Weak organic acid stress in Bacillus subtilis

Weak organic acids are commonly used food preservatives that protect food products from bacterial contamination. A variety of spore-forming bacterial species pose a serious problem to the food industry by causing extensive food spoilage or even food poisoning. Understanding the mechanisms of bacterial stress response and resistance to food preservatives is crucial for combating their growth. The spore-former Bacillus subtilis is one of the notorious food spoilers. As a result of over fifty years of research it is also the best characterized Gram-positive bacterium, which makes it an excellent model organism. The main goal of this study was to gain insight into B. subtilis response and resistance to weak organic acids by means of growth measurements, transcriptomics, mutagenesis and fluorescent-based intracellular pH measurements.
Chapter 1 introduces B. subtilis as a spore-forming bacterium as well as its threat to the food industry. The common stress responses of B. subtilis are indicated, including the SigB-mediated general stress response (GSR), sporulation, stringent response, biofilm formation and competence development for genetic transformation. Next, the GSR and sporulation are discussed in greater detail. Furthermore, background on weak organic acids is provided including the description of their presumed modes of action based on available studies from other organisms such as Saccharomyces cerevisiae and Escherichia coli. The antimicrobial activity of weak organic acids is related to their ability to uncouple the proton gradient, acidify the cytosol, cause osmotic stress by anion accumulation, induce metabolic perturbation by inactivation of specific enzymes and, last but not least, disrupt the plasma membrane. Thus, the structure and function of the plasma membrane of B. subtilis is introduced with a focus on composition and biosynthesis of its lipid and fatty acid components. Finally, the regulation of membrane lipid biosynthesis is discussed.
In Chapter 2 sorbic acid was shown to cause growth inhibition of exponentially growing B. subtilis at various pH values. The growth inhibition appeared to depend mostly, but not solely, on the undissociated form of the acid. Moreover, the time-resolved transcriptional response of B. subtilis to mild sorbic acid stress was elucidated. Hierarchical clustering and T-profiler analysis of the transcriptome data revealed that sorbic acid-stressed cells induce responses normally observed in cells that experience a nutrient limitation. This was indicated by the strong derepression of the CcpA, CodY, and Fur regulon, the induction of tricarboxylic acid cycle genes, SigL- and SigH-mediated genes, and the stringent response. Interestingly, competence, sporulation or the GSR was not induced upon sorbic acid stress. However, the transcriptional response to this weak organic acid indicated plasma membrane remodelling based on upregulation of fatty acid biosynthesis genes and the BkdR regulon. Finally, this result was further supported by the increased resistance of B. subtilis to fatty acid biosynthesis inhibitor cerulenin upon sorbic acid stress. 195
In Chapter 3 transposon mutagenesis was utilized to identify mutants with altered susceptibility to sorbic acid. Random mutant libraries created in B. subtilis wild-type strain PB2 were screened for sorbic acid sensitivity and resistance. Four sensitive and three resistant transposon mutants were identified in the screen. All seven mutants were subsequently tested for their susceptibility to four different stresses: acetic acid, carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), low pH and NaCl. The transposon mutant of unknown gene ymfI was shown to be sensitive to all tested stresses. Interestingly, YmfI shows high similarity to the essential 3-ketoacyl-(acyl-carrier-protein) reductase, FabG, committed to the elongation of fatty acids. The disruption of the unknown gene ymfM, coding for a possible transcriptional regulator, led to hypersensitivity for the weak organic acids: sorbic- and acetic- acid. This mutant was also found sensitive to low pH and NaCl stress, yet not to CCCP. Controlled reduction in the transcription of the downstream gene pgsA, using a Pspac-pgsA strain, demonstrated a similar phenotype as for that of the ymfM transposon mutant. Essential gene pgsA codes for phosphatidylglycerophosphate synthase and is committed to the biosynthesis of the anionic phospholipids: phosphatidylglycerol and cardiolipin. Finally, none of the genes identified in this screen were affected exclusively in sensitivity to sorbic acid.
The comparative physiological and transcriptional analysis of weak organic stresses (CCCP, sorbic acid and acetic acid) in B. subtilis was performed in Chapter 4. The concentration of each acid that was required to cause a similar growth reduction was shown to correlate negatively with their membrane solubility and positively with the concentration of the undissociated acid present. The contribution of the anion to growth inhibition was found to be most pronounced for CCCP out of all three stresses. The time-resolved microarray analysis of B. subtilis treated with sub-lethal concentrations of these weak organic acids revealed that all three acids activated transcriptional programs common for experiencing nutrient limitation, as demonstrated by derepression of CcpA-regulated genes and the induction of the stringent response. Additionally, all three stresses activated diverse responses that all indicated an adaptation of the cell envelope. As stated in Chapter 2 sorbic acid activated specifically the FapR and BkdR regulons. Conversely, acetic acid was found to repress genes regulated by FapR. The stress-specific repression of the YvrH, SigW, and SigX regulons was also observed. Interestingly, in contrast to the action of sorbic acid, both CCCP and acetic acid were found to induce the GSR. Only CCCP activated the expression of genes regulated by SigM and sodA, coding for superoxide dismutase. Finally, acetic acid-stressed cells showed a clear metabolic shift to acetate utilization as shown by the strong induction of the alsSD genes, acetyl-CoA synthetase acsA, and strong repression of the pdhABCD genes, encoding pyruvate dehydrogenase complex.
Chapter 5 presents the inducible ‘pH meter’, which was utilized to measure in vivo the intracellular pH (pHi) of B. subtilis. The constructed plasmid (pDG148-pHluorin) was utilized to test the effect of weak organic acids and hyperosmotic shock on the pHi of B. subtilis.
Lowering of the pHi of B. subtilis by the commonly used food preservatives potassium -sorbate, -acetate, and -benzoate was demonstrated. The uncoupler CCCP and hyperosmotic shock induced by NaCl were also shown to confer a decline in pHi. The main drop in pHi occurred for all stresses within the first six minutes and no recovery to the control pH value was observed during 2 h of stress. The growth inhibition percentage caused by all stresses related to the drop in pHi. However, no linear correlation was found, likely due to the buffering capacity of the cell. Comparison of the stresses revealed that CCCP gave the highest, and hyperosmotic shock the lowest pHi drop. Finally, we determined that a clear threshold value of the growth inhibition caused by NaCl was required before inducing a drop in pHi.
In Chapter 6 the phenomenon of physiological heterogeneity is introduced in the context of outgrowth of bacterial spores. The molecular mechanisms that likely contribute to observed heterogeneous behaviour of B. subtilis cells and spores towards the environment are discussed. Additionally, factors that influence a cell’s decision to sporulate and those that account for spore stress resistance are presented. In general, this work conveys the need for single-cell studies for enhancing the mechanistic basis of food preservation and spoilage models targeting bacterial spores.
Finally, Chapter 7 discusses the results obtained in this thesis and places them in the context of the established knowledge on weak organic acids and the recently published data on RodZ, a YmfM homologue and essential for maintaining bacterial cell shape. In conclusion, weak organic acids acidify the cytosolic pH and induce a generic transcriptional response that supports the uncoupling effect by the acids. Furthermore, the plasma membrane is an important component in the tolerance to weak organic acid stress. Depending on the food preservative used, the membrane is likely disrupted and consequently adapted. Finally, suggestions are given for future research. The data presented in this thesis can be used to search more specifically for targets in combating B. subtilis and consequently, to gain leads for a more general fight against (Gram-positive) bacterial food spoilers.