Structural basis and evolution of the photosystem I-light-harvesting supercomplex of cryptophyte algae.

Cryptophyte plastids originated from a red algal ancestor through secondary endosymbiosis. Cryptophyte photosystem I (PSI) associates with transmembrane alloxanthin-chlorophyll a/c proteins (ACPIs) as light-harvesting complexes (LHCs). Here, we report the structure of the photosynthetic PSI-ACPI supercomplex from the cryptophyte Chroomonas placoidea at 2.7-Å resolution obtained by crygenic electron microscopy. Cryptophyte PSI-ACPI represents a unique PSI-LHCI intermediate in the evolution from red algal to diatom PSI-LHCI. The PSI-ACPI supercomplex is composed of a monomeric PSI core containing 14 subunits, 12 of which originated in red algae, 1 diatom PsaR homolog, and an additional peptide. The PSI core is surrounded by 14 ACPI subunits that form 2 antenna layers: an inner layer with 11 ACPIs surrounding the PSI core and an outer layer containing 3 ACPIs. A pigment-binding subunit that is not present in any other previously characterized PSI-LHCI complexes, ACPI-S, mediates the association and energy transfer between the outer and inner ACPIs. The extensive pigment network of PSI-ACPI ensures efficient light harvesting, energy transfer, and dissipation. Overall, the PSI-LHCI structure identified in this study provides a framework for delineating the mechanisms of energy transfer in cryptophyte PSI-LHCI and for understanding the evolution of photosynthesis in the red lineage, which occurred via secondary endosymbiosis.

Mechanistic Insight into the Fragmentation of Type I Collagen Fibers into Peptides and Amino Acids by a Vibrio Collagenase.

Vibrio collagenases of the M9A subfamily are closely related to Vibrio pathogenesis for their role in collagen degradation during host invasion. Although some Vibrio collagenases have been characterized, the collagen degradation mechanism of Vibrio collagenase is still largely unknown. Here, an M9A collagenase, VP397, from marine Vibrio pomeroyi strain 12613 was characterized, and its fragmentation pattern on insoluble type I collagen fibers was studied. VP397 is a typical Vibrio collagenase composed of a catalytic module featuring a peptidase M9N domain and a peptidase M9 domain and two accessory bacterial prepeptidase C-terminal domains (PPC domains). It can hydrolyze various collagenous substrates, including fish collagen, mammalian collagens of types I to V, triple-helical peptide [(POG)10]3, gelatin, and 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-o-Arg (Pz-peptide). Atomic force microscopy (AFM) observation and biochemical analyses revealed that VP397 first assaults the C-telopeptide region to dismantle the compact structure of collagen and dissociate tropocollagen fragments, which are further digested into peptides and amino acids by VP397 mainly at the Y-Gly bonds in the repeating Gly-X-Y triplets. In addition, domain deletion mutagenesis showed that the catalytic module of VP397 alone is capable of hydrolyzing type I collagen fibers and that its C-terminal PPC2 domain functions as a collagen-binding domain during collagenolysis. Based on our results, a model for the collagenolytic mechanism of VP397 is proposed. This study sheds light on the mechanism of collagen degradation by Vibrio collagenase, offering a better understanding of the pathogenesis of Vibrio and helping in developing the potential applications of Vibrio collagenase in industrial and medical areas. IMPORTANCE Many Vibrio species are pathogens and cause serious diseases in humans and aquatic animals. The collagenases produced by pathogenic Vibrio species have been regarded as important virulence factors, which occasionally exhibit direct pathogenicity to the infected host or facilitate other toxins' diffusion through the digestion of host collagen. However, our knowledge concerning the collagen degradation mechanism of Vibrio collagenase is still limited. This study reveals the degradation strategy of Vibrio collagenase VP397 on type I collagen. VP397 binds on collagen fibrils via its C-terminal PPC2 domain, and its catalytic module first assaults the C-telopeptide region and then attacks the Y-Gly bonds in the dissociated tropocollagen fragments to release peptides and amino acids. This study offers new knowledge regarding the collagenolytic mechanism of Vibrio collagenase, which is helpful for better understanding the role of collagenase in Vibrio pathogenesis and for developing its industrial and medical applications.

Lack of N-Terminal Segment of the Flagellin Protein Results in the Production of a Shortened Polar Flagellum in the Deep-Sea Sedimentary Bacterium Pseudoalteromonas sp. Strain SM9913.

Bacterial polar flagella, comprised of flagellin, are essential for bacterial motility. Pseudoalteromonas sp. strain SM9913 is a bacterium isolated from deep-sea sediments. Unlike other Pseudoalteromonas strains that have a long polar flagellum, strain SM9913 has an abnormally short polar flagellum. Here, we investigated the underlying reason for the short flagellum and found that a single-base mutation was responsible for the altered flagellar assembly. This mutation leads to the fragmentation of the flagellin gene into two genes, PSM_A2281, encoding the core segment and the C-terminal segment, and PSM_A2282, encoding the N-terminal segment, and only gene PSM_A2281 is involved in the production of the short polar flagellum. When a chimeric gene of PSM_A2281 and PSM_A2282 encoding an intact flagellin, A2281::82, was expressed, a long polar flagellum was produced, indicating that the N-terminal segment of flagellin contributes to the production of a polar flagellum of a normal length. Analyses of the simulated structures of A2281 and A2281::82 and that of the flagellar filament assembled with A2281::82 indicate that due to the lack of two α-helices, the core of the flagellar filament assembled with A2281 is incomplete and is likely too weak to support the stability and movement of a long flagellum. This mutation in strain SM9913 had little effect on its growth and only a small effect on its swimming motility, implying that strain SM9913 can live well with this mutation in natural sedimentary environments. This study provides a better understanding of the assembly and production of bacterial flagella. IMPORTANCE Polar flagella, which are essential organelles for bacterial motility, are comprised of multiple flagellin subunits. A flagellin molecule contains an N-terminal segment, a core segment, and a C-terminal segment. The results of this investigation of the deep-sea sedimentary bacterium Pseudoalteromonas sp. strain SM9913 demonstrate that a single-base mutation in the flagellin gene leads to the production of an incomplete flagellin without the N-terminal segment and that the loss of the N-terminal segment of the flagellin protein results in the production of a shortened polar flagellar filament. Our results shed light on the important function of the N-terminal segment of flagellin in the assembly and stability of bacterial flagellar filament.

Internal pressure-induced formation of hemispherical poles in Bacillus subtilis.

The cell of a rod-shaped bacterium is composed of a cylinder and two hemispherical poles. In recent decades, the molecular mechanism of morphogenesis in rod-shaped bacteria has received extensive research. However, most works have focused on the morphogenesis of cylinders, and the morphogenesis of the hemispherical poles remains unclear. In the past, the pole of bacterial cell wall was considered as a rigid hemispherical structure. However, our work indicated that the pole in the isolated sacculi from Bacillus subtilis was a flat structure instead of a hemisphere form. Further works showed that internal pressure was responsible for shaping the hemispherical poles, indicating an elastic nature of the cell wall in poles. In addition, we found that the internal pressure was able to transform septa into hemispherical shape which is similar to normal poles. Based on our work, we proposed a model for the internal pressure-induced formation of hemispherical poles in B. subtilis, and this work may provide new clues into basic knowledge of the morphogenesis of rod-shaped bacteria.

Marinifaba aquimaris gen. nov., sp. nov., a novel chitin-degrading gammaproteobacterium in the family Alteromonadaceae isolated from seawater of the Mariana Trench.

A novel Gram-negative, rod-shaped, aerobic, oxidase-positive and catalase-negative bacterium, designated strain SM1970T, was isolated from a seawater sample collected from the Mariana Trench. Strain SM1970T grew at 15-37 oC and with 1-5% (w/v) NaCl. It hydrolyzed colloidal chitin, agar and casein but did not reduce nitrate to nitrite. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain SM1970T formed a distinct lineage close to the genus Catenovulum within the family Alteromonadaceae, sharing the highest sequence similarity (93.6%) with type strain of Catenovulum maritimum but < 93.0% sequence similarity with those of other known species in the class Gammaproteobacteria. The major fatty acids of strain SM1970T were summed feature 3 (C16: 1 ω7c and/or C16: 1 ω6c), C16: 0 and summed feature 8 (C18: 1 ω7c and/or C18: 1 ω6c). The major polar lipids of the strain included phosphatidylethanolamine and phosphatidylglycerol and its main respiratory quinone was ubiquinone 8. The draft genome of strain SM1970T consisted of 77 scaffolds and was 4,172,146 bp in length, containing a complete set of genes for chitin degradation. The average amino acid identity (AAI) values between SM1970T and type strains of known Catenovulum species were 56.6-57.1% while the percentage of conserved proteins (POCP) values between them were 28.5-31.5%. The genomic DNA G + C content of strain SM1970T was 40.1 mol%. On the basis of the polyphasic analysis, strain SM1970T is considered to represent a novel species in a novel genus of the family Alteromonadaceae, for which the name Marinifaba aquimaris is proposed with the type strain being SM1970T (= MCCC 1K04323T = KCTC 72844T).

Structural Visualization of Septum Formation in Staphylococcus warneri Using Atomic Force Microscopy.

Cell division of Staphylococcus adopts a "popping" mechanism that mediates extremely rapid separation of the septum. Elucidating the structure of the septum is crucial for understanding this exceptional bacterial cell division mechanism. Here, the septum structure of Staphylococcus warneri was extensively characterized using high-speed time-lapse confocal microscopy, atomic force microscopy, and electron microscopy. The cells of S. warneri divide in a fast popping manner on a millisecond timescale. Our results show that the septum is composed of two separable layers, providing a structural basis for the ultrafast daughter cell separation. The septum is formed progressively toward the center with nonuniform thickness of the septal disk in radial directions. The peptidoglycan on the inner surface of double-layered septa is organized into concentric rings, which are generated along with septum formation. Moreover, this study signifies the importance of new septum formation in initiating new cell cycles. This work unravels the structural basis underlying the popping mechanism that drives S. warneri cell division and reveals a generic structure of the bacterial cell.IMPORTANCE This work shows that the septum of Staphylococcus warneri is composed of two layers and that the peptidoglycan on the inner surface of the double-layered septum is organized into concentric rings. Moreover, new cell cycles of S. warneri can be initiated before the previous cell cycle is complete. This work advances our knowledge about a basic structure of bacterial cell and provides information on the double-layered structure of the septum for bacteria that divide with the "popping" mechanism.

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).

Antarcticimicrobium sediminis gen. nov., sp. nov., isolated from Antarctic intertidal sediment, transfer of Ruegeria lutea to Antarcticimicrobium gen. nov. as Antarcticimicrobium luteum comb. nov.

A Gram-stain-negative, aerobic, non-flagellated and rod- or ovoid-shaped bacterium, designated as strain S4J41T, was isolated from Antarctic intertidal sediment. The isolate grew at 0-37 °C and with 0.5-10 % (w/v) NaCl. It reduced nitrate to nitrite and hydrolysed Tween 80 and gelatin. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain S4J41T constituted a distinct phylogenetic line within the family Rhodobacteraceae and was closely related with some species in the genera Ruegeria, Phaeobacter, Pseudopuniceibacterium, Sulfitobacter, Puniceibacterium and Poseidonocella with 98.6-95.7 % 16S rRNA gene sequence similarities. The major cellular fatty acids were C16 : 0, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and C18 : 0 and the major polar lipids were phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol, phosphatidylethanolamine and one unidentified aminolipid. The sole respiratory quinone was Q-10. The genomic DNA G+C content of strain S4J41T was 60.3 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic data obtained in this study, strain S4J41T is considered to represent a novel species in a new genus within the family Rhodobacteraceae, for which the name Antarcticimicrobium sediminis gen. nov., sp. nov. is proposed. The type strain is S4J41T (=MCCC 1K03508T=KCTC 62793T). Moreover, the transfer of Ruegeria lutea Kim et al. 2019 to Antarcticimicrobium gen. nov. as Antarcticimicrobium luteum comb. nov. (type strain 318-1T=JCM 30927T=KCTC 72105T) is also proposed.

Pelagovum pacificum gen. nov., sp. nov., a novel member of the family Rhodobacteraceae isolated from surface seawater of the Mariana Trench.

A Gram-stain-negative, aerobic, ovoid-rod-shaped bacterium, designated strain SM1903T, was isolated from surface seawater of the Mariana Trench. The strain grew at 15-37 °C (optimum, 35 °C) and with 1-15 % (optimum, 4 %) NaCl. It hydrolysed aesculin but did not reduce nitrate to nitrite and hydrolyse Tween 80. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain SM1903T formed a separate lineage within the family Rhodobacteraceae, sharing the highest 16S rRNA gene sequence similarity with type strains of Pseudooceanicola antarcticus (95.7 %) and Roseisalinus antarcticus (95.7 %). In phylogenetic trees based on single-copy OCs and whole proteins sequences, strain SM1903T fell within a sub-cluster encompassed by Oceanicola granulosus, Roseisalinus antarcticus and Histidinibacterium lentulum and formed a branch adjacent to Oceanicola granulosus. The major cellular fatty acids were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C16 : 0 and 11-methyl-C18 : 1 ω7c. The polar lipids mainly comprised phosphatidylglycerol, phosphatidylcholine, one unidentified lipid, one unidentified aminolipid, and one unidentified glycolipid. The solo respiratory quinone was ubiquinone-10. The genomic DNA G+C content of strain SM1903T was 66.0 mol%. Based on the results of phenotypic, chemotaxonomic, and phylogenetic characterization for strain SM1903T, it is considered to represent a novel species of a novel genus in the family Rhodobacteraceae, for which the name Pelagovum pacificum gen. nov., sp. nov. is proposed. The type strain is SM1903T (=MCCC 1K03608T=KCTC 72046T).

Shewanella polaris sp. nov., a psychrotolerant bacterium isolated from Arctic brown algae.

A Gram-stain-negative, facultatively anaerobic, flagellated and rod-shaped bacterium, designated strain SM1901T, was isolated from a brown algal sample collected from Kings Bay, Svalbard, Arctic. Strain SM1901T grew at -4‒30 °C and with 0-7.0 % (w/v) NaCl. It reduced nitrate to nitrite and hydrolysed DNA and Tween 80. Results of phylogenetic analyses based on 16S rRNA gene sequences indicated that strain SM1901T was affiliated with the genus Shewanella, showing the highest sequence similarity to the type strain of Shewanella litoralis (97.5%), followed by those of Shewanella vesiculosa, Shewanella livingstonensis and Shewanella saliphila (97.3 % for all three). The major cellular fatty acids were summed feature 3 (C16 : 1 ω7с and/or C16 : 1 ω6с), C16 : 0, C18 : 0, iso-C15 : 0 and C17 : 1 ω8с and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The respiratory quinones were ubiquinones Q-7, Q-8, menaquinones MK-7(H) and MK-8. The genome of strain SM1901T was 4648537 nucleotides long and encoded a variety of cold adaptation related genes, providing clues for better understanding the ecological adaptation mechanisms of polar bacteria. The genomic DNA G+C content of strain SM1901T was 40.5 mol%. Based on the polyphasic evidence presented in this paper, strain SM1901T was considered to represent a novel species, constituting a novel psychrotolerant lineage out of the known SF clade encompassed by polar Shewanella species, within the genus Shewanella, for which the name Shewanella polaris sp. nov. is proposed. The type strain is SM1901T (=KCTC 72047T=MCCC 1K03585T).

Parvularcula marina sp. nov., isolated from surface water of the South China Sea, and emended description of the genus Parvularcula.

A Gram-stain-negative, aerobic, flagellated, rod-shaped bacterial strain, SM1705T, was isolated from a surface seawater sample collected from the South China Sea. The strain grew at 10-40 °C and with 0.5-13.0 % (w/v) NaCl. It hydrolysed Tweens 20, 40 and 60, but did not hydrolyse starch or Tween 80 nor reduce nitrate to nitrite. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1705T was affiliated with the genus Parvularcula, sharing the highest sequence similarity (96.0 %) with type strain of Parvularcula bermudensis and forming a coherent branch together with the latter within the clade of Parvularcula. The major cellular fatty acids were identified as summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0 and C18 : 0. Polar lipids included three unidentified glycolipids and one unidentified lipid. The major respiratory quinone of strain SM1705T was Q10. The genomic DNA G+C content of strain SM1705T was 59.3 mol%. Based on the polyphasic evidence presented in this paper, strain SM1705T represents a novel Parvularcula species, for which the name Parvularcula marina sp. nov. is proposed. The type strain is SM1705T (=KCTC 62795T=MCCC 1K03505T=CCTCC AB 2018345T).

Structural mechanism for bacterial oxidation of oceanic trimethylamine into trimethylamine N-oxide.

Trimethylamine (TMA) and trimethylamine N-oxide (TMAO) are widespread in the ocean and are important nitrogen source for bacteria. TMA monooxygenase (Tmm), a bacterial flavin-containing monooxygenase (FMO), is found widespread in marine bacteria and is responsible for converting TMA to TMAO. However, the molecular mechanism of TMA oxygenation by Tmm has not been explained. Here, we determined the crystal structures of two reaction intermediates of a marine bacterial Tmm (RnTmm) and elucidated the catalytic mechanism of TMA oxidation by RnTmm. The catalytic process of Tmm consists of a reductive half-reaction and an oxidative half-reaction. In the reductive half-reaction, FAD is reduced and a C4a-hydroperoxyflavin intermediate forms. In the oxidative half-reaction, this intermediate attracts TMA through electronic interactions. After TMA binding, NADP+ bends and interacts with D317, shutting off the entrance to create a protected micro-environment for catalysis and exposing C4a-hydroperoxyflavin to TMA for oxidation. Sequence analysis suggests that the proposed catalytic mechanism is common for bacterial Tmms. These findings reveal the catalytic process of TMA oxidation by marine bacterial Tmm and first show that NADP+ undergoes a conformational change in the oxidative half-reaction of FMOs.

Structural insights into the cold adaptation of the photosynthetic pigment-protein C-phycocyanin from an Arctic cyanobacterium.

The cold adaptation mechanism of phycobiliproteins, the major photosynthetic pigment-proteins in cyanobacteria and red algae, has rarely been studied. Here we reported the biochemical, structural, and molecular dynamics simulation study of the C-phycocyanin from Arctic cyanobacterial strain Pseudanabaena sp. LW0831. We characterized the phycobilisome components of LW0831 and obtained their gene sequences. Compared to the mesophilic counterpart from Arthrospira platensis (Ar-C-PC), LW0831 C-phycocyanin (Ps-C-PC) has a decreased thermostability (∆Tm of -16°C), one of the typical features of cold-adapted enzymes. To uncover its structural basis, we resolved the crystal structure of Ps-C-PC 1 at 2.04Å. Consistent with the decrease in thermostability, comparative structural analyses revealed decreased intra-trimer and inter-trimer interactions in Ps-C-PC 1, compared to Ar-C-PC. However, comparative molecular dynamics simulations indicated that Ps-C-PC 1 shows similar flexibilities to Ar-C-PC for both the (αß)3 trimer and (αß)6 hexamer. Therefore, the optimization mode is clearly different from cold-adapted enzymes, which usually have increased flexibilities. Detailed analyses demonstrated different optimization modes for the α and ß subunits and it was revealed that hydrophobic interactions are key to this difference, though salt bridges, hydrogen bonds, and surface hydrophobicity are also involved. This study is the first report of the structure of cold-adapted phycobiliproteins and provides insights into the cold-adaptation strategies of non-enzyme proteins.

Supramolecular architecture of photosynthetic membrane in red algae in response to nitrogen starvation.

The availability of nitrogen is one of the most important determinants that can limit the growth of photosynthetic organisms including plants and algae; however, direct observations on the supramolecular architecture of photosynthetic membranes in response to nitrogen stress are still lacking. Red algae are an important evolutionary group of algae which contain phycobilisomes (PBSs) on their thylakoid membranes, as do cyanobacteria. PBSs function not only as light-harvesting antennae but also as nitrogen storage. In this report, alterations of the supramolecular architecture of thylakoid membranes from red alga Porphyridium cruentum during nitrogen starvation were characterized. The morphology of the intact thylakoid membrane was observed to be round vesicles. Thylakoid membranes were reduced in content and PBSs were degraded during nitrogen starvation. The size and density of PBSs were both found to be reduced. PBS size decreased by less than one-half after 20days of nitrogen starvation, but their hemispherical morphology was retained. The density of PBSs on thylakoid membranes was more seriously affected as time proceeded. Upon re-addition of nitrogen led to increasing of PBSs on thylakoid membranes. This work reports the first direct observation on alterations in the supramolecular architecture of thylakoid membranes from a photosynthetic organism in response to nitrogen stress.

Structural and Mechanistic Insights into the Improvement of the Halotolerance of a Marine Microbial Esterase by Increasing Intra- and Interdomain Hydrophobic Interactions.

Halotolerant enzymes are beneficial for industrial processes requiring high salt concentrations and low water activity. Most halophilic proteins are evolved to have reduced hydrophobic interactions on the surface and in the hydrophobic cores for their haloadaptation. However, in this study, we improved the halotolerance of a thermolabile esterase, E40, by increasing intraprotein hydrophobic interactions. E40 was quite unstable in buffers containing more than 0.3 M NaCl, and its kcat and substrate affinity were both significantly reduced in 0.5 M NaCl. By introducing hydrophobic residues in loop 1 of the CAP domain and/or α7 of the catalytic domain in E40, we obtained several mutants with improved halotolerance, and the M3 S202W I203F mutant was the most halotolerant. ("M3" represents a mutation in loop 1 of the CAP domain in which residues R22-K23-T24 of E40 are replaced by residues Y22-K23-H24-L25-S26 of Est2.) Then we solved the crystal structures of the S202W I203F and M3 S202W I203F mutants to reveal the structural basis for their improved halotolerance. Structural analysis revealed that the introduction of hydrophobic residues W202 and F203 in α7 significantly improved E40 halotolerance by strengthening intradomain hydrophobic interactions of F203 with W202 and other residues in the catalytic domain. By further introducing hydrophobic residues in loop 1, the M3 S202W I203F mutant became more rigid and halotolerant due to the formation of additional interdomain hydrophobic interactions between the introduced Y22 in loop 1 and W204 in α7. These results indicate that increasing intraprotein hydrophobic interactions is also a way to improve the halotolerance of enzymes with industrial potential under high-salt conditions.IMPORTANCE Esterases and lipases for industrial application are often subjected to harsh conditions such as high salt concentrations, low water activity, and the presence of organic solvents. However, reports on halotolerant esterases and lipases are limited, and the underlying mechanism for their halotolerance is still unclear due to the lack of structures. In this study, we focused on the improvement of the halotolerance of a salt-sensitive esterase, E40, and the underlying mechanism. The halotolerance of E40 was significantly improved by introducing hydrophobic residues. Comparative structural analysis of E40 and its halotolerant mutants revealed that increased intraprotein hydrophobic interactions make these mutants more rigid and more stable than the wild type against high concentrations of salts. This study shows a new way to improve enzyme halotolerance, which is helpful for protein engineering of salt-sensitive enzymes.

Arcticibacterium luteifluviistationis gen. nov., sp. nov., isolated from Arctic seawater.

A Gram-staining-negative, aerobic, non-motile and yellow-pigmented bacterium, designated strain SM1504T, was isolated from Arctic seawater. It hydrolysed aesculin and gelatin but did not reduce nitrate to nitrite. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain SM1504T constituted a distinct phylogenetic line within the family Cytophagaceae and was closely related to species of the genera Lacihabitans, Emticicia, Fluviimonas and Leadbetterella, with respect to which low sequence similarities between 88.9 and 91.6 % were observed. The major fatty acids of strain SM1504T were summed feature 3 (comprising C16 : 1ω7c and/or iso-C15 : 0 2-OH) and iso-C15 : 0. The predominant polar lipids of strain SM1504T were phosphatidylethanolamine and one unidentified lipid. The only respiratory quinone detected in strain SM1504T was MK7. The DNA G+C content of strain SM1504T was 40.8 mol%. On the basis of the phylogenetic, chemotaxonomic and phenotypic characterization in this study, strain SM1504T is considered to represent a novel species in a new genus of the family Cytophagaceae, for which the name Arcticibacterium luteifluviistationis gen. nov., sp. nov. is proposed. The type strain is SM1504T (=KCTC 42716T=CCTCC AB 2015348T).

Flavobacterium arcticum sp. nov., isolated from Arctic seawater.

A Gram-reaction-negative, aerobic, non-flagellated, rod-shaped and yellow-pigmented bacterium, designated strain SM1502T, was isolated from Arctic seawater. The isolate grew at 10-40 °C and with 0-8.0 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate was affiliated with the genus Flavobacterium, with the highest sequence similarity (96.0 %) found with Flavobacterium suzhouense XIN-1T. The major fatty acids of strain SM1502T were iso-C15 : 0, iso-C17 : 1ω9c, iso-C15 : 1 G, C15 : 0, iso-C17 : 0 3-OH and unknown ECL 13.565. The major respiratory quinone of strain SM1502T was menaquinone-6 (MK-6). Polar lipids of strain SM1502T included phosphatidylethanolamine and one unidentified aminolipid and lipid. The genomic DNA G+C content of strain SM1502T was 37.0 mol%. Based on the polyphasic data obtained in this study, strain SM1502T is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium arcticum sp. nov. is proposed. The type strain is SM1502T (=KCTC 42668T=CCTCC AB 2015346T).

Molecular insight into bacterial cleavage of oceanic dimethylsulfoniopropionate into dimethyl sulfide.

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile DMS through the action of DMSP lyases and is important in the global sulfur and carbon cycles. When released into the atmosphere from the oceans, DMS is oxidized, forming cloud condensation nuclei that may influence weather and climate. Six different DMSP lyase genes are found in taxonomically diverse microorganisms, and dddQ is among the most abundant in marine metagenomes. Here, we examine the molecular mechanism of DMSP cleavage by the DMSP lyase, DddQ, from Ruegeria lacuscaerulensis ITI_1157. The structures of DddQ bound to an inhibitory molecule 2-(N-morpholino)ethanesulfonic acid and of DddQ inactivated by a Tyr131Ala mutation and bound to DMSP were solved. DddQ adopts a ß-barrel fold structure and contains a Zn(2+) ion and six highly conserved hydrophilic residues (Tyr120, His123, His125, Glu129, Tyr131, and His163) in the active site. Mutational and biochemical analyses indicate that these hydrophilic residues are essential to catalysis. In particular, Tyr131 undergoes a conformational change during catalysis, acting as a base to initiate the ß-elimination reaction in DMSP lysis. Moreover, structural analyses and molecular dynamics simulations indicate that two loops over the substrate-binding pocket of DddQ can alternate between "open" and "closed" states, serving as a gate for DMSP entry. We also propose a molecular mechanism for DMS production through DMSP cleavage. Our study provides important insight into the mechanism involved in the conversion of DMSP into DMS, which should lead to a better understanding of this globally important biogeochemical reaction.

Interdomain hydrophobic interactions modulate the thermostability of microbial esterases from the hormone-sensitive lipase family.

Microbial hormone-sensitive lipases (HSLs) contain a CAP domain and a catalytic domain. However, it remains unclear how the CAP domain interacts with the catalytic domain to maintain the stability of microbial HSLs. Here, we isolated an HSL esterase, E40, from a marine sedimental metagenomic library. E40 exhibited the maximal activity at 45 °C and was quite thermolabile, with a half-life of only 2 min at 40 °C, which may be an adaptation of E40 to the permanently cold sediment environment. The structure of E40 was solved to study its thermolability. Structural analysis showed that E40 lacks the interdomain hydrophobic interactions between loop 1 of the CAP domain and α7 of the catalytic domain compared with its thermostable homologs. Mutational analysis showed that the introduction of hydrophobic residues Trp(202) and Phe(203) in α7 significantly improved E40 stability and that a further introduction of hydrophobic residues in loop 1 made E40 more thermostable because of the formation of interdomain hydrophobic interactions. Altogether, the results indicate that the absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40. In addition, a comparative analysis of the structures of E40 and other thermolabile and thermostable HSLs suggests that the interdomain hydrophobic interactions between loop 1 and α7 are a key element for the thermostability of microbial HSLs. Therefore, this study not only illustrates the structural element leading to the thermolability of E40 but also reveals a structural determinant for HSL thermostability.

Structural and molecular basis for the novel catalytic mechanism and evolution of DddP, an abundant peptidase-like bacterial Dimethylsulfoniopropionate lyase: a new enzyme from an old fold.

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile dimethyl sulfide (DMS) and is an important step in global sulfur and carbon cycles. DddP is a DMSP lyase in marine bacteria, and the deduced dddP gene product is abundant in marine metagenomic data sets. However, DddP belongs to the M24 peptidase family according to sequence alignment. Peptidases hydrolyze C-N bonds, but DddP is deduced to cleave C-S bonds. Mechanisms responsible for this striking functional shift are currently unknown. We determined the structures of DMSP lyase RlDddP (the DddP from Ruegeria lacuscaerulensis ITI_1157) bound to inhibitory 2-(N-morpholino) ethanesulfonic acid or PO4 (3-) and of two mutants of RlDddP bound to acrylate. Based on structural, mutational and biochemical analyses, we characterized a new ion-shift catalytic mechanism of RlDddP for DMSP cleavage. Furthermore, we suggested the structural mechanism leading to the loss of peptidase activity and the subsequent development of DMSP lyase activity in DddP. This study sheds light on the catalytic mechanism and the divergent evolution of DddP, leading to a better understanding of marine bacterial DMSP catabolism and global DMS production.