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Lanthipeptides are ribosomally synthesized and post-translationally modified peptides characterized by the presence of lanthionine rings that provide stability and functionality. Genome mining techniques have shown their huge diversity and potential for the discovery of novel active molecules. However, in many cases, they are not easily produced under laboratory conditions. The heterologous expression of these molecules using well-characterized lanthipeptide biosynthetic enzymes is rising as an alternative system for the design and production of new lanthipeptides with biotechnological or clinical properties. Nevertheless, the substrate-enzyme specificity limits the complete modification of the desired peptides and hence, their full stability and/or biological activity. New low substrate-selective biosynthetic enzymes are therefore necessary for the heterologous production of new-to-nature peptides. Here, we have identified, cloned, and heterologously expressed in Lactococcus lactis the most promiscuous lanthipeptide synthetase described to date, i.e., SyncM from the marine cyanobacteria Synechococcus MITS9509. We have characterized the functionality of SyncM by the successful expression of 15 out of 18 different SyncA substrates, subsequently determining the dehydration and cyclization processes in six representatives of them. This characterization highlights the very relaxed substrate specificity of SyncM toward its precursors and the ability to catalyze the formation of exceptionally large rings in a variety of topologies. Our results suggest that SyncM could be an attractive enzyme to design and produce a wide variety of new-to-nature lanthipeptides with a broad range of ring topologies.

Whole-genome transcriptional analyses performed on microorganisms are traditionally based on a small number of samples. To map transient expression variations, and thoroughly characterize gene expression throughout the growth curve of the widely used model organism Lactococcus lactis MG1363, gene expression data were collected with unprecedented time resolution. The resulting gene expression patterns were globally analyzed in several different ways to demonstrate the richness of the data and the ease with which novel phenomena can be discovered. When the culture moves from one growth phase to another, gene expression patterns change to such an extent that we suggest that those patterns can be used to unequivocally distinguish growth phases from each other. Also, within the classically defined growth phases, subgrowth phases were distinguishable with a distinct expression signature. Apart from the global expression pattern shifts seen throughout the growth curve, several cases of short-lived transient gene expression patterns were clearly observed. These could help explain the gene expression variations frequently observed in biological replicates. A method was devised to estimate a measure of unnormalized/absolute gene expression levels and used to determine how global transcription patterns are influenced by nutrient starvation or acidification of the medium. Notably, we inferred that L. lactis MG1363 produces proteins with on average lower pIs and lower molecular weights as the medium acidifies and nutrients get scarcer. IMPORTANCE This data set is a rich resource for microbiologists interested in common mechanisms of gene expression, regulation and in particular the physiology of L. lactis. Thus, similar to the common use of genome sequence data by the scientific community, the data set constitutes an extensive data repository for mining and an opportunity for bioinformaticians to develop novel tools for in-depth analysis.

Lactococcus lactis is a Gram-positive bacterium that is widely used as a cell factory for the expression of heterologous proteins that are relevant in the pharmaceutical and nutraceutical fields. The signal peptide of the major secreted protein of L. lactis, Usp45, has been employed extensively in engineering strategies to secrete proteins of interest. However, the biological function of Usp45 has remained obscure despite more than 25 years of research. Studies on Usp45 homologs in other Gram-positive bacteria suggest that Usp45 may play a role in cell wall turnover processes. Here, we show the effect of inactivation and overexpression of the usp45 gene on L. lactis growth, phenotype, and cell division. Our results are in agreement with those obtained in streptococci and demonstrate that the L. lactis Usp45 protein is essential for proper cell division. We also show that the usp45 promoter is highly activated by galactose. Overall, our results indicate that Usp45 mediates cell separation, probably by acting as a peptidoglycan hydrolase. IMPORTANCE The cell wall, composed mainly of peptidoglycan, is key to maintaining the cell shape and protecting the cell from bursting. Peptidoglycan degradation by peptidoglycan hydrolysis and autolysins occurs during growth and cell division. Since peptidoglycan hydrolases are important for virulence, envelope integrity, and regulation of cell division, it is valuable to investigate their function and regulation. Notably, PcsB-like proteins such as Usp45 have been proposed as new targets for antimicrobial drugs and could also be target for the development of food-grade suicide systems. In addition, although various other expression and secretion systems have been developed for use in Lactococcus lactis, the most-used signal peptide for protein secretion in this bacterium is that of the Usp45 protein. Thus, elucidating the biological function of Usp45 and determining the factors affecting its expression would contribute to optimize several applications.

Efficient bacterial cell factories are important for the screening and characterization of potent antimicrobial peptides such as lantibiotics. Although lantibiotic production systems have been established in Lactococcus lactis and Escherichia coli, the industrial workhorse Bacillus subtilis has been left relatively unexplored as a lantibiotic production host. Therefore, we tested different B. subtilis strains for their ability to produce lantibiotic peptides by using the subtilin modification and transport enzymes derived from the natural subtilin producer B. subtilis ATCC 6633. Our study shows that although B. subtilis ATCC 6633 and 168 are able to produce various processed lantibiotic peptides, an evident advantage of using either the 8-fold protease-deficient strain WB800 or the genome-minimized B. subtilis 168 strain PG10 is the lack of extracellular serine protease activity. Consequently, leader processing of lantibiotic precursor peptides is circumvented and thus potential toxicity toward the production host is prevented. Furthermore, PG10 provides a clean secondary metabolic background and therefore appears to be the most promising B. subtilis lantibiotic production host. We demonstrate the production of various lantibiotic precursor peptides by PG10 and show different options for their in vitro activation. Our study thus provides a convenient B. subtilis-based lantibiotic production system, which facilitates the search for novel antimicrobial peptides.

Microbial lanthipeptides are formed by a two-step enzymatic introduction of (methyl)lanthionine rings. A dehydratase catalyzes the dehydration of serine and threonine residues, yielding dehydroalanine and dehydrobutyrine, respectively. Cyclase-catalyzed coupling of the formed dehydroresidues to cysteines forms (methyl)lanthionine rings in a peptide. Lanthipeptide biosynthetic systems allow discovery of target-specific, lanthionine-stabilized therapeutic peptides. However, the substrate specificity of existing modification enzymes impose limitations on installing lanthionines in non-natural substrates. The goal of the present study was to obtain a lanthipeptide dehydratase with the capacity to dehydrate substrates that are unsuitable for the nisin dehydratase NisB. We report high-throughput screening for tailored specificity of intracellular, genetically encoded NisB dehydratases. The principle is based on the screening of bacterially displayed lanthionine-constrained streptavidin ligands, which have a much higher affinity for streptavidin than linear ligands. The designed NisC-cyclizable high-affinity ligands can be formed via mutant NisB-catalyzed dehydration but less effectively via wild-type NisB activity. In Lactococcus lactis, a cell surface display precursor was designed comprising DSHPQFC. The Asp residue preceding the serine in this sequence disfavors its dehydration by wild-type NisB. The cell surface display vector was coexpressed with a mutant NisB library and NisTC. Subsequently, mutant NisB-containing bacteria that display cyclized strep ligands on the cell surface were selected via panning rounds with streptavidin-coupled magnetic beads. In this way, a NisB variant with a tailored capacity of dehydration was obtained, which was further evaluated with respect to its capacity to dehydrate nisin mutants. These results demonstrate a powerful method for selecting lanthipeptide modification enzymes with adapted substrate specificity.

Lanthipeptides belong to a family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) containing (methyl)lanthionine residues. Commonly, class I lanthipeptides are synthesized by a gene cluster encoding a precursor peptide (LanA), a biosynthetic machinery (LanBTC), a protease (LanP), a two-component regulatory system (LanRK), and an immunity system (LanI and LanFEG). Although nisin and subtilin are highly similar class I lanthipeptides, the cross-regulation by LanRK and the cross-immunity by LanI and LanFEG between the nisin and subtilin systems have been proven very low. Here, the possibility of the cross-functionality by LanBTC to modify and transport nisin precursor (NisA) and subtilin precursor (SpaS) was evaluated in Bacillus subtilis and Lactococcus lactis. Interestingly, we found that a promiscuous NisBC-SpaT complex is able to synthesize and export nisin precursor, as efficiently as the native nisin biosynthetic machinery NisBTC, in L. lactis, but not in B. subtilis. The assembly of the NisBC-SpaT complex at a single microdomain, close to the old cell pole, was observed by fluorescence microscopy in L. lactis. In contrast, such a complex was not formed in B. subtilis. Furthermore, the isolation of the NisBC-SpaT complex and its subcomplexes from the cytoplasmic membrane of L. lactis by pull-down assays was successfully conducted. Our work demonstrates that the association of LanBC with LanT is critical for the efficient biosynthesis and secretion of the lanthipeptide precursor with complete modifications, and suggests a cooperative mechanism between LanBC and LanT in the modification and transport processes. IMPORTANCE A multimeric synthetase LanBTC complex has been proposed for the in vivo production of class I lanthipeptides. However, it has been demonstrated that LanB, LanC, and LanT can perform their functionality in vivo and in vitro, independently of other Lan proteins. The role of protein-protein interactions, especially between the modification complex LanBC and the transport system LanT, in the biosynthesis process of lanthipeptides is still unclear. In this study, the importance of the presence of a well-installed LanBTC complex in the cell membrane for lanthipeptide biosynthesis and transport was reinforced. In L. lactis, the recruitment of SpaT from the peripheral cell membrane to the cell poles by the NisBC complex was observed, which may explain the mechanism by which secretion of premature peptide is prevented.

Nisin is synthesized by a putative membrane-associated lantibiotic synthetase complex composed of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT in Lactococcus lactis. Earlier work has demonstrated that NisB and NisT are linked via NisC to form such a complex. Here, we conducted for the first time the isolation of the intact NisBTC complex and NisT-associated subcomplexes from the cytoplasmic membrane by affinity purification. A specific interaction of NisT, not only with NisC but also with NisB, was detected. The cellular presence of NisB and/or NisC in complex with precursor nisin (NisA) was determined, which shows a highly dynamic and transient assembly of the NisABC complex via an alternating binding mechanism during nisin dehydration and cyclization. Mutational analyses, with cysteine-to-alanine mutations in NisA, suggest a tendency for NisA to lose affinity to NisC concomitant with an increasing number of completed lanthionine rings. Split NisBs were able to catalyze glutamylation and elimination reactions in an alternating way as efficiently as full-length NisB, with no significant influence on the following cyclization and transport. Notably, the harvest of the leader peptide in complex with the independent elimination domain of NisB points to a second leader peptide binding motif that is located in the C-terminal region of NisB, giving rise to a model where the leader peptide binds to different sites in NisB for glutamylation and elimination. Overall, these combined studies provide new insights into the cooperative biosynthesis mechanism of nisin and thereby lay a foundation for further structural and functional characterization of the NisBTC complex. IMPORTANCE Lantibiotics are ribosomally synthesized and posttranslationally modified peptide antibiotics. Although the membrane-associated lantibiotic biosynthesis machinery has long been proposed to exist, the isolation of such a complex has not been reported yet, which limits the elucidation of the processive mechanism of lantibiotic biosynthesis. In this work, we present direct evidence for the existence of the nisin biosynthetic complex at the cytoplasmic membrane of L. lactis, producing fully modified precursor nisin. By analyses of the interactions within the intact NisBTC complex and the modification machinery NisABC, we were able to elucidate the cooperative action for the modification and transport of nisin. Inspired by the natural and documented degradation process of NisB, artificial split-NisBs were made and thoroughly characterized, demonstrating a crucial clue to the evolution of the LanB family. Importantly, our study also suggests that the leader peptide of NisA binds to two different recognition motifs, i.e., one for glutamylation and one for elimination.

A membrane-associated lanthipeptide synthetase complex, consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT, has been described for nisin biosynthesis in the coccoid bacterium Lactococcus lactis. Here, we used advanced fluorescence microscopy to visualize the functional nisin biosynthesis machinery in rod-shaped cells and analyzed its spatial distribution and dynamics employing a platform we developed for heterologous production of nisin in Bacillus subtilis. We observed that NisT, as well as NisB and NisC, were all distributed in a punctate pattern along the cell periphery, opposed to the situation in coccoid cells. NisBTC proteins were found to be highly colocalized, being visualized at the same spots by dual fluorescence microscopy. In conjunction with the successful isolation of the biosynthetic complex NisBTC from the cell membrane, this corroborated that the visual bright foci were the sites for nisin maturation and transportation. A strategy of differential timing of expression was employed to demonstrate the in vivo dynamic assembly of NisBTC, revealing the recruitment by NisT of NisBC to the membrane. Additionally, by use of mutated proteins, the nucleotide binding domain (NBD) of NisT was found to function as a membrane anchor for NisB and/or NisC. We also show that the nisin biosynthesis sites are static and likely associated with proteins residing in lipid rafts. Based on these data, we propose a model for a three-phase production of modified precursor nisin in rod-shaped bacteria, presenting the assembly dynamics of NisBTC and emphasizing the crucial role of NisBC, next to NisT, in the process of precursor nisin translocation. IMPORTANCE Nisin is a model antimicrobial peptide for LanBC-modified lantibiotics that are modified and transported by a membrane synthetase complex. Although the subcellular localization and the assembly process of such a complex in L. lactis have been described in our recent work (J. Chen, A. J. van Heel, and O. P. Kuipers, mBio 11:e02825-20, 2020, https://doi.org/10.1128/mBio.02825-20), it proved difficult to gain a more detailed insight into the exact LanBTC assembly in the L. lactis system. Rod-shaped cells, especially B. subtilis, are better suited to study the assembly dynamics of these protein complexes. In this work, we present evidence for the existence of the lanthipeptide biosynthetic complex by visualizing and isolating the machinery in vivo. The dynamic behavior of the modification machinery and the transporter within the cells was characterized in depth, revealing the dependence of first LanB and LanC on each other and subsequent recruitment of them by LanT during the machinery assembly. Importantly, the elucidation of the dynamic assembly of the complex will facilitate future studies of lanthipeptide transport mechanisms and the structural characterization of the complete complex.

Metabolic engineering and synthetic biology approaches have prospered the field of biotechnology, in which the main focus has been on Escherichia coli and Saccharomyces cerevisiae as microbial workhorses. In more recent years, improving the Gram-positive bacteria Lactococcus lactis and Bacillus subtilis as production hosts has gained increasing attention. This review will demonstrate the different levels at which these bacteria can be engineered and their various application possibilities. For instance, engineered L. lactis strains show great promise for biomedical applications. Moreover, we provide an overview of recent synthetic biology tools that facilitate the use of these two microorganisms even more.

International audience; Plant material rich in anthocyanins has been historically used in traditional medicines, but only recently have the specific pharmacological properties of these compounds been the target of extensive studies. In addition to their potential to modulate the development of various diseases, coloured anthocyanins are valuable natural alternatives commonly used to replace synthetic colourants in food industry. Exploitation of microbial hosts as cell factories is an attractive alternative to extraction of anthocyanins and other flavonoids from plant sources or chemical synthesis. In this study, we present the lactic acid bacterium Lactococcus lactis as an ideal host for the production of high-value plant-derived bioactive anthocyanins using green tea as substrate. Besides the anticipated red-purple compounds cyanidin and delphinidin, orange and yellow pyranoanthocyanidins with unexpected methylation patterns were produced from green tea by engineered L. lactis strains. The pyranoanthocyanins are currently attracting significant interest as one of the most important classes of anthocyanin derivatives and are mainly formed during the aging of wine, contributing to both colour and sensory experience.

Noncanonical amino acids form a highly diverse pool of building blocks that can render unique physicochemical properties to peptides and proteins. Here, four methionine analogues with unsaturated and varying side chain lengths were successfully incorporated at four different positions in nisin in Lactococcus lactis through force feeding. This approach allows for residue-specific incorporation of methionine analogues into nisin to expand their structural diversity and alter their activity profiles. Moreover, the insertion of methionine analogues with biorthogonal chemical reactivity, e.g., azidohomoalanine and homopropargylglycine, provides the opportunity for chemical coupling to functional moieties and fluorescent probes as well as for intermolecular coupling of nisin variants. All resulting nisin conjugates retained antimicrobial activity, which substantiates the potential of this method as a tool to further study its localization and mode of action.

The sulfur-containing amino acids methionine and cysteine play an important role in food industry. These amino acids are used to confer a sulfur smell or meat-related aroma to food products. Besides their use as food additives, methionine and cysteine participate in flavor formation in dairy fermentations. For instance, the characteristic aroma of Cheddar cheeses is derived from methionine. Therefore, bacterial strains with the ability to overproduce and secrete these amino acids are relevant for the food industry. In addition, the quantification of these compounds in food matrices is a laborious task that involves sample preparation and specific analytical methods such as high-performance liquid chromatography. The ability of bacteria to naturally sense metabolites has successfully been exploited to develop biosensors. The presence of a specific metabolite is sensed by the biosensors, and it is subsequently translated into the expression of one or more reporter genes. In this study we aim to develop biosensors to detect methionine and cysteine, which are produced and secreted by wild-type Lactococcus lactis strains. We employed two strategies to create L. lactis biosensors, the first one is based on the methionine auxotrophy of this bacterium and the second strategy is based on a cysteine-responsive promoter. The characterization of the biosensors showed their specific response to the presence of these amino acids. Subsequently, we applied the methionine biosensor to quantify the presence of methionine in bacterial supernatants of wild-type L. lactis that naturally secretes methionine to benchmark the performance of our biosensors. The methionine biosensor responded linearly to the amounts of methionine present in the bacterial supernatants, i.e., the increases in the biosensor cell densities were proportional to the amounts of methionine present in the supernatants. The biosensors developed in this study tackle the limitations of amino acid quantification and the selection of strains with secretion of amino acids. These biosensors may eventually be used for screening of engineered strains to increase methionine and cysteine production, and may facilitate the detection of these amino acids in complex food matrices.

Lactococcus lactisis a lactic acid bacterium widely used as starter culture for the manufacture of fermented milk products like quark, buttermilk and cheese. Bacteriophage infection of starter cultures is one of the biggest causes of fermentation failure and, therefore, lactococcal phages have received great attention from the scientific community in the past decades. In this work we present evidence for the establishment of a carrier state life cycle (CSLC) by a bacteriophage belonging to the c2 species, in the model laboratory strainL. lactisMG1363. Our results show that infection ofL. lactisMG1363 with a second, dissimilar, c2 bacteriophage can induce the CSLC phage to enter an active lytic life cycle. The viral progeny obtained after this infection is a mixed population of phages with differences in their genome sequences and host ranges, indicative of an extremely rapid evolution process. We discuss the possible implications of this phage-host interaction, both with respect to bacteriophage evolution and phage adaptation to different hosts.IMPORTANCEOur results broaden the current know-how on the yet poorly investigated phage-host interaction mechanism of CSLC, propose a new bacteriophage evolution mechanism, and demonstrate that the outcome of phage infections is possibly more intricate than presently acknowledged.

Some secondary metabolites of fermentative bacteria are desired compounds for the food industry. Examples of these compounds are diacetyl and acetaldehyde, which are produced by species of the lactic acid bacteria (LAB) family. Diacetyl is an aromatic compound, giving the buttery flavor associated with dairy products, and acetaldehyde is the compound responsible for the yogurt flavor and aroma. The quantification of these compounds in food matrices is a laborious task that involves sample preparation and specific analytical methods. The ability of bacteria to naturally sense metabolites has successfully been exploited to develop biosensors that facilitate the identification and quantification of certain metabolites (Mahr and Frunzke, 2016). The presence of a specific metabolite is sensed by the biosensors, and it is subsequently translated into the expression of one or more reporter genes. In this study we aimed to develop fluorescence-based biosensors to detect diacetyl and acetaldehyde. Since the metabolic pathways for production and degradation of these compounds are present in Lactococcus lactis, the sensing mechanisms in this bacterium are expected. Thus, we identified diacetyl and acetaldehyde responsive promoters by performing transcriptome analyses in L. lactis. The characterization of the biosensors showed their response to the presence of these compounds, and a further analysis of the diacetyl-biosensors (its dynamics and orthogonality) was performed. Moreover, we attempted to produce natural diacetyl from producer strains, namely L. lactis subsp. lactis biovar diacetylactis, to benchmark the performance of our biosensors. The diacetyl-biosensors responded linearly to the amounts of diacetyl obtained in the bacterial supernatants, i.e., the increases in GFP expression were proportional to the amounts of diacetyl present in the supernatants of L. lactis subsp. lactis biovar diacetylactis MR3-T7 strain. The biosensors developed in this study may eventually be used to engineer strains or pathways for increased diacetyl and acetaldehyde production, and may facilitate the detection of these metabolites in complex food matrices.

Lactococcus lactis is a Gram-positive lactic acid bacterium commonly used in the dairy industry for the production of fermented foods such as buttermilk and a wide variety of cheeses. Here, we report the complete genome sequences of 28 bacteriophages infecting different L. lactis industrial starter strains isolated from dairy plants throughout the world.

Lactococcus lactis is a major microbe. This lactic acid bacterium (LAB) is used worldwide in the production of safe, healthy, tasteful and nutritious milk fermentation products. Its huge industrial importance has led to an explosion of research on the organism, particularly since the early 1970s. The upsurge in the research on L. lactis coincided not accidentally with the advent of recombinant DNA technology in these years. The development of methods to take out and re-introduce DNA in L. lactis, to clone genes and to mutate the chromosome in a targeted way, to control (over)expression of proteins and, ultimately, the availability of the nucleotide sequence of its genome and the use of that information in transcriptomics and proteomics research have enabled to peek deep into the functioning of the organism. Among many other things, this has provided an unprecedented view of the major gene regulatory pathways involved in nitrogen and carbon metabolism and their overlap, and has led to the blossoming of the field of L. lactis systems biology. All of these advances have made L. lactis the paradigm of the LAB. This review will deal with the exciting path along which the research on the genetics of and gene regulation in L. lactis has trodden.

Clostridium difficile has been reported as the most common cause of nosocomial diarrhea (antibiotic-associated diarrhea), resulting in significant morbidity and mortality in hospitalized patients. The resistance of the clostridia' spores to antibiotics and their side effects on the gut microbiota are two factors related to the emergence of infection and its relapses. Lantibiotics provide an innovative alternative for cell growth inhibition due to their dual mechanism of action (membrane pore-forming and cell wall synthesis inhibition) and low resistance rate. Based on the fact that bacteriocins are usually active against bacteria closely related to the producer strains, a new dual approach combining genome mining and synthetic biology was performed, by designing new lantibiotics with high activity and specificity toward Clostridium. We first attempted the heterologous expression of putative lantibiotics identified following Clostridium genome mining. Subsequently, we designed new hybrid lantibiotics combining the start or end of the putative clostridial peptides and the start or end parts of nisin. The designed peptides were cloned and expressed using the nisin biosynthetic machinery in Lactococcus lactis. From the 20 initial peptides, only 1 fulfilled the requirements established in this work to be considered as a good candidate: high heterologous production level and high specificity/activity against clostridial species. The high specificity and activity observed for the peptide AMV10 makes it an interesting candidate as an alternative to traditional antibiotics in the treatment of C. difficile infections, avoiding side effects and protecting the normal gut microbiota.

Lactococcus lactis is a Gram-positive bacterium widely used as starter culture for the production of different dairy products, especially a large variety of cheeses. Infection of lactococcal starter cultures by bacteriophages is one of the major causes of fermentation failure and often leads to production halt. Lactococcal bacteriophages belonging to the c2, 936 and P335 species are the most commonly isolated in dairy plants and have been extensively investigated in the past three decades. Information regarding bacteriophages belonging to less commonly isolated species is, on the other hand, less extensive, although these phages can also contribute to starter culture infection. Here we report the nucleotide sequence of the newly isolated L. lactis phage CHPC971, belonging to the rare 1706-species of lactococcal phages. We investigated the nature of the host receptor recognized by the phage and collected evidence strongly suggesting that it binds to a specific sugar moiety in the cell wall pellicle of its host. An in silico analysis of the genome of phage CHPC971 identified the hypothetical genes involved in receptor binding.Importance: Gathering information on how lactococcal bacteriophages recognize their host and proliferate in the dairy environment is of vital importance for the establishment of proper starter culture rotation plans, and to avoid fermentation failure and consequent great economic losses for dairy industries. We provide strong evidence on the type of receptor recognized by a newly isolated 1706-type lactococcal bacteriophage, increasing the knowledge on phage-host interactions relevant to dairying. This information can help to prevent phage infection events that, so far, are hard to predict and avoid.

Background A central theme in (micro)biology is understanding the molecular basis of fitness i.e. which strategies are successful under which conditions; how do organisms implement such strategies at the molecular level; and which constraints shape the trade-offs between alternative strategies. Highly standardized microbial laboratory evolution experiments are ideally suited to approach these questions. For example, prolonged chemostats provide a constant environment in which the growth rate can be set, and the adaptive process of the organism to such environment can be subsequently characterized. Results We performed parallel laboratory evolution of Lactococcus lactis in chemostats varying the quantitative value of the selective pressure by imposing two different growth rates. A mutation in one specific amino acid residue of the global transcriptional regulator of carbon metabolism, CcpA, was selected in all of the evolution experiments performed. We subsequently showed that this mutation confers predictable fitness improvements at other glucose-limited growth rates as well. In silico protein structural analysis of wild type and evolved CcpA, as well as biochemical and phenotypic assays, provided the underpinning molecular mechanisms that resulted in the specific reprogramming favored in constant environments. Conclusion This study provides a comprehensive understanding of a case of microbial evolution and hints at the wide dynamic range that a single fitness-enhancing mutation may display. It demonstrates how the modulation of a pleiotropic regulator can be used by cells to improve one trait while simultaneously work around other limiting constraints, by fine-tuning the expression of a wide range of cellular processes. Electronic supplementary material The online version of this article (10.1186/s12862-018-1331-x) contains supplementary material, which is available to authorized users.

Enterococcus faecalis is a lactic acid bacterium characterized by its tolerance of very diverse environmental conditions, a property that allows it to colonize many different habitats. This species can be found in food products, especially in fermented foods where it plays an important role as a biopreservative and influences the development of organoleptic characteristics. However, E. faecalis also produces the biogenic amines tyramine and putrescine. The consumption of food with high concentrations of these compounds can cause health problems. The present work reports the construction, via homologous recombination, of a double mutant of E. faecalis in which the clusters involved in tyramine and putrescine synthesis (which are located in different regions of the chromosome) are no longer present. Analyses showed the double mutant to grow and adhere to intestinal cells normally, and that the elimination of genes involved in the production of tyramine and putrescine has no effect on the expression of other genes., Tis work was performed with the fnancial support of AEI under grant number AGL2016-78708-R, (AEI/FEDER, UE) and the Plan for Science, Technology and Innovation 2013–2017 of the Principality of Asturias, co-funded by FEDER (GRUPIN IDI/2018/000114).

Introducing one or more intramolecular thioether bridges in a peptide provides a promising approach to create more stable molecules with improved pharmacodynamic properties and especially to protect peptides against proteolytic degradation. Lanthipeptides are compounds that naturally possess thioether bonds in their structure. The model lanthipeptide, nisin, is produced by Lactococcus lactis as a core peptide fused to a leader peptide. The modification machinery responsible for nisin production, including the Ser/Thr-dehydratase NisB and the cyclase NisC, can be applied for introducing a thioether bridge into peptides fused to the nisin leader peptide, e.g., to replace a disulfide bond. Vasopressin plays a key role in water homeostasis in the human body and helps to constrict blood vessels. There are two cysteine residues in the structure of wild type vasopressin, which form a disulfide bridge in the mature peptide. Here, we show it is possible to direct the biosynthesis of vasopressin variants in such a way that the disulfide bridge is replaced by a thioether bridge using the nisin modification machinery NisBTC, albeit at low efficiency. Vasopressin mutants were fused either to the nisin leader peptide directly (Type A), after the first three rings of nisin (Type B/C), or after full nisin (Type D). The type B strategy was optimal for expression. LC-MS/MS data verified the formation of a thioether bridge, which provides proof of principle for this modification in vasopressin. This is a first step prior to the necessary increase of the production yield and further purification of these peptides to finally test their biological activity in tissue and animal models., Chinese Scholarship Council (No. 201306770012), MM-L was supported by a grant of the EU FW7 Project SynPeptide.

Analysis of autolysis of derivatives of Lactococcus lactis subsp. cremoris MG1363 and subsp. lactis IL1403, both lacking the major autolysin AcmA, showed that L. lactis IL1403 still lysed during growth while L. lactis MG1363 did not. Zymographic analysis revealed that a peptidoglycan hydrolase activity of around 30 kDa is present in cell extracts of L. lactis IL1403 that could not be detected in strain MG1363. A comparison of all genes encoding putative peptidoglycan hydrolases of IL1403 and MG1363 led to the assumption that one or more of the 99 % homologous 27.9-kDa endolysins encoded by the prophages bIL285, bIL286 and bIL309 could account for the autolysis phenotype of IL1403. Induced expression of the endolysins from bIL285, bIL286 or bIL309 in L. lactis MG1363 resulted in detectable lysis or lytic activity. Prophage deletion and insertion derivatives of L. lactis IL1403 had a reduced cell lysis phenotype. RT-qPCR and zymogram analysis showed that each of these strains still expressed one or more of the three phage lysins. A homologous gene and an endolysin activity were also identified in the natural starter culture L. lactis subsp. cremoris strains E8, Wg2 and HP, and the lytic activity could be detected under growth conditions that were identical as those used for IL1403. The results presented here show that these endolysins of L. lactis are expressed during normal growth and contribute to autolysis without production of (lytic) phages. Screening for natural strains expressing homologous endolysins could help in the selection of strains with enhanced autolysis and, thus, cheese ripening properties. Electronic supplementary material The online version of this article (doi:10.1007/s00253-016-7822-z) contains supplementary material, which is available to authorized users.

Lactococcus lactis is a lactic acid bacterium widely used as a starter culture in the manufacture of dairy products, especially a wide variety of cheeses. Improved industrial strains would help to manufacture better food products that can meet the industry's and consumer's demands with respect to e.g. quality, taste, texture and shelf life. Bacteriophage infection of L. lactis starter cultures represents one of the main causes of fermentation failure and consequent economic losses for the dairy industry. In this study, however, we aim at employing bacteriophages for beneficial purposes. We developed an experimental setup to assess whether phage-mediated horizontal gene transfer could be used to enhance the genetic characteristics of L. lactis strains in accordance with the European law regarding the use of genetically modified organisms (GMOs) in the food industry. Although we could not show the transfer of chromosomal DNA we did successfully transduce two dissimilar plasmids from L. lactis strain MG1363 to one of its derivatives employing three different lactococcal bacteriophages.

Since the 1970s, galactose metabolism in Lactococcus lactis has been in debate. Different studies led to diverse outcomes making it difficult to conclude whether galactose uptake was PEP- or ATP- dependent and decide what the exact connection was between galactose and lactose uptake and metabolism. It was shown that some Lactococcus strains possess two galactose-specific systems - a permease and a PTS, even if they lack the lactose utilization plasmid, proving that a lactose-independent PTSGal exists. However, the PTSGal transporter was never identified. Here, with the help of transcriptome analyses and genetic knock-out mutants, we reveal the identities of two low-affinity galactose PTSs. A novel plant-niche-related PTS component Llmg_0963 forming a hybrid transporter Llmg_0963PtcBA and a glucose/mannose-specific PTS are shown to be involved in galactose transport in L. lactis MG1363.

Natural competence can be activated in Lactoccocus lactis subsp lactis and cremoris upon overexpression of ComX, a master regulator of bacterial competence. Herein, we demonstrate a method to activate bacterial competence by regulating the expression of the comX gene by using a nisininducible promoter in an L. lactis strain harboring either a chromosomal or plasmid-encoded copy of nisRK. Addition of moderate concentrations of the inducer nisin resulted in concomitant moderate levels of ComX, which led to an optimal transformation rate (1.0 x 10(-6) transformants/total cell number/g plasmid DNA). Here, a detailed description of the optimized protocol for competence induction is presented.

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) → ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell's resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

Nisin, an important bacteriocin from Lactococcus lactis subsp., is primarily active against various Gram-positive bacteria. Leucocin C, produced by Leuconostoc carnosum 4010, is a class IIa bacteriocin used to inhibit the growth of Listeria monocytogenes. Because two bacteriocins have different modes of action, the combined use of them could be a potential strategy for effective inhibition of foodborne pathogens. In this study, L. lactis N8-r-lecCI (N8 harboring lecCI gene) coexpressing nisin–leucocin C was constructed based on the food-grade carrier L. lactis N8. Production of both bacteriocins was stably maintained. Antimicrobial measurements showed that the recombinant strain is effectively against Listeria monocytogenes and Staphylococcus aureus and moderately against Salmonella enterica serovar Enteritidis and Escherichia coli because of its stronger antibacterial activity than the parental strain, this result first demonstrated that the co-expression of nisin and leucocin C results in highly efficient antimicrobial activity. The checkerboard assay showed that the antibacterial activity of L. lactis N8-r-lecCI supernatant was enhanced in the presence of low concentration of EDTA. Analysis of the scanning electron microscope image showed the biggest cellular morphology change in L. monocytogenes treated with a mixture of EDTA and L. lactis N8-r-lecCI supernatant. The practical effect was verified in pasteurized milk through time-kill assay. The L. lactis N8-r-lecCI strain expressing both nisin and leucocin C has a promising application prospect in pasteurized milk processing and preservation because of its strong antibacterial activity.

Nisin, an important bacteriocin from Lactococcus lactis subsp., is primarily active against various Gram-positive bacteria. Leucocin C, produced by Leuconostoc carnosum 4010, is a class IIa bacteriocin used to inhibit the growth of Listeria monocytogenes. Because two bacteriocins have different modes of action, the combined use of them could be a potential strategy for effective inhibition of foodborne pathogens. In this study, L. lactis N8-r-lecCI (N8 harboring lecCI gene) coexpressing nisin–leucocin C was constructed based on the food-grade carrier L. lactis N8. Production of both bacteriocins was stably maintained. Antimicrobial measurements showed that the recombinant strain is effectively against Listeria monocytogenes and Staphylococcus aureus and moderately against Salmonella enterica serovar Enteritidis and Escherichia coli because of its stronger antibacterial activity than the parental strain, this result first demonstrated that the co-expression of nisin and leucocin C results in highly efficient antimicrobial activity. The checkerboard assay showed that the antibacterial activity of L. lactis N8-r-lecCI supernatant was enhanced in the presence of low concentration of EDTA. Analysis of the scanning electron microscope image showed the biggest cellular morphology change in L. monocytogenes treated with a mixture of EDTA and L. lactis N8-r-lecCI supernatant. The practical effect was verified in pasteurized milk through time-kill assay. The L. lactis N8-r-lecCI strain expressing both nisin and leucocin C has a promising application prospect in pasteurized milk processing and preservation because of its strong antibacterial activity.

Lactococcus lactis subsp. cremoris strains typically carry many dairy niche-specific adaptations. During adaptation to the milk environment these former plant strains have acquired various pseudogenes and insertion sequence elements indicative of ongoing genome decay and frequent transposition events in their genomes. Here we describe the reactivation of a silenced plant sugar utilization cluster in an L. lactis MG1363 derivative lacking the two main cellobiose transporters, PtcBA-CelB and PtcBAC, upon applying selection pressure to utilize cellobiose. A disruption of the transcriptional repressor gene llmg_1239 by an insertion sequence (IS) element allows expression of the otherwise silent novel cellobiose transporter Llmg_1244 and leads to growth of mutant strains on cellobiose. Llmg_1239 was labeled CclR, for c ellobiose cl uster r epressor. IMPORTANCE Insertion sequences (ISs) play an important role in the evolution of lactococci and other bacteria. They facilitate DNA rearrangements and are responsible for creation of new genetic variants with selective advantages under certain environmental conditions. L. lactis MG1363 possesses 71 copies in a total of 11 different types of IS elements. This study describes yet another example of an IS-mediated adaptive evolution. An integration of IS 981 or IS 905 into a gene coding for a transcriptional repressor led to activation of the repressed gene cluster coding for a plant sugar utilization pathway. The expression of the gene cluster allowed assembly of a novel cellobiose-specific transporter and led to cell growth on cellobiose.

Dairy industry fermentative processes mostly use Lactococcus lactis as a starter. However, some dairy L. lactis strains produce putrescine, a biogenic amine that raises food safety and spoilage concerns, via the agmatine deiminase (AGDI) pathway. The enzymatic activities responsible for putrescine biosynthesis in this bacterium are encoded by the AGDI gene cluster. The role of the catabolic genes aguB, aguD, aguA, and aguC has been studied, but knowledge regarding the role of aguR (the first gene in the cluster) remains limited. In the present work, aguR was found to be a very low level constitutively expressed gene that is essential for putrescine biosynthesis and is transcribed independently of the polycistronic mRNA encoding the catabolic genes (aguBDAC). In response to agmatine, AguR acts as a transcriptional activator of the aguB promoter (PaguB), which drives the transcription of the aguBDAC operon. Inverted sequences required for PaguB activity were identified by deletion analysis. Further work indicated that AguR is a transmembrane protein which might function as a one-component signal transduction system that senses the agmatine concentration of the medium and, accordingly, regulates the transcription of the aguBDAC operon through a C-terminal cytoplasmic DNA-binding domain typically found in LuxR-like proteins., This work was performed with the financial support of the Spanish Ministry of Economy and Competitiveness (AGL2013-45431-R) and the Plan for Science, Technology and Innovation 2013–2017, funded by the European Regional Development Fund and the Principality of Asturias (GRUPIN14-137). D.M.L. and B.D.R. were the beneficiaries of JAE DOC contracts (CSIC).

Lantibiotics are potent antimicrobial peptides characterized by the presence of dehydrated amino acids, dehydroalanine and dehydrobutyrine, and (methyl)lanthionine rings. In addition to these posttranslational modifications, some lantibiotics exhibit additional modifications that usually confer increased biological activity or stability on the peptide. LtnJ is a reductase responsible for the introduction of d -alanine in the lantibiotic lacticin 3147. The conversion of l -serine into d -alanine requires dehydroalanine as the substrate, which is produced in vivo by the dehydration of serine by a lantibiotic dehydratase, i.e., LanB or LanM. In this work, we probe the substrate specificity of LtnJ using a system that combines the nisin modification machinery (dehydratase, cyclase, and transporter) and the stereospecific reductase LtnJ in Lactococcus lactis . We also describe an improvement in the production yield of this system by inserting a putative attenuator from the nisin biosynthesis gene cluster in front of the ltnJ gene. In order to clarify the sequence selectivity of LtnJ, peptides composed of truncated nisin and different mutated C-terminal tails were designed and coexpressed with LtnJ and the nisin biosynthetic machinery. In these tails, serine was flanked by diverse amino acids to determine the influence of the surrounding residues in the reaction. LtnJ successfully hydrogenated peptides when hydrophobic residues (Leu, Ile, Phe, and Ala) were flanking the intermediate dehydroalanine, while those in which dehydroalanine was flanked by one or two polar residues (Ser, Thr, Glu, Lys, and Asn) or Gly were either less prone to be modified by LtnJ or not modified at all. Moreover, our results showed that dehydrobutyrine cannot serve as a substrate for LtnJ.

Protein investment costs are considered a major driver for the choice of alternative metabolic strategies. We tested this premise in Lactococcus lactis, a bacterium that exhibits a distinct, anaerobic version of the bacterial Crabtree/Warburg effect; with increasing growth rates it shifts from a high yield metabolic mode [mixed-acid fermentation; 3 adenosine triphosphate (ATP) per glucose] to a low yield metabolic mode (homolactic fermentation; 2 ATP per glucose). We studied growth rate-dependent relative transcription and protein ratios, enzyme activities, and fluxes of L. lactis in glucose-limited chemostats, providing a high-quality and comprehensive data set. A three- to fourfold higher growth rate rerouted metabolism from acetate to lactate as the main fermentation product. However, we observed hardly any changes in transcription, protein levels and enzyme activities. Even levels of ribosomal proteins, constituting a major investment in cellular machinery, changed only slightly. Thus, contrary to the original hypothesis, central metabolism in this organism appears to be hardly regulated at the level of gene expression, but rather at the metabolic level. We conclude that L. lactis is either poorly adapted to growth at low and constant glucose concentrations, or that protein costs play a less important role in fitness than hitherto assumed.

The dairy strain Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) synthesizes the biogenic amine putrescine from agmatine via the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC, which encodes the proteins necessary for agmatine uptake and its conversion into putrescine [1,2]. The first gene of the cluster, aguR, encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC [2]. The catabolic operon aguBDAC is transcriptionally activated by agmatine [2] and transcriptionally regulated by carbon catabolite repression (CCR) via glucose, but not by other sugars such as lactose or galactose [1,3]. On the contrary, the transcription of the aguR regulatory gene is not subject to CCR regulation [1,3] nor is regulated by agmatine [2]. In this study we report the transcriptional profiling of L. lactis subsp. cremoris CECT 8666 grown in M17 medium with galactose (GalM17) as carbon source and supplemented with agmatine, compared to that of the strain grown in the same culture medium without agmatine. The transcriptional profiling data of agmatine-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under Accession no. GSE74808., This work was funded by the Spanish National Research Council (I-LINK 0380), the Spanish Ministry of Economy and Competitiveness (AGL2013-45431-R) and by the GRUPIN14-137 project, which is co-financed by the Plan for Science, Technology and Innovation of the Principality of Asturias 2013–2017 and the European Regional Development Funds.

Lantibiotics are ribosomally produced and posttranslationally modified peptides containing several lanthionine residues. They exhibit substantial antimicrobial activity against Gram-positive bacteria, including relevant pathogens. The production of the model lantibiotic nisin minimally requires the expression of the modification and export machinery. The last step during nisin maturation is the cleavage of the leader peptide. This liberates the active compound and is catalyzed by the cell wall-anchored protease NisP. Here, we report the production and purification of a soluble variant of NisP. This has enabled us to study its specificity and test its suitability for biotechnological applications. The ability of soluble NisP to cleave leaders from various substrates was tested with two sets of nisin variants. The first set was designed to investigate the influence of amino acid variations in the leader peptide or variations around the cleavage site. The second set was designed to study the influence of the lanthionine ring topology on the proteolytic efficiency. We show that the substrate promiscuity is higher than has previously been suggested. Our results demonstrate the importance of the arginine residue at the end of the leader peptide and the importance of lanthionine rings in the substrate for specific cleavage. Collectively, these data indicate that NisP is a suitable protease for the activation of diverse heterologously expressed lantibiotics, which is required to release active antimicrobial compounds.

In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis , natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis , natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX -driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK -harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies. IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo , and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two “superfolder” GFPs with codon adaptation specifically for Bacillus subtilis and Streptococcus pneumoniae and have benchmarked them against five other previously available variants of GFP in B. subtilis , S. pneumoniae , and Lactococcus lactis , using promoter- gfp fusions. Surprisingly, the best-performing GFP under our experimental conditions in B. subtilis was the one codon optimized for S. pneumoniae and vice versa . The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.

Here, we report a new zinc-inducible expression system for Lactococcus lactis , called Zirex, consisting of the pneumococcal repressor SczA and P czcD . P czcD tightly regulates the expression of green fluorescent protein in L. lactis . We show the applicability of Zirex together with the nisin-controlled expression system, enabling simultaneous but independent regulation of different genes.

Lanthipeptides are peptides that contain several post-translationally modified amino acid residues and commonly show considerable antimicrobial activity. After translation, the amino acid residues of these peptides are modified by a distinct set of modification enzymes. This process results in peptides containing one or more lanthionine rings and dehydrated Ser and Thr residues. Previously, an in vivo lanthipeptide production system based on the modification machinery of the model lantibiotic nisin was reported. Here, we present the addition of the modification enzymes LtnJ and GdrnD to this production system. With these enzymes we can now produce lanthipeptides that contain D-alanines or a C-terminal aminovinyl-cysteine. We show experimentally that the decarboxylase GdmD is responsible for the C-terminal decarboxylation. Our results demonstrate that different lanthipeptide modification enzymes can work together in an in vivo production system. This yields a plug-and-play system that can be used to select different sets of modification enzymes to work on diverse, specifically designed substrates.

This paper describes the molecular response of Lactococcus lactis NZ9700 to ethanol. This strain is a well-known nisin producer and a lactic acid bacteria (LAB) model strain. Global transcriptome profiling using DNA microarrays demonstrated a bacterial adaptive response to the presence of 2% ethanol in the culture broth and differential expression of 67 genes. The highest up-regulation was detected for those genes involved in arginine degradation through the arginine deiminase (ADI) pathway (20–40 fold up-regulation). The metabolic responses to ethanol of wild type L. lactis strains were studied and compared to those of regulator-deletion mutants MG∆argR and MG∆ahrC. The results showed that in the presence of 2% ethanol those strains with an active ADI pathway reached higher growth rates when arginine was available in the culture broth than in absence of arginine. In a chemically defined medium strains with an active ADI pathway consumed arginine and produced ornithine in the presence of 2% ethanol, hence corroborating that arginine catabolism is involved in the bacterial response to ethanol. This is the first study of the L. lactis response to ethanol stress to demonstrate the relevance of arginine catabolism for bacterial adaptation and survival in an ethanol containing medium., This work was supported by grant AGL2010-15466 of the Ministry of Research and Science of Spain and FEDER of the European Community. Lorena Diez was a contractual technician supported by the grant AGL2010-15466. Ana Solopova was supported by a Stichting Technische Wetenschappen grant in the scope of Project 10619 “Understanding Preculture- Dependent Growth and Acidification Rates of Lactococcus lactis as the Result of Population Heterogeneity”. Rocío Fenández was a Predoctoral Researcher of the Regional Autonomous Government of La Rioja.

International audience; To ensure optimal cell growth and separation and to adapt to environmental parameters, bacteria have to maintain a balance between cell wall (CW) rigidity and flexibility. This can be achieved by a concerted action of peptidoglycan (PG) hydrolases and PG-synthesizing/modifying enzymes. In a search for new regulatory mechanisms responsible for the maintenance of this equilibrium in Lactococcus lactis, we isolated mutants that are resistant to the PG hydrolase lysozyme. We found that 14% of the causative mutations were mapped in the guaA gene, the product of which is involved in purine metabolism. Genetic and transcriptional analyses combined with PG structure determination of the guaA mutant enabled us to reveal the pivotal role of the pyrB gene in the regulation of CW rigidity. Our results indicate that conversion of l-aspartate (l-Asp) to N-carbamoyl-l-aspartate by PyrB may reduce the amount of l-Asp available for PG synthesis and thus cause the appearance of Asp/Asn-less stem peptides in PG. Such stem peptides do not form PG cross-bridges, resulting in a decrease in PG cross-linking and, consequently, reduced PG thickness and rigidity. We hypothesize that the concurrent utilization of l-Asp for pyrimidine and PG synthesis may be part of the regulatory scheme, ensuring CW flexibility during exponential growth and rigidity in stationary phase. The fact that l-Asp availability is dependent on nucleotide metabolism, which is tightly regulated in accordance with the growth rate, provides L. lactis cells the means to ensure optimal CW plasticity without the need to control the expression of PG synthesis genes.

The Lactococcus lactis laboratory strain MG1363 has been described to be unable to utilize lactose. However, in a rich medium supplemented with lactose as the sole carbon source, it starts to grow after prolonged incubation periods. Transcriptome analyses showed that L. lactis MG1363 Lac + cells expressed celB , encoding a putative cellobiose-specific phosphotransferase system (PTS) IIC component, which is normally silent in MG1363 Lac − cells. Nucleotide sequence analysis of the cel cluster of a Lac + isolate revealed a change from one of the guanines to adenine in the promoter region. We showed here that one particular mutation, taking place at increased frequency, accounts for the lactose-utilizing phenotype occurring in MG1363 cultures. The G-to-A transition creates a −10 element at an optimal distance from the −35 element. Thus, a fully active promoter is created, allowing transcription of the otherwise cryptic cluster. Nuclear magnetic resonance (NMR) spectroscopy results show that MG1363 Lac + uses a novel pathway of lactose utilization.