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

Abstract

ABSTRACT Genes encoding key enzymes of the ectoine biosynthesis pathway in the halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z have been shown to be organized into an ectABC - ask operon. Transcription of the ect operon is initiated from two promoters, ectAp 1 and ectAp 2 ( ectAp 1 p 2 ), similar to the σ 70 -dependent promoters of Escherichia 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 ectAp 1 p 2 promoters in wild-type M. alcaliphilum 20Z and ectR1 mutant strains suggested that EctR1 is a negative regulator of the ectABC - ask operon. Purified recombinant EctR1-His 6 specifically binds as a homodimer to the putative −10 motif of the ectAp 1 promoter. 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 σ 70 -like promoter. The location of the EctR1 binding site between the transcriptional and translational start sites of the ectR1 gene suggests that EctR1 may regulate its own expression. The data presented suggest that in Methylomicrobium alcaliphilum 20Z, EctR1-mediated control of the transcription of the ect genes is not the single mechanism for the regulation of ectoine biosynthesis.