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Title: Adaptation mechanisms for growth under acid stress in Enterobacteriaceae
Other Titles: Adaptatiemechanismen voor groei onder zuurstress in Enterobacteriaceae
Authors: Vivijs, Bram; S0158120
Issue Date: 2-Jun-2015
Abstract: The Enterobacteriaceae family comprises several important foodborne pathogens and spoilage bacteria. To pass successfully through the food production chain, these bacteria have to endure a wide spectrum of stresses, such as cold, heat, desiccation, acid, or oxidative stress. Acid stress, in particular, exists in the form of the natural acidity of some food products, organic acids formed in fermented foods or added as a food acidulant or food preservative, and, finally, the extremely acidic environment of the human stomach. Two different aspects of acid stress are important with regard to microbiological food safety and stability. The first is the survival of extreme acid stress (pH 1.5 – 3.5), as experienced during passage through the stomach. Since adaptation to extreme acid stress is especially relevant for infective foodborne pathogens, most studies related to acid stress in enterobacteria have mainly focused on the survival (mechanisms) of important foodborne pathogens like Escherichia coli O157:H7 and Salmonella upon extreme acid challenge. However, a second important aspect of acid stress is the capability of bacteria to grow under moderate acid stress (pH 4.0 – 5.0), as experienced in mildly acidic foods. The mechanisms supporting growth under mild acid stress have received less attention, although this property can be anticipated to be also very important for the safety and stability of mildly acidic foods. Earlier research conducted in our laboratory demonstrated that 2,3-butanediol production prevents excessive acidification in Serratia plymuthica RVH1 and some other enterobacteria during fermentative growth, suggesting that this fermentation pathway might be important for enterobacteria to cope with acid stress. In the current work, we investigated the mechanisms that are required in enterobacteria to grow under moderate acid stress. We started with an analysis of the role of 2,3-butanediol fermentation, and subsequently conducted a mutational study in E. coli to identify additional mechanisms.
Although the 2,3-butanediol fermentation pathway consumes intracellular protons, similar to the amino acid decarboxylases which are involved in acid resistance in Enterobacteriaceae, this pathway did not protect against extreme acid stress and was not involved in the acid tolerance response (ATR) in S. plymuthica RVH1. On the other hand, 2,3-butanediol fermentation promoted growth of S. plymuthica RVH1 at moderately low pH in an acidified laboratory growth medium and in tomato juice. Our results demonstrate that the contribution to growth at moderately low pH may be a novel biological function of 2,3-butanediol fermentation, besides preventing excessive acidification, regulating the cellular NAD+/NADH ratio, and storage of carbon and energy.
Since the 2,3-butanediol fermentation pathway enhanced growth of S. plymuthica RVH1 under moderate acid stress, we investigated the impact of the acquisition of the first two steps of this pathway (designated further as the acetoin pathway, encoded by the budAB operon from S. plymuthica RVH1) by mixed-acid fermenting enterobacteria. In E. coli, acetoin production supported better growth and higher stationary-phase cell densities at a moderately low initial pH. Moreover, the budAB operon slightly reduced the minimal pH at which E. coli could initiate growth. On the other hand, an acetoin-producing E. coli strain was outcompeted by a nonproducing strain in a mixed-culture experiment at moderately low pH, suggesting a fitness cost associated with acetoin production. In initially neutral media containing sugars, introduction of the budAB operon conferred E. coli with the capacity to prevent lethal medium acidification and even to reverse acidification during stationary phase. The latter was also observed when the acetoin pathway was introduced in Salmonella, but not in Shigella, suggesting that stationary-phase deacidification was not (only) due to the consumption of protons accompanying acetoin production, but by activation of a pre-existing mechanism that does not exist or is not activated in Shigella. A random transposon mutagenesis approach identified this additional mechanism as the disproportionation of formate to H2 and CO2 by the formate hydrogen lyase (FHL) complex. Metabolite analysis in E. coli showed that introduction of the acetoin pathway reduces lactate and acetate production, but increases glucose consumption and formate and ethanol production.
In order to identify additional cellular functions required for growth of Enterobacteriaceae at moderately low pH, mutant screening was used to identify genes that are required for growth of E. coli MG1655 at moderately low pH in the presence of hydrochloric acid (pH 4.50), lactic acid (pH 4.80), or acetic acid (pH 5.50). Several genes involved in moderately low pH growth were identified, some of which could be linked to previously known mechanisms for the survival of extreme acid stress, such as the acid resistance system relying on intracellular lysine decarboxylation. The screening also revealed several additional cellular pathways and structures that had not previously been linked to acid stress, such as diadenosine tetraphosphate hydrolysis, an intact cell envelope, phosphate transport and DNA repair.
One of the proteins found to be involved in moderately low pH growth is MnmE, a tRNA-modifying enzyme. Since another tRNA-modifying enzyme, MnmA, was identified to be required for growth at low temperature in a mutant screening conducted in S. plymuthica RVH1 in our laboratory, the role of both enzymes in tolerance to different types of stress was investigated in more detail in E. coli and S. plymuthica. A mnmA mutant of S. plymuthica was found to be more susceptible to cold and oxidative stress, while mnmA and mnmE mutants, both in E. coli and S. plymuthica, were hampered in their ability to grow under moderate acid stress. Together with some other recent studies, our results support the hypothesis that tRNA modifications play a crucial role in the cellular stress response in a number of micro-organisms.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Centre for Food and Microbial Technology

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