Identification Of The Ribonuclease Invoved In Cleavage Of 5S Ribosomal RNA In Xanthomonas Campestris Pv.Campestris

Ribosomal RNA(rRNA)is the most abundant RNA molecule in the cell of all three domains of life,it is the key component of ribosome—the protein synthesis machine,and thus is essential for all living cells.In bacteria,there are three types of rRNA:23S,16S and 5S rRNA.It has been shown that,in Escherichia coli and some other bacteria,rRNA genes are usually organized as a co-transcribed gene cluster,and thus the matured 23S,16S and 5S rRNA are generated from a common rRNA precursor by ribonucleases(RNase).Genome sequencing and annotation revealed that,in the genome of Xanthomonas campestris pv.campestris strain 8004(Xcc8004),there are two rRNA gene clusters,indicating that the matured rRNAs are processed from a common rRNA precursor by RNase,but nothing is known about the RNase involving this process.The aim of this work is to identify the RNase that is involved in processing of 5S rRNA of Xcc8004.Firstly,we tested the matured 5S rRNA and its processing products by using Northern blotting.The results show that,in addition to the 119nt matured 5S rRNA,there are several smaller cleavage products were tested.Among which the signal of the ?100nt cleavage product is much stronger than others,and we consider it is the "the major cleavage product of 5S rRNA”.We hypothesis that the ?100nt cleavage product is resulted from cleavaging of the matured 5S rRNA by a unknown RNase.To identify the RNase,we tested the matured 5S rRNA and its cleavage products of the mutants of 20 RNase genes(Xcc8004 total has 22 RNase genes),and results show that mutations in rnD,a gene which encodes the RNase D,resulted in lost of the ?100nt cleavage product,indicating that rnD is involved in 5S rRNA processing.We further constructed the complementary strain of rnD mutant and the rnD overexpression strain,and we tested the matured 5S rRNA and its cleavage products in these strains.The results show that the ?100nt cleavage product can be complemented by rnD in trans,and rnD overexpression resulted in increased cleavaging of 5S rRNA.These results demonstrate that RNase D plays an important role in the cleavage of 5S rRNA in Xcc8004.To determine whether RNase D is directly involved in the 5S rRNA cleavage,the RNase D protein and the 5S rRNA were purified,and in intro cleavage of 5S rRNA by RNase D was tested.The resultd show that RNase D can cleavage 5S rRNA in intro,demonstrating that RNase D directly cleavage 5S rRNA.Since it has been shown previously that RNase D is enzyme for tRNA processing in bacteria,we tested its ability to cleavage two tRNAs from Xcc8004(XC4335 and XC4339)in vitro,the results show that RNase D can cleavage the two tRNAs in intro.These results demonstrated that the RNase D of Xcc8004 is able to cleavage both tRNA and 5S rRNA.To determine the biological function of rnD in Xcc8004,we tested and compared the phenotypes[including extracellular polysaccharides(EPS)production,extracellular emzyme activity,cell motility,biofilm formation and virulence]of the wild type strain,the rnD mutant and its complemented strains.The results show that mutation in rnD resulted in decreased EPS production and the phenotype is complemented by rnD in trans,demonstrating that rnD is required for the EPS production in Xcc8004.In summary,our study provide the first experimental evidence that RNase D is able to cleavage 5S rRNA,and expanded our understanding to the biochemical function of RNase D.