Identification and characterization of acoK, a regulatory gene of the Klebsiella pneumoniae acoABCD operon

Abstract

By using transposon insertional mutagenesis and deletion analyses, a recombinant clone containing the region upstream of the acoABCD operon of Klebsiella pneumoniae was found to be required for acetoin-inducible expression of the operon in Escherichia coli. The nucleotide sequence of the region was determined, and it displayed an open reading frame of 2,763 bp that is transcribed divergently to the acoABCD operon. This gene, designated acoK, is capable of encoding a protein with an overall 58.4% amino acid identity with MalT, the transcriptional activator of the E. coli maltose regulon. A conserved sequence for nucleotide binding at the N-terminal region, as well as a helix-turn-helix motif belonging to the LuxR family of transcriptional regulators at the C terminus, was also identified. Primer extension analysis identified two transcription initiation sites, S1 and S2, located 319 and 267 bp, respectively, upstream of the putative start codon of acoK. Several copies of NtrC recognition sequence to N 18 )-GTG] were found in the promoter regions of both the acoK gene and the acoABCD operon. Acetoin-dependent expression of the acoABCD operon could be restored in the E. coli acoK mutants by supplying a plasmid carrying an intact acoK, suggesting a transactivating function of the gene product. The AcoK protein overproduced in E. coli was approximately 100 kDa, which is in good agreement with the molecular mass deduced from the nucleotide sequence. A specific DNA binding property and an ATPase activity of the purified AcoK were also demonstrated.Many bacteria are able to utilize acetoin as a carbon source. In some of the bacteria, dissimilation of acetoin proceeds via an oxidative cleavage process which is catalyzed by an acetoin dehydrogenase enzyme system with acetaldehyde and acetyl coenzyme A as the end products (13, 22). The acetoin dehydrogenase enzyme system is composed of E1␣ and E1␤, an acetoin-dependent dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR); E2, a dihydrolipoamide acetyltransferase; and E3, a dihydrolipoamide dehydrogenase. Structural genes encoding E1␣, E1␤, E2, and E3 of the acetoin dehydrogenase enzyme system have recently been isolated from several bacterial species (11,19,25,28). In Alcaligenes eutrophus, the structural genes identified for E1␣ (acoA), E1␤ (acoB), and E2 (acoC) are clustered with acoX, which encodes a protein of unknown function in the order of acoXABC (28). The structural gene for E3 which is not found in A. eutrophus aco operon, however, is present as acoL in Pelobacter carbinolicus acoABCLS (25) and Clostridium magnum acoABXCL (19) operons and as acoD in the Klebsiella pneumoniae acoABCD operon (27). Amino acid sequences deduced from these genes exhibit significant homology to those of the respective component of various 2-oxo acid dehydrogenase complexes, suggesting an evolutionary relationship among these catabolic systems.Despite the wealth of information on acetoin dehydrogenases, how these aco operons are regulated is less understood. The acoR gene located upst...

Characterization and Analysis of the PikD Regulatory Factor in the Pikromycin Biosynthetic Pathway of Streptomyces venezuelae

“…Members of this family were proposed to operate by a similar biochemical mechanism. The less conserved middle region of the protein is proposed to be the possible site for inducer interaction, such as those found in MalT and AcoK, which require the coinducers maltotriose and acetoin, respectively (27,29).…”

STAND, a Class of P-Loop NTPases Including Animal and Plant Regulators of Programmed Cell Death: Multiple, Complex Domain Architectures, Unusual Phyletic Patterns, and Evolution by Horizontal Gene Transfer

“…Many of the bacterial STAND NTPases respond to the availability of simple nutrients in the environment. In particular, E. coli MalT is a positive regulator of the maltose regulon, 92 AcoK is required for the expression of the acetoin operon, 93 Bacillus GutR is the transcriptional activator of the glucitol operon, 45 Rhodococcus sp. M5 BpdS regulates biphenyl/polychlorobiphenyl metabolism, 74 and Streptomyces avermitilis LipR activates the LipA lipase, which apparently is involved in the utilization of oils present in the medium.…”

A new mechanism for the control of a prokaryotic transcriptional regulator: antagonistic binding of positive and negative effectors

“…Alone or in synergy with CRP (cAMP receptor protein), MalT activates all the operons comprising the Escherichia coli maltose regulon, which encodes proteins involved in the uptake and degradation of maltodextrins (Boos and Shuman, 1998). MalT (polypeptide size 103 kDa) is the best known member of an emerging family of homologous 100 kDa transcriptional activators (Wubbolts, 1994; Peng et al ., 1997). Transcription activation by MalT depends on the presence of ATP and maltotriose, two effectors of the protein (Raibaud and Richet, 1987; Richet and Raibaud, 1989), and involves MalT binding to an array of sites located upstream of the ‘−35’ region of the mal promoters (Boos and Shuman, 1998).…”