Single-cell enabled comparative genomics of a deep ocean SAR11 bathytype

Bacterioplankton of the SAR11 clade are the most abundant microorganisms in marine systems, usually representing 25% or more of the total bacterial cells in seawater worldwide. SAR11 is divided into subclades with distinct spatiotemporal distributions (ecotypes), some of which appear to be specific to deep water. Here we examine the genomic basis for deep ocean distribution of one SAR11 bathytype (depth-specific ecotype), subclade Ic. Four single-cell Ic genomes, with estimated completeness of 55%–86%, were isolated from 770 m at station ALOHA and compared with eight SAR11 surface genomes and metagenomic datasets. Subclade Ic genomes dominated metagenomic fragment recruitment below the euphotic zone. They had similar COG distributions, high local synteny and shared a large number (69%) of orthologous clusters with SAR11 surface genomes, yet were distinct at the 16S rRNA gene and amino-acid level, and formed a separate, monophyletic group in phylogenetic trees. Subclade Ic genomes were enriched in genes associated with membrane/cell wall/envelope biosynthesis and showed evidence of unique phage defenses. The majority of subclade Ic-specfic genes were hypothetical, and some were highly abundant in deep ocean metagenomic data, potentially masking mechanisms for niche differentiation. However, the evidence suggests these organisms have a similar metabolism to their surface counterparts, and that subclade Ic adaptations to the deep ocean do not involve large variations in gene content, but rather more subtle differences previously observed deep ocean genomic data, like preferential amino-acid substitutions, larger coding regions among SAR11 clade orthologs, larger intergenic regions and larger estimated average genome size.     

L-myo-inosose-1 as a probable intermediate in the reaction catalyzed by myo-inositol oxygenase

In previous investigations, it was necessary to have Fe(II) and cysteine present in order to assay the catalytic activity of purified hog kidney myo-inositol oxygenase. In the present study it was found that, if this purified nonheme iron enzyme is slowly frozen in solution with glutathione and stored at -20 degrees C, it is fully active in the absence of activators if catalase is present to remove adventitious H2O2. With this simpler assay system it was possible to clarify the effects of several variables on the enzymic reaction. Thus, the maximum velocity is pH-dependent with a maximum around pH 9.5, but the apparent Km for myo-inositol (air atmosphere) remains constant at 5.0 mM throughout a broad pH range. The enzyme is quite specific for its substrate myo-inositol, is very sensitive to oxidants and reductants, but is not affected by a variety of complexing agents, nucleotides, sulfhydryl reagents, etc. In other experiments it was found that L-myo-inosose-1, a potential intermediate in the enzymic reaction, is a potent competitive inhibitor (Ki = 62 microM), while other inososes and a solution thought to contain D-glucodialdehyde, another potential intermediate, are weak inhibitors. Also, both a kinetic deuterium isotope effect (kH/kD = 2.1) and a tritium isotope effect (kH/kT = 7.5) are observed for the enzymic reaction when [1-2H]- and [1-3H]-myo-inositol are used as reactants. These latter results are considered strong evidence that the oxygenase reaction proceeds by a pathway involving L-myo-inosose-1 as an intermediate rather than by an alternative pathway that would have D-glucodialdehyde as the intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Single-cell genomics shedding light on marine Thaumarchaeota diversification

Previous studies based on analysis of amoA, 16S ribosomal RNA or accA gene sequences have established that marine Thaumarchaeota fall into two phylogenetically distinct groups corresponding to shallow- and deep-water clades, but it is not clear how water depth interacts with other environmental factors, including light, temperature and location, to affect this pattern of diversification. Earlier studies focused on single-gene distributions were not able to link phylogenetic structure to other aspects of functional adaptation. Here, we analyzed the genome content of 46 uncultivated single Thaumarchaeota cells sampled from epi- and mesopelagic waters of subtropical, temperate and polar oceans. Phylogenomic analysis showed that populations diverged by depth, as expected, and that mesopelagic populations from different locations were well mixed. Functional analysis showed that some traits, including putative DNA photolyase and catalase genes that may be related to adaptive mechanisms to reduce light-induced damage, were found exclusively in members of the epipelagic clade. Our analysis of partial genomes has thus confirmed the depth differentiation of Thaumarchaeota populations observed previously, consistent with the distribution of putative mechanisms to reduce light-induced damage in shallow- and deep-water populations.     

乙型脑炎病毒SA14—14—2株E蛋白主要抗原片段的高效表达

在本实验室已构建的原核表达载体(含乙脑疫苗株SA14-14-2株E蛋白基因主要抗原片段)的基础上用巴斯德毕赤酵母系统表达,该片段长1113bp,编码371个氨基酸残基,将其亚克隆入酵母表达载体pPICZα-A,以电穿孔法转化酵母X-33,用Zeocin平板筛选重组子,经甲醇诱导表达后,SDS-PAGE和免疫印迹分析表达产物.由于糖基化不同,所表达产物有两种,其相对分子质量分别为44kDa和50kDa,表达量较高,约为290mg/L,经Western印迹验证,有较好的抗原性.在ELISA试验中,我们直接以PBS透析后的酵母上清包被,能够很明显地区分出乙脑阴阳性血清,与RT-PCR检测的相符率达95%,为制备JEV的诊断抗原和基因工程疫苗提供了依据.