Discovery of antiphage systems in the lactococcal plasmidome.

Until the late 2000s, lactococci substantially contributed to the discovery of various plasmid-borne phage defence systems, rendering these bacteria an excellent antiphage discovery resource. Recently, there has been a resurgence of interest in identifying novel antiphage systems in lactic acid bacteria owing to recent reports of so-called 'defence islands' in diverse bacterial genera. Here, 321 plasmid sequences from 53 lactococcal strains were scrutinized for the presence of antiphage systems. Systematic evaluation of 198 candidates facilitated the discovery of seven not previously described antiphage systems, as well as five systems, of which homologues had been described in other bacteria. All described systems confer resistance against the most prevalent lactococcal phages, and act post phage DNA injection, while all except one behave like abortive infection systems. Structure and domain predictions provided insights into their mechanism of action and allow grouping of several genetically distinct systems. Although rare within our plasmid collection, homologues of the seven novel systems appear to be widespread among bacteria. This study highlights plasmids as a rich repository of as yet undiscovered antiphage systems.

Phage defence loci of Streptococcus thermophilus-tip of the anti-phage iceberg?

Bacteria possess (bacterio)phage defence systems to ensure their survival. The thermophilic lactic acid bacterium, Streptococcus thermophilus, which is used in dairy fermentations, harbours multiple CRISPR-Cas and restriction and modification (R/M) systems to protect itself against phage attack, with limited reports on other types of phage-resistance. Here, we describe the systematic identification and functional analysis of the phage resistome of S. thermophilus using a collection of 27 strains as representatives of the species. In addition to CRISPR-Cas and R/M systems, we uncover nine distinct phage-resistance systems including homologues of Kiwa, Gabija, Dodola, defence-associated sirtuins and classical lactococcal/streptococcal abortive infection systems. The genes encoding several of these newly identified S. thermophilus antiphage systems are located in proximity to the genetic determinants of CRISPR-Cas systems thus constituting apparent Phage Defence Islands. Other phage-resistance systems whose encoding genes are not co-located with genes specifying CRISPR-Cas systems may represent anchors to identify additional Defence Islands harbouring, as yet, uncharacterised phage defence systems. We estimate that up to 2.5% of the genetic material of the analysed strains is dedicated to phage defence, highlighting that phage-host antagonism plays an important role in driving the evolution and shaping the composition of dairy streptococcal genomes.

Molecular mechanisms underlying the structural diversity of rhamnose-rich cell wall polysaccharides in lactococci.

In Gram-positive bacteria, cell wall polysaccharides (CWPS) play critical roles in bacterial cell wall homeostasis and bacterial interactions with their immediate surroundings. In lactococci, CWPS consist of two components: a conserved rhamnan embedded in the peptidoglycan layer and a surface-exposed polysaccharide pellicle (PSP), which are linked together to form a large rhamnose-rich CWPS (Rha-CWPS). PSP, whose structure varies from strain to strain, is a receptor for many bacteriophages infecting lactococci. Here, we examined the first two steps of PSP biosynthesis, using in vitro enzymatic tests with lipid acceptor substrates combined with LC-MS analysis, AlfaFold2 modeling of protein 3D-structure, complementation experiments, and phage assays. We show that the PSP repeat unit is assembled on an undecaprenyl-monophosphate (C55P) lipid intermediate. Synthesis is initiated by the WpsA/WpsB complex with GlcNAc-P-C55 synthase activity and the PSP precursor GlcNAc-P-C55 is then elongated by specific glycosyltransferases that vary among lactococcal strains, resulting in PSPs with diverse structures. Also, we engineered the PSP biosynthesis pathway in lactococci to obtain a chimeric PSP structure, confirming the predicted glycosyltransferase specificities. This enabled us to highlight the importance of a single sugar residue of the PSP repeat unit in phage recognition. In conclusion, our results support a novel pathway for PSP biosynthesis on a lipid-monophosphate intermediate as an extracellular modification of rhamnan, unveiling an assembly machinery for complex Rha-CWPS with structural diversity in lactococci.

Harnessing the endogenous Type I-C CRISPR-Cas system for genome editing in Bifidobacterium breve.

Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. The widespread presence of CRISPR-Cas systems in the B. breve genome makes endogenous CRISPR-based gene editing toolkits a promising tool. This study revealed that Type I-C CRISPR-Cas systems in B. breve can be divided into two groups based on the amino acid sequences encoded by cas gene clusters. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in five B. breve strains from both groups using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expands the toolkit for gene manipulation in B. breve and provides a new approach toward functional genome editing and analysis of B. breve strains.IMPORTANCEThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single-base substitution, gene insertion, and successive gene editing in B. breve. This study will facilitate discovery of functional genes and elucidation of molecular mechanisms of B. breve pertaining to health-associated benefits.

A multifaceted investigation of lactococcal strain diversity in undefined mesophilic starter cultures.

The dairy fermentation industry relies on the activity of lactic acid bacteria in robust starter cultures to accomplish milk acidification. Maintenance of the composition of these starter cultures, whether defined or undefined, is essential to ensure consistent and high-quality fermentation end products. To date, limited information exists regarding the microbial composition of undefined starter culture systems. Here, we describe a culture-based analysis combined with a metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, we describe a qPCR-based genotype detection assay, which is capable of discerning nine distinct lactococcal genotypes to characterize these undefined starter cultures, and which can be applied to monitor compositional changes in an undefined starter culture during a fermentation. IMPORTANCE: This study reports on the development of a combined culture-based analysis and metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, a novel qPCR-based genotype detection assay, capable of discerning nine distinct lactococcal genotypes (based on lactococcal cell wall polysaccharide biosynthesis gene clusters), was used to monitor compositional changes in an undefined starter culture following phage attack. These analytical approaches facilitate a multifaceted assessment of starter culture compositional stability during milk fermentation, which has become an important QC aspect due to the increasing demand for consistent and high-quality dairy products.

From raw milk cheese to the gut: investigating the colonization strategies of Bifidobacterium mongoliense.

The microbial ecology of raw milk cheeses is determined by bacteria originating from milk and milk-producing animals. Recently, it has been shown that members of the Bifidobacterium mongoliense species may become transmitted along the Parmigiano Reggiano cheese production chain and ultimately may colonize the consumer intestine. However, there is a lack of knowledge regarding the molecular mechanisms that mediate the interaction between B. mongoliense and the human gut. Based on 128 raw milk cheeses collected from different Italian regions, we isolated and characterized 10 B. mongoliense strains. Comparative genomics allowed us to unveil the presence of enzymes required for the degradation of sialylated host-glycans in B. mongoliense, corroborating the appreciable growth on de Man-Rogosa-Sharpe (MRS) medium supplemented with 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL). The B. mongoliense BMONG18 was chosen, due to its superior ability to utilize 3'-SL and mucin as representative strain, to investigate its behavior when co-inoculated with other bifidobacterial species. Conversely, members of other bifidobacterial species did not appear to benefit from the presence of BMONG18, highlighting a competitive scenario for nutrient acquisition. Transcriptomic data of BMONG18 reveal no significant differences in gene expression when cultivated in a gut simulating medium (GSM), regardless of whether cheese was included or not. Furthermore, BMONG18 was shown to exhibit high adhesion capabilities to HT29-MTX human cells, in line with its colonization ability of a human host.IMPORTANCEFermented foods are nourishments produced through controlled microbial growth that play an essential role in worldwide human nutrition. Research interest in fermented foods has increased since the 80s, driven by growing awareness of their potential health benefits beyond mere nutritional content. Bifidobacterium mongoliense, previously identified throughout the production process of Parmigiano Reggiano cheese, was found to be capable of establishing itself in the intestines of its consumers. Our study underscores molecular mechanisms through which this bifidobacterial species, derived from food, interacts with the host and other gut microbiota members.

The tal gene of lactococcal bacteriophage TP901-1 is involved in DNA release following host adsorption.

Temperate P335 phage TP901-1 represents one of the best-characterized Gram-positive phages regarding its structure and host interactions. Following its reversible adsorption to the polysaccharidic side-chain of the cell wall polysaccharide of its host Lactococcus cremoris 3107, TP901-1 requires a glucosylated cell envelope moiety to trigger its genome delivery into the host cytoplasm. Here, we demonstrate that three distinct single amino acid substitutions in the Tal protein of TP901-1 baseplate are sufficient to overcome the TP901-1 resistance of three L. cremoris 3107 derivatives, whose resistance is due to impaired DNA release of the phage. All of these Tal alterations are located in the N-terminally located gp27-like domain of the protein, conserved in many tailed phages. AlphaFold2 predictions of the Tal mutant proteins suggest that these mutations favor conformational changes necessary to reposition the Tal fiber and thus facilitate release of the tape measure protein from the tail tube and subsequent DNA ejection in the absence of the trigger otherwise required for phage genome release. IMPORTANCE: Understanding the molecular mechanisms involved in phage-host interactions is essential to develop phage-based applications in the food and probiotic industries, yet also to reduce the risk of phage infections in fermentations. Lactococcus, extensively used in dairy fermentations, has been widely employed to unravel such interactions. Phage infection commences with the recognition of a suitable host followed by the release of its DNA into the bacterial cytoplasm. Details on this latter, irreversible step are still very scarce in lactococci and other Gram-positive bacteria. We demonstrate that a component of the baseplate of the lactococcal phage TP901-1, the tail-associated lysin (Tal), is involved in the DNA delivery into its host, L. cremoris 3107. Specifically, we have found that three amino acid changes in Tal appear to facilitate structural rearrangements in the baseplate necessary for the DNA release process, even in the absence of an otherwise required host trigger.

Functional and practical insights into three lactococcal antiphage systems.

The persistent challenge of phages in dairy fermentations requires the development of starter cultures with enhanced phage resistance. Recently, three plasmid-encoded lactococcal antiphage systems, named Rhea, Aristaios, and Kamadhenu, were discovered. These systems were found to confer high levels of resistance against various Skunavirus members. In the present study, their effectiveness against phage infection was confirmed in milk-based medium, thus validating their potential to ensure reliable dairy fermentations. We furthermore demonstrated that Rhea and Kamadhenu do not directly hinder phage genome replication, transcription, or associated translation. Conversely, Aristaios was found to interfere with phage transcription. Two of the antiphage systems are encoded on pMRC01-like conjugative plasmids, and the Kamadhenu-encoding plasmid was successfully transferred by conjugation to three lactococcal strains, each of which acquired substantially enhanced phage resistance against Skunavirus members. Such advances in our knowledge of the lactococcal phage resistome and the possibility of mobilizing these protective functions to bolster phage protection in sensitive strains provide practical solutions to the ongoing phage problem in industrial food fermentations.IMPORTANCEIn the current study, we characterized and evaluated the mechanistic diversity of three recently described, plasmid-encoded lactococcal antiphage systems. These systems were found to confer high resistance against many members of the most prevalent and problematic lactococcal phage genus, rendering them of particular interest to the dairy industry, where persistent phage challenge requires the development of starter cultures with enhanced phage resistance characteristics. Our acquired knowledge highlights that enhanced understanding of lactococcal phage resistance systems and their encoding plasmids can provide rational and effective solutions to the enduring issue of phage infections in dairy fermentation facilities.

Selective human milk oligosaccharide utilization by members of the Bifidobacterium pseudocatenulatum taxon.

Human milk oligosaccharides (HMOs) are essentially unaffected by the digestive enzymes of the nursling and are known for their ability to enrich certain microbial species in the infant gut microbiota, in particular bifidobacteria. HMO metabolism has been studied in various bifidobacterial species such as B. breve, B. bifidum, and B. longum subsp. infantis. In the current study, we describe differential growth abilities elicited by twenty-three newly isolated Bifidobacterium pseudocatenulatum strains on particular HMOs, such as 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT). Through gene-trait matching and comparative genome analysis, we identified genes involved in the degradation of fucosylated HMOs in this strain set, while we employed a transcriptomic approach to facilitate the identification and characterization of genes and associated enzymes involved in LNT metabolism by strain B. pseudocatenulatum MM0196. A total of 252 publicly available genomes of the B. pseudocatenulatum taxon were screened for homologs of the glycosyl hydrolases (GHs) identified here as being required for selected HMO metabolism. From this analysis, it is clear that all members of this species possess homologs of the genes involved in LNT degradation, while genes required for degradation of fucosylated HMOs are variably present.IMPORTANCEOur findings allow a better understanding of the complex interaction between Bifidobacterium and its host and provide a roadmap toward future applications of B. pseudocatenulatum as a probiotic with a focus on infant health. Furthermore, our investigations have generated information on the role of HMOs in shaping the infant gut microbiota, thus also facilitating applications of HMOs in infant nutrition, with potential extension into the mature or adult gut microbiota. Supplementation of HMOs is known to result in the modulation of bacterial communities toward a higher relative abundance of bifidobacteria, which in turn enforces their ability to modulate particular immune functions and strengthen the intestinal barrier. This work may therefore inspire future studies to improve the formulation of neonatal nutritional products, aimed at facilitating the development of a healthy digestive and immune system and reducing the differences in gut microbiota composition observed between breastfed and formula-fed babies or full-term and preterm infants.

Ecology- and genome-based identification of the Bifidobacterium adolescentis prototype of the healthy human gut microbiota.

Bifidobacteria are among the first microbial colonizers of the human gut, being frequently associated with human health-promoting activities. In the current study, an in silico methodology based on an ecological and phylogenomic-driven approach allowed the selection of a Bifidobacterium adolescentis prototype strain, i.e., B. adolescentis PRL2023, which best represents the overall genetic content and functional features of the B. adolescentis taxon. Such features were confirmed by in vitro experiments aimed at evaluating the ability of this strain to survive in the gastrointestinal tract of the host and its ability to interact with human intestinal cells and other microbial gut commensals. In this context, co-cultivation of B. adolescentis PRL2023 and several gut commensals revealed various microbe-microbe interactions and indicated co-metabolism of particular plant-derived glycans, such as xylan.IMPORTANCEThe use of appropriate bacterial strains in experimental research becomes imperative in order to investigate bacterial behavior while mimicking the natural environment. In the current study, through in silico and in vitro methodologies, we were able to identify the most representative strain of the Bifidobacterium adolescentis species. The ability of this strain, B. adolescentis PRL2023, to cope with the environmental challenges imposed by the gastrointestinal tract, together with its ability to switch its carbohydrate metabolism to compete with other gut microorganisms, makes it an ideal choice as a B. adolescentis prototype and a member of the healthy microbiota of adults. This strain possesses a genetic blueprint appropriate for its exploitation as a candidate for next-generation probiotics.

The infant gut microbiota as the cornerstone for future gastrointestinal health.

The early postnatal period represents a critical window of time for the establishment and maturation of the human gut microbiota. The gut microbiota undergoes dramatic developmental changes during the first year of life, being influenced by a variety of external factors, with diet being a major player. Indeed, the introduction of complementary feeding provides novel nutritive substrates and triggers a shift from milk-adapted gut microbiota toward an adult-like bacterial composition, which is characterized by an enhancement in diversity and proportions of fiber-degrading bacterial genera like Ruminococcus, Prevotella, Eubacterium, and Bacteroides genera. Inadequate gut microbiota development in early life is frequently associated with concomitant and future adverse health conditions. Thus, understanding the processes that govern initial colonization and establishment of microbes in the gastrointestinal tract is of great importance. This review summarizes the actual understanding of the assembly and development of the microbial community associated with the infant gut, emphasizing the importance of mother-to-infant vertical transmission events as a fundamental arrival route for the first colonizers.

Isolation and identification of chlorate-reducing Hafnia sp. from milk.

Chlorate has become a concern in the food and beverage sector, related to chlorine sanitizers in industrial food production and water treatment. It is of particular concern to regulatory bodies due to the negative health effects of chlorate exposure. This study investigated the fate of chlorate in raw milk and isolated bacterial strains of interest responsible for chlorate breakdown. Unpasteurized milk was demonstrated to have a chlorate-reducing capacity, breaking down enriched chlorate to undetectable levels in 11 days. Further enrichment and isolation using conditions specific to chlorate-reducing bacteria successfully isolated three distinct strains of Hafnia paralvei. Chlorate-reducing bacteria were observed to grow in a chlorate-enriched medium with lactate as an electron donor. All isolated strains were demonstrated to reduce chlorate in liquid medium; however, the exact mechanism of chlorate degradation was not definitively identified in this study.

PBP2b Mutations Improve the Growth of Phage-Resistant Lactococcus cremoris Lacking Polysaccharide Pellicle.

Lactococcus lactis and Lactococcus cremoris are Gram-positive lactic acid bacteria widely used as starter in milk fermentations. Lactococcal cells are covered with a polysaccharide pellicle (PSP) that was previously shown to act as the receptor for numerous bacteriophages of the Caudoviricetes class. Thus, mutant strains lacking PSP are phage resistant. However, because PSP is a key cell wall component, PSP-negative mutants exhibit dramatic alterations of cell shape and severe growth defects, which limit their technological value. In the present study, we isolated spontaneous mutants with improved growth, from L. cremoris PSP-negative mutants. These mutants grow at rates similar to the wild-type strain, and based on transmission electron microscopy analysis, they exhibit improved cell morphology compared to their parental PSP-negative mutants. In addition, the selected mutants maintain their phage resistance. Whole-genome sequencing of several such mutants showed that they carried a mutation in pbp2b, a gene encoding a penicillin-binding protein involved in peptidoglycan biosynthesis. Our results indicate that lowering or turning off PBP2b activity suppresses the requirement for PSP and ameliorates substantially bacterial fitness and morphology. IMPORTANCE Lactococcus lactis and Lactococcus cremoris are widely used in the dairy industry as a starter culture. As such, they are consistently challenged by bacteriophage infections which may result in reduced or failed milk acidification with associated economic losses. Bacteriophage infection starts with the recognition of a receptor at the cell surface, which was shown to be a cell wall polysaccharide (the polysaccharide pellicle [PSP]) for the majority of lactococcal phages. Lactococcal mutants devoid of PSP exhibit phage resistance but also reduced fitness, since their morphology and division are severely impaired. Here, we isolated spontaneous, food-grade non-PSP-producing L. cremoris mutants resistant to bacteriophage infection with a restored fitness. This study provides an approach to isolate non-GMO phage-resistant L. cremoris and L. lactis strains, which can be applied to strains with technological functionalities. Also, our results highlight for the first time the link between peptidoglycan and cell wall polysaccharide biosynthesis.

Galacto-oligosaccharides as infant prebiotics: production, application, bioactive activities and future perspectives.

Galacto-oligosaccharides (GOS) are non-digestible oligosaccharides characterized by a mix of structures that vary in their degree of polymerization (DP) and glycosidic linkage between the galactose moieties or between galactose and glucose. They have enjoyed extensive scientific scrutiny, and their health-promoting effects are supported by a large number of scientific and clinical studies. A variety of GOS-associated health-promoting effects have been reported, such as growth promotion of beneficial bacteria, in particular bifidobacteria and lactobacilli, inhibition of pathogen adhesion and improvement of gut barrier function. GOS have attracted significant interest from food industries for their versatility as a bioactive ingredient and in particular as a functional component of infant formulations. These oligosaccharides are produced in a kinetically-controlled reaction involving lactose transgalactosylation, being catalyzed by particular ß-galactosidases of bacterial or fungal origin. Despite the well-established technology applied for GOS production, this process may still meet with technological challenges when employed at an industrial scale. The current review will cover relevant scientific literature on the beneficial physiological properties of GOS as a prebiotic for the infant gut microbiota, details of GOS structures, the associated reaction mechanism of ß-galactosidase, and its (large-scale) production.

Maternal gut microbiota Bifidobacterium promotes placental morphogenesis, nutrient transport and fetal growth in mice.

The gut microbiota plays a central role in regulating host metabolism. While substantial progress has been made in discerning how the microbiota influences host functions post birth and beyond, little is known about how key members of the maternal gut microbiota can influence feto-placental growth. Notably, in pregnant women, Bifidobacterium represents a key beneficial microbiota genus, with levels observed to increase across pregnancy. Here, using germ-free and specific-pathogen-free mice, we demonstrate that the bacterium Bifidobacterium breve UCC2003 modulates maternal body adaptations, placental structure and nutrient transporter capacity, with implications for fetal metabolism and growth. Maternal and placental metabolome were affected by maternal gut microbiota (i.e. acetate, formate and carnitine). Histological analysis of the placenta confirmed that Bifidobacterium modifies placental structure via changes in Igf2P0, Dlk1, Mapk1 and Mapk14 expression. Additionally, B. breve UCC2003, acting through Slc2a1 and Fatp1-4 transporters, was shown to restore fetal glycaemia and fetal growth in association with changes in the fetal hepatic transcriptome. Our work emphasizes the importance of the maternal gut microbiota on feto-placental development and sets a foundation for future research towards the use of probiotics during pregnancy.

Tap water as a natural vehicle for microorganisms shaping the human gut microbiome.

Fresh potable water is an indispensable drink which humans consume daily in substantial amounts. Nonetheless, very little is known about the composition of the microbial community inhabiting drinking water or its impact on our gut microbiota. In the current study, an exhaustive shotgun metagenomics analysis of the tap water microbiome highlighted the occurrence of a highly genetic biodiversity of the microbial communities residing in fresh water and the existence of a conserved core tap water microbiota largely represented by novel microbial species, representing microbial dark matter. Furthermore, genome reconstruction of this microbial dark matter from water samples unveiled homologous sequences present in the faecal microbiome of humans from various geographical locations. Accordingly, investigation of the faecal microbiota content of a subject that daily consumed tap water for 3 years provides proof for horizontal transmission and colonization of water bacteria in the human gut.

Designation of optimal reference strains representing the infant gut bifidobacterial species through a comprehensive multi-omics approach.

The genomic era has resulted in the generation of a massive amount of genetic data concerning the genomic diversity of bacterial taxa. As a result, the microbiological community is increasingly looking for ways to define reference bacterial strains to perform experiments that are representative of the entire bacterial species. Despite this, there is currently no established approach allowing a reliable identification of reference strains based on a comprehensive genomic, ecological, and functional context. In the current study, we developed a comprehensive multi-omics approach that will allow the identification of the optimal reference strains using the Bifidobacterium genus as test case. Strain tracking analysis based on 1664 shotgun metagenomics datasets of healthy infant faecal samples were employed to identify bifidobacterial strains suitable for in silico and in vitro analyses. Subsequently, an ad hoc bioinformatic tool was developed to screen local strain collections for the most suitable species-representative strain alternative. The here presented approach was validated using in vitro trials followed by metagenomics and metatranscriptomics analyses. Altogether, these results demonstrated the validity of the proposed model for reference strain selection, thus allowing improved in silico and in vitro investigations both in terms of cross-laboratory reproducibility and relevance of research findings.

Bipartite rgp Locus Diversity in Streptococcus thermophilus Corresponds to Backbone and Side Chain Differences of Its Rhamnose-Containing Cell Wall Polysaccharide.

The rhamnose-glucose polysaccharide (Rgp) of Streptococcus thermophilus represents a major cell wall component, and the gene cluster responsible for its biosynthesis (termed rgp) has recently been identified. Significant genetic diversity among these loci has previously been reported, with five distinct rgp genotypes identified (designated rgp1 through -5). In the present study, two additional genotypes were identified (designated rgp6 and rgp7) through comparative analysis of the rgp loci of 78 Streptococcus thermophilus genomes. The rgp locus of a given S. thermophilus strain encoded the biosynthetic machinery for a rhamnan-rich backbone and a variable side chain component, the latter being associated with the highly specific interactions with many bacteriophages that infect this species. The chemical structure of the Rgp from three S. thermophilus strains, representing the rgp2, -3, and -4 genotypes, was elucidated, and based on bioinformatic and biochemical analyses we propose a model for Rgp biosynthesis in dairy streptococci. Furthermore, we exploited the genetic diversity within the S. thermophilus bipartite rgp locus to develop a two-step multiplex PCR system to classify strains based on gene content associated with the biosynthesis of the variable side chain structure as well as the rhamnan backbone. IMPORTANCE Streptococcus thermophilus is present and applied in industrial and artisanal dairy fermentations for the production of various cheeses and yogurt. During these fermentations, S. thermophilus is vulnerable to phage predation, and recent studies have identified the rhamnose-glucose polymer (Rgp) as the definitive receptor for at least one problematic phage species. Detailed analysis of S. thermophilus rgp loci has revealed an unprecedented level of genetic diversity, particularly within the glycosyltransferase-encoding gene content of a given locus. Our study shows that this genetic diversity reflects the biochemical structure(s) of S. thermophilus Rgp. As such, we harnessed the genetic diversity of S. thermophilus rgp loci to develop a two-step multiplex PCR method for the classification of strain collections and, ultimately, the formation of phage-robust rational starter sets.

Brussowvirus SW13 Requires a Cell Surface-Associated Polysaccharide To Recognize Its Streptococcus thermophilus Host.

Four bacteriophage-insensitive mutants (BIMs) of the dairy starter bacterium Streptococcus thermophilus UCCSt50 were isolated following challenge with Brussowvirus SW13. The BIMs displayed an altered sedimentation phenotype. Whole-genome sequencing and comparative genomic analysis of the BIMs uncovered mutations within a family 2 glycosyltransferase-encoding gene (orf06955UCCSt50) located within the variable region of the cell wall-associated rhamnose-glucose polymer (Rgp) biosynthesis locus (designated the rgp gene cluster here). Complementation of a representative BIM, S. thermophilus B1, with native orf06955UCCSt50 restored phage sensitivity comparable to that of the parent strain. Detailed bioinformatic analysis of the gene product of orf06955UCCSt50 identified it as a functional homolog of the Lactococcus lactis polysaccharide pellicle (PSP) initiator WpsA. Biochemical analysis of cell wall fractions of strains UCCSt50 and B1 determined that mutations within orf06955UCCSt50 result in the loss of the side chain decoration from the Rgp backbone structure. Furthermore, it was demonstrated that the intact Rgp structure incorporating the side chain structure is essential for phage binding through fluorescence labeling studies. Overall, this study confirms that the rgp gene cluster of S. thermophilus encodes the biosynthetic machinery for a cell surface-associated polysaccharide that is essential for binding and subsequent infection by Brussowviruses, thus enhancing our understanding of S. thermophilus phage-host dynamics. IMPORTANCE Streptococcus thermophilus is an important starter culture bacterium in global dairy fermentation processes, where it is used for the production of various cheeses and yogurt. Bacteriophage predation of the species can result in substandard product quality and, in rare cases, complete fermentation collapse. To mitigate these risks, it is necessary to understand the phage-host interaction process, which commences with the recognition of, and adsorption to, specific host-encoded cell surface receptors by bacteriophage(s). As new groups of S. thermophilus phages are being discovered, the importance of underpinning the genomic elements that specify the surface receptor(s) is apparent. Our research identifies a single gene that is critical for the biosynthesis of a saccharidic moiety required for phage adsorption to its S. thermophilus host. The acquired knowledge provides novel insights into phage-host interactions for this economically important starter species.

Exploring the Ecological Effects of Naturally Antibiotic-Insensitive Bifidobacteria in the Recovery of the Resilience of the Gut Microbiota during and after Antibiotic Treatment.

Amoxicillin-clavulanic acid (AMC) is the most widely used antibiotic, being frequently prescribed to infants. Particular members of the genus Bifidobacterium are among the first microbial colonizers of the infant gut, and it has been demonstrated that they exhibit various activities beneficial for their human host, including promotion/maintenance of the human gut microbiota homeostasis. It has been shown that natural resistance of bifidobacteria to AMC is limited to a small number of strains. In the current study, we investigated the mitigation effects of AMC-resistant bifidobacteria in diversity preservation of the gut microbiota during AMC treatment. To this end, an in vitro coculture experiment based on infant fecal samples and an in vivo study employing a rodent model were performed. The results confirmed the ability of AMC-resistant bifidobacterial strains to bolster gut microbiota resilience, while specific covariance analysis revealed strain-specific and variable impacts on the microbiota composition by individual bifidobacterial taxa. IMPORTANCE The first microbial colonizers of the infant gut are members of the genus Bifidobacterium, which exhibit different activities beneficial to their host. Amoxicillin-clavulanic acid (AMC) is the most frequently prescribed antibiotic during infancy, and few strains of bifidobacteria are known to show a natural resistance to this antibiotic. In the present work, we evaluated the possible positive effects of AMC-resistant bifidobacterial strains in maintaining gut microbiota diversity during AMC exposure, performing an in vitro and in vivo experiment based on an infant gut model and a rodent model, respectively. Our results suggested the ability of AMC-resistant bifidobacterial strains to support gut microbiota restoration.