Staphylococcus equorum plasmid pKS1030-3 encodes auxiliary biofilm formation and trans-acting gene mobilization systems.

The foodborne bacterium Staphylococcus equorum strain KS1030 harbours plasmid pSELNU1, which encodes a lincomycin resistance gene. pSELNU1 undergoes horizontal transfer between bacterial strains, thus spreading antibiotic resistance. However, the genes required for horizontal plasmid transfer are not encoded in pSELNU1. Interestingly, a relaxase gene, a type of gene related to horizontal plasmid transfer, is encoded in another plasmid of S. equorum KS1030, pKS1030-3. The complete genome of pKS1030-3 is 13,583 bp long and encodes genes for plasmid replication, biofilm formation (the ica operon), and horizontal gene transfer. The replication system of pKS1030-3 possesses the replication protein-encoding gene repB, a double-stranded origin of replication, and two single-stranded origins of replication. The ica operon, relaxase gene, and a mobilization protein-encoding gene were detected in pKS1030-3 strain-specifically. When expressed in S. aureus RN4220, the ica operon and relaxase operon of pKS1030-3 conferred biofilm formation ability and horizontal gene transfer ability, respectively. The results of our analyses show that the horizontal transfer of pSELNU1 of S. equorum strain KS1030 depends on the relaxase encoded by pKS1030-3, which is therefore trans-acting. Genes encoded in pKS1030-3 contribute to important strain-specific properties of S. equorum KS1030. These results could contribute to preventing the horizontal transfer of antibiotic resistance genes in food.

Effects of Starter Candidates and NaCl on the Production of Volatile Compounds during Soybean Fermentation.

We inoculated different combinations of three starter candidates of Bacillus licheniformis, Staphylococcus succinus, and Tetragenococcus halophilus, into sterilized soybeans to predict their contributions to volatile compounds 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) reached almost their maximum growth in a day of incubation, while cell growth was delayed by increasing NaCl concentration in soybean cultures. The dominance of B. licheniformis and S. succinus in the mixed culture 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.

Urease Characteristics and Phylogenetic Status of Bacillus paralicheniformis.

In 2015, Bacillus paralicheniformis was separated from B. licheniformis on the basis of phylogenomic and phylogenetic studies, and urease activity was reported as a phenotypic property able to differentiate between the two species. Subsequently, we have found that the urease activity of B. paralicheniformis is strain-specific, and does not reliably discriminate between species, as strains having the same urease gene cluster were identified in B. licheniformis and B. sonorensis, the closest relatives of B. paralicheniformis. We developed a multilocus sequence typing scheme using eight housekeeping genes, adk, ccpA, glpF, gmk, ilvD, pur, spo0A, and tpi to clearly identify B. paralicheniformis from closely related Bacillus species and to find a molecular marker for the rapid identification of B. paralicheniformis. The scheme differentiated 33 B. paralicheniformis strains from 90 strains formerly identified as B. licheniformis. Among the eight housekeeping genes, spo0A possesses appropriate polymorphic sites for the design of a B. paralichenofomis-specific PCR primer set. The primer set designed in this study perfectly separated B. paralicheniformis from B. licheniformis and B. sonorensis.

Genomic insights into the non-histamine production and proteolytic and lipolytic activities of Tetragenococcus halophilus KUD23.

Tetragenococcus halophilus KUD23, a non-histamine producer, was isolated from a traditional Korean high-salt fermented soybean paste, doenjang. The strain was safe in terms of antibiotic susceptibility, hemolytic activity and biofilm formation. It could grow on De Man-Rogosa-Sharpe agar containing 21% (w/v) NaCl, exhibited acid production at 15% NaCl, and had strain-specific proteolytic and lipolytic activities under salt stress. Complete genome analysis of T. halophilus KUD23 and comparative genomic analysis shed light on the genetic background behind these phenotypic characteristics, including non-production of histamine and proteolytic and lipolytic activities.

Effects of the predominant bacteria from meju and doenjang on the production of volatile compounds during soybean fermentation.

We inoculated five starter candidates, Enterococcus faecium, Tetragenococcus halophilus, Bacillus licheniformis, Staphylococcus saprophyticus, and Staphylococcus succinus, into sterilized soybeans to predict their effectiveness for flavor production in fermented soybean foods. All of the starter candidates exhibited sufficient growth and acid production on soybean cultures. Twenty-two volatile compounds, such as acids, alcohols, carbonyls, esters, furans, and pyrazines, were detected from the control and starter candidate-inoculated soybean cultures. Principal component analysis of these volatile compounds concluded that E. faecium and T. halophilus produced a similar profile of volatile compounds to soybeans with no dramatic differences in soybean flavor. B. licheniformis and S. succinus produced the crucial volatile compounds that distinguish the flavor profiles of soybean. During soybean fermentation, phenylmethanol and 2,3,5,6-tetramethylpyrazine were determined as odor notes specific to B. licheniformis and 3-methylbutyl acetate as an odor note specific to S. succinus.