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RodZ and PgsA Play Intertwined Roles in Membrane Homeostasis of Bacillus subtilis and Resistance to Weak Organic Acid Stress.

ABSTRACT: Weak organic acids like sorbic and acetic acid are widely used to prevent growth of spoilage organisms such as Bacilli. To identify genes involved in weak acid stress tolerance we screened a transposon mutant library of Bacillus subtilis for sorbic acid sensitivity. Mutants of the rodZ (ymfM) gene were found to be hypersensitive to the lipophilic weak organic acid. RodZ is involved in determining the cell's rod-shape and believed to interact with the bacterial actin-like MreB cytoskeleton. Since rodZ lies upstream in the genome of the essential gene pgsA (phosphatidylglycerol phosphate synthase) we hypothesized that expression of the latter might also be affected in rodZ mutants and hence contribute to the phenotype observed. We show that both genes are co-transcribed and that both the rodZ::mini-Tn10 mutant and a conditional pgsA mutant, under conditions of minimal pgsA expression, were sensitive to sorbic and acetic acid. Both strains displayed a severely altered membrane composition. Compared to the wild-type strain, phosphatidylglycerol and cardiolipin levels were lowered and the average acyl chain length was elongated. Induction of rodZ expression from a plasmid in our transposon mutant led to no recovery of weak acid susceptibility comparable to wild-type levels. However, pgsA overexpression in the same mutant partly restored sorbic acid susceptibility and fully restored acetic acid sensitivity. A construct containing both rodZ and pgsA as on the genome led to some restored growth as well. We propose that RodZ and PgsA play intertwined roles in membrane homeostasis and tolerance to weak organic acid stress.

SUBMITTER: van Beilen J

PROVIDER: S-EPMC5073135 | biostudies-other | 2016

REPOSITORIES: biostudies-other

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RodZ and PgsA Play Intertwined Roles in Membrane Homeostasis of Bacillus subtilis and Resistance to Weak Organic Acid Stress.

van Beilen Johan J Blohmke Christoph J CJ Folkerts Hendrik H de Boer Richard R Zakrzewska Anna A Kulik Wim W Vaz Fred M FM Brul Stanley S Ter Beek Alexander A

Frontiers in microbiology 20161021

Weak organic acids like sorbic and acetic acid are widely used to prevent growth of spoilage organisms such as Bacilli. To identify genes involved in weak acid stress tolerance we screened a transposon mutant library of Bacillus subtilis for sorbic acid sensitivity. Mutants of the rodZ (ymfM) gene were found to be hypersensitive to the lipophilic weak organic acid. RodZ is involved in determining the cell's rod-shape and believed to interact with the bacterial actin-like Mre ...[more]

PMID: 27818647

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Project description:Intracellular pH (pHi) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pHi homeostasis. Unfortunately, accurate pHi quantification with existing methods is suboptimal, since measurements are averages across populations of cells, not taking into account interindividual heterogeneity. Yet, physiological heterogeneity in isogenic populations is well known to be responsible for differences in growth and division kinetics of cells in response to external stressors. To assess in this context the behavior of intracellular acidity, we have developed a robust method to quantify pHi at single-cell levels in Bacillus subtilis Bacilli spoil food, cause disease, and are well known for their ability to form highly stress-resistant spores. Using an improved version of the genetically encoded ratiometric pHluorin (IpHluorin), we have quantified pHi in individual B. subtilis cells, cultured at an external pH of 6.4, in the absence or presence of weak acid stresses. In the presence of 3 mM potassium sorbate, a decrease in pHi and an increase in the generation time of growing cells were observed. Similar effects were observed when cells were stressed with 25 mM potassium acetate. Time-resolved analysis of individual bacteria in growing colonies shows that after a transient pH decrease, long-term pH evolution is highly cell dependent. The heterogeneity at the single-cell level shows the existence of subpopulations that might be more resistant and contribute to population survival. Our approach contributes to an understanding of pHi regulation in individual bacteria and may help scrutinizing effects of existing and novel food preservation strategies.ImportanceThis study shows how the physiological response to commonly used weak organic acid food preservatives, such as sorbic and acetic acids, can be measured at the single-cell level. These data are key to coupling often-observed single-cell heterogeneous growth behavior upon the addition of weak organic acid food preservatives. Generally, these data are gathered in the form of plate counting of samples incubated with the acids. Here, we visualize the underlying heterogeneity in cellular pH homeostasis, opening up avenues for mechanistic analyses of the heterogeneity in the weak acid stress response. Thus, microbial risk assessment can become more robust, widening the scope of use of these well-known weak organic acid food preservatives.

Project description:A 5.9-kb region of the Bacillus subtilis chromosome is transcribed as a single transcript that is predicted to encode seven membrane-spanning proteins. Homologues of the first gene of this operon, for which the designation mrp (multiple resistance and pH adaptation) is proposed here, have been suggested to encode an Na+/H+ antiporter or a K+/H+ antiporter. In the present studies of the B. subtilis mrp operon, both polar and nonpolar mutations in mrpA were generated. Growth of these mutants was completely inhibited by concentrations of added Na+ as low as 0.3 M at pH 7.0 and 0.03 M at pH 8.3; there was no comparable inhibition by added K+. A null mutant that was constructed by full replacement of the mrp operon was even more Na+ sensitive. A double mutant with mutations in both mrpA and the multifunctional antiporter-encoding tetA(L) gene was no more sensitive than the mrpA mutants to Na+, consistent with a major role for mrpA in Na+ resistance. Expression of mrpA from an inducible promoter, upon insertion into the amyE locus, restored significant Na+ resistance in both the polar and nonpolar mrpA mutants but did not restore resistance in the null mutant. The mrpA disruption also resulted in an impairment of cytoplasmic pH regulation upon a sudden shift in external pH from 7.5 to 8.5 in the presence of Na+ and, to some extent, K+ in the range from 10 to 25 mM. By contrast, the mrpA tetA(L) double mutant, like the tetA(L) single mutant, completely lost its capacity for both Na+- and K+-dependent cytoplasmic pH regulation upon this kind of shift at cation concentrations ranging from 10 to 100 mM; thus, tetA(L) has a more pronounced involvement than mrpA in pH regulation. Measurements of Na+ efflux from the wild-type strain, the nonpolar mrpA mutant, and the complemented mutant indicated that inducible expression of mrpA increased the rate of protonophore- and cyanide-sensitive Na+ efflux over that in the wild-type in cells preloaded with 5 mM Na+. The mrpA and null mutants showed no such efflux in that concentration range. This is consistent with MrpA encoding a secondary, proton motive force-energized Na+/H+ antiporter. Studies of a polar mutant that leads to loss of mrpFG and its complementation in trans by mrpF or mrpFG support a role for MrpF as an efflux system for Na+ and cholate. Part of the Na+ efflux capacity of the whole mrp operon products is attributable to mrpF. Neither mrpF nor mrpFG expression in trans enhanced the cholate or Na+ resistance of the null mutant. Thus, one or more other mrp gene products must be present, but not at stoichiometric levels, for stability, assembly, or function of both MrpF and MrpA expressed in trans. Also, phenotypic differences among the mrp mutants suggest that functions in addition to Na+ and cholate resistance and pH homeostasis will be found among the remaining mrp genes.

Dataset Information

RodZ and PgsA Play Intertwined Roles in Membrane Homeostasis of Bacillus subtilis and Resistance to Weak Organic Acid Stress.

Publications

RodZ and PgsA Play Intertwined Roles in Membrane Homeostasis of <i>Bacillus subtilis</i> and Resistance to Weak Organic Acid Stress.

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