| Lipopolysaccharide(LPS) is the main composition of the outer membrane of Gram-negative bacterial cells, and consists of three parts: lipid A, core polysaccharide(core OS) and O-antigen. On one hand, LPS could protect the bacteria from the harsh environment and harmful substances, on the other hand, LPS would induced the immune response when the bacteria invade the human body, so it was also known as endotoxin. Compared to lipid A and O-antigen, the core OS has not been well studied. In order to understand relationship between the structure and function of LPS core OS, a set of LPS core OS mutant strains were constructed from Escherichia coli K12 strain W3110. The structure of cell membrane, cell function and cell metabolism were analyzed. The main results are listed below:(1) A set of mutants with the different core OS were constructed by deleting the relavent gene in the genome of E. coli W3110. According to the biosynthesis pathway of LPS core OS and the gene cluster involved in W3110, ten key glycosyl transferase and phosphate transferase were determined as the targets. The genes encoding these enzymes were deleted by RED recombination, and the resulting mutant strains were named as ?waaC, ?waaF, ?waaG, ?waaO, ?waaR, ?waaU, ?waaP, ?waaQ, ?waaY and ? waaB, respectively. LPS core OS structure changes in some mutants were confirmed by analyzing the extracted LPS. The structure of lipid A in different mutants was found no change. Growth curves of these strains showed that the structural variation of the core OS could promote the growth of most mutants within 12 hours.(2) The structure change of core OS can affect the outer membrane permeability, the flagellum and the synthesis of capsule polysaccharide. The study on the membrane permeability of mutant strains showed that the loss of core OS causes the increase of membrane permeability and antibiotic sensitivity, correlating with the length of the sugar chain. The loss of flagellum was found with the variation of the core OS by the observation of transmission electronic microscopy. By the determination of gene expression level involved the the synthesis of flagellum, it showed that the down regulation of gene expression about FliA and FliC, and induced the deficiency of flagellum. In the plate culture of ?waaF at 37 ?, the mucoid mutant was observed. Through the deletion of genes relavent to the biosynthesis of colanic acid, it was found that the high-level L-fucose synthesis caused the mucoid mutant. The content of capsule was determined by the fucose as target. The fucose levels in ?waaF and wild type cells were 3.48 and 1.12 mg/L, respectively. The transcriptional levels of cpsG and cpsB in ?waaF were 3.79 and 5.29 times higher than those in wild type, respectively; the transcriptional levels of genes wcaJ and wza related to the assembly and transport proteins in ?waaF were also up-reglulated.(3) The structural diversity of core OS can cause the differenec of glycometabolism in cell and the Intracellular accumulation of PHB. The parameter was measured after fermentation, the results showed that the content of intracellular protein was increased with the structure change of core OS, and it induced the improvement of biomass of strains. The pH, consumption of glucose, and the exocytosis of amino acid were found no change, but the intracellular amino acid, the metabolism of tyrosine and lysine was changed. The mutants of core OS was used for the biosynthesis of PHB by the expression of pBHR68, it was found that the accumulation of PHB in ?waaC, ?waaF, ?waaG, ?waaU, ?waaP and ?waaY strains, within the PHB proportion of 64.3% in ?waaU/pBHR68 and 65.1% in?waaY/pBHR68, it was 60% higher than wild strain W3110/pBHR68. By the analysis of fermentation parameter, the biomass of mutants also increased than W3110, and the pH also no change. The consumption of glucose was different in all strains, the consumption was decreased in ?waaC/pBHR68, ?waaG/pBHR68, ?waaP/pBHR68, and increased in ?waaF/pBHR68, ?waaU/pBHR68, ?waaY/pBHR68. It maybe caused by the mutative glycometabolism with the variation of core OS.(4) The structure change of core OS could affect cell-cell interaction such as biofilm formation and cell autoaggregation. The effect of oxygen and culture time on biofilm formation were confirmed by the test of biofilm formation under different condition. The variation of biofilm formation of different strains showed that the biofilm formation for the core OS mutants was weakened, and the variation was connect with the structure of core OS to some extent. The study on the autoaggregation characteristics of bacteria showed that the autoaggregation E. coli was independent of the environment temperature. By analysis of the expression and transcription level of Ag43, the relation between of autoaggregation and Ag43 of E. coli has been studied. By determining the hydrophobic property of the mutant cells, it was found that the deletion of the core OS has a reinforcing effect on the surface hydrophobicity, and the enhancement effect is related to the length of the sugar chain. The loss of phosphate group on the inner core could induce the cell surface hydrophobicity. By the analysis of the gene expression of signal molecular with quorum sensing, there was no obvious effect of the structure of core OS on the synthesis of signal molecular. The effect of flagellun on biofilm was confirmed, and it play a role with cell surface hydrophobicity and cell surface protein in the cell-cell interaction.(5) Transcriptome analysis showed that the synthesis and assembly of flagellum was affected by the core OS structure change. By analysing the differentially regulated genes in ?waaC(1382 genes), ?waaF(526 genes), ?waaG(965 genes), ?waa O(1979 genes), ?waaR(2005 genes), and ?waaU(1874 genes), it was found that LPS structure plays key role on the synthesis of flagellum and bacterial chemotaix. It was confirmed that the down-regulation of ?28, FlgM, and MotAB in response to the variation of core OS, and it was concluded that the regulation of flagellum by ?E, through the interaction with ?28 and FliGM. |
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