Host-Endosymbiont Genome Integration in a Deep-Sea Chemosymbiotic Clam.

Endosymbiosis with chemosynthetic bacteria has enabled many deep-sea invertebrates to thrive at hydrothermal vents and cold seeps, but most previous studies on this mutualism have focused on the bacteria only. Vesicomyid clams dominate global deep-sea chemosynthesis-based ecosystems. They differ from most deep-sea symbiotic animals in passing their symbionts from parent to offspring, enabling intricate coevolution between the host and the symbiont. Here, we sequenced the genomes of the clam Archivesica marissinica (Bivalvia: Vesicomyidae) and its bacterial symbiont to understand the genomic/metabolic integration behind this symbiosis. At 1.52 Gb, the clam genome encodes 28 genes horizontally transferred from bacteria, a large number of pseudogenes and transposable elements whose massive expansion corresponded to the timing of the rise and subsequent divergence of symbiont-bearing vesicomyids. The genome exhibits gene family expansion in cellular processes that likely facilitate chemoautotrophy, including gas delivery to support energy and carbon production, metabolite exchange with the symbiont, and regulation of the bacteriocyte population. Contraction in cellulase genes is likely adaptive to the shift from phytoplankton-derived to bacteria-based food. It also shows contraction in bacterial recognition gene families, indicative of suppressed immune response to the endosymbiont. The gammaproteobacterium endosymbiont has a reduced genome of 1.03 Mb but retains complete pathways for sulfur oxidation, carbon fixation, and biosynthesis of 20 common amino acids, indicating the host's high dependence on the symbiont for nutrition. Overall, the host-symbiont genomes show not only tight metabolic complementarity but also distinct signatures of coevolution allowing the vesicomyids to thrive in chemosynthesis-based ecosystems.

Thiosulfativibrio zosterae gen. nov., sp. nov., and Thiosulfatimonas sediminis gen. nov., sp. nov.

Aerobic, Gram-stain-negative, obligately chemolithoautotrophic thiosulfate-oxidizing bacteria, strains AkT22T and aks77T were isolated from a brackish lake in Japan. Strains AkT22T and aks77T were isolated from samples of eelgrass and sediment, respectively. Growth on sulfide, tetrathionate, elemental sulfur, and organic substrates was not observed for both strains. Growth of the strains was observed at 5 °C or higher temperature, with optimum growth at 22 °C. Strain AkT22T grew at a pH range of 5.8-8.0, with optimum growth at pH 6.7-7.8. Strain aks77T grew at a pH range of 5.8-8.5, with optimum growth at pH 7.0-7.9. Major cellular fatty acids (> 10% of total) of strain AkT22T were C16:1, C18:1, and C16:0. The sole respiratory quinone was ubiquinone-8 in both strains. The genome of strain AkT22T consisted of a circular chromosome, with size of approximately 2.6 Mbp and G + C content of 43.2%. Those values of the genome of strain aks77T were ca. 2.7 Mbp and 45.5%, respectively. Among cultured bacteria, Thiomicrorhabdus aquaedulcis HaS4T showed the highest sequence identities of the 16S rRNA gene, to strains AkT22T (94%) and aks77T (95%). On the basis of these results, Thiosulfativibrio zosterae gen. nov., sp. nov. and Thiosulfatimonas sediminis gen. nov., sp. nov. are proposed, with type strains of AkT22T (= BCRC 81184T = NBRC 114012T = DSM 109948T) and aks77T (= BCRC 81183T = NBRC 114013T), respectively.

Engineering the genetic components of a whole-cell catalyst for improved enzymatic CO2 capture and utilization.

Carbonic anhydrase (CA) is a diffusion-limited enzyme that rapidly catalyzes the hydration of carbon dioxide (CO2 ). CA has been proposed as an eco-friendly yet powerful catalyst for CO2 capture and utilization. A bacterial whole-cell biocatalyst equipped with periplasmic CA provides an option for a cost-effective CO2 -capturing system. However, further utilization of the previously constructed periplasmic system has been limited by its relatively low activity and stability. Herein, we engineered three genetic components of the periplasmic system for the construction of a highly efficient whole-cell catalyst: a CA-coding gene, a signal sequence, and a ribosome-binding site (RBS). A stable and halotolerant CA (hmCA) from the marine bacterium Hydrogenovibrio marinus was employed to improve both the activity and stability of the system. The improved secretion and folding of hmCA and increased membrane permeability were achieved by translocation via the Sec-dependent pathway. The engineering of RBS strength further enhanced whole-cell activity by improving both the secretion and folding of hmCA. The newly engineered biocatalyst displayed 5.7-fold higher activity and 780-fold higher stability at 60°C compared with those of the previously constructed periplasmic system, providing new opportunities for applications in CO2 capture and utilization.

Horizontal acquisition of hydrogen conversion ability and other habitat adaptations in the Hydrogenovibrio strains SP-41 and XCL-2.

BACKGROUND: Obligate sulfur oxidizing chemolithoauthotrophic strains of Hydrogenovibrio crunogenus have been isolated from multiple hydrothermal vent associated habitats. However, a hydrogenase gene cluster (encoding the hydrogen converting enzyme and its maturation/assembly machinery) detected on the first sequenced H. crunogenus strain (XCL-2) suggested that hydrogen conversion may also play a role in this organism. Yet, numerous experiments have underlined XCL-2's inability to consume hydrogen under the tested conditions. A recent study showed that the closely related strain SP-41 contains a homolog of the XCL-2 hydrogenase (a group 1b [NiFe]-hydrogenase), but that it can indeed use hydrogen. Hence, the question remained unresolved, why SP-41 is capable of using hydrogen, while XCL-2 is not. RESULTS: Here, we present the genome sequence of the SP-41 strain and compare it to that of the XCL-2 strain. We show that the chromosome of SP-41 codes for a further hydrogenase gene cluster, including two additional hydrogenases: the first appears to be a group 1d periplasmic membrane-anchored hydrogenase, and the second a group 2b sensory hydrogenase. The region where these genes are located was likely acquired horizontally and exhibits similarity to other Hydrogenovibrio species (H. thermophilus MA2-6 and H. marinus MH-110 T) and other hydrogen oxidizing Proteobacteria (Cupriavidus necator H16 and Ghiorsea bivora TAG-1 T). The genomes of XCL-2 and SP-41 show a strong conservation in gene order. However, several short genomic regions are not contained in the genome of the other strain. These exclusive regions are often associated with signs of DNA mobility, such as genes coding for transposases. They code for transport systems and/or extend the metabolic potential of the strains. CONCLUSIONS: Our results suggest that horizontal gene transfer plays an important role in shaping the genomes of these strains, as a likely mechanism for habitat adaptation, including, but not limited to the transfer of the hydrogen conversion ability.

New Zealand rickettsia-like organism (NZ-RLO) and Tenacibaculum maritimum: Distribution and phylogeny in farmed Chinook salmon (Oncorhynchus tshawytscha).

A total of 777 fish from three growing regions of New Zealand Chinook salmon farms comprising of five sites were tested. Quantitative PCR was used to determine the distribution of New Zealand rickettsia-like organism and Tenacibaculum maritimum. Genetic information from these bacteria were then compared with strains reported worldwide. Using this information, suggested associations of pathogens with clinically affected fish were made. NZ-RLO was detected in two of the three regions, and T. maritimum was detected in all regions. Three strains of NZ-RLO were identified during this study. Based on analysis of the ITS rRNA gene, NZ-RLO1 appears to be part of an Australasian grouping sharing high similarity with the Tasmanian RLO, NZ-RLO2 was shown to be the same as an Irish strain, and NZ-RLO3 was shown be closely related to two strains from Chile. Based on multi-locus sequence typing, the New Zealand T. maritimum was the same as Australian strains. NZ-RLOs were detected more frequently in fish with skin ulcers than fish without skin ulcers. While additional research is required to investigate the pathogenicity of these organisms, this is the first time that NZ-RLOs have been associated with the development of clinical infections in farmed Chinook salmon.

Diversity in CO2-Concentrating Mechanisms among Chemolithoautotrophs from the Genera Hydrogenovibrio, Thiomicrorhabdus, and Thiomicrospira, Ubiquitous in Sulfidic Habitats Worldwide.

Members of the genera Hydrogenovibrio, Thiomicrospira, and Thiomicrorhabdus fix carbon at hydrothermal vents, coastal sediments, hypersaline lakes, and other sulfidic habitats. The genome sequences of these ubiquitous and prolific chemolithoautotrophs suggest a surprising diversity of mechanisms for the uptake and fixation of dissolved inorganic carbon (DIC); these mechanisms are verified here. Carboxysomes are apparent in the transmission electron micrographs of most of these organisms but are lacking in Thiomicrorhabdus sp. strain Milos-T2 and Thiomicrorhabdus arctica, and the inability of Thiomicrorhabdus sp. strain Milos-T2 to grow under low-DIC conditions is consistent with the absence of carboxysome loci in its genome. For the remaining organisms, genes encoding potential DIC transporters from four evolutionarily distinct families (Tcr_0853 and Tcr_0854, Chr, SbtA, and SulP) are located downstream of carboxysome loci. Transporter genes collocated with carboxysome loci, as well as some homologs located elsewhere on the chromosomes, had elevated transcript levels under low-DIC conditions, as assayed by reverse transcription-quantitative PCR (qRT-PCR). DIC uptake was measureable via silicone oil centrifugation when a representative of each of the four types of transporter was expressed in Escherichia coli The expression of these genes in the carbonic anhydrase-deficient E. coli strain EDCM636 enabled it to grow under low-DIC conditions, a result consistent with DIC transport by these proteins. The results from this study expand the range of DIC transporters within the SbtA and SulP transporter families, verify DIC uptake by transporters encoded by Tcr_0853 and Tcr_0854 and their homologs, and introduce DIC as a potential substrate for transporters from the Chr family.IMPORTANCE Autotrophic organisms take up and fix DIC, introducing carbon into the biological portion of the global carbon cycle. The mechanisms for DIC uptake and fixation by autotrophic Bacteria and Archaea are likely to be diverse but have been well characterized only for "Cyanobacteria" Based on genome sequences, members of the genera Hydrogenovibrio, Thiomicrospira, and Thiomicrorhabdus have a variety of mechanisms for DIC uptake and fixation. We verified that most of these organisms are capable of growing under low-DIC conditions, when they upregulate carboxysome loci and transporter genes collocated with these loci on their chromosomes. When these genes, which fall into four evolutionarily independent families of transporters, are expressed in E. coli, DIC transport is detected. This expansion in known DIC transporters across four families, from organisms from a variety of environments, provides insight into the ecophysiology of autotrophs, as well as a toolkit for engineering microorganisms for carbon-neutral biochemistries of industrial importance.

The impact of the Deepwater Horizon blowout on historic shipwreck-associated sediment microbiomes in the northern Gulf of Mexico.

More than 2,000 historic shipwrecks spanning 500 years of history, rest on the Gulf of Mexico seafloor. Shipwrecks serve as artificial reefs and hotspots of biodiversity by providing hard substrate, something rare in deep ocean regions. The Deepwater Horizon (DWH) spill discharged crude oil into the deep Gulf. Because of physical, biological, and chemical interactions, DWH oil was deposited on the seafloor, where historic shipwrecks are present. This study examined sediment microbiomes at seven historic shipwrecks. Steel-hulled, World War II-era shipwrecks and wooden-hulled, 19th century shipwrecks within and outside of the surface oiled area and subsurface plume were examined. Analysis of 16S rRNA sequence libraries, sediment radiocarbon age data, sedimentation rates, and hydrocarbons revealed that the German U-boat U-166 and the wooden-hulled sailing vessel known as the Mardi Gras Wreck, both in the Mississippi Canyon leasing area, were exposed to deposited oil during a rapid sedimentation event. Impacts to shipwreck microbiomes included a significant increase in Piscirickettsiaceae-related sequences in surface sediments, and reduced biodiversity relative to unimpacted sites. This study is the first to address the impact of the spill on shipwreck-associated microbiomes, and to explore how shipwrecks themselves influence microbiome diversity in the deep sea.

Genomes of ubiquitous marine and hypersaline Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira spp. encode a diversity of mechanisms to sustain chemolithoautotrophy in heterogeneous environments.

Chemolithoautotrophic bacteria from the genera Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira are common, sometimes dominant, isolates from sulfidic habitats including hydrothermal vents, soda and salt lakes and marine sediments. Their genome sequences confirm their membership in a deeply branching clade of the Gammaproteobacteria. Several adaptations to heterogeneous habitats are apparent. Their genomes include large numbers of genes for sensing and responding to their environment (EAL- and GGDEF-domain proteins and methyl-accepting chemotaxis proteins) despite their small sizes (2.1-3.1 Mbp). An array of sulfur-oxidizing complexes are encoded, likely to facilitate these organisms' use of multiple forms of reduced sulfur as electron donors. Hydrogenase genes are present in some taxa, including group 1d and 2b hydrogenases in Hydrogenovibrio marinus and H. thermophilus MA2-6, acquired via horizontal gene transfer. In addition to high-affinity cbb3 cytochrome c oxidase, some also encode cytochrome bd-type quinol oxidase or ba3 -type cytochrome c oxidase, which could facilitate growth under different oxygen tensions, or maintain redox balance. Carboxysome operons are present in most, with genes downstream encoding transporters from four evolutionarily distinct families, which may act with the carboxysomes to form CO2 concentrating mechanisms. These adaptations to habitat variability likely contribute to the cosmopolitan distribution of these organisms.

Peculiar citric acid cycle of hydrothermal vent chemolithoautotroph Hydrogenovibrio crunogenus, and insights into carbon metabolism by obligate autotrophs.

The genome sequence of the obligate chemolithoautotroph Hydrogenovibrio crunogenus paradoxically predicts a complete oxidative citric acid cycle (CAC). This prediction was tested by multiple approaches including whole cell carbon assimilation to verify obligate autotrophy, phylogenetic analysis of CAC enzyme sequences and enzyme assays. Hydrogenovibrio crunogenus did not assimilate any of the organic compounds provided (acetate, succinate, glucose, yeast extract, tryptone). Enzyme activities confirmed that its CAC is mostly uncoupled from the NADH pool. 2-Oxoglutarate:ferredoxin oxidoreductase activity is absent, though pyruvate:ferredoxin oxidoreductase is present, indicating that sequence-based predictions of substrate for this oxidoreductase were incorrect, and that H. crunogenus may have an incomplete CAC. Though the H. crunogenus CAC genes encode uncommon enzymes, the taxonomic distribution of their top matches suggests that they were not horizontally acquired. Comparison of H. crunogenus CAC genes to those present in other 'Proteobacteria' reveals that H. crunogenus and other obligate autotrophs lack the functional redundancy for the steps of the CAC typical for facultative autotrophs and heterotrophs, providing another possible mechanism for obligate autotrophy.

Enhanced crude oil biodegradative potential of natural phytoplankton-associated hydrocarbonoclastic bacteria.

Phytoplankton have been shown to harbour a diversity of hydrocarbonoclastic bacteria (HCB), yet it is not understood how these phytoplankton-associated HCB would respond in the event of an oil spill at sea. Here, we assess the diversity and dynamics of the bacterial community associated with a natural population of marine phytoplankton under oil spill-simulated conditions, and compare it to that of the free-living (non phytoplankton-associated) bacterial community. While the crude oil severely impacted the phytoplankton population and was likely conducive to marine oil snow formation, analysis of the MiSeq-derived 16S rRNA data revealed dramatic and differential shifts in the oil-amended communities that included blooms of recognized HCB (e.g., Thalassospira, Cycloclasticus), including putative novel phyla, as well as other groups with previously unqualified oil-degrading potential (Olleya, Winogradskyella, and members of the inconspicuous BD7-3 phylum). Notably, the oil biodegradation potential of the phytoplankton-associated community exceeded that of the free-living community, and it showed a preference to degrade substituted and non-substituted polycyclic aromatic hydrocarbons. Our study provides evidence of compartmentalization of hydrocarbon-degrading capacity in the marine water column, wherein HCB associated with phytoplankton are better tuned to degrading crude oil hydrocarbons than that by the community of planktonic free-living bacteria.

Short-chain alkanes fuel mussel and sponge Cycloclasticus symbionts from deep-sea gas and oil seeps.

Cycloclasticus bacteria are ubiquitous in oil-rich regions of the ocean and are known for their ability to degrade polycyclic aromatic hydrocarbons (PAHs). In this study, we describe Cycloclasticus that have established a symbiosis with Bathymodiolus heckerae mussels and poecilosclerid sponges from asphalt-rich, deep-sea oil seeps at Campeche Knolls in the southern Gulf of Mexico. Genomic and transcriptomic analyses revealed that, in contrast to all previously known Cycloclasticus, the symbiotic Cycloclasticus appears to lack the genes needed for PAH degradation. Instead, these symbionts use propane and other short-chain alkanes such as ethane and butane as carbon and energy sources, thus expanding the limited range of substrates known to power chemosynthetic symbioses. Analyses of short-chain alkanes in the environment of the Campeche Knolls symbioses revealed that these are present at high concentrations (in the µM to mM range). Comparative genomic analyses revealed high similarities between the genes used by the symbiotic Cycloclasticus to degrade short-chain alkanes and those of free-living Cycloclasticus that bloomed during the Deepwater Horizon oil spill. Our results indicate that the metabolic versatility of bacteria within the Cycloclasticus clade is higher than previously assumed, and highlight the expanded role of these keystone species in the degradation of marine hydrocarbons.

In-situ incubation of iron-sulfur mineral reveals a diverse chemolithoautotrophic community and a new biogeochemical role for Thiomicrospira.

Sulfide mineral precipitation occurs at mid-ocean ridge (MOR) spreading centers, both in the form of plume particles and seafloor massive sulfide structures. A common constituent of MOR is the iron-bearing sulfide mineral pyrrhotite, which was chosen as a substrate for in-situ incubation studies in shallow waters of Catalina Island, CA to investigate the colonization of iron-oxidizing bacteria. Microbial community datasets were obtained from in-situ incubated pyrrhotite, allowing for direct comparison to microbial communities of iron-sulfides from active and inactive chimneys in deep-sea environments. Unclassified Gammaproteobacteria and Alphaproteobacteria (Magnetovibrio) largely dominated the bacterial community on pyrrhotite samples incubated in the water column while samples incubated at the surface sediment showed more even dominance by Deltaproteobacteria (Desulfobulbus), Gammaproteobacteria (Piscirickettsiaceae), Alphaproteobacteria (Rhodobacteraceae), and Bacteroidetes (Flavobacteriia). Cultivations that originated from pyrrhotite samples resulted in the enrichment of both, sheath-forming and stalk-forming Zetaproteobacteria. Additionally, a putative novel species of Thiomicrospira was isolated and shown to grow autotrophically with iron, indicating a new biogeochemical role for this ubiquitous microorganism.

Proteomic and Mutant Analysis of the CO2 Concentrating Mechanism of Hydrothermal Vent Chemolithoautotroph Thiomicrospira crunogena.

Many autotrophic microorganisms are likely to adapt to scarcity in dissolved inorganic carbon (DIC; CO2 + HCO3- + CO32-) with CO2 concentrating mechanisms (CCM) that actively transport DIC across the cell membrane to facilitate carbon fixation. Surprisingly, DIC transport has been well studied among cyanobacteria and microalgae only. The deep-sea vent gammaproteobacterial chemolithoautotroph Thiomicrospira crunogena has a low-DIC inducible CCM, though the mechanism for uptake is unclear, as homologs to cyanobacterial transporters are absent. To identify the components of this CCM, proteomes of T. crunogena cultivated under low- and high-DIC conditions were compared. Fourteen proteins, including those comprising carboxysomes, were at least 4-fold more abundant under low-DIC conditions. One of these proteins was encoded by Tcr_0854; strains carrying mutated copies of this gene, as well as the adjacent Tcr_0853, required elevated DIC for growth. Strains carrying mutated copies of Tcr_0853 and Tcr_0854 overexpressed carboxysomes and had diminished ability to accumulate intracellular DIC. Based on reverse transcription (RT)-PCR, Tcr_0853 and Tcr_0854 were cotranscribed and upregulated under low-DIC conditions. The Tcr_0853-encoded protein was predicted to have 13 transmembrane helices. Given the mutant phenotypes described above, Tcr_0853 and Tcr_0854 may encode a two-subunit DIC transporter that belongs to a previously undescribed transporter family, though it is widespread among autotrophs from multiple phyla.IMPORTANCE DIC uptake and fixation by autotrophs are the primary input of inorganic carbon into the biosphere. The mechanism for dissolved inorganic carbon uptake has been characterized only for cyanobacteria despite the importance of DIC uptake by autotrophic microorganisms from many phyla among the Bacteria and Archaea In this work, proteins necessary for dissolved inorganic carbon utilization in the deep-sea vent chemolithoautotroph T. crunogena were identified, and two of these may be able to form a novel transporter. Homologs of these proteins are present in 14 phyla in Bacteria and also in one phylum of Archaea, the Euryarchaeota Many organisms carrying these homologs are autotrophs, suggesting a role in facilitating dissolved inorganic carbon uptake and fixation well beyond the genus Thiomicrospira.

Genome-scale metabolic reconstruction for the insidious bacterium in aquaculture Piscirickettsia salmonis.

Piscirickettsia salmonis is a fish bacterium that causes the disease piscirickettsiosis in salmonids. This pathology is partially controlled by vaccines. The lack of knowledge has hindered its culture on laboratory and industrial scale. The study describes the metabolic phenotype of P. salmonis in culture. This study presents the first genome-scale model (iPF215) of the LF-89 strain of P. salmonis, describing the central metabolic pathway, biosynthesis and molecule degradation and transport mechanisms. The model was adjusted with experiment data, allowing the identification of the capacities that were not predicted by the automatic annotation of the genome sequences. The iPF215 model is comprised of 417 metabolites, 445 reactions and 215 genes, was used to reproduce the growth of P. salmonis (µmax 0.052±0.005h-1). The metabolic reconstruction of the P. salmonis LF-89 strain obtained in this research provides a baseline that describes the metabolic capacities of the bacterium and is the basis for developing improvements to its cultivation for vaccine formulation.

Analysis of defence systems and a conjugative IncP-1 plasmid in the marine polyaromatic hydrocarbons-degrading bacterium Cycloclasticus sp. 78-ME.

Marine prokaryotes have evolved a broad repertoire of defence systems to protect their genomes from lateral gene transfer including innate or acquired immune systems and infection-induced programmed cell suicide and dormancy. Here we report on the analysis of multiple defence systems present in the genome of the strain Cycloclasticus sp. 78-ME isolated from petroleum deposits of the tanker 'Amoco Milford Haven'. Cycloclasticus are ubiquitous bacteria globally important in polyaromatic hydrocarbons degradation in marine environments. Two 'defence islands' were identified in 78-ME genome: the first harbouring CRISPR-Cas with toxin-antitoxin system, while the second was composed by an array of genes for toxin-antitoxin and restriction-modification proteins. Among all identified spacers of CRISPR-Cas system only seven spacers match sequences of phages and plasmids. Furthermore, a conjugative plasmid p7ME01, which belongs to a new IncP-1θ ancestral archetype without any accessory mobile elements was found in 78-ME. Our results provide the context to the co-occurrence of diverse defence mechanisms in the genome of Cycloclasticus sp. 78-ME, which protect the genome of this highly specialized PAH-degrader. This study contributes to the further understanding of complex networks established in petroleum-based microbial communities.

Thiomicrospira hydrogeniphila sp. nov., an aerobic, hydrogen- and sulfur-oxidizing chemolithoautotroph isolated from a seawater tank containing a block of beef tallow.

A moderately psychrophilic, aerobic, hydrogen- and sulfur-oxidizing bacterium, designated strain MAS2T, was isolated from a tank containing coastal seawater from Tokyo Bay and a block of beef tallow added as organic material. Growth occurred under aerobic chemolithoautotrophic conditions in the presence of molecular hydrogen, thiosulfate, tetrathionate, elemental sulfur or sulfide as the sole energy source and bicarbonate as a carbon source. The isolate represented a Gram-staining-negative rod with a single polar flagellum and grew in artificial seawater medium with thiosulfate at 2-40 °C (optimum 30 °C). The isolate grew in media with thiosulfate at Na+ concentrations between 30 and 1380 mM (optimum 270 mM). MAS2T possessed C16 : 0, C16 : 1 and C18 : 1 as the major fatty acids. The G+C content of the genomic DNA was 39.6 mol%. The 16S rRNA gene sequence similarity analysis showed that the isolate represented a member of the genus Thiomicrospira within the class Gammaproteobacteria and was most closely related to Thiomicrospira frisia JB-A2T. On the basis of phenotypic and molecular properties, the isolate represents a novel species of the genus Thiomicrospira, for which the name Thiomicrospira hydrogeniphila sp. nov. is proposed (type strain, MAS2T=JCM 30760T=DSM 100274T).

Dissolved inorganic carbon uptake in Thiomicrospira crunogena XCL-2 is Δp- and ATP-sensitive and enhances RubisCO-mediated carbon fixation.

The gammaproteobacterium Thiomicrospira crunogena XCL-2 is an aerobic sulfur-oxidizing hydrothermal vent chemolithoautotroph that has a CO2 concentrating mechanism (CCM), which generates intracellular dissolved inorganic carbon (DIC) concentrations much higher than extracellular, thereby providing substrate for carbon fixation at sufficient rate. This CCM presumably requires at least one active DIC transporter to generate the elevated intracellular concentrations of DIC measured in this organism. In this study, the half-saturation constant (K CO2) for purified carboxysomal RubisCO was measured (276 ± 18 µM) which was much greater than the K CO2 of whole cells (1.03 µM), highlighting the degree to which the CCM facilitates CO2 fixation under low CO2 conditions. To clarify the bioenergetics powering active DIC uptake, cells were incubated in the presence of inhibitors targeting ATP synthesis (DCCD) or proton potential (CCCP). Incubations with each of these inhibitors resulted in diminished intracellular ATP, DIC, and fixed carbon, despite an absence of an inhibitory effect on proton potential in the DCCD-incubated cells. Electron transport complexes NADH dehydrogenase and the bc 1 complex were found to be insensitive to DCCD, suggesting that ATP synthase was the primary target of DCCD. Given the correlation of DIC uptake to the intracellular ATP concentration, the ABC transporter genes were targeted by qRT-PCR, but were not upregulated under low-DIC conditions. As the T. crunogena genome does not include orthologs of any genes encoding known DIC uptake systems, these data suggest that a novel, yet to be identified, ATP- and proton potential-dependent DIC transporter is active in this bacterium. This transporter serves to facilitate growth by T. crunogena and other Thiomicrospiras in the many habitats where they are found.

Genome sequence of obligate marine polycyclic aromatic hydrocarbons-degrading bacterium Cycloclasticus sp. 78-ME, isolated from petroleum deposits of the sunken tanker Amoco Milford Haven, Mediterranean Sea.

Cycloclasticus sp. 78-ME isolated from petroleum deposits of the sunken tanker "Amoco Milford Haven" (Gulf of Genoa, Ligurian Sea, Italy) could effectively degrade polycyclic aromatic hydrocarbons of up to five condensed rings. The genome of 78-ME was sequenced and analysed to gain insights into its remarkable degrading capacities. It comprises two circular replicons, the 2,613,078 bp chromosome and the plasmid of 42,347 bp, with 41.84% and 53.28% of the G + C content respectively. A total of 2585 protein-coding genes were obtained, and three large operons with more than fifteen enzymes belonging to four different classes of ring-cleavage dioxygenases were found.

Combining metagenomics with metaproteomics and stable isotope probing reveals metabolic pathways used by a naturally occurring marine methylotroph.

A variety of culture-independent techniques have been developed that can be used in conjunction with culture-dependent physiological and metabolic studies of key microbial organisms in order to better understand how the activity of natural populations influences and regulates all major biogeochemical cycles. In this study, we combined deoxyribonucleic acid-stable isotope probing (DNA-SIP) with metagenomics and metaproteomics to characterize an uncultivated marine methylotroph that actively incorporated carbon from (13) C-labeled methanol into biomass. By metagenomic sequencing of the heavy DNA, we retrieved virtually the whole genome of this bacterium and determined its metabolic potential. Through protein-stable isotope probing, the RuMP cycle was established as the main carbon assimilation pathway, and the classical methanol dehydrogenase-encoding gene mxaF, as well as three out of four identified xoxF homologues were found to be expressed. This proof-of-concept study is the first in which the culture-independent techniques of DNA-SIP and protein-SIP have been used to characterize the metabolism of a naturally occurring Methylophaga-like bacterium in the marine environment (i.e. Methylophaga thiooxydans L4) and thus provides a powerful approach to access the genome and proteome of uncultivated microbes involved in key processes in the environment.

A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a key enzyme of the Calvin cycle, which is responsible for most of Earth's primary production. Although research on RubisCO genes and enzymes in plants, cyanobacteria and bacteria has been ongoing for years, still little is understood about its regulation and activation in bacteria. Even more so, hardly any information exists about the function of metagenomic RubisCOs and the role of the enzymes encoded on the flanking DNA owing to the lack of available function-based screens for seeking active RubisCOs from the environment. Here we present the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. We constructed a metagenomic library from hydrothermal vent fluids and screened 1056 fosmid clones. Twelve clones exhibited RubisCO activity and the metagenomic fragments resembled genes from Thiomicrospira crunogena. One of these clones was further analyzed. It contained a 35.2 kb metagenomic insert carrying the RubisCO gene cluster and flanking DNA regions. Knockouts of twelve genes and two intergenic regions on this metagenomic fragment demonstrated that the RubisCO activity was significantly impaired and was attributed to deletions in genes encoding putative transcriptional regulators and those believed to be vital for RubisCO activation. Our new technique revealed a novel link between a poorly characterized gene and RubisCO activity. This screen opens the door to directly investigating RubisCO genes and respective enzymes from environmental samples.