Proteomic And O-antigen Analysis Of Escherichia Coli And Functional Genomic Research In Geobacillus Thermodenitrificans NG80-2

I Proteome research and O-antigen analysis of Escherichia coliBacteria like Escherichia coli spend much of the time under anaerobic conditions in the colon but also transits through aerobic environments. Extensive regulatory mechanisms exist for switching between aerobic and anaerobic conditions, but few studies have identified cellular components that limit this switch or identified the trade-off cost of shuttling between environments. To study E. coli adaptation to O2 limitation, we grew populations in the presence of glucose in continuous culture for four weeks and here identified the protein changes in two independent isolates under persistent O2 limitation. Based on proteomic comparisons, these lab-evolved strains share many differences to the ancestral E. coli K-12 strain under both aerobic and anaerobic conditions. The most striking shared difference is a reduction in ArcA, a central regulator of the adaptation to anaerobic conditions. There was also a reduction in fermentation, as suggested by reduced PflB, and increases in tricarboxylic acid cycle (TCA cycle) enzymes, suggesting ArcA is a burden under O2-limiting conditions. Another surprising difference was the unexpected up-regulation of multiple iron scavenging proteins, which suggests iron limitation was present in the selection condition, but which is consistent with the greater importance of the TCA cycle and respiration in O2-limited growth. Mutations in the experimental population also affect the outer membrane permeability for glucose, as suggested by up-regulation of OmpF, as well as changing the expression of many metabolic genes. The results are also consistent with multiple mutations, some unique to one the other strain, but a common reallocation of regulation with prolonged O2 limitation.The O-antigen is an essential component of lipopolysaccharide on the surface of Gram-negative bacteria and plays an important role in its pathogenicity. We investigated the chemical structure and characterized the gene cluster of E. coli 0161 O-antigen. Based on sugar and methylation analyses, and NMR spectroscopy, E. coli 0161 antigen has a trisaccharide O-unit containing a di-N-acyl derivative of legionaminic acid with the following structure: →8)-α-Legp5Ac7Ala-(2→4)-β-D-GlcpA-(1→3)-β-D-GlcpNAc-(1→The O-antigen gene cluster of E. coli 0161 was sequenced and characterized. The biosynthesis pathway of the legionaminic acid derivative was predicted, and further confirmed by sequencing the E. coli O61 O-antigen gene cluster and comparing the O-antigen genes of E. coli O161,O61 and O108, all of which contain di-N-acetyl sugar derivatives as O-antigen components.ⅡFunctional genomic research of Geobacillus thermodenitrificans NG80-2Geobacillus thermodenitrificans NG80-2, isolated from a deep-subsurface oil reservoir of Dagang oilfield, Northern China, is a described thermophilic bacterium (optimum 65℃) which can degrade long-chain (C15～C36) alkanes. It can utilize cured oil as the sole carbon sources and grow well in the anaerobic, deep subterranean oil-reserveoir environment with the ability to increase the ratio of light/heavy fraction of crude oils and enhance their fluidity. To investigate and utilize these abilities of NG80-2 will be very significant in the area of petroleum industry.An aldehyde dehydrogenase (ALDH) involved in alkane degradation was characterized in vitro. The ALDH was expressed heterologously in Escherichia coli and purified as a His-tagged homotetrameric protein with a subunit of 57 kDa based on SDS-PAGE and Native-PAGE analysis. The purified ALDH oxidized alkyl aldehydes ranging from formaldehyde (C1) to eicosanoic aldehyde (C20) with the highest activity on C1. It also oxidized several aromatic aldehydes including benzaldehyde, phenylacetaldehyde, o-chloro-benzaldehyde and o-phthalaldehyde. The ALDH uses only NAD+ as the cofactor, and has no reductive activity on acetate or hexadecanoic acid. Therefore, it is an irreversible NAD+-dependent aldehyde dehydrogenase. Kinetic parameters, temperature and pH optimum of the enzyme, and effects of metal ions, EDTA and Triton X-100 on the enzyme activity were investigated. Physiological roles of the ALDH for the survival of NG80-2 in oil reservoirs are discussed.