Physiological Characterization Of Covr/s And Affect Hyaluronic Acid Synthesis In Streptococcus Zooepidemicus

Streptococcus zooepidemicus is the main strain for industrial fermentation production of hyaluronic acid(HA),HA is found in the form of a lavish extracellular capsule in S.zooepidemicus.The response regulator CovR and CovS,which is a member of the two-component signal transduction regulatory systems.The promoter of HA capsule operon(has)can be bound by CovR and to effect the synthesis of HA.The gene of covR/S in group of A streptococcus and other strains have been well known,but still remain dark in S.zooepidemicus.Streptococcus equi subsp.zooepidemicus ATCC39920 was used as the object of study,to study the physiological characterization of covR/S in Streptococcus Zooepidemicus and affect hyaluronic acid synthesis,the main results were as follows:(1)Study covR/S gene location in Streptococcus zooepidemicus genome and its protein functionRetrieving covR/S gene in the NCBI database,find the location of the gene in Streptococcus zooepidemicus group and retrieve its protein sequence.We found that covR and covS is adjacent in position.Retrieving the amino acid sequence and founding that CovR protein function is TCS response regulator CovR-like protein and CovS protein function is TCS sensor kinase CovS-like protein in NCBI.CovS is co-transcribed with covR.(2)covR/S gene plays a positive regulatory role for the growth of S.zooepidemicus.we constructed ΔcovR,ΔcovS,ΔcovRΔcovS and covR/Op,covS/Op strains.Solid streak plate found the growth of ΔcovR,ΔcovS,ΔcovRΔcovS strains was significantly lower than wild-type strain,and covR/Op,covS/Op strains were significantly enhanced.To further explore this effect and conducted a shake flask fermentation experiments,we find that the growth ofΔcovR strain was significantly lower than wild-type strain,Δ covS strain was significantly lower thanΔ covR strain and higher thanA covRΔ covS strain and covR/Op strain was higher than wild-type strain,and lower than covS/Op strain.Based on the above results,covR/S gene plays a positive regulatory role for the growth of S.zooepidemicus,and the influenceis co vS higher than covR.(3)covR/S effect the synthesis of HA.The seven strains were fermented in a fermenter and got on later analysis.Delection of covR gene will give lessen to HA content,but delection of covS gene has a complex influence for HA content,a ΔcovS strain produced low level HA in the earlier stage of the whole fermentation process compared to the wild type and finally surpassed the wild type through a sluggish accumulation.Under the same conditions,HA generation was significantly higher than the wild-type strain in overexpression strain and covS/Op strain was higher than covR/Op strain,therefore the influenceis covS higher than covR for HA content.Simultaneous analysis of sucrose consumption and substance accumulation,sucrose consumption and material accumulation is also showing a positive correlation.(4)covR/S gene plays a positive regulatory role for the molecular weight of HA.An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution in seven strains,and used the Ukrainian-style viscometer roughly estimated the molecular weight hyaluronic acid.From the molecular weight of agarose gel electrophoresis molecular weight can be seen successively lower molecular weight in delection strains,and gradually increased in overexpression strains.From this we can conclude that covR/S gene plays a positive regulatory role for the molecular weight of HA,the influenceis covS higher than covR.(5)Temperature,calcium and magnesium ions impact on covR/S gene.Shaking flask fermentation analyze the impact of various environmental factors on covR/S gene.covS gene have a negative impact on temperature tolerance in Streptococcus zooepidemicus;magnesium ions has no effect on covR/S gene;covS gene for resistance to calcium ions is low.