Type II restriction modification system in Ureaplasma parvum OMC-P162 strain.

Heng Ning Wu,Ourlad Alzeus Gaddi Tantengco,Yukiko Nakura,Toshimitsu Takayanagi,Kiyoshi Yasukawa,Tomio Fujita,Michinobu Yoshimura,Itaru Yanagihara,Makoto Nomiyama,Chih-Horng Kuo

doi:10.1371/journal.pone.0205328

Abstract

Ureaplasma parvum serovar 3 strain, OMC-P162, was isolated from the human placenta of a preterm delivery at 26 weeks’ gestation. In this study, we sequenced the complete genome of OMC-P162 and compared it with other serovar 3 strains isolated from patients with different clinical conditions. Ten unique genes in OMC-P162, five of which encoded for hypothetical proteins, were identified. Of these, genes UPV_229 and UPV_230 formed an operon whose open reading frames were predicted to code for a DNA methyltransferase and a hypothetical protein, respectively. DNA modification analysis of the OMC-P162 genome identified N4-methylcytosine (m4C) and N6-methyladenine (m6A), but not 5-methylocytosine (m5C). UPV230 recombinant protein displayed endonuclease activity and recognized the CATG sequence, resulting in a blunt cut between A and T. This restriction enzyme activity was identical to that of the cultivated OMC-P162 strain, suggesting that this restriction enzyme was naturally expressed in OMC-P162. We designated this enzyme as UpaP162. Treatment of pT7Blue plasmid with recombinant protein UPV229 completely blocked UpaP162 restriction enzyme activity. These results suggest that the UPV_229 and UPV_230 genes act as a type II restriction-modification system in Ureaplasma OMC-P162.

Highlights

Ureaplasma spp. belong to the class Mollicutes [1] and have been implicated in the morbidity and mortality of newborns [2,3,4]
All of the U. parvum clinical strains used in this study and ATCC700970 were cultured at 37 ̊C in Ureaplasma medium [2], consisting of 2.4% Mycoplasma broth base, 2.5% yeast extract, 5% horse serum, 0.04% urea, 0.01% L-cysteine hydrochloride, 0.001% phenol red, and 1,000 U/ml penicillin
Four DNA fragments containing 32 genes were deleted from the genome without gene insertion in the OMC-P162 genome

Summary

Introduction

Ureaplasma spp. belong to the class Mollicutes [1] and have been implicated in the morbidity and mortality of newborns [2,3,4]. The inhibitory activity of UPV229 against the endonuclease activity of UPV230 was assessed by incubating purified UPV229 recombinant protein and 1 μg of closed circular pT7Blue plasmid or 250 ng of PCR products in a total volume of 20 μl containing methylation buffer (20 mM Tris-HCl [pH 7.5], 0.2 mM DTT, 0.5 mM EDTA, 5% glycerol, and 4 μM S-adenosylmethionine) at 37 ̊C for 1 h.

Results

Conclusion