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Tetragenococcus halophilus , a halophilic lactic acid bacterium (LAB), plays an important role in the production of high-salt fermented foods. Generally, formation of biofilm benefits the fitness of cells when faced with competitive and increasingly hostile fermented environments. In this work, the biofilm-forming capacity of T. halophilus was investigated. The results showed that the optimal conditions for biofilm formation by T. halophilus were at 3–9% salt content, 0–6% ethanol content, pH 7.0, 30°C, and on the surface of stainless steel. Confocal laser scanning microscopy (CLSM) analysis presented a dense and flat biofilm with a thickness of about 24 μm, and higher amounts of live cells were located near the surface of biofilm and more dead cells located at the bottom. Proteins, polysaccharides, extracellular-DNA (eDNA), and humic-like substances were all proved to take part in biofilm formation. Higher basic surface charge, greater hydrophilicity, and lower intracellular lactate dehydrogenase (LDH) activities were detected in T. halophilus grown in biofilms. Atomic force microscopy (AFM) imaging revealed that biofilm cultures of T. halophilus had stronger surface adhesion forces than planktonic cells. Cells in biofilm exhibited higher cell viability under acid stress, ethanol stress, heat stress, and oxidative stress. In addition, T. halophilus biofilms exhibited aggregation activity and anti-biofilm activity against Staphylococcus aureus and Salmonella Typhimurium. Results presented in the study may contribute to enhancing stress tolerance of T. halophilus and utilize their antagonistic activities against foodborne pathogens during the production of fermented foods. [ABSTRACT FROM AUTHOR]

Tetragenococcus halophilus, a halophilic lactic acid bacterium (LAB), plays an important role in the production of high-salt fermented foods. Generally, formation of biofilm benefits the fitness of cells when faced with competitive and increasingly hostile fermented environments. In this work, the biofilm-forming capacity of T. halophilus was investigated. The results showed that the optimal conditions for biofilm formation by T. halophilus were at 3–9% salt content, 0–6% ethanol content, pH 7.0, 30°C, and on the surface of stainless steel. Confocal laser scanning microscopy (CLSM) analysis presented a dense and flat biofilm with a thickness of about 24 μm, and higher amounts of live cells were located near the surface of biofilm and more dead cells located at the bottom. Proteins, polysaccharides, extracellular-DNA (eDNA), and humic-like substances were all proved to take part in biofilm formation. Higher basic surface charge, greater hydrophilicity, and lower intracellular lactate dehydrogenase (LDH) activities were detected in T. halophilus grown in biofilms. Atomic force microscopy (AFM) imaging revealed that biofilm cultures of T. halophilus had stronger surface adhesion forces than planktonic cells. Cells in biofilm exhibited higher cell viability under acid stress, ethanol stress, heat stress, and oxidative stress. In addition, T. halophilus biofilms exhibited aggregation activity and anti-biofilm activity against Staphylococcus aureus and Salmonella Typhimurium. Results presented in the study may contribute to enhancing stress tolerance of T. halophilus and utilize their antagonistic activities against foodborne pathogens during the production of fermented foods.

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.

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study explored the bacterial diversity of brines used for cheesemaking in Italy, as well as their physicochemical characteristics. In this context, 19 brines used to salt soft, semi-hard, and hard Italian cheeses were collected in 14 commercial cheese plants and analyzed using a culture-independent amplicon sequencing approach in order to describe their bacterial microbiota. Large NaCl concentration variations were observed among the selected brines, with hard cheese brines exhibiting the highest values. Acidity values showed a great variability too, probably in relation to the brine use prior to sampling. Despite their high salt content, brine microbial loads ranged from 2.11 to 6.51 log CFU/mL for the total mesophilic count. Microbial community profiling assessed by 16S rRNA gene sequencing showed that these ecosystems were dominated by Firmicutes and Proteobacteria, followed by Actinobacteria and Bacteroidetes. Cheese type and brine salinity seem to be the main parameters accountable for brine microbial diversity. On the contrary, brine pH, acidity and protein concentration, correlated to cheese brine age, did not have any selective effect on the microbiota composition. Nine major genera were present in all analyzed brines, indicating that they might compose the core microbiome of cheese brines. Staphylococcus aureus was occasionally detected in brines using selective culture media. Interestingly, bacterial genera associated with a functional and technological use were frequently detected. Indeed Bifidobacteriaceae, which might be valuable probiotic candidates, and specific microbial genera such as Tetragenococcus, Corynebacterium and non-pathogenic Staphylococcus, which can contribute to sensorial properties of ripened cheeses, were widespread within brines. [ABSTRACT FROM AUTHOR]