Plasmid-mediated horizontal gene mobilisation: Insights from two lactococcal conjugative plasmids.

The distinct conjugation machineries encoded by plasmids pNP40 and pUC11B represent the most prevalent plasmid transfer systems among lactococcal strains. In the current study, we identified genetic determinants that underpin pNP40- and pUC11B-mediated, high-frequency mobilisation of other, non-conjugative plasmids. The mobilisation frequencies of the smaller, non-conjugative plasmids and the minimal sequences required for their mobilisation were determined, owing to the determination of the oriT sequences of both pNP40 and pUC11B, which allowed the identification of similar sequences in some of the non-conjugative plasmids that were shown to promote their mobilisation. Furthermore, the auxiliary gene mobC, two distinct functional homologues of which are present in several plasmids harboured by the pNP40- and pUC11B-carrying host strains, was observed to confer a high-frequency mobilisation phenotype. These findings provide mechanistic insights into how lactococcal conjugative plasmids achieve conjugation and promote mobilisation of non-conjugative plasmids. Ultimately, these insights would be harnessed to optimise conjugation and mobilisation strategies for the rapid and predictable development of robust and technologically improved strains.

The substrate-binding domains of the osmoregulatory ABC importer OpuA transiently interact.

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.

Acute Cholecystitis Due to Lactococcus lactis and Single-Center Experience With Infections Due to Lactococcus spp.

Background: Lactococcus species are used to ferment milk to yogurt, cheese, and other products. The gram-positive coccus causes diseases in amphibia and fish and is a rare human pathogen. Patients and Methods: A 51-year-old male underwent laparoscopic cholecystectomy for acute and chronic calculous cholecystitis. Lactococcus lactis was isolated from pus from his gallbladder empyema. Results: Our institutional database was searched for other cases of Lactococcus spp. infections and four patients (2 males, 2 females; aged 51, 64, 78, and 80 years) were identified during a four-year period. The three other patients had positive blood cultures associated with pneumonia, toxic megacolon, and severe gastroenteritis. All isolates were monocultures with Lactococcus lactis (2), Lactococcus garvieae (1) and Lactococcus raffinolactis (1). Two patients died related to their sepsis. We report the second case of cholecystitis involving Lactococcus. Conclusions: Lactococcus is a very rare pathogen mainly causing blood stream infections but needs to be considered to cause serious surgical infections in humans.

Elucidation of the Catalytic Sequence of Dihydroorotate Dehydrogenase B from Lactoccocus lactis: Evidence for Accumulation of a Flavin Bisemiquinone State in Catalysis.

The physiological role of dihydroorotate dehydrogenase (DHOD) enzymes is to catalyze the oxidation of dihydroorotate to orotate in pyrimidine biosynthesis. DHOD enzymes are structurally diverse existing as both soluble and membrane-associated forms. The Family 1 enzymes are soluble and act either as conventional single subunit flavin-dependent dehydrogenases known as Class 1A (DHODA) or as unusual heterodimeric enzymes known as Class 1B (DHODB). DHODBs possess two active sites separated by ∼20 Å, each with a noncovalently bound flavin cofactor. NAD is thought to interact at the FAD containing site, and the pyrimidine substrate is known to bind at the FMN containing site. At the approximate center of the protein is a single Fe2S2 center that is assumed to act as a conduit, facilitating one-electron transfers between the flavins. We present anaerobic transient state analysis of a DHODB enzyme from Lactoccocus lactis. The data presented primarily report the exothermic reaction that reduces orotate to dihydroorotate. The reductive half reaction reveals rapid two-electron reduction that is followed by the accumulation of a four-electron reduced state when NADH is added in excess, suggesting that the initial two electrons acquired reside on the FMN cofactor. Concomitant with the first reduction is the accumulation of a long-wavelength absorption feature consistent with the blue form of a flavin semiquinone. Spectral deconvolution and fitting to a model that includes reversibility for the second electron transfer reveals equilibrium accumulation of a flavin bisemiquinone state that has features of both red and blue semiquinones. Single turnover reactions with limiting NADH and excess orotate reveal that the flavin bisemiquinone accumulates with reduction of the enzyme by NADH and decays with reduction of the pyrimidine substrate, establishing the bisemiquinone as a fractional state of the two-electron reduced intermediate observed.

Heterologous expression of P9 from Akkermansia muciniphila increases the GLP-1 secretion of intestinal L cells.

Glucagon-like peptide-1(GLP-1) is an incretin hormone secreted primarily from the intestinal L-cells in response to meals. GLP-1 is a key regulator of energy metabolism and food intake. It has been proven that P9 protein from A. muciniphila could increase GLP-1 release and improve glucose homeostasis in HFD-induced mice. To obtain an engineered Lactococcus lactis which produced P9 protein, mature polypeptide chain of P9 was codon-optimized, fused with N-terminal signal peptide Usp45, and expressed in L. lactis NZ9000. Heterologous secretion of P9 by recombinant L. lactis NZP9 were successfully detected by SDS-PAGE and western blotting. Notably, the supernatant of L. lactis NZP9 stimulated GLP-1 production of NCI-H716 cells. The relative expression level of GLP-1 biosynthesis gene GCG and PCSK1 were upregulated by 1.63 and 1.53 folds, respectively. To our knowledge, this is the first report on the secretory expression of carboxyl-terminal processing protease P9 from A. muciniphila in L. lactis. Our results suggest that genetically engineered L. lactis which expressed P9 may have therapeutic potential for the treatment of diabetes, obesity and other metabolic disorders.

A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration.

Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments. Riboswitches - like most structured RNAs - are believed to fold co-transcriptionally, subject to both ligand binding and transcription events; yet how these processes are orchestrated for robust regulation is poorly understood. Through a combination of single-molecule and bulk approaches, we discover how a single Mn2+ ion and the transcribing RNA polymerase (RNAP), paused immediately downstream by a DNA template sequence, are coordinated by the bridging switch helix P1.1 in the representative Lactococcus lactis riboswitch. This coordination achieves a heretofore-overlooked semi-docked global conformation of the nascent RNA, P1.1 base pair stabilization, transcription factor NusA ejection, and RNAP pause extension, thereby enforcing transcription readthrough. Our work demonstrates how a central, adaptable RNA helix functions analogous to a molecular fulcrum of a first-class lever system to integrate disparate signals for finely balanced gene expression control.

Microbes vary strategically in their metalation of mononuclear enzymes.

Studies have determined that nonredox enzymes that are cofactored with Fe(II) are the most oxidant-sensitive targets inside Escherichia coli. These enzymes use Fe(II) cofactors to bind and activate substrates. Because of their solvent exposure, the metal can be accessed and oxidized by reactive oxygen species, thereby inactivating the enzyme. Because these enzymes participate in key physiological processes, the consequences of stress can be severe. Accordingly, when E. coli senses elevated levels of H2O2, it induces both a miniferritin and a manganese importer, enabling the replacement of the iron atom in these enzymes with manganese. Manganese does not react with H2O2 and thereby preserves enzyme activity. In this study, we examined several diverse microbes to identify the metal that they customarily integrate into ribulose-5-phosphate 3-epimerase, a representative of this enzyme family. The anaerobe Bacteroides thetaiotaomicron, like E. coli, uses iron. In contrast, Bacillus subtilis and Lactococcus lactis use manganese, and Saccharomyces cerevisiae uses zinc. The latter organisms are therefore well suited to the oxidizing environments in which they dwell. Similar results were obtained with peptide deformylase, another essential enzyme of the mononuclear class. Strikingly, heterologous expression experiments show that it is the metal pool within the organism, rather than features of the protein itself, that determine which metal is incorporated. Further, regardless of the source organism, each enzyme exhibits highest turnover with iron and lowest turnover with zinc. We infer that the intrinsic catalytic properties of the metal cannot easily be retuned by evolution of the polypeptide.

Recombinant lactococcal-based oral vaccine for protection against Streptococcus agalactiae infections in tilapia (Oreochromis niloticus).

Streptococcosis outbreaks caused by Streptococcus agalactiae infection in tilapia aquaculture have been consistently reported and associated with high mortality and morbidity leading to significant economic losses. Existing vaccine candidates against Streptococcus spp. are designed for intraperitoneal injections that are not practical and labor-intensive which have prompted farmers to protect aquatic animals with antibiotics, thus encouraging the emergence of multidrug resistant bacteria. In this study, a live recombinant L. lactis vaccine expressing a 1403 bp surface immunogenic protein (SIP) and a 1100 bp truncated SIP (tSIP) gene was developed and evaluated against S. agalactiae infection in tilapia. Both SIP and tSIP sequences were cloned and transformed into L. lactis. The recombinant L.lactis vaccine was orally administered to juvenile tilapia for a month. Detection of SIP-specific serum IgM in vaccinated groups compared to control groups indicated that recombinant proteins expressed from L. lactis could elicit immunogenic reactions in tilapia. Fish immunized with the tSIP vaccine also showed the highest level of protection compared to other test groups, and the mortality rate was significantly reduced compared to both control groups. The relative percentage of survival (RPS) against S. agalactiae for both SIP and tSIP-vaccinated groups was 50 % and 89 %, respectively, at 14 days post-challenge. Significant up-regulation of IgM, IL-1ß, IL-10, TNF-α and IFN-γ were observed at day 34 between the vaccinated and control groups. These results indicated that the recombinant lactococcal tSIP vaccine can elicit both cell-mediated and humoral responses and is recommended as a potential oral vaccine against S. agalactiae infection. Future work will include further in vivo challenge assessments of this vaccine candidate fused with adjuvants to boost immunogenicity levels in tilapia.

The contribution of abortive infection to preventing populations of Lactococcus lactis from succumbing to infections with bacteriophage.

In the dairy industry bacteriophage (phage) contamination significantly impairs the production and quality of products like yogurt and cheese. To combat this issue, the strains of bacteria used as starter cultures possess mechanisms that make them resistant to phage infection, such as envelope resistance, or processes that render them immune to phage infection, such as restriction-modification and CRISPR-Cas. Lactococcus lactis, used to manufacture cheese and other dairy products, can also block the reproduction of infecting phages by abortive infection (Abi), a process in which phage-infected cells die before the phage replicate. We employ mathematical-computer simulation models and experiments with two Lactococcus lactis strains and two lytic phages to investigate the conditions under which Abi can limit the proliferation of phages in L. lactis populations and prevent the extinction of their populations by these viruses. According to our model, if Abi is almost perfect and there are no other populations of bacteria capable of supporting the replication of the L. lactis phages, Abi can protect bacterial populations from succumbing to infections with these viruses. This prediction is supported by the results of our experiment, which indicate that Abi can help protect L. lactis populations from extinction by lytic phage infections. However, our results also predict abortive infection is only one element of L. lactis defenses against phage infection. Mutant phages that can circumvent the Abi systems of these bacteria emerge. The survival of L. lactis populations then depends on the evolution of envelope mutants that are resistant to the evolved host-range phage.

Detection and classification of the integrative conjugative elements of Lactococcus lactis.

Lactococcus lactis is widely applied by the dairy industry for the fermentation of milk into products such as cheese. Adaptation of L. lactis to the dairy environment often depends on functions encoded by mobile genetic elements (MGEs) such as plasmids. Other L. lactis MGEs that contribute to industrially relevant traits like antimicrobial production and carbohydrate utilization capacities belong to the integrative conjugative elements (ICE). Here we investigate the prevalence of ICEs in L. lactis using an automated search engine that detects colocalized, ICE-associated core-functions (involved in conjugation or mobilization) in lactococcal genomes. This approach enabled the detection of 36 candidate-ICEs in 69 L. lactis genomes. By phylogenetic analysis of conserved protein functions encoded in all lactococcal ICEs, these 36 ICEs could be classified in three main ICE-families that encompass 7 distinguishable ICE-integrases and are characterized by apparent modular-exchangeability and plasticity. Finally, we demonstrate that phylogenetic analysis of the conjugation-associated VirB4 ATPase function differentiates ICE- and plasmid-derived conjugation systems, indicating that conjugal transfer of lactococcal ICEs and plasmids involves genetically distinct machineries. Our genomic analysis and sequence-based classification of lactococcal ICEs creates a comprehensive overview of the conserved functional repertoires encoded by this family of MGEs in L. lactis, which can facilitate the future exploitation of the functional traits they encode by ICE mobilization to appropriate starter culture strains.

New Genetic Determinants for qPCR Identification and the Enumeration of Selected Lactic Acid Bacteria in Raw-Milk Cheese.

Lactic acid bacteria (LAB) play an important role in the ripening of cheeses and contribute to the development of the desired profile of aroma and flavor compounds. Therefore, it is very important to monitor the dynamics of bacterial proliferation in order to obtain an accurate and reliable number of their cells at each stage of cheese ripening. This work aimed to identify and conduct a quantitative assessment of the selected species of autochthonous lactic acid bacteria from raw cow's milk cheese by the development of primers and probe pairs based on the uniqueness of the genetic determinants with which the target microorganisms can be identified. For that purpose, we applied real-time quantitative PCR (qPCR) protocols to quantify Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris cells in cheese directly after production and over three-month and six-month ripening periods. While L. lactis subsp. cremoris shows good acidification ability and the ability to produce antimicrobial compounds, L. delbrueckii subsp. bulgaricus has good proteolytic ability and produces exo-polysaccharides, and S. thermophilus takes part in the formation of the diacetyl flavor compound by metabolizing citrate to develop aroma, they all play an important role in the cheese ripening. The proposed qPCR protocols are very sensitive and reliable methods for a precise enumeration of L. delbrueckii subsp. bulgaricus, S. thermophilus, and L. lactis subsp. cremoris in cheese samples.

Bacterial proteome adaptation during fermentation in dairy environments.

The enzymatic repertoire of starter cultures belonging to the Lactococcus genus determines various important characteristics of fermented dairy products but might change in response to the substantial environmental changes in the manufacturing process. Assessing bacterial proteome adaptation in dairy and other food environments is challenging due to the high matrix-protein concentration and is even further complicated in particularly cheese by the high fat concentrations, the semi-solid state of that matrix, and the non-growing state of the bacteria. Here, we present bacterial harvesting and processing procedures that enable reproducible, high-resolution proteome determination in lactococcal cultures harvested from laboratory media, milk, and miniature Gouda cheese. Comparative proteome analysis of Lactococcus cremoris NCDO712 grown in laboratory medium and milk revealed proteome adaptations that predominantly reflect the differential (micro-)nutrient availability in these two environments. Additionally, the drastic environmental changes during cheese manufacturing only elicited subtle changes in the L. cremoris NCDO712 proteome, including modified expression levels of enzymes involved in flavour formation. The technical advances we describe offer novel opportunities to evaluate bacterial proteomes in relation to their performance in complex, protein- and/or fat-rich food matrices and highlight the potential of steering starter culture performance by preculture condition adjustments.

A high fiber diet or supplementation with Lactococcus lactis subspecies cremoris to pregnant mice confers protection against intestinal injury in adult offspring.

The diet during pregnancy, or antenatal diet, influences the offspring's intestinal health. We previously showed that antenatal butyrate supplementation reduces injury in adult murine offspring with dextran sulfate sodium (DSS)-induced colitis. Potential modulators of butyrate levels in the intestine include a high fiber diet or dietary supplementation with probiotics. To test this, we supplemented the diet of pregnant mice with high fiber, or with the probiotic bacteria Lactococcus lactis subspecies cremoris or Lactobacillus rhamnosus GG. We then induced chronic colitis with DSS in their adult offspring. We demonstrate that a high fiber antenatal diet, or supplementation with Lactococcus lactis subspecies cremoris during pregnancy diminished the injury from DSS-induced colitis in offspring. These data are evidence that antenatal dietary interventions impact offspring gut health and define the antenatal diet as a therapeutic modality to enhance offspring intestinal health.

Lactic acid bacteria modulate the CncC pathway to enhance resistance to ß-cypermethrin in the oriental fruit fly.

The gut microbiota of insects has been shown to regulate host detoxification enzymes. However, the potential regulatory mechanisms involved remain unknown. Here, we report that gut bacteria increase insecticide resistance by activating the cap "n" collar isoform-C (CncC) pathway through enzymatically generated reactive oxygen species (ROS) in Bactrocera dorsalis. We demonstrated that Enterococcus casseliflavus and Lactococcus lactis, two lactic acid-producing bacteria, increase the resistance of B. dorsalis to ß-cypermethrin by regulating cytochrome P450 (P450) enzymes and α-glutathione S-transferase (GST) activities. These gut symbionts also induced the expression of CncC and muscle aponeurosis fibromatosis. BdCncC knockdown led to a decrease in resistance caused by gut bacteria. Ingestion of the ROS scavenger vitamin C in resistant strain affected the expression of BdCncC/BdKeap1/BdMafK, resulting in reduced P450 and GST activity. Furthermore, feeding with E. casseliflavus or L. lactis showed that BdNOX5 increased ROS production, and BdNOX5 knockdown affected the expression of the BdCncC/BdMafK pathway and detoxification genes. Moreover, lactic acid feeding activated the ROS-associated regulation of P450 and GST activity. Collectively, our findings indicate that symbiotic gut bacteria modulate intestinal detoxification pathways by affecting physiological biochemistry, thus providing new insights into the involvement of insect gut microbes in the development of insecticide resistance.

Scrutinizing a Lactococcus lactis mutant with enhanced capacity for extracellular electron transfer reveals a unique role for a novel type-II NADH dehydrogenase.

Lactococcus lactis, a lactic acid bacterium used in food fermentations and commonly found in the human gut, is known to possess a fermentative metabolism. L. lactis, however, has been demonstrated to transfer metabolically generated electrons to external electron acceptors, a process termed extracellular electron transfer (EET). Here, we investigated an L. lactis mutant with an unusually high capacity for EET that was obtained in an adaptive laboratory evolution (ALE) experiment. First, we investigated how global gene expression had changed, and found that amino acid metabolism and nucleotide metabolism had been affected significantly. One of the most significantly upregulated genes encoded the NADH dehydrogenase NoxB. We found that this upregulation was due to a mutation in the promoter region of NoxB, which abolished carbon catabolite repression. A unique role of NoxB in EET could be attributed and it was directly verified, for the first time, that NoxB could support respiration in L. lactis. NoxB, was shown to be a novel type-II NADH dehydrogenase that is widely distributed among gut microorganisms. This work expands our understanding of EET in Gram-positive electroactive microorganisms and the special significance of a novel type-II NADH dehydrogenase in EET.IMPORTANCEElectroactive microorganisms with extracellular electron transfer (EET) ability play important roles in biotechnology and ecosystems. To date, there have been many investigations aiming at elucidating the mechanisms behind EET, and determining the relevance of EET for microorganisms in different niches. However, how EET can be enhanced and harnessed for biotechnological applications has been less explored. Here, we compare the transcriptomes of an EET-enhanced L. lactis mutant with its parent and elucidate the underlying reason for its superior performance. We find that one of the most significantly upregulated genes is the gene encoding the NADH dehydrogenase NoxB, and that upregulation is due to a mutation in the catabolite-responsive element that abolishes carbon catabolite repression. We demonstrate that NoxB has a special role in EET, and furthermore show that it supports respiration to oxygen, which has never been done previously. In addition, a search reveals that this novel NoxB-type NADH dehydrogenase is widely distributed among gut microorganisms.

Probing the stability and interdomain interactions in the ABC transporter OpuA using single-molecule optical tweezers.

Transmembrane transporter proteins are essential for maintaining cellular homeostasis and, as such, are key drug targets. Many transmembrane transporter proteins are known to undergo large structural rearrangements during their functional cycles. Despite the wealth of detailed structural and functional data available for these systems, our understanding of their dynamics and, consequently, how they function is generally limited. We introduce an innovative approach that enables us to directly measure the dynamics and stability of interdomain interactions of transmembrane proteins using optical tweezers. Focusing on the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis, we examine the mechanical properties and potential interactions of its substrate-binding domains. Our measurements are performed in lipid nanodiscs, providing a native-mimicking environment for the transmembrane protein. The technique provides high spatial and temporal resolution and allows us to study the functionally relevant motions and interdomain interactions of individual transmembrane transporter proteins in real time in a lipid bilayer.

Effect of a Lactococcus lactis culture supernatant on diarrhea and performance parameters of piglets in the post-weaning period and on expression of the faeG gene in vitro.

Objectives: To evaluate the effects of a cell-free supernatant from Lactococcus lactis (CFSM) on performance and diarrhearelated parameters and the presence of F4+ enterotoxigenic E. coli (ETEC) in piglets during post-weaning, and to evaluate the in vitro effect of the CFSM on faeG gene expression in an E. coli F4+. Animals and procedure: In 3 trials with 90 piglets per trial, pigs were assigned to receive a placebo or 1 of 2 CFSM treatments and observed for diarrhea and performance. Fecal swabs were taken to determine the presence of ETEC. Quantitative RT-PCR was used to assess faeG gene expression in E. coli 21259 after treatment with CFSM at 50 mg/mL. Results: The CFSM administered for 14 d at a dose of 24 mg/kg BW (2X) reduced diarrhea-related parameters compared to the placebo. Quantitative RT-PCR showed that, in E. coli 21259 treated with CFSM at 50 mg/mL, expression of the faeG gene was significantly repressed (P < 0.0001) relative to that in the untreated control. Conclusion: The evaluated CFSM reduced the frequency and prevalence of diarrhea in a field situation. The in vitro treatment had an inhibitory effect on the expression of the faeG gene in F4+ E. coli 21259.

Effet d'un surnageant de culture de Lactococcus lactis sur la diarrhée et les paramètres de performance des porcelets en période post-sevrage et sur l'expression du gène faeG in vitro. Objectifs: Évaluer les effets d'un surnageant acellulaire de Lactococcus lactis (CFSM) sur les paramètres de performance et de diarrhée et la présence d'E. coli entérotoxinogène F4+ (ETEC) chez les porcelets en post-sevrage, et évaluer l'effet in vitro du CFSM sur l'expression du gène faeG dans un E. coli F4+. Animaux et procédure: Dans 3 essais portant sur 90 porcelets par essai, les porcs ont reçu un placebo ou 1 des 2 traitements CFSM et ont été observés pour détecter la diarrhée et leurs performances. Des prélèvements fécaux ont été effectués pour déterminer la présence d'ETEC. La RT-PCR quantitative a été utilisée pour évaluer l'expression du gène faeG dans E. coli 21259 après traitement avec CFSM à 50 mg/mL. Résultats: Le CFSM administré pendant 14 jours à une dose de 24 mg/kg de poids corporel (2X) a réduit les paramètres liés à la diarrhée par rapport au placebo. La RT-PCR quantitative a montré que, chez E. coli 21259 traité avec CFSM à 50 mg/mL, l'expression du gène faeG était significativement réprimée (P < 0,0001) par rapport à celle du témoin non traité. Conclusion: Le CFSM évalué a réduit la fréquence et la prévalence de la diarrhée sur le terrain. Le traitement in vitro a eu un effet inhibiteur sur l'expression du gène faeG chez F4+ E. coli 21259.(Traduit par Dr Serge Messier).

Automated workflow for characterization of bacteriocin production in natural producers Lactococcus lactis and Latilactobacillus sakei.

BACKGROUND: Lactic acid bacteria are commonly used as protective starter cultures in food products. Among their beneficial effects is the production of ribosomally synthesized peptides termed bacteriocins that kill or inhibit food-spoiling bacteria and pathogens, e.g., members of the Listeria species. As new bacteriocins and producer strains are being discovered rapidly, modern automated methods for strain evaluation and bioprocess development are required to accelerate screening and development processes. RESULTS: In this study, we developed an automated workflow for screening and bioprocess optimization for bacteriocin producing lactic acid bacteria, consisting of microcultivation, sample processing and automated antimicrobial activity assay. We implemented sample processing workflows to minimize bacteriocin adsorption to producer cells via addition of Tween 80 and divalent cations to the cultivation media as well as acidification of culture broth prior to cell separation. Moreover, we demonstrated the applicability of the automated workflow to analyze influence of media components such as MES buffer or yeast extract for bacteriocin producers Lactococcus lactis B1629 and Latilactobacillus sakei A1608. CONCLUSIONS: Our automated workflow provides advanced possibilities to accelerate screening and bioprocess optimization for natural bacteriocin producers. Based on its modular concept, adaptations for other strains, bacteriocin products and applications are easily carried out and a unique tool to support bacteriocin research and bioprocess development is provided.

The strain-dependent cytostatic activity of Lactococcus lactis on CRC cell lines is mediated through the release of arginine deiminase.

BACKGROUND: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, posing a serious public health challenge that necessitates the development of new therapeutics, therapies, and prevention methods. Among the various therapeutic approaches, interventions involving lactic acid bacteria (LAB) as probiotics and postbiotics have emerged as promising candidates for treating and preventing CRC. While human-isolated LAB strains are considered highly favorable, those sourced from environmental reservoirs such as dairy and fermented foods are also being recognized as potential sources for future therapeutics. RESULTS: In this study, we present a novel and therapeutically promising strain, Lactococcus lactis ssp. lactis Lc4, isolated from dairy sources. Lc4 demonstrated the ability to release the cytostatic agent - arginine deiminase (ADI) - into the post-cultivation supernatant when cultured under conditions mimicking the human gut environment. Released arginine deiminase was able to significantly reduce the growth of HT-29 and HCT116 cells due to the depletion of arginine, which led to decreased levels of c-Myc, reduced phosphorylation of p70-S6 kinase, and cell cycle arrest. The ADI release and cytostatic properties were strain-dependent, as was evident from comparison to other L. lactis ssp. lactis strains. CONCLUSION: For the first time, we unveil the anti-proliferative properties of the L. lactis cell-free supernatant (CFS), which are independent of bacteriocins or other small molecules. We demonstrate that ADI, derived from a dairy-Generally Recognized As Safe (GRAS) strain of L. lactis, exhibits anti-proliferative activity on cell lines with different levels of argininosuccinate synthetase 1 (ASS1) expression. A unique feature of the Lc4 strain is also its capability to release ADI into the extracellular space. Taken together, we showcase L. lactis ADI and the Lc4 strain as promising, potential therapeutic agents with broad applicability.