Lactic acid bacteria (LAB) are associated with various plant, animal, and human niches and are also present in many fermented foods and beverages. Thus, they are subjected to several stress conditions and have developed advanced response mechanisms to resist, adapt, and grow. This work aimed to identify the genes involved in some stress adaptation mechanisms in LAB. For this purpose, global reverse genetics was applied by screening a library of 1287 Lactobacillus paracasei transposon mutants for mild monofactorial stresses. This library was submitted independently to heat (52°C, 30 min), ethanol (170 g.L−1, 30 min), salt (NaCl 0.8 M, 24 h), acid (pH 4.5, 24 h), and oxidative (2 mM H2O2, 24 h) perturbations which trigger mild monofactorial stresses compatible with bacterial adaptation. Stress sensitivity of mutants was determined either by evaluating viability using propidium iodide (PI) staining, or by following growth inhibition through turbidity measurement. The screening for heat and ethanol stresses lead respectively to the identification of 63 and 27 genes/putative promoters whose disruption lead to an increased sensitivity. Among them, 14 genes or putative promoters were common for both stresses. For salt, acid and oxidative stresses, respectively 8, 6, and 9 genes or putative promoters were identified as essential for adaptation to these unfavorable conditions, with only three genes common to at least two stresses. Then, RT-qPCR was performed on selected stress response genes identified by mutant screenings in order to evaluate if their expression was modified in response to stresses in the parental strain. Eleven genes (membrane, transposase, chaperone, nucleotide and carbohydrate metabolism, and hypothetical protein genes) were upregulated during stress adaptation for at least two stresses. Seven genes, encoding membrane functions, were upregulated in response to a specific stress and thus could represent potential transcriptomic biomarkers. The results highlights that most of the genes identified by global reverse genetics are specifically required in response to one stress and that they are not differentially transcribed during stress in the parental strain. Most of these genes have not been characterized as stress response genes and provide new insights into the adaptation of lactic acid bacteria to their environment.

No abstract

12Lactobacillus paracasei is able to persist in a variety of natural and technological 13 environments despite physico-chemical perturbations, in particular alternations between 14 desiccation and rehydration. However, the way in which it adapts to hydric fluctuations and 15 in particular the genetic determinants involved are not clearly understood. To identify the 16 genes involved in adaptation to desiccation, an annotated library of L. paracasei random 17 transposon mutants was screened for viability after desiccation (25% relative humidity, 18 25°C). Subsequently, the expression of the identified genes was measured at five stages of the 19 dehydration-rehydration process to formulate the chronology of gene expression. The 24 20 identified genes were related to metabolism and transport, membrane function and structure, 21 regulation of stress response, DNA related enzymes and environmental sensing. They were classified into four different transcriptomic profiles, in particular genes upregulated during 23 both desiccation and rehydration phases and genes upregulated during the desiccation phase 24 only. Thus, genetic response to hydric fluctuations seems to occur during desiccation and can 25 continue or not during rehydration. The genes identified should contribute to improving the 26 stabilization of lactobacillus starters in dry state. 27 28 Importance 29Since water is the fundamental component of all living organisms, desiccation and 30 rehydration alternation is one of the most prevalent and severe stresses for most 31 microorganisms. Adaptation to this stress occurs via a combination of mechanisms which 32 depend on the genetic background of the microorganism. In L. paracasei, we developed a 33 strategy to identify genes involved in the adaptation to hydric fluctuations using random 34 transposon mutagenesis and targeted transcriptomics. Both dehydration and rehydration were 35 studied to decipher the chronology of genetic mechanisms. We found 24 as yet unidentified 36 genes involved in this response. Most of them are linked to either the transport of molecules 37 or to cell wall structure and function. Our screening also identified genes for environment 38 sensing and two alarmones necessary for L. paracasei survival. Furthermore, our results show 39 that desiccation is a critical phase for inducing stress response in L. paracasei. 40 41 expression 43 44 45 46 47Water is essential for all living organisms as it contributes to the structure of cells, stabilizes 48 proteins, lipids and nucleic acids, and maintains vital metabolic systems and chemical 49 reactions (1). Desiccation leads to water exit from cells, induces structural modifications, and 50 causes osmotic and oxidative stresses (1-3). In addition, the rehydration phase could lead to 51 membrane alterations (4). The capacity to return to life after hydric fluctuations is not only 52 crucial for bacteria as a function of diurnal and seasonal cycles in natural environments but 53 also during food industry processes such as conventional drying, f...