Characterization of beer-spoiling Lactobacillus brevis and their associated bacteriophages

Certain bacteria have acquired the ability to survive and even grow in beer, despite the antimicrobial nature of this harsh liquid environment. Bacterial growth in beer is often attributed to lactic acid bacteria (LAB) that are found in many environments, yet are commonly associated with plant material. While most LAB species bestow positive attributes to foods and food fermentations, others have the potential to survive and even thrive in inhospitable environments, including beer. Lactobacillus brevis strains are among the most frequently encountered LAB isolates in spoiled beer. In finished beer products, the presence of Lb. brevis is considered undesirable as they typically impart undesirable organoleptic properties on the product including off-flavors, odors and changes in the physical appearance and viscosity. The goal of the current study was to identify and characterize the molecular players that allow certain Lb. brevis to grow in and spoil in beer. It was also aimed to isolate phages that infect strains of Lb. brevis in order to assess their potential application in the brewing industry for the purpose of preventing or diminishing bacterial spoilage. The findings described in this thesis have improved our understanding of Lb. brevis as a diverse species and as a beer-spoiling microorganism. The genomic features of 19 Lb. brevis strains (encompassing the genomes of six Lb. brevis strains sequenced in this study and thirteen Lb. brevis strains available in public databases) were evaluated in a comparative genome analysis of the species, paying particular attention to evolutionary aspects and adaptation to beer. Moreover, novel molecular players were identified revealing the importance of chromosomal genes for general stress response (pH tolerance), and the relevance of plasmid-encoded genes for beer-specific stress response (such as tolerance to hop compounds). The response of beer-spoiling Lb. brevis strains to stress factors associated with beer was assessed using a transcriptomic approach comparing growth in nutritive media with or without the imposition of various beer-associated stressors including ethanol, low pH and hops. This analysis allowed the identification of a chromosomal gene encoding a presumed manganese transporter involved in low pH tolerance in Lb. brevis. Furthermore, the importance of plasmids for beer-spoiling Lb. brevis strains was investigated resulting in the identification of a plasmid-encoded putative glycosyltransferase involved in hop tolerance and thus growth in beer. Furthermore, to alleviate the problem of beer spoilage associated with Lb. brevis, the possibility of employing lytic phages capable of infecting such strains can be envisaged as a bio-sanitation approach. This thesis reports on the isolation of five Lb. brevis-infecting virulent phages, which were shown to exhibit a high level of genetic and morphological diversity. Interestingly, certain phage isolates displayed activity against Lb. brevis beer-spoiling strains preventing them from growing optimally, thus providing a phage-mediated approach to control bacterial spoilage of beer. Moreover, the incidence of prophages among Lb. brevis strains was studied in order to understand their potential benefit for their bacterial carrier. Prophage-encoded phage resistance systems such as abortive infection system (Abi), confer to the host resistance to phage infection, thus increasing its overall fitness. Two adjacent genes encoding an Abi system were identified on the prophage sequence of a beer-spoiling Lb. brevis strain. This Abi system exhibits activity against phages infecting Lb. brevis and Lactococcus lactis strains. The presence of such prophage-encoded systems highlights the importance of temperate phages for Lb. brevis strains and, in some cases, may explain their resistance to phage infection. With the study and characterization of virulent and temperate Lb. brevis phages, we have significantly extended our knowledge on Lb. brevis phages. Furthermore, it has provided novel insights into the diversity of Lb. brevis phages and their potential application as a bio-sanitation tool in the beer brewing industry

Functional and structural dissection of the tape measure protein of lactococcal phage TP901-1

The tail tape measure protein (TMP) of tailed bacteriophages (also called phages) dictates the tail length and facilitates DNA transit to the cell cytoplasm during infection. Here, a thorough mutational analysis of the TMP from lactococcal phage TP901-1 (TMPTP901-1) was undertaken. We generated 56 mutants aimed at defining TMPTP901-1 domains that are essential for tail assembly and successful infection. Through analysis of the derived mutants, we determined that TP901-1 infectivity requires the N-terminal 154 aa residues, the C-terminal 60 residues and the first predicted hydrophobic region of TMPTP901-1 as a minimum. Furthermore, the role of TMPTP901-1 in tail length determination was visualized by electron microscopic imaging of TMP-deletion mutants. The inverse linear correlation between the extent of TMPTP901-1-encoding gene deletions and tail length of the corresponding virion provides an estimate of TMPTP901-1 regions interacting with the connector or involved in initiator complex formation. This study represents the most thorough characterisation of a TMP from a Gram-positive host-infecting phage and provides essential advances to understanding its role in virion assembly, morphology and infection

A plasmid-encoded putative glycosyltransferase is involved in hop tolerance and beer spoilage in Lactobacillus brevis

Lactobacillus brevis beer-spoiling strains harbor plasmids that contain genes such as horA, horC, and hitA which are known to confer hop tolerance. The L. brevis beer-spoiling strain UCCLBBS124, which possesses four plasmids, was treated with novobiocin, resulting in the isolation of UCCLBBS124 derivatives exhibiting hop sensitivity and an inability to grow in beer. One selected derivative was shown to have lost a single plasmid, here designated UCCLBBS124_D, which harbors the UCCLBBS124_pD0015 gene, predicted to encode a glycosyltransferase. Hop tolerance and growth in beer were restored when UCCLBBS124_pD0015 was introduced in one of these hop-sensitive derivatives on a plasmid. We hypothesize that this gene modifies the surface composition of the polysaccharide cell wall, conferring protection against hop compounds. Furthermore, the introduction of this gene in trans in L. brevis UCCLB521, a strain that cannot grow in and spoil beer, was shown to furnish the resulting strain with the ability to grow in beer, while its expression also conferred phage resistance. This study underscores how the acquisition of certain mobile genetic elements plays a role in hop tolerance and beer spoilage for strains of this bacterial species.IMPORTANCELactobacillus brevis is a member of the lactic acid bacteria and is often reported as the causative agent of food or beverage spoilage, in particular, that of beer. Bacterial spoilage of beer may result in product withdrawal or recall, with concomitant economic losses for the brewing industry. A very limited number of genes involved in beer spoilage have been identified and primarily include those involved in hop resistance, such as horA, hitA, and horC However, since none of these genes are universal, it is clear that there are likely (many) other molecular players involved in beer spoilage. Here, we report on the importance of a plasmid-encoded glycosyltransferase associated with beer spoilage by L. brevis that is involved in hop tolerance. The study highlights the complexity of the genetic requirements to facilitate beer spoilage and the role of multiple key players in this process

Biodiversity and classification of phages infecting Lactobacillus brevis

Lactobacillus brevis is a lactic acid bacterium that is known as a food and beverage spoilage organism, and more specifically as a beer-spoiler. Phages of L. brevis have been described, but very limited data is available regarding temperate phages of L. brevis. Temperate phages may exert benefits to the host, while they may also be employed to combat beer spoilage. The current study reports on the incidence of prophage sequences present in nineteen distinct L. brevis genomes. Prophage induction was evaluated using mitomycin C exposure followed by genome targeted-PCR, electron microscopy and structural proteome analysis. The morphological and genome sequence analyses revealed significant diversity among L. brevis prophages, which appear to be dominated by members of the Myoviridae phage family. Based on this analysis, we propose a classification of L. brevis phages into five groups

Comparative genome analysis of the Lactobacillus brevis species

Background: Lactobacillus brevis is a member of the lactic acid bacteria (LAB), and strains of L. brevis have been isolated from silage, as well as from fermented cabbage and other fermented foods. However, this bacterium is also commonly associated with bacterial spoilage of beer. Results: In the current study, complete genome sequences of six isolated L. brevis strains were determined. Five of these L. brevis strains were isolated from beer (three isolates) or the brewing environment (two isolates), and were characterized as beer-spoilers or non-beer spoilers, respectively, while the sixth isolate had previously been isolated from silage. The genomic features of 19 L. brevis strains, encompassing the six L. brevis strains described in this study and thirteen L. brevis strains for which complete genome sequences were available in public databases, were analyzed with particular attention to evolutionary aspects and adaptation to beer. Conclusions: Comparative genomic analysis highlighted evolution of the taxon allowing niche colonization, notably adaptation to the beer environment, with approximately 50 chromosomal genes acquired by L. brevis beer-spoiler strains representing approximately 2% of their total chromosomal genetic content. These genes primarily encode proteins that are putatively involved in oxidation-reduction reactions, transcription regulation or membrane transport, functions that may be crucial to survive the harsh conditions associated with beer. The study emphasized the role of plasmids in beer spoilage with a number of unique genes identified among L. brevis beer-spoiler strains

Spatial Distribution of Non-Immune Cells Expressing Glycoprotein A Repetitions Predominant in Human and Murine Metastatic Lymph Nodes

Several types of cancer spread through the lymphatic system via the sentinel lymph nodes (LNs). Such LN-draining primary tumors, modified by tumor factors, lead to the formation of a metastatic niche associated with an increased number of Foxp3+ regulatory T cells (Tregs). These cells are expected to contribute to the elaboration of an immune-suppressive environment. Activated Tregs express glycoprotein A repetitions predominant (GARP), which binds and presents latent transforming growth factor beta 1 (TGF-β1) at their surface. GARP is also expressed by other non-immune cell types poorly described in LNs. Here, we mapped GARP expression in non-immune cells in human and mouse metastatic LNs. The mining of available (human and murine) scRNA-Seq datasets revealed GARP expression by blood (BEC)/lymphatic (LEC) endothelial, fibroblastic, and perivascular cells. Consistently, through immunostaining and in situ RNA hybridization approaches, GARP was detected in and around blood and lymphatic vessels, in (αSMA+) fibroblasts, and in perivascular cells associated with an abundant matrix. Strikingly, GARP was detected in LECs forming the subcapsular sinus and high endothelial venules (HEVs), two vascular structures localized at the interface between LNs and the afferent lymphatic and blood vessels. Altogether, we here provide the first distribution maps for GARP in human and murine LNs

Spatial Distribution of Non-Immune Cells Expressing Glycoprotein A Repetitions Predominant in Human and Murine Metastatic Lymph Nodes.

Several types of cancer spread through the lymphatic system via the sentinel lymph nodes (LNs). Such LN-draining primary tumors, modified by tumor factors, lead to the formation of a metastatic niche associated with an increased number of Foxp3+ regulatory T cells (Tregs). These cells are expected to contribute to the elaboration of an immune-suppressive environment. Activated Tregs express glycoprotein A repetitions predominant (GARP), which binds and presents latent transforming growth factor beta 1 (TGF-β1) at their surface. GARP is also expressed by other non-immune cell types poorly described in LNs. Here, we mapped GARP expression in non-immune cells in human and mouse metastatic LNs. The mining of available (human and murine) scRNA-Seq datasets revealed GARP expression by blood (BEC)/lymphatic (LEC) endothelial, fibroblastic, and perivascular cells. Consistently, through immunostaining and in situ RNA hybridization approaches, GARP was detected in and around blood and lymphatic vessels, in (αSMA+) fibroblasts, and in perivascular cells associated with an abundant matrix. Strikingly, GARP was detected in LECs forming the subcapsular sinus and high endothelial venules (HEVs), two vascular structures localized at the interface between LNs and the afferent lymphatic and blood vessels. Altogether, we here provide the first distribution maps for GARP in human and murine LNs

Isolation and Characterization of <i>Lactobacillus brevis</i> Phages

Lactobacillus brevis has been widely used in industry for fermentation purposes. However, it is also associated with the spoilage of foods and beverages, in particular, beer. There is an increasing demand for natural food preservation methods, and in this context, bacteriophages possess the potential to control such spoilage bacteria. Just a few studies on phages infecting Lactobacillus brevis have been performed to date and in the present study, we report the isolation and characterization of five virulent phages capable of infecting Lb. brevis strains. The analysis reveals a high diversity among the isolates, with members belonging to both, the Myoviridae and Siphoviridae families. One isolate, designated phage 3-521, possesses a genome of 140.8 kb, thus representing the largest Lb. brevis phage genome sequenced to date. While the isolated phages do not propagate on Lb. brevis beer-spoiling strains, phages showed activity against these strains, impairing the growth of some Lb. brevis strains. The results highlight the potential of bacteriophage-based treatments as an effective approach to prevent bacterial spoilage of beer