The cis-acting regulatory element of the mvaAB operon of Pseudomonas mevalonii

Abstract

DNA upstream of the transcription start site of the mvaAB operon of Pseudomonas mevalonii, which encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.88) and HMG-CoA lyase (EC 4.1.3.4), contains a cis-acting regulatory element which functions in the response to mevalonate. The regulatory element resides within a 36-bp region located from 48 to 84 bp upstream of the transcription start site of mvaA.This location was inferred from the P-galactosidase activities of P. mevalonii harboring plasmid-encoded mvaA-lacZ fusions induced by mevalonate and by DNA gel retardation and competition assays. While protein from P. mevalonii grown on mevalonate produced a band shift, protein from cells grown on succinate gave no band shift, even when mevalonate was added. The operator contains three 10-bp direct repeats with the consensus sequence TGGGTACAGT, which may be important for regulation of the mvaAB operon.Mevalonate (3,5-dihydroxy-3-methylpentanoate) was initially isolated as a growth-stimulating factor for Lactobacillus acidophilus (23). In eukaryotes, mevalonate is converted to diverse isoprenoids, which include sterols, ubiquinone, isopentenyl adenine, and the farnesyl substituents of certain proteins. Several bacteria can utilize mevalonate as the sole source of carbon for growth (4,10,13,23). Growth of Pseudomonas mevalonii on mevalonate induces synthesis of an active transport system specific for R-mevalonate (12) and an NAD+-dependent 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase that converts R-mevalonate to S-HMG-CoA (13). Following HMG-CoA lyasecatalyzed cleavage of HMG-CoA to acetoacetate and acetylCoA (22), acetoacetate forms acetyl-CoA (21). P. mevalonii HMG-CoA reductase (13, 14) and HMG-CoA lyase (22) have been purified and characterized, and the genes mvaA (3) and mvaB (1) of the mvaAB operon that encode these enzymes have been cloned and sequenced. mvaA, which encodes HMG-CoA reductase, is the promoter-proximal gene. The promoter of the mvaAB operon has a -12, -24 consensus sequence (27) which in other bacteria is recognized by RNA polymerase holoenzymes which use factor (J4 (9). Gene transcription of this class of promoters has been proposed to be under positive control (16). We report here that a 36-bp region 48 bp upstream of the transcription start site of mvaAB contains a cis-acting element which responds to induction by mevalonate.(This research was carried out by Yuli Wang in partial fulfillment of the requirements for the Ph.D. degree from Purdue University, West Lafayette, Ind.) MATERIALS AND METHODS Bacterial strains, plasmids, bacteriophage, and culture conditions. Plasmids and phage M13 were propagated in Escherichia coli JM101 (8, 17). P. mevalonii was grown on ionic medium (12) containing the indicated sources of carbon and nitrogen. E. coli CSH26 harboring plasmid RP4 (2, 11) was grown on M9 medium (17) supplemented with 1 mM proline, * Corresponding author. t Journal paper 12791 from the Purdue University Agricultural Experiment Station. 50 ,ug of kana...

Biofilm Formation in Pseudomonas aeruginosa : Fimbrial cup Gene Clusters Are Controlled by the Transcriptional Regulator MvaT

“…MvaT was shown to bind to a region located at positions Ϫ48 to Ϫ84 upstream from the mvaA translational start codon (39). Analysis of the cupA1 promoter regions, as well as those of cupB1, cupC1, and lecA, analyzed since they also appeared to be down-regulated by MvaT, failed to identify any significant regions of homology with the mvaA cis-acting element.…”

Purification and characterization of the heteromeric transcriptional activator MvaT of the Pseudomonas mevalonii mvaAB operon

“…Activation of transcription requires a 36-bp cis-regulatory DNA element located at -48 to -84 bp upstream of the AUG start codon of the mvaA gene . Mobility shift assays revealed that this cis-regulatory DNA element is bound in a specific fashion by a P. mevalonii protein (Wang & Rodwell, 1991) that we term MvaT. Sequence of the double-stranded oligonucleotide Probe A formed from the complementary single-stranded oligonucleotides 1493D and 1494D.…”