Polysaccharide intercellular adhesin and proper phospholipid composition are important for aggregation in Tetragenococcus halophilus SL10.

Aggregating strains of Tetragenococcus halophilus tend to be trapped during soy sauce mash-pressing process and are, therefore, critical for clear soy sauce production. However, the precise molecular mechanism involved in T. halophilus aggregation remains elusive. In previous studies, we isolated a number of aggregating strains, including T. halophilus AB4 and AL1, and showed that a cell surface proteinaceous aggregation factor is responsible for their aggregation phenotype. In the present study, we explored the role of polysaccharide intercellular adhesin (PIA) in aggregate formation in T. halophilus SL10, isolated from soy sauce. SL10 exhibited similar aggregation to AB4 and AL1 but formed a non-uniform precipitate with distinctive wrinkles at the bottom of the test tube, unlike AB4 and AL1. Insertion sequence mutations in each gene of the ica operon diminished aggregation and PIA production, highlighting the critical role of IcaADBC-mediated PIA production in T. halophilus aggregation. Furthermore, two non-aggregating cardiolipin synthase (cls) gene mutants with intact ica operon did not produce detectable PIA. Phospholipid composition analysis in cls mutants revealed a decrease in cardiolipin and an increase in phosphatidylglycerol levels, highlighting the association between phospholipid composition and PIA production. These findings provide evidence for the pivotal role of cls in PIA-mediated aggregation and lay the foundation for future studies to understand the intricate networks of the multiple aggregation factors governing microbial aggregation.IMPORTANCEAggregation, commonly observed in various microbes, triggers biofilm formation in pathogenic variants and plays a beneficial role in efficient food production in those used for food production. Here, we showed that Tetragenococcus halophilus, a microorganism used in soy sauce fermentation, forms aggregates in a polysaccharide intercellular adhesin (PIA)-mediated manner. Additionally, we unveiled the relationship between phospholipid composition and PIA production. This study provides evidence for the presence of aggregation factors in T. halophilus other than the proteinaceous aggregation factor and suggests that further understanding of the coordinated action of these factors may improve clarified soy sauce production.

Vagococcus jeotgali sp. nov., isolated from cutlassfish jeotgal, a traditional Korean fermented seafood.

A novel, Gram-positive, facultatively anaerobic, and non-motile bacterial strain, designated B2T-5T, was isolated from jeotgal, a traditional Korean fermented seafood. Colonies grown on gifu anaerobic medium agar plates were cream-coloured, irregular, and umbonate with curled margins. Optimal growth of strain B2T-5T occurred at 20 °C, pH 8.0, and in the presence of 1% (w/v) NaCl. Strain B2T-5T was negative for oxidase and catalase activity. Hippurate was not hydrolysed and acetoin was not produced. The major cellular fatty acids were C18 : 1 ω9c and C16 : 0. The cell-wall peptidoglycan was of the A4α type containing l-Lys-d-Asp. The predominant respiratory quinone was menaquinone 7. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylcholine. According to the phylogenetic analysis based on 16S rRNA gene sequences, strain B2T-5T was most closely related to Vagococcus teuberi DSM 21459T, showing 98.2% sequence similarity. Genome sequencing of strain B2T-5T revealed a genome size of 2.0 Mbp and a G+C content of 33.8 mol%. The average nucleotide identities of strain B2T-5T with Vagococcus teuberi DSM 21459T, Vagococcus bubulae SS1994T, and Vagococcus martis D7T301T were 75.0, 74.7, and 75.1%, respectively. Based on the phenotypic, chemotaxonomic, and genotypic data, strain B2T-5T represents a novel species of the genus Vagococcus, for which the name Vagococcus jeotgali sp. nov. is proposed. The type strain is B2T-5T (=KCTC 21223T=JCM 35937T).

Sucrose contributed to the biofilm formation of Tetragenococcus halophilus and changed the biofilm structure.

Based on the previous research results that the addition of sucrose in the medium improved the biofilm formation of Tetragenococcus halophilus, the influence of sucrose on biofilm formation was explored. Moreover, the influence of exogenous expression of related genes sacA and galE from T. halophilus on the biofilm formation of L. lactis NZ9000 was investigated. The results showed that the addition of sucrose in the medium improved the biofilm formation, the resistance of biofilm cells to freeze-drying stress, and the contents of exopolysaccharides (EPS) and eDNA in the T. halophilus biofilms. Meanwhile, the addition of sucrose in the medium changed the monosaccharide composition of EPS and increased the proportion of glucose and galactose in the monosaccharide composition. Under 2.5% (m/v) salt stress condition, the expression of gene sacA promoted the biofilm formation and the EPS production of L. lactis NZ9000 with the sucrose addition in the medium and changed the EPS monosaccharide composition. The expression of gene galE up-regulated the proportion of rhamnose, galactose, and arabinose in the monosaccharide composition of EPS, and down-regulated the proportion of glucose and mannose. This study will provide a theoretical basis for regulating the biofilm formation of T. halophilus, and provide a reference for the subsequent research on lactic acid bacteria biofilms.

Identification of an operon and its regulator required for autoaggregation in Tetragenococcus halophilus.

IMPORTANCE: Tetragenococcus halophilus is a halophilic lactic acid bacterium generally used as a starter culture in fermenting soy and fish sauces. Aggregating strains can be useful in fermenting and obtaining clear soy sauce because cell clumps are trapped by the filter cake when the soy sauce mash is pressed. However, the genetic mechanisms of aggregation in T. halophilus are unknown. In this study, we identified genes encoding aggregation factor and its regulator. These findings may provide a foundation for developing improved T. halophilus starter cultures for soy sauce fermentation, leading to more efficient and consistent clear soy sauce production.

Targeted Screening for Spontaneous Insertion Mutations in a Lactic Acid Bacterium, Tetragenococcus halophilus.

Studies on the microorganisms used in food production are of interest because microbial genotypes are reflected in food qualities such as taste, flavor, and yield. However, several microbes are nonmodel organisms, and their analysis is often limited by the lack of genetic tools. Tetragenococcus halophilus, a halophilic lactic acid bacterium used in soy sauce fermentation starter culture, is one such microorganism. The lack of DNA transformation techniques for T. halophilus makes gene complementation and disruption assays difficult. Here, we report that the endogenous insertion sequence ISTeha4, belonging to the IS4 family, is translocated at an extremely high frequency in T. halophilus and causes insertional mutations at various loci. We developed a method named targeting spontaneous insertional mutations in genomes (TIMING), which combines high-frequency insertional mutations and efficient PCR screening, enabling the isolation of gene mutants of interest from a library. The method provides a reverse genetics and strain improvement tool, does not require the introduction of exogenous DNA constructs, and enables the analysis of nonmodel microorganisms lacking DNA transformation techniques. Our results highlight the important role of insertion sequences as a source of spontaneous mutagenesis and genetic diversity in bacteria. IMPORTANCE Genetic and strain improvement tools to manipulate a gene of interest are required for the nontransformable lactic acid bacterium Tetragenococcus halophilus. Here, we demonstrate that an endogenous transposable element, ISTeha4, is transposed into the host genome at an extremely high frequency. A genotype-based and non-genetically engineered screening system was constructed to isolate knockout mutants using this transposable element. The method described enables a better understanding of the genotype-phenotype relationship and serves as a tool to develop food-grade-appropriate mutants of T. halophilus.

Transcriptomic profiling reveals differences in the adaptation of two Tetragenococcus halophilus strains to a lupine moromi model medium.

BACKGROUND: Tetragenococcus (T.) halophilus is a common member of the microbial consortia of food fermented under high salt conditions. These comprises salty condiments based on soy or lupine beans, fish sauce, shrimp paste and brined anchovies. Within these fermentations this lactic acid bacterium (LAB) is responsible for the formation of lactic and other short chain acids that contribute to the flavor and lower the pH of the product. In this study, we investigated the transcriptomic profile of the two T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine moromi model medium supplied with galactose. To get further insights into which genomic trait is important, we used a setup with two strains. That way we can determine if strain dependent pathways contribute to the overall fitness. These strains differ in the ability to utilize L-arginine, L-aspartate, L-arabinose, D-sorbitol, glycerol, D-lactose or D-melibiose. The lupine moromi model medium is an adapted version of the regular MRS medium supplied with lupine peptone instead of casein peptone and meat extract, to simulate the amino acid availabilities in lupine moromi. RESULTS: The transcriptomic profiles of the T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine peptone-based model media supplied with galactose, used as simulation media for a lupine seasoning sauce fermentation, were compared to the determine potentially important traits. Both strains, have a great overlap in their response to the culture conditions but some strain specific features such as the utilization of glycerol, sorbitol and arginine contribute to the overall fitness of the strain TMW 2.2256. Interestingly, although both strains have two non-identical copies of the tagatose-6P pathway and the Leloir pathway increased under the same conditions, TMW 2.2256 prefers the degradation via the tagatose-6P pathway while TMW 2.2254 does not. Furthermore, TMW 2.2256 shows an increase in pathways required for balancing out the intracellular NADH/NADH+ ratios. CONCLUSIONS: Our study reveals for the first time, that both versions of tagatose-6P pathways encoded in both strains are simultaneously active together with the Leloir pathway and contribute to the degradation of galactose. These findings will help to understand the strain dependent features that might be required for a starter strain in lupine moromi.

Vagococcus luciliae sp. nov., isolated from the common green bottle fly Lucilia sericata.

The genus Vagococcus belongs to the family Enterococcaceae (order Lactobacillales) and is closely related to the genus Enterococcus. Currently, 19 species of the genus have been validly named. In this study, we isolated strain G314FT from the common green bottle fly Lucilia sericata collected in Germany. Sequencing of its almost-complete 16S rRNA gene revealed that the isolate belongs to the genus Vagococcus, being closely related to Vagococcus bubulae SS1994T with high sequence identity (99.50 %), followed by Vagococcus martis D7T301T (98.86 %), Vagococcus vulneris SS1995T (98.71 %), Vagococcus teuberi DSM 21459T (98.64 %), Vagococcus silagei 2B-2T (98.64 %) and Vagococcus penaei CD276T (98.64 %). Genome sequencing of strain G314FT was performed by a combination of Illumina and Oxford Nanopore technology, yielding a circular genome with a size of 2 139 468 bp and an 11 kb plasmid. Average nucleotide identity and digital DNA-DNA hybridization values were calculated between G314FT and its closest-related taxa, and found to be <91 % and <40 %, respectively, thus strongly supporting that strain G314FT represents a novel species of the genus Vagococcus. Phylogenetic and core protein-based phylogenomic trees revealed that G314FT was closely related to a group of three species, V. bubulae SS1994T, V. martis D7T301T and V. teuberi DSM 21459T. Comparatively, the genome of G314FT is the smallest in the group of the four related species, and the biochemical pathway comparison using BlastKOALA revealed that G314FT has lost some amino acid biosynthetic proteins; however, it has gained enzymes for carbohydrate metabolism. Phenotypically, G314FT was consistent with other species of the genus Vagococcus including a negative catalase reaction and non-motility. Using the polyphasic approach, our data supports that the isolate represents a new species, for which we propose the name Vagococcus luciliae G314FT (=DSM 112651T= CCM 9164T).

Comparative genome analysis of Vagococcus fluvialis reveals abundance of mobile genetic elements in sponge-isolated strains.

BACKGROUND: Vagococcus fluvialis is a species of lactic acid bacteria found both free-living in river and seawater and associated to hosts, such as marine sponges. This species has been greatly understudied, with no complete genome assembly available to date, which is essential for the characterisation of the mobilome. RESULTS: We sequenced and assembled de novo the complete genome sequences of five V. fluvialis isolates recovered from marine sponges. Pangenome analysis of the V. fluvialis species (total of 17 genomes) showed a high intraspecific diversity, with 45.5% of orthologous genes found to be strain specific. Despite this diversity, analyses of gene functions clustered all V. fluvialis species together and separated them from other sequenced Vagococcus species. V. fluvialis strains from different habitats were highly similar in terms of functional diversity but the sponge-isolated strains were enriched in several functions related to the marine environment. Furthermore, sponge-isolated strains carried a significantly higher number of mobile genetic elements (MGEs) compared to previously sequenced V. fluvialis strains from other environments. Sponge-isolated strains carried up to 4 circular plasmids each, including a 48-kb conjugative plasmid. Three of the five strains carried an additional circular extrachromosomal sequence, assumed to be an excised prophage as it contained mainly viral genes and lacked plasmid replication genes. Insertion sequences (ISs) were up to five times more abundant in the genomes of sponge-isolated strains compared to the others, including several IS families found exclusively in these genomes. CONCLUSIONS: Our findings highlight the dynamics and plasticity of the V. fluvialis genome. The abundance of mobile genetic elements in the genomes of sponge-isolated V. fluvialis strains suggests that the mobilome might be key to understanding the genomic signatures of symbiosis in bacteria.

Selective extracellular secretion of small double-stranded RNA by Tetragenococcus halophilus.

Small double-stranded RNAs (dsRNAs) abundantly produced by lactic acid bacteria demonstrate immunomodulatory activity and antiviral protective immunity. However, the extracellular secretion of dsRNA from lactic acid bacteria and their compositional and functional differences compared to the intracellular dsRNA is unknown. In this study, we compared the intracellular and secreted extracellular dsRNA of the lactic acid bacteria, Tetragenococcus halophilus, commonly present in fermented foods, by growing in RNA-free and RNase-free media. We used RNA deep sequencing and in-silico analysis to annotate potential regulatory functions for the comparison. A time series sampling of T. halophilus culture demonstrated growth phase-dependent dynamics in extracellular dsRNA secretion with no major change in the intracellular dsRNA profile. The RNA deep sequencing resulted in thousands of diverse dsRNA fragments with 14-21 nucleotides in size from T. halophilus culture. Over 70% of the secreted extracellular dsRNAs were unique in their sequences compared to the intracellular dsRNAs. Furthermore, the extracellular dsRNA abundantly contains sequences that are not T. halophilus genome encoded, not detected intracellularly and showed higher hits on human transcriptome during in-silico analysis, which suggests the presence of extrachromosomal mobile regulatory elements. Further analysis showed significant enrichment of dsRNA target genes of human transcriptome on cancer pathways and transcription process, indicating the extracellular dsRNA of T. halophilus is different not only at the sequence level but also in function. Studying the bacterial extracellular dsRNA is a promising area of future research, particularly for developing postbiotic fermented functional foods and understanding the impact of commensal gut bacteria on human health.

Development of duplex qPCR targeting Carnobacterium maltaromaticum and Vagococcus salmoninarum.

In November 2018, Vagococcus salmoninarum was identified as the causative agent of a chronic coldwater streptococcosis epizootic in broodstock brook trout (Salvelinus fontinalis) at the Iron River National Fish Hatchery in Wisconsin, USA. By February 2019, the epizootic spread to adjacent raceways containing broodstock lake trout (Salvelinus namaycush), whereby fish were found to be coinfected with Carnobacterium maltaromaticum and V. salmoninarum. To differentiate these two pathogens and determine the primary cause of the lake trout morbidity, a quantitative real-time PCR (qPCR) was developed targeting the C. maltaromaticum phenylalanyl-tRNA synthase alpha subunit (pheS) gene. The qPCR was combined with a V. salmoninarum qPCR, creating a duplex qPCR assay that simultaneously quantitates C. maltaromaticum and V. salmoninarum concentrations in individual lake trout tissues, and screens presumptive isolates from hatchery inspections and wild fish from national fish hatchery source waters throughout the Great Lakes basin. Vagococcus salmoninarum and C. maltaromaticum were co-detected in broodstock brook trout from two tribal hatcheries and C. maltaromaticum was present in wild fish in source waters of several national fish hatcheries. This study provides a powerful new tool to differentiate and diagnose two emerging Gram-positive bacterial pathogens.

Investigation of Melissococcus plutonius isolates from 3 outbreaks of European foulbrood disease in commercial beekeeping operations in western Canada.

European foulbrood (EFB) disease is an economically important bacterial disease of honey bee larvae caused by enteric infection with Melissococcus plutonius. In this study, we investigated 3 clinical outbreaks of EFB disease in commercial beekeeping operations in western Canada in the summer of 2020 and characterized the Melissococcus plutonius isolates cultured from these outbreaks according to genetic multi-locus sequence type and i n vitro larval pathogenicity. We isolated M. plutonius sequence type 19 from EFB outbreaks in British Columbia and Alberta, and a novel M. plutonius sequence type 36 from an EFB outbreak in Saskatchewan. In vitro larval infection with each M. plutonius isolate was associated with decreased larval survival in vitro by 58.3 to 70.8% (P < 0.001) compared to non-infected controls. Further elucidation of mechanisms of virulence of M. plutonius, paired with epidemiologic investigation, is imperative to improve EFB management strategies and mitigate risks of EFB outbreaks in western Canada.

Enquête sur des isolats de Melissococcus plutonius provenant de trois éclosions de loque e uropéenne dans des exploitations apicoles commerciales de l'Ouest canadien. La loque européenne (EFB) est une maladie bactérienne économiquement importante des larves d'abeilles mellifères causée par une infection entérique par Melissococcus plutonius. Dans cette étude, nous avons enquêté sur trois éclosions cliniques de la maladie EFB dans des exploitations apicoles commerciales dans l'ouest du Canada à l'été 2020 et caractérisé les isolats de Melissococcus plutonius cultivés à partir de ces éclosions selon le typage génomique multilocus et la pathogénicité larvaire in vitro. Nous avons isolé le type de séquence 19 de M. plutonius des éclosions d'EFB en Colombie-Britannique et en Alberta, et une nouvelle séquence de type 36 de M. plutonius d'une éclosion d'EFB en Saskatchewan. L'infection larvaire in vitro avec chaque isolat de M. plutonius était associée à une diminution de la survie larvaire in vitro de 58,3 à 70,8 % (P < 0,001) par rapport aux témoins non infectés. Une élucidation plus poussée des mécanismes de virulence de M. plutonius, associée à une enquête épidémiologique, est impérative pour améliorer les stratégies de gestion de l'EFB et atténuer les risques d'épidémies d'EFB dans l'Ouest canadien.(Traduit par Dr Serge Messier).

The diversity among the species Tetragenococcus halophilus including new isolates from a lupine seed fermentation.

BACKGROUND: Tetragenococcus (T.) halophilus can be isolated from a variety of fermented foods, such as soy sauce, different soy pastes, salted fish sauce and from cheese brine or degraded sugar beet thick juice. This species contributes by the formation of short chain acids to the flavor of the product. Recently, T. halophilus has been identified as a dominant species in a seasoning sauce fermentation based on koji made with lupine seeds. RESULTS: In this study we characterized six strains of T. halophilus isolated from lupine moromi fermentations in terms of their adaptation towards this fermentation environment, salt tolerance and production of biogenic amines. Phylogenic and genomic analysis revealed three distinctive lineages within the species T. halophilus with no relation to their isolation source, besides the lineage of T. halophilus subsp. flandriensis. All isolated strains from lupine moromi belong to one lineage in that any of the type strains are absent. The strains form lupine moromi could not convincingly be assigned to one of the current subspecies. Taken together with strain specific differences in the carbohydrate metabolism (arabinose, mannitol, melibiose, gluconate, galactonate) and amino acid degradation pathways such as arginine deiminase pathway (ADI) and the agmatine deiminase pathway (AgDI) the biodiversity in the species of T. halophilus is greater than expected. Among the new strains, some strains have a favorable combination of traits wanted in a starter culture. CONCLUSIONS: Our study characterized T. halophilus strains that were isolated from lupine fermentation. The lupine moromi environment appears to select strains with specific traits as all of the strains are phylogenetically closely related, which potentially can be used as a starter culture for lupine moromi. We also found that the strains can be clearly distinguished phylogenetically and phenotypically from the type strains of both subspecies T. halophilus subsp. halophilus and T. halophilus subsp. flandriensis.

Co-culture with Tetragenococcus halophilus improved the ethanol tolerance of Zygosaccharomyces rouxii by maintaining cell surface properties.

The accumulation of ethanol has a negative effect on the viability and fermentation performance of microorganisms during the production of fermented foods because of its toxicity. In this study, we investigated the effect of co-culture with Tetragenococcus halophilus on ethanol stress resistance of Zygosaccharomyces rouxii. The result showed that co-culture with T. halophilus promoted cell survival of Z. rouxii under ethanol stress, and the tolerance improved with increasing co-culture time when ethanol content was 8%. Physiological analysis showed that the co-cultured Z. rouxii cells maintained higher intracellular content of trehalose and amino acids including tyrosine, tryptophan, arginine and proline after 8% ethanol stress for 90 min. The membrane integrity analysis and biophysical analysis of the cell surface indicated that the presence of ethanol resulted in cell membrane damage and changes of Young's modulus value and roughness of cell surface. While the co-cultured Z. rouxii cells exhibited better membrane integrity, stiffer and smoother cell surface than single-cultured cells under ethanol stress. As for transcriptomic analyses, the genes involved in unsaturated fatty acid biosynthesis, trehalose biosynthesis, various types of N-glycan biosynthesis, inositol phosphate metabolism, MAPK signaling pathway and tight junction had higher expression in co-cultured Z. rouxii cells with down-regulation of majority of gene expression after stress. And these genes may function in the improvement of ethanol tolerance of Z. rouxii in co-culture.

Different impacts of pMP19 on the virulence of Melissococcus plutonius strains with different genetic backgrounds.

Virulence factors responsible for bacterial pathogenicity are often encoded by plasmids. In Melissococcus plutonius, the causative agent of European foulbrood of honey bees, a putative virulence plasmid (pMP19) possessing mtxA, which encodes a putative insecticidal toxin, was found by comparative genome analyses. However, as the role of pMP19 in the pathogenesis of European foulbrood remains to be elucidated, we generated pMP19 cured-M. plutonius from representative strains of the three genetically distinct groups (CC3, CC12 and CC13) and compared their virulence against Apis mellifera larvae using our in vitro infection model. Under the conditions tested, the loss of pMP19 abrogated the pathogenicity in CC3 strains, and > 94% of pMP19-cured CC3 strain-infected larvae became adult bees, suggesting that pMP19 is a virulence determinant of CC3 strains. However, introduction of mtxA on its own did not increase the virulence of pMP19-cured strains. In contrast to CC3 strains, the representative CC12 strain remained virulent even in the absence of pMP19, whereas the representative CC13 strain was avirulent even in the presence of the plasmid. Thus, pMP19 plays a role in the virulence of M. plutonius; however, its impact on the virulence varies among strains with different genetic backgrounds.

Transcriptional regulator SpxA1a controls the resistance of the honey bee pathogen Melissococcus plutonius to the antimicrobial activity of royal jelly.

Royal jelly (RJ), a brood food of honey bees, has strong antimicrobial activity. Melissococcus plutonius, the causative agent of European foulbrood of honey bees, exhibits resistance to this antimicrobial activity and infects larvae orally. Among three genetically distinct groups (CC3, CC12 and CC13) of M. plutonius, CC3 strains exhibit the strongest RJ resistance. In this study, to identify genes involved in RJ resistance, we generated an RJ-susceptible derivative from a highly RJ-resistant CC3 strain by UV mutagenesis. Genome sequence analysis of the derivative revealed the presence of a frameshift mutation in the putative regulator gene spxA1a. The deletion of spxA1a from a CC3 strain resulted in increased susceptibility to RJ and its antimicrobial component 10-hydroxy-2-decenoic acid. Moreover, the mutant became susceptible to low-pH and oxidative stress, which may be encountered in brood foods. Differentially expressed gene analysis using wild-type and spxA1a mutants revealed that 45 protein-coding genes were commonly upregulated in spxA1a-positive strains. Many upregulated genes were located in a prophage region, and some highly upregulated genes were annotated as universal/general stress proteins, oxidoreductase/reductase, chaperons and superoxide dismutase. These results suggest that SpxA1a is a key regulator to control the tolerance status of M. plutonius against stress in honey bee colonies.

Characterization of the two nonidentical ArgR regulators of Tetragenococcus halophilus and their regulatory effects on arginine metabolism.

The halophilic lactic acid bacterium Tetragenococcus halophilus has been widely used in high-salinity fermentation processes of food. Previous studies have indicated that the catabolism of arginine may contribute to the osmotic stress adaptation of T. halophilus. Unusually, in the chromosome of T. halophilus, preceding the arginine deiminase (ADI) operon, locate two co-transcribed genes, both encoding an ArgR regulator; similar structure was rarely found and the roles of the regulators have not been demonstrated. In the current study, regulatory roles of these two nonidentical ArgR regulators on the arginine metabolism of T. halophilus were investigated. The results show that these two regulators play different roles in arginine metabolism, ArgR1 acts as a negative regulator of the ADI pathway by binding to the promoter sequences and repressing the transcription of genes, and the addition of arginine or hyper-osmotic stress conditions can abolish the ArgR1 repression, whereas ArgR2 negatively regulates the genes involved in arginine biosynthesis. Our study found that despite the commonly known roles of the ArgR regulators as the activator of arginine catabolism and the repressor of arginine biosynthesis, which are found in most studied bacteria possessed one ArgR regulator, the two nonidentical ArgR regulators of T. halophilus both act as repressors, and the repression by which is regulated when sensing changes of environments. By revealing the regulation of arginine metabolism, the current study provides molecular insights and potential tools for future applications of halophiles in biotechnology. KEY POINTS: • The expression of the ADI pathway of T. halophilus is regulated by carbon sources and osmotic stress. • The arginine metabolism process of T. halophilus is fine-tuned by the two ArgR regulators. • The ADI pathway may contribute to the osmotic stress adaptation by generating more energy and accumulating citrulline which acts as compatible solute.

Identification of the putative N-acetylglucosaminidase CseA associated with daughter cell separation in Tetragenococcus halophilus.

The lactic acid bacterium Tetragenococcus halophilus, which is used as a starter to brew soy sauce, comprises both cluster-forming strains and dispersed strains. The cluster-forming strains are industrially useful for obtaining clear soy sauce, because the cell clusters are trapped by filter cloth when the soy sauce mash is pressed. However, the molecular mechanism underlying cell cluster formation is unknown. Whole genome sequence analysis and subsequent target sequence analysis revealed that the cluster-forming strains commonly have functional defects in N-acetylglucosaminidase CseA, a peptidoglycan hydrolase. CseA is a multimodular protein that harbors a GH73 domain and six peptidoglycan-binding LysM domains. Recombinant CseA hydrolyzed peptidoglycan and promoted cell separation. Functional analysis of truncated CseA derivatives revealed that the LysM domains play an important role in efficient peptidoglycan degradation and cell separation. Taken together, the results of this study identify CseA as a factor that greatly affects the cluster formation in T. halophilus.

Conjugative Delivery of CRISPR-Cas9 for the Selective Depletion of Antibiotic-Resistant Enterococci.

The innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) that are recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiont Enterococcus faecalis is associated with HAIs, and some strains are MDR. Therefore, novel strategies to control E. faecalis populations are needed. We previously characterized an E. faecalis type II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here, we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers to E. faecalis for the selective removal of antibiotic resistance genes. Using in vitro competition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistant E. faecalis by several orders of magnitude. Finally, we show that E. faecalis donor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinants in vivo Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.

LRE-Finder, a Web tool for detection of the 23S rRNA mutations and the optrA, cfr, cfr(B) and poxtA genes encoding linezolid resistance in enterococci from whole-genome sequences.

OBJECTIVES: In enterococci, resistance to linezolid is often mediated by mutations in the V domain of the 23S rRNA gene (G2576T or G2505A). Furthermore, four genes [optrA, cfr, cfr(B) and poxtA] encode linezolid resistance in enterococci. We aimed to develop a Web tool for detection of the two mutations and the four genes encoding linezolid resistance in enterococci from whole-genome sequence data. METHODS: LRE-Finder (where LRE stands for linezolid-resistant enterococci) detected the fraction of Ts in position 2576 and the fraction of As in position 2505 of the 23S rRNA and the cfr, cfr(B), optrA and poxtA genes by aligning raw sequencing reads (fastq format) with k-mer alignment. For evaluation, fastq files from 21 LRE isolates were submitted to LRE-Finder. As negative controls, fastq files from 1473 non-LRE isolates were submitted to LRE-Finder. The MICs of linezolid were determined for the 21 LRE isolates. As LRE-negative controls, 26 VRE isolates were additionally selected for linezolid MIC determination. RESULTS: LRE-Finder was validated and showed 100% concordance with phenotypic susceptibility testing. A cut-off of 10% mutations in position 2576 and/or position 2505 was set in LRE-Finder for predicting a linezolid resistance phenotype. This cut-off allows for detection of a single mutated 23S allele in both Enterococcus faecalis and Enterococcus faecium, while ignoring low-level sequencing noise. CONCLUSIONS: A Web tool for detection of the 23S rRNA mutations (G2576T and G2505A) and the optrA, cfr, cfr(B) and poxtA genes from whole-genome sequences from enterococci is now available online.

Transposition of IS4 Family Insertion Sequences ISTeha3, ISTeha4, and ISTeha5 into the arc Operon Disrupts Arginine Deiminase System in Tetragenococcus halophilus.

Tetragenococcus halophilus, a halophilic lactic acid bacterium, is often used as a starter culture in the manufacturing of soy sauce. T. halophilus possesses an arginine deiminase system, which is responsible for the accumulation of citrulline, the main precursor of the potential carcinogen ethyl carbamate. In this study, we generated five derivatives lacking arginine deiminase activity from T. halophilus NBRC 12172 by UV irradiation. Using these derivatives as a fermentation starter prevented arginine deimination in soy sauce. DNA sequence analysis of the derivatives revealed that novel IS4 family insertion sequences, designated ISTeha3, ISTeha4, and ISTeha5, were transposed into the region around the arginine deiminase (arc) operon in the mutants. These insertion sequences contain a single open reading frame encoding a putative transposase and 13- to 15-bp inverted repeats at both termini, which are adjacent to 7- to 9-bp duplications of the target sequence. Investigation of wild strains isolated from soy sauce mash incapable of arginine deimination also indicated that insertion sequences are involved in the disruption of the arginine deiminase system in T. halophilusIMPORTANCE Insertion sequences play important roles in bacterial evolution and are frequently utilized in mutagenesis systems. However, the intrinsic insertion sequences of tetragenococci are not well characterized. Here, we identified three active insertion sequences of T. halophilus by transposition into the region around the arc operon. This report provides an example of insertion sequence-mediated generation and evolution of T. halophilus and primary information about their characteristics.