New Genes Involved in Mild Stress Response Identified by Transposon Mutagenesis in Lactobacillus paracasei

Abstract

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.

Identification and expression ofLactobacillus paracaseigenes for adaptation to desiccation and rehydration

“…Twenty-four desiccation-sensitive mutants were identified from the L. paracasei mutant library screening after desiccation followed by rapid rehydration. Contrary to our recent screening of this library for mild stress sensitivity (33), no mutants for putative promoters were identified as sensitive to desiccation. Half of the identified genes were specific of the L. casei/paracasei/rhamnosus group.…”

“…Finally, three hypothetical proteins exhibited a transmembrane domain (LSEI_0040, LSEI_0806 and LSEI_1316). Interestingly LSEI_0040 was involved in the adaptation of L. paracasei to thermal, ethanol and oxidative stresses (33) and LSEI_0806 to oxidative stress. Both the latter gene and LSEI_1316 were also required for gut establishment (29).…”

“…The RNR mutant was particularly sensitive (mean viability 10-fold less than WT). RNR is an essential enzyme since its mediates the synthesis of deoxyribonucleotides, the precursors of DNA synthesis (51), and we have previously reported that the RNR gene was involved in general response to mild stresses in L. paracasei (33). SAICAR synthase is involved in de novo purine biosynthesis (52).…”

“…5 µg/mL erythromycin (Em) was used to select mutants. The mutants correspond to the 1287 genic and intergenic mutants already described (33) (29). For each mutant, the putative inactivated function was assigned thanks to the genome annotation of this strain (24).…”

“…Recently, using this approach we determined five Lactobacillus pentosus mutants sensitive to olive brine due to multifactorial stress (32). In addition, new genetic determinants were identified during the early stage of L. paracasei establishment in the gut (29) and during monofactorial perturbations of mild intensities (33). In the present work, the L. paracasei transposon mutant library was screened to identify genes involved in the adaptation of this LAB to desiccation and rehydration.…”