Structure of cryptophyte photosystem II-light-harvesting antennae supercomplex.

Cryptophytes are ancestral photosynthetic organisms evolved from red algae through secondary endosymbiosis. They have developed alloxanthin-chlorophyll a/c2-binding proteins (ACPs) as light-harvesting complexes (LHCs). The distinctive properties of cryptophytes contribute to efficient oxygenic photosynthesis and underscore the evolutionary relationships of red-lineage plastids. Here we present the cryo-electron microscopy structure of the Photosystem II (PSII)-ACPII supercomplex from the cryptophyte Chroomonas placoidea. The structure includes a PSII dimer and twelve ACPII monomers forming four linear trimers. These trimers structurally resemble red algae LHCs and cryptophyte ACPI trimers that associate with Photosystem I (PSI), suggesting their close evolutionary links. We also determine a Chl a-binding subunit, Psb-γ, essential for stabilizing PSII-ACPII association. Furthermore, computational calculation provides insights into the excitation energy transfer pathways. Our study lays a solid structural foundation for understanding the light-energy capture and transfer in cryptophyte PSII-ACPII, evolutionary variations in PSII-LHCII, and the origin of red-lineage LHCIIs.

Gilvimarinus xylanilyticus sp. nov., a novel 1,3-xylanase-secreting bacterium isolated from a marine green alga.

1,3-xylan, an important organic carbon in the ocean, is peculiar to marine algae. 1,3-xylanase-secreting bacteria and their extracellular 1,3-xylanases play pivotal roles in the degradation and biomass conversion of 1,3-xylan. However, only a few 1,3-xylanase-secreting bacteria and 1,3-xylanases have been reported. Here, we identified a novel marine bacterium capable of secreting 1,3-xylanases, designated as strain HB14T. Phylogenetic analysis revealed that strain HB14T clustered tightly with known species of the genus Gilvimarinus, showing the highest 16S rRNA gene sequence similarity (97.7%) with the type strain of Gilvimarinus chinensis. Based on phylogenetic, genomic, chemotaxonomic and phenotypic studies, strain HB14T was classified as a representative of a novel species in the genus Gilvimarinus, for which the name Gilvimarinus xylanilyticus sp. nov. was proposed. The type strain is HB14T (=CCTCC AB 2022109T = KCTC 92379T). Four 1,3-xylanases secreted by strain HB14T were identified based on genome and secretome analyses, and the two (Xyn65 and Xyn80) with relatively higher abundance in secretome were successfully expressed in Escherichia coli and biochemically characterized. They showed the highest activity at pH 6.0-7.0 and 40°C and released mainly 1,3-xylobiose and 1,3-xylotriose from 1,3-xylan. These data suggest that strain HB14T acts as a player in marine 1,3-xylan degradation and recycling and that its extracellular 1,3-xylanases may have a good potential in 1,3-xylooligosaccharides preparation.

A preliminary report of exploration of the exosomal shuttle protein in marine invertebrate Chlamys farreri.

Exosomes are extracellular vesicles secreted by diverse cell under normal or abnormal physiological conditions, which could carry a range of bioactive molecules and play significant roles in biological processes, such as intercellular communication and immune response. In the current study, a preliminary study was performed to investigate the exosomal shuttle protein in Chlamys farreri (designated as CfesPro) and to predict the potential function of exosomes in scallop innate immunity. The serum derived exosomes (designated as CfEVs) were obtained from lipopolysaccharide (LPS)-stimulated C. farreri and untreated ones. After confirmation and characterization by transmission electron microscopy (TEM), nano-HPLC-MS/MS spectrometry was performed on CfEVs using a label-free quantitative method. Totally 2481 exosomal shuttle proteins were identified in CfEVs proteomic data, which included many innate immune related proteins. GO and KOG functional annotation showed that CfesPro participated in cellular processes, metabolism reactions, signaling transductions, immune responses and so on. Moreover, 1421 proteins in CfesPro were enriched to 324 pathways by KEGG analysis, including several immune-related pathways, such as autophagy, apoptosis and lysosome pathway. Meanwhile, eight autophagy-related proteins were initially identified in CfesPro, indicating that CfEVs had a potential role with autophagy. All these findings showed that CfEVs were involved in C. farreri innate immune defenses. This research would enrich the protein database of marine exosomes and provide a basis for the exploration of immune defense systems in marine invertebrates.

Proteomic analysis of exosomes in pacific oyster Crassostrea gigas during bacterial stimulation.

Exosomes are 30-150 nm-sized extracellular vesicles of endocytic origin that are released into the extracellular environment and play roles in cell-cell communication. Accumulating research achievements demonstrated that exosomes could act as innate immune effectors that contribute to the host defense mechanism. To better understand the immune functions of exosomes in Crassostrea gigas against bacterial stimulation, the iTRAQ LC-MS/MS approach was applied to identifying differentially expressed proteins (DEPs) of exosomes in oyster post Staphylococcus aureus and Vibrio splendidus stimulation. A total of 9467 unique peptides corresponding to 1634 proteins were identified. Among them, 99 proteins were upregulated and 152 were downregulated after S. aureus infection. After V. splendidus infection, 431 proteins were identified as differentially abundant, including 76 that were upregulated and 355 were downregulated. Several proteins related to apoptosis, including E3 ubiquitin-protein ligase, eukaryotic translation initiation factor 3, and protein kinase C delta type were found up-regulated in the S. aureus stimulation group, indicating that the apoptosis process was involved in the response to S. aureus stimulation. Thirty up-regulated and 123 down-regulated proteins were identified as differentially abundant after both bacterial stimuli. Among them, some proteins related to the actin-myosin cytoskeleton process were down-regulated, indicating that phagocytosis may be inhibited in both bacterial stimuli. This study would enrich the C. gigas proteome database and provide information for further understanding the immune functions of oyster exosomes against bacterial infection.

Biogeography of culturable marine bacteria from both poles reveals that 'everything is not everywhere' at the genomic level.

Based on 16S rRNA gene analyses, the same bacterial operational taxonomic units (OTUs) are common to both the Arctic and Antarctic oceans, supporting the concept 'everything is everywhere'. However, whether the same OTUs from both poles have identical genomes, i.e. whether 'everything is still everywhere' at the genomic level has not yet been examined systematically. Here, we isolated, sequenced and compared the genomes of 45 culturable marine bacteria belonging to three genera of Salinibacterium, Psychrobacter and Pseudoalteromonas from both polar oceans. The bacterial strains with identical 16S rRNA genes were common to both poles in every genus, and four identical genomes were detected in the genus Salinibacterium from the Arctic region. However, no identical genomes were observed from opposite poles in this study. Our data, therefore, suggest that 'everything is not everywhere' at the genomic level. The divergence time between bacteria is hypothesized to exert a strong impact on the bacterial biogeography at the genomic level. The geographical isolation between poles was observed for recently diverged, highly similar genomes, but not for moderately similar genomes. This study thus improves our understanding of the factors affecting the genomic-level biogeography of marine microorganisms isolated from distant locations.

Extracellular Enzyme Activity and Its Implications for Organic Matter Cycling in Northern Chinese Marginal Seas.

Extracellular enzymes, initiating the degradation of organic macromolecules, are important functional components of marine ecosystems. Measuring in situ seawater extracellular enzyme activity (EEA) can provide fundamental information for understanding the biogeochemical cycling of organic matter in the ocean. Here we investigate the patterns of EEA and the major factors affecting the seawater EEA of Chinese marginal seas. The geographic distribution of EEA along a latitudinal transect was examined and found to be associated with dissolved organic carbon. Compared with offshore waters, inshore waters had higher enzyme activity. All the tested substrates were hydrolyzed at different rates and phosphatase, ß-glucosidase and protease contributed greatly to summed hydrolysis rates. For any particular enzyme activity, the contribution of dissolved to total EEA was strongly heterogenous between stations. Comparisons of hydrolysis rates of the polymers and their corresponding oligomers suggest that molecule size does not necessarily limit the turnover of marine organic matter. In addition, several typical enzyme-producing clades, such as Bacteroidetes, Planctomycetes, Chloroflexi, Roseobacter, Alteromonas, and Pseudoalteromonas, were detected in the in situ environments. These enzyme-producing clades may be responsible for the production of different enzymes. Overall, each enzyme was found to flexibly respond to environmental conditions and were linked to microbial community composition. It is likely that this activity will profoundly affect organic matter cycling in the Chinese marginal seas.

Development of a Cold-Adapted Pseudoalteromonas Expression System for the Pseudoalteromonas Proteins Intractable for the Escherichia coli System.

Although the Escherichia coli expression system is the most commonly used expression system, some proteins are still difficult to be expressed by this system, such as proteins with high thermolability and enzymes that cannot mature by autoprocessing. Therefore, it is necessary to develop alternative expression systems. In this study, a cold-adapted Pseudoalteromonas expression system was developed. A shuttle vector was constructed, and a conjugational transfer system between E. coli and psychrophilic strain Pseudoalteromonas sp. SM20429 was established. Based on the shuttle vector, three reporter vectors were constructed to compare the strength of the cloned promoters at low temperature. The promoter of xylanase gene from Pseudoalteromonas sp. BSi20429 was chosen due to its high activity at 10-15°C. An expression vector pEV containing the chosen promoter, multiple cloning sites and a His tag was constructed for protein expression and purification. With pEV as expression vector and SM20429 as the host, a cold-adapted protease, pseudoalterin, which cannot be maturely expressed in E. coli, was successfully expressed as an active extracellular enzyme when induced by 2% oat spelt xylan at 15°C for 48 h. Recombinant pseudoalterin purified from the culture by Ni affinity chromatography had identical N-terminal sequence, similar molecular mass and substrate specificity as the native pseudoalterin. In addition, another two cold-adapted enzymes were also successfully expressed by this system. Our results indicate that this cold-adapted Pseudoalteromonas expression system will provide an alternative choice for protein expression, especially for the Pseudoalteromonas proteins intractable for the E. coli system.

Filamentous phages prevalent in Pseudoalteromonas spp. confer properties advantageous to host survival in Arctic sea ice.

Sea ice is one of the most frigid environments for marine microbes. In contrast to other ocean ecosystems, microbes in permanent sea ice are space confined and subject to many extreme conditions, which change on a seasonal basis. How these microbial communities are regulated to survive the extreme sea ice environment is largely unknown. Here, we show that filamentous phages regulate the host bacterial community to improve survival of the host in permanent Arctic sea ice. We isolated a filamentous phage, f327, from an Arctic sea ice Pseudoalteromonas strain, and we demonstrated that this type of phage is widely distributed in Arctic sea ice. Growth experiments and transcriptome analysis indicated that this phage decreases the host growth rate, cell density and tolerance to NaCl and H2O2, but enhances its motility and chemotaxis. Our results suggest that the presence of the filamentous phage may be beneficial for survival of the host community in sea ice in winter, which is characterized by polar night, nutrient deficiency and high salinity, and that the filamentous phage may help avoid over blooming of the host in sea ice in summer, which is characterized by polar day, rich nutrient availability, intense radiation and high concentration of H2O2. Thus, while they cannot kill the host cells by lysing them, filamentous phages confer properties advantageous to host survival in the Arctic sea ice environment. Our study provides a foremost insight into the ecological role of filamentous phages in the Arctic sea ice ecosystem.

Comparative genomics of the marine bacterial genus Glaciecola reveals the high degree of genomic diversity and genomic characteristic for cold adaptation.

To what extent the genomes of different species belonging to one genus can be diverse and the relationship between genomic differentiation and environmental factor remain unclear for oceanic bacteria. With many new bacterial genera and species being isolated from marine environments, this question warrants attention. In this study, we sequenced all the type strains of the published species of Glaciecola, a recently defined cold-adapted genus with species from diverse marine locations, to study the genomic diversity and cold-adaptation strategy in this genus.The genome size diverged widely from 3.08 to 5.96 Mb, which can be explained by massive gene gain and loss events. Horizontal gene transfer and new gene emergence contributed substantially to the genome size expansion. The genus Glaciecola had an open pan-genome. Comparative genomic research indicated that species of the genus Glaciecola had high diversity in genome size, gene content and genetic relatedness. This may be prevalent in marine bacterial genera considering the dynamic and complex environments of the ocean. Species of Glaciecola had some common genomic features related to cold adaptation, which enable them to thrive and play a role in biogeochemical cycle in the cold marine environments.

Puniceibacterium antarcticum gen. nov., sp. nov., isolated from seawater.

A Gram-reaction-negative, aerobic, non-flagellated, rod-shaped bacterium, designated strain SM1211T, was isolated from Antarctic seawater. The isolate grew at 4-35 °C and with 0-10% (w/v) NaCl. It could produce bacteriochlorophyll a, but did not reduce nitrate to nitrite or hydrolyse DNA. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain SM1211T constituted a distinct phylogenetic line within the family Rhodobacteraceae and was closely related to species in the genera Litorimicrobium, Leisingera, Seohaeicola and Phaeobacter with 95.1-96.0% similarities. The predominant cellular fatty acid was C18:1ω7c. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid and two unidentified phospholipids. The genomic DNA G+C content of strain SM1211T was 60.7 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic data obtained in this study, strain SM1211T is considered to represent a novel species in a new genus within the family Rhodobacteraceae, for which the name Puniceibacterium antarcticum gen. nov., sp. nov. is proposed. The type strain of Puniceibacterium antarcticum is SM1211T (=CCTCC AB 2013147T=KACC 16875T).

Development of a genetic system for the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913.

BACKGROUND: Pseudoalteromonas species are a group of marine gammaproteobacteria frequently found in deep-sea sediments, which may play important roles in deep-sea sediment ecosystem. Although genome sequence analysis of Pseudoalteromonas has revealed some specific features associated with adaptation to the extreme deep-sea environment, it is still difficult to study how Pseudoalteromonas adapt to the deep-sea environment due to the lack of a genetic manipulation system. The aim of this study is to develop a genetic system in the deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913, making it possible to perform gene mutation by homologous recombination. RESULTS: The sensitivity of Pseudoalteromonas sp. SM9913 to antibiotic was investigated and the erythromycin resistance gene was chosen as the selective marker. A shuttle vector pOriT-4Em was constructed and transferred into Pseudoalteromonas sp. SM9913 through intergeneric conjugation with an efficiency of 1.8 × 10-3, which is high enough to perform the gene knockout assay. A suicide vector pMT was constructed using pOriT-4Em as the bone vector and sacB gene as the counterselective marker. The epsT gene encoding the UDP-glucose lipid carrier transferase was selected as the target gene for inactivation by in-frame deletion. The epsT was in-frame deleted using a two-step integration-segregation strategy after transferring the suicide vector pMT into Pseudoalteromonas sp. SM9913. The ΔepsT mutant showed approximately 73% decrease in the yield of exopolysaccharides, indicating that epsT is an important gene involved in the EPS production of SM9913. CONCLUSIONS: A conjugal transfer system was constructed in Pseudoalteromonas sp. SM9913 with a wide temperature range for selection and a high transfer efficiency, which will lay the foundation of genetic manipulation in this strain. The epsT gene of SM9913 was successfully deleted with no selective marker left in the chromosome of the host, which thus make it possible to knock out other genes in the same host. The construction of a gene knockout system for Pseudoalteromonas sp. SM9913 will contribute to the understanding of the molecular mechanism of how Pseudoalteromonas adapt to the deep-sea environment.

Diversity of both the cultivable protease-producing bacteria and bacterial extracellular proteases in the coastal sediments of King George Island, Antarctica.

Protease-producing bacteria play a vital role in degrading sedimentary organic nitrogen. However, the diversity of these bacteria and their extracellular proteases in most regions remain unknown. In this paper, the diversity of the cultivable protease-producing bacteria and of bacterial extracellular proteases in the sediments of Maxwell Bay, King George Island, Antarctica was investigated. The cultivable protease-producing bacteria reached 10(5) cells/g in all 8 sediment samples. The cultivated protease-producing bacteria were mainly affiliated with the phyla Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria, and the predominant genera were Bacillus (22.9%), Flavobacterium (21.0%) and Lacinutrix (16.2%). Among these strains, Pseudoalteromonas and Flavobacteria showed relatively high protease production. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria were serine proteases or metalloproteases. These results begin to address the diversity of protease-producing bacteria and bacterial extracellular proteases in the sediments of the Antarctic Sea.

Genome sequence of proteorhodopsin-containing sea ice bacterium Glaciecola punicea ACAM 611T.

Here, we report the draft genome sequence of Antarctic sea ice bacterium Glaciecola punicea ACAM 611(T), the type species of the genus Glaciecola. A blue-light-absorbing proteorhodopsin gene is present in the 3.08-Mb genome. This genome sequence can facilitate the study of the physiological metabolisms and ecological roles of sea ice bacteria.

Characterization of a cryptic plasmid pSM429 and its application for heterologous expression in psychrophilic Pseudoalteromonas.

BACKGROUND: Pseudoalteromonas is an important genus widespread in marine environment, and a lot of psychrophilic Pseudoalteromonas strains thrive in deep sea and polar sea. By now, there are only a few genetic systems for Pseudoalteromonas reported and no commercial Pseudoalteromonas genetic system is available, which impedes the study of Pseudoalteromonas, especially for psychrophilic strains. The aim of this study is to develop a heterologous expression system for psychrophilic Pseudoalteromonas. RESULTS: A cryptic plasmid pSM429 isolated from psychrophilic Pseudoalteromonas sp. BSi20429 from the Arctic sea ice, was sequenced and characterized. The plasmid pSM429 is 3874 bp in length, with a G+C content of 28%. Four putative open reading frames (ORFs) were identified on pSM429. Based on homology, the ORF4 was predicted to encode a replication initiation (Rep) protein. A shuttle vector (Escherichia coli, Pseudoalteromonas), pWD, was constructed by ligating pSM429 and pUC19 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. To determine the minimal replicon of pSM429 and to check the functionality of identified ORFs, various pWD derivatives were constructed. All derivatives except the two smallest ones were shown to allow replication in Pseudoalteromonas sp. SM20429, a plasmid-cured strain of Pseudoalteromonas sp. BSi20429, suggesting that the orf4 and its flanking intergenic regions are essential for plasmid replication. Although not essential, the sequence including some repeats between orf1 and orf2 plays important roles in segregational stability of the plasmid. With the aid of pWD-derived plasmid pWD2, the erythromycin resistance gene and the cd gene encoding the catalytic domain of a cold-adapted cellulase were successfully expressed in Pseudoalteromonas sp. SM20429. CONCLUSIONS: Plasmid pSM429 was isolated and characterized, and the regions essential for plasmid replication and stability were determined, helping the development of pSM429-based shuttle vectors. The shuttle vectors pWD and its derivatives could be used as cloning vectors for Pseudoalteromonas, offering new perspectives in the genetic manipulation of Pseudoalteromonas strains. With the aid of pWD-derived vector and its host, the erythromycin resistance gene and the cd gene of a cold-adapted protein were successfully expressed, indicating that the potential use of this system for recombinant protein production, especially for cold-adapted proteins.

Myroides profundi sp. nov., isolated from deep-sea sediment of the southern Okinawa Trough.

A Gram-negative, nonmotile, aerobic and oxidase- and catalase-positive bacterium, designated D25T, was isolated from the deep-sea sediments of the southern Okinawa Trough area. Phylogenetic analyses of 16S rRNA gene sequences showed that strain D25T fell within the genus Myroides, with 99.2%, 96.0% and 93.4% sequence similarities to the only three recognized species of Myroides. However, the DNA-DNA similarity value between strain D25T and its nearest neighbour Myroides odoratimimus JCM 7460T was only 49.9% (<70%). Several phenotypic properties could be used to distinguish strain D25T from other Myroides species. The main cellular fatty acids of strain D25T were iso-C15:0, iso-C17:1omega9c, iso-C17:03-OH and Summed Feature 3 (comprising C16:1omega7c and/or iso-C15:02-OH). The major respiratory quinone was MK-6. The DNA G+C content was 33.0 mol%. The results of the polyphasic taxonomy analysis suggested that strain D25T represents a novel species of the genus Myroides, for which the name Myroides profundi sp. nov. is proposed. The type strain is D25T (=CCTCC M 208030T=DSM 19823T).

Wangia profunda gen. nov., sp. nov., a novel marine bacterium of the family Flavobacteriaceae isolated from southern Okinawa Trough deep-sea sediment.

An orange-pigmented, Gram-negative, nonmotile, strictly aerobic and oxidase- and catalase-positive bacterium (SM-A87(T)) was isolated from the deep-sea sediment of the southern Okinawa Trough area. The main fatty acids were i15 : 0, i17 : 0 3OH, i15 : 1 G, i17 : 1 omega 9c, 15 : 0, i15 : 0 3OH and summed feature 3 (comprising i-15 : 0 2OH and/or 16 : 1 omega 7c). MK-6 was the predominant respiratory quinone. DNA G+C content was 35.8 mol%. Flexirubin-type pigments were absent. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain SM-A87(T) formed a distinct lineage within the family Flavobacteriaceae, with <93% sequence similarity to the nearest strain of genus Salegentibacter. Moreover, strain SM-A87(T) could be distinguished from the nearest phylogenetic neighbors by a number of chemotaxonomic and phenotypic properties. On the basis of polyphasic analyses, it is proposed that strain SM-A87(T) be classified in a novel genus and a new species in the family Flavobacteriaceae, designated Wangia profunda gen. nov., sp. nov. The type strain is SM-A87(T) (CCTCC AB 206139(T)=DSM 18752).