Effect Of Lipopolysaccharide Structure On Colanic Acid Biosynthesis In Escherichia Coli

Lipopolysaccharide(LPS) and colanic acid(CA) belong to the extracellular polymeric substance. LPS, which plays a role in stabilizing the cell membrane, is an important component of wall-membrane system in gram-negative bacteria. CA is a widely existing extracellular heteropolysaccharide in Enterobacter and has an important role in helping bacteria resist dry, hypertonic and other adverse environment. However, the relationship between the two components is unclear. Based on series of core-oligosaccharide mutants, this research explored the relationship between the core-oligosaccharide and CA biosynthesis. The main conclusions are as follows:(1) When 10 Escherichia coli mutants with defects in LPS core biosynthesis were grown on agar medium at 30 oC, four of these, ΔwaaF, ΔwaaG, ΔwaaP, and ΔwaaB, secreted extracellular polysaccharide(EPS), while the other six, ΔwaaU, ΔwaaR, ΔwaaO, ΔwaaC, ΔwaaQ, and ΔwaaY, did not. However, the EPS production in these mutants are also effected by multiple factors. The ΔwaaF mutant produced the highest amounts of the exopolysaccharide, regardless of whether it was grown on agar or in liquid medium.(2) The exopolysaccharides were isolated from the liquid growth medium of ΔwaaF cells, hydrolyzed, analyzed using high-performance ion-exchange chromatography, and found that the exopolysaccharides are colanic acids. When key genes wza, wzb, wzc, and wcaA related to the biosynthesis of colanic acids were deleted in ΔwaaF, the exopolysaccharides could not be produced any more, further confirming they are colanic acids.(3) RT-PCR suggests the change of LPS structure stimulated the signal transduction in Rcs system. Colanic acids could not be produced when genes rcsA, rcsB, rcsD, or rcsF was deleted in ΔwaaF, but could be partially produced when the gene rcsC was deleted in ΔwaaF, suggesting that structure change of LPS of ΔwaaF cells might be sensed by Rcs phosphorelay system, leading to the production of colanic acids.The membrane protein RcsD, regulatory factor RcsB, auxiliary factor RcsA and outer membrane lipoprotein RcsF are all required for CA secretion in ΔwaaF mutants.(4) In hypertonic conditions, lacking CA in ΔwaaF background will further growth delay. Either LPS change or CA deficiency will strengthen the self-aggregation and hydrophobic ability. These suggest LPS and CA may have a complementary function in the high osmotic pressure and other adverse environments.In conclusion, E. coli cells could activate CA production through Rcs phosphorelay system in response to the structure deficiency of LPS, among them, the Hep-Kdo2-lipid A structure of ΔwaaF has a maximum CA secretion. This study also analyzed the effects for CA production from multiple perspectives, which provides experimental basis for the research of EPS mutual effect.