Transcriptome analysis of the hepatopancreas from the Litopenaeus vannamei infected with different flagellum types of Vibrio alginolyticus strains.

Vibrio alginolyticus, one of the prevalently harmful Vibrio species found in the ocean, causes significant economic damage in the shrimp farming industry. Its flagellum serves as a crucial virulence factor in the invasion of host organisms. However, the processes of bacteria flagella recognition and activation of the downstream immune system in shrimp remain unclear. To enhance comprehension of this, a ΔflhG strain was created by in-frame deletion of the flhG gene in V. alginolyticus strain HN08155. Then we utilized the transcriptome analysis to examine the different immune responses in Litopenaeus vannamei hepatopancreas after being infected with the wild type and the mutant strains. The results showed that the ΔflhG strain, unlike the wild type, lost its ability to regulate flagella numbers negatively and displayed multiple flagella. When infected with the hyperflagella-type strain, the RNA-seq revealed the upregulation of several immune-related genes in the shrimp hepatopancreas. Notably, two C-type lectins (CTLs), namely galactose-specific lectin nattectin and macrophage mannose receptor 1, and the TNF receptor-associated factor (TRAF) 6 gene were upregulated significantly. These findings suggested that C-type lectins were potentially involved in flagella recognition in shrimp and the immune system was activated through the TRAF6 pathway after flagella detection by CTLs.

Characterization of exogenous lactate addition on the growth, photosynthetic performance, and biochemical composition of four bait microalgae strains.

AIMS: To quickly obtain the biomass of bait microalgae with high value-added products, researchers have examined the influence of biochemical and environmental factors on the growth rates and biochemical composition of microalgae. Previous studies have shown that lactate plays an important role in metabolic regulation in Phaeodactylum tricornutum. In this study, we investigated the effect of exogenous lactate on the growth rates, photosynthetic efficiency, and biochemical composition of four commonly used bait microalgae in aquaculture. METHODS AND RESULTS: The optical density of the algal cultures at specific time points, YII, Fv/Fm, and the total lipid, protein, soluble sugar, insoluble sugar, chlorophyll a, and carotenoid content of P. tricornutum, Isochrysis galbana (I. galbana), Chaetoceros muelleri, and Cylindrotheca fusiformis were determined. In I. galbana, the growth rate was enhanced with the addition of lactate, even though higher concentrations of lactate were associated with a decrease in YII and Fv/Fm. In general, the total lipid content of these microalgal strains increased gradually in a concentration-dependent manner over the range of lactate concentrations. In addition, higher concentrations of lactate also induced significant changes in the total soluble and insoluble sugar levels in all microalgal strains. However, chlorophyll a and carotenoid contents increased at lower but decreased at higher concentrations of lactate in all microalgal strains. The total protein content was significantly elevated at all concentrations of lactate in P. tricornutum, whereas there were no significant differences in that of C. fusiformis. CONCLUSIONS: Lactate effective influences in the growth, metabolism, and synthesis of important biochemical components in the four microalgal strains under investigation.

Screening of bioflocculant and cellulase-producing bacteria strains for biofloc culture systems with fiber-rich carbon source.

The biofloc technology (BFT) system has been widely applied in the shrimp and fish culture industry for its advantages in water-saving, growth improvement, and water quality purification. However, The BFT system usually takes a long time to establish, and the extra carbon source input increases the maintenance cost of the system. In this study, we aimed to develop a low-cost and high-efficient BFT system for Litopenaeus vannamei by applying bacteria that could promote the formation of BFT and utilize cheap carbon sources. Three bioflocculant-producing bacteria strains (M13, M15, and M17) have been screened from a cellulolytic strain collection. All three strains have been identified as Bacillus spp. and can use sugarcane bagasse (SB) as a carbon source, which is a cheap byproduct of the sucrose industry in the tropic area of China. Compared to sucrose, the addition of SB and the three strains could improve the biofloc formation rate, biofloc size distribution, ammonia removal rate, and the growth performance of the shrimps. These results suggest that the bioflocculant and cellulase-producing bacteria strains could promote the biofloc formation and the growth of shrimps by using SB as an economic substitute carbon source in the BFT shrimp culture system.

Prevalence of Bacterial Coinfections with Vibrio harveyi in the Industrialized Flow-through Aquaculture Systems in Hainan Province: A Neglected High-Risk Lethal Causative Agent to Hybrid Grouper.

Vibrio harveyi is one of the most serious bacterial pathogens to aquatic animals worldwide. Evidence is mounting that coinfections caused by multiple pathogens are common in nature and can alter the severity of diseases in marine animals. However, bacterial coinfections involving V. harveyi have received little attention in mariculture. In this study, the results of pathogen isolation indicated that bacterial coinfection was a common and overlooked risk for hybrid groupers (♀ Epinephelus polyphekadion × â™‚ E. fuscoguttatus) reared in an industrialized flow-through pattern in Hainan Province. The artificial infection in hybrid groupers revealed that coinfections with V. harveyi strain GDH11385 (a serious lethal causative agent to groupers) and other isolated pathogens resulted in higher mortality (46.67%) than infection with strain GDH11385 alone (33.33%), whereas no mortality was observed in single infection with other pathogens. Furthermore, the intestine, liver and spleen of hybrid groupers are target organs for bacterial coinfections involving V. harveyi. Based on the infection patterns found in this study, we propose that V. harveyi may have a specific spatiotemporal expression pattern of virulence genes when infecting the host. Taken together, bacterial coinfection with V. harveyi is a neglected high-risk lethal causative agent to hybrid groupers in the industrialized flow-through aquaculture systems in Hainan Province.

Analysis of the Antioxidant Composition of Low Molecular Weight Metabolites from the Agarolytic Bacterium Alteromonas macleodii QZ9-9: Possibilities for High-Added Value Utilization of Macroalgae.

Agar accounts for ~60% of the dry weight of some red macroalgae, and the breakdown of this kind of polysaccharide releases high-value compounds; therefore, the resource utilization of agar is of great significance to improve the added value of these macroalgae. Herein, Alteromonas macleodii QZ9-9 isolated from tropical Gracilaria hainanensis in Hainan Island was characterized as an agarolytic bacterium, which displayed a high agar-degrading activity. The highest diameters of the degradation zones of the A. macleodii QZ9-9 and its extracellular-agarase (12.16 U/mL) were 41.46 mm and 22.89 mm, respectively, and the first-order degradation rate constants of those were 0.02 h-1 and 0.77 U-1, respectively. Importantly, the fermentation products of A. macleodii QZ9-9 exhibited antioxidant activity, and the peak of DPPH scavenging activity of 50 h fermentation products of this strain was up to 50.79% in the reaction for 1 h; the DPPH scavenging activity of low molecule metabolites (≤3 kDa) in particular was up to ~85.85%. A total of 766 metabolites were detected in the low molecule metabolites by metabolomics. The peptide-like metabolites, such as prolyl-histidine, isoleucyl-histidine, isoleucyl-proline and arginyl-proline, and the antioxidant maculosin were found in the top 20 metabolites with relatively high abundance. Additionally, the antioxidant activity of maculosin was further verified in this work. We concluded that the low molecule metabolites of A. macleodii QZ9-9 with relatively high antioxidant activity are interesting candidates for preparing desirable non-toxic antioxidants, thereby facilitating the high value-added utilization of macroalgae in the fields of cosmetic, food preservation, and pharmaceutical industries.

Multi-Costimulatory Pathways Drive the Antagonistic Pseudoalteromonas piscicida against the Dominant Pathogenic Vibrio harveyi in Mariculture: Insights from Proteomics and Metabolomics.

Vibrio harveyi is the dominant pathogen in mariculture, and biocontrol of this pathogen using antagonistic probiotics is a long-standing biological challenge. Here, Pseudoalteromonas piscicida WCPW15003 as a probiotic effectively antagonized dominant pathogenic V. harveyi in a mariculture, with a growth-of-inhibition ratio of 6.3 h-1. The antagonistic activities of cells and intracellular components of WCPW15003 made a greater contribution to the antagonistic process than did extracellular metabolites and caused the dominance of WCPW15003 during the antagonistic process in vitro. WCPW15003 was safe for the pearl gentian grouper (♀ Epinephelus fuscoguttatus × â™‚ Epinephelus lanceolatus) and, as a consequence of the antagonistic effect on V. harveyi, protected the fish from an immune response in vivo. A comprehensive combined proteomics and metabolomics analysis of antagonistic WCPW15003 and pathogenic V. harveyi in a coculture compared to a monoculture was performed to investigate the antagonistic molecular mechanisms. The results showed that during the antagonistic process, WCPW15003 in a coculture had significantly downregulated metabolic pathways for histidine metabolism, arginine biosynthesis, and phenylalanine metabolism, and upregulated glycerophospholipid metabolism, leading to a competitive advantage against the co-occurring species, V. harveyi. This defined a mechanism by which multi-costimulatory pathways drove P. piscicida WCPW15003 against V. harveyi. IMPORTANCE V. harveyi as a dominant pathogen has become a major hazard in mariculture development and seafood safety, and biocontrol of this pathogen using antagonistic probiotic agents is a long-standing biological challenge. P. piscicida WCPW15003 has promise as a novel, safe, and effective bioagent for specifically inhibiting dominant pathogenic V. harveyi and protects mariculture animals from infection by this pathogen by moderating the host immune response, which is heavily driven by multi-costimulatory pathways in a coculture of WCPW15003 and V. harveyi. This work identified a direction for comprehensively elucidating the molecular mechanism of WCPW15003 antagonism against the dominant pathogen in mariculture using modern molecular biology techniques and provided deep insights into the advantages and potential of this antagonistic probiotic against V. harveyi for the construction of an environmentally friendly, recirculating mariculture system.

Cross-habitat distribution pattern of Bacillus communities and their capacities of producing industrial hydrolytic enzymes in Paracel Islands: Habitat-dependent differential contributions of the environment.

Bacillus as a predominant genus of enzyme-producing bacteria presents desirable features to fulfill the vast demand of specific industries, whereas the knowledge of the Bacillus communities and their capacities of producing industrial hydrolytic enzymes across the microhabitats of the Paracel Islands is limited. Herein, a total of 193 culturable Bacillus strains belonging to 19 species were isolated across the microhabitats of seawater, sediment, coral and seagrass, covering 39 stations of the Paracel Islands. Each microhabitat displayed its unique species, while the species of Bacillus paramycoides besides being the dominant species with an abundance of 54.94% also was the only species shared by all microhabitats of the Paracel Islands. Of the Bacillus communities, 97.41% of the isolates exhibited the capacity of producing one-or-more types of enzymes with comparatively higher and broader ranges of enzyme activities, including 163 protease-, 27 cellulase-, 118 alginate lyase-, 140 K-carrageenase- and 158 agarose-producing strains. By the correlation analyses of "Bacillus-environmental factors" and "Enzyme-producing Bacillus-environmental factors", the cross-habitat distribution and enzyme-producing capacity pattern of the Bacillus communities were strongly driven by habitat type, and the environmental factors made habitat-dependent differential contributions to that in the Paracel Islands. It's worth noting that the cellulase-producing strain wasn't detected in seagrass due to its survival strategy to prevent cellulose degradation by inhibiting cellulase-producing bacteria, while coral contained more stable microbial metabolic functions to protect against environmental fluctuations. These findings besides providing large quantities of promising enzyme-producing candidates for specific industrial desires, also facilitate the development and utilization of marine microbial resources and the environmental policy- and/or law-making according to environmental features across the microhabitats of the Paracel Islands.

Effects of Taurine on Primary Metabolism and Transcription in a Coral Symbiodinium sp.

Coral reefs belong to the marine ecosystems and host the richest biodiversity of marine organisms. Coral reefs are formed as a result of the symbiotic relationship between the host coral animal and photosynthetic dinoflagellates, namely Symbiodinium sp. Coral animals induce the release of carbon fixation products of symbiotic Symbiodinium sp. through secreting host release factors (HRFs) such as taurine. To study the potential effect of taurine on photosynthesis and release of carbon fixation products of Symbiodinium sp., we compared the growth of Symbiodinium sp. under control and taurine-stimulated conditions. Photosynthesis parameters were detected to monitor the photosynthetic efficiency. Biomass and the contents of total soluble sugar, total insoluble sugar, total protein, total lipids, chlorophyll a were analyzed. Metabolome and transcriptome analyses were performed to analyze the potential effect of taurine on primary metabolism and mRNA transcription. The results revealed that taurine significantly increased the growth, photosynthesis efficiency, total soluble sugar, chlorophyll a, and chlorophyll b and free amino acid content of Symbiodinium sp. while decreased the content of total insoluble sugar. Results of metabolome and transcriptome analyses suggested that taurine might affect metabolic pathways in Symbiodinium sp. by altering the permeability of the algal cell membrane, diverting photosynthetically fixed carbon from storage compounds to translocated compounds, releasing a signal of low concentrations of nitrogen to initiate a series of response mechanisms, and controlling the density of Symbiodinium sp. through the quorum sensing effect. These results help to explore how corals control carbon metabolism in Symbiodinium sp. and to provide theoretical guidance for furthering our understanding of Symbiodinium sp. biology and coral-algal symbiosis.

Euendolithic Cyanobacteria and Proteobacteria Together Contribute to Trigger Bioerosion in Aquatic Environments.

Shellfish, mussels, snails, and other aquatic animals, which assimilate limestone (calcium carbonate, CaCO3) to build shells and skeletons, are effective carbon sinks that help mitigate the greenhouse effect. However, bioerosion, the dissolution of calcium carbonate and the release of carbon dioxide, hinders carbon sequestration process. The bioerosion of aquatic environments remains to be elucidated. In this study, the bioerosion of Bellamya spp. shells from the aquatic environment was taken as the research object. In situ microbial community structure analysis of the bioerosion shell from different geographical locations, laboratory-level infected culture, and validated experiments were conducted by coupling traditional observation and 16S rRNA sequencing analysis method. Results showed that bioeroders can implant into the CaCO3 layer of the snail shell, resulting in the formation of many small holes in the shell, which reduced the shell's density and made the shell fragile. Results also showed that bioeroders were distributed in two major phyla, namely, Cyanobacteria and Proteobacteria. Cluster analysis showed that Cyanobacteria sp. and two unidentified genera (Burkholderiaceae and Raistonia) were the key bioeroders. Moreover, results suggested that the interaction of Cyanobacteria and other bacteria promoted the biological function of "shell bioerosion." This study identified the causes of "shell bioerosion" in aquatic environments and provided some theoretical basis for preventing and controlling it in the aquatic industry. Results also provided new insights of cyanobacterial bioerosion of shells and microalgae carbon sequestration.

Metabolomic, proteomic and lactylated proteomic analyses indicate lactate plays important roles in maintaining energy and C:N homeostasis in Phaeodactylum tricornutum.

BACKGROUND: Phaeodactylum tricornutum accumulates lipids while the growth also increases under high CO2, shedding light on its potential application in the reduction of CO2 emissions and at the same time acquiring biodiesel raw materials. However, the sensing and transducing of high C:N signals and the related response mechanism(s) remained unknown. RESULTS: In this study, a multiple omics analysis was performed with P. tricornutum under low nitrogen (LN) and high CO2 (HC) conditions. The results indicated that 2-oxoglutarate was significantly increased under both LN and HC. Meanwhile, proteins involved in carbon concentration mechanism decreased, indicated that 2-oxoglutarate might regulate C:N balance through suppressing carbon fixation. Lactate, which acts in energy metabolism, signal transduction and 'LactoylLys' modification on proteins, was the most upregulated metabolite under both LN and HC conditions. Meanwhile, proteins involved in carbon, nitrogen and energy metabolisms were significantly regulated. Western blotting analysis suggested that non-histone L-lactylation modification was enhanced under LN and HC. Moreover, lactylated proteins were enriched in photosynthesis, central carbon metabolism, nitrogen metabolism, fatty acid synthesis and oxidative phosphorylation. CONCLUSION: It is suggested that lactate might play important roles in energy homeostatic maintenance and C:N balance regulation in P. tricornutum through protein lactylation modification.

Two (p)ppGpp Synthetase Genes, relA and spoT, Are Involved in Regulating Cell Motility, Exopolysaccharides Production, and Biofilm Formation of Vibrio alginolyticus.

The stringent response mediated by the signal molecule (p)ppGpp is involved in response to multiple environmental stresses and control of various physiological processes. Studies have revealed that (p)ppGpp strongly affects the formation and maintenance of several bacterial biofilms. However, the specific regulatory roles of (p)ppGpp in biofilms, especially in the expression of genes related to cell motility and exopolysaccharides (EPSs) production, remain poorly understood. We recently reported two (p)ppGpp synthetase genes relA and spoT from the epizootic pathogen Vibrio alginolyticus. Herein, we found that the (p)ppGpp synthetase genes of V. alginolyticus contributed to biofilm formation at low cell density and biofilm detachment at high cell density, respectively, in polystyrene microtiter plates. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that the expression levels of both EPSs and motility associated genes were consistent with the development of biofilms. Besides, the (p)ppGpp synthetase gene spoT was found to be closely involved in the regulation of flagellum, smooth/translucent colony morphology and spotty pellicle at the air-liquid interface. Interestingly, pleiotropic phenotypes of ΔrelAΔspoT were similar to that of the rpoN (σ54) deletion mutant. Meanwhile, the absence of (p)ppGpp synthetase genes significantly reduced the expression levels of rpoN at low cell density, suggesting that (p)ppGpp may mediate the formation via positively affecting the alternative sigma factor RpoN. These findings allow us to propose (p)ppGpp as a crucial regulator for biofilm development in V. alginolyticus, in view of the regulatory roles of relA and spoT in cell motility and EPSs production.

Effects of Lactobacillus pentosus combined with Arthrospira platensis on the growth performance, immune response, and intestinal microbiota of Litopenaeus vannamei.

Litopenaeus vannamei is one of the most productive shrimp species in the world. However, shrimp farming is suffering from adverse environmental conditions and disease outbreaks. Typically, Lactobacillus pentosus and Arthrospira platensis are used as substitutes for some antibiotics. In the present study, we assessed the effects of dietary supplements along with living bacteria or cell-free extracts of L. pentosus combined with A. platensis on the growth performance, immune response, intestinal microbiota, and disease resistance of L. vannamei against Vibrio alginolyticus. Shrimp fed L. pentosus live bacteria combined with A. platensis showed the best growth performance and lowest feed conversion rate. The supplementation diet with L. pentosus live bacteria and A. platensis could significantly enhance the trypsin activity in shrimp after the feeding trial. Given the lowest feed conversion rate in shrimp fed L. pentosus live bacteria combined with A. platensis, we reasonably speculated that the decrease in feed conversion rate may be related to the increase in trypsin activity. In addition, dietary cell-free extracts of L. pentosus combined with A. platensis enhanced the expression of immune-related genes after the feeding trial or challenge test. Moreover, results of the bacterial challenge test indicated that the shrimp fed cell-free extracts of L. pentosus combined with A. platensis diet resulted in the highest survival rate, which suggested that cell-free extracts of L. pentosus and A. platensis could improve the disease resistance against V. alginolyticus by up-regulating the expressions of immune-related genes. Dietary L.pentosus or A. platensis, or their combination, reduced the abundance of harmful bacteria, including Proteobacteria in shrimp intestine, which suggested that L. pentosus and A. platensis could improve the growth performance and health of shrimp by regulating the structure of the intestinal microbiota. The findings of this study demonstrated that L. pentosus live bacteria and A. platensis exerted synergistic effects on the growth performance and digestion in shrimp, while cell-free extracts of L. pentosus and A. platensis showed synergistic effects on the immune response and disease resistance of shrimp against V. alginolyticus.

Proteomic and biochemical responses to different concentrations of CO2 suggest the existence of multiple carbon metabolism strategies in Phaeodactylum tricornutum.

BACKGROUND: Diatoms are well known for high photosynthetic efficiency and rapid growth rate, which are not only important oceanic primary producer, but also ideal feedstock for microalgae industrialization. Their high success is mainly due to the rapid response of photosynthesis to inorganic carbon fluctuations. Thus, an in-depth understanding of the photosynthetic carbon fixation mechanism of diatoms will be of great help to microalgae-based applications. This work directed toward the analysis of whether C4 photosynthetic pathway functions in the model marine diatom Phaeodactylum tricornutum, which possesses biophysical CO2-concentrating mechanism (CCM) as well as metabolic enzymes potentially involved in C4 photosynthetic pathway. RESULTS: For P. tricornutum, differential proteome, enzyme activities and transcript abundance of carbon metabolism-related genes especially biophysical and biochemical CCM-related genes in response to different concentrations of CO2 were tracked in this study. The upregulated protein abundance of a carbonic anhydrases and a bicarbonate transporter suggested biophysical CCM activated under low CO2 (LC). The upregulation of a number of key C4-related enzymes in enzymatic activity, transcript and protein abundance under LC indicated the induction of a mitochondria-mediated CCM in P. tricornutum. Moreover, protein abundance of a number of glycolysis, tricarboxylic acid cycle, photorespiration and ornithine-urea cycle related proteins upregulated under LC, while numbers of proteins involved in the Calvin cycle and pentose phosphate pathway were downregulated. Under high CO2 (HC), protein abundance of most central carbon metabolism and photosynthesis-related proteins were upregulated. CONCLUSIONS: The proteomic and biochemical responses to different concentrations of CO2 suggested multiple carbon metabolism strategies exist in P. tricornutum. Namely, LC might induce a mitochondrial-mediated C4-like CCM and the improvement of glycolysis, tricarboxylic acid cycle, photorespiration and ornithine-urea cycle activity contribute to the energy supply and carbon and nitrogen recapture in P. tricornutum to cope with the CO2 limitation, while P. tricornutum responds to the HC environment by improving photosynthesis and central carbon metabolism activity. These findings can not only provide evidences for revealing the global picture of biophysical and biochemical CCM in P. tricornutum, but also provide target genes for further microalgal strain modification to improve carbon fixation and biomass yield in algal-based industry.

Tropical Cellulolytic Bacteria: Potential Utilization of Sugarcane Bagasse as Low-Cost Carbon Source in Aquaculture.

Sugarcane bagasse (SB), as a major by-product of sugarcane, is one of the most abundant organic matter and characterized by cheap and easily available carbon source in Hainan Island, China. The objective of this study was to isolate tropical cellulolytic bacteria from Hainan Island and demonstrate their prospects of utilization of SB as a low-cost carbon source to greatly reduce the cost of aquaculture. A total of 97 cellulolytic marine bacteria were isolated, of which, 58 cellulolytic marine bacteria displayed the hydrolysis capacity (HC) of more than 1, while 28 cellulolytic marine bacteria displayed more than 2. Of the 28 tropical cellulolytic bacterial strains with HC more than 2, Microbulbifer sp. CFW-C18 and Vibrio sp. MW-M19 exhibited excellent SB decomposition in a small-scale laboratory simulation of shrimp aquaculture, up to 75.31 and 74.35%, respectively, and both of them were safe for shrimps. Meanwhile, both of CFW-C18 and MW-M19 besides displaying low multiple antibiotic resistance (MAR) index, also increased the C/N ratio (CFW-C18: C/N ratio of 14.34; MW-M19: C/N ratio of 14.75) of the small-scale laboratory simulation of shrimp aquaculture by decreasing the nitrogen content after a supplement of SB for 15 days. More importantly, CFW-C18 and MW-M19 displayed a relatively low MAR index, 0.47 and 0.1, respectively, especially MW-M19, with the lowest MAR index (0.1), which was resistant to only three antibiotics, streptomycin, amikacin, and levofloxacin, indicating that this strain was safe and non-drug resistance for further use. Overall, tropical cellulolytic bacteria isolated from Hainan Island, especially CFW-C18 and MW-M19, will provide the proficient candidates as probiotics for further construction of the recirculating aquaculture system based on the supplement of low-cost external carbon source-SB.

Stress adaptation and virulence in Vibrio alginolyticus is mediated by two (p)ppGpp synthetase genes, relA and spoT.

Vibrio alginolyticus belongs to gram-negative opportunistic pathogen realm infecting humans and aquatic animals causing severe economic losses. The (p)ppGpp-mediated stringent response is corroborated to stress adaptation and virulence of pathogenic mechanisms. Limited reports are documented for the intricate assessment of (p)ppGpp synthetase genes in combating various stress adaptation and elucidation of virulence in V. alginolyticus remains unraveled. The present assessment comprises of generation of deletion mutants in the (p)ppGpp-deficient strains, ΔrelA (relA gene single mutant) and ΔrelAΔspoT (relA and spoT genes double mutant), and the complemented strains, ΔrelA+ and ΔrelAΔspoT+, were constructed to investigate the pivotal roles of (p)ppGpp synthetase genes in V. alginolyticus, respectively. Amino acid sequence alignment analysis initially revealed that RelA and SpoT possess relatively conserved domains and synthetase activity. Hydrolase activity was emancipated by SpoT alone showing variant mode of action. Compared with the wild type and complemented strains, the relA-deficient strain was more sensitive to amino acid starvation and mupirocin. Interestingly, the deletion of spoT resulted in a significant growth deficiency supplemented with bile salts, 3 % ethanol and heat shock. Rapid growth was observed in the stationary phase upon exposure to cold stress and lower doses of ethanol. Subsequently, disruption of (p)ppGpp synthetase genes caused the decline in swimming motility, enhanced biofilm formation, cell aggregation of V. alginolyticus, and reduced mortality of Litopenaeus vannamei. The expression levels of some virulence-associated genes were quantified affirming consistency established by pleiotropic phenotypes. The results are evident for putative roles of (p)ppGpp synthetase genes attributing essential roles for environmental adaption and virulence regulation in V. alginolyticus.

Multi-Strain Tropical Bacillus spp. as a Potential Probiotic Biocontrol Agent for Large-Scale Enhancement of Mariculture Water Quality.

Aquaculture is suffering from long-term water eutrophication in intensive models, whereas the knowledge of multi-strain/specie for improving water quality is extremely limited. Herein, we aimed to develop multi-strain tropical Bacillus spp. as a potential probiotic biocontrol agent for large-scale enhancement of mariculture water quality. Given the practical application, the optimum multi-strain tropical Bacillus spp. (B. flexus QG-3, B. flexus NS-4, and B. licheniformis XCG-6 with the proportion 5: 5: 4) as a probiotic biocontrol agent was screened and obtained, which effectively improved water quality by removing chemical oxygen demand (COD), ammonia-nitrogen, and nitrate and significantly inhibited Vibrio spp. even at relatively low bacterial concentrations (104 CFU/ml) in artificial feed wastewater and large-scale shrimp aquaculture ponds. More importantly, we found that the initial proportion of these three Bacillus sp. strains of multi-strain tropical Bacillus spp. markedly affected the final purification effects, whereas the initial concentration of that only influenced the purification rates at the early stage (0-48 h) instead of final purification effects. We reason that this multi-strain tropical Bacillus spp. as a good probiotic biocontrol agent could perform multiple actions, such as COD-degrading, nitrifying, denitrifying, and antagonistic actions, for large-scale enhancement of tropical aquaculture water. Additionally, the multi-strain tropical Bacillus spp. was safe for shrimp and could be stored for at least 240 days in spore form at room temperature. This multi-strain probiotic biocontrol agent may facilitate its adoption for further marine recirculating aquaculture system development and large-scale commercial application.

Roles of rpoN in biofilm formation of Vibrio alginolyticus HN08155 at different cell densities.

RpoN (δ54) as a global regulator controls crucialvirulence-associated phenotype, which can regulate flagellum and exopolysaccharides (EPS) during pathogenic biofilm formation. However, the knowledge of the roles of rpoN in biofilm formation of V. alginolyticus is limited, especially at different cell densities. Herein, deletion mutant strain ΔrpoN, complementary strain ΔrpoN-C and negative control strain ΔrpoN-Z were constructed to investigate the effects of rpoN on biofilm formation of V. alginolyticus HN08155 based on flagellum and EPS at different cell density conditions. The results showed that all of strains can form biofilm, and biofilms of strains with rpoN were formed at low cell density (LCD) and detached at high cell density (HCD), while those of ΔrpoN and ΔrpoN-Z were absent at LCD and accumulated excessively with a spotty pellicle at HCD without detaching. The EPS contents of strains with rpoN was greater than that of ΔrpoN and ΔrpoN-Z at LCD, while the opposite trends were observed at HCD. The expression levels of rpoN were quantified, which were consistent with the trend of biofilm formation. It's worth noting that absence of rpoN resulted in the failure of biofilm detachment, lacking of flagellum and decreasing motility, indicating that rpoN was not necessary for biofilm formation, but it was essential for biofilm detachment.

Community Structure of Protease-Producing Bacteria Cultivated From Aquaculture Systems: Potential Impact of a Tropical Environment.

Protease-producing bacteria play vital roles in degrading organic matter of aquaculture system, while the knowledge of diversity and bacterial community structure of protease-producing bacteria is limited in this system, especially in the tropical region. Herein, 1,179 cultivable protease-producing bacterial strains that belonged to Actinobacteria, Firmicutes, and Proteobacteria were isolated from tropical aquaculture systems, of which the most abundant genus was Bacillus, followed by Vibrio. The diversity and relative abundance of protease-producing bacteria in sediment were generally higher than those in water. Twenty-one genera from sediment and 16 genera from water were identified, of which Bacillus dominated by Bacillus hwajinpoensis in both and Vibrio dominated by Vibrio owensii in water were the dominant genera. The unique genera in sediment or water accounted for tiny percentage may play important roles in the stability of community structure. Eighty V. owensii isolates were clustered into four clusters (ET-1-ET-4) at 58% of similarity by ERIC-PCR (enterobacterial repetitive intergenic consensus-polymerase chain reaction), which was identified as a novel branch of V. owensii. Additionally, V. owensii strains belonged to ET-3 and ET-4 were detected in most aquaculture ponds without outbreak of epidemics, indicating that these protease-producing bacteria may be used as potential beneficial bacteria for wastewater purification. Environmental variables played important roles in shaping protease-producing bacterial diversity and community structure in aquaculture systems. In sediment, dissolved oxygen (DO), chemical oxygen demand (COD), and salinity as the main factors positively affected the distributions of dominant genus (Vibrio) and unique genera (Planococcus and Psychrobacter), whereas temperature negatively affected that of Bacillus (except B. hwajinpoensis). In water, Alteromonas as unique genus and Photobacterium were negatively affected by NO3 --N and NO2 --N, respectively, whereas pH as the main factor positively affected the distribution of Photobacterium. These findings will lay a foundation for the development of protease-producing bacterial agents for wastewater purification and the construction of an environment-friendly tropical aquaculture model.

Elevated CO2 improves both lipid accumulation and growth rate in the glucose-6-phosphate dehydrogenase engineered Phaeodactylum tricornutum.

BACKGROUND: Numerous studies have shown that stress induction and genetic engineering can effectively increase lipid accumulation, but lead to a decrease of growth in the majority of microalgae. We previously found that elevated CO2 concentration increased lipid productivity as well as growth in Phaeodactylum tricornutum, along with an enhancement of the oxidative pentose phosphate pathway (OPPP) activity. The purpose of this work directed toward the verification of the critical role of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme in the OPPP, in lipid accumulation in P. tricornutum and its simultaneous rapid growth rate under high-CO2 (0.15%) cultivation. RESULTS: In this study, G6PDH was identified as a target for algal strain improvement, wherein G6PDH gene was successfully overexpressed and antisense knockdown in P. tricornutum, and systematic comparisons of the photosynthesis performance, algal growth, lipid content, fatty acid profiles, NADPH production, G6PDH activity and transcriptional abundance were performed. The results showed that, due to the enhanced G6PDH activity, transcriptional abundance and NAPDH production, overexpression of G6PDH accompanied by high-CO2 cultivation resulted in a much higher of both lipid content and growth in P. tricornutum, while knockdown of G6PDH greatly decreased algal growth as well as lipid accumulation. In addition, the total proportions of saturated and unsaturated fatty acid, especially the polyunsaturated fatty acid eicosapentaenoic acid (EPA; C20:5, n-3), were highly increased in high-CO2 cultivated G6PDH overexpressed strains. CONCLUSIONS: The successful of overexpression and antisense knockdown of G6PDH well demonstrated the positive influence of G6PDH on algal growth and lipid accumulation in P. tricornutum. The improvement of algal growth, lipid content as well as polyunsaturated fatty acids in high-CO2 cultivated G6PDH overexpressed P. tricornutum suggested this G6PDH overexpression-high CO2 cultivation pattern provides an efficient and economical route for algal strain improvement to develop algal-based biodiesel production.

Provision of carbon skeleton for lipid synthesis from the breakdown of intracellular protein and soluble sugar in Phaeodactylum tricornutum under high CO2.

BACKGROUND: Increasing CO2 emissions have resulted in ocean acidification, affecting marine plant photosynthesis and changing the nutrient composition of marine ecosystems. The physiological and biochemical processes of marine phytoplankton in response to ocean acidification have been reported, but have been mainly focused on growth and photosynthetic physiology. To acquire a thorough knowledge of the molecular regulation mechanisms, model species with clear genetic background should be selected for systematic study. Phaeodactylum tricornutum is a pennate diatom with the characteristics of small genome size, short generation cycle, and easy to transform. Furthermore, the genome of P. tricornutum has been completely sequenced. RESULTS AND DISCUSSION: In this study, P. tricornutum was cultured at high and normal CO2 concentrations. Cell composition changes during culture time were investigated. The 13C isotope tracing technique was used to determine fractional labeling enrichments for the main cellular components. The results suggested that when lipid content increased significantly under high CO2 conditions, total protein and soluble sugar contents decreased. The 13C labeling experiment indicated that the C skeleton needed for fatty acid C chain elongation in lipid synthesis under high CO2 conditions is not mainly derived from NaHCO3 (carbon fixed by photosynthesis). CONCLUSION: This study indicated that breakdown of intracellular protein and soluble sugar provide C skeleton for lipid synthesis under high CO2 concentration.