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.

Diversity and dynamics of sourdough lactic acid bacteriota created by a slow food fermentation system.

Sourdough is a naturally fermented dough that is used worldwide to produce a variety of baked foods. Various lactic acid bacteria (LAB), which can determine the quality of sourdough baked foods by producing metabolites, have been found in the sourdough ecosystem. However, spontaneous fermentation of sourdough leads to unpredictable growth of various micro-organisms, which result in unstable product quality. From an ecological perspective, many researchers have recently studied sourdough LAB diversity, particularly the elucidation of LAB community interactions and the dynamic mechanisms during the fermentation process, in response to requests for the control and design of a desired sourdough microbial community. This article reviews recent advances in the study of sourdough LAB diversity and its dynamics in association with unique characteristics of the fermentation system; it also discusses future perspectives for better understanding of the complex sourdough microbial ecosystem, which can be attained efficiently by both in vitro and in situ experimental approaches.

Transcriptome profile of carbon catabolite repression in an efficient l-(+)-lactic acid-producing bacterium Enterococcus mundtii QU25 grown in media with combinations of cellobiose, xylose, and glucose.

Enterococcus mundtii QU25, a non-dairy lactic acid bacterium of the phylum Firmicutes, is capable of simultaneously fermenting cellobiose and xylose, and is described as a promising strain for the industrial production of optically pure l-lactic acid (≥ 99.9%) via homo-fermentation of lignocellulosic hydrolysates. Generally, Firmicutes bacteria show preferential consumption of sugar (usually glucose), termed carbon catabolite repression (CCR), while hampering the catabolism of other sugars. In our previous study, QU25 exhibited apparent CCR in a glucose-xylose mixture phenotypically, and transcriptional repression of the xylose operon encoding initial xylose metabolism genes, likely occurred in a CcpA-dependent manner. QU25 did not exhibit CCR phenotypically in a cellobiose-xylose mixture. The aim of the current study is to elucidate the transcriptional change associated with the simultaneous utilization of cellobiose and xylose. To this end, we performed RNA-seq analysis in the exponential growth phase of E. mundtii QU25 cells grown in glucose, cellobiose, and/or xylose as either sole or co-carbon sources. Our transcriptomic data showed that the xylose operon was weakly repressed in cells grown in a cellobiose-xylose mixture compared with that in cells grown in a glucose-xylose mixture. Furthermore, the gene expression of talC, the sole gene encoding transaldolase, is expected to be repressed by CcpA-mediated CCR. QU25 metabolized xylose without using transaldolase, which is necessary for homolactic fermentation from pentoses using the pentose-phosphate pathway. Hence, the metabolism of xylose in the presence of cellobiose by QU25 may have been due to 1) sufficient amounts of proteins encoded by the xylose operon genes for xylose metabolism despite of the slight repression of the operon, and 2) bypassing of the pentose-phosphate pathway without the TalC activity. Accordingly, we have determined the targets of genetic modification in QU25 to metabolize cellobiose, xylose and glucose simultaneously for application of the lactic fermentation from lignocellulosic hydrolysates.

Kunkecin A, a New Nisin Variant Bacteriocin Produced by the Fructophilic Lactic Acid Bacterium, Apilactobacillus kunkeei FF30-6 Isolated From Honey Bees.

Apilactobacillus kunkeei FF30-6 isolated from healthy honey bees synthesizes the bacteriocin, which exhibits antimicrobial activity against Melissococcus plutonius. The bacteriocin, kunkecin A, was purified through three-step chromatography, and mass spectrometry revealed that its relative molecular mass was 4218.3. Edman degradation of purified kunkecin A showed only the N-terminal residue, isoleucine. Hence, alkaline alkylation made the subsequent amino acid residues accessible to Edman degradation, and 30 cycles were sequenced with 11 unidentified residues. Whole genome sequencing of A. kunkeei FF30-6, followed by Sanger sequencing, revealed that the genes encoding the proteins involved in lantibiotic biosynthesis were within the plasmid, pKUNFF30-6. Most of the identified proteins exhibited significant sequence similarities to the biosynthetic proteins of nisin A and its variants, such as subtilin. However, the kunkecin A gene cluster lacked the genes corresponding to nisI, nisR, and nisK of the nisin A biosynthetic gene cluster. A comparison of the gene products of kukA and nisA (kunkecin A and nisin A structural genes, respectively) suggested that they had similar post-translational modifications. Furthermore, the structure of kunkecin A was proposed based on a comparison of the observed and calculated relative molecular masses of kunkecin A. The structural analysis revealed that kunkecin A and nisin A had a similar mono-sulfide linkage pattern. Purified kunkecin A exhibited a narrow antibacterial spectrum, but high antibacterial activity against M. plutonius. Kunkecin A is the first bacteriocin to be characterized in fructophilic lactic acid bacteria and is the first nisin-type lantibiotic found in the family Lactobacillaceae.

Processing and secretion of non-cognate bacteriocins by EnkT, an ABC transporter from a multiple-bacteriocin producer, Enterococcus faecium NKR-5-3.

EnkT is an ATP-binding cassette (ABC) transporter produced by Enterococcus faecium NKR-5-3, which is responsible for the secretion of multiple bacteriocins; enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z). EnkT has been shown to possess a tolerant recognition mechanism that enables it to secrete the mature Ent53C from a chimeric precursor peptide containing the leader peptide moieties that are derived from different heterologous bacteriocins. In this study, to further characterize EnkT, we aimed to investigate the capacity of EnkT to recognize, process, and secrete non-cognate bacteriocins, which belong to different subclasses of class II. For this, the non-cognate bacteriocin precursor peptides, including enterocin A, pediocin PA-1, lactococcin Q, lactococcin A, and lacticin Q were co-expressed with EnkT, and thereafter, the production of the mature forms of these non-cognate bacteriocins was assessed. Our results revealed that EnkT could potentially recognize, process, and secrete the non-cognate bacteriocins with an exception of the leaderless bacteriocin, lacticin Q. Moreover, the processing and secretion efficiencies of these heterologous non-cognate bacteriocins by EnkT were further enhanced when the leader peptide moiety was replaced with the Ent53C leader peptide (derived from a native NKR-5-3 bacteriocin). The findings of this study describe the wide substrate tolerance of this ABC transporter, EnkT, that can be exploited in the future in establishing effective bacteriocin production systems adaptive to complex fermentation conditions common in many food systems.

Protection of gut microbiome from antibiotics: development of a vancomycin-specific adsorbent with high adsorption capacity.

The fraction of administered antibiotics that reach the cecum and colon causes dysbiosis of the gut microbiome, resulting in various diseases. Protection of the gut microbiome from antibiotics using antibiotic adsorbents in the cecum and colon is a promising method to overcome this issue. Previously, activated charcoal (AC) has been reported to protect the gut microbiome of host animals. AC is an adsorbent that is widely used to capture toxic compounds and overdosed drugs in the gastrointestinal tract. The specificity of adsorbents for antibiotics is critical to avoid the risk of unexpected side effects caused by nonspecific adsorption of biological compounds in the intestinal fluid, such as bile acids and essential micronutrients. Here, we have developed specific adsorbents for vancomycin (VCM), which is known to cause gut dysbiosis. The adsorbents were composed of polyethyleneglycol-based microparticles (MPs) in which a specific ligand for VCM, D-Ala-D-Ala-OH, was attached via dendrons of D-lysine to raise the content of the ligand in the MPs. The MPs successfully protected Staphylococcus lentus from VCM in vitro because of the adsorption of VCM in the culture media. Pre-administration of MPs to mice reduced the amount of free VCM in the feces to an undetectable level. This treatment minimized the effect of VCM on gut microbiota and provided protection against Clostridioides difficile infection after oral challenge with spores.

Impact of pH on succession of sourdough lactic acid bacteria communities and their fermentation properties.

Sourdough, a traditional fermented dough, is made via natural fermentation by lactic acid bacteria (LAB). Its pH changes from near neutral to acid during the subculture process. However, the product quality of subcultured sourdough depends on the unpredictable succession of LAB communities, the influential factors of which are still unclear. To elucidate one end of the LAB community succession mechanism, we evaluated the effect of pH by designing four subculture experiments using a model medium adjusted to pH 6.7, 5.5, and 4.5, as well as a natural sourdough subculture. All experiments began by inoculating a sourdough LAB mixture, and both bacterial successions and fermentative properties were monitored until ten subculture steps. In media subcultures, lactic acid production was higher in higher pH media. Three LAB genera, Weissella, Pediococcus, and Lactobacillus, each represented by one operational taxonomic unit (OTU), were successively detected in all subcultures. In later steps with lower pH media, an OTU closely related to Lactobacillus brevis dominated, replacing an OTU closely related to the Weissella cibaria-confusa group that was more dominant than the L. brevis OTU in the near-neutral pH medium. In the sourdough subculture, the three genera were also detected, while Lactobacillus was dominant in earlier steps due to the emergence of an OTU closely related to Lactobacillus sanfranciscensis. These results suggest that a lower pH is favorable for the sequence of sourdough bacterial community evolution finalizing with Lactobacillus domination. Further research is needed to elucidate additional factors other than pH that influence the pattern of LAB community shift.

Lowering effect of viable Pediococcus pentosaceus QU 19 on the rise in postprandial glucose.

In the present study, we investigated the glucose-decreasing action of lactic acid bacteria (LAB). The finding of this study could be helpful for people in controlling their blood sugar levels. The LAB candidate was isolated from a Japanese fermented food and identified as Pediococcus pentosaceus by an analysis of its genome sequence. Postprandial blood glucose elevation was investigated using oral starch tolerance tests in mice. Normal mice were fed starch and lyophilized cells of P. pentosaceus QU 19 at the same time. Even without pre-administration of P. pentosaceus QU 19, elevation of the blood glucose level was significantly suppressed by the intake of P. pentosaceus QU 19 at the same time as oral administration of starch. According to the results for its survival in simulated digestive juice and the reduction of blood glucose level in mice, P. pentosaceus QU 19 has potential hypoglycemic activity. In vitro measurements revealed that the glucose-decreasing action of P. pentosaceus QU 19 is probably caused by the glucose assimilation of the strain, not the inhibition of carbohydrate-splitting enzymes which has been reported for other LABs previously. These findings indicate that specific strains of LAB, especially P. pentosaceus QU 19, and foods fermented by LAB may be beneficial for people who must manage glucose ingestion.

Non-carbon loss long-term continuous lactic acid production from mixed sugars using thermophilic Enterococcus faecium QU 50.

In this study, a non-sterile (open) continuous fermentation (OCF) process with no-carbon loss was developed to improve lactic acid (LA) productivity and operational stability from the co-utilization of lignocellulose-derived sugars by thermophilic Enterococcus faecium QU 50. The effects of different sugar mixtures on LA production were firstly investigated in conventional OCF at 50°C, pH 6.5 and a dilution rate of 0.20 hr-1 . The xylose consumption ratio was greatly lower than that of glucose in fermentations with glucose/xylose mixtures, indicating apparent carbon catabolite repression (CCR). However, CCR could be efficiently eliminated by feeding solutions containing the cellobiose/xylose mixture. In OCF at a dilution rate ca. 0.10 hr-1 , strain QU 50 produced 42.6 g L-1 of l-LA with a yield of 0.912 g g-1 -consumed sugars, LA yield of 0.655 g g-1 based on mixed sugar-loaded, and a productivity of 4.31 g L-1 hr-1 from simulated energy cane hydrolyzate. In OCF with high cell density by cell recycling, simultaneous and complete co-utilization of sugars was achieved with stable LA production at 60.1 ± 3.25 g L-1 with LA yield of 0.944 g g-1 -consumed sugar and LA productivity of 6.49 ± 0.357 g L-1 hr-1 . Besides this, a dramatic increase in LA yield of 0.927 g g-1 based on mixed sugar-loaded with prolonged operational stability for at least 500 hr (>20 days) was established. This robust system demonstrates an initial green step with a no-carbon loss under energy-saving toward the feasibility of sustainable LA production from lignocellulosic sugars.

Mosaic Cooperativity in Slow Polypeptide Topological Isomerization Revealed by Residue-Specific NMR Thermodynamic Analysis.

Slow polypeptide conformational changes on time scales of >1 s are generally assumed to be highly cooperative two-state transitions, reflecting the high energy barrier. However, few experimental characterizations have tested the validity of this assumption. We performed residue-specific NMR thermodynamic analysis of the 27-residue lantibiotic peptide, nukacin ISK-1, to characterize the isomerization between two topological states on the second time scale. Unexpectedly, the thermal transition behaviors were distinct among peptide regions, indicating that the topological isomerization process is a mosaic of different degrees of cooperativity. The conformational change path between the two NMR structures was deduced by a targeted molecular dynamics simulation. The unique side-chain threading motions through the monosulfide rings are the structural basis of the high energy barrier, and the nonlocal interactions in the hydrophobic core are the structural basis of the cooperativity. Taken together, we provide an energetic description of the topological isomerization of nukacin ISK-1.

Molecular characterization of the possible regulation of multiple bacteriocin production through a three-component regulatory system in Enterococcus faecium NKR-5-3.

Enterococcus faecium NKR-5-3 produces multiple-bacteriocins, enterocins NKR-5-3A, B, C, D, and Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). However, the biosynthetic mechanisms on how their productions are regulated are yet to be fully understood. In silico analysis revealed putative promoters and terminators in the enterocin NKR-5-3ACDZ gene cluster, and the putative direct repeats (5'-ATTTTAGGATA-3') were conserved upstream of each promoter. Transcriptional analysis by quantitative real-time polymerase chain reaction (PCR) of the biosynthetic genes for the enterocins NKR-5-3 suggested that an inducing peptide (Ent53D) regulates the transcription of the structure genes and corresponding biosynthetic genes of enterocins NKR-5-3, except for Ent53B (a circular bacteriocin), thus consequently regulating their production. Moreover, transcriptional analysis of some knock-out mutants showed that the production of Ent53A, C, D and Z is controlled by a three-component regulatory system (TCS) consisting of Ent53D, EnkR (response regulator), and EnkK (histidine kinase). The production of the circular bacteriocin Ent53B appeared to be independent from this TCS. Nevertheless, disrupting the TCS by deletion of a single component (enkD, enkR and enkK) resulted in a slight increase of enkB transcription and consequently the production of Ent53B, presumably, as an indirect consequence of the increase of available energy to the strain NKR-5-3. Here, we demonstrate the regulatory control of the multiple bacteriocin production of strain NKR-5-3 likely through the TCS consisting of Ent53D, EnkR, and EnkK. The information of the sharing of the regulatory machinery between bacteriocins in strain NKR-5-3 can be useful in its future application such as designing strategies to effectively dispense its multiple bacteriocin arsenal.