Opposing genetic polymorphisms of two ABC transporters contribute to the variation of nukacin resistance in Streptococcus mutans.

Streptococcus mutans is a cariogenic bacterium that produces a variety of bacteriocins and retains resistance to these bacteriocins. In this study, we investigated the susceptibility of 127 S. mutans strains to nukacins produced by Staphylococcus spp., which are commensal bacteria in humans. We detected diverse susceptibilities among strains. Nineteen strains had a disrupted LctF (type I), which is responsible for nukacin susceptibility, whereas the remaining 108 strains had an intact LctF (type II) and displayed resistance to nukacins. However, the type I strains still showed resistance to nukacins to some extent. Interestingly, 18/19 (94.7%) type I strains carried a mukA-T locus, which is related to the synthesis of mutacin K8, and mukFEG, an ABC transporter. In contrast, among type II strains, only 6/108 strains (5.6%) had both the mukA-T locus and mukFEG, 19/108 strains (17.6%) carried only mukFEG, and 83/108 strains (76.9%) harbored neither mukA-T nor mukFEG. We also found that MukF had two variants: 305 amino acids (type α) and 302 amino acids (type ß). All type I strains showed a type α (MukFα), whereas most type II strains with mukFEG (22/25 strains) had a type ß (MukFß). Then, we constructed a mukFEG-deletion mutant complemented with MukFαEG or MukFßEG and found that only MukFαEG was involved in nukacin resistance. The nukacin resistance capability of type II-LctFEG was stronger than that of MukFαEG. In conclusion, we identified a novel nukacin resistance factor, MukFEG, and either LctFEG or MukFEG was active in most strains via genetic polymorphisms depending on mukA-T genes. IMPORTANCE: Streptococcus mutans is an important pathogenic bacterium not only for dental caries but also for systemic diseases. S. mutans is known to produce a variety of bacteriocins and to retain resistance these bacteriocins. In this study, two ABC transporters, LctFEG and MukFEG, were implicated in nukacin resistance and each ABC transporter has two subtypes, active and inactive. Of the two ABC transporters, only one ABC transporter was always resistant, while the other ABC transporter was inactivated by genetic mutation. Interestingly, this phenomenon was defined by the presence or absence of the mutacin K8 synthesis gene region, one of the bacteriocins of S. mutans. This suggests that the resistance acquisition is tightly controlled in each strain. This study provides important evidence that the insertion of bacteriocin synthesis genes is involved in the induction of genetic polymorphisms and suggests that bacteriocin synthesis genes may play an important role in bacterial evolution.

Cyclic pairwise interaction representing a rock-paper-scissors game maintains the population of the vulnerable yeast Saccharomyces cerevisiae within a multispecies sourdough microbiome.

IMPORTANCE: Traditionally, multispecies consisting of lactic acid bacteria and yeasts collaboratively engage sourdough fermentation, which determines the quality of the resulting baked goods. Nonetheless, the successive transfer of these microbial communities can result in undesirable community dynamics that prevent the formation of high-quality sourdough bread. Thus, a mechanistic understanding of the community dynamics is fundamental to engineer sourdough complex fermentation. This study describes the population dynamics of five species of lactic acid bacteria-yeast communities in vitro using a generalized Lotka-Volterra model that examines interspecies interactions. A vulnerable yeast species was maintained within up to five species community dynamics by obtaining support with a cyclic interspecies interaction. Metaphorically, it involves a rock-paper-scissors game between two lactic acid bacteria species. Application of the generalized Lotka-Volterra model to real food microbiomes including sourdoughs will increase the reliability of the model prediction and help identify key microbial interactions that drive microbiome dynamics.

Identification of a Novel Bioactive Peptide Derived from Frozen Chicken Breast Hydrolysate and the Utilization of Hydrolysates as Biopreservatives.

Frozen chicken breast was hydrolyzed by treatment with thermolysin enzyme to obtain a chicken hydrolysate containing bioactive peptides. After that, a peptide was purified from the chicken hydrolysate utilizing a Sep-Pak C18 cartridge and reversed-phase high-performance liquid chromatography (RP-HPLC). The molecular weight of the chicken peptide was 2766.8. Protein sequence analysis showed that the peptide was composed of 25 amino acid residues. The peptide, designated as C25, demonstrated an inhibitory action on the angiotensin-converting enzyme (ACE) with a half maximal inhibitory concentration (IC50) value of 1.11 µg/mL. Interestingly, C25 showed antimicrobial activity against multi-drug resistant bacteria Proteus vulgaris F24B and Escherichia coli JM109, both with MIC values of 24 µg/mL. The chicken hydrolysate showed antioxidant activity with an IC50 value of 348.67 µg/mL. Furthermore, the proliferation of aerobic bacteria and Enterobacteriaceae as well as lipid oxidation were significantly reduced when the chicken hydrolysate was used as a natural preservative during cold storage of chicken breasts. Hydrolysates derived from muscle sources have the potential to be used in formulated food products and to contribute positively to human health.

Multiple bacteriocin production in lactic acid bacteria.

Bacteriocin production in lactic acid bacteria (LAB) has always been considered as a highly desirable trait as it enhances the strain's utility in different industrial applications. Bacteriocin producing LAB strains are considered to have higher bacterial fitness as they are able to easily establish themselves into target microbial niche and hence are more effective starter cultures in food fermentation and/or probiotic strains. The rapid advancement in genomic research revealed the true bacteriocin producing capacity of some select novel LAB strains capable of producing multiple bacteriocins which further improves their utility in different application systems. What is common to these novel strains is the remarkable sharing of some elements in the biosynthetic process enabling them to accomplish the extraordinary feat of producing multiple bacteriocins without exhausting its energy. Contrary to the common understanding that biosynthetic enzymes are specific to their cognate bacteriocins, multiple bacteriocin producing strains employ shared biosynthetic elements between their multiple bacteriocins. The quorum-sensing three-component regulatory system, bacteriocin maturation and transport mechanisms are shared among multiple bacteriocins in these strains. Nevertheless, although these novel strains possess enormous application potential, their safety with regards to their potential virulence and pathogenicity needs to be confirmed through comprehensive genotypic characterization. Here, we compile the occurrence of multiple bacteriocin production in some novel LAB strains and highlight specific examples of the unique sharing mechanism of its biosynthetic machinery because a good understanding how these novel strains synthesize their multiple bacteriocins can aid in maximizing their application potential.

Purification, amino acid sequence, and characterization of bacteriocin GA15, a novel class IIa bacteriocin secreted by Lactiplantibacillus plantarum GCNRC_GA15.

The bacteriocins produced by lactic acid bacteria (LAB) are attracting attention due to their promising applications in food and pharmaceuticals fields. Hence, a LAB strain, GCNRC_GA15, was isolated from Egyptian goat cheese, and molecularly identified as Lactiplantibacillus plantarum. This strain showed a wide antimicrobial spectrum, which was found to be of proteineous nature, suggesting that L. plantarum GCNRC_GA15 is a bacteriocin-producer. This bacteriocin (bacteriocin GA15) was partially purified using cation exchange, and hydrophobic interaction chromatography. Tricine SDS-PAGE analysis for the fraction showing bacteriocin activity has estimated the molecular mass to be 4369 Da. Furthermore, amino acid sequencing of this peptide has detected 34 amino acids, and comparing its amino acid sequence with those of some pediocin-like bacteriocins revealed that bacteriocin GA15 has the conserved sequence (YYGNGV/L) in its N-terminal region which identified bacteriocin GA15 as a pediocin-like bacteriocin. Bacteriocin GA15 showed good heat and pH stabilities, and its activity was enhanced after treatment with Tween 80 or Triton X-100. Bacteriocin production medium was statistically optimized using the Plackett-Burman and Central Composite designs. As a result, bacteriocin production increased from 800 to 12,800 AU/ml using the optimized medium in comparison with result recorded for the un-optimized medium.

Identification and characterization of bacteriocin biosynthetic gene clusters found in multiple bacteriocins producing Lactiplantibacillus plantarum PUK6.

The multiple bacteriocins produced by Lactiplantibacillus plantarum PUK6 isolated from misozuke-tofu (tofu pickled in miso) were identified as plantaricins A, EF, and NC8. The pln locus (21,847 bp) containing the three plantaricin structural genes and another newly found putative bacteriocin structural genes (orf1 and orf2) were determined, and a biosynthesis mechanism was proposed. Reverse transcription-PCR analysis revealed that orf1 and orf2, the putative two-peptide bacteriocin structural genes, were expressed after 8 h (logarithmic growth phase) and 20 h (stationary growth phase) of cultivation of the PUK6 strain. Additionally, the growth inhibition profile obtained using the chemically synthesized mature peptides of Orf1 and Orf2 (referred to as mOrf1 and mOrf2) showed that the equimolar mixture of mOrf1 and mOrf2 exhibited bactericidal effect against the indicator strain Latilactobacillus sakei subsp. sakei JCM 1157T. Furthermore, fluorescence microscopic analysis revealed disruption of the cell membranes. These findings indicate that orf1 and orf2 are structural genes encoding class IIb bacteriocins consisting of two peptides. Therefore, the novel bacteriocin encoded by plnPUK6α (orf1) and plnPUK6ß (orf2) genes was named plantaricin PUK6. Since the PUK6 strain produces multiple bacteriocins, when used as a starter culture, it could contribute to improving the shelf life of fermented foods and preventing the appearance of bacteriocin-resistant bacteria.

Characterization of the Biosynthetic Gene Cluster of Enterocin F4-9, a Glycosylated Bacteriocin.

Enterocin F4-9 belongs to the glycocin family having post-translational modifications by two molecules of N-acetylglucosamine ß-O-linked to Ser37 and Thr46. In this study, the biosynthetic gene cluster of enterocin F4-9 was cloned and expressed in Enterococcus faecalis JH2-2. Production of glycocin by the JH2-2 expression strain was confirmed by expression of the five genes. The molecular weight was greater than glycocin secreted by the wild strain, E. faecalis F4-9, because eight amino acids from the N-terminal leader sequence remained attached. This N-terminal extension was eliminated after treatment with the culture supernatant of strain F4-9, implying an extracellular protease from E. faecalis F4-9 cleaves the N-terminal sequence. Thus, leader sequences cleavage requires two steps: the first via the EnfT protease domain and the second via extracellular proteases. Interestingly, the long peptide, with N-terminal extension, demonstrated advanced antimicrobial activity against Gram-positive and Gram-negative bacteria. Furthermore, enfC was responsible for glycosylation, a necessary step prior to secretion and cleavage of the leader peptide. In addition, enfI was found to grant self-immunity to producer cells against enterocin F4-9. This report demonstrates specifications of the minimal gene set responsible for production of enterocin F4-9, as well as a new biosynthetic mechanism of glycocins.

Mechanistic Insight into Yeast Bloom in a Lactic Acid Bacteria Relaying-Community in the Start of Sourdough Microbiota Evolution.

The spontaneous microbiota of wheat sourdough, often comprising one yeast species and several lactic acid bacteria (LAB) species, evolves over repeated fermentation cycles, which bakers call backslopping. The final product quality largely depends on the microbiota functions, but these fluctuate sometimes during the initial months of fermentation cycles due to microbiota evolution in which three phases of LAB relay occur. In this study, the understanding of yeast-LAB interactions in the start of the evolution of the microbiota was deepened by exploring the timing and trigger interactions when sourdough yeast entered a preestablished LAB-relaying community. Monitoring of 32 cycles of evolution of 6 batches of spontaneous microbiota in wheat sourdoughs revealed that sourdough yeasts affected the LAB community when the 2nd- or 3rd-relaying types of LAB genera emerged. In in vitro pairwise cocultures, all 12 LAB strains containing the 3 LAB-relaying types arrested the growth of a Saccharomyces cerevisiae strain, a frequently found species in sourdoughs, to various extents by sugar-related interactions. These findings suggest competition due to different affinities of each LAB and a S. cerevisiae strain for each sugar. In particular, maltose was the driver of S. cerevisiae growth in all pairwise cocultures. The functional prediction of sugar metabolism in sourdough LAB communities showed a positive correlation between maltose degradation and the yeast population. Our results suggest that maltose-related interactions are key factors that enable yeasts to enter and then settle in the LAB-relaying community during the initial part of evolution of spontaneous sourdough microbiota. IMPORTANCE Unpredictable evolution of spontaneous sourdough microbiota sometimes prevents bakers from making special-quality products because the unstable microbiota causes the product quality to fluctuate. Elucidation of the evolutionary mechanisms of the sourdough community, comprising yeast and lactic acid bacteria (LAB), is fundamental to control fermentation performance. This study investigated the mechanisms by which sourdough yeasts entered and settled in a bacterial community in which a three-phase relay of LAB occurred. Our results showed that all three layers of LAB restricted the cohabiting yeast population by competing for the sugar sources, particularly maltose. During the initial evolution of spontaneous sourdough microbiota, yeasts tended to grow synchronously with the progression of the lactic acid bacterial relay, which was predictably associated with changes in the maltose degradation functions in the bacterial community. Further study of ≥3 species' interactions while considering yeast diversity can uncover additional interaction mechanisms driving the initial evolution of sourdough microbiota.

Transition and regulation mechanism of bacterial biota in Kishu saba-narezushi (mackerel narezushi) during its fermentation step.

The transition of the bacterial biota of Kishu saba-narezushi (mackerel-narezushi) in the Hidaka region of Wakayama prefecture, Japan, was analyzed using amplicon sequencing based on the V3-V4 variable region of the 16S rRNA gene. In the non-fermented sample (0 day), the major genus with the highest abundance ratio was Staphylococcus. In the early stage (fermentation for 2 days), however, the genus Lactococcus became a dominant species, and in the later stage (fermentation for 5 days), the abundance ratio of the genus Lactobacillus increased significantly. Lactococcus lactis strains isolated from the narezushi samples had the ability to suppress the growth of not only Staphylococcus genera but also Lactobacillus. Moreover, the isolates produced a bacteriocin, which was identified as nisin Z. On the basis of these results, it is concluded that L. lactis plays an important role in preparing the fermentation conditions of Kishu saba-narezushi in the early stage by suppressing unwanted microorganisms using lactic acid and nisin Z.