Stimulatory Effect And Molecular Mechanism Of Glucose On Pseudomonas Stutzeri A1501 Exopolysaccharide

Bacteria synthesize and secrete exopolysaccharides,which participate in the important activities such as bacterial pathogenicity,antifungal properties,and host immunity.Meanwhile,exopolysaccharides are also one of the main components of biofilm.In the course of long-term evolution,bacteria have developed a complex regulatory system containing multiple enzymes and transport systems involved in the synthesis of exopolysaccharides.Pseudomonas stutzeri A1501 is a root-associated nitrogen-fixing bacterium colonizing in the rhizosphere of gramineous plants,its biofilm formation is helpful to colonization and competitive advantage.Previous studies have found that glucose can affect the formation ability of A1501 biofilm,but the mechanism is not clear.In this study,the mechanism of glucose stimulated exopolysaccharide synthesis was studied by mutant construction,phenotype analysis,exopolysaccharide determination and transcriptome analysis.The main findings are as follow:(1)The growth curve showed that A1501 only grew slowly in glucose compared with other sugars,and its growth ability was far weaker than that of organic acids and amino acids,indicating that glucose is a non-preferred carbon source of A1501.Only glucose could stimulate biofilm formation when sugars were added to normal culture medium,and the biofilm promotion effect was independent of glucose concentration.It was speculated that glucose was a environmental influencing factor of A1501 biofilm.(2)Transcriptome analysis of glucose-induced changes showed that 186 genes were up-regulated,including exopolysaccharide synthesis,glucose metabolism,transport and chemotaxis,and 75 genes were down regulated,mainly involved in iron transport.It is suggested that glucose induces exopolysaccharide formation at the transcriptional level.(3)GC-MS was used to detect the exopolysaccharide components of A1501.The results showed that the exopolysaccharide was mainly composed of glucose,mannose,ribose,galactose and rhamnose.Further analysis showed that the monosaccharide content increased significantly by glucose,speculating that glucose may induce monosaccharide synthesis to form exopolysaccharide.(4)qRT-PCR analysis revealed that glucose can induce the expression of alg A,rfb D,gal U and psl A more than twice.These genes were involved in the synthesis of GDP-mannose,TDP-rhamnose,UDP-glucose and glycosyl transfer.The related mutants were constructed,all of the four mutant strains lost the glucose stimulation effect,which indicated that glucose stimulated the synthesis and secretion of exopolysaccharide by inducing the expression of alg A,rfb D,gal U and psl A.(5)In order to determine the location of glucose action,the transport system was studied.The expression of Gts ABCD related genes was up-regulated nearly 100 folds by glucose.Gts A mutation leads to complete loss of glucose utilization ability of A1501 and can not promote the formation of biofilm,indicating that glucose needs to enter the cell through Gts ABCD to participate in biofilm formation.In summary,non-preferred carbon source glucose enters the cell through Gts ABCD transport system,induces the expression of exopolysaccharide synthesis related genes,activates exopolysaccharide secretion protein,then promotes the exopolysaccharide production and affects the formation of A1501 biofilm.