Identification And Functional Analysis Of A Mip-like Gene In Xanthomonas Campestris Pv. Campestris

Mip (Macrophage infectivity potentiator) is an important virulence factor of the human bacterial pathogen Legionella pneumophila, it belongs to the FKBP (FK506-binding proteins) type of peptidylprolyl cis-trans isomerase (PPIase). Mips or Mip-like proteins are also the virulence factors of several other human and animal pathogenic organisms such as Chlamydia trachomatis and Salmonella typhimurium Copenhagen. However, the existence and the biological function of Mips or Mip-like proteins in plant pathogens are still unclear. The only mip or mip-like gene reported for a plant parasite so far is the one annotated in the genome of Xylella fastidiosa. In this work, by using the amino acid sequence of the putative product of mip-like gene of X. fastidiosa to search the genome of Xanthomonas campestris pathovar campestris (Xcc), the causal agent of the black rot disease of cruciferous plants, and found that the ORF XC2699 (GenBank accession number NC₀04556), annotated as a 'peptidyl-prolyl cis-trans isomerase', shows 85% and 49% similarity to the mip-like gene of X. fastidiosa and mip gene of L. pneumophila at the amino acid level, respectively. Accordingly, we renamed XC2699 as mip-like.To determine if Xcc Mip-like protein has PPIase activity and its type, the mip-like gene XC2699 was overexpressed in E. coli strain BL21 (DE3)pLysS, and the fusion protein with a (His)6 tag was purified using a Ni-column. The PPIase activity of the purified protein was determined by the protease-coupled assay as described using N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide as substrate. Result showed that the purified Xcc (His)6-Mip-like protein exhibits the PPIase activity, and the K_m and K_cat of the enzyme were determined to be 21.72 mM and 7.2 × 10⁴ S^-1 and the K_cat/K_m was calculated to be 3.29 × 10⁶ M^-1S^-1. The effect of the immunosuppressants FK506 and Cyclosporin A on the PPIase activity of the enzyme was tested, and found that the PPIase activity of Xcc (His)6-Mip-like protein is specifically inhibited by FK506 but not affected by Cyclosporin A. This result demonstrated that Xcc Mip-like protein is a FKBP type of PPIase.To investigate the biological function of the mip-like gene in Xcc, the nonpolar mutant (NK2699) of this gene was constructed by homologous suicide plasmid pK18mob integration. The virulence of the mutant NK2699 was tested on the host plant Chinese radish (Raphanus sativus L.var.radiculus Pers.) by the leaf clipping method. Ten days postinoculation, a lesion length with a mean of 2.3 mm was observed on the leaves inoculated with the mutant NK2699, while a lesion length with a mean of 15.1 mm was observed on leaves inoculated with the wild type strain 8004, and the attenuated virulence of the mutant could be restored by the wild typemip-like gene XC2699 in trans. These results revealed that the mip-like gene is required for the full virulence of X. campestris pv. campestris. For further understand the role of mip-like in Xcc pathogenesis, we tested the effects of mip-like mutation on the growth and production of virulence fators of Xcc, and found that, in addition to reduction in virulence, mutation in Xcc mip-like resulted in several other phenotype changes: (i) grow poorly in minimal medium;(ii) reduction in extracellular polysaccharide (EPS) production and;(iii) completely abolished the extracellular protease activity and reduction in the activity of extracellular cellulase and amylase. This result indicates that the mip-like gene is involved in the growth and production of exopolysaccharide and extracellular enzymes protease, endoglucanase and amylase of Xcc.To determine the contribution of the PPIase activity to the function of Xcc Mip-like protein, firstly, we determined three essential residues for PPIase activity in this protein by site-directed mutagenesis;and then plasmids expressing the wild-type and the mutagenized Mip-like proteins without PPIase activity or with reduced PPIase activity were used to complement the Xcc mip-like mutant NK2699. Phenotypic analysis result showed that plasmids expressing the mutant Mip-like protein with an undetectable PPIase activity could fully restore the reduced virulence of the Xcc mip-like mutant, indicating that the PPIase activity is not essential for the virulence function of Xcc Mip-like protein.