JB Accepts, published online ahead of print on 6 November 2009

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J. Bacteriol. doi:10.1128/JB.00553-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Identification and Characterization of EctR1, a New Transcriptional Regulator of the Ectoine Biosynthesis Genes in the Halotolerant Methanotroph Methylomicrobium alcaliphilum 20Z

Ildar I. Mustakhimov, Alexander S. Reshetnikov, Anatoly S. Glukhov, Valentina N. Khmelenina, Marina G. Kalyuzhnaya^*, and Yuri A. Trotsenko

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Institute of Protein Research, RAS, Pushchino, Moscow Region, 142290, Russia; Department of Microbiology, University of Washington, Seattle, WA, USA

^* To whom correspondence should be addressed. Email: mkalyuzh{at}u.washington.edu.

Genes encoding key enzymes of the ectoine biosynthesis pathway in halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z were shown to be organized into an ectABCask operon. Transcription of the ect operon is initiated from two promoters ectAp1 and ectAp2 similar to the ⁷⁰-dependent promoters of E. coli. Upstream of the gene cluster, an open reading frame (ectR1) encoding a MarR-like transcriptional regulator was identified. Investigation of the influence of EctR1 on the activity of the ectAp1p2 promoters in wild type and ectR1^- mutant strains of M.alcaliphilum 20Z suggested that EctR1 is a negative regulator of the ectABCask operon. Purified recombinant EctR1-His₆ specifically binds as homodimer to the putative -10 motif of the promoter ectAp1. The EctR1 binding site contains a pseudopalindromic sequence (TATTTAGT-GT-ACTATATA) composed of 8-bp half-sites separated by 2 bp. Transcription of the ectR1 gene is initiated from a single ⁷⁰-like promoter. Location of the EctR1 binding site between transcriptional and translational start sites of the ectR1 gene suggests that EctR1 may regulate its own expression. Presented data suggest that in Methylomicrobium alcaliphilum 20Z EctR1-mediated control of transcription of the ect-genes is not the single mechanism for regulation of ectoine biosynthesis.