Polyamine stress at high pH in Escherichia coli K-12

BMC Microbiology Polyamine stress at high pH in Escherichia coli K-12 Elizabeth Yohannes 0 Amy E Thurber 0 Jessica C Wilks 0 Daniel P Tate 0 Joan L Slonczewski 0 0 Address: Department of Biology, Kenyon College , Gambier, OH 43022 Background: Polyamines such as spermine and spermidine are required for growth of Escherichia coli; they interact with nucleic acids, and they bind to ribosomes. Polyamines block porins and decrease membrane permeability, activities that may protect cells in acid. At high concentrations, however, polyamines impair growth. They impair growth more severely at high pH, probably due to their increased uptake as membrane-permeant weak bases. The role of pH is critical in understanding polyamine stress. Results: The effect of polyamines was tested on survival of Escherichia coli K-12 W3110 in extreme acid or base (pH conditions outside the growth range). At pH 2, 10 mM spermine increased survival by 2-fold, and putrescine increased survival by 30%. At pH 9.8, however, E. coli survival was decreased 100-fold by 10 mM spermine, putrescine, cadaverine, or spermidine. At pH 8.5, spermine decreased the growth rate substantially, whereas little effect was seen at pH 5.5. Spermidine required ten-fold higher concentrations to impair growth. On proteomic 2-D gels, spermine and spermidine caused differential expression of 31 different proteins. During log-phase growth at pH 7.0, 1 mM spermine induced eight proteins, including PykF, GlpK, SerS, DeaD, OmpC and OmpF. Proteins repressed included acetate-inducible enzymes (YfiD, Pta, Lpd) as well as RapA (HepA), and FabB. At pH 8.5, spermine induced additional proteins: TnaA, OmpA, YrdA and NanA (YhcJ) and also repressed 17 proteins. Four of the proteins that spermine induced (GlpK, OmpA, OmpF, TnaA) and five that were repressed (Lpd, Pta, SucB, TpiA, YfiD) show similar induction or repression, respectively, in base compared to acid. Most of these base stress proteins were also regulated by spermidine, but only at ten-fold higher concentration (10 mM) at high pH (pH 8.5). Conclusion: Polyamines increase survival in extreme acid, but decrease E. coli survival in extreme base. Growth inhibition by spermine and spermidine requires neutral or higher pH. At or above pH 7, spermine and spermidine regulate specific proteins, many of which are known to be regulated by base stress. High pH amplifies polyamine stress; and naturally occurring polyamines may play an important role in base stress. - Background Polyamines are required for the normal cell growth of Escherichia coli, although their functions are poorly understood [1-3]. Polyamines bind nucleic acids and ribosomes, where they are needed for optimal function [4,5]. Excessive intracellular concentrations, however, retard protein synthesis and cell growth [6]. Polyamine metabolism is stimulated by a variety of environmental stresses such as heat shock [7]. The major polyamine of bacteria, putrescine [NH2(CH2)4NH2], is synthesized by biosynthetic decarboxylation of arginine and/or ornithine [8,9]. Putrescine is metabolized to spermidine [NH2(CH2)3NH(CH2)4NH2]. Spermine [NH2(CH2)3NH(CH2)4NH(CH2)3NH2], a longer polyamine commonly produced by eukaryotes, is not produced by E. coli. Nevertheless, uptake of exogenous spermine fullfills the bacterial requirement for polyamines [4,5]. Spermine and spermidine do not undergo catabolism by E. coli, although excess concentrations are acetylated by polyamine acetyltransferase [10,11]. In the human colon, bacteria excrete putrescine and cadaverine during digestion of high-protein foods. Exposure of colonic epithelium to these polyamines stimulates human cell proliferation and leads to colonic tumors [12], which can be treated by drugs that deplete polyamine content [13]. Thus the modulation of polyamine metabolism under conditions of the gut is an important medical concern. The inhibition of growth by excess polyamines is amplified at high pH [14]. Polyamine stress enhances translation of the growth phase sigma RpoS [15], whose role in stationary-phase survival involves high pH [16]. A possible explanation for the amplification of polyamine stress at high pH is that polyamines become deprotonated and neutralized, thus capable of crossing the cell membane as membrane-permeant weak bases. Base-dependent uptake could augment the uptake through transporters [17]. The uptake of amines leads to their accumulation proportional to the transmembrane pH difference (ten-fold for each pH unit). Only a small fraction of an amine needs to be unprotonated (less than 1%) in order for significant membrane passage to occur. The pKa values of linear polyamines range from pKa = 8.3 to 11.6; for example, Ref [18] reports for 30 mM spermidine values of pKa1 = 8.6, pKa2 = 10.0, pKa3 = 11.1. However, literature reports vary for different conditions, and polyamine protonation levels under biological conditions are further influenced by complexing with fatty acids and phospholipids. cytoplasmic production of membrane-permeant amines can enhance bacterial growth. A pH-dependent source of amines in E. coli is the degradative arginine and ornithine decarboxylases (AdiA and SpeF respectively) and lysine decarboxylase (CadA), which are induced anaerobically at low pH [19]. The generation of putrescine (by AdiA) or cadaverine (by CadA), followed by excretion via cotranscribed transporters, neutralizes the acidic external environment [20]; for review, see [21,22]. At low-to-neutral pH, E. coli polyamines block the porins OmpF and OmpC, decreasing membrane permeability [23-25]. It was proposed that polyamines contribute to E. coli survival in extreme acid, below the growth range [24]; a phenomenon termed acid resistance or acid survival [21,22]. An OmpC mutant in which the porin fails to be blocked by cadaverine shows decreased survival at low pH (acid resistance) in the presence of cadaverine [24]. To our knowledge, it has not been shown directly that exogenous polyamines enhance acid resistance of wild-type cells. If polyamines do enhance survival in extreme acid, they could assist E. coli and other enteric pathogens during their passage through the stomach [26]. The interactions between pH and polyamines however remain poorly characterized, and are often discounted in studies of polyamine stress. For example, a recent major study of polyamine-mediated gene regulation does not address pH [3]. We report the effect of exogenous spermine and other polyamines on E. coli survival in extreme acid or extreme base. We also present protein profiles of E. coli exposed to exogenous polyamines under neutral and alkaline pH conditions, in the context of known pHdependent expression profiles [27,28]. Results Survival at extreme pH E. coli grown at moderate pH values possess mechanisms of protection against more extreme pH; these mechanisms are typically induced during stationary phase, or during growth near the acid or base end of their pH range [21,22]. We (...truncated)