Project description:Enterobacter sp. FY-07 can produce bacterial cellulose (BC) under aerobic and anaerobic conditions. Three potential BC synthesis gene clusters (bcsI, bcsII and bcsIII) of Enterobacter sp. FY-07 have been predicted using genome sequencing and comparative genome analysis, in which bcsIII was confirmed as the main contributor to BC synthesis by gene knockout and functional reconstitution methods. Protein homology, gene arrangement and gene constitution analysis indicated that bcsIII had high identity to the bcsI operon of Enterobacter sp. 638; however, its arrangement and composition were same as those of BC synthesizing operon of G. xylinum ATCC53582 except for the flanking sequences. According to the BC biosynthesizing process, oxygen is not directly involved in the reactions of BC synthesis, however, energy is required to activate intermediate metabolites and synthesize the activator, c-di-GMP. Comparative transcriptome and metabolite quantitative analysis demonstrated that under anaerobic conditions genes involved in the TCA cycle were downregulated, however, genes in the nitrate reduction and gluconeogenesis pathways were upregulated, especially, genes in three pyruvate metabolism pathways. These results suggested that Enterobacter sp. FY-07 could produce energy efficiently under anaerobic conditions to meet the requirement of BC biosynthesis.
Project description:Study of the potential of Antarctic microorganisms for use in bioremediation is of increasing interest due to their adaptations to harsh environmental conditions and their metabolic potential in removing a wide variety of organic pollutants at low temperature. In this study, the psychrotolerant bacterium <i>Rhodococcus</i> sp. strain AQ5-07, originally isolated from soil from King George Island (South Shetland Islands, maritime Antarctic), was found to be capable of utilizing phenol as sole carbon and energy source. The bacterium achieved 92.91% degradation of 0.5 g/L phenol under conditions predicted by response surface methodology (RSM) within 84 h at 14.8 °C, pH 7.05, and 0.41 g/L ammonium sulphate. The assembled draft genome sequence (6.75 Mbp) of strain AQ5-07 was obtained through whole genome sequencing (WGS) using the Illumina Hiseq platform. The genome analysis identified a complete gene cluster containing <i>catA</i>, <i>catB</i>, <i>catC</i>, <i>catR</i>, <i>pheR</i>, <i>pheA2,</i> and <i>pheA1</i>. The genome harbours the complete enzyme systems required for phenol and catechol degradation while suggesting phenol degradation occurs via the ?-ketoadipate pathway. Enzymatic assay using cell-free crude extract revealed catechol 1,2-dioxygenase activity while no catechol 2,3-dioxygenase activity was detected, supporting this suggestion. The genomic sequence data provide information on gene candidates responsible for phenol and catechol degradation by indigenous Antarctic bacteria and contribute to knowledge of microbial aromatic metabolism and genetic biodiversity in Antarctica.
Project description:BACKGROUND:Heterogeneity of oil-bearing formations is one of major contributors to low oil recovery efficiency globally. Long-term water flooding will aggravate this heterogeneity by resulting in many large channels during the exploitation process. Thus, injected water quickly flows through these large channels rather than oil-bearing areas, which ultimately leads to low oil recovery. This problem can be solved by profile control using polymer plugging. However, non-deep profile control caused by premature plugging is the main challenge. Here, a conditional bacterial cellulose-producing strain, namely Enterobacter sp. FY-0701, was constructed for deep profile control to solve the problem of premature plugging. Its deep profile control and oil displacement capabilities were subsequently identified and assessed. RESULTS:The conditional bacterial cellulose-producing strain Enterobacter sp. FY-0701 was constructed by knocking out a copy of fructose-1, 6-bisphosphatase (FBP) encoding gene in Enterobacter sp. FY-07. Scanning electron microscope observation showed this strain produced bacterial cellulose using glucose rather than glycerol as the sole carbon source. Bacterial concentration and cellulose production at different locations in core experiments indicated that the plugging position of FY-0701 was deeper than that of FY-07. Moreover, enhanced oil recovery by FY-0701 was 12.09%, being 3.86% higher than that by FY-07 in the subsequent water flooding process. CONCLUSIONS:To our knowledge, this is the first report of conditional biopolymer-producing strains used in microbial enhance oil recovery (MEOR). Our results demonstrated that the conditional bacterial cellulose-producing strain can in situ produce biopolymer far from injection wells and plugs large channels, which increased the sweep volume of injection water and enhance oil recovery. The construction of this strain provides an alternative strategy for using biopolymers in MEOR.
Project description:Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (also known as CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A and F740L and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering three PB1 and PA mutations into the parental CA/07 strain, we demonstrated that these mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation.