The Regulatory Role Of (p)ppGpp In Biofilm Formation Of Pseudomonas Putida KT2440

As a model bacterium for environmental microbiology research,Pseudomonas putida KT2440 is a certified biosafety strain and it has been studied for multiple purposes,including degradation of aromatic compounds,tolerance to heavy metals,plant-bacterial interactions and regulation of bacterial motility and biofilm.Forming biofilm is a strategy for bacteria to survive,and it can effectively enhance the biodegradability and the tolerance to stressful environment,leading improved adaptability under a variety of challenging conditions.Thus,investigating the regulation of biofilm is helpful for understanding the relationship between bacteria and environments.The signaling alarmone(p)pp Gpp,which plays a global regulatory role in bacteria,is produced in response to nutrient starvation and some other environmental stresses.It is also important for bacteria to survive in changeful environment.The roles of(p)pp Gpp in biofilm have been studied in some pathogenic and enteric bacterial strains,however,little is known about its roles in biofilm regulation in environmental bacteria.In this study,we focus on the regulatory role of(p)pp Gpp in biofilm formation and the regulation of exopolysaccharide gene cluster pea.The main results are as follows:1.The(p)pp Gpp level in KT2440 is controlled by Rel A and Spo T.Rel A has strong activity to synthesize(p)pp Gpp while Spo T displays both(p)pp Gpp hydrolase and synthase activity.We found that deleting rel A alone would not interfere with the phenotype of biofilm,but when both rel A and spo T were interrupted,KT2440 showed enhanced ability to form biofilm on solid surface.Conversely,the pellicle of this mutant formed at the air-liquid interface lost the robust structure and became frail.After prolonged incubation,the biofilm of rel A-spo T mutant did not disperse normally.Besides,the rel A-spo T mutant had improved ability to attach to coverslip,but it is defective in microcolony formation.The attachment and development of biofilm and the construction of biofilm structure are closely related to exopolysaccharides and adhesins.Through the quantification of exopolysaccharides,we found the rel A mutant and the rel A-spo T mutant can produce more exopolysaccharides.Although the polysaccharide production of rel A mutant and rel A-spo T mutant was similar,only the rel A-spo T mutant displayed smooth and mucoid colony morphology.Increased production of exopolysaccharides seems not to be the reason for such morphological change.We then measured the expression levels of four exopolysaccharide synthesis genes in wild-type and variants.The deletion of(p)pp Gpp synthase genes increased the expression of peb and bcs but repressed the expression of pea strongly,and these changes were stronger in rel A-spo T mutant than in rel A mutant,suggesting that smooth and mucoid colony in rel A-spo T mutant might be due to a lack of the polysaccharide Pea.Additionally,we also found the expression level of lap A was significantly increased,while that of lap F showed the opposite trend.According to function of each kind of exopolysaccharide and adhesin,the enhanced ability to form biofilm in rel A-spo T mutant might be attributed to overproduction of Lap A,and the failure of this mutant to form structural pellicle might be caused by the decreased production of Lap F and Pea.To better illustrate the effect of(p)pp Gpp on expression of genes for extracellular matrix synthesis,we tested the related regulators and found that Fle Q expression was significantly increased in the rel A-spo T mutant,while rpo S expression dropped notably.When(p)pp Gpp was absent,the expression of ? factor Rpo S is suppressed,resulting in a decline in lap F expression level and enhanced expression of fle Q which transcriptionally depends on ? factor Rpo D.Subsequently,increased expression of Fle Q promotes lap A transcription,although Fle Q can inhibit the expression of bcs,high level of c-di-GMP can relieve this inhibition and promote the expression of bcs.2.Previous studies have shown that exopolysaccharide Pea plays roles in the stability of biofilm and formation of air-liquid interface pellicle,but the expression pattern of Pea has not been elucidated.In order to explain the changes of phenotype and pea expression level in rel A-spo T mutant,we explored the function of Pea and regulation of the gene cluster for its synthesis.We identified that the pea gene cluster was transcribed from one site,which located at the 25 bp upstream of the gene cluster,and subsequently proved that transcription of pea was dependent on Rpo S.The reduced expression of pea in rel A-spo T mutant could be attributed to the decreased expression of Rpo S.When Rpo S was absent,another sigma factor,likely Rpo D,could also activate pea transcription but at a low level.The results from biofilm assay showed that deletion of pea did not influence the initial stage of biofilm formation,but affected the development of biofilm in the subsequent stage,leading decreased production of biofilm.In addition,pea mutant completely lost the ability to form a pellicle at air-liquid interface,but deletion of lap F which also transcriptionally depended on Rpo S did not lead obvious difference compared to wild-type in biofilm phenotype,suggesting that Pea was a key component for forming a pellicle.The rel A-spo T mutant could not form a perfect pellicle might be attributed to that it did not have a full production of Pea.Pea is also necessary for development of the c-di-GMP-dependent wrinkly colony.The rpo S mutant with elevated c-di-GMP exhibited wrinkly morphology,but it was weaker than that of the wild-type strain,because pea transcription may also be mediated by Rpo D and that the low expression level of pea may also contribute to c-di-GMP-dependent wrinkly morphology.In addition,we also identify that the transcription of pea is inhibited by the regulatory factor Amr Z.Amr Z directly binds to the region between the-10 and-35 elements of the pea promoter and interferes with the ability of RNA polymerase to bind the promoter,thus resulting in restriction of initiation of pea transcription.