A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition.

Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of foreign nucleic acids (spacers) into genomic CRISPR arrays. Cas6 proteins then process CRISPR array transcripts into spacer-derived RNAs (CRISPR RNAs; crRNAs) that target Cas nucleases to matching invaders. We find that a Marinomonas mediterranea fusion protein combines three enzymatic domains (Cas6, reverse transcriptase [RT], and Cas1), which function in both crRNA biogenesis and spacer acquisition from RNA and DNA. We report a crystal structure of this divergent Cas6, identify amino acids required for Cas6 activity, show that the Cas6 domain is required for RT activity and RNA spacer acquisition, and demonstrate that CRISPR-repeat binding to Cas6 regulates RT activity. Co-evolution of putative interacting surfaces suggests a specific structural interaction between the Cas6 and RT domains, and phylogenetic analysis reveals repeated, stable association of free-standing Cas6s with CRISPR RTs in multiple microbial lineages, indicating that a functional interaction between these proteins preceded evolution of the fusion.

Marinomonas mediterranea synthesizes an R-type bacteriocin.

Prophages integrated into bacterial genomes can become cryptic or defective prophages, which may evolve to provide various traits to bacterial cells. Previous research on Marinomonas mediterranea MMB-1 demonstrated the production of defective particles. In this study, an analysis of the genomes of three different strains (MMB-1, MMB-2, and MMB-3) revealed the presence of a region named MEDPRO1, spanning approximately 52 kb, coding for a defective prophage in strains MMB-1 and MMB-2. This prophage seems to have been lost in strain MMB-3, possibly due to the presence of spacers recognizing this region in an I-F CRISPR array in this strain. However, all three strains produce remarkably similar defective particles. Using strain MMB-1 as a model, mass spectrometry analyses indicated that the structural proteins of the defective particles are encoded by a second defective prophage situated within the MEDPRO2 region, spanning approximately 13 kb. This finding was further validated through the deletion of this second defective prophage. Genomic region analyses and the detection of antimicrobial activity of the defective prophage against other Marinomonas species suggest that it is an R-type bacteriocin. Marinomonas mediterranea synthesizes antimicrobial proteins with lysine oxidase activity, and the synthesis of an R-type bacteriocin constitutes an additional mechanism in microbial competition for the colonization of habitats such as the surface of marine plants.IMPORTANCEThe interactions between bacterial strains inhabiting the same environment determine the final composition of the microbiome. In this study, it is shown that some extracellular defective phage particles previously observed in Marinomonas mediterranea are in fact R-type bacteriocins showing antimicrobial activity against other Marinomonas strains. The operon coding for the R-type bacteriocin has been identified.

Biodetoxification mercury by using a marine bacterium Marinomonas sp. RS3 and its merA gene expression under mercury stress.

Mercury (Hg) pollution in water has been a problem for the ecosystem and human health, thus eco-friendly remediation methods are gaining traction around the world. In this study, a bacterial strain designated as RS3 isolated from the Red Sea (Saudi Arabia) has shown tolerance to more than 250 mg/L of Hg2+ on minimum inhibitory studies. The isolate RS3 was identified as Marinomonas sp., (Accession No: OK271312) using 16s rRNA sequencing. Tracing the growth curve for the RS3 showed that maximum growth attained at 72 h and only 10% reduction than the control medium for 50 mg/L HgCl2 supplemented seawater medium, which continued to reduce as 21% to 60 with the increment of HgCl2 from 100 to 350 mg/L. The Hg2+ removal potential of RS3 is observed to be 78% at 50 mg/L HgCl2/72 h, which is significantly altered with the addition of carbon source such as glucose (84.5%) > fructose (79.8%) > control (78%) > citrate (73.4%) > acetate (60.2%) > maltose (54.7%). Box-Behnken design (BBD) well proposed a model with R2 value of 0.8922, which predict a utmost Hg2+ removal of 89.5% by RS2 at favorable conditions (pH-7; NaC 1% and glucose 5%) at 72 h. Mercuric reductase enzyme encoded merA gene expression was found to be high in RS3 isolates cultivated in 100 mg/L of HgCl2 in comparison with other variables. Thus the seawater isolate Marinomonas sp. RS3 expressed a significant tolerance and removal potential towards the Hg2+, which would make it is a noteworthy applicant for effective mercury remediation practices.

Bacterial sugar-binding protein as a one-step affinity purification tag on dextran-containing resins.

Marinobacter hydrocarbonoclasticus is an oil-eating bacterium that possesses a large adhesion protein (MhLap) with the potential to bind extracellular ligands. One of these ligand-binding modules is the ~20-kDa PA14 domain (MhPA14) that has affinity for glucose-based carbohydrates. Previous studies showed this sugar-binding domain is retained on dextran-based size-exclusion resins during chromatography, requiring the introduction of glucose or EDTA to remove the protein from the column. Given the ready availability of such size-exclusion resins in biochemistry laboratories, this study explores the use of MhPA14 as an affinity tag for recombinant protein purification. Two different fusion proteins were tested: 1) Green fluorescent protein (GFP) linked to the N-terminus of the MhPA14 tag; and 2) the ice-binding domain from the Marinomonas primoryensis ice-binding protein (MpIBD) linked to the MhPA14 C-terminus by a TEV cut site. The GFP_MhPA14 fusion visibly bound to Superdex, Sephadex, and Sephacryl resins, but did not bind to Sepharose. Using Superdex resin, dextran-affinity purification proved to be an effective one-step purification strategy for both proteins, superior to even nickel-affinity chromatography. Dextran-affinity chromatography was also the most effective method of separating the MhPA14 tag from MpIBD following TEV proteolysis, as compared to both nickel-affinity and ice-affinity methods. These results indicate that MhPA14 has potential for widespread use in recombinant protein purification.

Insights into Thiotemplated Pyrrole Biosynthesis Gained from the Crystal Structure of Flavin-Dependent Oxidase in Complex with Carrier Protein.

Sequential enzymatic reactions on substrates tethered to carrier proteins (CPs) generate thiotemplated building blocks that are then delivered to nonribosomal peptide synthetases (NRPSs) to generate peptidic natural products. The underlying diversity of these thiotemplated building blocks is the principal driver of the chemical diversity of NRPS-derived natural products. Structural insights into recognition of CPs by tailoring enzymes that generate these building blocks are sparse. Here we present the crystal structure of a flavin-dependent prolyl oxidase that furnishes thiotemplated pyrrole as the product, in complex with its cognate CP in the holo and product-bound states. The thiotemplated pyrrole is an intermediate that is well-represented in natural product biosynthetic pathways. Our results delineate the interactions between the CP and the oxidase while also providing insights into the stereospecificity of the enzymatic oxidation of the prolyl heterocycle to the aromatic pyrrole. Biochemical validation of the interaction between the CP and the oxidase demonstrates that NRPSs recognize and bind to their CPs using interactions quite different from those of fatty acid and polyketide biosynthetic enzymes. Our results posit that structural diversity in natural product biosynthesis can be, and is, derived from subtle modifications of primary metabolic enzymes.

Interaction of GoxA with Its Modifying Enzyme and Its Subunit Assembly Are Dependent on the Extent of Cysteine Tryptophylquinone Biosynthesis.

GoxA is a glycine oxidase bearing a protein-derived cysteine tryptophylquinone (CTQ) cofactor that is formed by posttranslational modifications catalyzed by a flavoprotein, GoxB. Two forms of GoxA were isolated: an active form with mature CTQ and an inactive precursor protein that lacked CTQ. The active GoxA was present as a homodimer with no detectable affinity for GoxB, whereas the precursor was isolated as a monomer in a tight complex with one GoxB. Thus, the interaction of GoxA with GoxB and subunit assembly of mature GoxA are each dependent on the extent of CTQ biosynthesis.

Different recombinant forms of polyphenol oxidase A, a laccase from Marinomonas mediterranea.

Polyphenol oxidase from the marine bacterium Marinomonas mediterranea (MmPPOA) is a membrane-bound, blue, multi-copper laccase of 695 residues. It possesses peculiar properties that distinguish it from known laccases, such as a broad substrate specificity (common to tyrosinases) and a high redox potential. In order to push the biotechnological application of this laccase, the full-length enzyme was overexpressed in Escherichia coli cells with and without a C-terminal His-tag. The previous form, named rMmPPOA-695-His, was purified to homogeneity by HiTrap chelating chromatography following solubilization by 1% SDS in the lysis buffer with an overall yield of ≈1 mg/L fermentation broth and a specific activity of 1.34 U/mg protein on 2,6-dimethoxyphenol as substrate. A truncated enzyme form lacking 58 residues at the N-terminus encompassing the putative membrane binding region, namely rMmPPOA-637-His, was successfully expressed in E. coli as soluble protein and was purified by using the same procedure set-up as for the full-length enzyme. Elimination of the N-terminal sequence decreased the specific activity 15-fold (which was partially restored in the presence of 1 M NaCl) and altered the secondary and tertiary structures and the pH dependence of optimal stability. The recombinant rMmPPOA-695-His showed kinetic properties on catechol higher than for known laccases, a very high thermal stability, and a strong resistance to NaCl, DMSO, and Tween-80, all properties that are required for specific, targeted industrial applications.

An in silico, in vitro and in vivo investigation of indole-3-carboxaldehyde identified from the seawater bacterium Marinomonas sp. as an anti-biofilm agent against Vibrio cholerae O1.

Biofilm formation is a major contributing factor in the pathogenesis of Vibrio cholerae O1 (VCO1) and therefore preventing biofilm formation could be an effective alternative strategy for controlling cholera. The present study was designed to explore seawater bacteria as a source of anti-biofilm agents against VCO1. Indole-3-carboxaldehyde (I3C) was identified as an active principle component in Marinomonas sp., which efficiently inhibited biofilm formation by VCO1 without any selection pressure. Furthermore, I3C applications also resulted in considerable collapsing of preformed pellicles. Real-time PCR studies revealed the down-regulation of virulence gene expression by modulation of the quorum-sensing pathway and enhancement of protease production, which was further confirmed by phenotypic assays. Furthermore, I3C increased the survival rate of Caenorhabditis elegans when infected with VCO1 by significantly reducing in vivo biofilm formation, which was corroborated by a survivability assay. Thus, this study revealed, for the first time, the potential of I3C as an anti-biofilm agent against VCO1.

LodB is required for the recombinant synthesis of the quinoprotein L-lysine-ε-oxidase from Marinomonas mediterranea.

Marinomonas mediterranea is a marine gamma-proteobacterium that synthesizes LodA, a novel L-lysine-ε-oxidase (E.C. 1.4.3.20). This enzyme oxidizes L-lysine generating 2-aminoadipate 6-semialdehyde, ammonium, and hydrogen peroxide. Unlike other L-amino acid oxidases, LodA is not a flavoprotein but contains a quinone cofactor. LodA is encoded by an operon with two genes, lodA and lodB. In the native system, LodB is required for the synthesis of a functional LodA. In this study, we report the recombinant expression of LodA in Escherichia coli using vectors that allow its expression and accumulation in the cytoplasm. To reveal the L-lysine-ε-oxidase activity using the Amplex Red method for hydrogen peroxide detection, it is necessary to first remove the E. coli cytoplasmic catalases. The flavoprotein LodB is the only M. mediterranea protein required in the recombinant system for the generation of the cofactor of LodA. In the absence of LodB, LodA does not contain the quinone cofactor and remains in an inactive form. The results presented indicate that LodB participates in the posttranslational modification of LodA that generates the quinone cofactor.

Marinomonas brasilensis sp. nov., isolated from the coral Mussismilia hispida, and reclassification of Marinomonas basaltis as a later heterotypic synonym of Marinomonas communis.

A Gram-negative, aerobic bacterium, designated strain R-40503(T), was isolated from mucus of the reef-builder coral Mussismilia hispida, located in the São Sebastião Channel, São Paulo, Brazil. Phylogenetic analyses revealed that strain R-40503(T) belongs to the genus Marinomonas. The 16S rRNA gene sequence similarity of R-40503(T) was above 97 % with the type strains of Marinomonas vaga, M. basaltis, M. communis and M. pontica, and below 97 % with type strains of the other Marinomonas species. Strain R-40503(T) showed less than 35 % DNA-DNA hybridization (DDH) with the type strains of the phylogenetically closest Marinomonas species, demonstrating that it should be classified into a novel species. Amplified fragment length polymorphism (AFLP), chemotaxonomic and phenotypic analyses provided further evidence for the proposal of a novel species. Concurrently, a close genomic relationship between M. basaltis and M. communis was observed. The type strains of these two species showed 78 % DDH and 63 % AFLP pattern similarity. Their phenotypic features were very similar, and their DNA G+C contents were identical (46.3 mol%). Collectively, these data demonstrate unambiguously that Marinomonas basaltis is a later heterotypic synonym of Marinomonas communis. Several phenotypic features can be used to discriminate between Marinomonas species. The novel strain R-40503(T) is clearly distinguishable from its neighbours. For instance, it shows oxidase and urease activity, utilizes l-asparagine and has the fatty acid C(12 : 1) 3-OH but lacks C(10 : 0) and C(12 : 0). The name Marinomonas brasilensis sp. nov. is proposed, with the type strain R-40503(T) ( = R-278(T)  = LMG 25434(T)  = CAIM 1459(T)). The DNA G+C content of strain R-40503(T) is 46.5 mol%.

Non-specific porins of Gram-negative bacteria as proteins containing intrinsically disordered regions with amyloidogenic potential.

Features of the structure and functional activity of bacterial outer membrane porins, coupled with their dynamic "behavior," suggests that intrinsically disordered regions (IDPRs) are contained in their structure. Using bioinformatic analysis, the quantitative content of amyloidogenic regions in the amino acid sequence of non-specific porins inhabiting various natural niches was determined: from terrestrial bacteria of the genus Yersinia (OmpF and OmpC proteins of Y. pseudotuberculosis and Y. ruckeri) and from the marine bacterium Marinomonas primoryensis (MpOmp). It was found that OmpF and OmpC porins can be classified as moderately disordered proteins, while MpOmp can be classified as highly disordered protein. Mapping of IDPRs, performed using 3D structures of monomers of the proteins, showed that the regions of increased conformational plasticity fall on the regions, the functional importance of which has been reliably confirmed as a result of numerous experimental studies. The revealed correlation made it possible to explain the differences in the physicochemical characteristics and properties of not only porins from terrestrial and marine bacteria, but also non-specific porins of different types, OmpF and OmpC proteins. First of all, this concerns the flexible outer loops that form the pore vestibule, as well as regions of the barrel with an increased "ability" for aggregation, the so-called "hot spots" of aggregation. The abnormally high content of IDPRs in the MpOmp structure made it possible to suggest that the high adaptive potential of bacteria may correlate with an increase in the number of IDPRs and/or regions with increased conformational variability.

Structural Basis of Ligand Selectivity by a Bacterial Adhesin Lectin Involved in Multispecies Biofilm Formation.

Carbohydrate recognition by lectins governs critical host-microbe interactions. MpPA14 (Marinomonas primoryensis PA14 domain) lectin is a domain of a 1.5-MDa adhesin responsible for a symbiotic bacterium-diatom interaction in Antarctica. Here, we show that MpPA14 binds various monosaccharides, with l-fucose and N-acetylglucosamine being the strongest ligands (dissociation constant [Kd ], ∼150 µM). High-resolution structures of MpPA14 with 15 different sugars bound elucidated the molecular basis for the lectin's apparent binding promiscuity but underlying selectivity. MpPA14 mediates strong Ca2+-dependent interactions with the 3,4-diols of l-fucopyranose and glucopyranoses, and it binds other sugars via their specific minor isomers. Thus, MpPA14 only binds polysaccharides like branched glucans and fucoidans with these free end groups. Consistent with our findings, adhesion of MpPA14 to diatom cells was selectively blocked by l-fucose, but not by N-acetyl galactosamine. The MpPA14 lectin homolog present in a Vibrio cholerae adhesin was produced and was shown to have the same sugar binding preferences as MpPA14. The pathogen's lectin was unable to effectively bind the diatom in the presence of fucose, thus demonstrating the antiadhesion strategy of blocking infection via ligand-based antagonists.IMPORTANCE Bacterial adhesins are key virulence factors that are essential for the pathogen-host interaction and biofilm formation that cause most infections. Many of the adhesin-driven cell-cell interactions are mediated by lectins. Our study reveals for the first time the molecular basis underlying the binding selectivity of a common bacterial adhesin lectin from the marine bacterium Marinomonas primoryensis, homologs of which are found in both environmental and pathogenic species. The lectin-ligand interactions illustrated at the atomic level guided the identification of a ligand that serves as an inhibitor to block bacterium-host adhesion. With conventional bactericidal antibiotics losing their potency due to resistance, our work gives critical insight into an antiadhesion strategy to treat bacterial infections.

Complete genome sequence of Marinomonas arctica BSI20414, a giant antifreeze protein-producing bacterium isolated from Arctic sea ice.

Sea ice in the polar oceans is a dynamic and challenging environment for life to survive, with extreme gradients of temperature, salinity and nutrients etc., as well as formation of ice crystals. Bacteria surviving in sea ice attract broad attention from academia and industry, due to fascinating mechanisms for adaptation. Here we described the complete genome sequence of Marinomonas arctica BSI20414, isolated from Arctic sea ice. The strain tolerated high salinity and low temperature. Genetic features commonly related to adaptation to oxidative stress, osmotic stress and cold stress were detected in the genome. In addition, a large adhesion protein containing a putative antifreeze protein (AFP) domain was detected in the genome, similar with the giant AFP MpIBP from M. primoryensis. The presence of the putative AFP could facilitate M. arctica BSI20414 to bind to sea ice for favorable conditions and protect it from freezing. The genome sequence and the AFP reported here can provide insights into adaptation to sea ice and can be explored further for biotechnological applications.

A histidine kinase and a response regulator provide phage resistance to Marinomonas mediterranea via CRISPR-Cas regulation.

CRISPR-Cas systems are used by many prokaryotes to defend against invading genetic elements. In many cases, more than one CRISPR-Cas system co-exist in the same cell. Marinomonas mediterranea MMB-1 possesses two CRISPR-Cas systems, of type I-F and III-B respectively, which collaborate in phage resistance raising questions on how their expression is regulated. This study shows that the expression of both systems is controlled by the histidine kinase PpoS and a response regulator, PpoR, identified and cloned in this study. These proteins show similarity to the global regulators BarA/UvrY. In addition, homologues to the sRNAs CsrB and CsrC and the gene coding for the post-transcriptional repressor CsrA have been also identified indicating the conservation of the elements of the BarA/UvrY regulatory cascade in M. mediterranea. RNA-Seq analyses have revealed that all these genetics elements are regulated by PpoS/R supporting their participation in the regulatory cascade. The regulation by PpoS and PpoR of the CRISPR-Cas systems plays a role in phage defense since mutants in these proteins show an increase in phage sensitivity.

Isethionate formation from taurine in Chromohalobacter salexigens: purification of sulfoacetaldehyde reductase.

Bacterial generation of isethionate (2-hydroxyethanesulfonate) from taurine (2-aminoethanesulfonate) by anaerobic gut bacteria was established in 1980. That phenomenon in pure culture was recognized as a pathway of assimilation of taurine-nitrogen. Based on the latter work, we predicted from genome-sequence data that the marine gammaproteobacterium Chromohalobacter salexigens DSM 3043 would exhibit this trait. Quantitative conversion of taurine to isethionate, identified by mass spectrometry, was confirmed, and the taurine-nitrogen was recovered as cell material. An eight-gene cluster was predicted to encode the inducible vectorial, scalar and regulatory enzymes involved, some of which were known from other taurine pathways. The genes (Csal_0153-Csal_0156) encoding a putative ATP-binding-cassette (ABC) transporter for taurine (TauAB(1)B(2)C) were shown to be inducibly transcribed by reverse transcription (RT-) PCR. An inducible taurine : 2-oxoglutarate aminotransferase [EC 2.6.1.55] was found (Csal_0158); the reaction yielded glutamate and sulfoacetaldehyde. The sulfoacetaldehyde was reduced to isethionate by NADPH-dependent sulfoacetaldehyde reductase (IsfD), a member of the short-chain alcohol dehydrogenase superfamily. The 27 kDa protein (SDS-PAGE) was identified by peptide-mass fingerprinting as the gene product of Csal_0161. The putative exporter of isethionate (IsfE) is encoded by Csal_0160; isfE was inducibly transcribed (RT-PCR). The presumed transcriptional regulator, TauR (Csal_0157), may autoregulate its own expression, typical of GntR-type regulators. Similar gene clusters were found in several marine and terrestrial gammaproteobacteria, which, in the gut canal, could be the source of not only mammalian, but also arachnid and cephalopod isethionate.

Isolation and characterization marine bacteria capable of degrading lignin-derived compounds.

Lignin, a characteristic component of terrestrial plants. Rivers transport large amounts of vascular plant organic matter into the oceans where lignin can degrade over time; however, microorganisms involved in this degradation have not been identified. In this study, several bacterial strains were isolated from marine samples using the lignin-derived compound vanillic acid (4-hydroxy-3-methoxybenzoic acid) as the sole carbon and energy source. The optimum growth temperature for all isolates ranged from 30 to 35°C. All isolates grew well in a wide NaCl concentration range of 0 to over 50 g/L, with an optimum concentration of 22.8 g/L, which is the same as natural seawater. Phylogenetic analysis indicates that these strains are the members of Halomonas, Arthrobacter, Pseudoalteromonas, Marinomonas, and Thalassospira. These isolates are also able to use other lignin-derived compounds, such as 4-hydroxybenzoic acid, ferulic acid, syringic acid, and benzoic acid. Vanillic acid was detected in all culture media when isolates were grown on ferulic acid as the sole carbon source; however, no 4-hydroxy-3-methoxystyrene was detected, indicating that ferulic acid metabolism by these strains occurs via the elimination of two side chain carbons. Furthermore, the isolates exhibit 3,4-dioxygenase or 4,5-dioxygenase activity for protocatechuic acid ring-cleavage, which is consistent with the genetic sequences of related genera. This study was conducted to isolate and characterize marine bacteria of degrading lignin-derived compounds, thereby revealing the degradation of aromatic compounds in the marine environment and opening up new avenues for the development and utilization of marine biological resources.

Horizontal gene transfer and silver nanoparticles production in a new Marinomonas strain isolated from the Antarctic psychrophilic ciliate Euplotes focardii.

We isolated a novel bacterial strain from a prokaryotic consortium associated to the psychrophilic marine ciliate Euplotes focardii, endemic of the Antarctic coastal seawater. The 16S rDNA sequencing and the phylogenetic analysis revealed the close evolutionary relationship to the Antarctic marine bacterium Marinomonas sp. BSw10506 and the sub antarctic Marinomonas polaris. We named this new strain Marinomonas sp. ef1. The optimal growth temperature in LB medium was 22 °C. Whole genome sequencing and analysis showed a reduced gene loss limited to regions encoding for transposases. Additionally, five genomic islands, e.g. DNA fragments that facilitate horizontal gene transfer phenomena, were identified. Two open reading frames predicted from the genomic islands coded for enzymes belonging to the Nitro-FMN-reductase superfamily. One of these, the putative NAD(P)H nitroreductase YfkO, has been reported to be involved in the bioreduction of silver (Ag) ions and the production of silver nanoparticles (AgNPs). After the Marinomonas sp. ef1 biomass incubation with 1 mM of AgNO3 at 22 °C, we obtained AgNPs within 24 h. The AgNPs were relatively small in size (50 nm) and had a strong antimicrobial activity against twelve common nosocomial pathogenic microorganisms including Staphylococcus aureus and two Candida strains. To our knowledge, this is the first report of AgNPs biosynthesis by a Marinomonas strain. This biosynthesis may play a dual role in detoxification from silver nitrate and protection from pathogens for the bacterium and potentially for the associated ciliate. Biosynthetic AgNPs also represent a promising alternative to conventional antibiotics against common pathogens.

Arsenic resistance and removal by marine and non-marine bacteria.

Arsenic resistance and removal was evaluated in nine bacterial strains of marine and non-marine origins. Of the strains tested, Marinomonas communis exhibited the second-highest arsenic resistance with median effective concentration (EC(50)) value of 510 mg As l(-1), and was capable of removing arsenic from culture medium amended with arsenate. Arsenic accumulation in cells amounted to 2290 microg As g(-1) (dry weight) when incubated on medium containing 5 mg As l(-1) of arsenate. More than half of the arsenic removed was related to metabolic activity: 45% of the arsenic was incorporated into the cytosol fraction and 10% was found in the lipid-bound fraction of the membrane, with the remaining arsenic considered to be adsorbed onto the cell surface. Potential arsenic resistance and removal were also examined in six marine and non-marine environmental water samples. Of the total bacterial colony counts, 28-100% of bacteria showed arsenic resistance. Some of the bacterial consortia, especially those from seawater enriched with arsenate, exhibited higher accumulated levels of arsenic than M. communis under the same condition. These results showed that arsenic resistant and/or accumulating bacteria are widespread in the aquatic environment, and that arsenic-accumulating bacteria such as M. communis are potential candidates for bioremediation of arsenic contaminated water.

Unusually high mechanical stability of bacterial adhesin extender domains having calcium clamps.

To gain insight into the relationship between protein structure and mechanical stability, single molecule force spectroscopy experiments on proteins with diverse structure and topology are needed. Here, we measured the mechanical stability of extender domains of two bacterial adhesins MpAFP and MhLap, in an atomic force microscope. We find that both proteins are remarkably stable to pulling forces between their N- and C- terminal ends. At a pulling speed of 1 µm/s, the MpAFP extender domain fails at an unfolding force Fu = 348 ± 37 pN and MhLap at Fu = 306 ± 51 pN in buffer with 10 mM Ca2+. These forces place both extender domains well above the mechanical stability of many other ß-sandwich domains in mechanostable proteins. We propose that the increased stability of MpAFP and MhLap is due to a combination of both hydrogen bonding between parallel terminal strands and intra-molecular coordination of calcium ions.

Precipitation of minerals by 22 species of moderately halophilic bacteria in artificial marine salts media: influence of salt concentration.

Precipitation of minerals was shown by 22 species of moderately halophilic bacteria in both solid and liquid artificial marine salts media at different concentration and different Mg2+-to-Ca2+ ratio. Precipitation of minerals was observed for all the bacteria used. When salt concentration increased, the quantity and the size of bioliths decreased, the time required for precipitation being increased. The precipitated minerals were calcite, magnesian calcite, aragonite, dolomite, monohydrocalcite, hydromagnesite and struvite in variable proportions, depending on the bacterial species, the salinity and the physical state of the medium; the Mg content of the magnesian calcite also varied according to the same parameters. The precipitated minerals do not correspond exactly to those which could be precipitated inorganically according to the saturation indices. Scanning electron microscopy showed that the formation of the bioliths is initiated by grouping of calcified cells and that the dominant final morphologies were spherulitic with fibrous radiated interiors. It was demonstrated that moderately halophilic bacteria play an active role in the precipitation of carbonates and we hypothesize about this process of biomineralization.