT pathways under Cu(II) stress, including those involving sulfur, cysteine, and methionine, are likely required for high-affinity efflux systems, while flagella formation and chemotaxis were over-represented under Cd(II) stress. Consistent with these differences, STEM-EDX analysis revealed that polyphosphate-like granules (pPLG), the formation of CdS particles, and the periplasmic space are crucial for Cd(II) sequestration. Overall, this study provides new insights regarding metal-specific adaptations of Campylobacterota to deep-sea hydrothermal vent environments.

Adaptations to high pressure of Nautilia sp. strain PV-1, a piezophilic Campylobacterium (aka Epsilonproteobacterium) isolated from a deep-sea hydrothermal vent.

Physiological and gene expression studies of deep-sea bacteria under pressure conditions similar to those experienced in their natural habitat are critical for understanding growth kinetics and metabolic adaptations to in situ conditions. The Campylobacterium (aka Epsilonproteobacterium) Nautilia sp. strain PV-1 was isolated from hydrothermal fluids released from an active deep-sea hydrothermal vent at 9° N on the East Pacific Rise. Strain PV-1 is a piezophilic, moderately thermophilic, chemolithoautotrophic anaerobe that conserves energy by coupling the oxidation of hydrogen to the reduction of nitrate or elemental sulfur. Using a high-pressure-high temperature continuous culture system, we established that strain PV-1 has the shortest generation time of all known piezophilic bacteria and we investigated its protein expression pattern in response to different hydrostatic pressure regimes. Proteogenomic analyses of strain PV-1 grown at 20 and 5 MPa showed that pressure adaptation is not restricted to stress response or homeoviscous adaptation but extends to enzymes involved in central metabolic pathways. Protein synthesis, motility, transport, and energy metabolism are all affected by pressure, although to different extents. In strain PV-1, low-pressure conditions induce the synthesis of phage-related proteins and an overexpression of enzymes involved in carbon fixation.

Structural studies reveal a ring-shaped architecture of deep-sea vent phage NrS-1 polymerase.

NrS-1 is the first known phage that can infect Epsilonproteobacteria, one of the predominant primary producers in the deep-sea hydrothermal vent ecosystems. NrS-1 polymerase is a multidomain enzyme and is one key component of the phage replisome. The N-terminal Prim/Pol and HBD domains are responsible for DNA polymerization and de novo primer synthesis activities of NrS-1 polymerase. However, the structure and function of the C-terminus (CTR) of NrS-1 polymerase are poorly understood. Here, we report two crystal structures, showing that NrS-1 CTR adopts one unique hexameric ring-shaped conformation. Although the central helicase domain of NrS-1 CTR shares structural similarity with the superfamily III helicases, the folds of the Head and Tail domains are completely novel. Via mutagenesis and in vitro biochemical analysis, we identified many residues important for the helicase and polymerization activities of NrS-1 polymerase. In addition to NrS-1 polymerase, our study may also help us identify and understand the functions of multidomain polymerases expressed by many NrS-1 related phages.

Epsilonproteobacterial hydroxylamine oxidoreductase (εHao): characterization of a 'missing link' in the multihaem cytochrome c family.

Members of the multihaem cytochrome c family such as pentahaem cytochrome c nitrite reductase (NrfA) or octahaem hydroxylamine oxidoreductase (Hao) are involved in various microbial respiratory electron transport chains. Some members of the Hao subfamily, here called εHao proteins, have been predicted from the genomes of nitrate/nitrite-ammonifying bacteria that usually lack NrfA. Here, εHao proteins from the host-associated Epsilonproteobacteria Campylobacter fetus and Campylobacter curvus and the deep-sea hydrothermal vent bacteria Caminibacter mediatlanticus and Nautilia profundicola were purified as εHao-maltose binding protein fusions produced in Wolinella succinogenes. All four proteins were able to catalyze reduction of nitrite (yielding ammonium) and hydroxylamine whereas hydroxylamine oxidation was negligible. The introduction of a tyrosine residue at a position known to cause covalent trimerization of Hao proteins did neither stimulate hydroxylamine oxidation nor generate the Hao-typical absorbance maximum at 460 nm. In most cases, the εHao-encoding gene haoA was situated downstream of haoC, which predicts a tetrahaem cytochrome c of the NapC/NrfH family. This suggested the formation of a membrane-bound HaoCA assembly reminiscent of the menaquinol-oxidizing NrfHA complex. The results indicate that εHao proteins form a subfamily of ammonifying cytochrome c nitrite reductases that represents a 'missing link' in the evolution of NrfA and Hao enzymes.

Sulfurovum denitrificans sp. nov., an obligately chemolithoautotrophic sulfur-oxidizing epsilonproteobacterium isolated from a hydrothermal field.

A novel marine sulfur-oxidizing bacterium, designated strain eps51T, was isolated from a surface rock sample collected from the hydrothermal field of Suiyo Seamount on the Izu-Bonin Arc in the Western Pacific Ocean. This bacterium was Gram-staining-negative, non-motile and rod-shaped. Strain eps51T grew chemolithoautotrophically, by sulfur-oxidizing respiration with elemental sulfur and thiosulfate as electron donors and used only carbon dioxide as a carbon source. Oxygen and nitrate were used as its electron acceptors. The isolate grew optimally at 30 °C, at pH 7.0 and with 3 % NaCl. The predominant fatty acids were C16 : 1ω7c, C18 : 1ω7c and C16 : 0. The respiratory quinone was menaquinone-6 and the genomic DNA G+C content was 40.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequence revealed that eps51T represented a member of the genus Sulfurovum and the closest relative was Sulfurovum aggregans (96.7 %). Based on its phylogenetic position along with its physiological and chemotaxonomic characteristics, the name Sulfurovum denitrificans sp. nov. is proposed, with the type strain eps51T (=NBRC 102602T=DSM 19611T).

Metagenomic Signatures of Microbial Communities in Deep-Sea Hydrothermal Sediments of Azores Vent Fields.

The organisms inhabiting the deep-seafloor are known to play a crucial role in global biogeochemical cycles. Chemolithoautotrophic prokaryotes, which produce biomass from single carbon molecules, constitute the primary source of nutrition for the higher organisms, being critical for the sustainability of food webs and overall life in the deep-sea hydrothermal ecosystems. The present study investigates the metabolic profiles of chemolithoautotrophs inhabiting the sediments of Menez Gwen and Rainbow deep-sea vent fields, in the Mid-Atlantic Ridge. Differences in the microbial community structure might be reflecting the distinct depth, geology, and distance from vent of the studied sediments. A metagenomic sequencing approach was conducted to characterize the microbiome of the deep-sea hydrothermal sediments and the relevant metabolic pathways used by microbes. Both Menez Gwen and Rainbow metagenomes contained a significant number of genes involved in carbon fixation, revealing the largely autotrophic communities thriving in both sites. Carbon fixation at Menez Gwen site was predicted to occur mainly via the reductive tricarboxylic acid cycle, likely reflecting the dominance of sulfur-oxidizing Epsilonproteobacteria at this site, while different autotrophic pathways were identified at Rainbow site, in particular the Calvin-Benson-Bassham cycle. Chemolithotrophy appeared to be primarily driven by the oxidation of reduced sulfur compounds, whether through the SOX-dependent pathway at Menez Gwen site or through reverse sulfate reduction at Rainbow site. Other energy-yielding processes, such as methane, nitrite, or ammonia oxidation, were also detected but presumably contributing less to chemolithoautotrophy. This work furthers our knowledge of the microbial ecology of deep-sea hydrothermal sediments and represents an important repository of novel genes with potential biotechnological interest.

Diversity patterns and isolation of Planctomycetes associated with metalliferous deposits from hydrothermal vent fields along the Valu Fa Ridge (SW Pacific).

The microbial diversity associated with diffuse venting deep-sea hydrothermal deposits is tightly coupled to the geochemistry of the hydrothermal fluids. Previous 16S rRNA gene amplicon sequencing (metabarcoding) of marine iron-hydroxide deposits along the Arctic Mid Ocean Ridge, revealed the presence of diverse bacterial communities associated with these deposits (Storesund and Øvreås in Antonie van Leeuwenhoek 104:569-584, 2013). One of the most abundant and diverse phyla detected was the enigmatic Planctomycetes. Here we report on the comparative analyses of the diversity and distribution patterns of Planctomycetes associated with metalliferous deposits from two diffuse-flow hydrothermal vent fields (Mariner and Vai Lili) from the Valu Fa Ridge in the Southwestern Pacific. Metabarcoding of 16S rRNA genes showed that the major prokaryotic phyla were Proteobacteria (51-73% of all 16S rRNA gene reads), Epsilonbacteraeota (0.5-19%), Bacteriodetes (5-17%), Planctomycetes (0.4-11%), Candidatus Latescibacteria (0-5%) and Marine Benthic Group E (Hydrothermarchaeota) (0-5%). The two different sampling sites differed considerably in overall community composition. The abundance of Planctomycetes also varied substantially between the samples and the sites, with the majority of the sequences affiliated with uncultivated members of the classes Planctomycetacia and Phycisphaerae, and other deep branching lineages. Seven different strains affiliated with the order Planctomycetales were isolated, mostly from the Vai Lili samples, where also the highest Planctomycetales diversity was seen. Most of the isolates were affiliated with the genera Gimesia, Rhodopirellula and Blastopirellula. One isolate was only distantly related to known cultured, but uncharacterized species within the Pir4 group. This study shows that the deep-sea Planctomycetes represent a very heterogeneous group with a high phylogenetic diversity and a substantial potential for novel organism discovery in these deep ocean environments.

Discovery of a Novel Alginate Lyase from Nitratiruptor sp. SB155-2 Thriving at Deep-sea Hydrothermal Vents and Identification of the Residues Responsible for Its Heat Stability.

Extremophiles are expected to represent a source of enzymes having unique functional properties. The hypothetical protein NIS_0185, termed NitAly in this study, was identified as an alginate lyase-homolog protein in the genomic database of ϵ-Proteobacteria Nitratiruptor sp. SB155-2, which was isolated from deep-sea hydrothermal vents at a water depth of 1,000 m. Among the characterized alginate lyases in the polysaccharide lyase family 7 (PL-7), the amino acid sequence of NitAly showed the highest identity (39%) with that of red alga Pyropia yezoensis alginate lyase PyAly. Recombinant NitAly (rNitAly) was successfully expressed in Escherichia coli Purified rNitAly degraded alginate in an endolytic manner. Among alginate block types, polyM was preferable to polyG and polyMG as a substrate, and its end degradation products were mainly tri-, tetra-, and penta-saccharides. The optimum temperature and pH values were 70 °C and around 6, respectively. A high concentration of NaCl (0.8-1.4 m) was required for maximum activity. In addition, a 50% loss of activity was observed after incubation at 67 °C for 30 min. Heat stability was decreased in the presence of 5 mm DTT, and Cys-80 and Cys-232 were identified as the residues responsible for heat stability but not lyase activity. Introducing two cysteines into PyAly based on homology modeling using Pseudomonas aeruginosa alginate lyase PA1167 as the template enhanced its heat stability. Thus, NitAly is a functional alginate lyase, with its unique optimum conditions adapted to its environment. These insights into the heat stability of NitAly could be applied to improve that of other PL-7 alginate lyases.

Success of chemolithoautotrophic SUP05 and Sulfurimonas GD17 cells in pelagic Baltic Sea redox zones is facilitated by their lifestyles as K- and r-strategists.

Chemolithoautotrophic sulfur-oxidizing and denitrifying Gamma- (particularly the SUP05 cluster) and Epsilonproteobacteria (predominantly Sulfurimonas subgroup GD17) are assumed to compete for substrates (electron donors and acceptors) in marine pelagic redox gradients. To elucidate their ecological niche separation we performed 34 S0 , 15 NO3- and H13 CO3- stable-isotope incubations with water samples from Baltic Sea suboxic, chemocline and sulfidic zones followed by combined phylogenetic staining and high-resolution secondary ion mass spectrometry of single cells. SUP05 cells were small-sized (0.06-0.09 µm3 ) and most abundant in low-sulfidic to suboxic zones, whereas Sulfurimonas GD17 cells were significantly larger (0.26-0.61 µm3 ) and most abundant at the chemocline and below. Together, SUP05 and GD17 cells accumulated up to 48% of the labelled substrates but calculation of cell volume-specific rates revealed that GD17 cells incorporated labelled substrates significantly faster throughout the redox zone, thereby potentially outcompeting SUP05 especially at high substrate concentrations. Thus, in synopsis with earlier described features of SUP05/GD17 we conclude that their spatially overlapping association in stratified sulfidic zones is facilitated by their different lifestyles: whereas SUP05 cells are streamlined, non-motile K-strategists adapted to low substrate concentrations, GD17 cells are motile r-strategists well adapted to fluctuating substrate and redox conditions.

Microbial potential for carbon and nutrient cycling in a geogenic supercritical carbon dioxide reservoir.

Microorganisms catalyze carbon cycling and biogeochemical reactions in the deep subsurface and thus may be expected to influence the fate of injected supercritical (sc) CO2 following geological carbon sequestration (GCS). We hypothesized that natural subsurface scCO2 reservoirs, which serve as analogs for the long-term fate of sequestered scCO2 , harbor a 'deep carbonated biosphere' with carbon cycling potential. We sampled subsurface fluids from scCO2 -water separators at a natural scCO2 reservoir at McElmo Dome, Colorado for analysis of 16S rRNA gene diversity and metagenome content. Sequence annotations indicated dominance of Sulfurospirillum, Rhizobium, Desulfovibrio and four members of the Clostridiales family. Genomes extracted from metagenomes using homology and compositional approaches revealed diverse mechanisms for growth and nutrient cycling, including pathways for CO2 and N2 fixation, anaerobic respiration, sulfur oxidation, fermentation and potential for metabolic syntrophy. Differences in biogeochemical potential between two production well communities were consistent with differences in fluid chemical profiles, suggesting a potential link between microbial activity and geochemistry. The existence of a microbial ecosystem associated with the McElmo Dome scCO2 reservoir indicates that potential impacts of the deep biosphere on CO2 fate and transport should be taken into consideration as a component of GCS planning and modelling.

CO2 assimilation strategies in stratified lakes: Diversity and distribution patterns of chemolithoautotrophs.

While mechanisms of different carbon dioxide (CO2 ) assimilation pathways in chemolithoautotrohic prokaryotes are well understood for many isolates under laboratory conditions, the ecological significance of diverse CO2 fixation strategies in the environment is mostly unexplored. Six stratified freshwater lakes were chosen to study the distribution and diversity of the Calvin-Benson-Bassham (CBB) cycle, the reductive tricarboxylic acid (rTCA) cycle, and the recently discovered archaeal 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) pathway. Eleven primer sets were used to amplify and sequence genes coding for selected key enzymes in the three pathways. Whereas the CBB pathway with different forms of RubisCO (IA, IC and II) was ubiquitous and related to diverse bacterial taxa, encompassing a wide range of potential physiologies, the rTCA cycle in Epsilonproteobacteria and Chloribi was exclusively detected in anoxic water layers. Nitrifiying Nitrosospira and Thaumarchaeota, using the rTCA and HP/HB cycle respectively, are important residents in the aphotic and (micro-)oxic zone of deep lakes. Both taxa were of minor importance in surface waters and in smaller lakes characterized by an anoxic hypolimnion. Overall, this study provides a first insight on how different CO2 fixation strategies and chemical gradients in lakes are associated to the distribution of chemoautotrophic prokaryotes with different functional traits.

Capturing Compositional Variation in Denitrifying Communities: a Multiple-Primer Approach That Includes Epsilonproteobacteria.

Denitrifying Epsilonproteobacteria may dominate nitrogen loss processes in marine habitats with intense redox gradients, but assessment of their importance is limited by the currently available primers for nitrite reductase genes. Nine new primers targeting the nirS gene of denitrifying Epsilonproteobacteria were designed and tested for use in sequencing and quantitative PCR on two microbial mat samples (vent 2 and vent 4) from the Calypso hydrothermal vent field, Bay of Plenty, New Zealand. Commonly used nirS and nirK primer sets nirS1F/nirS6R, cd3aF/R3cd, nirK1F/nirK5R, and F1aCu/R3Cu were also tested to determine what may be missed by the common single-primer approach to assessing denitrifier diversity. The relative importance of Epsilonproteobacteria in these samples was evaluated by 16S rRNA gene sequencing. Epsilonproteobacteria represented up to 75.6% of 16S rRNA libraries, but nirS genes from this group were not found with commonly used primers. Pairing of the new primer EPSnirS511F with either EPSnirS1100R or EPSnirS1105R recovered nirS sequences from members of the genera Sulfurimonas, Sulfurovum, and Nitratifractor. The new quantitative PCR primers EPSnirS103F/EPSnirS530R showed dominance of denitrifying Epsilonproteobacteria in vent 4 compared to vent 2, which had greater representation by "standard" denitrifiers measured with the cd3aF/R3cd primers. Limited results from commonly used nirK primers suggest biased amplification between primers. Future application of multiple nirS and nirK primers, including the new epsilonproteobacterial nirS primers, will improve the detection of denitrifier diversity and the capability to identify changes in dominant denitrifying communities.IMPORTANCE Estimating the potential for increasing nitrogen limitation in the changing global ocean is reliant on understanding the microbial community that removes nitrogen through the process of denitrification. This process is favored under oxygen limitation, which is a growing global-ocean phenomenon. Current methods use the nitrite reductase genes nirS and nirK to assess denitrifier diversity and abundance using primers that target only a few known denitrifiers and systematically exclude denitrifying Epsilonproteobacteria, a group known to dominate in reducing environments, such as hydrothermal vents and anoxic basins. As oxygen depletion expands in the oceans, it is important to study denitrifier community dynamics within those areas to predict future global ocean changes. This study explores the design and testing of new primers that target epsilonproteobacterial nirS and reveals the varied success of existing primers, leading to the recommendation of a multiple-primer approach to assessing denitrifier diversity.

Diversity of Total Bacterial Communities and Chemoautotrophic Populations in Sulfur-Rich Sediments of Shallow-Water Hydrothermal Vents off Kueishan Island, Taiwan.

Shallow-water hydrothermal vents (HTVs) are an ecologically important habitat with a geographic origin similar to that of deep-sea HTVs. Studies on shallow-water HTVs have not only facilitated understanding of the influences of vents on local ecosystems but also helped to extend the knowledge on deep-sea vents. In this study, the diversity of bacterial communities in the sediments of shallow-water HTVs off Kueishan Island, Taiwan, was investigated by examining the 16S ribosomal RNA gene as well as key functional genes involved in chemoautotrophic carbon fixation (aclB, cbbL and cbbM). In the vent area, Sulfurovum and Sulfurimonas of Epsilonproteobacteria appeared to dominate the benthic bacterial community. Results of aclB gene analysis also suggested involvement of these bacteria in carbon fixation using the reductive tricarboxylic acid (rTCA) cycle. Analysis of the cbbM gene showed that Alphaproteobacterial members such as the purple non-sulfur bacteria were the major chemoautotrophic bacteria involving in carbon fixation via the Calvin-Benson-Bassham (CBB) cycle. However, they only accounted for <2% of the total bacterial community in the vent area. These findings suggest that the rTCA cycle is the major chemoautotrophic carbon fixation pathway in sediments of the shallow-water HTVs off Kueishan Island.

Gifted microbes for genome mining and natural product discovery.

Actinomycetes are historically important sources for secondary metabolites (SMs) with applications in human medicine, animal health, and plant crop protection. It is now clear that actinomycetes and other microorganisms with large genomes have the capacity to produce many more SMs than was anticipated from standard fermentation studies. Indeed ~90 % of SM gene clusters (SMGCs) predicted from genome sequencing are cryptic under conventional fermentation and analytical analyses. Previous studies have suggested that among the actinomycetes with large genomes, some have the coding capacity to produce many more SMs than others, and that strains with the largest genomes tend to be the most gifted. These contentions have been evaluated more quantitatively by antiSMASH 3.0 analyses of microbial genomes, and the results indicate that many actinomycetes with large genomes are gifted for SM production, encoding 20-50 SMGCs, and devoting 0.8-3.0 Mb of coding capacity to SM production. Several Proteobacteria and Firmacutes with large genomes encode 20-30 SMGCs and devote 0.8-1.3 Mb of DNA to SM production, whereas cultured bacteria and archaea with small genomes devote insignificant coding capacity to SM production. Fully sequenced genomes of uncultured bacteria and archaea have small genomes nearly devoid of SMGCs.

Hydrodynamics and collective behavior of the tethered bacterium Thiovulum majus.

The ecology and dynamics of many microbial systems, particularly in mats and soils, are shaped by how bacteria respond to evolving nutrient gradients and microenvironments. Here we show how the response of the sulfur-oxidizing bacterium Thiovulum majus to changing oxygen gradients causes cells to organize into large-scale fronts. To study this phenomenon, we develop a technique to isolate and enrich these bacteria from the environment. Using this enrichment culture, we observe the formation and dynamics of T. majus fronts in oxygen gradients. We show that these dynamics can be understood as occurring in two steps. First, chemotactic cells moving up the oxygen gradient form a front that propagates with constant velocity. We then show, through observation and mathematical analysis, that this front becomes unstable to changes in cell density. Random perturbations in cell density create oxygen gradients. The response of cells magnifies these gradients and leads to the formation of millimeter-scale fluid flows that actively pull oxygenated water through the front. We argue that this flow results from a nonlinear instability excited by stochastic fluctuations in the density of cells. Finally, we show that the dynamics by which these modes interact can be understood from the chemotactic response of cells. These results provide a mathematically tractable example of how collective phenomena in ecological systems can arise from the individual response of cells to a shared resource.