Characterization of the LlaCI methyltransferase from Lactococcus lactis subsp. cremoris W15 provides new insights into the biology of type II restriction–modification systems

Author:

Mruk Iwona1,Cichowicz Magdalena1,Kaczorowski Tadeusz1

Affiliation:

1. Department of Microbiology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland

Abstract

The gene encoding the LlaCI methyltransferase (M.LlaCI) fromLactococcus lactissubsp.cremorisW15 was overexpressed inEscherichia coli. The enzyme was purified to apparent homogeneity using three consecutive steps of chromatography on phosphocellulose, blue-agarose and Superose 12HR, yielding a protein ofMr31 300±1000 under denaturing conditions. The exact position of the start codon AUG was determined by protein microsequencing. This enzyme recognizes the specific palindromic sequence 5′-AAGCTT-3′. Purified M.LlaCI was characterized. Unlike many other methyltransferases, M.LlaCI exists in solution predominantly as a dimer. It modifies the first adenine residue at the 5′ end of the specific sequence toN6-methyladenine and thus is functionally identical to the corresponding methyltransferases of the HindIII (Haemophilus influenzaeRd) and EcoVIII (Escherichia coliE1585-68) restriction–modification systems. This is reflected in the identity of M.LlaCI with M.HindIII and M.EcoVIII noted at the amino acid sequence level (50 % and 62 %, respectively) and in the presence of nine sequence motifs conserved amongN6-adenineβ-class methyltransferases. However, polyclonal antibodies raised against M.EcoVIII cross-reacted with M.LlaCI but not with M.HindIII. Restriction endonucleases require Mg2+for phosphodiester bond cleavage. Mg2+was shown to be a strong inhibitor of the M.LlaCI enzyme and its isospecific homologues. This observation suggests that sensitivity of the M.LlaCI to Mg2+may strengthen the restriction activity of the cognate endonuclease in the bacterial cell. Other biological implications of this finding are also discussed.

Publisher

Microbiology Society

Subject

Microbiology

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3