Deletion of hepatic growth hormone receptor (GHR) alters the mouse gut microbiota by affecting bile acid metabolism.

Both growth hormone (GH) and gut microbiota play significant roles in diverse physiological processes, but the crosstalk between them is poorly understood. Despite the regulation of GH by gut microbiota, study on GH's influence on gut microbiota is limited, especially on the impacts of tissue specific GH signaling and their feedback effects on the host. In this study, we profiled gut microbiota and metabolome in tissue-specific GHR knockout mice in the liver (LKO) and adipose tissue (AKO). We found that GHR disruption in the liver rather than adipose tissue affected gut microbiota. It changed the abundance of Bacteroidota and Firmicutes at phylum level as well as abundance of several genera, such as Lactobacillus, Muribaculaceae, and Parasutterella, without affecting α-diversity. Moreover, the impaired liver bile acid (BA) profile in LKO mice was strongly associated with the change of gut microbiota. The BA pools and 12-OH BAs/non-12-OH BAs ratio were increased in the LKO mice, which was due to the induction of CYP8B1 by hepatic Ghr knockout. Consequently, the impaired BA pool in cecal content interacted with gut bacteria, which in turn increased the production of bacteria derived acetic acid, propionic acid, and phenylacetic acid that were possible to participate in the impaired metabolic phenotype of the LKO mice. Collectively, our findings suggested that the liver GH signaling regulates BA metabolism by its direct regulation on CYP8B1, which is an important factor influencing gut microbiota. Our study is significant in exploring gut microbiota modification effects of tissue-specific GH signaling as well as its involvement in gut microbiota-host interaction.

Exosomal LINC00460/miR-503-5p/ANLN positive feedback loop aggravates pancreatic cancer progression through regulating T cell-mediated cytotoxicity and PD-1 checkpoint.

BACKGROUND: Long non-coding RNA (lncRNA) LINC00460 is an onco-lncRNA in a variety of cancers, including pancreatic cancer (PC). This study is aimed to investigate the regulatory mechanisms of LINC00460 in PC. METHODS: The tumor and adjacent normal tissues were collected from 73 PC patients. The expression of LINC00460, miR-503-5p, and ANLN was detected using qRT-PCR. We then analyzed the proliferation, migration, invasion, and apoptosis/cell cycle of PC cells by performing the MTT/EdU, transwell, and flow cytometry assays, respectively. The xenograft tumor model were utilized to confirm the effect of LINC00460 knockdown on PC through anti-PD-1 therapy in vivo, and the sensitivity of PANC-1 cells to the cytotoxicity of CD8+ T cells in vitro. Western blotting was used to determine the protein levels. A co-culture model was utilized to explore the effects of exosomes on macrophages. RESULTS: LINC00460 was up-regulated in PC tissues and cells. LINC00460 knockdown suppressed cell proliferation, migration, and invasion, facilitated cell apoptosis and G0/G1 phase arrest, and inhibited the tumor growth through anti-PD-1 therapy. Both miR-503-5p down-regulation and ANLN up-regulation reversed the effects of LINC00460 knockdown on inhibiting the proliferation, migration and invasion, and on promoting the apoptosis, G0/G1 phase arrest, and the sensitivity of PC cells to the cytotoxicity of CD8+ T cells. Exosomes were uptaken by the ambient PC cells. PANC-1 cells-derived exosomal LINC00460-induced M2 macrophage polarization accelerates the cell migration and invasion. CONCLUSIONS: LINC00460 silencing attenuates the development of PC by regulating the miR-503-5p/ANLN axis and exosomal LINC00460-induced M2 macrophage polarization accelerates the migration and invasion of PANC-1 cells, thus LINC00460 may act as a possible therapeutic target for treating PC.

Microalgae oil from Schizochytrium sp. alleviates obesity and modulates gut microbiota in high-fat diet-fed mice.

Omega-3 PUFAs rich in fish oil are believed to prevent obesity by improving lipid metabolism and regulating gut microbiota. Microalgae oil is considered as an alternative source of omega-3 PUFAs owing to diminishing fish resources. Schizochytrium microalgae oil (SMO), with a high DHA proportion, is a promising source for commercial DHA production. However, its weight-loss and gut microbiota-regulating properties are not well studied. Here we compared the obesity reducing effects of SMO, commercial fish oil (FO) and a weight-loss drug, Orlistat (OL), in a high-fat diet (HFD) induced obesity mouse model. We found that SMO is comparable to commercial FO and OL with regard to weight loss, and it even exhibits the weight-loss effects earlier than FO and OL. It can efficiently inhibit the expression of lipogenesis-related genes and induce the expression of lipolysis-related genes. Moreover, SMO has different gut microbiota modulating effects from those of FO and OL. It does not influence the diversity of bacterial community, but does increase the abundance of several beneficial SCFAs-producing bacteria and inhibits obesity-promoting Desulfovibrio and several pathogens. We also found that SMO recovers the HFD-disturbed metabolic capability of gut microbiota. It can increase the abundance of several metabolism-related pathways, such as those of amino acids, SCFAs and bile acid, and decrease the level of the LPS biosynthesis pathway, which probably contributes to an improvement of lipid metabolism and restoration of the colonic mucosal barrier impaired by HFD. Our data suggest that SMO can be used as a superior dietary supplement for alleviating obesity.

Dynamics of phytoplankton community in scallop farming waters of the Bohai Sea and North Yellow Sea in China.

BACKGROUND: As the major suppliers of food for higher consumers, phytoplankton are closely related to the yield, nutritional ingredients and even toxin contents of mariculture animals, potentially influencing the human health when they are consumed. With the increase of shellfish culture density, phytoplankton in the coastal waters have been excessively consumed in recent years, and the nutrients they depend on are becoming more and more limited, which severely restrict the shellfish mariculture and threaten the coastal ecosystems. RESULTS: In the present study, nutrient concentrations, phytoplankton assemblages and scallop growth status were investigated in the main bay scallop farming waters of the Bohai Sea (Qinhuangdao site) and North Yellow Sea (Zhuanghe site) in 2018. Both phosphate and silicate limitations were observed at the two sites, with the major determinant of phytoplankton assemblages being silicate in Qinhuangdao and phosphate in Zhuanghe, respectively. The phytoplankton assemblages at the two sites displayed different community structures and succession patterns. The phytoplankton community was dominated by dinoflagellates and diatoms in Qinhuangdao, while dinoflagellates were the most abundant group in Zhuanghe, which accounted for 41.9% of the total phytoplankton abundance. The dominant genera of diatoms in Qinhuangdao were Skeletonema, Thalassiosira and Leptocylindrus, while those in Zhuanghe were Thalassiosira and Cyclotella. Greater biomass and more appropriate structure of phytoplankton contributed to higher growth rate and glycogen content of cultured bay scallops. CONCLUSIONS: Our study characterized the relationship between nutrient concentration, phytoplankton community and scallop mariculture in the main bay scallop farming waters in northern China. The results suggest that, as nutrient limitation intensified, dinoflagellates are becoming the dominant phytoplankton species in the scallop farming waters of the Bohai Sea and the North Yellow Sea, which is harmful to the coastal mariculture.

Ajuba functions as a co-activator of C/EBPß to induce expression of PPARγ and C/EBPα during adipogenesis.

Adipogenesis is regulated by a complicated network of transcription factors among which PPARγ and C/EBP family members are the major regulators. During adipogenesis, C/EBPß is induced early and then transactivates PPARγ and C/EBPα, which cooperatively induce genes whose expressions give rise to the mature adipocyte phenotype. Identifying the factors that influence the expression and activity of C/EBPß should provide additional insight into the mechanisms regulating adipogenesis. Here, we demonstrate that depletion of Ajuba in 3T3-L1 cells significantly decreases mRNA and protein levels of PPARγ and C/EBPα and impairs adipocyte differentiation, while overexpression increases expression of these genes and promotes adipocyte differentiation. Moreover, restoration of C/EBPα or PPARγ expression in Ajuba-deficient 3T3-L1 cells improves the impaired lipid accumulation. Mechanistically, Ajuba interacts with C/EBPß and recruits CBP to facilitate the binding of C/EBPß to the promoter of PPARγ and C/EBPα, resulting in increased H3 histone acetylation and target gene expression. Collectively, these data indicate that Ajuba functions as a co-activator of C/EBPß, and may be an important therapeutic target for combating obesity-related diseases.

Distinct microbiota assembly mechanisms revealed in different reconstruction stages during gut regeneration in the sea cucumber Apostichopus japonicus.

Apostichopus japonicus is a useful model for studying organ regeneration, and the gut microbiota is important for host organ regeneration. However, the reconstruction process and the mechanisms of gut microbiota assembly during gut regeneration in sea cucumbers have not been well studied. In the present study, gut regeneration was induced (via evisceration) in A. japonicus, and gut immune responses and bacterial diversity were investigated to reveal gut microbiota assembly and its possible mechanisms during gut regeneration. The results revealed that bacterial community reconstruction involved two stages with distinct assembly mechanisms, where the reconstructed community was initiated from the bacterial consortium in the residual digestive tract and tended to form a novel microbiota in the later stage of reconstruction. Together, the results of immunoenzyme assays, community phylogenetic analysis, and source tracking suggested that the host deterministic process was stronger in the initial stage than in the later stage. The bacterial interactions that occurred were significantly different between the two stages. Positive interactions dominated in the initial stage, while more complex and competitive interactions developed in the later stage. Such a dynamic bacterial community could provide the host with energetic and immune benefits that promote gut regeneration and functional recovery. The results of the present study provide insights into the processes and mechanisms of gut microbiota assembly during intestinal regeneration that are valuable for understanding gut regeneration mechanisms mediated by the microbiota.

Long noncoding RNA KIF9-AS1 promotes cell apoptosis by targeting the microRNA-148a-3p/suppressor of cytokine signaling axis in inflammatory bowel disease.

OBJECTIVE: Inflammatory bowel disease (IBD) is a chronic intestinal disease. This study was attempted to investigate the effects of long noncoding RNA KIF9-AS1 (KIF9-AS1) on the development of IBD and its underlying mechanism of action. METHODS: Quantitative real time PCR (qRT-PCR) was implemented to examine the expression of KIF9-AS1 and microRNA-148a-3p (miR-148a-3p). The IBD mouse model was induced by dextran sulfate sodium (DSS). The body weight, disease activity index (DAI) score, colon length and histological injury were used to evaluate the colon injury. The levels of proinflammatory cytokines were measured by ELISA. In vitro, IBD was simulated by DSS treatment in colonic cells. Then the apoptosis of colonic cells was detected by flow cytometry assay. Furthermore, a dual-luciferase reporter assay was used to demonstrate the interactions among KIF9-AS1, miR-148a-3p and suppressor of cytokine signaling (SOCS3). RESULTS: KIF9-AS1 expression was upregulated in IBD patients, DSS-induced IBD mice and DSS-induced colonic cells, whereas miR-148a-3p expression was downregulated. KIF9-AS1 silencing attenuated the apoptosis of DSS-induced colonic cells in vitro and alleviated colon injury and inflammation in DSS-induced IBD mice in vivo. Additionally, the mechanical experiment confirmed that KIF9-AS1 and SOCS3 were both targeted by miR-148a-3p with the complementary binding sites at 3'UTR. Moreover, miR-148a-3p inhibition or SOCS3 overexpression reversed the suppressive effect of KIF9-AS1 silencing on the apoptosis of DSS-induced colonic cells. CONCLUSION: KIF9-AS1 silencing hampered the colon injury and inflammation in DSS-induced IBD mice in vivo, and restrained the apoptosis of DSS-induced colonic cells by regulating the miR-148a-3p/SOCS3 axis in vitro, providing a new therapeutic target for IBD.

Close homolog of L1-deficient ameliorates inflammatory bowel disease by regulating the balance of Th17/Treg.

OBJECTIVE: The aim of this study is to investigate the role of close homolog of L1 (CHL1) on inflammatory bowel disease (IBD), and the correlation with the balance of Th17/Treg. METHODS: Dextran sodium sulfate (DSS)-induced IBD mice model was established. CHL1 knockout (KO) mice and CHL1 wild-type (WT) mice were subjected to DSS. CHL1 expression was detected using qRT-PCR. Weight was recorded daily, and disease activity index (DAI) score was assessed. The colon length and histological changes were measured. The number of neutrophils, macrophages and T cells was observed by immunohistochemistry. The expression of inflammatory cytokines and the proportion of Th17/Treg cells were detected by qRT-PCR and flow cytometry. The expression of RORγt, STAT3 and Foxp3 was detected by using immunohistochemistry and Western blot. RESULTS: CHL1 expression was upregulated in DSS-induced IBD mice. DSS-CHLl-KO mice exhibited less weight loss than the DSS-CHLl-WT mice. The DAI score and histological score were decreased in DSS-CHLl-KO mice compared with DSS-CHLl-WT mice, while colon length was increased. Number of neutrophils, macrophages and T cells, and expression of TNF-α, IL-6, IL-17A, IL-21 and IL-23 were decreased in DSS-CHLl-KO mice, while IL-10 expression was increased. Moreover, CHL1-deficient inhibited Th17 cells differentiation and promoted Treg cells differentiation in IBD mice. CHL1-deficient also inhibited the expression of RORγt and STAT3, and promoted the expression of Foxp3 in IBD mice. CONCLUSION: CHL1-deficient reduces the inflammatory response by regulating the balance of Th17/Treg in mice with IBD. CHL1 is expected to be a new target for the treatment of IBD.

miR-802 participates in the inflammatory process of inflammatory bowel disease by suppressing SOCS5.

The present study aims to reveal the detailed molecular mechanism of microRNA (miR)-802 in the progression of inflammatory bowel disease (IBD). IBD tissues were obtained from IBD patients, followed by CD4+ cells isolation. Then, qRT-PCR and ELISA were used to detect the expression of miR-802, suppressor of cytokine signaling 5 (SOCS5), interleukin (IL)-17A and tumor necrosis factor (TNF)-α. Transfection of miR-802 mimics and miR-802 inhibitor in CD4+ cells was detected by Western blot. TargetScan and luciferase reporter assay were used to detect the relationship between SOCS5 and miR-802. Finally, colitis mice model was established to verify whether miR-802 inhibitor was involved in the protective effect of colonic mucosa. The miR-802 was highly expressed in inflamed mucosa and PBMC cells of IBD. The highest expression of miR-802 was observed in CD4+ T cells based on different immune cell subsets analysis. SOCS5 was the target gene of miR-802. The mice model experiments showed that blockade of miR-802 could alleviate mice colitis. Our study suggests that up-regulation of miR-802 plays an important role in inflammatory process of IBD via targeting SOCS5. Moreover, the differentiation of Th17 and secretion of TNF-α in IBD could be stimulated by miR-802.

Beyond the state/market dichotomy: Institutional innovations in China's electricity industry reform.

China's electricity industry has gone through three decades of market-oriented reform, and this process is often interpreted as a tug of war between marketization and state control. However, this paper argues that a dichotomous conception of the state-market relationship cannot explain the evolution and complexity of China's electricity governance. By systematically analyzing the institutions of China's electricity reform, this paper identifies a sequence of innovations that align well with both the history of China's economic transition and the institutional underpinnings of China's economic governance. Also, by interpreting the respective roles of state and market in China's reformed electricity industry, this paper shows that state control and marketization are complementary rather than contradictory, and that their complementarity is best exercised in addressing issues like market fragmentation and renewable energy curtailment. Moreover, this paper presents two case studies - both on China's most recent electricity market experimentation - to elaborate how state and market jointly serve sectoral functions as well as broader economic and political goals.

A tandem-repeat galectin-1 from Apostichopus japonicus with broad PAMP recognition pattern and antibacterial activity.

Galectins belong to the family of carbohydrate-binding proteins and play major roles in the immune and inflammatory responses of both vertebrates and invertebrates. In the present study, one novel galectin-1 protein named AjGal-1 was identified from Apostichopus japonicas with an open reading frame of 1179 bp encoding a polypeptide of 392 amino acids. The deduced amino acids sequence of AjGal-1 contained three carbohydrate recognition domains (CRDs) which shared 34-37% identity with that of other galectin proteins from echinodermata, fishes, and birds. In the phylogenetic tree, AjGal-1 was closely clustered with galectins from Mesocentrotus nudus and Paracentrotus lividus. The mRNA transcripts of AjGal-1 were ubiquitously expressed in all the detected tissues, including gut, longitudinal muscle, gonad, coelomocytes, respiratory tree, tentacle and body wall, with the highest expression level in coelomocytes. After Vibrio splendidus stimulation, the mRNA expression levels of AjGal-1 in coelomocytes were significantly increased at 6 and 12 h (P < 0.01) compared with that in control group, and went back to normal level at 72 h. The recombinant protein of AjGal-1 (rAjGal-1) could bind various PAMPs including d-galactose, lipopolysaccharide (LPS), peptidoglycan (PGN) and mannose (Man), and exhibited the highest affinity to d-galactose. Meanwhile, rAjGal-1 could also bind and agglutinate different kinds of microorganisms, including gram-negative bacteria (V. splendidus and Escherichia coli), gram-positive bacteria (Micrococus leteus), and fungi (Pichia pastoris). rAjGal-1 also exhibited anti-microbial activity against V. splendidus and E. coli. All these results suggested that AjGal-1 could function as an important PRR with broad spectrum of microbial recognition and anti-microbial activity against the invading pathogen in A. japonicas.

The differences of bacterial communities in the tissues between healthy and diseased Yesso scallop (Patinopecten yessoensis).

The tissues of marine invertebrates are colonized by species-rich microbial communities. The dysbiosis of host's microbiota is tightly associated with the invertebrate diseases. Yesso scallop (Patinopecten yessoensis), one of the most important maricultured scallops in northern China, has recently suffered massive summer mortalities, which causes huge production losses. The knowledge about the interactions between the Yesso scallop and its microbiota is important to develop the strategy for the disease prevention and control. In the present study, the bacterial communities in hemolymph, intestine, mantle and adductor muscle were compared between the healthy and diseased Yesso scallop based on the high-throughput sequencing of 16S rRNA gene. The results indicated obvious difference of the composition rather than the diversity of the bacterial communities between the healthy and diseased Yesso scallop. Vibrio, Francisella and Photobacterium were found to overgrow and dominate in the mantle, adductor muscle and intestine of the diseased scallops, respectively. The prediction of bacterial community metagenomes and the variations of KEGG pathways revealed that the proportions of the pathways related with neurodegenerative diseases and carbohydrate metabolism both increased significantly in the mantle and hemolymph of the diseased scallops. The abundance of the metabolism pathways including carbohydrate metabolism, lipid metabolism and amino acid metabolism decreased significantly in the intestine of diseased scallops. The results suggested that the changes of bacterial communities might be closely associated with the Yesso scallop's disease, which was helpful for further investigation of the pathogenesis as well as prevention and control of the disease in Yesso scallop.

A novel caspase-associated recruitment domain (CARD) containing protein (CgCARDCP-1) involved in LPS recognition and NF-κB activation in oyster (Crassostrea gigas).

Caspase-associated recruitment domain (CARD) containing proteins play critical roles in molecular interaction and regulation of various signaling pathways, such as the activation of caspase and NF-κB singling pathway in the process of apoptosis or inflammation. In the present study, a novel CARD containing protein (designed CgCARDCP-1) was identified and characterized from oyster Crassostrea gigas. Molecular feature analysis revealed that, the open reading frame (ORF) of CgCARDCP-1 gene was 759 bp encoding a polypeptide of 253 amino acids with a conserved N-terminal CARD domain and two transcriptional coactivator p15 (PC4) domains in C-terminus. Homologous alignment showed that the amino acid sequence of CgCARDCP-1 shared 30%-46% identity with that of caspase-2. By RT-PCR detection, the mRNA transcripts of CgCARDCP-1 were found to be widely distributed in various tissues of oyster with the highest expression level in hemocytes and mantle. And CgCARDCP-1 protein was mostly distributed in the cytoplasm of oyster hemocytes as shown by immunohistochemistry. Moreover, the CgCARDCP-1 mRNA expression level in hemocytes was significantly up-regulated after lipopolysaccharide (LPS) and Vibrio splendidus stimulations. The recombinant CgCARDCP-1 displayed strong binding activity with LPS in vitro. In addition, after transfected into the HEK-293T cell with luciferase reporter system, CgCARDCP-1 could significantly promote the NF-κB activation (1.29-fold, p < 0.05) compared to that in the control group. These results collectively demonstrated that the CgCARDCP-1 might serve as a recognition molecule for LPS and a regulator of NF-κB activation in the immune response of oyster.

A GTP-dependent Phosphoenolpyruvate Carboxykinase from Crassostrea gigas Involved in Immune Recognition.

Phosphoenolpyruvate carboxykinase (PEPCK) is well known as a key enzyme involved in the metabolic pathway of gluconeogenesis in organisms, but the information about its involvement in immune response is still very limited. In the present study, a novel PEPCK homolog named CgPEPCK was identified from oyster Crassostrea gigas. The deduced amino acid sequence of CgPEPCK shared 52%-74% similarities with those from other known PEPCKs. There were one conserved guanosine triphosphate (GTP) binding site, one substrate binding site, one metal binding site and one active site in CgPEPCK. The mRNA transcripts of CgPEPCK were constitutively expressed in all the tested tissues including hemolymph, mantle, gill, muscle, gonad and hepatopancreas. CgPEPCK proteins were mainly distributed in adductor muscle, gonad, gill and mantle, and rarely detected in hepatopancreas by using immunohistochemical analysis. After the stimulations with lipopolysaccharide (LPS), peptidoglycan (PGN), Vibrio splendidus and V. anguillarum, CgPEPCK transcripts in hemocytes were significantly up-regulated and peaked at 6 h (LPS, 9.62-fold, p < 0.01), 9 h (PGN, 4.25-fold, p < 0.01), 12 h (V. splendidus, 5.72-fold, p < 0.01), 3 h (V. anguillarum, 2.87-fold, p < 0.01), respectively. The recombinant CgPEPCK protein (rCgPEPCK) exhibited Mn2+/Mg2+ dependent GTP binding activity, and the activities to bind LPS and PGN, but not ß-1,3-glucan (GLU), lipoteichoic acid (LTA), mannan (MAN) nor polyinosinic-polycytidylic (Poly I: C). It could also bind Escherichia coli, Staphylococcus aureus, Micrococcus luteus and significantly inhibit their growth. All these results collectively suggested that CgPEPCK could not only exert GTP binding activity involved in gluconeogenesis, but also mediate the bacteria recognition and clearance in immune response of oysters.

The self-activation and LPS binding activity of executioner caspase-1 in oyster Crassostrea gigas.

Executioner caspases play important roles in apoptotic pathway and immune defense, which is considered to coordinate the execution phase of apoptosis by cleaving multiple structural and repair proteins. However, the knowledge about the activation mechanism and function of executioner caspases in mollusks, especially marine bivalves is limited. In the present study, the full-length cDNA sequence of caspase-1 was cloned from oyster Crassostrea gigas, which encoded a predicted protein containing a small subunit (p10) and large subunit (p20) with a conserved caspase active site QACRG similar to that of human executioner caspase-3/7. SDS-polyacrylamide gel electrophoresis and western blot results demonstrated that the CgCaspase-1 zymogen could be cleaved into p20p10, p20 and p10 in prokaryotic expression systems, and the C-terminus of CgCaspase-1 was also cleaved into p20 and p10. Both of the recombinant CgCaspase-1 (rCgCaspase-1) and the C-terminus of CgCaspase-1 (rCgCaspase-1-C) exhibited similar caspase activity towards proteolytic substrate Ac-DMQD-pNA and Ac-DEVD-pNA. However, the recombinant N-terminus of CgCaspase-1 (rCgCaspase-1-N) did not display any caspase activity. Moreover, the inhibitor of both caspase-3/7 and pan-caspase could significantly inhibit the proteolytic activity of rCgCaspase-1. The strong binding activities towards lipopolysaccharide (LPS) of both rCgCaspase-1 and rCgCaspase-1-C were revealed by ELISA techniques and western blotting. A high level of CgCaspase-1 mRNA transcripts was detected in the gills and hemocytes by quantitative real-time PCR, and the CgCaspase-1 protein was mainly located in the cytoplasm of oyster hemocytes by immunofluorescence assay. These results collectively suggested that CgCaspase-1 was a homolog of executioner caspase-3/7, which could be self-activated through proteolytic cleavage in prokaryotic expression systems, and performed caspase and LPS binding activities in the innate immune response of oyster.

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.

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.

Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas.

Pseudoalteromonas is commonly found throughout the world's oceans, and has gained increased attention due to the ecological and biological significance. Although over fifty Pseudoalteromonas genomes have been sequenced with an aim to explore the adaptive strategies in different habitats, in vivo studies are hampered by the lack of effective genetic manipulation systems for most strains in this genus. Here, nine Pseudoalteromonas strains isolated from different habitats were selected and used as representative strains to develop a universal genetic manipulation system. Erythromycin and chloramphenicol resistance were chosen as selection markers based on antibiotics resistance test of the nine strains. A conjugation protocol based on the RP4 conjugative machinery in E. coli WM3064 was developed to overcome current limitations of genetic manipulation in Pseudoalteromonas. Two mobilizable gene expression shuttle vectors (pWD2-oriT and pWD2Ery-oriT) were constructed, and conjugation efficiency of pWD2-oriT from E. coli to the nine Pseudoalteromonas strains ranged from 10(-6) to 10(-3) transconjugants per recipient cells. Two suicide vectors, pK18mobsacB-Cm and pK18mobsacB-Ery (with sacB for counter-selection), were constructed for gene knockout. To verify the feasibility of this system, we selected gene or operon that may lead to phenotypic change once disrupted as targets to facilitate in vivo functional confirmation. Successful deletions of two genes related to prodigiosin biosynthesis (pigMK) in P. rubra DSM 6842, one biofilm related gene (bsmA) in P. sp. SM9913, one gene related to melanin hyperproduction (hmgA) in P. lipolytica SCSIO 04301 and two flagella-related genes (fliF and fliG) in P. sp. SCSIO 11900 were verified, respectively. In addition, complementation of hmgA using shuttle vector pWD2-oriT was rescued the phenotype caused by deletion of chromosomal copy of hmgA in P. lipolytica SCSIO 04301. Taken together, we demonstrate that the vectors and the conjugative protocol developed here have potential to use in various Pseudoalteromonas strains.

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.

Oceanisphaera profunda sp. nov., a marine bacterium isolated from deep-sea sediment, and emended description of the genus Oceanisphaera.

A Gram-stain-negative, aerobic, oxidase- and catalase-positive, flagellated, rod-shaped bacterial strain, designated SM1222(T), was isolated from the deep-sea sediment of the South China Sea. The strain grew at 4-35 °C and with 0.5-8 % NaCl (w/v). Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain SM1222(T) was affiliated with the genus Oceanisphaera in the class Gammaproteobacteria. It shared the highest sequence similarity with the type strain of Oceanisphaera ostreae (96.8 %) and 95.4-96.6 % sequence similarities with type strains of other species of the genus Oceanisphaera with validly published names. Strain SM1222(T) contained summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), C18 : 1ω7c, C16 : 0, C12 : 0 and summed feature 2 (C14 : 0 3-OH and/or iso-C16 : 1 I) as the major fatty acids and ubiquinone Q-8 as the predominant respiratory quinone. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The genomic DNA G+C content of strain SM1222(T) was 51.5 mol%. On the basis of the evidence presented in this study, strain SM1222(T) represents a novel species of the genus Oceanisphaera, for which the name Oceanisphaera profunda sp. nov. is proposed. The type strain of Oceanisphaera profunda is SM1222(T) ( = CCTCC AB 2013241(T) = KCTC 32510(T)). An emended description of the genus Oceanisphaera Romanenko et al. 2003 emend. Choi et al. 2011 is also proposed.