Abstract:[Background] Nonpathogenic Epsilonproteobacteria globally distributes in diverse natural environments, especially extreme environments like deep sea hydrothermal vents, are usually detected in microbial communities as a dominant microbial group. Only few hydrothermal Epsilonproteobacteria species have been isolated due to the limitation of current culture technology, which significantly influences our further understanding about their physiological features, metabolic pathways and ecological roles. [Objective] Gaining insight into the phylogenetic position, metabolic potentials and putative roles of uncultured Epsilonproteobacteria in the deep sea hydrothermal communities and vent ecosystems. [Methods] We analyzed the phylogeny and metabolic pathway of four Epsilonproteobacteria reconstructed from the East Pacific Rise deep sea hydrothermal chimney sample based on metagenomic Binning technology. [Results] Bin189 is phylogenetically independent from all other known Epsilonproteobacteria, whereas the other three reconstructed genomes have closed relationship with Nitratiruptor sp. SB155-2. In metabolic potentials, all of reconstructed genomes have the sqr and rTCA carbon fixation pathway related genes, besides they all have the lipopolysaccharide exporter system and multiple secretion system. However, Bin189 has the extra organic matter and amino acid transporters, all of the other three genomes have the complete denitrification pathway and two of them also have the Sox system, hydrogenase as well as the flagella system. [Conclusion] Bin189 very likely is a kind of novel heterotrophic Epsilonproteobacteria detected in the deep sea hydrothermal environments, whereas the other three chemoautotrophic taxa are capable of oxidizing diverse reduced sulfur compounds and hydrogen as energy source. Consequently, these Epsilonproteobacteria are supposed to play crucial roles in the colonization and development of the hydrothermal microbial communities as well as the deep sea geochemical element cycling.

Abstract:[Background] Alginate oligosaccharides produced by enzymatic hydrolysis of alginate have diverse biological functions such as anti-oxidation, anti-tumor, induction of immune regulation, and regulation of plant growth, and exhibit broad application prospects in the fields of food and medicine. Mass screening of alginate-degrading bacteria is beneficial to obtain new structures and functions of alginate oligosaccharides, which is conducive to actively promoting the industrial progress of oligosaccharides. [Objective] High-efficiency screening was carried out for alginate-degrading strains and exploring the in situ microbial resources of seaweeds with development prospects. [Methods] The only carbon source of sodium alginate was used to select the microorganisms on the surface of brown seaweed. The chromogenic reaction of gram iodine solution indicated the characteristics of microorganisms to degrade alginate. The activity of alginate lyase was determined by Oxford cup method. [Results] A total of 81 colonies were obtained from the surface of the algae samples. Twenty-eight strains were screened by clear circle staining. Seven strains of alginate-degrading bacteria were identified by 16S rRNA gene sequencing analysis. They belonged to the genus Bacillus (3/7), Arthrobacter (1/7), Desemzia (1/7), Brachybacterium (1/7) and Streptosporangium (1/7). The genera Arthrobacter, Desemzia, Brachybacterium and Streptosporangium have not been previously reported to produce alginate lyase. Further analysis showed that T-1 strain (Bacillus) had maximum enzyme production and highest enzyme activity. [Conclusion] Seven alginate lyase-excreting strains were isolated, and the enzyme activities of the strains were compared by Gram’s iodine coloration combined with the Oxford cup method. This indicates that the screening method is simple, efficient and suitable for large-scale screening of alginate lyase-excreting microorganisms.

Abstract:[Background] Using amoA gene as marker for analyzing the diversity of ammonia-oxidizing archaea (AOA) has stronger specificity and higher resolution than 16S rRNA gene, it can more accurately reflect the community structure and distribution pattern of AOA in environmental samples. However, there are two limitations in the analysis of amoA gene sequence with high-throughput sequencing: one is the lack of corresponding amoA gene reference taxonomic database; and the other is no determined species-level cut-off value for operational taxonomic units (OTUs) clustering. [Objective] The aim of this article was to develop a method for ammonia-oxidizing archaea diversity analysis based on amoA gene sequence with high-throughput sequencing, providing a reference method to analyze the functional microbial diversity based on high-throughput sequencing. [Methods] amoA gene sequences of 34 AOA strains from pure or enrichment culture were used as seed sequences. Uncultured amoA gene sequences from environmental samples were downloaded from functional gene database. All sequences were used to construct taxonomic database for amoA gene sequences. By pairwise comparison of 16S rRNA gene and archaeal amoA identities of all recognized species of AOA, we determined the cut-off value for OTU clustering at species-level. We used the established reference database and determined cut-off value to analyze the diversity for water samples?in a vertical profile of the South China Sea with the MOTHUR software. [Results] We constructed a reference taxonomic database containing 26 091 amoA gene sequences, and determined 89% sequence identity as cut-off value for OTU clustering at species-level. The diversity analysis of AOA sufficiently showed the community structure and phylogenetic relationship, and effectively revealed the AOA vertical distribution differences in the South China Sea. [Conclusion] We developed a method for ammonia-oxidizing archaea diversity analysis based on amoA gene sequence with high-throughput sequencing. This method can effectively analyze the diversity of AOA in environmental samples.

Abstract:[Background] Coral bleaching events occur frequently which causes the coral reef undergoing severe degeneration in recent years. Disease caused by pathogens is one of the reasons for coral bleaching. Vibrio shilonii was identified to be a pathogen that is able to infect the coral Oculina patagonica and cause its bleaching. However, its epidermology remains largely unknown due to the lack of a rapid diagnostic tool. [Objective] To establish a rapid and simple method for detection of V. shilonii using Loop-mediated isothermal amplification (LAMP) technique. [Methods] A set of 6 specific primers based on the target of rpoD sequence was designed for LAMP amplification. The sensitivity and specification of the primers were tested and conditions for LAMP amplification were optimized. The products of LAMP were visualized with fluorescent reagent of calcein and agarose gel electrophoresis respectively. The limitation of the method was compared with conventional PCR and Real-time PCR. [Results] The result showed that this method can specifically detect V. shilonii without showing cross-reaction with closely related vibrio species or other bacteria. The detection limit is visualized with calcein was 3.641×103 cps/mL of the plasmids carrying rpoD, which was 1 000 times more sensitive than conventional PCR and similar to that of Real-time PCR method; Meanwhile, this method was shown to be able to directly detect the pathogen in the seawater with a minimum concentration of 1.3×102 cfu/mL. [Conclusion] Overall, this study revealed that the method we developed has high specificity and sensitivity for diagnosis of V. shilonii. It is more sensitive and more convenient than the conventional PCR or real-time PCR. Therefore, it has the potential to be used for diagnosis in the field.

Abstract:[Background] Marine-derived fungi are prolific resources of natural products due to their diversity and ability to produce novel bioactive compounds. [Objective] To obtain the bioactive secondary metabolites from the fungus Parengyodontium album SCSIO 40430, which isolated from a Montipora foliosa sample from the South China Sea, and to evaluate for their antibacterial activity. [Methods] The compounds were isolated and purified by silica gel column, Sephadex LH-20 column and semi-prepartive HPLC chromatography. Their structures were identified by HR-ESI-MS, 1H, 13C NMR, and comparison with the data of literatures. [Results] Three compounds were isolated from the butanone extract and structurely identified as deoxyphomalone (1), 2-ethyl-3,5-dihydroxy-11-methylchroman- 9-one (2) and 2-ethyl-3-hydroxy-5-methoxy-11-methylchroman-9-one (3). [Conclusion] Three chromanone compounds were aquired, and compounds 2 displayed antibacterial activities of Methicillin-resistant Stphylococcus and Bacillus thuringiensis SCSIO BT01 (MIC 16 μg/mL).

Abstract:[Background] Streptomyces sp. OUCMDZ-3434 was isolated from Enteromorpha prolifera collected from the Zhanqiao Beach, Qingdao, Shandong, and it produced wailupemycins with good α-glycosidase inhibitory activity, but at very low yield. [Objective] To develop the genetic system of marine-derived Streptomyces sp. OUCMDZ-3434 and investigate the role of wblA gene on wailupemycins production. [Methods] Escherichia coli-Streptomyces intergeneric conjugation manipulation was established for Streptomyces sp. OUCMDZ-3434 using pSET152. wblA gene was inactivated by using PCR-targeting strategy. After confirmation by PCR, the fermentation broths of the wild-type and mutant strains were analyzed by HPLC, and morphological differentiation was observed by using phase contrast microscope. [Results] The conjugation method for Streptomyces sp. OUCMDZ-3434 was established and the wblA gene inactivation mutant was constructed. The yield of wailupemycin G in the wblA inactivation mutant was increased by 3-fold compared to that of the wild-type strain. Additionally, the wblA gene mutant lost the ability of sporulation. [Conclusion] The wblA gene in Streptomyces sp. OUCMDZ-3434 negatively regulates the biosynthesis of wailupemycins and is involved in regulation of sporulation as well. These results laid the foundation for yield improvement of wailupemycins by genetic manipulation.

Abstract:[Background] Naphthopyrones possess various bioactivities that are characteristic secondary metabolites of the fungus Aspergillus niger. [Objective] To illuminate the structures and activities of naphthopyrones from fungus A. niger isolated from mud flat. [Methods] Guided by TLC analysis, we used combined chromatographic and spectrographic techniques to identify naphthopyrones. The activities against pathogen bacteria and Artemia salina were tested by micro-dilution method. [Results] Six naphthopyrones: Rubrofusarin B (1), Flavasperone (2), Aurasperone A (3), Asperpyrones C (4), Asperpyrones B (5) and Fonsecinone A (6) were isolated from A. niger. In bioassays, compounds 1 through 6 showed different levels of antibacterial activities against pathogen bacteria. Flavasperone (2) and Asperpyrones C (4) showed potent antibacterial activities against S. aureus ATCC33591 with MIC values 43.7 and 21.9 μmol/L, respectively. Aurasperone A (3) exhibited significant activities against E. faecium ATCC35667 with MIC values 21.9 μmol/L. Compounds 1 through 6 displayed toxicity on brine shrimps larvae, the LD50 values of Flavasperone (2) and Aurasperone A (3) were 35.0 and 8.8 μmol/L, respectively. [Conclusion] Diverse naphthopyrones were produced by strain XJJ-3. Compounds 1 through 6 showed different levels of activities against pathogen bacteria and Artemia salina. This study may provide reference for the development of antibacterial and cytotoxic drugs.

Abstract:[Background] Deep-sea environment is complex and diverse with abundant fungi that provide new opportunities to study the chemical and biological activity of deep-sea fungal metabolites. [Objective] To isolate, identify and evaluate fungi from the South China Sea deep-sea sediment, to discover new potential drug source fungi and lay the foundation for further utilization of deep-sea fungi from the South China Sea. [Methods] Based on intergenic transcribed spacer sequencing, 52 fungi isolated from deep-sea sediment were identified. All fungal crude extracts were tested for antibacterial, brine shrimp lethal and antioxidative activities by using Kirby-Bauer test, Solis’ method and DPPH radical scavenging method. [Results] These fungi could be clarified into 16 genera, in which Cladosporium sp. and Aspergillus sp. were in majority, accounting for 25.00% and 23.08%, respectively; 32 strains inhibited 1 indicative bacterium at least, 8 of which inhibited all 4 indicative bacteria; 23 strains exhibited medium brine shrimp lethality, accounting for 44.23% of all isolated strains, 2 of which exhibited obvious activities with IC50 of 68.59, 78.83 μg/mL; 30 strains showed DPPH radical scavenging activity, accounting for 57.69%, 9 of which with EC50 values lower than 100 μg/mL. [Conclusion] This study revealed the distribution and metabolite activities of deep-sea fungi from South China Sea and found a number of potential active fungi, and provides support for subsequent investigation on chemical diversities and biological activities of deep-sea fungi.

Abstract:[Background] Favia favus is one of the main reef-building corals in the South China Sea, To our knowledge, to date no study has been performed on the symbiotic zooxanthellae in F. favus. [Objective] To investigate the phylogenetic diversity of the Symbiodinium in F. favus at different life stages, and to gain insights into potential acquisition mode of the Symbiodinium. [Methods] Fluorescence microscopy was used to detect the presence of symbiotic zooxanthellae in the eggs, larvae and adults of F. favus, respectively. Meanwhile, the clone library based on ITS2 sequence was constructed to assess the diversity of Symbiodinium. [Results] By means of direct examination with fluorescence microscopy, zooxanthellae was absent in the eggs and larvae but present in the adults of F. favus, which was assigned to Symbiodinium type C1 based on the ITS2 sequence analysis. [Conclusion] Only Symbiodinium type C1 was present in adults rather than eggs and larvae, suggesting that the symbiotic zooxanthellae in F. favus were probably acquired by horizontal transmission from the environment instead of vertical transmission.

Abstract:[Background] The highly intensification of shrimp culture industry is facing an increasingly serious problem in water pollution. Simultaneously and high-efficiently degradation of organic matter, ammonia and nitrite in shrimp wastewater is one of important guarantees for the sustainable and healthy development of aquaculture. [Objective] In order to promote sustainable development of Litopenaeus vannamei culture in China, high-efficiently degradation of organic matter, ammonia and nitrite of aquaculture wastewater was developed by combination of immobilized nitrifying bacterial consortia (NBC) and selected Bacillus population. [Methods] High-throughput analysis of 16S rRNA gene was applied to study NBC’s community, which was enriched from the bottom mud of local shrimp ponds. We screened the strain with the highest COD (chemical oxygen demand) degrading capability from five Bacillus strains, and selected nontoxic immobilizing materials with good absorption capacity and ball performance. Further, the immobilized formula was optimized by orthogonal test to improve the mechanical strength. The optimum use concentrations of NBC and selected Bacillus strain were evaluated for high-efficiently degradation of COD, ammonia and nitrite in shrimp wastewater, respectively. [Results] High-throughput analysis of 16S rRNA gene showed that NBC was dominated by Proteobacteria (61.10%), and the abundance of autotrophic nitrifying bacteria in NBC was 12.69% with high diversity. NBC also contained denitrifying or potential denitrifying bacteria with an abundance of 47.44% and photosynthetic bacteria with an abundance of 12.85%. It is an important complement for nitrification under high organic loading and nitrogen removal through denitrification. Bacillus amyloliquefacien YL-10 has the highest COD degradation rate 100% in 48 h. The optimal formula for immobilization is 5% of shell powder, 3% of sodium alginate and 4% of calcium chloride. The mechanical strength of the embedding pellets is 129.68 mN. The immobilization significantly increased the degradation rate of ammonia and nitrite by 128.13% and 130.11%, respectively, but had no obvious effect on COD degradation. The optimum concentration of NBC and YL-10 was 1×108 CFU/mL. Under the joint action of immobilized NBC and YL-10, the concentrations of ammonia, nitrite and COD in shrimp farming wastewater were significantly reduced from (6.32±0.12), (5.69±0.11) and (65.29±1.14) mg/L to (0.03±0.03), (0.06±0.01) and 0 mg/L in 48 h (P<0.05), respectively. The degradation rates were 99.57%, 99.03% and 100%, respectively. [Conclusion] Organic matter, ammonia and nitrite in shrimp wastewater were biodegraded simultaneously and efficiently by combination of immobilized NBC and selected Bacillus population, which provides the scientific data reference for the large-scale practical application in high-density culture of L. vannamei.

Abstract:[Background] In recent years, the increasingly polluted environment of Bohai Bay has impacted the sustainability of offshore ecosystems. [Objective] To explore the distribution characteristics of bacterial communities in surface sediments and their response to environmental factors, we selected surface sediment samples from 21 stations for study. [Methods] We investigated the microbial community structure by sequencing the V3?V4 hypervariable regions of bacterial 16S rRNA gene using the Illumina HiSeq 2500. Bacterial community composition, spatial distribution characteristics and environmental factors were combined and analyzed in an attempt to understand the key interactions. [Results] The 16S rRNA gene clone library analysis indicated that the most common phyla that were widely distributed in the 21 sites were: Proteobacteria (56.8%), Acidobacteria (8.9%), Chloroflexi (8.1%), Bacteroidetes (6.2%). Moreover, γ- Proteobacteria and δ- Proteobacteria were the most dominant classes in Proteobacteria. Total organic carbon and total organic nitrogen content in the near shore sediments is high due to the input of anthropogenic materia and gradually decreased offshore. The most significant influence on species composition and community structure within the study area were found to be from particle size (content<4 μm) and organic nitrogen load. [Conclusion] The microbial diversity in the inlet of Bohai Bay is very high and is significantly related to a variety of environmental parameters. Human activities play an important role in structuring the microbial diversity, community structure and distribution.

Abstract:[Background] Sustainable development of coastal ecosystem has become one of the most important concerns for people nowadays. Riverine output and anthropogenic interrupt have important impacts on the coastal environment. [Objective] In this study, we collected 12 samples from three transects including Bohai Bay, Liaodong Bay and Laizhou Bay to explore the microbial community and diversity in summer of 2015. [Methods] DNA was extracted from water samples by using DNA extraction kit. Samples were analyzed by Illumina HiSeq sequencing technology. We compared the differences among these three transects according to the analysis results. [Results] The diversity index and rarefaction curves showed significant differences among these three transects. The order of diversity value was Laizhou Bay>Bohai Bay>Liaodong Bay. The distribution of the dominant community was as follows: the proportion of Proteobacteria, Bacteroidetes, Cyanobacteria, Actinobacteria and Planctomycetes in the Bohai Bay was 39.8%, 25.7%, 22.4%, 5.85% and 4.38%, respectively. The dominant community proportion in Liaodong bay was Proteobacteria (37.8%), Bacteroidetes (25.7%), Cyanobacteria (17.8%), Actinobacteria (10.4%) and Planctomycetes (5.64%). While in Laizhou Bay there were only four dominant communities as follows: Proteobacteria (59.0%), Bacteroidetes (17.5%), Cyanobacteria (8.2%), Actinobacteria (7.88%). By using the principal component analysis (PCA) and Heatmap correlation analysis, we found that environmental factors were key roles in controlling the microbial diversity in the Bohai Sea. Among them, the concentration of nitrate was particularly significant according to the Mantel test analysis. [Conclusion] The microbial diversity in the three bays of Bohai Sea was very rich and multifarious. The population structure and species in the Laizhou Bay is the most complex and abundant among these three bays, and then it is Bohai Bay and Liaodong Bay. There was a significant correlation among microbial diversity, environmental factors and the spatial distribution. Above all, this study will provide a theoretical basis for further protection and ecological development of Bohai Sea.

Abstract:[Background] The Synechococcus is a kind of single celled cyanobacteria grown in ocean. Because of its rapid growth, it is used to purify sewage. In order to reduce the cost, the immobilized method has been adopted in production. However, what does happen in physiological and biochemical characteristics of Synechococcus after be immobilized? There is no report yet. [Objective] This paper studied the changes of physiological and biochemical characteristic and purifying sewage ability of Synechococcus after immobilization in order to provide scientific basis for promoting the application of Synechococcus. [Methods] The Synechococcus was immobilized by sodium alginate and calcium chloride. The growth rate of Synechococcus was determined by microscopic observation. The net photosynthetic efficiency of Synechococcus was detected by dissolved oxygen analyzer. The nitric oxide (NO) content was detected by fluorescence method. The content of chlorophyll and protein, the activity of nitrate reductase (NR) and Rubisco carboxylase and the index of water-quality were detected by spectrophotometer method. [Results] The maximum specific growth rate of Synechococcus was decreased by 24.30% by immobilization. The net photosynthetic rate of immobilized Synechococcus decreased from 9.10% to 29.10% in comparison with that of free Synechococcus. However, there was no significant difference in chlorophyll content between free and immobilizing Synechococcus. The activity of Rubisco carboxylase and NR were decreased by 25.70% and 40% respectively and the NO content was decreased by 32.10% by immobilization process. The removing to total nitrogen, total phosphorus, ammonia nitrogen and nitrite in Takifug rubripes culturing wastewater by Synechococcus were decreased by 30.00%, 17.70%, 20.20% and 21.20% respectively by immobilization process, but it had no effect on removing nitrate and chemical oxygen demand (COD). It is speculated that the immobilization process decreased Rubisco carboxylase activity in Synechococcus by decreasing NO content which regulating Rubisco carboxylase activity by post transcriptional modification. [Conclusion] The activity of NR was inhibited by immobilization process, which resulted in the decrease of NO content, and the latter inhibited the Rubisco activity, akey enzyme of photosynthesis, by posttranscriptional modification. The decrease of Rubisco activity led to the decrease of photosynthetic efficiency, and finally results in the decrease in growth rate and purifying sewage ability of immobilizing Synechococcus.

Abstract:[Background] The bacterial biofilm has gained widespread application in waste water treatment field. However, the bacterial strains used in marine aquaculture are lack of the adapation to the high salt environment. Marichromatium gracile YL28 isolated mangrove is a true salt-requirement marine bacterium. Strain YL28 not only exhibits effective inorganic nitrogen removal ability, moreover, it has better biofilm-forming traits in response to illumination. [Objective] This work aims to investigate the characterizations of biofilm formation in marine purple sulfur bacterium Marichromatium gracile YL28 with the basis for the development and application of biofilm bioreactor in marine aquaculture. [Methods] The characteristics of biofilm formation and nitrogen removal of YL28 were examined by measuring biomass, dehydrogenase activity, polysaccharide content, protein content and inorganic nitrogen removal in simulated seawater system under anaerobically in the light condition. [Results] The planktonic bacterial biomass in culture suspension increased gradually with increasing time at 4 000 lx and followed by a rapid decrease in biomass before stationary period, and the biofilm-forming bacterial biomass reached a stabilization after stationary period, indicating that the planktonic bacteria had phototaxis and formed biofilm on the vessel wall. The biofilm forming rate of YL28 was highest (71.21%) under lower light illumination condition (500 lx) for 4 d. The light illumination in the range from 1 000?4 000 lx benefited for biofilm formation (0.60?0.80 mg/cm2 dry weight) although its biofilm forming rate (54.64%?68.66%) was not higher than low light condition. The light illumination of 5 000 lx had negative effect on the dehydrogenase activity of biofilm-forming bacteria. The activity of dehydrogenase of both the biofilm-forming bacteria and planktonic bacteria increased with the increasing light intensity, no significant difference in dehydrogenase activity between biofilm-forming bacteria and planktonic bacteria. The biofilm formation limited the illumination penetration into the vessel reactor, however, dehydrogenase activity of planktonic bacteria had not been greatly affected, indicating that the planktonic bacteria in culture suspension mainly grow on the surface of the biofilm and diffused freely to the culture fluid. With the changes in light intensity (1 000?5 000 lx) and incubation time (4?10 d), protein contents in extracellular polymeric substances (EPS) varied significantly. The highest protein content was observed at 3 000 lx, while polysaccharide content had little change. At 4 000 lx, the removal efficiency to ammonium and nitrite had no obvious difference between biofilm-forming bacteria and planktonic bacteria in culture suspension, whereas the nitrate removal efficiency by biofilm-forming bacteria was higher than that by planktonic bacteria. [Conclusion] YL28 had better biofilm-forming traits, and the mechanism of biofilm formation was that bacteria grew on the vessel wall in response to illumination. The biofilm-forming bacteria were capable of efficiently removing amonium and nitrite. This study shows great application potential for eliminating nitrogen pollution in marine aquaculture.

Abstract:[Background] Although microbial community in polyculture environment and animal intestines have been reported in previous studies, relationship of microbial community between intestinal microflora and polyculture environment needs to be deeply studied. [Objective] To study microbial communities of shrimp and clam intestine associated with polyculture environment. [Methods] Illumina sequencing was used to identify shift of taxonomic diversity among samples collected from two polyculture ponds in Putian. [Results] Bacterial community of shrimp intestine had very high similarity with clam intestine collected from two different polyculture pond. Shannon and Chao1 estimator supported the diversity results and suggested that the bacterial community in pond sediments was richer than those in pond water and animal intestine. Furthermore, bacterial community of animal intestines was dominated by Firmicute and Gammaproteobacteria, and culture water was dominated by Actinobacteria, Alphaproteobacteria and Bacteroidete, as well as by Gammaproteobacteria and Deltaproteobacteria in the underlying sediment. High abundance of potential probiotics, such as Lactococcus and Pseudomonas, was detected in animal intestine. Conditional pathogenic bacteria (such as Flavobacterium) had high abundance in polyculture environment, whereas little pathogenic bacteria were detected in animal intestine. Finally, high abundances of sulfate-reducing bacteria were found in the pond sediments, which may cause serious harm to aquaculture environment through producing hydrogen sulfide. [Conclusion] This study explores the significant difference of microbial community between polyculture environment and animal intestines and revealed the effect of polyculture environment on intestinal microflora. This study will provide reference for the optimization of polyculture structure and the diseases prevention of aquaculture animals.

Abstract:[Background] Agarases belong to glycoside hydrolases with broad application potentials in scientific research, health-food, pharmaceutical and cosmetic industries. Although a β-agarase YM01-3 from Catenovulum agarivorans YM01T was obtained in previous studies with an activity up to 1.14×104 U/mg, its catalytic mechanism is still unclear. [Objective] In order to clarify the catalytic mechanism of YM01-3, the key sites affecting the activity should be identified. [Methods] A random mutation library in Bacillus brevis was constructed by error-prone PCR to select mutants that completely lost enzyme activity. In total 227 mutants showing lower activity were selected from about 10 000 clones and 80 mutants were sequenced and analyzed. The putative key sites were confirmed by mutation construction. [Results] Compared to the original enzyme YM01-3, two mutants at 137 and 237 sites lost more than 90% catalytic activity. Y137 and D237, located in the catalytic cavity, were proved to be the novel key sites closely related to YM01-3 hydrolytic activity. [Conclusion] Our findings provide reference for further studying catalytic mechanism and molecular modification of β-agarase.

Abstract:[Background] L-isoleucine (L-Ile) and L-allo-isoleucine (L-allo-Ile) are two isomers in nature. The anti-infective antibiotic desotamides contain an L-allo-Ile moiety, and the aminotransferase DsaD and isomerase DsaE in their biosynthetic pathway catalyze the interconversion between L-Ile and L-allo-Ile. [Objective] To overexpress the aminotransferase DsaD and isomerase DsaE as a fusion protein DsaDE, and assay its activity to catalyze the interconversion between L-Ile and L-allo-Ile. [Methods] The coding region of dsaE and dsaD-linker (coding region of dasD containing 114 bp linker) were amplified by PCR; the purified DNA fragments were digested and ligated into pET28a(+), and the resulting recombinant plasmid was transformed into Escherichia coli BL21(DE3) to overexpress the fusion protein; the fusion protein DsaDE was purified by Ni-NTA affinity chromatography, then the activity of the fusion protein DsaDE was assayed using L-Ile or L-allo-Ile as substrate, respectively; the reaction mixtures were analyzed by high performance liquid chromatography (HPLC). [Results] PCR analyses, restriction endonuclease digestion and sequencing demonstrated that dasE and dsaD-linker were correctly ligated into pET28a(+); the fusion protein DsaDE with His6 tags at both the N-terminus and C-terminus was solubly produced in E. coli BL21(DE3) and purified to ~95% homogeneity. The purified fusion protein can catalyze the interconversion between L-Ile and L-allo-Ile efficiently in vitro. [Conclusion] The aminotransferase DsaD and isomerase DsaE were successfully overexpressed as a fusion protein; the fusion protein was readily purified by one-step Ni-NTA affinity chromatography; and purified fusion protein can catalyze the interconversion between L-Ile and L-allo-Ile efficiently, laying the foundation for further research on the industrial production of L-allo-Ile.

Abstract:[Background] Phosphopantetheinyl transferases (PPTases) are known to catalyze the transformation of peptidyl carrier proteins (PCPs) in nonribosomal peptide synthetases (NRPSs) from inactive apo-form to active holo-form, so that initiating the synthesis of nonribosomal polypeptides. [Objective] To identify the Sfp-type PPTases Bap from the marine sponge Dysidea avara symbiont Bacillus atrophaeus C89 and to confirm the function of Bap in activating PCPs in NRPSs. [Methods] Sfp-type PPTase Bap from B. atrophaeus C89 was identified by BLAST and amino acid sequences alignment. The bap gene was heterologously expressed in the sfp gene mutant strain Bacillus subtilis 168. The nonribosomal polypeptide surfactin was detected from the metabolites of the recombinant strains B. subtilis 168-bap. [Results] Bap was identified to be Sfp-type PPTases, and the production of surfactin was detected in recombinant strains B. subtilis 168-bap. [Conclusion] Our findings provide basis for heterologous expression of NRPSs gene clusters from marine B. atrophaeus.

Abstract:[Background] Cold-adapted enzymes from Antarctic and Arctic microorganisms offer several advantages over their mesophilic and thermophilic counterparts, and their properties make them valuable alternatives with industrial and biotechnological applications. Recent studies have shown that these cold environments represent an important reservoir of cold-adapted, complex polysaccharide-degrading marine bacteria that are a major source of novel polysaccharidases and diverse biological active compounds. In general, screening medium is not the best fermentation condition for producing enzyme bacteria, in order to improve enzyme production for research and application, the culture conditions of enzyme producing bacteria need to be optimized. [Objective] To identify a producing carrageenase Antarctic bacterium and optimize the fermentation conditions for carrageenase production. [Methods] The producing carrageenase Antarctic bacterium R11-5 was identified by 16S rRNA gene sequence and the fermentation conditions for carrageenase production of R11-5 was optimized using the method of response surface methodology. [Results] The 16S rRNA gene sequence analysis results showed that the Antarctic bacterium belonged to Alteromonas sp. and was named as Alteromonas sp. R11-5. In order to optimize the fermentation conditions of Alteromonas sp. R11-5, 7 single factors (inoculation amount, temperature, pH, carbon source, nitrogen source, carrageenan concentration and metal ions) were selected by single test. Four important factors (temperature, beef extract, carrageenan and calcium ion) influencing carrageenase production, which identified by initial experimental design of Plackett-Burman by Design-Expert software. Then Box-Behnken design and response surface analysis were adopted to further study the interaction among the variables and obtain optimal values that bring maximum carrageenase production. The optimal fermentation conditions were as follows: Temperature 15.0 °C, beef extract concentration 11.0 g/L, carrageenan concentration 3.0 g/L, Ca2+ concentration 5.0 mmol/L. The supernatant carrageenanse activity of optimized fermentation reached 87.193 U/mL, 1.8 times higher than that before optimization. [Conclusion] Carrageenanse production of Antarctic bacterium Alteromonas sp. R11-5 was improved after the fermentation conditions optimized using the method of response surface methodology. R11-5 was a promising candidate for industrial application.

Abstract:海洋占地球表面积的71%，海洋微生物对海洋及人类的生活有重大影响。由于特殊的生存环境，海洋微生物能产生陆栖微生物所不能产生的结构新颖、作用特殊的生物活性物质，有潜力应用于医疗、食品、环境保护等各个领域。海洋微生物研究充满了新的机遇，同时也面临着新的挑战。《微生物学通报》本期推出了“海洋微生物学主题刊”，旨在展现我国海洋微生物学研究的最新进展和成果，促进我国海洋微生物学的交流和发展。

Abstract:Marine animals are rich in microorganisms, which can help animal hosts synthesize some nutrients for the growth of hosts or compounds against other animals. Many compounds isolated from these microorganisms have good bioactivities and some are used in clinical treatment. Only few microorganisms were isolated from marine animals in the common conditions. Therefore, novel and efficient approaches are urgently required to promote the isolation and cultivation of uncultured microbes from marine animals. In this review, we provide an overview of the novel cultivation-based approaches while documenting their outcomes and limitations, including in situ method for cultivating microorganisms, electrical retrieval method and the refinement of culture media, and focusing on diffusion growth chambers, I-tip method, and micro-encapsulation approach, etc. These new approaches may lead to valuable advances in obtaining more new microbial strains and novel secondary metabolites, understanding the relationship between microorganisms and animal hosts, and making use of marine microbial resources.

Abstract:Algae and bacteria are inseparable in the ocean and have sophisticated interactions with each other, including mutualism, commensalism, antagonism, competition and parasitism. They are both major players in regulating the structure and function of marine ecosystem. Around a single algae cell, there usually exists a special microenvironment called “phycosphere” inhabited by specific microbial communities. The phycosphere is the main location for the interactions between algae and bacteria. The formation of specific microbial assembly within algal phycosphere has certain regularity. In natural ecosystem, the algae-bacteria interactions affect the dynamic process of the red tide and play important roles in water quality restoration. Moreover, algae and bacteria are the major drivers for marine carbon fixation and sequestration, their interactions have important impacts on marine carbon cycling. In view of the current research status of marine algae-bacteria interactions, several research prospects are discussed, for example, the role of viruses in regulating the relationship between algae and bacteria is poorly understood and awaiting a deep study.

Abstract:Recent studies have demonstrated that fucoidans and their degradation products possess various biological activities. Thus, the enzymes to hydrolyze fucoidans attract more attention. This review summarizes the recent achievements in the discovery, detection of activity, properties and application of fucoidan-degrading enzymes derived from marine microorganisms. Also, the approaches to understand fucoidan-degrading enzymes from marine microorganisms are introduced. It is expected that the rapid development of modern omics and structural biology will promote the research of novel fucoidan-degrading enzymes derived from marine microorganisms.