The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor

Abstract

The growth of the soil bacterium Pseudomonas putida KT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual cells. Fluorescence microscopy and flow cytometry, as well as the analysis of the temporal start of growth in single-cell cultures, revealed that adoption of a glycerol-metabolizing regime was not the result of a gradual change in the whole population but rather reflected a time-dependent bimodal switch between metabolically inactive (i.e., nongrowing) and fully active (i.e., growing) bacteria. A transcriptional Φ(glpD-gfp) fusion (a proxy of the glycerol-3-phosphate [G3P] dehydrogenase activity) linked the macroscopic phenotype to the expression of the glp genes. Either deleting glpR (encoding the G3P-responsive transcriptional repressor that controls the expression of the glpFKRD gene cluster) or altering G3P formation (by overexpressing glpK, encoding glycerol kinase) abolished the bimodal glpD expression. These manipulations eliminated the stochastic growth start by shortening the otherwise long lag phase. Provision of glpR in trans restored the phenotypes lost in the ΔglpR mutant. The prolonged nongrowth regime of P. putida on glycerol could thus be traced to the regulatory device controlling the transcription of the glp genes. Since the physiological agonist of GlpR is G3P, the arrangement of metabolic and regulatory components at this checkpoint merges a positive feedback loop with a nonlinear transcriptional response, a layout fostering the observed time-dependent shift between two alternative physiological states.

A Metabolic Widget Adjusts the Phosphoenolpyruvate-Dependent Fructose Influx in Pseudomonas putida

“…When using Glu or Suc as the carbon source, the three strains followed rather similar growth patterns, and the differences in the kinetic parameters analyzed were not significant. In fact, the specific growth rates/characteristic doubling times and the duration of the lag phase in these cultures were consistent with previous findings (11, 35, 39), Suc promoting the fastest growth among the conditions tested. A different situation, however, was noted when Fru was used as the substrate.…”

“…This situation ensures the availability of enough of the PEP needed for the phosphorylation of the PTS components all the way up to exogenous Fru during shifts between conditions characterized by different levels of hexose availability. This situation is reminiscent of the regulatory phenomenon observed when P. putida cells grow on glycerol as the carbon source (39). Under these conditions, this bacterium displays a sort of memory, ruled by the GlpR regulator, that enables the cells to better adapt to environmental situations in which glycerol is highly abundant.…”

Population collapse and adaptive rescue during long‐term chemostat fermentation

“…GlpR is the repressor of sn-glycerol-3-phosphate regulon which is composed of several operons (Schweizer, Boos & Larson, 1985;Yang, Gerhardt, & Larson, 1997) responsible for the metabolism of glycerol and glycerol-3-phosphate. Interestingly, a recent study demonstrated that when Pseudomonas putida is grown in glycerol supplemented growth media, it showed bimodal growth/nogrowth patterns, with GlpR responsible for bimodal growth (Nikel, Romero-Campero, Zeidman, Goñi-Moreno, & de Lorenzo, 2015). The second gene gatZ is a member of the gat operon driving the galactitol metabolism (Nobelmann & Lengeler, 1996).…”

The global regulator Crc orchestrates the metabolic robustness underlying oxidative stress resistance in Pseudomonas aeruginosa

“…Negative controls were run by adding 2 μl of 5 mM CCCP to the cell suspension. The RSG fluorescence was quantified at 400 nm and 447 nm as the excitation and emission wavelengths, respectively, according to the protocol previously developed for P. putida (Nikel et al, 2015b).…”