Utilization of plant derived lactic acid bacteria for efficient bioconversion of brewers' spent grain into acetoin.

Brewers' spent grain (BSG) is a major side-stream from the beer industry, with an annual estimated production of 39 million tons worldwide. Due to its high nutritional value, high abundance and low price, it has been proposed as an ingredient in human food. Here we investigated the ability of different lactic acid bacteria to produce the flavor molecule acetoin in liquid BSG extract, in order to broaden the possibilities of utilization of BSG in human food. All the investigated lactic acid bacteria (LAB) covering the Leuconostoc, Lactobacillus and Lactoccocus species were able to convert the fermentable sugars in liquid BSG into acetoin. Production levels varied significantly between the different LAB species, with Leuconostoc pseudomesenteroides species reaching the highest titers of acetoin with only acetate as the main byproduct, while also being the fastest consumer of the fermentable sugars present in liquid BSG. Surprisingly, the currently best investigated LAB for acetoin production, L. lactis, was unable to consume the maltose fraction of liquid BSG and was therefore deemed unfit for full conversion of the sugars in BSG into acetoin. The production of acetoin in Leu. pseudomesenteroides was pH dependent as previously observed in other LAB, and the conversion of BSG into acetoin was scalable from shake flasks to 1 L bioreactors. While all investigated LAB species produced acetoin under aerobic conditions, Leu. pseudomesenteroides was found to be an efficient and scalable organism for bioconversion of liquid BSG into a safe acetoin rich food additive.

Leuconostoc performance in soy-based fermentations - Survival, acidification, sugar metabolism, and flavor comparisons.

Leuconostoc spp. is often regarded as the flavor producer, responsible for the production of acetoin and diacetyl in dairy cheese. In this study, we investigate seven plant-derived Leuconostoc strains, covering four species, in their potential as a lyophilized starter culture for flavor production in fermented soy-based cheese alternatives. We show that the process of lyophilization of Leuconostoc can be feasible using a soy-based lyoprotectant, with survivability up to 63% during long term storage. Furthermore, the storage in this media improves the subsequent growth in a soy-based substrate in a strain specific manner. The utilization of individual raffinose family oligosaccharides was strain dependent, with Leuconostoc pseudomesenteroides NFICC99 being the best consumer. Furthermore, we show that all investigated strains were able to produce a range of volatile flavor compounds found in dairy cheese products, as well as remove certain dairy off-flavors from the soy-based substrate like hexanal and 2-pentylfuran. Also here, NFICC99 was strain producing most cheese-related volatile flavor compounds, followed by Leuconostoc mesenteroides NFICC319. These findings provide initial insights into the development of Leuconostoc as a potential starter culture for plant-based dairy alternatives, as well as a promising approach for generation of stable, lyophilized cultures.

The circular bacteriocin enterocin NKR-5-3B has an improved stability profile over nisin.

Bacteriocins are a large family of bacterial peptides that have antimicrobial activity and potential applications as clinical antibiotics or food preservatives. Circular bacteriocins are a unique class of these biomolecules distinguished by a seamless circular topology, and are widely assumed to be ultra-stable based on this constraining structural feature. However, without quantitative studies of their susceptibility to defined thermal, chemical, and enzymatic conditions, their stability characteristics remain poorly understood, limiting their translational development. Here, we produced the circular bacteriocin enterocin NKR-5-3B (Ent53B) in mg/L quantities using a heterologous Lactococcus expression system, and characterized its thermal stability by NMR, chemical stability by circular dichroism and analytical HPLC, and enzymatic stability by analytical HPLC. We demonstrate that Ent53B is ultra-stable, resistant to temperatures approaching boiling, acidic (pH 2.6) and alkaline (pH 9.0) conditions, the chaotropic agent 6 M urea, and following incubation with a range of proteases (i.e., trypsin, chymotrypsin, pepsin, and papain), conditions under which most peptides and proteins degrade. Ent53B is stable across a broader range of pH conditions and proteases than nisin, the most widely used bacteriocin in food manufacturing. Antimicrobial assays showed that differences in stability correlated with differences in bactericidal activity. Overall, this study provides quantitative support for circular bacteriocins being an ultra-stable class of peptide molecules, suggesting easier handling and distribution options available to them in practical applications as antimicrobial agents.

Cyclic-di-AMP signalling in lactic acid bacteria.

Cyclic dimeric adenosine monophosphate (cyclic-di-AMP) is a nucleotide second messenger present in Gram-positive bacteria, Gram-negative bacteria and some Archaea. The intracellular concentration of cyclic-di-AMP is adjusted in response to environmental and cellular cues, primarily through the activities of synthesis and degradation enzymes. It performs its role by binding to protein and riboswitch receptors, many of which contribute to osmoregulation. Imbalances in cyclic-di-AMP can lead to pleiotropic phenotypes, affecting aspects such as growth, biofilm formation, virulence, and resistance to osmotic, acid, and antibiotic stressors. This review focuses on cyclic-di-AMP signalling in lactic acid bacteria (LAB) incorporating recent experimental discoveries and presenting a genomic analysis of signalling components from a variety of LAB, including those found in food, and commensal, probiotic, and pathogenic species. All LAB possess enzymes for the synthesis and degradation of cyclic-di-AMP, but are highly variable with regards to the receptors they possess. Studies in Lactococcus and Streptococcus have revealed a conserved function for cyclic-di-AMP in inhibiting the transport of potassium and glycine betaine, either through direct binding to transporters or to a transcriptional regulator. Structural analysis of several cyclic-di-AMP receptors from LAB has also provided insights into how this nucleotide exerts its influence.

Assessment of post-infarct ventricular septal defects through 3D printing and statistical shape analysis.

Background: Post-infarct ventricular septal defect (PIVSD) is a serious complication of myocardial infarction. We evaluated 3D-printing models in PIVSD clinical assessment and the feasibility of statistical shape modeling for morphological analysis of the defects. Methods: Models (n = 15) reconstructed from computed tomography data were evaluated by clinicians (n = 8). Statistical shape modeling was performed on 3D meshes to calculate the mean morphological configuration of the defects. Results: Clinicians' evaluation highlighted the models' utility in displaying defects for interventional/surgical planning, education/training and device development. However, models lack dynamic representation. Morphological analysis was feasible and revealed oval-shaped (n = 12) and complex channel-like (n = 3) defects. Conclusion: 3D-PIVSD models can complement imaging data for teaching and procedural planning. Statistical shape modeling is feasible in this scenario.

Following heart attacks, the heart muscle becomes scarred and weaker, making it prone to tearing under high pressures. These tears are known as 'post-infarct ventricular septal defects'. Their shape varies greatly as the heart beats. The approach to fixing these can range from plugging them with a device or patching them by open heart surgery. We created 15 3D-printed models of hearts with these kinds of defects and made digital reconstructions of the tears to see the different sizes/shapes that they can have. Doctors agreed that 3D-printed models could help in planning repairs and training other doctors. The digital reconstructions of the tears showed that many were round, but some had irregular shapes which would mean devices used to fix them may not fit.

Evaluation of the fermentation potential of lactic acid bacteria isolated from herbs, fruits and vegetables as starter cultures in nut-based milk alternatives.

Fermentation of plant-based milk alternatives (PBMAs), including nut-based products, has the potential to generate new foods with improved sensorial properties. In this study, we screened 593 lactic acid bacteria (LAB) isolates from herbs, fruits and vegetables for their ability to acidify an almond-based milk alternative. The majority of the strongest acidifying plant-based isolates were identified as Lactococcus lactis, which were found to lower the pH of almond milk faster than dairy yoghurt cultures. Whole genome sequencing (WGS) of 18 plant-based Lc. lactis isolates revealed the presence of sucrose utilisation genes (sacR, sacA, sacB and sacK) in the strongly acidifying strains (n = 17), which were absent in one non-acidifying strain. To confirm the importance of Lc. lactis sucrose metabolism in efficient acidification of nut-based milk alternatives, we obtained spontaneous mutants defective in sucrose utilisation and confirmed their mutations by WGS. One mutant containing a sucrose-6-phosphate hydrolase gene (sacA) frameshift mutation was unable to efficiently acidify almond, cashew and macadamia nut milk alternatives. Plant-based Lc. lactis isolates were heterogeneous in their possession of the nisin gene operon near the sucrose gene cluster. The results of this work show that sucrose-utilising plant-based Lc. lactis have potential as starter cultures for nut-based milk alternatives.

Co-culture with Acinetobacter johnsonii enhances benzalkonium chloride resistance in Salmonella enterica via triggering lipid A modifications.

Salmonella enterica is one of the leading causes of foodborne gastroenteritis worldwide. In the food production environment, many bacterial species co-exist on surfaces in biofilm structures, which can act as reservoirs of microbial contamination of food products. Polymicrobial biofilms have been shown to have greater tolerance to antimicrobials, such as disinfectants, however the mechanistic basis of this is poorly understood. In this study, S. enterica subsp. enterica serovar Liverpool was co-cultured in mixed-species biofilms with bacteria isolated from the food production environment and challenged with the cationic biocide disinfectant, benzalkonium chloride (BC). Co-culture with the common environmental bacterium Acinetobacter johnsonii resulted in >200-fold higher resistance of S. Liverpool to BC, compared to mono-culture biofilms. The transcriptional response of S. enterica to biofilm co-culture was determined using a dual RNA-seq strategy. Genes controlled by the PhoPQ and PmrAB two-component systems, involved in lipid A modification and associated with cationic antimicrobial peptide resistance (CAMP) of S. Liverpool, were significantly upregulated. Deletion of either the phoP or pmrA genes resulted in an increase in susceptibility to BC, suggesting that activation of their regulons during co-culture enhances BC resistance. S. Liverpool lipid A profiles changed significantly upon co-culturing, with greater incorporation of both phosphoethanolamine and palmitate, which was dependent upon activation of PhoPQ and PmrAB. We conclude that when grown in the presence of A. johnsonii, S. Liverpool increases its tolerance to cationic BC disinfection by remodelling its cell envelope including reducing the net negative charge of lipid A and increasing lipid A acyl density.

Post-infarction ventricular septal defect: percutaneous or surgical management in the UK national registry.

AIMS: Post-infarction ventricular septal defect (PIVSD) is a mechanical complication of acute myocardial infarction (AMI) with a poor prognosis. Surgical repair is the mainstay of treatment, although percutaneous closure is increasingly undertaken. METHODS AND RESUTS: Patients treated with surgical or percutaneous repair of PIVSD (2010-2021) were identified at 16 UK centres. Case note review was undertaken. The primary outcome was long-term mortality. Patient groups were allocated based upon initial management (percutaneous or surgical). Three-hundred sixty-two patients received 416 procedures (131 percutaneous, 231 surgery). 16.1% of percutaneous patients subsequently had surgery. 7.8% of surgical patients subsequently had percutaneous treatment. Times from AMI to treatment were similar [percutaneous 9 (6-14) vs. surgical 9 (4-22) days, P = 0.18]. Surgical patients were more likely to have cardiogenic shock (62.8% vs. 51.9%, P = 0.044). Percutaneous patients were substantially older [72 (64-77) vs. 67 (61-73) years, P < 0.001] and more likely to be discussed in a heart team setting. There was no difference in long-term mortality between patients (61.1% vs. 53.7%, P = 0.17). In-hospital mortality was lower in the surgical group (55.0% vs. 44.2%, P = 0.048) with no difference in mortality after hospital discharge (P = 0.65). Cardiogenic shock [adjusted hazard ratio (aHR) 1.97 (95% confidence interval 1.37-2.84), P < 0.001), percutaneous approach [aHR 1.44 (1.01-2.05), P = 0.042], and number of vessels with coronary artery disease [aHR 1.22 (1.01-1.47), P = 0.043] were independently associated with long-term mortality. CONCLUSION: Surgical and percutaneous repair are viable options for management of PIVSD. There was no difference in post-discharge long-term mortality between patients, although in-hospital mortality was lower for surgery.

Heat, cold, acid, and bile salt induced differential proteomic responses of a novel potential probiotic Lactococcus garvieae C47 isolated from camel milk.

Probiotics encounter various stresses during food processing and digestion. This study evaluated the differential proteomic responses of a newly identified potential probiotic lactic acid bacteria, Lactococcus garvieae, isolated from camel milk. Lc. garvieae C47 was exposed to heat, cold, acid, and bile conditions, and stress-responsive proteins were identified. The proteomic analysis was done using 2D-IEF SDS PAGE and nano-LC-MS/MS. Out of 91 differentially expressed proteins, 20 upregulated and 27 downregulated proteins were shared among the stresses. The multivariate data analysis revealed abundance of elongation factor Ts (spot C42), uridine phosphorylase, fructose-bisphosphate aldolase, peptidase T, cobalt ECF transporter T component CbiQ, UDP-N-acetylmuramate-l-alanine ligase, uncharacterized protein, aspartokinase, chaperone protein DnaK, IGP synthase cyclase subunit, probable nicotinate-nucleotide adenylyltransferase, NADH-quinone oxidoreductase, holo-[acyl-carrier-protein] synthase, l-lactate dehydrogenase, and uncharacterized protein. The maximum number of differentially expressed proteins belonged to carbohydrate and protein metabolism, which indicates Lc. garvieae shifts towards growth and energy metabolism for resistance against stress conditions.

Potential Probiotic Pediococcus pentosaceus M41 Modulates Its Proteome Differentially for Tolerances Against Heat, Cold, Acid, and Bile Stresses.

Probiotics containing functional food confer health benefits in addition to their nutritional properties. In this study, we have evaluated the differential proteomic responses of a potential novel probiotic Pediococcus pentosaceus M41 under heat, cold, acid, and bile stress conditions. We identified stress response proteins that could provide tolerances against these stresses and could be used as probiotic markers for evaluating stress tolerance. Pediococcus pentosaceus M41 was exposed for 2 h to each condition: 50°C (heat stress), 4°C (cold stress), pH 3.0 (acid stress) and 0.05% bile (bile stress). Proteomic analysis was carried out using 2D-IEF SDS PAGE and LC-MS/MS. Out of 60 identified proteins, 14 upregulated and 6 downregulated proteins were common among all the stress conditions. These proteins were involved in different biological functions such as translation-related proteins, carbohydrate metabolism (phosphoenolpyruvate phosphotransferase), histidine biosynthesis (imidazole glycerol phosphate synthase) and cell wall synthesis (tyrosine-protein kinase CapB). Proteins such as polysaccharide deacetylase, lactate oxidase, transcription repressor NrdR, dihydroxyacetone kinase were upregulated under three out of the four stress conditions. The differential expression of these proteins might be responsible for tolerance and protection of P. pentosaceus M41 against different stress conditions.

Invited review: Characterization of new probiotics from dairy and nondairy products-Insights into acid tolerance, bile metabolism and tolerance, and adhesion capability.

The selection of potential probiotic strains that possess the physiological capacity of performing successfully in the gastrointestinal tract (GIT) is a critical challenge. Probiotic microorganisms must tolerate the deleterious effects of various stresses to survive passage and function in the human GIT. Adhesion to the intestinal mucosa is also an important aspect. Recently, numerous studies have been performed concerning the selection and evaluation of novel probiotic microorganisms, mainly probiotic bacteria isolated from dairy and nondairy products. Therefore, it would be crucial to critically review the assessment methods employed to select the potential probiotics. This article aims to review and discuss the recent approaches, methods used for the selection, and outcomes of the evaluation of novel probiotic strains with the main purpose of supporting future probiotic microbial assessment studies. The findings and approaches used for assessing acid tolerance, bile metabolism and tolerance, and adhesion capability are the focus of this review. In addition, probiotic bile deconjugation and bile salt hydrolysis are explored. The selection of a new probiotic strain has mainly been based on the in vitro tolerance of physiologically related stresses including low pH and bile, to ensure that the potential probiotic microorganism can survive the harsh conditions of the GIT. However, the varied experimental conditions used in these studies (different types of media, bile, pH, and incubation time) hamper the comparison of the results of these investigations. Therefore, standardization of experimental conditions for characterizing and selecting probiotics is warranted.

Cyclic di-AMP Oversight of Counter-Ion Osmolyte Pools Impacts Intrinsic Cefuroxime Resistance in Lactococcus lactis.

The broadly conserved cyclic di-AMP (c-di-AMP) is a conditionally essential bacterial second messenger. The pool of c-di-AMP is fine-tuned through diadenylate cyclase and phosphodiesterase activities, and direct binding of c-di-AMP to proteins and riboswitches allows the regulation of a broad spectrum of cellular processes. c-di-AMP has a significant impact on intrinsic ß-lactam antibiotic resistance in Gram-positive bacteria; however, the reason for this is currently unclear. In this work, genetic studies revealed that suppressor mutations that decrease the activity of the potassium (K+) importer KupB or the glutamine importer GlnPQ restore cefuroxime (CEF) resistance in diadenylate cyclase (cdaA) mutants of Lactococcus lactis Metabolite analyses showed that glutamine is imported by GlnPQ and then rapidly converted to glutamate, and GlnPQ mutations or c-di-AMP negatively affects the pools of the most abundant free amino acids (glutamate and aspartate) during growth. In a high-c-di-AMP mutant, GlnPQ activity could be increased by raising the internal K+ level through the overexpression of a c-di-AMP-insensitive KupB variant. These results demonstrate that c-di-AMP reduces GlnPQ activity and, therefore, the level of the major free anions in L. lactis through its inhibition of K+ import. Excessive ion accumulation in cdaA mutants results in greater spontaneous cell lysis under hypotonic conditions, while CEF-resistant suppressors exhibit reduced cell lysis and lower osmoresistance. This work demonstrates that the overaccumulation of major counter-ion osmolyte pools in c-di-AMP-defective mutants of L. lactis causes cefuroxime sensitivity.IMPORTANCE The bacterial second messenger cyclic di-AMP (c-di-AMP) is a global regulator of potassium homeostasis and compatible solute uptake in many Gram-positive bacteria, making it essential for osmoregulation. The role that c-di-AMP plays in ß-lactam resistance, however, is unclear despite being first identified a decade ago. Here, we demonstrate that the overaccumulation of potassium or free amino acids leads to cefuroxime sensitivity in Lactococcus lactis mutants partially defective in c-di-AMP synthesis. It was shown that c-di-AMP negatively affects the levels of the most abundant free amino acids (glutamate and aspartate) in L. lactis Regulation of these major free anions was found to occur via the glutamine transporter GlnPQ, whose activity increased in response to intracellular potassium levels, which are under c-di-AMP control. Evidence is also presented showing that they are major osmolytes that enhance osmoresistance and cell lysis. The regulatory reach of c-di-AMP can be extended to include the main free anions in bacteria.

Comparison between cage and free-range egg production on microbial composition, diversity and the presence of Salmonella enterica.

The microbial composition of the food production environment plays an important role in food safety and quality. This study employed both 16 S rRNA gene sequencing technology and culture-based techniques to investigate the bacterial microbiota of an egg production facility comprising of both free-range and conventional cage housing systems. The study also aimed to detect the presence of Salmonella enterica and determine whether its presence was positively or negatively associated with other taxa. Our findings revealed that microbiota profiles of free-range and cage houses differ considerably in relation to the relative abundance and diversity with a number of taxa unique to each system and to individual sampling sites within sheds. Core to each housing system were known inhabitants of the poultry gastrointestinal tracts, Romboutsia and Turicibacter, as well as common spoilage bacteria. Generally, free-range samples contained fewer taxa and were dominated by Staphylococcus equorum, differentiating them from the cage samples. Salmonella enterica was significantly associated with the presence of a taxa belonging to the Carnobacteriaceae family. The results of this study demonstrate that the diversity and composition of the microbiota is highly variable across egg layer housing systems, which could have implications for productivity, food safety and spoilage.

Isolation and Evaluation of Anti-Listeria Lactococcus lactis from Vegetal Sources.

This chapter describes methods used to isolate, identify, and partially characterize lactic acid bacteria (LAB) which exhibit inhibitory activity against Listeria monocytogenes from foods. Vegetal (plant based) sources are rich in naturally occurring LAB and therefore provide an easily accessible source of strains with potential antimicrobial activity for use in food-processing applications. From our previous work, the majority of LAB with inhibitory activity against L. monocytogenes were identified as generally recognized as safe (GRAS) Lactococcus lactis. Although these bacteria are most commonly known for their role in industrial dairy fermentations, they are believed to have originally derived from natural plant-based habitats. These isolates with anti-Listeria activity were all found to carry the genes involved in the production of nisin, which is an approved food-grade preservative (E234). These isolates may find various applications for in situ production of nisin allowing control of L. monocytogenes in various fermented and non-fermented foods and other environments.

Updates on understanding of probiotic lactic acid bacteria responses to environmental stresses and highlights on proteomic analyses.

Probiotics are defined as live microorganisms that improve the health of the host when administered in adequate quantities. Nonetheless, probiotics encounter extreme environmental conditions during food processing or along the gastrointestinal tract. This review discusses different environmental stresses that affect probiotics during food preparation, storage, and along the alimentary canal, including high temperature, low temperature, low and alkaline pH, oxidative stress, high hydrostatic pressure, osmotic pressure, and starvation. The understanding of how probiotics deal with environmental stress and thrive provides useful information to guide the selection of the strains with enhanced performance in specific situations, in food processing or during gastrointestinal transit. In most cases, multiple biological functions are affected upon exposure of the cell to environmental stress. Sensing of sublethal environmental stress can allow for adaptation processes to occur, which can include alterations in the expression of specific proteins.

Engineering Escherichia coli for propionic acid production through the Wood-Werkman cycle.

Native to propionibacteria, the Wood-Werkman cycle enables propionate production via succinate decarboxylation. Current limitations in engineering propionibacteria strains have redirected attention toward the heterologous production in model organisms. Here, we report the functional expression of the Wood-Werkman cycle in Escherichia coli to enable propionate and 1-propanol production. The initial proof-of-concept attempt showed that the cycle can be used for production. However, production levels were low (0.17 mM). In silico optimization of the expression system by operon rearrangement and ribosomal-binding site tuning improved performance by fivefold. Adaptive laboratory evolution further improved performance redirecting almost 30% of total carbon through the Wood-Werkman cycle, achieving propionate and propanol titers of 9 and 5 mM, respectively. Rational engineering to reduce the generation of byproducts showed that lactate (∆ldhA) and formate (∆pflB) knockout strains exhibit an improved propionate and 1-propanol production, while the ethanol (∆adhE) knockout strain only showed improved propionate production.

Isolation, identification, and potential probiotic characterization of isolated lactic acid bacteria and in vitro investigation of the cytotoxicity, antioxidant, and antidiabetic activities in fermented sausage.

BACKGROUND: Probiotic bacteria can provide health benefits when delivered in functional foods. This study involved isolation of lactic acid bacteria (LAB) from traditionally dried and salted anchovy fish and characterization of their survival in simulated gastrointestinal digestion. Promising strains were used to prepare fermented fish sausages which were then evaluated for cytotoxicity activity against two cancer cell-lines, antidiabetic activity as determined by α-amylase and α-glucosidase inhibition, and antioxidant and proteolytic activities in vitro, as compared to non-fermented control sausages. RESULTS: Out of 85 LAB obtained, 13 isolates with high tolerance to simulated gastrointestinal digestion were obtained, which were identified as Enterococcus spp. Four E. faecium strains, one E. faecalis, and one E. durans were used separately to make fermented fish sausages. The α-amylase and α-glucosidase inhibition from fish sausages fermented by Enterococcus spp. ranged from 29.2 to 68.7% and 23.9 to 41.4%, respectively, during 21 days of storage. The cytotoxicity activities against Caco2 and MCF-7 cells of fish sausages fermented with Enterococcus spp. ranged from 18.0 to 24% and 13.9 to 27.9%, respectively. Cytotoxicity activities correlated positively with proteolysis and antioxidant activities, α-amylase and α-glucosidase inhibition activities, but negatively with the pH in fermented fish sausages. Strains also exhibited antimicrobial activity against foodborne pathogens and presented no significant concerns with regards to antibiotic resistance or virulence gene content. CONCLUSIONS: Fish sausages fermented by potential probiotic isolates of Enterococcus spp. from dried fish had valuable health-promoting benefits compared with non-fermented control sausages.

Onward and [K+]Upward: a New Potassium Importer under the Spell of Cyclic di-AMP.

Cyclic di-AMP (c-di-AMP) is a second messenger which plays a major role in osmotic homeostasis in bacteria. In work by Quintana et al. (I. M. Quintana, J. Gibhardt, A. Turdiev, E. Hammer, et al., J Bacteriol 201:e00028-19, 2019, https://doi.org/10.1128/jb.00028-19), two Kup homologs from Lactococcus lactis were identified as high-affinity K+ importers whose activities are inhibited by direct binding of c-di-AMP. The results broaden the scope of K+ level regulation by c-di-AMP, with Kup homologs found in a number of pathogenic, commensal, and industrial bacteria.

Enhanced uptake of potassium or glycine betaine or export of cyclic-di-AMP restores osmoresistance in a high cyclic-di-AMP Lactococcus lactis mutant.

The broadly conserved bacterial signalling molecule cyclic-di-adenosine monophosphate (c-di-AMP) controls osmoresistance via its regulation of potassium (K+) and compatible solute uptake. High levels of c-di-AMP resulting from inactivation of c-di-AMP phosphodiesterase activity leads to poor growth of bacteria under high osmotic conditions. To better understand how bacteria can adjust in response to excessive c-di-AMP levels and to identify signals that feed into the c-di-AMP network, we characterised genes identified in a screen for osmoresistant suppressor mutants of the high c-di-AMP Lactococcus ΔgdpP strain. Mutations were identified which increased the uptake of osmoprotectants, including gain-of-function mutations in a Kup family K+ importer (KupB) and inactivation of the glycine betaine transporter transcriptional repressor BusR. The KupB mutations increased the intracellular K+ level while BusR inactivation increased the glycine betaine level. In addition, BusR was found to directly bind c-di-AMP and repress expression of the glycine betaine transporter in response to elevated c-di-AMP. Interestingly, overactive KupB activity or loss of BusR triggered c-di-AMP accumulation, suggesting turgor pressure changes act as a signal for this second messenger. In another group of suppressors, overexpression of an operon encoding an EmrB family multidrug resistance protein allowed cells to lower their intracellular level of c-di-AMP through active export. Lastly evidence is provided that c-di-AMP levels in several bacteria are rapidly responsive to environmental osmolarity changes. Taken together, this work provides evidence for a model in which high c-di-AMP containing cells are dehydrated due to lower K+ and compatible solute levels and that this osmoregulation system is able to sense and respond to cellular water stress.

The genetic basis underlying variation in production of the flavour compound diacetyl by Lactobacillus rhamnosus strains in milk.

Diacetyl and the closely related compound acetoin impart desirable buttery flavour and odour to many foods including cheese and are generated through the metabolism of citrate by lactic acid bacteria (LAB). To increase the levels of these compounds, adjunct cultures capable of producing them can be added to cheese fermentations. In this study, we compared the diacetyl and acetoin producing abilities of 13 Lactobacillus rhamnosus strains from cheese sources. Diacetyl and acetoin production was found to be a common feature of Lb. rhamnosus grown in milk, with 12 strains producing these compounds. Whole genome sequencing of four strains revealed that genes encoding the citrate metabolising pathway present in other LAB are conserved in Lb. rhamnosus. One strain was, however, totally defective in diacetyl and acetoin production. This was likely due to an inability to produce the diacetyl/acetoin precursor compound acetolactate resulting from a frameshift mutation in the acetolactate synthase (als) gene. Complementation of this defective strain with a complete als gene from a diacetyl producing strain restored production of diacetyl and acetoin to levels equivalent to naturally high producing strains. Introduction of the same als-containing plasmid into the probiotic Lb. rhamnosus strain GG also increased diacetyl and acetoin levels. In model cheesemaking experiments, the als-complemented strain produced very high levels of diacetyl and acetoin over 35days of ripening. These findings identify the genetic basis for natural variation in production of a key cheese flavour compound in Lb. rhamnosus strains.