Mol Gen Genet (1981) 181:491~496 © Springer-Verlag 1981 Acetohydroxy Acid Synthase lsoenzymes of Escherichia coli K-12: A trans-Acting Regulatory Locus for ilvHl Gene Expression Matilde Valeria Ursini 1, Paolo Arcari 2, and Maurilio De Felice 1 International Institute of Genetics and Biophysics, C.N.R., Via Marconi 10, 1-80125 Naples, Italy 2 Primo Istituto di Chimica Biologica, Seconda Facolt/t di Medicina e Chirurgia, Universitfi di Napoli, 1-80131 Naples, Italy Summary. We isolated an Escherichia coli K-12 regulatory muta- tion affecting the acetohydroxy acid synthase III isoenzyme. This mutation was found to lie outside the structural genes ilvHI for this isoenzyme and was designated lrs-1. A strain carrying this mutation was found to be altered in the leucine-mediated control of ilvHI mRNA and acetohydroxy acid synthase III synthesis observed in the isogenic Irs + strain. These alterations appeared to be a consequence of a reduced intracellular concen- tration of a single one of five tRNA Leu isoaccepting species. Introduction Acetohydroxy acid synthase (AHAS) III is one of the two isoen- zymes (La Cara and De Felice 1979) which catalyze the first step common to the biosynthesis of isoleucine, leucine and valine in Escherichia coli K-12. The other isoenzyme is AHAS I (the ilvB gene product). AHAS III is composed of two subunits, whose structural genes ilvH and ilvI are closely linked to each other and located at 2 rain on the E. coli K-12 linkage map (De Felice etal. 1979; Bachmann and Low 1980). These two genes constitute an operon which is transcribed into an approxi- mately 2,500 nucleotide long mRNA molecule (C. Squires et al., submitted for publication). Expression of the ilvHI genes is nega- tively affected by leucine (De Felice and Levinthal 1977), but neither the molecular mechanism of the leucine effect nor the nature and the role of other possible effectors is known. This is, at least in part, a consequence of the unavailability of regulato- ry mutations. In the work reported here we took advantage of the leucine- sensitive phenotype of a strain lacking AHAS I to isolate mutants altered in the mechanism of AHAS III synthesis. We performed an analysis of one of these mutants, from which it was possible to identify a locus whose product is involved in the negative control of ilvHI gene expression. Table 1. Bacterial strains ~ Strain Genotype Origin or reference Ca85 HfrH thi-1 lac amber CGSC5346 F thi-1 thr-1 leu-6 lacY1 tonA21 JF193 F4 (thr + ara + leu + ilvHI + lrs + proA +)/thi-1 thr leu proA arg his rec-13 str X478 F thi-1 metE proC purE trp lysA leuB xyl azi ara lacZ str MI148e F- thi-1 metE proC purE trp lysA xyl azi lacZ str MI148f MI316 MI316/pCV7 PS1035 LR4 LR16 LRI6a RA16 F-thi-1 metE proC purE trp lysA leuB xyl azi ara lacZ str hal HfrH thi-J glyA ara bglR20 ilvB619 ilvH612 ilvI614 pCV7 (ilvH+I+)/thi-1 glyA ara bglR20 ilvB619 ilvH612 ilv[614 HfrH thi-1 glyA bglR20 ilvB619 HfrH thi-1 glyA bglR20 ilvB619 Irs-2 HfrH thi-1 glyA bglR20 ilvB619 Irs-1 ara HfrH thi-1 glyA bglR20 ilvB619 lrs-1 ara J. Beckwith J.M. Calvo M. Iaccarino P. Berg Ara + Leu + transduc- taut of X478 with P1 grown on Ca85 Spontaneous deriva- tive of X478 resis- tant to 20 gg/ml nalidixic acid This laboratory This laboratory De Felice et al., 1978a This paper This paper ilvH+I + ara- lrs-1 derivative of MI316 with Plphage grown on LR16 F4 (thr + ara + leu + ilvH+ I + lrs * This paper proA +)/thi-1 glyA bglR20 ilvB619 lrs-1 ara a Genetic symbols are those used by Bachmann and Low (1980), except for lrs, which is used for the first time in the present paper Materials and Methods Bacterial Strains The E. coli K-12 strains used are listed in Table 1. Media and Growth Conditions Minimal medium was the one described by Vogel and Bonner (1956). The carbon source was generally 0.4% glucose and in some specific For offprints contact." Dr. M. De Felice cases (outlined in the text) 0.2% arabinose. Suppiements, when re- quired, were added at a final concentration of 50 gg/ml with the excep- tion of adenine (20/ag/ml), arginine (100 gg/ml), thiamine (10 ~tg/ml) and valine (100 gg/ml). LC medium contained 10 g Bacto-tryptone, 5 g yeast extract, 8 g NaC1, 0.3 g CaClz and 4 g glucose per liter H20. Tryptone plates contained: 5 g NaC1, 10 g Bacto-tryptone, 20 g Bacto-agar per liter H20. Since strains carrying an ilvB mutation have a partial requirement for c~-, e-diaminopimelic acid and a temperature sensitive phenotype (De Felice et al. 1977), all experiments were performed at 30 33°C in media containing 100 ~tg/ml c~-, e-diaminopimelic acid. 0026-8925/81/0181/0491/$01.20