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.

A Novel Subfamily Esterase with a Homoserine Transacetylase-like Fold but No Transferase Activity.

Microbial esterases play important roles in deep-sea organic carbon degradation and cycling. Although they have similar catalytic triads and oxyanion holes, esterases are hydrolases and homoserine transacetylases (HTAs) are transferases. Because two HTA homologs were identified as acetyl esterases, the HTA family was recently divided into the bona fide acetyltransferase subfamily and the acetyl esterase subfamily. Here, we identified and characterized a novel HTA-like esterase, Est22, from a deep-sea sedimentary metagenomic library. Est22 could efficiently hydrolyze esters with acyl lengths of up to six carbon atoms but had no transacetylase activity, which is different from HTAs and HTA-like acetyl esterases. Phylogenetic analysis also showed that Est22 and its homologs form a separate branch of the HTA family. We solved the structures of Est22 and its L374D mutant and modeled the structure of the L374D mutant with p-nitrophenyl butyrate. Based on structural, mutational, and biochemical analyses, Phe71 and Met176 in the oxyanion hole and Arg294 were revealed to be the key substrate-binding residues. A detailed structural comparison indicated that differences in their catalytic tunnels lead to the different substrate specificities of Est22 and the other two HTA subfamilies. Biochemical and sequence analyses suggested that Est22 homologs may have the same substrate recognition and catalysis mechanisms as Est22. Due to the significant differences in sequences, structures, and substrate specificities between Est22 (and its homologs) and the other two HTA subfamilies, we suggest that Est22 and its homologs represent a new subfamily in the HTA family.IMPORTANCE Microbial esterases play important roles in the turnover of organic carbon in the deep sea. Esterases and HTAs represent two groups of α/ß hydrolases. Esterases catalyze the hydrolysis of simple esters and are widely used in the pharmaceutical and agrochemical industries, while HTAs catalyze the transfer of an acetyl group from acetyl-coenzyme A (CoA) to homoserine and are essential for microbial growth. Here, we report on a novel HTA-like esterase, Est22, from a deep-sea sediment. Because of the significant differences in sequences, structures, and substrate specificities of HTAs and HTA-like acetyl esterases, Est22 and its homologs represent a new subfamily in the HTA family. This study offers new knowledge regarding marine esterases.

Changchengzhania lutea gen. nov., sp. nov., a new member of the family Flavobacteriaceae isolated from Antarctic intertidal sediment.

A Gram-negative, aerobic, yellow pigmented, non-flagellated, non-gliding, rod-shaped bacterial strain, designated SM1355T, was isolated from Antarctic intertidal sediment collected near the Chinese Antarctic Great Wall Station. The strain grew at 4-35 °C and with 0.5-7.0 % (w/v) NaCl. It hydrolysed aesculin but didn't reduce nitrate to nitrite. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1355T formed a distinct phylogenetic lineage within the family Flavobacteriaceae, sharing the highest 16S rRNA gene sequence similarity with Flaviramulus ichthyoenteri (96.3 %) and fairly high sequence similarities (95.0-96.0 %) with over 20 recognized species in eight genera of the family Flavobacteriaceae. The predominant fatty acids were anteiso-C15 : 0, iso-C15 : 0 and iso-C15 : 1 G. The major polar lipids were phosphatidylethanolamine and one unidentified lipid. The genomic DNA G+C content of strain SM1355T was 36.2 mol%. Based on the results of the polyphasic characterization for strain SM1355T, it is identified as the representative of a novel species in a new genus of the family Flavobacteriaceae, for which the name Changchengzhania lutea gen. nov., sp. nov. is proposed. The type strain of Changchengzhania lutea is SM1355T (=JCM 30336T=CCTCC AB 2014246T).

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.

Pilot-Scale Production and Thermostability Improvement of the M23 Protease Pseudoalterin from the Deep Sea Bacterium Pseudoalteromonas sp. CF6-2.

Pseudoalterin is the most abundant protease secreted by the marine sedimental bacterium Pseudoalteromonas sp. CF6-2 and is a novel cold-adapted metalloprotease of the M23 family. Proteases of the M23 family have high activity towards peptidoglycan and elastin, suggesting their promising biomedical and biotechnological potentials. To lower the fermentive cost and improve the pseudoalterin production of CF6-2, we optimized the fermentation medium by using single factor experiments, added 0.5% sucrose as a carbon source, and lowered the usage of artery powder from 1.2% to 0.6%. In the optimized medium, pseudoalterin production reached 161.15 ± 3.08 U/mL, 61% greater than that before optimization. We further conducted a small-scale fermentation experiment in a 5-L fermenter and a pilot-scale fermentation experiment in a 50-L fermenter. Pseudoalterin production during pilot-scale fermentation reached 103.48 ± 8.64 U/mL, 77% greater than that before the medium was optimized. In addition, through single factor experiments and orthogonal tests, we developed a compound stabilizer for pseudoalterin, using medically safe sugars and polyols. This stabilizer showed a significant protective effect for pseudoalterin against enzymatic thermal denaturation. These results lay a solid foundation for the industrial production of pseudoalterin and the development of its biomedical and biotechnological potentials.

Mechanistic Insight into Trimethylamine N-Oxide Recognition by the Marine Bacterium Ruegeria pomeroyi DSS-3.

UNLABELLED: Trimethylamine N-oxide (TMAO) is an important nitrogen source for marine bacteria. TMAO can also be metabolized by marine bacteria into volatile methylated amines, the precursors of the greenhouse gas nitrous oxide. However, it was not known how TMAO is recognized and imported by bacteria. Ruegeria pomeroyi DSS-3, a marine Roseobacter, has an ATP-binding cassette transporter, TmoXWV, specific for TMAO. TmoX is the substrate-binding protein of the TmoXWV transporter. In this study, the substrate specificity of TmoX of R. pomeroyi DSS-3 was characterized. We further determined the structure of the TmoX/TMAO complex and studied the TMAO-binding mechanism of TmoX by biochemical, structural, and mutational analyses. A Ca(2+) ion chelated by an extended loop in TmoX was shown to be important for maintaining the stability of TmoX. Molecular dynamics simulations indicate that TmoX can alternate between "open" and "closed" states for binding TMAO. In the substrate-binding pocket, four tryptophan residues interact with the quaternary amine of TMAO by cation-π interactions, and Glu131 forms a hydrogen bond with the polar oxygen atom of TMAO. The π-π stacking interactions between the side chains of Phe and Trp are also essential for TMAO binding. Sequence analysis suggests that the TMAO-binding mechanism of TmoX may have universal significance in marine bacteria, especially in the marine Roseobacter clade. This study sheds light on how marine microorganisms utilize TMAO. IMPORTANCE: Trimethylamine N-oxide (TMAO) is an important nitrogen source for marine bacteria. The products of TMAO metabolized by bacteria are part of the precursors of the greenhouse gas nitrous oxide. It is unclear how TMAO is recognized and imported by bacteria. TmoX is the substrate-binding protein of a TMAO-specific transporter. Here, the substrate specificity of TmoX of Ruegeria pomeroyi DSS-3 was characterized. The TMAO-binding mechanism of TmoX was studied by biochemical, structural, and mutational analyses. Moreover, our results suggest that the TMAO-binding mechanism may have universal significance in marine bacteria. This study sheds light on how marine microorganisms utilize TMAO and should lead to a better understanding of marine nitrogen cycling.

Structural insights into the multispecific recognition of dipeptides of deep-sea gram-negative bacterium Pseudoalteromonas sp. strain SM9913.

UNLABELLED: Peptide uptake is important for nutrition supply for marine bacteria. It is also an important step in marine nitrogen cycling. However, how marine bacteria absorb peptides is still not fully understood. DppA is the periplasmic dipeptide binding protein of dipeptide permease (Dpp; an important peptide transporter in bacteria) and exclusively controls the substrate specificity of Dpp. Here, the substrate binding specificity of deep-sea Pseudoalteromonas sp. strain SM9913 DppA (PsDppA) was analyzed for 25 different dipeptides with various properties by using isothermal titration calorimetry measurements. PsDppA showed binding affinities for 8 dipeptides. To explain the multispecific substrate recognition mechanism of PsDppA, we solved the crystal structures of unliganded PsDppA and of PsDppA in complex with 4 different types of dipeptides (Ala-Phe, Met-Leu, Gly-Glu, and Val-Thr). PsDppA alternates between an "open" and a "closed" form during substrate binding. Structural analyses of the 4 PsDppA-substrate complexes combined with mutational assays indicate that PsDppA binds to different substrates through a precise mechanism: dipeptides are bound mainly by the interactions between their backbones and PsDppA, in particular by anchoring their N and C termini through ion-pair interactions; hydrophobic interactions are important in binding hydrophobic dipeptides; and Lys457 is necessary for the binding of dipeptides with a C-terminal glutamic acid or glutamine. Additionally, sequence alignment suggests that the substrate recognition mechanism of PsDppA may be common in Gram-negative bacteria. All together, our results provide structural insights into the multispecific substrate recognition mechanism of marine Gram-negative bacterial DppA, which provides a better understanding of the mechanisms of marine bacterial peptide uptake. IMPORTANCE: Peptide uptake plays a significant role in nutrition supply for marine bacteria. It is also an important step in marine nitrogen cycling. However, how marine bacteria recognize and absorb peptides is still unclear. This study analyzed the substrate binding specificity of deep-sea Pseudoalteromonas sp. strain SM9913 DppA (PsDppA; the dipeptide-binding protein of dipeptide permease) and solved the crystal structures of unliganded PsDppA and PsDppA in complex with 4 different types of dipeptides. The multispecific recognition mechanism of PsDppA for dipeptides is explained based on structural and mutational analyses. We also find that the substrate-binding mechanism of PsDppA may be common in Gram-negative bacteria. This study sheds light on marine Gram-negative bacterial peptide uptake and marine nitrogen cycling.

Characterization of a novel subtilisin-like protease myroicolsin from deep sea bacterium Myroides profundi D25 and molecular insight into its collagenolytic mechanism.

Collagen is an insoluble protein that widely distributes in the extracellular matrix of marine animals. Collagen degradation is an important step in the marine nitrogen cycle. However, the mechanism of marine collagen degradation is still largely unknown. Here, a novel subtilisin-like collagenolytic protease, myroicolsin, which is secreted by the deep sea bacterium Myroides profundi D25, was purified and characterized, and its collagenolytic mechanism was studied. Myroicolsin displays low identity (<30%) to previously characterized subtilisin-like proteases, and it contains a novel domain structure. Protein truncation indicated that the Pro secretion system C-terminal sorting domain in the precursor protein is involved in the cleavage of the N-propeptide, and the linker is required for protein folding during myroicolsin maturation. The C-terminal ß-jelly roll domain did not bind insoluble collagen fiber, suggesting that myroicolsin may degrade collagen without the assistance of a collagen-binding domain. Myroicolsin had broad specificity for various collagens, especially fish-insoluble collagen. The favored residue at the P1 site was basic arginine. Scanning electron microscopy and atomic force microscopy, together with biochemical analyses, confirmed that collagen fiber degradation by myroicolsin begins with the hydrolysis of proteoglycans and telopeptides in collagen fibers and fibrils. Myroicolsin showed strikingly different cleavage patterns between native and denatured collagens. A collagen degradation model of myroicolsin was proposed based on our results. Our study provides molecular insight into the collagen degradation mechanism and structural characterization of a subtilisin-like collagenolytic protease secreted by a deep sea bacterium, shedding light on the degradation mechanism of deep sea sedimentary organic nitrogen.

Structural basis for dimerization and catalysis of a novel esterase from the GTSAG motif subfamily of the bacterial hormone-sensitive lipase family.

Hormone-sensitive lipases (HSLs) are widely distributed in microorganisms, plants, and animals. Microbial HSLs are classified into two subfamilies, an unnamed new subfamily and the GDSAG motif subfamily. Due to the lack of structural information, the detailed catalytic mechanism of the new subfamily is not yet clarified. Based on sequence analysis, we propose to name the new subfamily as the GTSAG motif subfamily. We identified a novel HSL esterase E25, a member of the GTSAG motif subfamily, by functional metagenomic screening, and resolved its structure at 2.05 Å. E25 is mesophilic (optimum temperature at 50 °C), salt-tolerant, slightly alkaline (optimum pH at 8.5) for its activity, and capable of hydrolyzing short chain monoesters (C2-C10). E25 tends to form dimers both in the crystal and in solution. An E25 monomer contains an N-terminal CAP domain, and a classical α/ß hydrolase-fold domain. Residues Ser(186), Asp(282), and His(312) comprise the catalytic triad. Structural and mutational analyses indicated that E25 adopts a dimerization pattern distinct from other HSLs. E25 dimer is mainly stabilized by an N-terminal loop intersection from the CAP domains and hydrogen bonds and salt bridges involving seven highly conserved hydrophilic residues from the catalytic domains. Further analysis indicated that E25 also has some catalytic profiles different from other HSLs. Dimerization is essential for E25 to exert its catalytic activity by keeping the accurate orientation of the catalytic Asp(282) within the catalytic triad. Our results reveal the structural basis for dimerization and catalysis of an esterase from the GTSAG motif subfamily of the HSL family.

Molecular insight into the role of the N-terminal extension in the maturation, substrate recognition, and catalysis of a bacterial alginate lyase from polysaccharide lyase family 18.

Bacterial alginate lyases, which are members of several polysaccharide lyase (PL) families, have important biological roles and biotechnological applications. The mechanisms for maturation, substrate recognition, and catalysis of PL18 alginate lyases are still largely unknown. A PL18 alginate lyase, aly-SJ02, from Pseudoalteromonas sp. 0524 displays a ß-jelly roll scaffold. Structural and biochemical analyses indicated that the N-terminal extension in the aly-SJ02 precursor may act as an intramolecular chaperone to mediate the correct folding of the catalytic domain. Molecular dynamics simulations and mutational assays suggested that the lid loops over the aly-SJ02 active center serve as a gate for substrate entry. Molecular docking and site-directed mutations revealed that certain conserved residues at the active center, especially those at subsites +1 and +2, are crucial for substrate recognition. Tyr(353) may function as both a catalytic base and acid. Based on our results, a model for the catalysis of aly-SJ02 in alginate depolymerization is proposed. Moreover, although bacterial alginate lyases from families PL5, 7, 15, and 18 adopt distinct scaffolds, they share the same conformation of catalytic residues, reflecting their convergent evolution. Our results provide the foremost insight into the mechanisms of maturation, substrate recognition, and catalysis of a PL18 alginate lyase.

Deep RNA sequencing reveals a high frequency of alternative splicing events in the fungus Trichoderma longibrachiatum.

BACKGROUND: Alternative splicing is crucial for proteome diversity and functional complexity in higher organisms. However, the alternative splicing landscape in fungi is still elusive. RESULTS: The transcriptome of the filamentous fungus Trichoderma longibrachiatum was deep sequenced using Illumina Solexa technology. A total of 14305 splice junctions were discovered. Analyses of alternative splicing events revealed that the number of all alternative splicing events (10034), intron retentions (IR, 9369), alternative 5' splice sites (A5SS, 167), and alternative 3' splice sites (A3SS, 302) is 7.3, 7.4, 5.1, and 5.9-fold higher, respectively, than those observed in the fungus Aspergillus oryzae using Illumina Solexa technology. This unexpectedly high ratio of alternative splicing suggests that alternative splicing is important to the transcriptome diversity of T. longibrachiatum. Alternatively spliced introns had longer lengths, higher GC contents, and lower splice site scores than constitutive introns. Further analysis demonstrated that the isoform relative frequencies were correlated with the splice site scores of the isoforms. Moreover, comparative transcriptomics determined that most enzymes related to glycolysis and the citrate cycle and glyoxylate cycle as well as a few carbohydrate-active enzymes are transcriptionally regulated. CONCLUSIONS: This study, consisting of a comprehensive analysis of the alternative splicing landscape in the filamentous fungus T. longibrachiatum, revealed an unexpectedly high ratio of alternative splicing events and provided new insights into transcriptome diversity in fungi.

Physiological and genetic analyses reveal a mechanistic insight into the multifaceted lifestyles of Pseudoalteromonas sp. SM9913 adapted to the deep-sea sediment.

Although bacteriobenthos play a major role in the degradation of particulate organic matter in marine sediment, knowledge of the sediment-adapted lifestyles of bacteriobenthos is still scarce. Here, the particle-associated, swimming and swarming lifestyles of the benthonic bacterium Pseudoalteromonas sp. SM9913 (SM9913) were illustrated. SM9913 had a clay particle-associated lifestyle, and its exopolysaccharide played an important role in this lifestyle. SM9913 also had swimming and swarming motilities, indicating that it may have swimming and swarming lifestyles in the sediment. The lateral flagella were responsible for the swarming motility, and the polar flagella were responsible for the swimming motility. Iron limitation was an indispensable inductive signal of the swarming motility. An analysis of the motilities of SM9913 and its mutants in clay demonstrated that SM9913 moved in clay by both swimming and swarming motilities. Genomic analysis suggests that having two flagella systems is most likely a common adaptation of some bacteriobenthos to the sediment environment. Our results reveal the lifestyles of benthonic SM9913, providing a better understanding of the environmental adaptation of benthonic bacteria.

Bizionia arctica sp. nov., isolated from Arctic fjord seawater, and emended description of the genus Bizionia.

A Gram-stain-negative, yellow-pigmented, aerobic, non-flagellated, non-gliding bacterial strain, designated SM1203(T), was isolated from surface seawater of Kongsfjorden, Svalbard. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1203(T) was affiliated with the genus Bizionia in the family Flavobacteriaceae. The strain shared the highest 16S rRNA gene sequence similarity (>96%) with the type strains of Formosa spongicola (96.8%), Bizionia paragorgiae (96.3%), B. saleffrena (96.3%) and B. echini (96.1%) and 95.4-95.7% sequence similarity with the type strains of other known species of the genus Bizionia. The strain grew at 4-30 °C and in the presence of 1.0-5.0% (w/v) NaCl. The major fatty acids of strain SM1203(T) were iso-C15 : 0, iso-C15 : 1, anteiso-C15 : 0 and C15 : 0 and the main polar lipids were phosphatidylethanolamine and an unidentified lipid. The major respiratory quinone of strain SM1203(T) was menaquinone 6 (MK-6). The genomic DNA G+C content of strain SM1203(T) was 34.8 mol%. Based on the polyphasic characterization of strain SM1203(T) in this study, the strain represents a novel species in the genus Bizionia, for which the name Bizionia arctica sp. nov. is proposed. The type strain is SM1203(T) ( = CGMCC 1.12751(T) = JCM 30333(T)). An emended description of the genus Bizionia is also given.

Mechanistic insight into the elastin degradation process by the metalloprotease myroilysin from the deep-sea bacterium Myroides profundi D25.

Elastases have been widely studied because of their important uses as medicine and meat tenderizers. However, there are relatively few studies on marine elastases. Myroilysin, secreted by Myroides profundi D25 from deep-sea sediment, is a novel elastase. In this study, we examined the elastin degradation mechanism of myroilysin. When mixed with insoluble bovine elastin, myroilysin bound hydrophobically, suggesting that this elastase may interact with the hydrophobic domains of elastin. Consistent with this, analysis of the cleavage pattern of myroilysin on bovine elastin and recombinant tropoelastin revealed that myroilysin preferentially cleaves peptide bonds with hydrophobic residues at the P1 and/or P1' positions. Scanning electron microscopy (SEM) of cross-linked recombinant tropoelastin degraded by myroilysin showed preferential damages of spherules over cross-links, as expected for a hydrophobic preference. The degradation process of myroilysin on bovine elastin fibres was followed by light microscopy and SEM, revealing that degradation begins with the formation of crevices and cavities at the fibre surface, with these openings increasing in number and size until the fibre breaks into small pieces, which are subsequently fragmented. Our results are helpful for developing biotechnological applications for myroilysin.

Culture Condition Optimization and Pilot Scale Production of the M12 Metalloprotease Myroilysin Produced by the Deep-Sea Bacterium Myroides profundi D25.

The protease myroilysin is the most abundant protease secreted by marine sedimental bacterium Myroides profundi D25. As a novel elastase of the M12 family, myroilysin has high elastin-degrading activity and strong collagen-swelling ability, suggesting its promising biotechnological potential. Because myroilysin cannot be maturely expressed in Escherichia coli, it is important to be able to improve the production of myroilysin in the wild strain D25. We optimized the culture conditions of strain D25 for protease production by using single factor experiments. Under the optimized conditions, the protease activity of strain D25 reached 1137 ± 53.29 U/mL, i.e., 174% of that before optimization (652 ± 23.78 U/mL). We then conducted small scale fermentations of D25 in a 7.5 L fermentor. The protease activity of strain D25 in small scale fermentations reached 1546.4 ± 82.65 U/mL after parameter optimization. Based on the small scale fermentation results, we further conducted pilot scale fermentations of D25 in a 200 L fermentor, in which the protease production of D25 reached approximately 1100 U/mL. These results indicate that we successfully set up the small and pilot scale fermentation processes of strain D25 for myroilysin production, which should be helpful for the industrial production of myroilysin and the development of its biotechnological potential.

A proposed genus boundary for the prokaryotes based on genomic insights.

Genomic information has already been applied to prokaryotic species definition and classification. However, the contribution of the genome sequence to prokaryotic genus delimitation has been less studied. To gain insights into genus definition for the prokaryotes, we attempted to reveal the genus-level genomic differences in the current prokaryotic classification system and to delineate the boundary of a genus on the basis of genomic information. The average nucleotide sequence identity between two genomes can be used for prokaryotic species delineation, but it is not suitable for genus demarcation. We used the percentage of conserved proteins (POCP) between two strains to estimate their evolutionary and phenotypic distance. A comprehensive genomic survey indicated that the POCP can serve as a robust genomic index for establishing the genus boundary for prokaryotic groups. Basically, two species belonging to the same genus would share at least half of their proteins. In a specific lineage, the genus and family/order ranks showed slight or no overlap in terms of POCP values. A prokaryotic genus can be defined as a group of species with all pairwise POCP values higher than 50%. Integration of whole-genome data into the current taxonomy system can provide comprehensive information for prokaryotic genus definition and delimitation.