Key genes expression of reductive tricarboxylic acid cycle from deep-sea hydrothermal chemolithoautotrophic Caminibacter profundus in response to salinity, pH and O_2

CO2 fixation pathway of Caminibacter profundus, a chemolithoautotrophic e-Proteobacteria from deep-sea hydrothermal vent, was determined and characterized by genetic and enzymatic analyses. Gene expression of key enzymes for CO2 fixation in response to salinity, pH and O2 in Medium 829 were also investigated. The results demonstrate that C. profundus contained aclB, porA and oorA, the genes encoding key enzymes of reductive tricarboxylic acid （rTCA） cycle. However, genes fragments of cbbL and cbbMencoding key enzyme of Calvin cycle were not recovered. Key enzymatic activities of ATP citrate lyase （ACL）, pyruvate： ferredoxin oxidoreductase （POR） and 2-oxoglutarate： ferredoxin oxidoreductase （OOR） were also present in C. profun- dus. The combination of genetic and enzymatic analyses confirm that C. profundus adopted rTCA cycle for carbon assimilation. The results of aclB and oorA relative expressions of C. profundus demonstrate that the ranges of environmental factors for high genes expression were sea salt 3.0%-5.0% （optimum 3.0%）, pH 5.0-6.5（optimum pH 6.5）, anaerobic to microaerobic conditions （optimum 1.0% 02）. Gene expression pat- terns under different conditions show similar patterns with bacterial growth, revealing that key rTCA cycle genes provided molecular basis for bacterial growth and propagation. Our results suggest that C. profun- dus could regulate key genes of rTCA cycle for carbon assimilation and energy metabolism in response to environmental fluctuations in hydrothermal vent.

Screening and Characterization of the Alkaline Protease Isolated from PLI-1, a Strain of Brevibacillus sp. Collected from Indonesia's Hot Springs

A total of 69 strains of thermophilic bacteria were isolated from water, soil and sediment samples from three Indonesia's hot spring areas (Pantai cermin, Kalianda and Banyu wedang) by using Minimal Synthetic Medium (MSM). The extreme thermophile Brevibacillus sp. PLI-1 was found to produce extracellular thermophilic alkaline protease with optimal activity at 70℃ and pH 8.0-9.0. The molecular weight of the protease was estimated to be around 56 kD by SDS-PAGE. The maximum activity of the protease was 26.54 U mL-1. The protease activity did not decrease after 30 min and still retained more than 70% of relative activity after 60 min at 70℃ and pH 8.0. The ion Mg2+ was found to promote protease activity at both low and high concentrations, whereas Cu2+ and Zn2+ could almost completely inhibit the activity. Divalent cation chelator EDTA inhibited the enzyme activity by 55.06% ± 0.27%, while the inhibition caused by PMSF, Leupeptin, Pepstain A and Benzamidine were 66.78% ± 3.25%, 52.37% ± 0.25%, 62.47% ± 2.96% and 50.99% ± 0.24%, respectively. Based on these observations, the enzyme activity was conspicuously sensitive to the serine and cysteine protease inhibitors. All these results indicated that the protease isolated from the strain PLI-1 was a thermophilic protease and had a high-temperature stability and a pH stability.

Expression and characterization of a bifunctional alginate lyase named Al163 from the Antarctic bacterium Pseudoalteromonas sp. NJ-21

In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amino acid sequence encoded by al163 belongs to the polysaccharide lyase 6(PL-6) family and has a molecular mass of about 80 kDa. Recombinant enzyme was purified by Ni-Sepharose affinity chromatography. Recombinant Al163 exhibited maximum activity(258 U/mg) at pH 7.0 and 40℃, and thermal stability assays showed retention of almost 90% activity after incubation at 30℃ for 30 min. Al163 activity was stimulated by Cd^(2+), Ca^(2+), Fe^(3+), and Mn^(2+), but inhibited by Cu^(2+), Si^(2+), Fe^(2+), and Ni^(2+). Thin-layer chromatographic analysis indicated that Al163 degraded sodium alginate, poly M, and poly G, generating disaccharides and trisaccharides as the final products. Only a few bacterial strains that produce a bifunctional alginate lyase have been reported. Our results indicate that recombinant Al163 exhibits broad substrate specificity and its products exhibit low degrees of polymerization. Both properties imply high potential for use of the enzyme in several industrial fields, including cosmetics and pharmaceuticals, based on the high demand for biologically active oligosaccharides.

Changes of proteins in the Antarctic ice microalga Chlamydomonas sp. cultured under UV-B radiation stress

Antarctic ice microalga Chlamydomonas sp. can thrive undisturbed under high UV radiation in the Antarctic ice layer. However, it is unknown that the initial adaptation mechanisms in protein level occurring in response to high UV radiation. Global-expression profiling of proteins in response to stress was analyzed by two-dimensional electrophoresis （2-DE） and image analysis. In the 2-DE analysis, protein preparation is the key step. Three different protein extract methods were compared, and the results showed that the trichloroacetic acid （TCA）-acetone fractional precipitation method was the fittest one. At the same time, the proteins in Chlamydomonas sp. were compared in 2-DE way, and the synthesis of seven protein spots was found disappeared and 18 decreased after exposed to UV-B radiation. In addition, 14 protein spots were enhanced or induced, among which two new peptides （20 and 21 kDa） appeared whose isoelectric point （pI） was 7.05 and 4.60 respectively. These changed proteins might act as key role in the acclimation of Antarctic ice microalga to UV-B radiation

Transcriptomic analysis reveals the effect of the exopolysaccharide of Psychrobacter sp.B-3 on gene expression in RAW264.7 macrophage cells

B-3 exopolysaccharide is extracted from the Antarctic psychrophilic bacterium Psychrobacter sp. B-3. We have previously shown that it activates macrophages and affects their immunoregulatory activities. To determine what genes are affected during this process, we detected the genes differentially expressed in cells of RAW264.7 macrophages treated with B-3 exopolysaccharide by transcriptomic analysis. B-3 exopolysaccharide treatment caused differential expression of 420 genes, of which 178 were up-regulated and 242 were down-regulated. These genes were shown to be involved in many aspects of cell function, mainly metabolism and immunity. Genes were enriched in multiple immune-related pathways, and the most significantly enriched genes were involved in antigen processing and presentation pathways. The pathway in which differentially expressed genes were the most significantly enriched was the metabolic pathway; specifically, the expression of many metabolic enzyme genes was altered by B-3 exopolysaccharide treatment. Additionally, the genes involved in metabolisms of amino acids, carbohydrates, lipids and nucleotides, varied to certain degrees. B-3 exopolysaccharide, therefore, appears to directly affect the immune function of RAW264.7 macrophages as an immunostimulant, or to indirectly change intracellular metabolism. This is the first study to determine the effect of an Antarctic psychrophilic bacterial exopolysaccharide on RAW264.7 macrophages. Our findings provide an important reference for research into the regulation of macrophage immune function by different polysaccharides.

Identification of SNARE proteins in fish—Tilapia Oreochromis niloticus

SNARE proteins are a group of membrane-associated proteins involved in exocytosis, secretion and membrane trafficking events in eukaryotic cells. Research on SNARE protein biology has become a more attractive field in recent years, which is applied to marine biology specifically to the fish Tilapia （Oreochromis niloticus）. Plasma membrane fractions of different tissues of Tilapia, including brain, liver-pancreas, intestine, skin and muscle, were extracted, and immuno-decorated with isoform-specific antibodies to the SNARE families and associated proteins. The presence of Syntaxins - 1 A, 2 and 3, SNAP - 23 and SNAP - 25, VAMP - 2, Munc - 18 - 1 and Munc - 13 in the brain was identified, which were differentially distributed in the other organ tissues of the fish Tilapia. The distinct distribution of SNARE and associated proteins will serve as the basis for further investigation into their special secretory function in these tissues of the fish.

Microbial community structure of Arctic seawater as revealed by pyrosequencing

This study aimed to determine the microbial community structure of seawater in（ICE-1） and out（FUBIAO） of the pack ice zone in the Arctic region.Approximate 10 L seawater was filtrated by 0.2 μm Whatman nuclepore filters and the environmental genomic DNA was extracted.We conducted a detailed census of microbial communities by pyrosequencing.Analysis of the microbial community structures indicated that these two samples had high bacterial,archaeal and eukaryotic diversity.Proteobacteria and Bacteroidetes were the two dominant members of the bacterioplankton community in both samples,and their relative abundance were 51.29% and 35.39%,72.95%and 23.21%,respectively.Euryarchaeota was the most abundant archaeal phylum,and the relative abundance was nearly up to 100% in FUBIAO and 60% in ICE-1.As for the eukaryotes,no_rank_Eukaryota,Arthropoda and no_rank_Metazoa were the most abundant groups in Sample FUBIAO,accounting for 85.29% of the total reads.The relative abundance of the most abundant phylum in Sample ICE-1,no_rank_Eukaryota and no_rank_Metazoa,was up to 90.69% of the total reads.Alphaproteobacteria,Flavobacteria and Gammaproteobacteria were the top three abundant classes in the two samples at the bacterial class level.There were also differences in the top ten abundant bacterial,archaeal and eukaryotic OTUs at the level of 97% similarity between the two samples.

Transcriptome analysis of the Antarctic psychrotrophic bacterium Psychrobacter sp. G in response to temperature stress

The key functional genes involved in temperature adaption of the Antarctic psychrotrophic bacterium Psychrobacter sp. G. were identified by transcriptomic sequencing. We analyzed the global transcriptional profile of Psychrobacter sp. G under cold stress（0°C） and heat stress（30°C）, with the optimal growth temperature 20°C as the control. There were large alterations of the transcriptome profile, including significant upregulation of 11 and 12 transcripts as well as significant downregulation of 47 and 42 transcripts in the cold and heat stress groups, respectively, compared to the control. The expression of various genes encoding enzymes and transcriptional regulators, including Pfp I and Tet R family transcriptional regulators under heat stress, as well as the expression of DEAD/DEAH box helicase and the Icl R family of transcriptional regulators under cold stress, were upregulated significantly. The expression of several genes, most affiliated with Ton Bdependent receptor and siderophore receptor, was downregulated significantly under both heat and cold stress. Many of the genes associated with the metabolism of fatty acid and ABC transporters were regulated differentially under different temperature stress. The results of this survey of transcriptome and temperature stress-relevant genes contribute to our understanding of the stress-resistant mechanism in Antarctic bacteria.

Cloning and characterization of a new endo-type polyG-specific alginate lyase from bacteria Vibrio sp. QD-5

Seven bacterial clones with alginate-utilizing activity were isolated from rotten kelp. By activity test, the Vibrio sp. QD-5 with the potential alginate-degrading capability was chosen to carry out the draft genome sequencing, and the result showed that the Vibrio sp. QD-5 containing an alginate lyase gene cluster. One of these genes, aly-IV, was cloned and characterized for the first time. After overexpression, Aly-IV, with a molecular mass of about 62 kDa and a theoretical isoelectric point (pI) of 5.12, was purified to a specific activity of 1 256.78 U/mg and showed highest activity at 35°C in the Tris-HCl buffer at pH of 8.9. Moreover, the enzyme activity was enhanced by the metal ions of Na+, K+ and Mg2+ under certain concentration. Aly-IV degraded favorably polyG blocks in an endo-type, yielding monomer and dimer as the main products. Due to its high substrate specificity, Aly-IV could be used as a potential tool for production of polyG oligosaccharides with low degree of polymerization (DP) and for determining the fine structure of alginate.

Activation of macrophages by an exopolysaccharide isolated from Antarctic P sychrobacter sp.B-3

An exopolysaccharide(EPS) was isolated and purified from an Antarctic psychrophilic bacterium B-3,identified as Psychrobacter sp.,and the activation of RAW264.7 cells by B-3 EPS was investigated.The results show that B-3 EPS,over a certain concentration range,promoted cell viability,nitric oxide production,tumor necrosis factor(TNF)a secretion,and phagocytic ability.Furthermore,TAK-242,an inhibitor of the toll-like receptor 4(TLR4) significantly reduced nitric oxide production by these cells after stimulation with B-3 EPS.Moreover,B-3 EPS induced p65 phosphorylation and IκBα degradation in these cells.In conclusion,B-3 EPS might have activated RAW264.7 cells by combining with TLR4 on cell surface and triggering activation of NF-κB signaling pathways,implying that this EPS could activate macrophages and regulate initial immune response.

The Effect of Organic Carbon on Soil Bacterial Diversity in an Antarctic Lake Region

This study assessed the effects of changes in organic carbon content on soil bacterial community composition and diversity in the Antarctic Fildes Peninsula.16 S rRNA gene sequencing was performed to investigate bacterial community composition.Firstly,we found that organic carbon(OrC)and nutrients showed an increasing trend in the lake area.Secondly,soil geochemistry changes affected microbial composition in the soil.Specifically,we found 3416 operational taxonomical units(OTUs)in 300 genera in five main phyla:Proteobacteria,Actinobacteria,Acidobacteria,Chloroflexi,and Bacteroidetes.Although the diversity was similar among the four sites,the composition was different.Among them,Hungateii content changed very significantly,from 16.67% to 33.33%.Canonical correspondence analysis showed that most measured geochemical factors were relevant in structuring microbiomes,and organic carbon concentration showed the highest correlation,followed by NO3^--N.Hungateii was significantly correlated with the content of organic carbon.Our finding suggested organic carbon played an important role in soil bacterial communities of the Antarctic coastal lake region.

Genome-wide transcriptional response of the Arctic bacteriumPseudoalteromonas sp. A2 to oxidative stress induced byhydrogen peroxide

Oxidative stress is one of the major challenges faced by Arctic marine bacteria due to the high oxygen concentration of seawater, low temperatures and UV radiations. Transcriptome sequencing was performed to obtain the key functional genes involved in the adaptation to oxidative stress induced by hydrogen peroxide in the Arctic bacterium Pseudoalteromonas sp. A2. Exposure to 1 mmol/L H2O2 resulted in large alterations of the transcriptome profile, including significant up-regulation of 109 genes and significant down-regulation of 174 genes. COG functional classification revealed that among the significantly regulated genes with known function categories, more genes belonging to posttranslational modification, protein turnover and chaperones were significantly up-regulated, and more genes affiliated with chaperones and amino acid transport and metabolism were significantly down-regulated. It was notable that the expressions of eighteen genes affiliated with flagella and four genes affiliated with heat shock proteins were significantly up-regulated. Meanwhile, the expression of nine genes belonging to cytochrome and cytochrome oxidase, and five genes belonging to Ton B-dependent receptor,were significantly down-regulated. Among the eighteen genes with antioxidant activity categorized by GO analysis, the expression of one gene was significantly up-regulated; however, the expressions of two genes were significantly down-regulated. Briefly, RNA-Seq indicated that, except for the classical anti-oxidative genes and stress proteins, genes affiliated with flagella and function unknown played important roles in coping with oxidative stress in Pseudoalteromonas sp. A2. This overall survey of transcriptome and oxidative stress-relevant genes can contribute to understand the adaptive mechanism of Arctic bacteria.

Bacterial diversity in Arctic marine sediment determined by culture-dependent and-independent approaches

Bacterial diversity in surface sediment from the Arctic Ocean was investigated by culture-dependent and-independent approaches. Conventional culture-dependent techniques revealed 11 strains based on their distinct morphological characteristics on marine Zobell 2216E agar plates. Phylogenetic analysis showed that these isolates belonged to three major lineages of the Bacteria,γ-proteobacteria, Bacteroidetes and Actinobacteria, and that they included 10 genera. Most isolates were psychrotrophic, and NaCl was not necessary for their growth. Furthermore, they exhibited activity of at least one extracellular hydrolytic enzyme at 4＆#176;C and had various abilities to assimilate carbon sources. A total of 67 phylotypes were detected among 142 clones based on the 16S rRNA library of the total community DNA and grouped into nine major lineages of bacteria. Phylotypes afifliated withγ-,δ-andε-proteobacteria accounted for 36.7%, 21.8%and 16.9%of the total clones, respectively. The rest of the clones belonged to Bacteroidetes,α-proteobacteria, Actinobacteria, Fusobacteria, Nitrospirae and an unclassiifed group.

Effects of Crop Development on the Emission of Volatiles in Leaves of Lycopersicon esculentum and Its Inhibitory Activity to Botrytis cinerea and Fusarium oxysporum

Volatiles emitted from the leaves of Lycopersicon esculentum at the two-, ten-leaf and anthesis periods were collected by a gas absorbing method and analyzed by gas chromatography （GC）-mass spectrometry. In total, 33 compounds of volaUles emitted from three developmental stage plants were separated and identified, and quantitatively analyzed by the internal standard addition method. All of the samples of volatile were found to be rich in monoterpenes and sesquiterpenes.β-phellandrene and caryophyllene predominated in the volatiles of the leaves of plants at the two- and ten-leaf stages. Furthermore, （E）-2-hexenal were the dominant components in the volatiles emitted from anthesis plants. The results of volatiles analyzed show that the compositions varied depending on the developmental stages. The volatiles emitted from crushed tomato leaves of plants at the anthesis stage had the most strongly inhibitory activity against the spore germination and hyphal growth of Botrytis cinerea and Fusarium oxysporum, followed by ten- and two-leaf plants. However, the activity of volatiles, emitted from the leaves of plants at the two-leaf stage, in inhibiting F. oxysporum was greater than B. cinerea.

Activities of principal photosynthetic enzymes in green macroalga Ulva linza:functional implication of C_4 pathway in CO_2 assimilation

The green-tide-forming macroalga Ulva linza was profiled by transcriptome sequencing to ascertain whether the alga carries both C3 and C4 photosynthesis genes.The key enzymes involved in C4 metabolism including pyruvate orthophosphate dikinase(PPDK),phosphoenolpyruvate carboxylase(PEPC),and phosphoenolpyruvate carboxykinase(PCK) were found.When measured under normal and different stress conditions,expression of rbcL was higher under normal conditions and lower under the adverse conditions,whereas that of PPDK was higher under some adverse conditions,namely desiccation,high salinity,and low salinity.Both ribulose-1,5-biphosphate carboxylase(RuBPCase) and PPDK were found to play a role in carbon fixation,with significantly higher PPDK activity across the stress conditions.These results suggest that elevated PPDK activity alters carbon metabolism in U.linza leading to partial operation of the C4 carbon metabolism,a pathway that,under stress conditions,probably contributes to the hardy character of U.linza and thus to its wide distribution.

Differential physiological responses of the coastal cyanobacterium Synechococcus sp. PCC7002 to elevated pCO2 at lag, exponential,and stationary growth phases

We studied the effects of expected end-of-the-century p CO_2(1000 ppm)on the photosynthetic performance of a coastal marine cyanobacterium Synechococcus sp.PCC7002 during the lag,exponential,and stationary growth phases.Elevated p CO_2significantly stimulated growth,and enhanced the maximum cell density during the stationary phase.Under ambient p CO_2conditions,the lag phase lasted for 6 days,while elevated p CO_2shortened the lag phase to two days and extended the exponential phase by four days.The elevated p CO_2increased photosynthesis levels during the lag and exponential phases,but reduced them during the stationary phase.Moreover,the elevated p CO_2reduced the saturated growth light(Ik)and increased the light utilization efficiency(α)during the exponential and stationary phases,and elevated the phycobilisome:chlorophyll a(Chl a)ratio.Furthermore,the elevated p CO_2reduced the particulate organic carbon(POC):Chl a and particulate organic nitrogen(PON):Chl a ratios during the lag and stationary phases,but enhanced them during the exponential phase.Overall,Synechococcus showed differential physiological responses to elevated p CO_2during different growth phases,thus providing insight into previous studies that focused on only the exponential phase,which may have biased the results relative to the effects of elevated p CO_2in ecology or aquaculture.