Phylogenetic analysis of bacterial community in deep-sea sediment from the western Pacific “warm pool”

A depth profile of bacterial community structure in one deep-sea sediment core of the western Pacific ＂warm pool＂ （WP） was investigated and compared with that in a sediment sample from the eastern Pacific （EP） by phylogenetic analysis of 16S rDNA fragments. Five bacterial 16S rDNA clone libraries were constructed, and 133 clones with different restriction fragment length polymorphism （RFLP） patterns were sequenced. A phylogenetic analysis of these sequences revealed that the bacterial diversity in a sample from the WP was more abundant than that in the EP sample. The bacterial population in the sediment core of WP was composed of eight major lineages of the domain bacteria. Among them the γ-Proteobacteria was the predominant and most diverse group in each section of WP sediment core, followed by the α-Proteobacteria. The genus Colwellia belonging to γ-Proteobacteria was predominant in this sample. The shift of bacterial communities among different sections of the WP sediment core was δ-, ε-Proteobacteria, and Cytopahga-Flexibacteria-Bacteroides （CFB） group. The ratios between them in the bacterial communities all showed inversely proportional to the depth of sediment. The sequences related to sulphate reducing bacteria （SRB） were detected in every section. The bacterial community structure in this sediment core might be related to the environmental characteristics of the surface seawater of the western Pacific WP.

Phylogenetic diversity of TypeⅠpolyketide synthase genes from sediments of Ardley Island in Antarctica

The diversity of modular polyketide synthase （PKS） genes in sediments of Ardley Island in Antarctica, was studied by restriction fragment length polymorphism （RFLP） analysis. Phylogenetic analysis of 14 amino acid （AA） sequences indicates that the identified ketosynthase （KS） domains were clustered with those from diverse bacterial groups, including Cyanobacteria, γ-Proteobacteria, Actinobacteria, Firmicutes, and some unidentified microorganisms from marine sponge, bryozoan and other environmental samples. The obtained KS domains showed 43%–81% similarity at the AA level to reference sequences in GenBank. Six identified KS domains showed diverse sequences of the motif （VQTACSTS） that was used to identify the hybrid PKS/nonribosomal peptide synthetase （NRPS） enzyme complex, and formed a new branch. These results reveal a high diversity and novelty of PKS genes in antarctic sediments.

Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp.547: enzyme characterization and gene cloning

A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15~C and 30~C, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35~C and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride （PMSF） and hydrochloride 4-（2-aminoethyl）-benzenesulfonyl fluoride （AEBSF）, indicating that it was a serine protease. The presence of Cae＋ and Mnz＋ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid （AA） residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.

Coextraction of microbial metagenomic DNA and RNA from deep-sea sediment

A protocol to coextract the microbial metagenomic DNA and RNA from deep-sea sediment was developed for the microbiological study of environmental samples. The obtained pure metagenomic DNA with the size larger than 23 kb and stable RNA could be used directly for PCR and reverse transcription - PCR （ RT - PCR） respectively. The direct lysis including the treatments of SDS, proteinase and lysozyme was applied to acquiring the metagenomic DNA and RNA furthest. Prior to the lysis treatment, the glass bead and denaturing solution were added to enhance the lysis efficiency and keep the integrity of RNA respectively. Denaturing gradient gel electrophoresis （DGGE） was applied in accessing the microbial 16S rRNA diversity by PCR and RT -PCR amplification from a single extraction. The pattern obtained by this analysis revealed some differences between them, indicating the efficiency of the protocol in extracting the metagenomic DNA and total RNA from deep-sea sediment.

Phylogenetically diverse,acetaldehyde-degrading bacterial community in the deep sea water of the West Pacific Ocean

As a major aldehyde pollutant widely existing in industry and our daily life, acetaldehyde is more and more harmful to human health. As characteristic habitat niche, bacteria from deep sea environments are abundant and distinctive in heredity, physiology and ecological functions. Thus, the development of acetaldehyde-degrading bacteria from deep sea provides a new method to harness acetaldehyde pollutant. Firstly, in this study,acetaldehyde-degrading bacteria in the deep sea water of the West Pacific Ocean were enriched in situ and in the laboratory respectively, and then the diversity of uncultured bacteria was studied by using 16 S r RNA genes. Then acetaldehyde-degrading strains were isolated from two samples, including enrichment in situ and enrichment in laboratory samples of deep sea water from the West Pacific Ocean using acetaldehyde as the sole carbon source,and then the ability of acetaldehyde degradation was detected. Our results showed that the main uncultured bacteria of two samples with different enrichment approaches were similar, including Proteobacteria,Actinobacteria, Firmicutes, Cyanobacteria, but the structure of bacterial community were significant different.Four subgroups, α, γ, δ and ε, were found in Proteobacteria group. The γ-Proteobacteria was dominant（63.5%clones in laboratory enriched sample, 75% clones in situ enriched sample）. The species belonged to γ-Proteobacteria and their proportion was nearly identical between the two enrichment samples, and Vibrio was the predominant genus（45% in laboratory enriched sample, 48.5% in situ enriched sample）, followed by Halomonas（9% in situ enriched sample） and Streptococcus（6% in laboratory enriched sample）. A total of 12 acetaldehyde-degrading strains were isolated from the two samples, which belonged to Vibrio, Halomonas,Pseudoalteromonas, Pseudomonas and Bacillus of γ-Proteobacteria. Strains ACH-L-5, ACH-L-8 and ACH-S-12,belonging to Vibrio and Halomonas, have strong ability of acetaldehyde degradation, which could tolerate 1.5 g/L acetaldehyde and degrade 350 mg/L acetaldehyde within 24 hours. Our results indicated that bacteria of γ-Proteobacteria may play an important role in carbon cycle of deep sea environments, especial the bacteria belonging to Vibrio and Halomonas and these strains was suggested for their potentials in government of aldehyde pollutants.

Enhanced a novel β-agarase production in recombinant Escherichia coli BL21(DE3) through induction mode optimization and glycerol feeding strategy

Agarases are hydrolytic enzymes that act on the hydrolysis of agar and have a broad range of applications in food,cosmetics and pharmaceutical industries. In this study, a glycerol feeding strategy based on induction mode optimization for high cell density and β-agarase production was established, which could effectively control acetate yield. First, exponential feeding strategy of glycerol with different overall specific growth rates（μ） was applied in the pre-induction phase. The results showed that the low μ（μ=0.2） was suggested to be the optimal for cell growth and β-agarase production. Second, the effects of induction temperature and the inducer concentration on cell growth and β-agarase production were investigated in the post-induction phase. When induced by isopropyl-β-d-thiogalactoside（IPTG）, the strategy of 0.8 mmol/L IPTG induction at 20℃ was found to be optimal for β-agarase production. When cultivation was induced by continuous lactose feeding strategy of 1.0 g/（L·h）, the β-agarase activity reached 112.5 U/mL, which represented the highest β-agarase production to date.Furthermore, the β-agarase was capable of degrading G. lemaneiformis powder directly to produce neoagarooligosaccharide, and the hydrolysates were neoagarotetraose（NA4） and neoagarohexaose（NA6）. The overall research may be useful for the industrial production and application of β-agarase.

需钠弧菌WPAGA4菌株3条琼胶酶基因克隆、表达与酶学性质分析

【目的】本文拟从西太平洋深海沉积物中分离得到的需钠弧菌(Vibrio natriegens) WPAGA4菌株中克隆并表达3条β-琼胶酶基因agaW3418、agaW3419和agaW3472,并对其酶学性质进行研究。【方法】通过克隆表达技术将得到的3条琼胶酶基因在大肠杆菌BL21(DE3)细胞中进行表达,通过二硝基水杨酸(DNS)法测定重组琼胶酶的酶活,并对其中活性最优的一种琼胶酶进行热稳定性和产酶条件优化。【结果】三种琼脂酶均为属于GH50家族。AgaW3418、AgaW3419和AgaW3472在温度分别为50、60和30℃以及pH值分别为6.0、7.0和7.0条件下,发挥作用的能力最强。其中琼胶酶AgaW3472表现出最优的酶活性质,在20℃下保持良好的稳定性,且在SOB (super optimal broth)培养基条件下以1%(质量体积分数)的乳糖作为碳源,同时添加20 mmol/L MgCl_(2),并将诱导温度和异丙基-1硫代-β-D-半乳糖苷(isopropyl-1 thio-β-D-galactoside, IPTG)浓度分别设置为37℃和0.1 mmol/L时能够获得最高的表达量。【结论】三条重组酶具有琼胶酶活性,为WPAGA4菌株代谢琼胶多糖并参与海洋碳循环过程提供物质基础。琼胶酶AgaW3472具有较高的酶活性、低温适应性与稳定性,为琼胶降解相关产业的发展提供了潜在的生物工具酶。