Poly(A) Polymerase Participates In The Regulatory Pathway Of Indole In Pantoea Agglomerans YS19

Bacteria evolve a lot of signal regulatory mechanisms among individuals inconfronting diverse environmental stresses, of which indole-based regulation attractsmore attention. As a kind of signal molecule which works in both intraspecies andinterspecies, indole regulates a variety of bacterial behaviors such as cell aggregation,etc., but the regulatory mechanism is still under investigation. The formation ofbacterial multicellular aggregate structure is a typical performance of adaptability andsurvivability, whose formation mechanism is a hot research area of the bacteriology.Obtaining the key proteins in indole regulatory pathway is significant forunderstanding its regulatory mechanism and also the formation mechanisms ofbacterial aggregation.This study selected Pantoea agglomerans YS19, a rice edophyte beingcharactered to form a special aggregate structure called symplasmata, as the modelstrain to construct a mutant library using the mTn5transposon mutagenesis method.From the mutant library, a positive mutant that is inhibited by indole was obtained,where the expression of the reportor gene decreased by32.6%when indole was added.By TAIL-PCR, it is demonstrated that the mutational site is pcnB, which codes thepoly(A) polymerase to catalyze the polyadenylation of RNAs. The full-length of thepcnB sequence is1332bp and phylogenetic analysis showed that pcnB is highconservative among strains in the species of P. agglomerans. It is revealed that indolesignificantly inhibited (by36.6%) the expression of pcnB by RT-PCR.Many physiological behaviors were changed in the pcnB mutant. In LB medium,the mutation of pcnB does not affect the growth of the bacterium inexponential growth phase, but increases the rate of decay in the late stationary growthphase. The cell concentration of the mutant decreased by24.6%in comparation to thewild type in36h. Meanwhile, the symplasmata formation ratio and average size wereonly19.6%and37.9%of that in the wild type at40h of cultivation, repectively.These data suggested that poly(A) polymerase played an important regulatory role inconfronting the environmental stresses and the symplasmata formation, although it isnot essential for the normal growth.The swimming and swarming abilities decreased by29.5%and11.1%comparedwith the wild strain, respectively, which might be one of the reasons that affect thecellular aggregation behaviors. The relationship between pcnB and the gene transcription of CsrA, a carbon storage regulator that is closely related to indoleregulation and symplasmata formation was further explored. It was revealed thatindole inhibites the expression of csrA, whose relative expression was inhibited by74.8%in comparison with the control at12h of cultivation. The deletion of pcnB alsoinhibited the transcription of csrA, especially in stationary phase. The relativeexpression of csrA was only41.3%of the control at40h. However, indole alsoinhibited the transcription of csrA by73.3%at12h. In other words, the deletion ofpcnB does not affect the the regulation pathway of indole on csrA expression,indicating that indole may cooperate with poly(A) polymerase by independentpathways to inhibit the csrA expression or regulate the csrA expression by severalpathways with poly(A) polymerase being involved in, which however need to befurther studied. Besides, SDS-PAGE examination on the whole cellular protein of thewild type and the mutant revealed that the expression of30-kD in the mutant wasupregulated.To explore the role of poly(A) polymerase in the plant-endophytes associationsystem, gnotobiotic cultivation of rice seedling was carried out on EPA medium.Inoculation of the wild strain and△pcnB improved the fresh weight of the21-d-cultivated host rice seedlings by36.1%and20.1%, respectively, suggesting thatthe deletion of pcnB inhibited the growth promoting effect of YS19. Meanwhile,indole improved the promoting effect of YS19. The fresh weight of the overgroundrice seedlings in the exogenous indole supplementary group increased by27.2%and21.6%, respectively, in comparison with the none supplemented groups. Thecolonization ability of YS19was also inhibited by the deletion of pcnB. Comparedwith the wild type, the total colonization number of△pcnB decrased by16.6%.Regardless of the mutation of pcnB, indole improved the of colonization ability YS19.The colonization number of the wild type and△pcnB mutant were increased by34.7%and25%, respectively, when indole was added.This study discussed the indole regulatory pathways from the point of RNApost-transcriptional modification, which enriched our knowledges of polyadenylationand expanded the research ideas of indole regulation.