Nucleotide sequence of the Escherichia coli cad operon: a system for neutralization of low extracellular pH

Abstract

Lysine decarboxylase ofEscherichia coli has been the subject of enzymological studies, and the gene encoding lysine decarboxylase (cadA4) and a regulatory gene (cadR) have been mapped. This enzyme is induced at low pH in the presence of lysine and achieves maximal level under anaerobic conditions. The induction of lysine decarboxylase increases the pH of the extracellular medium and provides a distinctive marker in tests of clinical strains. We report the sequence of the cad operon encoding lysine decarboxylase, a protein of 715 amino acids, and another protein, CadB, of 444 amino acids. The amino acid sequence of lysine decarboxylase showed high homology to that of the lysine decarboxylase of Hafnia alvei with less homology to the sequence of speC, which encodes the biosynthetic ornithine decarboxylase of E. coli. The cadA and cadB genes were separately cloned and placed under the control of lac and tac promoters, respectively, to facilitate independent study of their physiological effects. The cadB gene product had a mobility characteristic of a smaller protein on protein gels, analogous to that found for some other membrane proteins. The CadB sequence showed homology to that of ArcD of Pseudomonas aeruginosa, encoding an arginine/ornithine antiporter. Excretion studies of various strains, the coinduction of cadB and cadA, and the attractive physiological role for an antiport system led to a model for the coupled action of cadA and cadB in uptake of lysine, the reduction of H+ concentration, and excretion of cadaverine.Two types of amino acid decarboxylases exist in Escherichia coli. Biosynthetic enzymes are involved in the synthesis of polyamines and are expressed at low levels regardless of pH variation (56). Biodegradative enzymes such as lysine decarboxylase are strongly induced at acidic pH (5,49,56) and hence have a possible role in maintaining pH homeostasis or extending the growth period by detoxification of the extracellular medium (20). Although it has been stated elsewhere that the cadaverine generated from decarboxylation of L-lysine is not further metabolized for energy production but is excreted out of the cell as part of a countermeasure to acidic environment (49), no direct detailed analysis of this aspect has been reported. Unlike arginine and ornithine decarboxylases, two types of enzymes that have been well characterized previously (56), only the inducible form of lysine decarboxylase has been analyzed previously (49, 50), although some evidence has indicated the presence of a pH-constitutive form of lysine decarboxylase in E. coli (24,64).Mutants of cadA have been isolated, and the gene was first mapped to around 92 min on the E. coli genome (57). Subsequent detailed mapping of in vivo Mu dX operon fusion strains deficient in lysine decarboxylase activity and mini-Mu clones revealed that cadA4 was at 93.7 min (5). In studies of protein expression from the plasmid pLC4-5, it was found that cadA4 and lysU were both expressed (59); however, the orientation and order relative to the surrounding...

Excretion and Uptake of Putrescine by the PotE Protein in Escherichia coli

“…These proteins play important roles in the generation of a proton motive force (45), neutralization of low extracellular pH (44), and supply of carbon dioxide (46). In this study, we clarified that the ornithine-putrescine antiporter also functions as a putrescine uptake system that is dependent on the membrane potential.…”

“…It has been reported that there are three basic amino acid/ decarboxylated derivative antiporters on prokaryotic membranes, they are the ornithine-putrescine (15), lysine-cadaverine (44), and histidine-histamine (45) antiporters. These proteins play important roles in the generation of a proton motive force (45), neutralization of low extracellular pH (44), and supply of carbon dioxide (46).…”

Inhibition of Shigella flexneri-induced transepithelial migration of polymorphonuclear leucocytes by cadaverine

“…We determined previously that BS529, the wild-type S.¯exneri 2a strain transformed with pCADA, produces 225±300 mM cadaverine under the growth conditions we used here (Maurelli et al, 1998). Similarly, wildtype strains of E. coli K-12 produce up to 350 mM cadaverine when grown in LB at pH 5.8 (Meng and Bennett, 1992;Samartzidou and Delcour, 1999). It should also be emphasized that the concentration of cadaverine in the immediate vicinity of a lysine decarboxylase-producing bacteria colonizing the intestinal mucosal surface would probably be signi®cantly higher than the global concentration that we measured in a liquid culture of the same bacteria.…”

Secondary solute transport in bacteria

“…On the basis of the homology between ArcD, LysI and a putative membrane protein (CadB), the latter protein has been proposed to be a lysine/cadaverine exchanger [98]. Lysine and cadaverine carry one and two net positive charges, respectively, and lysine/ cadaverine exchange is therefore expected to be counteracted by the membrane potential (inside negative).…”