• Journal of Microbiology and Biotechnology
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• v.33 no.2
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• pp.211-218
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• 2023

A cryptic plasmid (pTH32) was characterized from Tetragenococcus halophilus 32, an isolate from jeotgal, Korean traditional fermented seafood. pTH32 is 3,198 bp in size with G+C content of 35.84%, and contains 4 open reading frames (ORFs). orf1 and orf2 are 456 bp and 273 bp in size, respectively, and their translation products showed 65.16% and 69.35% similarities with RepB family plasmid replication initiators, respectively, suggesting the rolling-circle replication (RCR) mode of pTH32. orf3 and orf4 encodes putative hypothetical protein of 186 and 76 amino acids, respectively. A novel Tetragenococcus-Escherichia coli shuttle vector, pMJ32E (7.3 kb, Em^r), was constructed by ligation of pTH32 with pBluescript II KS(+) and an erythromycin resistance gene (ErmC). pMJ32E successfully replicated in Enterococcus faecalis 29212 and T. halophilus 31 but not in other LAB species. A pepA gene, encoding aminopeptidase A (PepA) from T. halophilus CY54, was successfully expressed in T. halophilus 31 using pMJ32E. The transformant (TF) showed higher PepA activity (49.8 U/mg protein) than T. halophilus 31 cell (control). When T. halophilus 31 TF was subculturd in MRS broth without antibiotic at 48 h intervals, 53.8% of cells retained pMJ32E after 96 h, and only 2.4% of cells retained pMJ32E after 14 days, supporting the RCR mode of pTH32. pMJ32E could be useful for the genetic engineering of Tetragenococcus and Enterococcus species.

• Journal of Microbiology and Biotechnology
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• v.29 no.10
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• pp.1591-1602
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• 2019

To shed light on the genetic basis of salt tolerance in Enterococcus faecium, Enterococcus faecalis, and Tetragenococcus halophilus, we performed comparative genome analysis of 10 E. faecalis, 11 E. faecium, and three T. halophilus strains. Factors involved in salt tolerance that could be used to distinguish the species were identified. Overall, T. halophilus contained a greater number of potassium transport and osmoprotectant synthesis genes compared with the other two species. In particular, our findings suggested that T. halophilus may be the only one among the three species capable of synthesizing glycine betaine from choline, cardiolipin from glycerol and proline from citrate. These molecules are well-known osmoprotectants; thus, we propose that these genes confer the salt tolerance of T. halophilus.

• Journal of Microbiology and Biotechnology
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• v.33 no.3
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• pp.371-377
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• 2023

In this study, a pepA gene encoding glutamyl (aspartyl)-specific aminopeptidase (PepA; E.C. 3.4.11.7) was cloned from Tetragenococcus halophilus CY54. The translated PepA from T. halophilus CY54 showed very low similarities with PepAs from Lactobacillus and Lactococcus genera. The pepA from T. halophilus CY54 was overexpressed in E. coli BL21(DE3) using pET26b(+). The recombinant PepA was purified by using an Ni- NTA column. The size of the recombinant PepA was 39.13 kDa as determined by SDS-PAGE, while its optimum pH and temperature were pH 5.0 and 60℃, respectively. In addition, the PepA was completely inactivated by 1 mM EDTA, indicating its metallopeptidase nature. The Km and Vmax of the PepA were 0.98 ± 0.006 mM and 0.1 ± 0.002 mM/min, respectively, when Glu-pNA was used as the substrate. This is the first report on PepA from Tetragenococcus species.

• Journal of Microbiology and Biotechnology
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• v.27 no.9
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• pp.1681-1691
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• 2017

This study investigated the effect of proline addition on the salt tolerance of Tetragenococcus halophilus. Salt stress led to the accumulation of intracellular proline in T. halophilus. When 0.5 g/l proline was added to hyperhaline medium, the biomass increased 34.6% (12% NaCl) and 27.7% (18% NaCl) compared with the control (without proline addition), respectively. A metabolomic approach was employed to reveal the cellular metabolic responses and protective mechanisms of proline upon salt stress. The results showed that both the cellular membrane fatty acid composition and metabolite profiling responded by increasing unsaturated and cyclopropane fatty acid proportions, as well as accumulating some specific intracellular metabolites (environmental stress protector). Higher contents of intermediates involved in glycolysis, the tricarboxylic acid cycle, and the pentose phosphate pathway were observed in the cells supplemented with proline. In addition, addition of proline resulted in increased concentrations of many organic osmolytes, including glutamate, alanine, citrulline, N-acetyl-tryptophan, and mannitol, which may be beneficial for osmotic homeostasis. Taken together, results in this study suggested that proline plays a protective role in improving the salt tolerance of T. halophilus by regulating the related metabolic pathways.

• Microbiology and Biotechnology Letters
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• v.51 no.1
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• pp.26-31
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• 2023

A map gene encoding methionyl-specific aminopeptidase (MAP; EC 3.4.11.18) was cloned from Tetragenococcus halophilus CY54. Translated amino acid sequence of CY54 MAP showed high similarities with those from Enterococcus faecalis (83.8%) and Streptococcus salivarius (62.2%) but low similarities with MAPs from Lactobacillus and Lactococcus genera. The map gene was overexpressed in E. coli BL21(DE3) using pET26b(+),pET26b(+), and the recombinant MAP was purified by using an Ni-NTA column. The size of recombinant MAP was 29 kDa as determined by SDS-PAGE. The optimum pH and temperature of CY54 MAP were pH 5.0 and 60℃, respectively. The activity of CY54 MAP was most significantly increased by Co²⁺ ion (159%), and showed the highest activity at 12% NaCl. K_m and V_max were 0.64 ± 0.006 mM and 10.12 ± 0.014 U/mg protein, respectively when met-pNA was used as the substrate. This is the first report on a MAP from Tetragenococcus species.

• Microbiology and Biotechnology Letters
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• v.51 no.2
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• pp.147-156
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• 2023

Tetragenococcus halophilus CY54, an isolate from jeotgal, grows best in media with 5% NaCl and can grow at 18% and higher salt concentration. Three different doenjang samples were prepared with multiple starters including T. halophilus CY54. TBZA doenjang was prepared with T. halophilus, Bacillus subtilis, Zygosaccharomyces rouxii and Aspergillus oryzae. BZA doenjang was prepared with the same 3 starters except T. halophilus. KACC doenjang was prepared with a single starter, B. subtilis KACC16750. During 16 weeks of fermentation at 25℃, the viable counts were maintained in the range of 7-8 log CFU/g in all 3 samples. As fermentation progressed, pH decreased and titratable acidity (TA) gradually increased. Crude protein contents decreased slightly. TBZA doenjang showed higher amino-type nitrogen (ANN) and volatile basic nitrogen (VBN) contents, and KACC doenjang showed higher ammonia-type nitrogen (AMN) content. TBZA doenjang showed higher fibrinolytic and protease activity than other doenjang samples. Metabolites analyses by GC/MS showed that doenjang samples were separated from each other by partial least squares-discriminant analysis (PLS-DA) analysis. Seventeen major metabolites involved in the differences between samples were identified and they included organic acids, amino acids, sugars, fatty acids and alcohols. TBZA doenjang showed higher contents for most metabolites responsible for flavor and taste of fermented foods including doenjang. These results showed that T. halophilus could be useful as a starter for doenjang and can improve the product quality by accelerating the fermentation processes.

• Microbiology and Biotechnology Letters
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• v.46 no.4
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• pp.313-319
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• 2018

Halophilic lactic acid bacteria (LAB) were isolated from myeolchi-jeotgal (23% NaCl, w/v) fermented in jangdok (Korean earthenware) located outside a house. They were identified as Tetragenococcus halophilus by 16S rRNA and recA gene sequencing. Four T. halophilus isolates showing high protease activities were selected for further studies. Four strains grew well, reaching $OD_{600}$ values of 0.75-0.92 at 18% NaCl content (w/v) and 0.28-0.44 at 23% salt. They showed rapid growth, attaining $OD_{600}$ values of 1.1-1.2 at $20-30^{\circ}C$, but did not grow at $4^{\circ}C$. At $15^{\circ}C$, the highest $OD_{600}$ values, which exceeded 0.6, were observed at 20 days, and were higher than those of cultures at $37^{\circ}C$ and $42^{\circ}C$ (approximately 0.5). Four isolates grew best in broth where the initial pH was adjusted to 8 and did not grow at $pH{\leq}4$. T. halophilus BS2-36 showed the highest survival ratio of 18.7% after 2 hours of exposure at pH 3. BS2-36 showed the highest survival ratio (1.29%) in presence of 0.3% bile salts. T. halophilus BS2-36 seems a promising candidate as a starter for jeotgal and other fermented foods with high salinities.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.191-199
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• 2019

We inoculated different combinations of three starter candidates, Bacillus licheniformis, Staphylococcus succinus, and Tetragenococcus halophilus, into sterilized soybeans to predict their contributions to volatile compound production through soybean fermentation. Simultaneously, we added NaCl to soybean cultures to evaluate its effect on the volatile compounds profile. Cells in soybean cultures (1.5% NaCl) nearly reached their maximum growth in a day of incubation, while cell growth was delayed by increasing NaCl concentrations in soybean cultures. The dominance of B. licheniformis and S. succinus in the mixed cultures of three starter candidates switched to T. halophilus as the NaCl concentration increased from 1.5% to 14% (w/w). Seventeen volatile compounds were detected from the control and starter candidate-inoculated soybean cultures with and without the addition of NaCl. Principal component analysis of these volatile compounds concluded that B. licheniformis and S. succinus made major contributions to producing a specific volatile compound profile from soybean cultures where both species exhibited good growth. 3-Hydroxybutan-2-one, butane-2,3-diol, and 2,3,5,6-tetramethylpyrazine are specific odor notes for B. licheniformis, and 3-methylbutyl acetate and 2-phenylethanol are specific for S. succinus. Octan-3-one and 3-methylbutan-1-ol were shown to be decisive volatile compounds for determining the involvement of S. succinus in the soybean culture containing 7% NaCl. 3-Methylbutyl acetate and 3-methylbutan-1-ol were also produced by T. halophilus during soybean fermentation at an appropriate level of NaCl. Although S. succinus and T. halophilus exhibited growth on the soybean cultures containing 14% NaCl, species-specific volatile compounds determining the directionality of the volatile compounds profile were not produced.

• Journal of Microbiology and Biotechnology
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• v.24 no.5
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• pp.648-660
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• 2014

Doenjang, a traditional Korean fermented soybean paste, is made by mixing and ripening meju with high salt brine (approximately 18%). Meju is a naturally fermented soybean block prepared by soaking, steaming, and molding soybean. To understand living bacterial community migration and the roles of bacteria in the manufacturing process of doenjang, the diversity of culturable bacteria in meju and doenjang was examined using media supplemented with NaCl, and some physiological activities of predominant isolates were determined. Bacilli were the major bacteria involved throughout the entire manufacturing process from meju to doenjang; some of these bacteria might be present as spores during the doenjang ripening process. Bacillus siamensis was the most populous species of the genus, and Bacillus licheniformis exhibited sufficient salt tolerance to maintain its growth during doenjang ripening. Enterococcus faecalis and Enterococcus faecium, the major lactic acid bacteria (LAB) identified in this study, did not continue to grow under high NaCl conditions in doenjang. Enterococci and certain species of coagulase-negative staphylococci (CNS) were the predominant acid-producing bacteria in meju fermentation, whereas Tetragenococcus halophilus and CNS were the major acid-producing bacteria in doenjang fermentation. We conclude that bacilli, LAB, and CNS may be the major bacterial groups involved in meju fermentation and that these bacterial communities undergo a shift toward salt-tolerant bacilli, CNS, and T. halophilus during the doenjang fermentation process.

• Journal of Microbiology and Biotechnology
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• v.28 no.7
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• pp.1112-1121
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• 2018

Jeotgal is a Korean traditional fermented seafood with a high concentration of salt. In this study, we isolated lactic acid bacteria (LAB) from galchi (Trichiurus lepturus, hairtail) and myeolchi (Engraulis japonicas, anchovy) jeotgal on MRS agar and MRS agar containing 5% NaCl (MRS agar+5% NaCl), and identified them by using 16S rRNA gene sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as culture-dependent methods. We also performed polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) as a culture-independent method to identify bacterial communities. Five samples of galchi-jeotgal and seven samples of myeolchi-jeotgal were collected from different regions in Korea. A total of 327 and 395 colonies were isolated from the galchi- and myeolchi-jeotgal samples, respectively. 16S rRNA gene sequencing and MALDI-TOF MS revealed that the genus Pediococcus was predominant on MRS agar, and Tetragenococcus halophilus on MRS agar+5% NaCl. PCR-DGGE revealed that T. halophilus, Tetragenococcus muriaticus, and Lactobacillus sakei were predominant in both types of jeotgal. T. halophilus was detected in all samples. Even though the same species were identified by both culture-dependent and -independent methods, many species identified by the culture-dependent methods were not in the bacterial list identified by the culture-independent methods. The distribution of bacteria in galchi-jeotgal was more diverse than in myeolchi-jeotgal. The diverse LAB in galchi- and myeolchi-jeotgals can be further studied as candidates for starter cultures to produce fermented foods.