Specific DNA binding of a potential transcriptional regulator, inosine 5'-monophosphate dehydrogenase-related protein VII, to the promoter region of a methyl coenzyme m reductase I-encoding operon retrieved from Methanothermobacter thermautotrophicus strain DeltaH

Abstract

Two methyl coenzyme M reductases (MCRs) encoded by the mcr and mrt operons of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus DeltaH are expressed in response to H(2) availability. In the present study, cis elements and trans-acting factors responsible for the gene expression of MCRs were investigated by using electrophoretic mobility shift assay (EMSA) and affinity particle purification. A survey of their operator regions by EMSA with protein extracts from mrt-expressing cultures restricted them to 46- and 41-bp-long mcr and mrt upstream regions, respectively. Affinity particle purification of DNA-binding proteins conjugated with putative operator regions resulted in the retrieval of a protein attributed to IMP dehydrogenase-related protein VII (IMPDH VII). IMPDH VII is predicted to have a winged helix-turn-helix DNA-binding motif and two cystathionine beta-synthase domains, and it has been suspected to be an energy-sensing module. EMSA with oligonucleotide probes with unusual sequences showed that the binding site of IMPDH VII mostly overlaps the factor B-responsible element-TATA box of the mcr operon. The results presented here suggest that IMPDH VII encoded by MTH126 is a plausible candidate for the transcriptional regulator of the mcr operon in this methanogen.

FIG. 1.

Time course of optical density (circles) and H₂ concentration (squares) during 2 h of continued cultivation of M. thermautotrophicus ΔH in 100-ml (closed) and 500-ml (open) cultures. The optical density (600 nm) of the cultures at the starting point (0 min) was 0.1, and the headspace was refreshed with H₂-CO₂ (80:20) at 0.2 MPa before continued cultivation. Data were obtained from triplicate cultures, and error bars indicate standard deviations. Detailed cultivation conditions are described in Materials and Methods.

FIG. 2.

Northern blot analysis of transcripts from the mcr and mrt operons of cells grown in different culture volumes (100 to 500 ml). Twenty micrograms of total RNA was used for the analysis. Transcripts were simultaneously detected with each operon-specific probe prepared with the primers listed in Table 1. Only the cells grown in 100- and 500-ml cultures were used for the following studies as mrt-expressing (mcr-repressed) and mcr-expressing (mrt-repressed) cultures, respectively.

FIG. 3.

Survey for operator sites of the mcr and mrt operons by EMSA. The probes used in the survey and the sequence structures of putative operator sites of the mcr (a) and mrt (b) upstream regions are shown. Black and gray lines show the regions of shifted and nonshifted probes, respectively. The sequences in bold are the final restricted regions expected to contain operator sites. Transcriptional start sites and palindromic sequences previously reported (22) are represented by asterisks and arrows, respectively. The TATA box and the BRE are boxed. Electropherogram of an EMSA with fragmented upstream regions of the mcr and mrt operons (c). Lane 1, unbound probes (60 fg); lane 2, probes reacted with protein extract prepared from an mrt-expressing culture (20 μg); lane 3, probes reacted with protein extract and unlabeled probes (60 pg). All reactions were performed at 4°C for 2 h. Mobility shifts are clearly observed in reaction mixtures with probes 1168-05 and 1132-12, as indicated by the arrowhead.

FIG. 4.

DNA-binding protein purified from mrt-expressing cultures and its recombinant protein. (a) SDS-PAGE of purified proteins with affinity particles conjugated with the putative operator regions of the mcr and mrt operons. (b) SDS-PAGE of His-tagged (H) and His-tagged and cleaved (C) recombinant IMPDH VII (0.5 μg each). M indicates the molecular mass standard. Gels were stained with Coomassie brilliant blue. The molecular mass of the purified protein is consistent with that of recombinant IMPDH VII (32.4 kDa).

FIG. 5.

Specific binding of IMPDH VII to the putative mcr and mrt operator regions. Lane 1, free probe; lanes 2 to 4, 0.5, 1, and 2 μg of IMPDH VII, respectively; lane 5, 2 μg of IMPDH VII and 1 μg of poly(dI-dC); lane 6, 2 μg of IMPDH VII and 60 pg of unlabeled probes. Forty-five femtograms of DIG-labeled probes was used for the reactions. All reactions were carried out at 4°C for 2 h.

FIG. 6.

Influence of base changes in the putative mcr operator region to unnatural sequences. (a) Probes and their corresponding sites in the putative mcr operator region. The BRE, TATA box, and transcriptional start site are indicated by the boxes and the asterisk, respectively. Palindromic sequences are indicated by arrows. (b) Electropherogram of an EMSA with probes harboring unnatural sequences. A minus or a plus sign indicates the absence or presence, respectively, of recombinant IMPDH VII. All EMSA reactions were carried out at 4°C for 2 h.