Heat Adaptation Induced Cross Protection Against Ethanol Stress in Tetragenococcus halophilus: Physiological Characteristics and Proteomic Analysis

Ethanol is a toxic factor that damages membranes, disturbs metabolism, and may kill the cell. Tetragenococcus halophilus, considered as the cell factory during the manufacture of traditional fermented foods, encounters ethanol stress, which may affect the viability and fermentative performance of cells. In order to improve the ethanol tolerance of T. halophilus, a strategy based on cross protection was proposed in the current study. The results indicated that cross protection induced by heat preadaptation (45°C for 1.5 h) could significantly improve the stress tolerance (7.24-fold increase in survival) of T. halophilus upon exposure to ethanol (10% for 2.5 h). Based on this result, a combined analysis of physiological approaches and TMT-labeled proteomic technology was employed to investigate the protective mechanism of cross protection in T. halophilus. Physiological analysis showed that the heat preadapted cells exhibited a better surface phenotype, higher membrane integrity, and higher amounts of unsaturated fatty acids compared to unadapted cells. Proteomic analysis showed that a total of 163 proteins were differentially expressed in response to heat preadaptation. KEGG enrichment analysis showed that energy metabolism, membrane transport, peptidoglycan biosynthesis, and genetic information processing were the most abundant metabolic pathways after heat preadaptation. Three proteins (GpmA, AtpB, and TpiA) involved in energy metabolism and four proteins (ManM, OpuC, YidC, and HPr) related to membrane transport were up-regulated after heat preadaptation. In all, the results of this study may help understand the protective mechanisms of preadaptation and contribute to the improvement of the stress resistance of T. halophilus during industrial processes.

Comparative Genomics of Closely Related Tetragenococcus halophilus Strains Elucidate the Diversity and Microevolution of CRISPR Elements

Tetragenococcus halophilus – a halophilic lactic acid bacterium – is frequently used as a starter culture for manufacturing fermented foods. Tetragenococcus is sometimes infected with bacteriophages during fermentation for soy sauce production; however, bacteriophage infection in starter bacteria is one of the major causes of fermentation failure. Here, we obtained whole-genome sequences of the four T. halophilus strains YA5, YA163, YG2, and WJ7 and compared them with 18 previously reported genomes. We elucidated five types of clustered regularly interspaced short palindromic repeat (CRISPR) loci in seven genomes using comparative genomics with a particular focus on CRISPR elements. CRISPR1 was conserved in the four closely related strains 11, YA5, YA163, and YG2, and the spacer sequences were partially retained in each strain, suggesting that partial deletions and accumulation of spacer sequences had occurred independently after divergence of each strain. The host range for typical bacteriophages is narrow and strain-specific thus these accumulation/deletion events may be responsible for differences in resistance to bacteriophages between bacterial strains. Three CRISPR elements, CRISPR1 in strains 11, YA5, YA163, and YG2, CRISPR2 in strain WJ7, and CRISPR2 in strain MJ4, were inserted in almost the same genomic regions, indicating that several independent insertions had occurred in this region. As these elements belong to class 1 type I-C CRISPR group, the results suggested that this site is a hotspot for class 1, type I-C CRISPR loci insertion. Thus, T. halophilus genomes may have acquired strain-specific bacteriophage-resistance through repeated insertion of CRISPR loci and accumulation/deletion events of their spacer sequences.