TY - JOUR
AU - van Mastrigt,, Oscar
AB - ABSTRACT Lactococcus lactis serves as a paradigm organism for the lactic acid bacteria (LAB). Extensive research into the molecular biology, metabolism and physiology of several model strains of this species has been fundamental for our understanding of the LAB. Genomic studies have provided new insights into the species L. lactis, including the resolution of the genetic basis of its subspecies division, as well as the control mechanisms involved in the fine-tuning of growth rate and energy metabolism. In addition, it has enabled novel approaches to study lactococcal lifestyle adaptations to the dairy application environment, including its adjustment to near-zero growth rates that are particularly relevant in the context of cheese ripening. This review highlights various insights in these areas and exemplifies the strength of combining experimental evolution with functional genomics and bacterial physiology research to expand our fundamental understanding of the L. lactis lifestyle under different environmental conditions. lactic acid bacteria, Lactococcus lactis, metabolism, adaptation, environmental fitness, dairy application INTRODUCTION: LACTOCOCCUS LACTIS A PARADIGM LAB SPECIES Lactococcus lactis plays an important role in the fermentation industry where it is applied in the production of a range of fermented products. Due to its importance in industrial food fermentations, L. lactis has been the subject of fundamental research for over a century. Initially described as Bacterium lactis, it was first reclassified as Streptococcus lactis and later as Lactococcus lactis (van Hylckama Vlieg et al. 2006). Many of the L. lactis applications in food fermentation are within the dairy sector, using milk as the raw food material that is converted by lactococcal fermentation to a range of products in different regions of the world (for a review, see Cavanagh, Fitzgerald and McAuliffe 2015). The primary role of lactococci in these fermentations, acidification, is dependent on efficient conversion of the milk-sugar lactose to lactic acid, which contributes to the extended shelf life of fermented milk products by preventing the outgrowth of pathogenic and spoilage organisms. Additionally, L. lactis contributes to the texture and flavour characteristics of the fermented products. Texture properties of fermented milk products are not only dependent on the changes in physical chemical properties of the milk proteins that are elicited by the fermentative acidification but also influenced by the lactococcal production of extracellular polysaccharides that contribute to the rheological properties of fermented milk products (for a review, see Kleerebezem et al. 1999; Zeidan et al. 2017). The importance of flavour formation by L. lactis is especially prominent in a variety of cheeses of which the production includes a long ripening period during which milk constituents are converted to flavour compounds that determine the taste of the cheese. The lactococci play a central role in flavour formation by the degradation of milk proteins and the conversion of the resulting amino acids into volatile aroma components. Extracellular proteolysis, peptide and amino acid import and intracellular peptidolysis by peptidases are of obvious importance in this process, and the proteins involved as well as the regulation of their expression have extensively been studied in L. lactis (for reviews, see Kunji et al. 1996; Savijoki, Ingmer and Varmanen 2006; Kok et al. 2017). Nevertheless, recent studies underpin that our understanding of amino acid import can still be refined. This is exemplified by the recent discovery of the co-existence of two isogenic L. lactis subpopulations that depend on either high- or low-affinity import systems of the essential amino acid methionine, which is controlled by a riboswitch present in the promoter region of the genes encoding the high-affinity importer (Hernandez-Valdes, van Gestel and Kuipers 2020). Similarly, the transport-coordinating role of the peculiar presence of two distinct and competing substrate binding domains in the L. lactis GlnPQ importer for asparagine, glutamine and glutamic acid was only recently unravelled (Gouridis et al. 2015; Fulyani et al. 2016; Schuurman-Wolters et al. 2018). Amino acid catabolism by L. lactis plays an essential role in flavour formation and although the metabolic activities that are linked to flavour formation are not fully deciphered, a number of pathways and key enzymes involved in this process have been described (for a review, see Smid and Kleerebezem 2014). For a long time, L. lactis has served as a paradigm organism in research of the lactic acid bacteria (LAB), where it contributed tremendously to our present understanding of the molecular biology, genetics, gene regulation and metabolism of this group of bacteria (for reviews, see Mahony et al. 2016; Papadimitriou et al. 2016; Kok et al. 2017; Price et al. 2019). Especially since the 1970s, the research on L. lactis genetics and the introduction of genetic engineering have contributed to our molecular insight in key functions of L. lactis in dairy fermentations, serving as an example and source of inspiration for similar research in a variety of other LAB. This exemplary role of L. lactis research is underpinned by many common strategies, procedures and tools that are used in genetic engineering of LAB that were pioneered in L. lactis (for reviews, see Mills 2001; Daniel et al. 2011; Landete 2017; Bron et al. 2019). In the last couple of decades, the scientific investigations of L. lactis have progressed to embrace the wealth of the post-genomic approaches. Simultaneously, post-genomic research of other LAB species also accelerated, leading to an explosion of information related to the genetics, diversity and evolution of the LAB in general. To some extent, this development has diminished the status of L. lactis as paradigm species, although it is still among the most intensely investigated LAB. Below, we will discuss a selection of comparative genomics-derived insights in the species L. lactis, including the resolution of the genomic basis of its subspecies division, the understanding of lactococcal metabolism through metabolic and genetic engineering and genome-scale metabolic models, and the investigations of its capacity to adjust its lifestyle, including the use of experimental evolution approaches. Finally, we will discuss our current knowledge of the adaptation of L. lactis to dairy application environments, including its adjustment during dormancy and near-zero growth rates that are particularly relevant in the context of cheese ripening. LACTOCOCCUS LACTIS SUBSPECIES AND GENOMIC REFLECTIONS OF NICHE ADAPTATION Within the species L. lactis, the two subspecies (spp.) lactis and cremoris are considered the most important in biotechnological applications. On basis of the sequence of their 16S rRNA encoding gene, these subspecies were estimated to have diverged ∼17 million years ago (Bolotin et al. 2004a) and are traditionally discriminated by subspecies-specific phenotypic traits (Schleifer et al. 1985; Kim, Ren and Dunn 1999; van Hylckama Vlieg et al. 2006; Fernandez et al. 2011). Strains belonging to the subspecies lactis can produce ammonia from arginine, utilise maltose and grow at 40°C and in the presence of 4% sodium chloride, whereas strains of ssp. cremoris lack these properties. Several studies have described the distinction of L. lactis genotype lineages that were also designated cremoris and lactis, which can be discriminated by various genetic typing methods like multi-locus sequence typing, lineage-specific PCR and high-resolution genetic fingerprinting (Beimfohr, Ludwig and Schleifer 1997; Pu et al. 2002; Kutahya et al. 2011; Laroute et al. 2017). However, although the names ‘cremoris’ and ‘lactis’ are used for both genetic and phenotypic discrimination of the L. lactis strains, these classifications are not entirely congruent because several strains that belong to the cremoris genetic lineage display a subspecies lactis phenotype (van Hylckama Vlieg et al. 2006; Kelly, Ward and Leahy 2010; Parapouli et al. 2013; Cavanagh, Fitzgerald and McAuliffe 2015). Despite various attempts, including detailed analysis of selected genes and comparative genomics, the genetic basis of the lactis and cremoris phenotype–genotype disparity was only recently resolved. The phylogenetic analysis of single nucleotide polymorphisms in the core genome of 43 L. lactis strains indicated that strains displaying the incongruent lactis phenotype and cremoris genotype share a common ancestor with L. lactis subspecies lactis and that strains with a congruent cremoris genotype and phenotype evolved only once (Wels et al. 2019). The genetic repertoire that allowed the resolution of the lactis-cremoris phenotype–genotype discrepancy encoded not only a variety of hypothetical proteins, transporters and regulators of unknown specificity but also several functions associated with heat and osmotic stress response that may explain the corresponding differences in growth at elevated temperature and salt concentrations. Moreover, erosion of the arginine deiminase pathway was associated with strains displaying the spp. cremoris phenotype, whereas redundancy of the maltose utilisation gene cluster was found in strains with the spp. lactis phenotype (Wels et al. 2019). Interestingly, whereas the vast majority of phenotype cremoris, genotype cremoris strains are found in dairy environments, the strains displaying the ssp. lactis phenotype are isolated from both dairy and non-dairy environments, in particular from plant materials (Kelly, Ward and Leahy 2010). Two dairy-derived model strains have been used extensively for fundamental research in L. lactis. Strain IL1403 is a plasmid free derivative of IL594, which was originally isolated from a cheese starter culture (Bolotin et al. 2001). One of the plasmids of IL594 allows it to metabolise citrate, which classifies this strain as a L. lactis subspecies lactis biovar diacetylactis. Strain MG1363 was generated by curing of prophages and plasmids from strain NCDO712, a strain isolated from a dairy starter culture (Gasson 1983; Wegmann et al. 2007). It has a subspecies cremoris-like genotype and a subspecies lactis-like phenotype. While plasmid and prophage curation of these strains was an important step to enable genetic engineering using plasmid-based approaches, the fact that IL594 and NCDO712, respectively, harboured 7 and 6 plasmids (Gorecki et al. 2011; Tarazanova et al. 2016) hints at their importance in the habitual environment of these strains. This was further corroborated by the observation that in both strains several plasmid encoded functions are critical for efficient growth in the dairy environment, including lactose utilisation, and an extracellular protease required for milk-protein proteolysis as well as peptide transporters and proteins involved in stress resistance (Gorecki et al. 2011; Tarazanova et al. 2016). Recent comparative analyses of plasmid encoded functions in a larger panel of L. lactis strains further confirmed the richness of genes with biotechnological importance in the lactococcal plasmid complement (Ainsworth et al. 2014, Kelleher et al. 2019). A striking example is the examination of 12 plasmids in L. lactis FM03P, which revealed that no less than 10 of these plasmids encode genes that are predicted to be important for growth and survival in the dairy environment (van Mastrigt et al. 2018c). The plasmid encoded L. lactis repertoire includes not only a variety of metabolic functions (e.g. lactose utilisation, proteolysis and peptide import), but also genes involved in conjugation, bacteriocin production, exopolysaccharide synthesis and phage resistance (Kelleher et al. 2019). Intriguingly, the latter category not only included a variety of abortive infection functions (Ainsworth, Mahony and van Sinderen 2014; Kelleher et al. 2019), but also a range of genes encoding restriction modification systems (van Mastrigt et al. 2018c), as well as the first CRISPR-Cas system for adaptive phage resistance described for this species (Millen et al. 2012, 2019). Moreover, recent studies have identified plasmid encoded sortase C dependent pili, forming proteinaceous appendages on the lactococcal cell surface. Pilin formation in lactococci was previously associated with clinical- and plant-derived isolates (Oxaran et al. 2012). Engineered expression of pili in L. lactis strongly affected the cell's adhesion properties to different surfaces as well as human intestinal epithelial cells (Meyrand et al. 2013; Castelain et al. 2016). Although the latter observation does not reflect a normal habitat for L. lactis, this finding may indicate a role of the pili in adhesion to niche-specific surfaces, suggesting that lifestyle and niche adaptation may involve adjustments of pilin expression. Intriguingly, expression of pili drastically altered cell-surface hydrophobicity (Tarazanova et al. 2018) and influenced aggregation properties, which was reflected in the ∼15-fold increased efficiency of conjugation of mobile genetic elements between pilin expressing strains (Tarazanova et al. 2016). Moreover, milk fermentation by such strains was shown to lead to increased viscosity and gel firmness of the fermented product and also increased the retention of L. lactis cells in curd rather than whey during the cheese making process (Tarazanova et al. 2018), which may both reflect the enhanced aggregation and/or milk–matrix interaction displayed by these pilin-expressing cells. Taken together these findings underpin the importance of plasmid complement encoded functions in the dairy applications of the lactococci. Importantly, a recent analysis of the ‘pan-plasmidome’ of L. lactis revealed that at present this field has not reached saturation, implying that there are many plasmid-encoded functions that remain to be discovered in this species (Ainsworth et al. 2014; Kelleher et al. 2019). Analogously, a recent comparative genomics analysis of another class of mobile genetic elements in L. lactis, the integrative conjugative elements (ICEs), revealed that lactococcal ICEs display a high degree of genetic plasticity and can be classified in seven ICE families (van der Els et al. 2020b). Similar to the lactococcal plasmids, the ICEs detected in L. lactis encode a variety of industrially relevant functions, including phage abortive infection, antimicrobial production and carbohydrate utilisation (van der Els et al. 2020a), further supporting the biotechnological importance of functions encoded by mobile genetic elements in the dairy lactococci. A third model strain that has been used as a representative of plant-derived L. lactis spp. lactis is strain KF147, of which the genome analysis clearly reflected its adaptation to the plant environment. Among these adaptations, the most prominent is the presence of a large panel of genes encoding glycoside-utilisation pathways in this plant isolate compared with the dairy-derived strains (Siezen et al. 2008; Siezen et al. 2010). Growth of KF147 on plant-derived medium (Arabidopsis thaliana leaf tissue lysate) was shown to involve the expression of a large repertoire of these pathways, including the utilisation of xylose, arabinose, cellobiose and hemicellulose, reflecting the availability of these nutrient sources in habitats associated with plants (Golomb and Marco 2015). Additionally, this strain encodes a mega-enzyme system composed of nonribosomal peptide synthetases and polyketide synthases (hybrid NRPS-PKS) predicted to produce a hexapeptide-malonyl hybrid molecule (Leu-DLeu-Asp-DAsn-Gly-Malonyl3-DAsp; Khayatt et al. 2020) that contributes to oxidative stress tolerance, and could enhance growth under oxidative conditions in plant-associated environments (Golomb et al. 2018). THE FOUNDATION OF ACIDIFICATION, CARBON METABOLISM IN L. LACTIS Fuelled by its important role in food preservation by fermentation, carbon metabolism and lactic acid formation in L. lactis has been a prominent subject of research. Contrary to the plant isolates, most dairy isolates of L. lactis display a relatively limited capacity to use different carbohydrates as substrates for growth, including glucose, mannose, fructose, galactose, lactose, maltose and cellobiose (Siezen et al. 2008; Wels et al. 2019). This has been proposed to reflect genome-decay associated with prolonged cultivation in nutrient-rich niches (Wels et al. 2019), such as milk, which has also been detected in the genome analyses of several other LAB that serve as workhorses in the dairy fermentation industry like Streptococcus thermophilus and Lactobacillus helveticus (Bolotin et al. 2004b, Hols et al. 2005; Callanan et al. 2008). Expression and activity of carbohydrate import systems in L. lactis is subject to catabolite control exerted by carbon catabolite protein A (CcpA) and HPr, which enables preferential utilisation of sugars in environments that contain more than one sugar (Fig. 1). This leads to diauxic growth that includes a lag phase during which cells adapt to the utilisation of alternative sugars once the preferred sugar is consumed. Intriguingly, the analysis of diauxic growth of L. lactis in media containing glucose and cellobiose revealed that only a fraction of the culture proceeds to utilise cellobiose once the glucose is depleted, illustrating that diauxic shifts may include aspects of culture heterogeneity and bet-hedging (Solopova et al. 2014). Figure 1. Open in new tabDownload slide Schematic representation of the glucose and lactose utilisation pathway in L. lactis, including the differential pyruvate dissipation pathways known in this species. The scheme highlights the dualistic role of HPr and its differential phosphorylation in facilitating transport (HPr-HP) and catabolite regulation (HPr-SP) in concert with CcpA. The glycolytic pathway is highlighted with a grey background. Figure 1. Open in new tabDownload slide Schematic representation of the glucose and lactose utilisation pathway in L. lactis, including the differential pyruvate dissipation pathways known in this species. The scheme highlights the dualistic role of HPr and its differential phosphorylation in facilitating transport (HPr-HP) and catabolite regulation (HPr-SP) in concert with CcpA. The glycolytic pathway is highlighted with a grey background. HPr plays a dualistic role in control of carbohydrate metabolism (for a review, see Deutscher, Francke and Postma 2006). On the one hand, it participates in the canonical EnzymeI-HPr phosphorelay that couples the glycolytic intermediate phosphoenolpyruvate (PEP) via phosphorylation on histidine residue 15 of HPr (HPr-HP) to substrate-specific phosphotransferase systems (PTS) that concurrently phosphorylate and import sugar. On the other hand, HPr can be phosphorylated on serine residue 46 (HPr-SP) by HPr kinase. Under conditions that support high glycolytic flux, the intracellular levels of the glycolytic intermediate fructose 1,6-bisphosphate (FBP) and ATP are increased, resulting in activation of HPr kinase activity leading to increased levels of HPr-SP. HPr-SP forms a complex with CcpA that plays a pleiotropic role in genome-wide regulation of carbohydrate utilisation (for reviews, see Titgemeyer and Hillen 2002; Warner and Lolkema 2003). In addition to global carbon catabolite control, carbohydrate utilisation systems are additionally controlled by dedicated regulator proteins that often act as repressors, whose repressor function is eliminated when they bind a specific intermediate of the utilisation pathway of that carbohydrate (for a review, see Kok et al. 2017). Since lactose is the main carbon source encountered by L. lactis when growing in milk, its utilisation pathway has been studied in detail (Fig. 1). As indicated above, in many lactococcal strains lactose utilisation genes are plasmid encoded and a well-characterised example is present on the lactose mini-plasmid pMG820. The lac-operon on this plasmid encodes a lactose PTS import system (lacEF), a phospho-β-galactosidase (lacG) and the tagatose pathway (lacABCD) (de Vos et al. 1990; van Rooijen, van Schalkwijk and de Vos 1991), and its expression is controlled by the divergently transcribed repressor LacR, which relieves repression upon binding to the tagatose pathway intermediate tagatose-6-phosphate (van Rooijen, Gasson and de Vos 1992). Once internalised, in L. lactis carbohydrates are processed through the Embden–Meyerhof–Parnas glycolytic pathway with different carbohydrates entering at different points in the pathway (Fig. 1). The enzymes involved in the pathway are generally highly expressed in L. lactis, which is in agreement with the genomic localisation of the encoding genes in close proximity of the genome origin of replication (Cocaign-Bousquet et al. 2002). In most growth conditions the glycolytic enzymes constitute a large proportion (reported to be up to 20%) of the overall cytoplasmic proteome (Puri 2014a; Goel et al. 2015). Expression of several glycolytic enzymes in L. lactis is regulated by the CcpA-HPr-SP complex, which upregulates the expression of phosphofructokinase (PFK) and pyruvate kinase (PYK) (Luesink et al. 1998; Zomer et al. 2007; Kok et al. 2017), as well as phosphoglucose isomerase (PGI), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (Guedon, Jamet and Renault 2002). The control of glycolytic flux has been extensively studied and various control scenarios have been proposed that integrate gene expression regulation with allosteric regulation of glycolytic enzymes by glycolytic intermediates as well as by intracellular levels or ratios of ATP/ADP + Pi and NADH/NAD+. Overall, it has become clear that the lactococcal glycolysis runs at a very high (maximal) rate during balanced growth in batch cultures and remains highly active also in non-growing cells, demonstrating the uncoupling of central metabolism and growth (for reviews, see Koebmann et al. 2002; Liu et al. 2019). The glycolytic pathway leads to the formation of pyruvate, which under most conditions is almost exclusively dissipated to lactic acid involving lactate dehydrogenase (LDH), resulting in homolactic fermentation. However, pyruvate dissipation can also lead to the formation of other metabolic end-products like acetic acid, formic acid, ethanol (i.e. mixed acid fermentation), acetoin and butanediol (Fig. 1). These alternative metabolic end-products have been reported for specific growth conditions, including carbon limitation (Thomas, Ellwood and Longyear 1979), diminished rates of sugar catabolism (Cocaign-Bousquet et al. 1996) and aerobic conditions (Lopez de Felipe et al. 1998). The linkage between glycolysis and pyruvate dissipation to lactic acid is reflected by the genetic linkage of the LDH encoding gene with the genes encoding the glycolytic enzymes PFK and PYK in the so-called las operon. Although the ldh gene is considered to be part of the lasoperon (i.e. the last of the three genes), a monocistronic ldh transcript has also been detected, which was initially proposed to derive from post-transcriptional processing of the polycistronic las operon transcript (Luesink et al. 1998), but was later proposed to depend on a separate promoter sequence upstream of the ldh gene (Bachmann et al. 2010). As described above, the las operon is subject to carbon catabolite regulation (Luesink et al. 1998; Zomer et al. 2007), aligning the expression of LDH and two key enzymes in the glycolytic pathway. In parallel, carbon catabolite regulation represses the expression of genes that encode functions involved in mixed acid fermentation like acetate kinase and pyruvate dehydrogenase (Zomer et al. 2007; Lopez de Felipe and Gaudu 2009). The consequences of CcpA-HPr-SP mediated regulation of pyruvate dissipation were underpinned by the observed reduced growth rate and mixed acid fermentation profile in a CcpA-deficient mutant of L. lactis MG1363 (Luesink et al. 1998). Thereby, carbon catabolite regulation plays a pleiotropic role in controlling carbon metabolism and fermentation in L. lactis that exceeds its canonical role in carbohydrate import regulation described in various bacteria (for reviews, see Titgemeyer and Hillen 2002; Warner and Lolkema 2003). PYRUVATE DISSIPATION CONTROL The main difference between homolactic and mixed acid fermentation lies in the energy yield, where pyruvate dissipation towards acetate yields an extra ATP in addition to the two ATP gained by the glycolytic pathway. It is quite striking that under anaerobic conditions and at higher growth rates L. lactis mainly displays ‘energy-inefficient’ lactic acid fermentation, despite the high energy demand of fast growth (Thomas, Ellwood and Longyear 1979; Flahaut et al. 2013; Goel et al. 2015). One would expect that at high growth rates lactococcal cells would employ their most energy-efficient metabolic pathway, which is also predicted by stoichiometry-based genome-scale metabolic models of L. lactis that predict carbon-flux distribution using highest energy and biomass yield as an objective (Teusink et al. 2009; Flahaut et al. 2013). These observations raise questions about the mechanism underlying the switching between homolactic and mixed acid fermentation in L. lactis, and imply that these bacteria have not evolved to optimised energy and biomass yield but rather towards maximised acidification rates (Teusink et al. 2009). The intracellular redox balance, reflected by the NADH/NAD+ ratio, plays an important role in controlling pyruvate dissipation in L. lactis (Garrigues et al. 1997; Lopez de Felipe et al. 1998; Lopez de Felipe and Gaudu 2009). The LDH-catalysed reduction of pyruvate to lactic acid regenerates NAD+ from the glycolytically formed NADH, thereby, maintaining redox balance and sustaining high carbon flux and growth rate (Fig. 1). Indeed, LDH deficient mutants of L. lactis grow ∼4- to 5-fold slower but reach a higher final cell density in batch cultures as compared with their parental strain under anaerobic conditions, where pyruvate is dissipated predominantly to acetoin/2,3-butanediol, ethanol and acetic acid (Platteeuw et al. 1995; Bongers et al. 2003). Moreover, LDH-deficient mutants also accumulate mannitol, which is formed from the glycolytic intermediate fructose-6-phosphate, involving mannitol-1-phosphate dehydrogenase that contributes to NAD+ regeneration in these cells (Neves et al. 2000). However, when grown under aerobic conditions the LDH-deficient strain grows at almost the same rate as the wild-type strain, which is dependent on the activity of the endogenous water-forming NADH oxidase that regenerates NAD+ and restores redox balance, utilising molecular oxygen as electron acceptor. In the presence of oxygen, the LDH-deficient strain produces predominantly acetic acid and acetoin, while ethanol formation and mannitol accumulation are to a large extent eliminated (Neves et al. 2000; Bongers et al. 2003). These results support that alternative NADH-utilising pathways are crucial in maintenance of redox balance in strains where lactic acid formation is compromised. Ethanol formation by alcohol dehydrogenase (ADH) plays such a role (Fig. 1), which was supported by the improved glucose fermentation observed in LDH-deficient lactococcal cells as a consequence of acetate co-metabolisation and its conversion to ethanol, with the concomitant NAD+ regeneration (Hols et al. 1999b). Moreover, so-called ‘redox engineering’ by overexpression of NADH oxidase in L. lactis was shown to be an effective strategy to metabolically engineer wild-type (i.e. containing LDH) L. lactis towards almost exclusive production of acetic acid and acetoin (Lopez de Felipe et al. 1998). Employing the same strategy in an α-acetolactate decarboxylase deficient strain led to increased production levels of the butter aroma component diacetyl (Neves et al. 2000), whereas co-expression with pyruvate decarboxylase (derived from Zymomonas mobilis) increased the production levels of the yoghurt aroma component acetaldehyde (Bongers, Hoefnagel and Kleerebezem 2005). Collectively, these findings underpin the importance of the redox-balance reflected by the NADH/NAD+ ratio in controlling lactococcal pyruvate metabolism. The importance of LDH in the lactococcal lifestyle is further supported by the redundancy of genes encoding this function (Bongers et al. 2003; Gaspar et al. 2011). This is especially remarkable in the light of the small genome size of the lactococci and the fact that it has 3–4 LDH encoding genes. However, only the ldh gene encoded within the las operon is expressed at a high level, although it was reported that two of the alternative ldh genes (ldhB and ldhX) were also expressed at a low level (Gaspar et al. 2011). However, the expression of ldhB in L. lactis MG1363 was shown to become activated by promoter-up mutations upon prolonged anaerobic growth of a strain mutated in the lasoperon encoded ldh. In many cases, this expression activation depended on IS981 mobilisation and its directional and precisely spaced insertion upstream of the ldhB gene, providing this gene with a restored promoter that depends on a IS981-derived −35 promoter region (Bongers et al. 2003). Regained LDH activity by ldhB expression recovers lactic acid production that under anaerobic conditions coincides with the regain of growth rates comparable to the wild-type strain and loss of the increased biomass yield (Bongers et al. 2003). Engineered mutant strains of L. lactis MG1363 that lack the ldh gene of the las operon, or also the redundant ldhB and ldhX genes, have been extensively used for metabolic engineering to achieve highly efficient cell-factories for the production of various reduced end-products like alanine, mannitol and various 2,3-butanediol isomers (Hols et al. 1999a; Gaspar et al. 2011; Kandasamy et al. 2016; Liu et al. 2016, 2017, 2019). Taken together, these findings highlight the critical role of LDH in sustaining efficient and fast growth in L. lactis under anaerobic conditions. Finally, this notion is further supported by a study that employed a water-in-oil emulsion microdroplet technology to experimentally evolve L. lactis MG1363 towards maximised cell- and biomass yield rather than the maximised-growth and -acidification rate lifestyle of the wild-type strain. The evolved strains grew slower and reached higher culture densities driven by mutations that led to reduced rates of glucose import and glycolytic flux, which elicited a switch from homolactic to mixed acid fermentation leading to increased metabolic efficiency and biomass yield (Bachmann et al. 2013; Bachmann et al. 2017). In addition, a recent study revealed that these biomass-yield optimised strains also had an activated arginine utilisation pathway, further enhancing their metabolic capacity for energy generation to support biomass formation (Nugroho, Kleerebezem and Bachmann 2020). Notably, the reduced growth rate, increased biomass yield and acetic acid production resemble the physiological characteristics of ldh mutants of L. lactis, further supporting the pivotal role of lactic acid production by LDH in maintenance of the redox balance required to sustain high glycolytic flux and maximal growth (and acidification) rate. The above indicates that the lifestyle of L. lactis is apparently optimised for fast growth and lactic acid production, a combination that is likely providing a competitive advantage in different environmental niches by the suppression of growth of competing microbes by rapid lactic acid accumulation and pH lowering. This characteristic is of fundamental importance for the fermentation industry, where fast preservation and shelf-life extension of the food raw-materials by acidification is one of the primary objectives. DO PROTEOME CONSTRAINTS PLAY A ROLE IN ENVIRONMENTAL ADAPTATION? To maintain optimal environmental fitness, bacteria need to adapt to changing conditions by changing their proteome composition. Proteome changes are required because overall protein synthesis and maintenance capacity of a cell is limited by cellular resources, disallowing the expression of optimal levels of all proteins at the same time. Such constraints can lead to fitness trade-offs, where optimisation of fitness-enhancing traits under one specific condition comes at the expense of fitness-enhancing traits under another condition. This results in so-called Pareto fronts that cannot be crossed (for a review, see Emmerich and Deutz 2018) forcing cells to regulate the adaptation of their proteome. Bacterial proteome changes are predominantly driven by the adjustment of gene expression patterns, which is governed by regulatory networks involving single or multiple regulator proteins that respond to environmental cues. These regulatory networks have been extensively studied in L. lactis, in particular in model strain MG1363, and large amounts of gene regulation studies have contributed to the deciphering of the lactococcal ‘genome-wide’ gene regulation network (de Jong et al. 2013; Kok et al. 2017; Omony et al. 2019). In addition, several studies involving proteome analyses have examined and modelled the determinants of protein levels, translation efficiency and metabolic costs associated with protein synthesis and maintenance (Dressaire et al. 2009; Dressaire et al. 2010; Lahtvee et al. 2011; Lahtvee et al. 2014). Basic models of self-replicating systems indicated that a trade-off between enzyme costs and metabolic benefits, i.e. ATP yield, could explain the apparent contradiction of energetically inefficient homolactic fermentation observed in fast-growing and thereby high-energy-demanding L. lactis (Molenaar et al. 2009). Importantly, the model took the costs related to the synthesis of enzymes required for a specific metabolic pathway into account. In contrast, traditional approaches such as Flux Balance Analysis of genome-scale metabolic models only use reaction stoichiometry, thereby disregarding the costs of production of the enzyme involved in the underlying metabolic pathways. Although the model was constructed to generically represent self-replicating systems, its extrapolation to L. lactis predicted that the ‘extra’ investment involved in biosynthesis of the acetic acid formation pathway is unfavourable at high growth rates (Molenaar et al. 2009). Notably, similar proteome efficiency effects that can be expressed as the amount of ATP produced per amount of protein, were later shown to explain overflow metabolism in Escherichia coli. (Basan et al. 2015). However, proteome investigation of L. lactis MG1363 at different growth rates established that the enzymes involved in acetic acid production were present at equal levels in homolactic fast-growing cells and slow-growing cells that displayed a mixed acid fermentation profile, which appeared to falsify the protein cost-benefit prediction by the model (Goel et al. 2015). It is important to note that the proteome study employed glucose-limited chemostat cultivation conditions that are expected to have led to relief of carbon catabolite repression, which may have influenced the levels of expression of the acetic acid and ethanol formation pathways (see above). Nevertheless, the obtained results establish that, although present, the acetic acid formation pathway remains unemployed by L. lactis during fast growth, suggesting that growth rate is not strictly constrained by proteome optimisation under these conditions. Do these findings indicate that constraints in resource allocation and proteome optimisation are not applicable as the predominant determinant of growth rate and/or fitness trade-offs in L. lactis? The most likely answer to this question is ‘no’. A possible explanation could be that primary constraint is not the cytoplasmic proteome of L. lactis, but rather the membrane proteome and/or the cellular transport capacities. This notion appears to be supported by several experimental evolution studies performed with L. lactis MG1363 (Smith et al. 2012; Bachmann et al. 2013; Price et al. 2019). An interesting example is the selection of L. lactis mutants with an improved heat-stress resistance phenotype, which coincided with the appearance of a salt-hypersensitivity phenotype (Smith et al. 2012). The evolved strain carried a mutation in the gene encoding GdpP, which is a membrane bound protein involved in stress signalling via its cyclic dimeric AMP (c-di-AMP)-phosphodiesterase activity that modulates the cytoplasmic levels of c-di-AMP (Smith et al. 2012). The c-di-AMP molecule serves as a second messenger that plays a critical role in regulation of a variety of phenotypes, including osmotic-stress tolerance, in various bacteria (Yin et al. 2020). In L. lactis c-di-AMP was shown to affect osmotic homeostasis by controlling transporters associated with potassium homeostasis and import of osmoprotectants (Pham et al. 2018; Pham and Turner 2019) as well as by its connection to peptidoglycan biosynthesis (Zhu et al. 2016). The mechanism underlying the effect of GdpP-mutation on heat-stress tolerance remains unclear, but it is intriguing that in Streptococcus pneumoniae mutations in two heat shock suppressors were shown to induce changes of c-di-AMP levels and undermine the toxicity associated with this second messenger (Zarrella, Metzger and Bai 2018). The finding that both stress tolerance systems are connected to c-di-AMP in L. lactis, combined with the prominent role of this compound in controlling membrane proteome function, would support the hypothesis that membrane proteome constraints underlie the observed heat- and salt-stress fitness trade-off. In addition, experimental evolution to obtain L. lactis MG1363 mutants that were either optimised for biomass yield rather than growth rate (Bachmann et al. 2013) or displaying improved fitness under glucose limitation (Price et al. 2019) led to the isolation of mutants with an altered membrane proteome that resulted in opposing adjustments of glucose import capacity. The biomass-yield optimised strains that were selected using a water-in-oil droplet culturing regime displayed a typical mixed acid fermentation that coincided with an ∼25–40% reduced acidification rate. The study identified an amino-acid change in a component of the only high-affinity glucose PTS (Castro et al. 2009), PtnD (F65L), that led to reduced glucose import rates observed in this strain (Bachmann et al. 2013). These findings agree with the hypothesised membrane proteome constraint underlying the investigated trade-off between growth rate and biomass yield. Mutation analysis of the evolved strains selected for improved fitness under glucose limitation, identified amino acid substitution in CcpA (M19I or M19T) as the causative mutation. The mutation altered the affinity of CcpA for specific sequence-variants of its target cis-acting element, the cre-box. The CcpA affinity change led to a modified pattern of expression, including both increased and decreased transcriptional activity of multiple genes belonging to the CcpA regulon, particularly affecting the transcriptional landscape of genes encoding carbohydrate transport systems. The transcriptional changes included the elevated expression of the ptn operon encoded high-affinity glucose PTS, in parallel with reduced expression of the ptc operon encoded low-affinity glucose PTS (Castro et al. 2009). These changes in glucose import system expression led to a 3-fold accelerated glucose import rate in the evolved strain, which explained the improved fitness under glucose-limiting conditions. The pleiotropic effects on the CcpA-regulon were also shown to negatively affect the capacity of the evolved strain to utilise alternative carbon sources due to reduced expression levels of the import systems for these sugars (Price et al. 2019). Thereby this evolved strain has become more specialised for conditions with limited glucose availability, but at the same time has apparently lost part of its adaptability to environmental conditions that contain alternative carbon sources. These experimental evolution studies agree with the hypothesis that constraints in resource allocation, proteome composition and transport functions are associated with boundaries in the membrane proteome of L. lactis. ADAPTATION TO THE DAIRY NICHE The adaptation of L. lactis to the dairy niche has been studied in many ways, each of them addressing specific aspects of the L. lactis lifestyle during its most prevalent biotechnological application. The comparative analysis of L. lactis genomes confirms the hypothesis that dairy isolates have evolved from plant isolates, which is exemplified by the dairy isolates are characterised by the loss of several glycoside utilisation gene clusters that are dispensable in the dairy niche but are important for growth on plant material (Siezen et al. 2008; Passerini et al. 2010; Bachmann et al. 2012; Price et al. 2012; Cavanagh, Fitzgerald and McAuliffe 2015; Golomb and Marco 2015; Wels et al. 2019). In general, the genomic adaptations to the dairy environment appear to be predominated by events of loss of function, which is contrasted by the observation that gain of function in dairy adapted L. lactis strains is often associated with the acquisition of mobile genetic elements, i.e. plasmids (see also above). As an example of dairy-associated loss-of function, it was recently established that engineered expression of the regulator ComX could activate the competence phenotype in specific L. lactis plant isolates (David et al. 2017; Mulder et al. 2017), whereas a variety of disruptive mutations in late-competence genes was found in all L. lactis strains isolated from the dairy environment (Wydau et al. 2006; Mulder et al. 2017). This finding suggests that natural competence could compromise environmental fitness of L. lactis in the dairy environment, which is a striking contrast with the consistent presence and functionality of natural competence associated genes in another dairy LAB-workhorse, Streptococcus thermophilus (Fontaine et al. 2010; Fontaine et al. 2013). The loss of competence in dairy adapted L. lactis strains is possibly related to the activation of competence expression under carbon-starvation conditions (Redon, Loubiere and Cocaign-Bousquet 2005; Ercan et al. 2015a) that may occur during cheese production (see also below). This suggestion is supported by the reported activation of comX expression during cheese production (Bachmann et al. 2010). Although the precise environmental trigger and regulatory mechanism involved in competence activation in L. lactis remain to be deciphered, competence activation during dairy fermentation could lead to genomic instability, which could provide the selective pressure that favours the loss of this function. The genome adaptations of plant-derived L. lactis isolates to the dairy environment (e.g. a process termed ‘domestication’) were investigated by experimental evolution that involved 1000 generations of growth of the plant isolate L. lactis subspecies lactis KF147 in milk. The original strain (Siezen et al. 2010) does not grow efficiently in milk but prolonged cultivation in this medium allowed the isolation of mutant derivatives that displayed a significantly improved growth rate in milk (Bachmann et al. 2012). The genome analysis of these ‘domesticated’ mutants revealed gain of function mutations in amino acid biosynthesis and transport functions as well as the loss of an integrated conjugative element (ICE) that encodes functions associated with growth on plant derived materials (Bachmann et al. 2012). Moreover, comparative transcriptome analyses of the original KF147 strain, its milk-adapted derivatives and a typical dairy isolate of L. lactis confirmed that the ‘domesticated’ mutants displayed an increased transcriptome-resemblance with the dairy isolate. Relative to its parental strain KF147, the transcriptional changes observed in the milk-adapted mutants reflected its adaptation to the dairy niche, including the activation of various nitrogen metabolism functions and the repression of many carbohydrate utilisation pathways (Bachmann et al. 2012). These genomic adaptation signatures clearly illustrate that experimental domestication approaches can recapitulate some of the genome differences detected by comparative genomics of L. lactis strains isolated from different environmental niches. However, it should be noted that this experimental evolution experiment ‘only’ encompassed 1000 generations of growth in milk, while the hypothesised progressive loss of glycoside utilisation genes is likely to require prolonged cultivation in milk. ADAPTIVE RESPONSES DURING DAIRY APPLICATIONS The transcriptional responses of L. lactis during growth in milk and cheese production conditions have been investigated. Lactococcus lactis is especially important in the production of semi-hard cheeses, where it plays a key role in texture and flavour properties of the fermented product (see above). The environmental conditions encountered during cheese production and ripening are highly dynamic and depend on the specific protocol employed for production of different semi-hard cheeses like Cheddar or Gouda. These protocols expose the lactococci to variable temperature regimes, rapid decreases in pH by conversion of lactose to lactate, followed by carbon starvation during ripening, as well as increases in osmolarity during brining, and decreases in water activity after curd separation from whey (Fig. 2). Figure 2. Open in new tabDownload slide Schematic representation of starter culture production for direct vat inoculations and a Gouda-type cheese manufacturing protocol. The generations column is an estimate of the cumulative number to generations made by the starter culture during cheese making. Starter culture cells are exposed to rapid environmental changes such as temperature, pH, salt, water availability and sugar concentrations (periods of change are indicated). Figure 2. Open in new tabDownload slide Schematic representation of starter culture production for direct vat inoculations and a Gouda-type cheese manufacturing protocol. The generations column is an estimate of the cumulative number to generations made by the starter culture during cheese making. Starter culture cells are exposed to rapid environmental changes such as temperature, pH, salt, water availability and sugar concentrations (periods of change are indicated). Various studies focused on the response and coping mechanisms of L. lactis using a variety of stress conditions that are considered important during cheese production processes. This has led to a relatively comprehensive view of overall stress responses in this species (for a review, see Papadimitriou et al. 2016). However, most stress-response studies addressed a single environmental stress and often employed laboratory media rather than milk. Although these studies may adequately reflect part(s) of the lactococcal response to dairy applications, they fail to recapitulate the application's environmental complexity and its impact on global gene expression control. Several studies have described the responses of L. lactis to growth and acidification in milk using transcriptome (Raynaud et al. 2005; de Jong et al. 2013; Larsen, Brosted Werner and Jespersen 2016a, Larsen et al. 2016b) and proteome (Gitton et al. 2005) analyses. These studies highlighted dynamic changes in expression of carbohydrate-, nitrogen- and nucleotide-metabolism as well as ion-transport and stress response functions, indicating the wide-ranging impact of growth in milk on the overall physiology of L. lactis. The observed responses included the anticipated increased expression of peptide transport systems, peptidolytic enzymes and amino-acid biosynthesis and interconversion functions, which were especially apparent at later timepoints of fermentation (Gitton et al. 2005; de Jong et al. 2013; Larsen et al. 2016b). In addition, several studies highlight the important transcriptional consequences of oxygen presence in milk and its depletion during early stages of lactococcal growth (de Jong et al. 2013; Cretenet et al. 2014; Larsen, Brosted Werner and Jespersen 2016a, Larsen et al. 2016b). However, the results of these studies are difficult to compare because they employ different milk-medium preparations, culturing regimes and sampling timepoints. Moreover, and maybe most importantly, these studies use different reference samples for the normalisation of the data obtained during growth in milk. Finally, only one study presents an integrative view of the transcriptome profiles during growth in milk and uses the data to reconstruct a time-resolved gene-regulation network (de Jong et al. 2013). In contrast, the other studies investigated transcriptional profiles at only a few timepoints during growth in milk, which provides important, but limited, insights in the dynamics of milk-adaptive behaviour of L. lactis. To decipher the adaptive response of L. lactis during cheese production and ripening, an adapted recombinase-based in vivo expression technology (R-IVET) screening (for a review, see de Vos, Bron and Kleerebezem 2004) was implemented in model strain MG1363. The screening system combined the typical R-IVET antibiotic-resistance based selection of in situ activated promoters with the downstream quantitative in situ analysis of promoter activity in a time-resolved manner, employing the bacterial luciferase encoding luxAB genes as promoter probe (Bachmann, Kleerebezem and van Hylckama Vlieg 2008). This R-IVET approach was employed in different LAB to identify promoters that are activated during dairy application conditions (Bachmann et al. 2010; Junjua et al. 2014), including the time-resolved analysis of promoter activation in L. lactis MG1363 during Gouda-type cheese production conditions (Fig. 2) using a miniaturised, 96-well-based cheese production set-up (Bachmann et al. 2009, 2010). The study identified close to 100 promoter elements in the L. lactis MG1363 genome that were activated during cheese production and ripening compared with laboratory culture conditions. Analysis of the genes under control of these in situ activated promoters highlighted adaptation in carbon metabolism, in particular the activation of the pentose-phosphate pathway, as well as the activated expression of nucleotide import systems, indicative of nucleotide starvation during the application in cheese. The data also prominently supported the activation of amino acid and peptide transport as well as several amino acid biosynthesis and interconversion pathways (Bachmann et al. 2010), which were proposed to be controlled by the global nitrogen metabolism regulator CodY (for a review, see Kok et al. 2017) and further confirmed the importance of adaptation of nitrogen metabolism during cheese production. Moreover, brining activation was observed for promoters located upstream of genes involved in acid tolerance that have been reported to be inducible by chloride exposure (Sanders et al. 1998), confirming the accuracy of the R-IVET screening model to reflect the environmental adaptations of the gene expression repertoire in these cells. Even though L. lactis MG1363 lacks several functions, disallowing it to grow autonomously in milk, this study started to unravel the in situ behaviour of L. lactis in an unprecedented time-resolved manner, which provided a first glimpse at this species’ adaptive behaviour during cheese production. With the decreasing costs involved in high-throughput sequencing, recent studies have started to assess the population dynamics and in situ gene expression of complex cheese starters during cheese production, using metagenomic and metatranscriptomic approaches. Several studies reported on the LAB-population dynamics during industrial and artisanal cheese production, detecting not only the starter culture-derived bacteria but also the non-starter LAB that are particularly important in several cheese types. These studies have targeted a variety of different cheese products including the Dutch Gouda (Erkus et al. 2013; Erkus et al. 2016), and Maasdam (Swiss-type) cheeses (Duru et al. 2018; Mataragas 2020), as well as different (artisanal) cheeses from other geographic origins, including Italian (De Filippis et al. 2016), French (Dugat-Bony et al. 2015), Mexican (Escobar-Zepeda, Sanchez-Flores and Quirasco Baruch 2016) and Belgian (Delcenserie et al. 2014) cheeses. In several of these systems, (multiple) L. lactis strains are dominating the microbial community during cheese production and metagenome analysis offers insight in their abundance over time, while metatranscriptome analysis can assess their gene expression over time. Comparative metatranscriptome analysis to decipher the general lactococcal gene expression profiles during cheese production is complicated by the different strains used in these processes, as well as the variable production regimes of the different cheese-types. Moreover, analogous to the transcriptome studies performed for L. lactis grown in milk (see above), the number of timepoints that is generally analysed in these studies is quite limited and provides not more than a snapshot of the lactococcal activity profile. For example, the study of the lactococcal gene expression profile during Maasdam cheese production employed triplicate samples taken at two stages of the ripening period, i.e. after 12 days of the 30-day initial ‘warm-room’ (20°C) ripening and 7 days after the transfer to the second stage 30-day ripening period in the ‘cold-room’ (5°C) (Duru et al. 2018). Although the study does not allow true time-resolved expression analysis, it did reveal the sustained expression of central carbon metabolism associated genes in L. lactis, also during the cold-room ripening period. This sharply contrasted the observed decline of activity seen for the other members of the microbial community involved in the production of this type of cheese, which include Lactobacillus rhamnosus, Lactobacillus helveticus and Propionibacterium freudenreichii that collectively represent 10–20% of the overall cheese microbiota (Duru et al. 2018). This finding implies that the propionic acid fermentation that is of importance in the ripening of this type of cheese appears to be constrained to the ripening period at 20°C where the responsible organism (Propionibacterium freudenreichii) is still actively expressing its metabolic functions. Despite the limitations of the presently available studies, the pioneered metagenome and metatranscriptome approaches are appropriate to unravel the adaptive behaviour of starter cultures during the dynamic environmental conditions encountered during cheese production processes. It is important to align approaches and technologies applied in different cheese model systems, to enable the comparative analysis of lactococcal adaptation to different cheese environments and to eventually decipher the common and/or product-specific responses of the species to these dairy applications. Improved understanding of the activity profiles of L. lactis and other LAB during cheese ripening may offer new approaches to use ripening marker-genes for the selection of novel starter strains with improved ripening performance, or could provide clues towards alterations in the production process conditions that modulate or accelerate ripening (De Filippis et al. 2016). THE IMPORTANCE OF MICROBIAL DORMANCY IN CHEESE PRODUCTION In natural reservoirs, L. lactis cells probably spend more time in a non-growing state than in intermittent periods of (fast) growth. Similarly, during most of the cheese ripening period, L. lactis is also in a senescent or dormant state, which is due to the low-level availability and rapid depletion of lactose during cheese production (Fig. 2; see also below). There is only limited knowledge of the physiology of lactococcal cells in such a dormant state. Most of our knowledge about L. lactis physiology is based on studies employing batch cultures grown in laboratory media that are rich in carbon sources and allow rapid acidification, and where the eventual arrest of growth is caused by the lowering of pH in combination with the accumulation of organic acid. The dormancy state in bacterial cultures is characterised by two commonly emerging physiological states, e.g. the persister and the viable-but-not-culturable (VBNC) (sub)populations. VBNC (sub)populations retain metabolic activity but are unable to spontaneously resume growth when transferred to growth-supporting conditions, whereas persister (sub)populations are able to do so (for reviews, see Hayes and Low 2009; Oliver 2010; Ayrapetyan, Williams and Oliver 2018). Dormancy states have been described for many microorganisms and are considered the prevalent state in various natural environments, which is exemplified by the reported fraction of dormant cells in soil samples that can exceed 80% (Lennon and Jones 2011). Microbes have been described to enter this dormancy state upon the exposure to stressful conditions, such as an unfavourable temperature or pH, the presence of antibiotics or starvation (Ayrapetyan, Williams and Oliver 2018). The evolutionary relevance of dormancy states is probably related to the improved stress resistance of dormant cells, whereby fitness and survival is improved in environments that are variable and include transient and/or long-term harsh conditions that are incompatible with growth. This is underpinned by the observation that VBNC L. lactis cells are able to survive for long periods of time, i.e. for >10 years (Weimer 2011). VBNC cells have been described to emerge in cultures of L. lactis and other LAB in response to carbon starvation and low pH (Ganesan, Stuart and Weimer 2007; El Arbi et al. 2011; van Mastrigt et al. 2018a) and during food production processes such as cheese ripening (Ruggirello, Dolci and Cocolin 2014). It has been demonstrated that carbon starved VBNC cells can still transcribe genes and utilise amino acids as carbon and energy source, which leads to the production of flavour compounds such as branched-chain fatty acids (Ganesan, Dobrowolski and Weimer 2006). In addition, the contribution of VBNC cells to bioconversion of raw materials has been demonstrated, where for example, it was shown that VBNC Lactobacillus hilgardii can contribute to malolactic fermentation occurring during apple cider production (Quirós et al. 2009). Recently, it was described that exposure of L. lactis MG1363 to ampicillin leads to the emergence of both VBNC and persister subpopulations (van Tatenhove-Pel et al. 2019). Ampicillin-induced persister cells represented only a minor subpopulation (0.3%) when exponentially growing cultures were used, but increased substantially (7.6%) when cells were obtained from the stationary phase of growth (van Tatenhove-Pel et al. 2019). The simultaneous co-occurrence of VBNC and persister subpopulations has also been described for other organisms (Orman and Brynildsen 2013; Ayrapetyan et al. 2015) and mechanisms that have been proposed to explain this heterogeneous culture response include the involvement of toxin–antitoxin systems, ppGpp-mediated stringent responses and intracellular ATP levels. However, none of these mechanistic explanations adequately explains the dormancy continuum that includes VBNC and persister subpopulation formation in L. lactis (van Tatenhove-Pel et al. 2019). Taken together, these findings indicate that it is important to better understand the physiology of dormant L. lactis cells that can emerge during cheese ripening and/or carbon starvation, or by near-zero growth rate conditions (van Mastrigt et al. 2018a). Therefore, the investigation of L. lactis cells that are in a dormant state or growing at extremely low rates could provide an appropriate impression of the lactococcal physiology during cheese production and ripening. Mimicking such conditions in the laboratory may serve as a proxy for the quantitative assessment of lactococcal metabolic activities under cheese ripening conditions, including its capacity for flavour formation. ADAPTIVE RESPONSES TO ZERO GROWTH; RETENTOSTAT CULTIVATION Retentostat cultivation provides an approach to study the adaptation of microorganisms to long periods of nutrient limitation and extremely low growth rates. A retentostat is a modification of the well-established chemostat cultivation method, in which a biomass filter is connected to the effluent line to retain the biomass inside the bioreactor. Upon prolonged retentostat cultivation, the culture reaches a stage where all nutrient-derived energy is allocated to maintenance and growth completely stagnates, which is referred to as zero growth. The physiological adaptation to zero growth has been investigated in several LAB, including different strains of L. lactis, but also Leuconostoc mesenteroides and Lactobacillus plantarum (Goffin et al. 2010; Ercan, Smid and Kleerebezem 2013; van Mastrigt et al. 2018a, 2019a). During long-term retentostat cultivation the growth rate of L. lactis decreased to 0.0006–0.001 h−1, corresponding to generation times of a month to even a year. These cultures consistently retained a high level of viability according to live/dead staining analysis, although part of the population appeared to have entered a VBNC state (Ercan, Smid and Kleerebezem 2013; van Mastrigt et al. 2018a). Remarkable differences in maintenance energy requirement were reported for the L. lactis plant isolate KF147 and the dairy isolate FM03-V1. The common approach to estimate maintenance energy requirements of a microbe is based on the extrapolation of the energy requirements in chemostat cultivations at different growth rates. For L. lactis KF147 this estimate was very similar to the maintenance energy determined by prolonged retentostat cultivation (Ercan, Smid and Kleerebezem 2013), whereas for L. lactis FM03-V1 this estimate was >6-fold higher than that determined when cells were in a zero growth state. This observation was further confirmed by growing L. lactis FM03-V1 at a rate below 0.025 h−1 using a partial cell-recycling chemostat (van Mastrigt et al. 2019b). Comparison of the maximum biomass yield and maintenance requirements of the plant and dairy L. lactis strains revealed that the plant-isolate KF147 has an ∼2-fold reduced energy requirement for biomass formation and spends ∼4-fold less energy on maintenance when cultured at growth rates higher than 0.1 h−1. Nevertheless, the maintenance energy requirement in a zero growth state was very similar for both strains (Ercan, Smid and Kleerebezem 2013; van Mastrigt et al. 2018a). Another difference observed during retentostat cultivation of the two L. lactis strains was their pyruvate dissipation profile, where L. lactis FM03-V1 consistently displayed the mixed acid fermentation profile that is expected during low growth rate and glucose limitation (van Mastrigt et al. 2018a), whereas L. lactis KF147 displayed an unstable pyruvate dissipation profile that fluctuated between homolactic and mixed acid fermentation over time (Ercan, Smid and Kleerebezem 2013), which remains to be understood. These strain-specific energy requirements for growth and maintenance imply that the plant isolate L. lactis KF147 is continuously in an energy-saving mode, whereas the dairy isolate L. lactis FM03-V1 only enters such mode in a zero growth state. This physiological difference may reflect the long-term adaptation of the plant isolate to the energy-poor plant environment and/or the dairy isolate to the energy-rich dairy environment. The latter suggestion is supported by the observation that the energy household of another dairy-isolated LAB, Leuconostoc mesenteroides FM06, resembled that of L. lactis FM03-V1 (van Mastrigt et al. 2018a, 2019a). PROTEOME ADAPTATION AND MANAGEMENT DURING ZERO GROWTH It has been proposed that protein turnover is the main determinant of energy requirement in L. lactis (Lahtvee et al. 2014; Kempes et al. 2017). Based on studies of the behaviour of L. lactis during retentostat cultivation, it was hypothesised that energy-saving adaptations in these bacteria are driven by changing proteome management from the energetically costly breakdown and subsequent re-synthesis of damaged proteins, towards disaggregation of damaged proteins and their chaperone-assisted refolding (Mogk, Bukau and Kampinga 2018). This hypothesis is supported by time-resolved transcriptome studies of L. lactis KF147 during more than a month of retentostat cultivation (Ercan et al. 2015b), revealing the progressive upregulation of stress response functions. These functions included the chaperones ClpB, DnaK, DnaJ and GrpE that cooperate in disaggregation and refolding of proteins, as well as in the upregulation of the so-called hibernation-promoting factor YfiA (Puri et al. 2014b) that increases ribosome dimerisation and thereby lowers translation (Fig. 3). This reduced translation capacity is in agreement with the reduced expression during zero growth of components of the translational machinery, including ribosomal protein and amino acyl tRNA synthases. Notably, stress response activation and reduced translation capacity appears to be a conserved response to retentostat cultivation in various microbes, including Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus niger (Jørgensen et al. 2010; Boender et al. 2011; Overkamp et al. 2015; Ercan et al. 2015c). Refolding of damaged proteins and slowing-down protein translation reduces the energy requirements associated with de novo protein biosynthesis and is beneficial during long periods of nutrient limitation. However, these adaptations have a trade-off because they reduce the adaptability of the proteome that is required to enable growth in new environments, which may (in part) underlie the observed inability of the VBNC population to form colonies when plated on rich media. However, this trade-off is (in part) compensated by the progressive relief of repression of several operons involved in carbohydrate import and utilisation that occurs during prolonged retentostat cultivation of L. lactis KF147, and which was shown to support the accelerated utilisation of these carbohydrates when they become available (Ercan et al. 2015a,b). This kind of lifestyle adaptation towards a more generalised phenotype has also been observed in other microorganisms during prolonged retentostat growth (Ercan et al. 2015c) and is a typical adjustment observed in microbes that live in environments that are notoriously poor in nutrients (Egli 2010; Hobbie and Hobbie 2013). In the context of energy-saving adaptations that occur in proteome management during zero growth conditions, it is quite remarkable that L. lactis FM03-V1 appears to maintain its plasmid complement with unaltered copy-numbers under these conditions, which underpins the autonomy of the replication of these extrachromosomal elements (van Mastrigt et al. 2018d). Figure 3. Open in new tabDownload slide Proteome management at near-zero growth rates is shifting towards repairing damaged proteins rather than breakdown and de novo synthesis. Panel (A): Expression of genes related to protein turnover, e.g. protein synthesis, disaggregation, (re)folding, degradation during retentostat cultivation of L. lactis KF147 (Ercan et al. 2015b). Colours represent the relative expression (log2 scaled) compared with the initiation of retentostat cultivation where cells grow at a rate of 0.025 h−1. Panel (B): Schematic representation of the proposed model of protein turnover adaptation in L. lactis at high and near-zero growth rates. The thickness of the arrows intends to represent the relative conversion rates. At near-zero growth rates, bacteria are proposed to switch from protein degradation and de novo re-synthesis (steps v and vi) to disaggregation and refolding (steps iv and i) of misfolded proteins, which is supported by the induction of the ClpB-DnaKJ system as well as ribosome hibernation factor YfiA (Puri et al. 2014b) (see panel A). Arrow numbers indicate the different steps in protein turnover: (i) protein folding or refolding, (ii) unfolding, (iii) aggregation, (iv) disaggregation, (v) breakdown to amino acids and (vi) de novo synthesis. Figure 3. Open in new tabDownload slide Proteome management at near-zero growth rates is shifting towards repairing damaged proteins rather than breakdown and de novo synthesis. Panel (A): Expression of genes related to protein turnover, e.g. protein synthesis, disaggregation, (re)folding, degradation during retentostat cultivation of L. lactis KF147 (Ercan et al. 2015b). Colours represent the relative expression (log2 scaled) compared with the initiation of retentostat cultivation where cells grow at a rate of 0.025 h−1. Panel (B): Schematic representation of the proposed model of protein turnover adaptation in L. lactis at high and near-zero growth rates. The thickness of the arrows intends to represent the relative conversion rates. At near-zero growth rates, bacteria are proposed to switch from protein degradation and de novo re-synthesis (steps v and vi) to disaggregation and refolding (steps iv and i) of misfolded proteins, which is supported by the induction of the ClpB-DnaKJ system as well as ribosome hibernation factor YfiA (Puri et al. 2014b) (see panel A). Arrow numbers indicate the different steps in protein turnover: (i) protein folding or refolding, (ii) unfolding, (iii) aggregation, (iv) disaggregation, (v) breakdown to amino acids and (vi) de novo synthesis. The transcriptome analysis of L. lactis K147 during retentostat cultivation revealed that besides the elevated expression of the dnaKJgrpE operon also other stress responses were induced, including heat shock (groELS) membrane-associated stress (hemG, lmrA, rmaB, yhbF), acid (uvrB, uspA, rcfB), cold shock (cspA1, cspB1, cspC1, cspD, cspA2, cspB2, cspC2) and membrane-associated stress (hemG, lmrA, rmaB, yhbF) (Ercan et al. 2015b). Stress tolerance analysis indicated that these responses corresponded to >1000-fold higher resistance to heat and acid stress and an ∼10-fold higher resistance to oxidative stress (Ercan 2014). Notably, the expression level of these stress-related genes displayed a strong inverse correlation with the growth rate of L. lactis KF147, exemplifying the trade-off between growth rate and stress robustness in Lactococcus lactis (Ercan 2014). ZERO GROWTH CONDITIONS AS A REFLECTION OF CHEESE RIPENING PHYSIOLOGY Cheese ripening involves long-term incubation periods that are required for the development of flavour volatile profiles that are determining the taste of the cheese. Ripening may take several years, e.g. up to 3 years for the production of Parmigiano-Reggiano cheese. Thereby, the contribution of starter cultures, including L. lactis, to the formation of these flavour volatiles is largely dependent on cells that are in a zero growth state. Importantly, the conversion of amino acids into cheese-relevant flavour volatiles was reported to be higher in non-growing compared with growing L. lactis cells (van de Bunt et al. 2014), and the amino-acid catabolic pathways involved were shown to be induced under starvation conditions that coincide with the emergence of VBNC (sub)populations (Ganesan, Stuart and Weimer 2007). Therefore, analysis of flavour formation capacities during zero growth could provide more insight in the flavour-forming capacities of L. lactis during cheese ripening. Zero growth cultures of L. lactis FM03-V1 produced several aroma compounds that are also present in cheese produced with this strain, while fast-growing cells of this strain failed to do so. These flavour compounds included several amino acid catabolism-derived metabolites of which the production rate was progressively increasing during prolonged retentostat cultivation (van Mastrigt et al. 2018a,b). Lactococcus lactis KF147 displayed a progressively declining amino acid consumption level during the initial 3 weeks of retentostat cultivation, which is likely to reflect the decreased amino acid requirement for protein synthesis when growth rate declines towards near-zero growth rates (Ercan et al. 2015a,b). However, prolongment of retentostat cultivation led to elevated consumption of amino acids, which coincided with the appearance of VBNC subpopulations in the culture (Ercan, Smid and Kleerebezem 2013). Unfortunately, the formation of amino acid catabolism-derived flavour compounds was not determined during L. lactis KF147 retentostat cultivation (Ercan, Smid and Kleerebezem 2013), but it is tempting to speculate that the elevated amino acid consumption during long-term retentostat cultivation coincides with the formation of aroma components analogous to what was found for L. lactis FM03-V1. Notably, prolonged retentostat cultivation of L. lactis FM03-V1 did not lead to the production of branched-chain fatty acids that were previously reported to be produced by starvation-induced VBNC subpopulations of different L. lactis strains (Ganesan, Dobrowolski and Weimer 2006; Ganesan, Stuart and Weimer 2007). Although these differences agree with the notion that different strains produce different flavour compound profiles, they could also imply that regulation of amino acid catabolism is differentially regulated by the conditions used to induce growth stagnation and VBNC formation. Therefore, in order to predict the functional properties and flavour forming potential of L. lactis strains during cheese ripening, it is important to further elucidate the mechanisms underlying the regulation of amino acid catabolism during zero growth and/or VBNC lifestyles. FUTURE PERSPECTIVES This review highlights genomic and lifestyle differences of L. lactis inhabiting different environmental niches and explores our understanding of mechanisms involved in lifestyle adaptations when these bacteria migrate between different environments. Lactococcal gene regulation and metabolic control have been extensively studied in several model strains, mostly employing laboratory media and conditions that support relatively high growth rates. Taken together these studies indicate that the metabolism of L. lactis is optimised for maximal acidification rather than maximal growth rate and biomass yield. This optimisation is controlled by multiple regulatory mechanisms that are strongly intertwined, including the interplay between gene-regulation networks, carbon source import systems, glycolytic-flux, redox-balance and pyruvate dissipation pathways. Despite the recognition of the role of the multiple factors involved, our understanding of the observed switch between homolactic and mixed acid fermentation remains incomplete. Further development of advanced metabolic models that include the integration of cost-benefit calculations for the synthesis of required proteins as well as constraints in membrane transport capacities may help to achieve accurate predictions of the metabolic lifestyle of L. lactis under different conditions. Some observations suggest that the history and niche adaptation of L. lactis strains are reflected in its general lifestyle as well as its adaptive responses during changes in environmental conditions. This is exemplified by the distinct characteristics in carbon metabolism and energy household observed for dairy and plant isolates of L. lactis during retentostat cultivation. These differences indicate that there is a need to expand our understanding of the regulation of gene expression and metabolism to different L. lactis strains in order to prevent building a knowledge base that is detailed and accurate for a model strain like L. lactis MG1363, but fails to represent other strains of the species. Consequently, the construction of a generic metabolic model for the species L. lactis would require the inclusion of some form of modelling flexibility that allows the accommodation of strain-specific physiological characteristics and its adaptation during environmental changes. Experimental evolution has been shown to be instrumental in revealing constraints that govern lifestyle adjustments in L. lactis, as well as the phenotypic trade-offs that are the consequences of these constraints. Detection of such trade-offs and the exploration of the constraints that cause them could pave the way to unravelling the ‘design principles’ underlying the lactococcal lifestyle and its adaptability under dynamic conditions, including those that are encountered during dairy fermentation processes. Similar to what is concluded above, studies targeting such constraints and their consequential phenotypic trade-offs should preferably be expanded beyond the often-used model strains. Recent advances highlighted the importance of dormancy states of L. lactis subpopulations during the ripening stages of cheese production, where these bacteria play a critical role in flavour formation. Retentostat cultivation that aims to achieve near-zero growth rates appears to be a suitable laboratory approach to mimic the lifestyle of L. lactis during the ripening of cheese. Deciphering the mechanisms that regulate the adaptation to these conditions, including the control of amino acid catabolic pathways, can provide novel selection criteria for the identification of improved flavour-forming strains and/or increased control of flavour formation during cheese production. Moreover, understanding the regulatory process that controls the phenotype generalisation in zero growth cultures, which include the expansion of carbohydrate utilisation repertoires and increased stress robustness, may reveal mechanisms that are conserved in many microbes because analogous phenotypic responses are common during nutrient limitation and zero growth. Conflicts of interest None declared. References Ainsworth S , Mahony J, van Sinderen D. The plasmid complement of Lactococcus lactis UC509.9 encodes multiple bacteriophage resistance systems . Appl Environ Microbiol . 2014 ; 80 : 4341 – 9 . 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TI - Lifestyle, metabolism and environmental adaptation in Lactococcus lactis
JF - FEMS Microbiology Reviews
DO - 10.1093/femsre/fuaa033
DA - 2020-11-24
UR - https://www.deepdyve.com/lp/oxford-university-press/lifestyle-metabolism-and-environmental-adaptation-in-lactococcus-b01Zbi00Nj
SP - 804
EP - 820
VL - 44
IS - 6
DP - DeepDyve
ER -