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

Abstract

The mvaAB operon of Pseudomonas mevalonii encodes HMG-CoA reductase (EC 1.1.1.88) and HMG-CoA lyase (EC 4.1.3.4), enzymes that catalyze the initial reactions of mevalonate catabolism in this organism. Expression of this operon is regulated by the constitutively expressed transcriptional activator protein MvaT that binds in vitro to an upstream regulatory element. Mevalonate is essential for activation of transcription in vivo, and in vitro data demonstrated that MvaT binds to the rnvaAB cis-regulatory element in the absence of mevalonate with a Kd,app of 2 nM. Purification of MvaT enriched for two polypeptides of approximate molecular mass 15 kDa and 16 kDa, designated P15 and P16. MvaT, assayed by its DNA-binding activity, comigrated with PI5 and P16 during DNA-affinity chromatography, size-exclusion chromatography, and sucrose density gradient centrifugation. PI5 and P16 also comigrated during denaturing isoelectric focusing of purified MvaT. Treatment of MvaT with dimethylsuberimidate formed a 31-kDa polypeptide complex that contained N-terminal sequences from P15 and P16. The apparent association of P15 and P16 in solution and their copurification with MvaT activity strongly suggests that MvaT is comprised of these two subunits. Size-exclusion chromatography gave an estimated molecular mass for MvaT of 33 kDa. A partial DNA sequence of the P16 gene was obtained using PCR employing degenerate primers directed against the N-termini of P15 and P16. P16 appears to be comprised of at least 128 aminoacyl residues having a predicted molecular mass of 14.3 kDa.

MvaT proteins in Pseudomonas spp.: a novel class of H-NS-like proteins

“…The comparison of the Pseudomonas strain On: Fri, 11 May 2018 05:53:00 Y1000 H-NS-like protein amino acid sequence with all sequences present in databases revealed approximately 50 % identity with MvaT, a transcriptional activator that participates in the control of mevalonate metabolism in Pseudomonas mevalonii (Rosenthal et al, 1998). By scanning complete and unfinished bacterial genome sequences, we identified at least one homologous protein in each Pseudomonas species.…”

“…All these proteins display at least 47 % amino acid identity. Except for the MvaT proteins of P. mevalonii and P. aeruginosa, which possess a regulatory role in mevalonate metabolism and in virulence gene expression, respectively (Rosenthal et al, 1998;Diggle et al, 2002), the function of the Pseudomonas H-NS-related proteins remains unknown and some have been annotated as hypothetical proteins. Overexpression of the hns-like gene of Pseudomonas strain Y1000 in the same strain from the low-copy-number vector pDIA584, i.e.…”

The Pseudomonas aeruginosa global regulator MvaT specifically binds to the ptxS upstream region and enhances ptxS expression

“…2a). A search of the P. aeruginosa genome with the amino acid sequences obtained from amino terminus sequencing showed that the 9 kDa protein is the P. aeruginosa HU protein, which is encoded by the hupB gene (Delic-Attree et al, 1995), while the 16 kDa protein is the P. aeruginosa transcriptional regulator MvaT (Diggle et al, 2002), a homologue of the MvaT beta subunit in Pseudomonas mevalonii (Rosenthal & Rodwell, 1998). Three other column fractions produced gel shift bands that varied in their migration distance when compared to the 0?6 M KCl fraction (data not shown).…”

Novel Physiological Modulation of the Pu Promoter of TOL Plasmid

“…Identification and in Silico Analysis of TurA Protein-The amino acid sequence of P16 amino-terminal end was determined, searched through the KT2440 deduced protein data base at The Institute for Genomic Research (TIGR) Comprehensive Microbial Resource (31,32), and found at the aminoterminal of a small deduced protein of 125 amino acids, accession number PP1366, annotated as sharing a high degree of similarity with the transcriptional regulator MvaT, P16 subunit (33). Standard BLAST search (34) also found in KT2440 genome four open reading frames (loci PP3765, PP0017, PP3693, PP2947, respectively) that code for protein paralogues of P16.…”