Effects Of Rsh Gene Deletion On Acyl-Homoserine Lactone Accumulation In Two Strains In Sphingomonads

As a kind of bacteria that can degrade a wide range of aromatic compounds,such as polycyclic aromatic hydrocarbon(PAH),sphingomonads has good potential for bioremediation.However,bacteria often encounter different environmental stress factors including high temperature,drought,salinity and poor nutrition,affecting the growth and survival of the bacteria.As a result,the efficiency of pollution remediation will decrease.The stringent response mediated by guanosine tetraphosphate and guanosine pentaphosphate((p)ppGpp)can regulate metabolic activities of bacteria and help bacteria survive under environmental stress.In sphingomonads,(p)ppGpp is synthesized and hydrolyzed by Rsh protein.As quorum sensing(QS)can also regulate various physiological activities in bacteria,the study of the effect of rsh gene on the accumulation of QS signal molecules in sphingomonads can provide the theory basis for the future application of sphingomonads in pollutant degradation in harsh conditions.The main QS signal molecule in sphingomonads is acyl-homoserine lactone(AHL).So far,there is a large gap in the study of the relationship between the rsh gene and the AHL accumulation in sphingomonads.Although there are several pairs of luxI/R homologs in sphingomonads genomes,the relationship between the rsh gene and the AHL accumulation in the strains containing several pairs of luxI/R homologs has never been studied.Novosphingobium pentaromativorans US6-1 was chosen to study the effect of rsh gene deletion on the AHL accumulation.Next,qRT-PCR,AHL degradation experiment and RNA-Seq were used to study the specific molecular mechanism.Considering that sphingomonads often contains several luxI/R homologs,Sphingobium sp.SYK6 was selected as the research object,and the effect of rsh gene deletion on AHL accumulation was studied under the nutrient-rich and nutrient-limiting conditions.The difference between the effects of rsh gene deletion on the strains containing one pair or several pairs of luxI/R homologs was investigated.The main conclusions in this paper are as follows:(1)? rshUS6-1 could not accumulate AHL signal molecules in the biofilm and static culture conditions,indicating that rshUS6-1 was a necessary gene for the AHL accumulation.RshUS6-1 regulated the AHL accumulation via ppGpp.The reason why ? rshUS6-1 could not accumulate AHL was the decreased expression of novIUS6-1 and nowRUS6-1 during the exponential growth period.(2)According to the RNA-Seq analyses of WTUS6-1 and ?rshUS6-1 the down-regulated expression of gene coding extracytoplasmic function sigma factor?Z and the increased expression of dksA and genes involved in RNA degrading and Lon protease enzyme might cause the down-regulation of 1novIUS6-1 and novRUS6-1.The proposed molecular mechanism can give insight to the regulation of stringent response on QS.Besides,although RshUS6-1 negatively regulated the expression of genes involved in PAH degradation,it can help bacteria adapt to the environmental stress and support the survival,thus indirectly improving the degradation efficiency of PAH.Whether RshUS6-1 affected the PAH degradation via AHL signal molecules needed further study.(3)Strain SYK6 produced four kinds of AHL signal molecules(3-OH-C6-HSL,3-OH-C8-HSL,C8-HSL,C14-HSL)under nutrient-rich condition,of which 3-OH-C8-HSL was the main signal molecule.Under nutrient-limiting condition,only two kinds of AHL(3-OH-C8-HSL,C14-HSL)were produced,of which C14-HSL was the main signal molecule.This indicated that SYK6 accumulated different AHL in different nutrition conditions,which might be beneficial to its survival.In these two conditions,? rshSYK6 accumulated AHL.Under the nutrient-rich condition,there was no difference in the concentration ratio of AHL between ?rshSYK6 and WTSYK6 but the AHL concentration in ? rshSYK6 was higher,which might result from more biomass.Under the nutrient-limiting condition,the kinds of AHL were more and the AHL concentration was higher in WTSYK6.The expression of luxI/R homologs was also studied.rshSYK6 deletion decreased the gene expression but most genes were down-regulated more in oligotrophic nutrition.Besides,the most down-regulated gene was different in these nutritional environments:sphRl under the nutrient-rich condition and sphll in the oligotrophic environment.The expression of sphI2 was similar in these two conditions,which indicated that it might not be regulated by RshSYK6.The above results showed that AHL accumulation was affected more by rshSYK6 deletion in the nutrition-limiting condition.By comparing the effects of rsh gene deletion on the AHL accumulation in strain US6-1,SYK6 and previously reported strains in sphingomonads,it was showed that the effects of rsh gene deletion were different in strains containing one pair or several pairs of luxI/R homologs genes.For strains with one pair of luxI/R homolog,the rsh gene was necessary for the AHL accumulation.For the strain with several pairs of luxI/R homologs,although the deletion of rsh decreased the expression of luxI/R homologs,the rsh deletion mutant still could accumulate AHL,which might result from luxI/R homologs not regulated by rsh.More experiments are needed to find if the results are common in sphingomonads.