Mechanism for Regulation of the Putrescine Utilization Pathway by the Transcription Factor PuuR in Escherichia coli K-12

Abstract

bIn Escherichia coli, putrescine is metabolized to succinate for use as a carbon and nitrogen source by the putrescine utilization pathway (Puu pathway). One gene in the puu gene cluster encodes a transcription factor, PuuR, which has a helix-turn-helix DNA-binding motif. DNA microarray analysis of an E. coli puuR mutant, in which three amino acid residues in the helix-turnhelix DNA binding motif of PuuR were mutated to alanine to eliminate DNA binding of PuuR, suggested that PuuR is a negative regulator of puu genes. Results of gel shift and DNase I footprint analyses suggested that PuuR binds to the promoter regions of puuA and puuD. The binding of wild-type PuuR to a DNA probe containing PuuR recognition sites was diminished with increasing putrescine concentrations in vitro. These results suggest that PuuR regulates the intracellular putrescine concentration by the transcriptional regulation of genes in the Puu pathway, including puuR itself. The puu gene cluster is found in E. coli and closely related enterobacteria, but this gene cluster is uncommon in other bacterial groups. E. coli and related enterobacteria may have gained the Puu pathway as an adaptation for survival in the mammalian intestine, an environment in which polyamines exist at relatively high concentrations.

Engineering tunable biosensors for monitoring putrescine in Escherichia coli

“…Next, we designed a series of synthetic promoters by inserting PuuR operator sequence into strong promoters, such as the tac , phage lambda , and phage T7 promoters (Lutz & Bujard, ). Earlier, the PuuR operator (PuuO) with a length of 20 bp has been identified from the puuA promoter region of E. coli (Nemoto et al, ). In order to examine the effects of varying the copy number and position of PuuO, synthetic promoters were made with either a single PuuO downstream of transcriptional start site or with an additional one between the −35 and the −10 region of the phage T7 , phage lambda and tac promoters; the AR2 , LR2 , and TacR2 promoters, each with dual PuuO sequences, and the single PuuO‐harboring AR3 , LR3 , and TacR3 promoters were thus constructed (Figure a).…”

Elimination of polyamine N-acetylation and regulatory engineering improved putrescine production by Corynebacterium glutamicum

“…Secretion of polyamines has been described as alternative way to polyamine N-acetylation for regulation of intracellular polyamine levels in living cells (Rhee et al, 2007). In E. coli, six polyamine transport systems have been identified so far: PotABCD and PotFGHI for specific spermidine and putrescine uptake, respectively (Igarashi and Kashiwagi, 1999), CadB as cadaverine/lysine antiporter (Soksawatmaekhin et al, 2006), PotE as putrescine/ornithine antiporter (Kashiwagi, 1996), and the putrescine uptake systems YcjJ and Puu (Kurihara et al, 2005;Nemoto et al, 2012). In Bacillus subtilis, spermidine is exported by the multidrug exporter Blt, which is encoded in the blt-bltD operon together with the spermine/spermidine N-acetyltransferase BltD (Woolridge et al, 1997).…”

Model-driven discovery of underground metabolic functions in Escherichia coli

“…S2B). The genes puuR and puuC are both in the same operon as the isozyme puuE explored in this study, with puuR acting as a repressor for this operon under conditions of low putrescine concentration (28).…”

“…The puuR DNA-binding transcriptional repressor consists of two domains, namely, a helixturn-helix DNA-binding domain and a Cupin-family domain (28). Using the high number of homologous templates available, a homology model was constructed from the amino acid sequence of the puuR gene via the available toolkits (29-31) (with an average confidence of 97% across templates; Fig.…”