Cellular choline and glycine betaine pools impact osmoprotection and phospholipase C production in Pseudomonas aeruginosa

Abstract

Choline is abundantly produced by eukaryotes and plays an important role as a precursor of the osmoprotectant glycine betaine. In Pseudomonas aeruginosa, glycine betaine has additional roles as a nutrient source and an inducer of the hemolytic phospholipase C, PlcH. The multiple functions for glycine betaine suggested that the cytoplasmic pool of glycine betaine is regulated in P. aeruginosa. We used (13)C nuclear magnetic resonance ((13)C-NMR) to demonstrate that P. aeruginosa maintains both choline and glycine betaine pools under a variety of conditions, in contrast to the transient glycine betaine pool reported for most bacteria. We were able to experimentally manipulate the choline and glycine betaine pools by overexpression of the cognate catabolic genes. Depletion of either the choline or glycine betaine pool reduced phospholipase production, a result unexpected for choline depletion. Depletion of the glycine betaine pool, but not the choline pool, inhibited growth under conditions of high salt with glucose as the primary carbon source. Depletion of the choline pool inhibited growth under high-salt conditions with choline as the sole carbon source, suggesting a role for the choline pool under these conditions. Here we have described the presence of a choline pool in P. aeruginosa and other pseudomonads that, with the glycine betaine pool, regulates osmoprotection and phospholipase production and impacts growth under high-salt conditions. These findings suggest that the levels of both pools are actively maintained and that perturbation of either pool impacts P. aeruginosa physiology.

Fig 1

¹³C-NMR of cell extracts showing choline and glycine betaine (GB) pools during growth on different carbon sources in the presence of high or low concentrations of salt. The carbon source for each column is noted at the top of the figure. The top row is low salt (50 mM NaCl, standard MOPS medium), and the bottom row is high salt (MOPS plus 700 mM NaCl). The x axis represents ppm. Under all conditions, cells were grown in the presence of 5 mM [1,2-¹³C]choline. For panels A and E, additional unlabeled choline was added for a total of 20 mM choline; therefore, the labeled compound is only one-quarter of the pool in these cells (as observed by the peak heights compared to that of the methanol standard). Abbreviations: c, choline; g, glycine betaine (GB); me, [¹³C]methanol; s, sarcosine. Each panel shows results representative of at least three experiments.

Fig 2

¹³C-NMR of P. aeruginosa cell extracts after exposure to [1,2-¹³C]choline, with choline as the sole carbon source. Cells were grown as described in Materials and Methods in the presence of gentamicin, l-arabinose (0.05%), and labeled choline for 6 h. (A) Cells maintaining the pMQ80 empty vector (expresses green fluorescent protein [GFP] during arabinose induction under p_BAD control); (B) cells expressing the betBA genes under p_BAD control; (C) cells expressing the gbcBA genes under p_BAD control. These spectra are representative of at least three biological replicates. Abbreviations: cho, choline; GB, glycine betaine; Me, [¹³C]methanol standard; sarc, sarcosine.

Fig 3

PLC activity measured by NPPC hydrolysis activity in culture supernatants. Cells were grown in MOPS pyruvate with or without choline and with or without NaCl for 24 h as noted below the x axis. Enzyme activity was calculated as described in Materials and Methods and was normalized to that of a culture OD₆₀₀. These data are representative of three independent experiments each containing three biological replicates. Error bars represent standard deviations.

Fig 4

Effect of catabolic enzyme overexpression on P. aeruginosa growth in increasing salt concentrations. (A) P. aeruginosa carrying the specified vector grown on MOPS glucose with gentamicin and 0.05% l-arabinose for 24 h. Growth conditions were with or without 250 μM choline and with or without added NaCl, as noted below the x axis. Cells exposed to these high-salt conditions were unable to grow without added choline. (B) P. aeruginosa growth in 750 mM NaCl when cells were grown with choline as the sole carbon source for 40 h. Both panels are representative of the results of at least three experiments each containing three biological replicates. Error bars represent standard deviations.

Fig 5

Accumulation of choline and GB in a set of Proteobacteria capable of aerobic choline catabolism as measured by ¹³C-NMR when the organisms were grown on choline as a sole carbon source. P. aeruginosa (P.a.) and Burkholderia cepacia (B.c.) were grown at both 30°C and 37°C. P. putida (P.p.), P. fluorescens (P.f.), P. syringae (P.s.), and Sinorhizobium meliloti (S.m.) were grown at 30°C. Panel B is identical to Fig. 1A and is our representative image for this condition in P. aeruginosa. Data are representative of at least three independent experiments. Abbreviations: c, choline; g, glycine betaine; me, [¹³C]methanol. Results in each panel are representative of at least three experiments.