Comparative genomics reveals a deep-sea sediment-adapted life style of Pseudoalteromonas sp. SM9913

Deep-sea sediment is one of the most important microbial-driven ecosystems, yet it is not well characterized. Genome sequence analyses of deep-sea sedimentary bacteria would shed light on the understanding of this ecosystem. In this study, the complete genome of deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913 (SM9913) is described and compared with that of the closely related Antarctic surface sea-water ecotype Pseudoalteromonas haloplanktis TAC125 (TAC125). SM9913 has fewer dioxygenase genes than TAC125, indicating a possible sensitivity to reactive oxygen species. Accordingly, experimental results showed that SM9913 was less tolerant of H₂O₂ than TAC125. SM9913 has gene clusters related to both polar and lateral flagella biosynthesis. Lateral flagella, which are usually present in deep-sea bacteria and absent in the related surface bacteria, are important for the survival of SM9913 in deep-sea environments. With these two flagellar systems, SM9913 can swim in sea water and swarm on the sediment particle surface, favoring the acquisition of nutrients from particulate organic matter and reflecting the particle-associated alternative lifestyle of SM9913 in the deep sea. A total of 12 genomic islands were identified in the genome of SM9913 that may confer specific features unique to SM9913 and absent from TAC125, such as drug and heavy metal resistance. Many signal transduction genes and a glycogen production operon were also present in the SM9913 genome, which may help SM9913 respond to food pulses and store carbon and energy in a deep-sea environment.     

Molecular analysis of the gene encoding a cold-adapted halophilic subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913: cloning, expression, characterization and function analysis of the C-terminal PPC domains

Only a few cold-adapted halophilic proteases have been reported. Here, the gene mcp03 encoding a cold-adapted halophilic protease MCP-03 was cloned from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913, which contains a 2,130-bp ORF encoding a novel subtilase precursor. The recombinant MCP-03, expressed in Escherichia coli BL21 and purified from fermented broth, is a multi-domain protein with a catalytic domain and two PPC domains. Compared to mesophilic subtilisin Carlsberg, MCP-03 had characteristics of a typical cold-adapted enzyme (e.g., higher activity at low temperatures, lower optimum temperature and higher thermolability). MCP-03 also exhibited good halophilic ability with maximal activity at 3 M NaCl/KCl and good stability in 3 M NaCl. Deletion mutagenesis showed that the C-terminal PPC domains were unnecessary for enzyme secretion but had an inhibitory effect on MCP-03 catalytic efficiency and were essential for keeping MCP-03 thermostable. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. X.-L. Chen and B.-Q. Yan contributed equally to this work.     

Differences in malate dehydrogenases from the obligately piezophilic deep-sea bacterium Moritella sp. strain 2D2 and the psychrophilic bacterium Moritella sp. strain 5710

The gene encoding malate dehydrogenase (MDH) of the obligately piezophilic deep-sea bacterium Moritella sp. strain 2D2 was cloned and sequenced. There were two positions [close to the active site (Ala-180) and in the subunit interaction site (His-229)] with 2D2-specific substitutions. The MDH genes of strain 2D2 and a psychrophilic bacterium Moritella sp. strain 5710 exhibiting the highest sequence similarity were overexpressed in Escherichia coli. The 2D2 MDH was more heat-stable than the 5710 MDH. The apparent Km value at 62.1 MPa for NADH of the 2D2 MDH was higher than that of the 5710 MDH. The 2D2 MDH in which a His-Gln substitution was introduced at position 229 decreased the thermal stability and Km value at 62.1 MPa. The 5710 MDH that was substituted Gln-229 with His increased the thermal stability and Km value at 62.1 MPa. These results indicate that the His residue at position 229 of the 2D2 MDH may play a role in the thermal stability and the MDH function at high pressure.     

Bio-removal of cadmium by growing deep-sea bacterium Pseudoalteromonas sp. SCSE709-6

Effective bio-removal of heavy metals is important for water treatment. Although a number of microorganism species demonstrated the ability of living cells to remove cadmium, most of them were tested at fixed concentration of metals, salinity, and temperature. This paper reported a research on the screening and performance of a newly developed deep-sea bacterium, Pseudoalteromonas sp. SCSE709-6, for Cd(II) removal by growing cells under a range of experimental conditions: 0–50 mg/L of Cd(II), 15–30 °C of incubation temperatures, 6.5–8.0 of initial pH, and 1.5–5.0 % of salinity. Study results revealed that Pseudoalteromonas sp. SCSE709-6 could remove more than 96 % of Cd(II) on growth. The Cd(II) bioremoval was in correlation but not in accordance with biomass. As cadmium concentrations increased, the Cd(II) removal by cell adsorption played an increasingly important role compared with that of intracellular accumulation. For the removal mechanism, Fourier transform infrared spectroscopy revealed that carboxyl, amido and hydroxyl of saccharides, and proteins in the extracellular polymeric substances are the most active groups for Cd(II) absorption. The bacterium reported in this study offers a new microbe strain for Cd(II) bioremediation.     

Characterization and gene cloning of a cold-active cellulase from a deep-sea psychrotrophic bacterium Pseudoalteromonas sp. DY3

The celX gene encoding an extracellular cold-active cellulase was isolated from a psychrotrophic bacterium, which was isolated from deep-sea sediment and identified as a Pseudoalteromonas species. It encoded a protein consisting of 492 amino acids with a calculated molecular mass of 52.7 kDa. The CelX consisted of an N-terminal catalytic domain belonging to glycoside hydrolase family 5 and a C-terminal cellulose-binding domain belonging to carbohydrate-binding module family 5. The long linker sequence connecting both domains was composed of 105 residues. The optimal temperature for cellulase activity of CelX was 40°C. The enzyme was most active at pH 6–7 and showed better resistance to alkaline condition. The zymogram activity analysis indicated that the CelX consisted of single enzyme component. The cellobiose was main hydrolysate of CelX.     

Structural and functional characterization of mature forms of metalloprotease E495 from Arctic sea-ice bacterium Pseudoalteromonas sp. SM495

E495 is the most abundant protease secreted by the Arctic sea-ice bacterium Pseudoalteromonas sp. SM495. As a thermolysin family metalloprotease, E495 was found to have multiple active forms in the culture of strain SM495. E495-M (containing only the catalytic domain) and E495-M-C1 (containing the catalytic domain and one PPC domain) were two stable mature forms, and E495-M-C1-C2 (containing the catalytic domain and two PPC domains) might be an intermediate. Compared to E495-M, E495-M-C1 had similar affinity and catalytic efficiency to oligopeptides, but higher affinity and catalytic efficiency to proteins. The PPC domains from E495 were expressed as GST-fused proteins. Both of the recombinant PPC domains were shown to have binding ability to proteins C-phycocyanin and casein, and domain PPC1 had higher affinity to C-phycocyanin than domain PPC2. These results indicated that the domain PPC1 in E495-M-C1 could be helpful in binding protein substrate, and therefore, improving the catalytic efficiency. Site-directed mutagenesis on the PPC domains showed that the conserved polar and aromatic residues, D26, D28, Y30, Y/W65, in the PPC domains played key roles in protein binding. Our study may shed light on the mechanism of organic nitrogen degradation in the Arctic sea ice.     

Antibiofilm activity of an exopolysaccharide from marine bacterium Vibrio sp. QY101

Bacterial exopolysaccharides have always been suggested to play crucial roles in the bacterial initial adhesion and the development of complex architecture in the later stages of bacterial biofilm formation. However, Escherichia coli group II capsular polysaccharide was characterized to exert broad-spectrum biofilm inhibition activity. In this study, we firstly reported that a bacterial exopolysaccharide (A101) not only inhibits biofilm formation of many bacteria but also disrupts established biofilm of some strains. A101 with an average molecular weight of up to 546 KDa, was isolated and purified from the culture supernatant of the marine bacterium Vibrio sp. QY101 by ethanol precipitation, iron-exchange chromatography and gel filtration chromatography. High performance liquid chromatography traces of the hydrolyzed polysaccharides showed that A101 is primarily consisted of galacturonic acid, glucuronic acid, rhamnose and glucosamine. A101 was demonstrated to inhibit biofilm formation by a wide range of Gram-negative and Gram-positive bacteria without antibacterial activity. Furthermore, A101 displayed a significant disruption on the established biofilm produced by Pseudomonas aeruginosa, but not by Staphylococcus aureus. Importantly, A101 increased the aminoglycosides antibiotics' capability of killing P. aeruginosa biofilm. Cell primary attachment to surfaces and intercellular aggregates assays suggested that A101 inhibited cell aggregates of both P. aeruginosa and S. aureus, while the cell-surface interactions inhibition only occurred in S. aureus, and the pre-formed cell aggregates dispersion induced by A101 only occurred in P. aeruginosa. Taken together, these data identify the antibiofilm activity of A101, which may make it potential in the design of new therapeutic strategies for bacterial biofilm-associated infections and limiting biofilm formation on medical indwelling devices. The found of A101 antibiofilm activity may also promote a new recognition about the functions of bacterial exopolysaccharides.     

Gene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipX_His with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipX_His is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipX_His lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.     

Hydrolysis of insoluble collagen by deseasin MCP-01 from deep-sea Pseudoalteromonas sp. SM9913: collagenolytic characters, collagen-binding ability of C-terminal polycystic kidney disease domain, and implication for its novel role in deep-sea sedimentary particulate organic nitrogen degradation

Collagens are the most abundant proteins in marine animals and their degradation is important for the recycling of marine nitrogen. However, it is rather unclear how marine collagens are degraded because few marine collagenolytic proteases are studied in detail. Deseasins are a new type of multidomain subtilases. Here, the collagenolytic activity of deseasin MCP-01, the type example of deseasins, was studied. MCP-01 had broad substrate specificity to various type collagens from terrestrial and marine animals. It completely decomposed insoluble collagen into soluble peptides and amino acids, and was more prone to degrade marine collagen than terrestrial collagen. Thirty-seven cleavage sites of MCP-01 on bovine collagen chains were elucidated, showing the cleavage is various but specific. As the main extracellular cold-adapted protease from deep-sea bacterium Pseudoalteromonas sp. SM9913, MCP-01 displayed high activity at low temperature and alkaline range. Our data also showed that the C-terminal polycystic kidney disease (PKD) domain of MCP-01 was able to bind insoluble collagen and facilitate the insoluble collagen digestion by MCP-01. Site-directed mutagenesis demonstrated that Trp-36 of the PKD domain played a key role in its binding to insoluble collagen. It is the first time that the structure and function of a marine collagenolytic protease, deseasin MCP-01, has been studied in detail. Moreover, the PKD domain was experimentally proven to bind to insoluble protein for the first time. These results imply that MCP-01 would play an important role in the degradation of deep-sea sedimentary particulate organic nitrogen.     

Membrane lipids of a psychrophilic and barophilic deep-sea bacterium

In a psychrophilic and barophilic marine bacterial isolate of the genusAlteromonas, the ratio of total unsaturated versus saturated fatty acids in the membrane lipids increased when the organism was grown at increasing hydrostatic pressures and decreasing temperatures. This regulatory capacity, as well as the presence of relatively large amounts of 20:5 polyunsaturated fatty acid, appear to be functional in maintaining membrane fluidity within a range of pressures distinctly below and above the specific optimum and at typical deep sea temperatures.     

Structural determination of the exopolysaccharide of Pseudoalteromonas strain HYD 721 isolated from a deep-sea hydrothermal vent.

The structure of the exopolysaccharide produced by Pseudoalteromonas reference strain HYD 721 recovered from a deep-sea hydrothermal vent has been investigated. By means of methylation and beta-elimination analysis, selective degradation of the uronic acids, partial depolymerization and NMR studies, the repeating unit of the polymer was deduced to be a branched octasaccharide with the structure shown. [formula: see text]     

Structural investigation on the lipooligosaccharide fraction of psychrophilic Pseudoalteromonas haloplanktis TAC 125 bacterium.

The core structure of the cell-wall lipooligosaccharide (LOS) fraction of an Antarctic Gram-negative bacterium, Pseudoalteromonas haloplanktis TAC 125 strain, was determined to be deacetylated alditols. These were obtained from native LOS fraction by O-deacylation, dephosphorylation, reduction and finally N-deacylation. Two novel structures were detected, the more highly represented molecule consisting of the following hexasaccharide chain: alpha-D-ManpNH(2)-(1-->3)-beta-D-Galp-(1-->4)-alpha-L-glycero-D-manno-Hepp-(1-->5)-alpha-D-Kdo-(2-->6)-beta-D-GlcpNH(2)-(1-->6)-D-GlcNH(2)(ol) while the corresponding pentasaccharide, lacking the ManpNH(2) residue, was less abundant. To the best of our knowledge, the structural investigation presented here, mainly performed by NMR and MS methods, is the first report of the lipopolysaccharide fraction of a psychrophilic bacterium.     

Structure of an acidic O-specific polysaccharide of the marine bacterium Pseudoalteromonas sp. KMM 634

An acidic O-specific polysaccharide containing D-glucuronic acid (D-GlcA), 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (D-GlcNAc3NAcA), 2,3-diacetamido-2,3-dideoxy-D-mannuronoyl-L-alanine (D-ManNAc3NAcA6Ala), and 2-acetamido-2,4, 6-trideoxy-4-[(S)-3-hydroxybutyramido]-D-glucose (D-QuiNAc4NAcyl) was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Pseudoalteromonas sp. KMM 634 followed by gel-permeation chromatography. The polysaccharide was cleaved selectively with a new solvolytic agent, trifluoromethanesulfonic acid, to give a disaccharide and a trisaccharide with D-GlcNAc3NAcA at the reducing end. The borohydride-reduced oligosaccharides and the initial polysaccharide were studied by GLC-MS and 1H- and 13C-NMR spectroscopy, and the following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established: -->3)-alpha-D-QuipNAc4Ac4NAcyl-(1-->4)-beta-D-ManpNAc3NAcA6Ala+ ++-(1-->4)-b eta-D-GlcpNAc3NAc3NAcA-(1-->4)-beta-D-GlcpA-(1-->.     

A new high-alkaline alginate lyase from a deep-sea bacterium Agarivorans sp.

A high-alkaline, salt-activated alginate lyase is produced by Agarivorans sp. JAM-A1m from a deep-sea sediment off Cape Nomamisaki on Kyushu Island, Japan. Purified to homogeneity, as judged by SDS-PAGE, the enzyme (A1m) had a molecular mass of approximately 31 kDa. The optimal pH was around 10 in glycine–NaOH buffer, and the activity was increased to 1.8 times by adding 0.2 M NaCl. However, when the optimal pH in the presence of 0.2 M NaCl was shifted to pH 9.0, the activity was more than 10 times compared with that at pH 9 in the absence of NaCl. A1m showed the optimal temperature at around 30°C and was stable to incubation between pH 6 and 9. The enzyme degraded favorably mannuronate–guluronate and guluronate-rich fragments in alginate. Shotgun cloning and sequencing of the gene for A1m revealed a 930-bp open reading frame, which encoded a mature enzyme of 289 amino acids (32,295 Da) belonging to polysaccharide lyase family 7. The deduced amino acid sequence showed the highest similarity to that of a Klebsiella enzyme, with only 54% identity.     

Acetobacterium tundrae sp. nov., a new psychrophilic acetogenic bacterium from tundra soil

A new psychrophilic, anaerobic, acetogenic bacterium from the tundra wetland soil of Polar Ural is described. The organism fermented H2/CO2, formate, methanol, and several sugars to acetate as the sole end-product. The temperature range for growth was 1-30 degrees C with an optimum at 20 degrees C. The bacterium showed no growth at 32 degrees C. Cells were gram-positive, oval-shaped, flagellated rods 0.7-1.l x 1.1-4.0 microm in size when grown at 1-20 degrees C. At 25-30 degrees C, the cell size increased up to 2-3 x 10-15 microm due to a defect in cell division. The DNA G+C content of the organism was 39.2 mol%. Based upon 16S rDNA analysis and DNA-DNA reassociation studies, the organism was classified in the genus Acetobacterium as a new species, for which the name Acetobacterium tundrae sp. nov. is proposed. The type strain is Z-4493 (=DSM 9173T).     

Rhodoferax antarcticus sp. nov., a moderately psychrophilic purple nonsulfur bacterium isolated from an Antarctic microbial mat

A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15-18 degrees C with a growth temperature range of 0-25 degrees C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.     

Molecular characterization of a cryptic plasmid from the psychrotrophic antarctic bacterium Pseudoalteromonas sp. 643A

We report the identification and nucleotide sequence analysis of pKW1, a plasmid of the psychrotrophic bacterium Pseudoalteromonas sp. 643A isolated from the stomach of Antarctic krill Euphasia superba. pKW1 consists of 4583 bp, has a G+C content of 43% and seven putative open reading frames (ORFs). The deduced amino acid sequence from ORF-1 shared significant similarity with the plasmid replicase protein of Psychrobacter cryohalolentis, strain K5. The DNA region immediately downstream of the ORF-1 showed some homology with the Rep-binding sequence of the theta-replicating ColE2-type plasmids. The ORF-3 amino acid sequence revealed amino acid sequence homology with the mobilization protein of Psychrobacter sp. PRwf-1 and Moraxella catarrhalis, with identities of 28% and 25%, respectively. The ORF-4 showed 46% amino acid sequence homology with the putative relaxase/mobilization nuclease MobA of Hafnia alvei and 44% homology with the putative mobilization protein A of Pasterulla multocida. The copy number of pKW1 in Pseudoalteromonas sp. 643A was estimated of 15 copies per chromosome.