Mol Gen Genet (1989) 220:69-72 © Springer-Verlag 1989 Down regulation of the mercury resistance operon by the most promoter-distal gene merD Giuseppina Nueifora, Simon Silver, and Tapan K. Misra Department of Microbiology and Immunology, University of Illinois College of Medicine, P.O. Box 6998, Chicago, IL 60680, USA Summary. The effect of the merD gene on the expression of the mer operon was determined from the rates of accu- mulation of merA-lacZ fusion protein in the presence and absence of an active merD gene in trans. In the presence of the merD gene, fl-galactosidase activity was 2- to 4-fold lower. The merD gene was cloned in a T7 promoter expres- sion vector and the MerD protein product was visualized by autoradiography. Key words: Mercury operon - merD regulation - merR reg- ulation - Metal regulation - Operon regulation Introduction merD is an open reading frame of 366 nucleotides (363 in the Tn21 mer operon) in the structural gene cluster of the mercury resistance operons of transposons Tn501 and Tn21 (Brown et al. 1986) and plasmid pDU1358 (Griffin et al. 1987). Mutants in merD were first obtained by inser- tion of Tn5 in the mercury resistance operon of Tn21 (Ni'Bhriain et al. 1983), and later by deletion of part of the merD open reading frame in the mercury resistance operon of Tn501 (Itoh and Haas 1985). In both cases, the resulting mutant operon gave a lower level of mercury resis- tance to the cell compared to the intact operon (Brown et al. 1986). Gambill and Summers (1989) have recently detected a merD-lacZ protein fusion product. However, although there is a very strong DNA sequence homology among the three merD open reading frames (pDUI358 and Tn501, 91% identical nucleotides; pDU1358 and Tn21, 81%; Tn501 and Tn21, 83%) which suggests function(s) for the merD gene product, no protein corresponding to the pre- dicted MerD polypeptide has been identified in cells ex- pressing the mer operon (Ni'Bhriain and Foster 1986). It is possible that the reduced mer operon function associated with merD mutations results from effects at the nucleic acid level (mRNA synthesis or stability; translational control) and not from effects of the translated merD protein prod- uct. We have investigated the possible role of the merD gene from the broad-spectrum mer operon of plasmid pDU1358 in the regulation of the mer operon. The effect of the merD gene was measured in trans as induction of the reporter gene product fl-galactosidase produced from Offprint requests to: G. Nucifora a merA-lacZ transcriptional fusion. The results of these ex- periments, reported in this paper, suggest that the merD gene product may have regulatory functions. Materials and methods Bacterial strains and plasmid constructions. Escherichia coli strains used have been described elsewhere. Strain MC1061 (Miller 1987) was used as plasmid recipient in Hg 2+ resis- tance measurements, strain K-38 containing plasmid pGPI- 2 (Tabor and Richardson 1985) was used in protein expres- sion experiments, and strain DU511 containing plasmid pDU1161 (Ni'Bhriain et al. 1983) was used in complemen- ration assays. The plasmids containing the merD reading frame in the orientation of the T7 promoter (pGN117-1) or in the oppo- site orientation (pGN117-2) were constructed by ligating a 1.1 kb NsiI-PstI fragment containing the entire reading frame of merD including 25 nucleotides upstream of the predicted merD ribosome binding site (Griffin et al. 1987) into the PstI site of the polylinker region of plasmid pGEM3 (Promega Biotechnology, Madison, Wis). The con- struction of plasmids pGN120 and pGN107-2, used in the complementation assays, is outlined in Fig. 1. pGN120 was obtained by cloning the BglII-PstI fragment of priG103 (containing the entire broad-spectrum mercury resistance determinant; Griffin etal. 1987) into the ScaI site of pBR322 after polishing the ends of the fragment with Klenow DNA polymerase. Plasmid pGNI07-2 was gener- ated in two steps: first, a merD deleted operon was made by partial digestion of mGN103 (containing the BglII-PstI fragment of priG103 cloned in the M13 derivative mTM010; Misra 1987) with AvaII (which cuts in the merB gene of the operon) and complete digestion with PstI (which has a single site in the 3' distal polylinker region of the mTM010 vector). After polishing the ends with Klenow DNA polymerase, the linearized phage was ligated. The merD deleted mer determinant was then separated from the phage mTM010 vector by digestion with SalI, which has recognition sites distal to merR (at the 5' end of the operon) and in the phage polylinker region (3' to the operon). After polishing the ends, the fragment was cloned into the ScaI site of pBR322. In vivo expression of MerD. The T7 RNA polymerase ex- pression system (Tabor and Richardson 1985) was used