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.

Marinomonas rhodophyticola sp. nov. and Marinomonas phaeophyticola sp. nov., isolated from marine algae.

Two Gram-stain-negative, facultatively aerobic, and motile rod bacteria, designated as strains KJ51-3T and 15G1-11T, were isolated from marine algae collected in the Republic of Korea. Both strains exhibited catalase- and oxidase-positive activities. Optimum growth conditions for strain KJ51-3T were observed at 30 °C and pH 6.0-8.0, with 1.0-7.0 % (w/v) NaCl, whereas strain 15G1-11T exhibited optimal growth at 30 °C, pH 7.0, and 1.0-5.0 % NaCl. Major fatty acids detected in both strains included C16 : 0, C10 : 0 3-OH and summed features 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). As for polar lipids, strain KJ51-3T contained phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol, and two unidentified phospholipids, whereas strain 15G1-11T had PE, PG, and an unidentified aminolipid. Ubiquinone-8 was the predominant respiratory quinone in both strains, with minor detection of ubiquinone-9 in strain KJ51-3T. The genomic DNA G+C contents were 44.0 mol% for strain KJ51-3T and 40.5 mol% for strain 15G1-11T. Phylogenetic analyses based on both 16S rRNA gene and genome sequences placed strains KJ51-3T and 15G1-11T into distinct lineages within the genus Marinomonas, most closely related to Marinomonas arctica 328T (98.6 %) and Marinomonas algicola SM1966T (98.3 %), respectively. Strains KJ51-3T and 15G1-11T exhibited a 94.6 % 16S rRNA gene sequence similarity and a 70.7 % average nucleotide identity (ANI), with ANI values of 91.9 and 79.3 % between them and M. arctica 328T and M. algicola SM1966T, respectively, indicating that they represent novel species. In summary, based on their phenotypic, chemotaxonomic, and phylogenetic properties, strains KJ51-3T and 15G1-11T are proposed to represent novel species within the genus Marinomonas, for which the names Marinomonas rhodophyticola sp. nov. (KJ51-3T=KACC 22756T=JCM 35591T) and Marinomonas phaeophyticola sp. nov. (15G1-11T=KACC 22593T=JCM 35412T) are respectively proposed.

Psychroserpens ponticola sp. nov. and Marinomonas maritima sp. nov., isolated from seawater.

Two Gram-stain-negative, catalase- and oxidase-positive, aerobic non-motile and motile rod bacteria, strains MSW6T and RSW2T, were isolated from surface seawater. Strain MSW6T optimally grew at 20 °C, pH 7.0 and 3 % NaCl, while strain RSW2T optimally grew at 25 °C, pH 7.0-8.0 and 2 % NaCl. Strain MSW6T possessed menaquinone-6 as the major respiratory quinone, and its major fatty acids were iso-C15 : 1 G, iso-C15 : 0 and iso-C15 : 0 3-OH. The major polar lipid identified in strain MSW6T was phosphatidylethanolamine (PE). On the other hand, strain RSW2T had ubiquinone-8 as the predominant respiratory quinone, and its major fatty acids consisted of summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and C16 : 0. The major polar lipids identified in strain RSW2T were PE and phosphatidylglycerol. As the sole respiratory quinone, strain MSW6T possessed menaquinone-6, while strain RSW2T had ubiquinone-8. The DNA G+C contents of strains MSW6T and RSW2T were 31.9 and 43.4 mol%, respectively. Phylogenetic analyses based on 16S rRNA and core gene sequences showed that strain MSW6T formed a phylogenic lineage with Psychroserpens mesophilus KOPRI 13649T, while strain RSW2T formed a phylogenic lineage with Marinomonas primoryensis KMM 3633T. Strain MSW6T shared 97.9 % 16S rRNA gene sequence similarity and 80.7 % average nucleotide identity (ANI) ith P. mesophilus KOPRI 13649T, and strain RSW2T shared 99.1 % 16S rRNA gene sequence similarity and 93.1 % ANI with M. primoryensis KMM 3633T. Based on the results of phenotypic, chemotaxonomic and phylogenetic analyses, strains MSW6T and RSW2T represent novel species of the genera Psychroserpens and Marinomonas, respectively, for which the names Psychroserpens ponticola sp. nov. and Marinomonas maritima sp. nov. are proposed, respectively. The type strain of P. ponticola is MSW6T (=KACC 22338T=JCM 35022T) and the type strain of M. maritima is RSW2T (=KACC 22716T=JCM 35550T).

Marinomonas transparens sp. nov. and Marinomonas sargassi sp. nov., isolated from marine alga.

Two Gram-stain-negative, rod-shaped, non-spore-forming, strictly aerobic, motile bacteria with a single polar flagellum, designated strains C1424T and C2222T, were isolated from marine alga collected from the sea shore at Yantai, PR China. Strain C1424T grew at 4-37 °C and in the presence of 1-9 % (w/v) NaCl, while strain C2222T grew at 4-32 °C with 1-6 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences and concatenated amino acid sequences of 120 ubiquitous single-copy proteins showed that both strains C1424T and C2222T belonged to the genus Marinomonas, showing highest 16S rRNA gene sequence similarities to the type strains of Marinomonas primoryensis (98.1 %) and Marinomonas dokdonensis (98.1 %), respectively. The major fatty acids of the two strains were C18 : 1 ω6c and/or C18 : 1 ω7c, C16 : 1 ω6c and/or C16 : 1 ω7c and C16 : 0, their predominant polar lipids were phosphatidylethanolamine and phosphatidylglycerol, and their sole respiratory quinone was Q8. On the basis of polyphasic analyses, strains C1424T and C2222T are considered to represent two novel species within the genus Marinomonas, for which the names Marinomonas transparens sp. nov. and Marinomonas sargassi sp. nov. are proposed. The type strains are C1424T (=KCTC 72119T=MCCC 1K03601T) and C2222T (=KCTC 72120T=MCCC 1K03602T), respectively.

TnpAREP and REP sequences dissemination in bacterial genomes: REP recognition determinants.

REP, diverse palindromic DNA sequences found at high copy number in many bacterial genomes, have been attributed important roles in cell physiology but their dissemination mechanisms are poorly understood. They might represent non-autonomous transposable elements mobilizable by TnpAREP, the first prokaryotic domesticated transposase associated with REP. TnpAREP, fundamentally different from classical transposases, are members of the HuH superfamily and closely related to the transposases of the IS200/IS605 family. We previously showed that Escherichia coli TnpAREP processes cognate single stranded REP in vitro and that this activity requires the integrity of the REP structure, in particular imperfect palindromes interrupted by a bulge and preceded by a conserved DNA motif. A second group of REPs rather carry perfect palindromes, raising questions about how the latter are recognized by their cognate TnpAREP. To get insight into the importance of REP structural and sequence determinants in these two groups, we developed an in vitro activity assay coupled to a mutational analysis for three different TnpAREP/REP duos via a SELEX approach. We also tackled the question of how the cleavage site is selected. This study revealed that two TnpAREP groups have co-evolved with their cognate REPs and use different strategies to recognize their REP substrates.

Comparative genomic analysis of the genus Marinomonas and taxonomic study of Marinomonas algarum sp. nov., isolated from red algae Gelidium amansii.

Members of the genus Marinomonas are known for their environmental adaptation and metabolically versatility, with abundant proteins associated with antifreeze, osmotic pressure resistance, carbohydrase and multiple secondary metabolites. Comparative genomic analysis focusing on secondary metabolites and orthologue proteins was conducted with 30 reference genome sequences in the genus Marinomonas. In this study, a Gram-stain-negative, rod-shaped, non-flagellated and strictly aerobic bacterium, designated as strain E8T, was isolated from the red algae (Gelidium amansii) in the coastal of Weihai, China. Optimal growth of the strain E8T was observed at temperatures 25-30 °C, pH 6.5-8.0 and 1-3% (w/v) NaCl. The DNA G + C content was 42.8 mol%. The predominant isoprenoid quinone was Q-8 and the major fatty acids were C16:0, summed feature 3 and summed feature 8. The major polar lipids were phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). Based on data obtained from this polyphasic taxonomic study, strain E8T should be considered as a novel species of the genus Marinomonas, for which the name Marinomonas algarum is proposed. The type strain is E8T (= KCTC 92201T = MCCC 1K07070T).

Light-induced expression of a novel marine laccase in Escherichia coli from Marinomonas profundimaris and its application in synthetic dye decolorization.

Laccases (EC 1.10.3.2) are green biocatalysts with a considerable potential in numerous environmental and industrial applications due to their abilities to oxidize a wide range of substrates, such as aromatic amines, while reducing molecular oxygen to water. In this study, a putative laccase, LacMp1, encoding a protein of 48.3 kDa and belonging to the Cu-oxidase_3 superfamily, was cloned and overexpressed in Escherichia coli with a light-induced expression system. High-level expression of recombinant protein LacMp1 was achieved under the light intensity of 6500 ± 200 lx from a white light-emitting diode (LED) belt. The purified LacMp1 showed high activity toward various laccase substrates, with the lowest Km value and highest kcat/Km value for syringaldazine at the optimal temperature and pH of 50 °C and 7.5. Dimethyl sulfoxide, ethanol, and metal ions such as Co2+, Ca2+, K+, Li+, Zn2+, Mn2+, Fe3+, and Ni2+ did not significantly inhibit the activity of LacMp1. Furthermore, LacMp1 showed tolerance to NaCl and kept 66.67 ± 2.24% of its initial activity at concentrations lower than 400 mM. Moreover, LacMp1 exhibited wide-spectrum decolorization ability towards indigoid, anthraquinonic, and azo dyes without the aid of redox mediators at pHs ranging from 5.0 to 9.0. It decolorized 99.83 ± 0.12% of indigo carmine, 99.54 ± 0.43% of Congo red, 88.41 ± 3.22% of Eriochrome black T, and 51.61 ± 1.82% of Reactive blue 4, respectively. These unusual properties demonstrated that LacMp1 had potential in specific industrial or environmental applications.

Marinomonas lutimaris sp. nov., isolated from a tidal flat sediment of the East China Sea.

A Gram-stain-negative bacterial strain, designated as E165T, was isolated from a tidal flat sediment of the East China Sea. Strain E165T grew optimally at pH 6, at 32 °C and with 1-2 % (w/v) NaCl. The 16S rRNA gene sequence similarity results revealed that strain E165T was most closely related to Marinomonas rhizomae IVIA-Po-145T, Marinomonas polaris CK13T, Marinomonas foliarum IVIA-Po-155T, Marinomonas hwangdonensis HDW-15T, Marinomonas pontica 46-16T, Marinomonas mangrovi B20-1T and Marinomonas shanghaiensis DSL-35T with values of 97.0-98.5 %. The digital DNA-DNA hybridization and average nucleotide identity values between strain E165T and the reference strains were 21.9-34.3 % and 77.6-87.3 %, respectively. The DNA G+C content of the isolate was 42.9 mol%. Strain E165T contained Q-8 as the sole ubiquinone and C16 : 0, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) as the major fatty acids. The major polar lipids of strain E165T were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, aminolipid and aminophospholipid. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness, a novel species, Marinomonas lutimaris sp. nov., is proposed with E165T (=MCCC 1K06241T=KCTC 82809T) as the type strain.

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.

Marinomonas vulgaris sp. nov., a marine bacterium isolated from seawater in a coastal intertidal zone of Zhoushan island.

A Marinomonas-like, Gram-stain-negative, strictly aerobic and rod to ovoid-shaped bacterium, designated as strain A79T, was isolated from the seawater mixtures of oyster shells and brown algae in a coastal intertidal zone of Zhoushan, China. The strain was positive for oxidase and catalase. Colonies grown on marine agar for 48 h were round, milky white, smooth and moist with the diameter of 2-3 mm. Growth was observed at 15-30 °C (optimum, 25℃), pH 5.5-9.5 (optimum, pH 8.5) and with 0.5-8% (w/v) NaCl (optimum, 2-2.5%). The G + C content based on the genome sequence was 46.0%. The only respiratory quinone was Q-8. The main polar lipids contained phosphatidylglycerol, phosphatidylethanolamine, unidentified glycolipids, unidentified phospholipid and three unidentified lipids. The major fatty acids (> 10%) were C16:0, Summed feature 3 (comprising C16:1 ω6c and/or C16:1 ω7c) and summed feature 8 (comprising C18:1 ω6c and/or C18:1 ω7c). The 16S rRNA gene sequence similarity between strain A79T and Marinomonas pollencensis IVIA-Po-185T was 97.4%, the similarities with other type strains of the genus Marinomonas were 93.8-96.7%. Based on the results, Marinomonas vulgaris sp. nov. was proposed as a novel species. The type strain is A79T (= MCCC 1K05799T = KCTC 82519T = JCM 34473T).

Maribrevibacterium harenarium gen. nov., sp. nov., represented by a marine strain of the family Oceanospirillaceae.

A Gram-stain-negative, non-motile, facultatively anaerobic, short rod-shaped bacterium, designated HB171799T, was isolated from seacoast sandy soil collected at Qishui Bay, Hainan, PR China. The chemotaxonomic analysis revealed that the respiratory quinones were Q-8 and Q-7, and the major cellular fatty acids were summed feature 8 (comprising C18 : 1 ω7c and/or C18 : 1 ω6c), C16 : 0 and C18 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid and an unidentified lipid. The size of the draft genome was 3.68 Mb with a DNA G+C content of 48.0 mol%. Results of phylogenetic analyses based on 16S rRNA gene and genome sequences showed that the novel isolate belonged to the family Oceanospirillaceae and formed a distinct subcluster at the base of the radiation of the genus Marinomonas. The highest sequence similarity (96.0 %) of the novel isolate was found to the type strains of Marinomonas fungiae JCM 18476T and Marinomonas ostreistagni DSM23425T. The whole genome-based phylogeny and differences in cellular fatty acids and polar lipids readily distinguished strain HB171799T from all the closely related validly published type strains. Strain HB171799T is therefore suggested to represent a novel species of a new genus in the family Oceanospirillaceae, for which the name Maribrevibacterium harenarium gen. nov., sp. nov. is proposed. The type strain is HB171799T (=CGMCC 1.16727T=JCM 33332T).

Effect of pH on the activity of ice-binding protein from Marinomonas primoryensis.

The ability of an ice-binding protein (IBP) from Marinomonas primoryensis (MpIBP) to influence ice crystal growth and structure in nonphysiological pH environments was investigated in this work. The ability for MpIBP to retain ice interactivity under stressed environmental conditions was determined via (1) a modified splat assay to determine ice recrystallization inhibition (IRI) of polycrystalline ice and (2) nanoliter osmometry to evaluate the ability of MpIBP to dynamically shape the morphology of a single ice crystal. Circular dichroism (CD) was used to relate the IRI and DIS activity of MpIBP to secondary structure. The results illustrate that MpIBP secondary structure was stable between pH 6 and pH 10. It was found that MpIBP did not interact with ice at pH ≤ 4 or pH ≥ 13. At 6 ≤ pH ≥ 12 MpIBP exhibited a reduction in grain size of ice crystals as compared to control solutions and demonstrated dynamic ice shaping at 6 ≤ pH ≥ 10. The results substantiate that MpIBP retains some secondary structure and function in non-neutral pH environments; thereby, enabling its potential utility in nonphysiological materials science and engineering applications.

Heterologous expression of a cryptic gene cluster from Marinomonas fungiae affords a novel tricyclic peptide marinomonasin.

The ω-ester-containing peptides (OEPs) are a group of ribosomally synthesized and post-translationally modified peptides (RiPPs). The biosynthetic gene clusters of ω-ester-containing peptides commonly include ATP-grasp ligase coding genes and are distributed over the genomes of a wide variety of bacteria. A new biosynthetic gene cluster of ω-ester-containing peptides was found in the genome sequence of the marine proteobacterium Marinomonas fungiae. Heterologous production of a new tricyclic peptide named marinomonasin was accomplished using the biosynthetic gene cluster in Escherichia coli expression host strain BL21(DE3). By ESI-MS and NMR experiments, the structure of marinomonasin was determined to be a tricyclic peptide 18 amino acids in length with one ester and two isopeptide bonds in the molecule. The bridging patterns of the three intramolecular bonds were determined by the interpretation of HMBC and NOESY data. The bridging pattern of marinomonasin was unprecedented in the ω-ester-containing peptide group. The results indicated that the ATP-grasp ligase for the production of marinomonasin was a novel enzyme possessing bifunctional activity to form one ester and two isopeptide bonds. KEY POINTS: • New tricyclic peptide marinomonasin was heterologously produced in Escherichia coli. • Marinomonasin contained one ester and two isopeptide bonds in the molecule. • The bridging pattern of intramolecular bonds was novel.

Heterotrophic nitrification-aerobic denitrification characteristics and antibiotic resistance of two bacterial consortia from Marinomonas and Halomonas with effective nitrogen removal in mariculture wastewater.

Heterotrophic nitrification-aerobic denitrification (HNAD) characteristics and antibiotic resistance of two bacterial consortia, Marinomonas communis & Halomonas titanicae (MCH) and Marinomonas aquimarina & Halomonas titanicae (MAH), and their single isolates (MC, MA, and H) were determinated in this study. When cultured in sole and mixed N-source media (NH4+-N and/or NO2--N of 10 mg/L), MCH and MAH exhibited greater efficiency and stability of inorganic-N removal than single isolates, and these strains preferred to remove NH4+-N by simultaneous HNAD in mixed N-source media. Meanwhile, 45%-70% of NH4+-N and/or NO2--N was mainly converted to organic nitrogen (15%-25%) and gaseous nitrogen (30%-40%) by these strains, and more inorganic-N was transformed to intracellular-N by MCH and MAH via assimilation instead of gaseous-N production by denitrification. Both isolates and their consortia had the maximal NH4+-N or NO2--N removal efficiency above 95% under the optimum conditions including temperature of 20-30 °C, C/N ratios of 15-20, and sucrose as carbon source. Interestingly, bacterial consortia performed greater nitrogen removal than single isolates under the low temperature of 10 °C or C/N ratios of 2-5. In real mariculture wastewater, MCH and MAH also showed higher NH4+-N removal efficiency (65%-68%) and more stable cell quantity (4.2-5.2 × 108 CFU/mL) than single strains, due to the interspecific coexistence detected by bacterial quantitation with indirect immunoassay. Additionally, these isolates and consortia had stronger resistances to polypeptides, tetracyclines, sulfonamides, furanes, and macrolides than other antibiotics. These findings will be conducive to the applications of HNAD bacteria of Marinomonas and Halomonas on reducing nitrogen pollution in mariculture or other saline environments.

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.

Marinomonas agarivorans sp. nov., an agar-degrading marine bacterium isolated from red algae.

A Gram-stain-negative, aerobic and curved-rod-shaped bacterium, designated QM202T, was isolated from red algae (Gracilaria blodgettii). Cells of strain QM202T were 0.2-0.3 µm wide and 1.0-2.5 µm long, catalase-negative and oxidase-positive. The strain exhibited an agar-degrading activity. It was motile by means of a single polar flagellum. Optimal growth occurred at 28-30 °C, pH 7.0-7.5 and in the presence of 2.0-3.0 % (w/v) NaCl. The DNA G+C content was 41.4 mol%. The isoprenoid quinone was identified as Q-8. Phophatidylethanolamine and phosphatidylglycerol were the predominant phospholipids. The dominant fatty acids were C18:1ω7c, C16:0 and C16:1ω7c and/or iso-C15:0 2-OH. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain QM202T belonged to the genus Marinomonas. The closest described neighbour in terms of 16S rRNA gene sequence identity was Marinomonas blandensis MED121T (95.5 %). The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness indicated that strain QM202T can be considered to represent a novel species, Marinomonas agarivorans sp. nov. The type strain is QM202T (=KCTC 52475T=MCCC 1H00145T).

Marinomonas profundi sp. nov., isolated from deep seawater of the Mariana Trench.

A Gram-stain-negative, aerobic, polarly flagellated, straight or curved rod-shaped bacterium, designated strain M1K-6T, was isolated from deep seawater samples collected from the Mariana Trench. The strain grew at -4 to 37 °C (optimum, 25-30 °C), at pH 5.5-10.0 (optimum, pH 7.0) and with 0.5-14.0  % (w/v) NaCl (optimum, 2.0 %). It did not reduce nitrate to nitrite nor hydrolyse gelatin or starch. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain M1K-6T was affiliated with the genus Marinomonas, sharing 93.1-97.0  % sequence similarity with the type strains of recognized Marinomonas species. The major cellular fatty acids were summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c), summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), C16 : 0, C10 : 0 3-OH and C18 : 0. The predominant respiratory quinone was ubiquinone-8. Polar lipids of strain M1K-6T included phosphatidylethanolamine, phosphatidylglycerol and two unidentified lipids. The genomic G+C content of strain M1K-6T was 46.0 mol%. Based on data from the present polyphasic study, strain M1K-6T was considered to represent a novel species within the genus Marinomonas, for which the name Marinomonas profundi sp. nov. is proposed. The type strain is M1K-6T (=KCTC 72501T=MCCC 1K03890T).

Porin from Marine Bacterium Marinomonas primoryensis KMM 3633T: Isolation, Physico-Chemical Properties, and Functional Activity.

Marinomonas primoryensis KMM 3633T, extreme living marine bacterium was isolated from a sample of coastal sea ice in the Amursky Bay near Vladivostok, Russia. The goal of our investigation is to study outer membrane channels determining cell permeability. Porin from M. primoryensis KMM 3633T (MpOmp) has been isolated and characterized. Amino acid analysis and whole genome sequencing were the sources of amino acid data of porin, identified as Porin_4 according to the conservative domain searching. The amino acid composition of MpOmp distinguished by high content of acidic amino acids and low content of sulfur-containing amino acids, but there are no tryptophan residues in its molecule. The native MpOmp existed as a trimer. The reconstitution of MpOmp into black lipid membranes demonstrated its ability to form ion channels whose conductivity depends on the electrolyte concentration. The spatial structure of MpOmp had features typical for the classical gram-negative porins. However, the oligomeric structure of isolated MpOmp was distinguished by very low stability: heat-modified monomer was already observed at 30 °C. The data obtained suggest the stabilizing role of lipids in the natural membrane of marine bacteria in the formation of the oligomeric structure of porin.

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.