Functional Analysis Of The RnD_Xcc Gene In Xanthomonas Campestris PV. Campestris

Ribonuclease D(RNase D)is an exoribonuclease widespread in bacteria.RNase D,encoded by rnD,plays an important role in 3'-end processing of tRNAs.Although rnD has been found in almost all bacteria whose genome has been sequenced,the function of rnD in bacteria has not been identified except for Escherichia coli.Genome sequencing and gene annotation showed that a single copy of rnD(XC1809)(here renamed rnDXcc)existed in the genome of Xanthomonas campestris pathovar campestris 8004(Xcc8004).The purpose of this study is investigating the biological function of rnDXcc and the biochemical function of its product RnDXcc,by using genetic,molecular biology and biochemical methods.The transposon and plasmid insertion mutants of rnDXcc have been obtained in our laboratory previously.Unfortunately,both of them have polarity effects.To determine the function of rnDXcc precisely,this study constructed the deletion mutant of rnDXcc(?rnDXcc)and its complementary strain(C?rnDXcc),as well as the overexpression strain of rnDXcc(OErnDXcc).Phenotypic analysis showed that,the tolerance of the deletion mutant to SDS is significantly decreased compare to that of the wild-type strain,while the tolerance of the complementary strain to SDS is similar to that of the wild-type strain.These results demonstrte that rnDXcc plays an important role in the resistance to SDS stress.No significant difference in growth,pathogenicity,extracellular polysaccharide production and extracellular enzyme activity were observed in the rnDXcc deletion mutant and the wild-type strain.Phenotypic analysis of the rnDXcc overexpression strain showed that,compared to the wild-type strain carrying the empty overexpressed plasmid pBBad(pB/WT),cell growth,extracellular amylase activity,cell swimming motility and pathogenicity of the overexpression strain are significantly decreased,suggesting that over production of RnDXcc resulted in negative effects on these physiological processes.It has been well known that the substrate of RNase D is tRNA.However,our laboratory previously found that RnDXcc may be able to cut 5S rRNA.To determine whether RnDXcc really has 5S rRNA-degrading or processing function,5S rRNA was detected and compared in the wild-type strain,rnDXcc deletion mutant,the complementary strain and the overexpression strain by Northern blotting.The results showed that only the mature 5S rRNA(?120 nt)was detected in the rnDXcc deletion mutant,however,in addition to a mature 5S rRNA,a strong processing product(?110 nt)and several smaller(<100 nt)weak signal bands were detected in the wild-type and the complemented strain.The hybridization band of 5S rRNA displayed a diffuse state in the overexpression strain,indicating that the 5S rRNA has been over-cutting.These results demonstrate that RnDXcc has the ability to degrade or processing 5S rRNA in vivo.To determine whether RnDXcc can cleavage 5S rRNA in vitro,the recombinant RnDXcc protein(RnDXcc-His6),in which a 6xHis tag was added et the C-terminal,was expressed and purified in E.coli.Meanwhile,the 5S rRNA of Xcc8004 was prepared by in vitro transcription.The ability of RnDXcc-His6 to cleavage 5S rRNA was tested.The results showed that RnDXcc-His6 is able to cleave 5S rRNA in vitro.Further in vitro cleavage experiments also showed that RnDXcc-His6 is can cleavage tRNA,but not 16S rRNA and 23 S rRNA.In summary,in this study,the biological function of RnDXcc and the biochemical function of RnDXcc were investigated,and the results showed that:(1)RnDXcc plays an important role in the resistance of Xcc to SDS stress;(2)Over-production of RnDXcc significantly decrease the cell growth,extracellular amylase activity,cell swimming motility and pathogenicity of Xcc;(3)RnDXcc not only can cleave tRNA but also 5S rRNA.These results expand our understanding of the function of RNase D.