Showing papers on "Lactococcus lactis published in 2005"
TL;DR: Various enzymes and pathways involved in flavour formation will be highlighted and the impact of these findings for the development of industrial starter cultures will be discussed.
Abstract: Flavour development in dairy fermentations, most notably cheeses, results from a series of (bio)chemical processes in which the starter cultures provide the enzymes. Particularly the enzymatic degradation of proteins (caseins) leads to the formation of key-flavour components, which contribute to the sensory perception of dairy products. More specifically, caseins are degraded into peptides and amino acids and the latter are major precursors for volatile aroma compounds. In particular, the conversion of methionine, the aromatic and the branched-chain amino acids are crucial. A lot of research has focused on the degradation of caseins into peptides and free amino acids, and more recently, enzymes involved in the conversion of amino acids were identified. Most data are generated on Lactococcus lactis, which is the predominant organism in starter cultures used for cheese-making, but also Lactobacillus, Streptococcus, Propionibacterium and species used for surface ripening of cheeses are characterised in their flavour-forming capacity. In this paper, various enzymes and pathways involved in flavour formation will be highlighted and the impact of these findings for the development of industrial starter cultures will be discussed.
749 citations
TL;DR: An extensive overview is provided of the different applications in lactococci and other Gram-positive bacteria: over-expression of homologous and heterologous genes for functional studies and to obtain large quantities of specific gene products, metabolic engineering, and large-scale applications.
Abstract: Lactococcus lactis is a Gram-positive lactic acid bacterium that, in addition to its traditional use in food fermentations, is increasingly used in modern biotechnological applications. In the last 25 years great progress has been made in the development of genetic engineering tools and the molecular characterization of this species. A new versatile and tightly controlled gene expression system, based on the auto-regulation mechanism of the bacteriocin nisin, was developed 10 years ago-the NIsin Controlled gene Expression system, called NICE. This system has become one of the most successful and widely used tools for regulated gene expression in Gram-positive bacteria. The review describes, after a brief introduction of the host bacterium L. lactis, the fundaments, components and function of the NICE system. Furthermore, an extensive overview is provided of the different applications in lactococci and other Gram-positive bacteria: (1) over-expression of homologous and heterologous genes for functional studies and to obtain large quantities of specific gene products, (2) metabolic engineering, (3) expression of prokaryotic and eukaryotic membrane proteins, (4) protein secretion and anchoring in the cell envelope, (5) expression of genes with toxic products and analysis of essential genes and (6) large-scale applications. Finally, an overview is given of growth and induction conditions for lab-scale and industrial-scale applications.
542 citations
TL;DR: A genome-scale flux model able to simulate and analyze network capabilities and whole-cell function under aerobic and anaerobic continuous cultures and for integration with other types of 'omic' data is developed to assist in finding new information on cellular organization and function.
Abstract: Background
Genome-scale flux models are useful tools to represent and analyze microbial metabolism. In this work we reconstructed the metabolic network of the lactic acid bacteria Lactococcus lactis and developed a genome-scale flux model able to simulate and analyze network capabilities and whole-cell function under aerobic and anaerobic continuous cultures. Flux balance analysis (FBA) and minimization of metabolic adjustment (MOMA) were used as modeling frameworks.
271 citations
TL;DR: Data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins.
Abstract: Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species.
222 citations
Journal Article•
TL;DR: Nisin is a natural, toxicologically safe, antibacterial food preservative as mentioned in this paper, which is a polypeptide produced by certain strains of the food-grade lactic acid bacterium Lactococcus lactis subsp.
Abstract: Nisin is a natural, toxicologically safe, antibacterial food preservative. It is regarded as natural because it is a polypeptide produced by certain strains of the food-grade lactic acid bacterium Lactococcus lactis subsp. lactis (hereafter referred to as L. lactis), during fermentation. Nisin exhibits antimicrobial activity towards a wide range of Gram positive bacteria, and is particularly effective against spores. It shows little or no activity against Gram negative bacteria, yeasts, and moulds.
197 citations
TL;DR: It is shown that CodY regulates its own synthesis and requires the CodY-box and branched-chain amino acids to interact with its promoter, and it is presented that this 15-bp cis-sequence, AATTTTCWGAAAATT, serves as a high affinity binding site for CodY.
183 citations
TL;DR: In vivo nuclear magnetic resonance has proven a very useful technique to monitor non-invasively the dynamics of intracellular metabolite and co-factor pools following a glucose pulse and the use of this information to direct metabolic engineering strategies is illustrated.
Abstract: The wide application of lactic acid bacteria in the production of fermented foods depends to a great extent on the unique features of sugar metabolism in these organisms. The relative metabolic simplicity and the availability of genetic tools made Lactococcus lactis the organism of choice to gain insight into metabolic and regulatory networks. In vivo nuclear magnetic resonance has proven a very useful technique to monitor non-invasively the dynamics of intracellular metabolite and co-factor pools following a glucose pulse. Examples of the application of this methodology to identify metabolic bottlenecks and regulatory sites are presented. The use of this information to direct metabolic engineering strategies is illustrated.
178 citations
TL;DR: The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme, and the highest activity was observed with branched-chain α-keto acids, indicating that the enzyme might be active under cheese ripening conditions.
Abstract: The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain alpha-keto acid decarboxylase (KdcA) The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains By using a random mutagenesis approach, the gene encoding KdcA in L lactis B1157 was identified The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L lactis IL1403 Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3' terminus of the ipd gene encoding a truncated nonfunctional decarboxylase The kdcA gene was overexpressed in L lactis for further characterization of the decarboxylase enzyme Of all of the potential substrates tested, the highest activity was observed with branched-chain alpha-keto acids Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 63, indicating that the enzyme might be active under cheese ripening conditions
175 citations
TL;DR: These results describe for the first time the genes encoding a histidine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.
Abstract: Histamine production from histidine in fermented food products by lactic acid bacteria results in food spoilage and is harmful to consumers. We have isolated a histamine-producing lactic acid bacterium, Lactobacillus hilgardii strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions. The hdcA gene, coding for the histidine decarboxylase of L. hilgardii IOEB 0006, was located on an 80-kb plasmid that proved to be unstable. Sequencing of the hdcA locus disclosed a four-gene cluster encoding the histidine decarboxylase, a protein of unknown function, a histidyl-tRNA synthetase, and a protein, which we named HdcP, showing similarities to integral membrane transporters driving substrate/product exchange. The gene coding for HdcP was cloned downstream of a sequence specifying a histidine tag and expressed in Lactococcus lactis. The recombinant HdcP could drive the uptake of histidine into the cell and the exchange of histidine and histamine. The combination of HdcP and the histidine decarboxylase forms a typical bacterial decarboxylation pathway that may generate metabolic energy or be involved in the acid stress response. Analyses of sequences present in databases suggest that the other two proteins have dispensable functions. These results describe for the first time the genes encoding a histamine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.
168 citations
TL;DR: Data show that lantibiotic enzymes can be applied for the modification of peptides, thereby enabling the biotechnological production of dehydroresidue-containing and/or thioether-bridged therapeutic peptides with enhanced stability and/ or modulated activities.
Abstract: Post-translationally introduced dehydroamino acids often play an important role in the activity and receptor specificity of biologically active peptides. In addition, a dehydroamino acid can be coupled to a cysteine to yield a cyclized peptide with increased biostability and resistance against proteolytic degradation and/or modified specificity. The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. Its post-translational enzymatic modification involves NisB-mediated dehydration of serines and threonines and NisC-catalyzed coupling of cysteines to dehydroresidues, followed by NisT-mediated secretion. Here, we demonstrate that a L. lactis strain containing the nisBTC genes effectively dehydrates and secretes a wide range of medically relevant nonlantibiotic peptides among which variants of adrenocorticotropic hormone, vasopressin, an inhibitor of tripeptidyl peptidase II, enkephalin, luteinizing hormone-releasing hormone, angiotensin, and erythropoietin. For most of these peptides, ring formation was demonstrated. These data show that lantibiotic enzymes can be applied for the modification of peptides, thereby enabling the biotechnological production of dehydroresidue-containing and/or thioether-bridged therapeutic peptides with enhanced stability and/or modulated activities.
157 citations
TL;DR: This is the first antimicrobial peptide to be isolated from any oyster species and it is named American oyster defensin (AOD), which had high sequence homology to arthropod defensins, including those from other bivalves, the mussels MyTilus edulis and Mytilus galloprovincialis.
TL;DR: Because of the antimicrobial activity of pediOCin PA-1, its production in situ by strains of LAB growing efficiently in milk would extend the application of this bacteriocin in cheese manufacture.
TL;DR: Newly identified plasmid-encoded functions could facilitate the uptake of various cations, while the pabA and pabB genes could be essential for folate biosynthesis.
Abstract: Lactococcus lactis strains are known to carry plasmids encoding industrially important traits. L. lactis subsp. cremoris SK11 is widely used by the dairy industry in cheese making. Its complete plasmid complement was sequenced and found to contain the plasmids pSK11A (10,372 bp), pSK11B (13,332 bp), pSK11L (47,165 bp), and pSK11P (75,814 bp). Six highly homologous repB-containing replicons were found, all belonging to the family of lactococcal theta-type replicons. Twenty-three complete insertion sequence elements segment the plasmids into numerous modules, many of which can be identified as functional units or containing functionally related genes. Plasmid-encoded functions previously known to reside on L. lactis SK11 plasmids were now mapped in detail, e.g., lactose utilization (lacR-lacABCDFEGX), the proteolytic system (prtM-prtP, pepO, pepF), and the oligopeptide permease system (oppDFBCA). Newly identified plasmid-encoded functions could facilitate the uptake of various cations, while the pabA and pabB genes could be essential for folate biosynthesis. A competitive advantage could be obtained by using the putative flavin adenine dinucleotide-dependent d-lactate dehydrogenase and oxalate:formate antiporter for enhanced ATP synthesis, while the activity of the predicted alpha-acetolactate decarboxylase may contribute to the formation of an additional electron sink. Various stress response proteins are plasmid encoded, which could enhance strain robustness. A substantial number of these "adaptation" genes have not been described before on L. lactis plasmids. Moreover, several genes were identified for the first time in L. lactis, possibly reflecting horizontal gene transfer.
TL;DR: The different methods used to detect starter lysis in situ and current knowledge concerning the impact of lysis on cheese ripening are described, particularly the increase of free amino acids due to early lysis and the reduction of bitterness by hydrolysis of large hydrophobic peptides.
TL;DR: The majority of the observed resistance appeared to be either intrinsic or nonspecific, although some strains of Lactococcus lactis, Enterococcus spp.
Abstract: In the production of the Spanish traditional blue-veined Cabrales cheese, lactic acid bacteria strains free of antibiotic resistance that have a transferrable capacity are necessary as components of a specific starter. To select for these bacteria, the minimum inhibitory concentration (MIC) of 12 antibiotics and 2 mixtures (containing beta-lactamase inhibitor and penicillin) were determined by microbroth and agar dilution techniques in 146 strains belonging to the genera Lactococcus, Enterococcus, Lactobacillus, and Leuconostoc. The antibiotic-resistance profiles of Lactococcus and Enterococcus species were different from those of Lactobacillus and Leuconostoc, but clear genus- or species-associated patterns were not observed. Cefoxitin and metronidazole were not effective against bacteria of these genera. The MICs of beta-lactam antibiotics for lactobacilli and leuconostoc isolates were higher than those for lactococci and enterococci, but no strain was clinically resistant. All lactobacilli and leuconostoc isolates were resistant to high levels of vancomycin, a type of resistance not seen among the tested members of the genera Lactococcus and Enterococcus. The majority of the observed resistance appeared to be either intrinsic or nonspecific, although some strains of Lactococcus lactis, Enterococcus spp., and Lactobacillus spp. were resistant to antibiotics, such as chloramphenicol, erythromycin, clindamycin, or tetracycline.
TL;DR: The direct role of CodY in the regulation of several target genes was demonstrated by gel retardation experiments, and a conserved 15 nt palindromic sequence, the so-called CodY-box, located in the vicinity of the -35 box of target promoter regions was identified.
Abstract: CodY, a pleiotropic transcriptional regulator conserved in low G+C species of Gram-positive bacteria, was previously described to be the central regulator of proteolysis in Lactococcus lactis. In this study, over 100 potential CodY targets were identified by DNA-microarray analysis. Complementary transcriptional analysis experiments were carried out to validate the newly defined CodY regulon. Moreover, the direct role of CodY in the regulation of several target genes was demonstrated by gel retardation experiments. Interestingly, 45 % of CodY-dependent genes encode enzymes involved in amino acid biosynthesis pathways, while most of the other genes are involved in functions related to nitrogen supply. CodY of L. lactis represents the first example of a regulator in Gram-positive bacteria that globally controls amino acid biosynthesis. This global control leads to growth inhibition in several amino-acid-limited media containing an excess of isoleucine. A conserved 15 nt palindromic sequence (AATTTTCNGAAAATT), the so-called CodY-box, located in the vicinity of the −35 box of target promoter regions was identified. Relevance of the CodY-box as an operator for CodY was demonstrated by base substitutions in gel retardation experiments. This motif is also frequently found in the promoter region of genes potentially regulated by CodY in other Gram-positive bacteria.
TL;DR: The antimicrobial peptide, nisin, produced by several strains of Lactococcus lactis, which belongs to the Class I bacteriocins called lantibiotics, is a small, 34-amino acid, cationic, hydrophobic peptide and has the five characteristic (β-methyl)lanthionine rings formed by significant post-translational modification.
Abstract: The antimicrobial peptide, nisin, produced by several strains of Lactococcus lactis, which belongs to the Class I bacteriocins called lantibiotics, is a small (3.4 kDa), 34-amino acid, cationic, hydrophobic peptide and has the five characteristic (β-methyl)lanthionine rings formed by significant post-translational modification. A cluster of 11 genes has been involved in the biosynthesis of nisin and are proposed to be transcriptionally arranged as nisA(Z)BTCIP, nisRK, and nisFEG. The biosynthesis of nisin is regulated in a growth-phase-dependent manner including nisin-mediated induction which occurs via NisRK two-component regulatory system. This review outlines some of the more recent developments in the properties, regulation and applications of nisin biosynthesis.
TL;DR: AcmA, the major autolysin of Lactococcus lactis MG1363 is a modular protein consisting of an Nterminal active site domain and a C-terminal peptidoglycan-binding domain this paper.
Abstract: AcmA, the major autolysin of Lactococcus lactis MG1363 is a modular protein consisting of an N-terminal active site domain and a C-terminal peptidoglycan-binding domain. The active site domain is homologous to that of muramidase-2 of Enterococcus hirae, however, RP-HPLC analysis of muropeptides released from Bacillus subtilis peptidoglycan, after digestion with AcmA, shows that AcmA is an N-acetylglucosaminidase. In the C-terminus of AcmA three highly similar repeated regions of 45 amino acid residues are present, which are separated by short nonhomologous sequences. The repeats of AcmA, which belong to the lysine motif (LysM) domain family, were consecutively deleted, removed, or, alternatively, one additional repeat was added, without destroying the cell wall-hydrolyzing activity of the enzyme in vitro, although AcmA activity was reduced in all cases. In vivo, proteins containing no or only one repeat did not give rise to autolysis of lactococcal cells, whereas separation of the producer cells from the chains was incomplete. Exogenously added AcmA deletion derivatives carrying two repeats or four repeats bound to lactococcal cells, whereas the derivative with no or one repeat did not. In conclusion, these results show that AcmA needs three LysM domains for optimal peptidoglycan binding and biological functioning.
TL;DR: This is the first example that shows that nisin-regulated gene expression in L. lactis can be used at industrial scale to produce large amounts of a target protein, such as lysostaphin.
Abstract: The NI sin-C ontrolled gene E xpression system NICE of Lactococcus lactis is one of the most widespread used expression systems of Gram-positive bacteria. It is used in more than 100 laboratories for laboratory-scale gene expression experiments. However, L. lactis is also a micro-organism with a large biotechnological potential. Therefore, the aim of this study was to test whether protein production in L. lactis using the NICE system can also effectively be performed at the industrial-scale of fermentation. Lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) from S. simulans biovar. Staphylolyticus, was used as a model system. Food-grade lysostaphin expression constructs in L. lactis were grown at 1L-, 300-L and 3000-L scale and induced with nisin for lysostaphin production. The induction process was equally effective at all scales and yields of about 100 mg/L were obtained. Up-scaling was easy and required no specific effort. Furthermore, we describe a simple and effective way of downstream processing to obtain a highly purified lysostaphin, which has been used for clinical phase I trials. This is the first example that shows that nisin-regulated gene expression in L. lactis can be used at industrial scale to produce large amounts of a target protein, such as lysostaphin. Downstream processing was simple and in a few steps produced a highly purified and active enzyme.
TL;DR: Protein production with the NICE gene expression system in L. lactis strongly depends on the medium composition, the fermentation parameters and the amount of nisin added for induction, leading to a significant increase in the yield of the target protein.
Abstract: Background
The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria. Despite its widespread use, no optimization of the culture conditions and nisin induction has been carried out to obtain maximum yields. As a model system induced production of lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) produced by S. simulans biovar. Staphylolyticus, was used. Three main areas need optimization for maximum yields: cell density, nisin-controlled induction and protein production, and parameters specific for the target-protein.
TL;DR: Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8 and confers arsenate and arsenite resistance, which can be used as selective markers.
Abstract: Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the pWCFS101 replicon includes Lactobacillus species and Lactococcus lactis, while that of the pWCFS102 replicon also includes Carnobacterium maltaromaticum and Bacillus subtilis. The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to L. plantarum. Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8. It confers arsenate and arsenite resistance, which can be used as selective markers.
TL;DR: The use of probiotics such as Lactococcus lactis and other lactic acid bacteria (LAB) has been proposed for the management of food allergy but no experimental study has clearly demonstrated any preventive or therapeutic inhibition of an allergen‐specific IgE response.
Abstract: Summary
Background The use of probiotics such as Lactococcus lactis and other lactic acid bacteria (LAB) has been proposed for the management of food allergy. However, no experimental study has clearly demonstrated any preventive or therapeutic inhibition of an allergen-specific IgE response.
Objective We aimed to study the immunomodulatory effect of recombinant L. lactis expressing bovine β-lactoglobulin (BLG), a major cow's milk allergen, in a validated mouse model of allergy.
Methods Six-week-old female Balb/c mice received five repeated doses of BLG, of L. lactis plus BLG, or of recombinant L. lactis by gavage. Different recombinant strains were inoculated, which corresponded to BLG doses ranging from 4 to 70 μg/mice. Mice were then sensitized by intra-peritoneal injection of BLG emulsified in incomplete Freund's adjuvant to induce high IgE concentrations.
Results Pre-treatment with natural L. lactis plus BLG allowed induction of BLG-specific T-helper type 1 (Th1) response, and abrogated the oral tolerance induced by BLG alone, demonstrating the adjuvant effect of this non-colonizing LAB. Moreover, pre-treatment with some of the recombinant strains favoured the development of a Th1 response inhibiting the Th2 one: it induced a significant decrease of specific IgE response, and an intense increase of specific IgG2a and IFN-γ productions. The most efficient strains that inhibited the IgE response were those producing the highest amounts of the BLG protein.
Conclusion Oral administration of some recombinant L. lactis was demonstrated to induce a specific Th1 response down-regulating a further Th2 one. Prophylaxis protocols will thus be evaluated using the most efficient strains.
TL;DR: Data support the hypothesis that the mode of action for lacticin 3147 involves a lipid II binding step (by the mersacidin-likeLtnA1 peptide, which would explain its independent inhibitory activity), followed by insertion of the more linear LtnA2 peptide into the target membrane, resulting in pore formation and ultimate cell death.
Abstract: Lacticin 3147 is a two-peptide (LtnA1 and LtnA2) lantibiotic produced by Lactococcus lactis subsp. lactis DPC3147 and has inhibitory activity against all gram-positive microorganisms tested. In this study the specific activities of the component peptides (alone or in combination) were determined by using L. lactis subsp. cremoris HP as the target strain. Lacticin 3147 exhibited an MIC50 of 7 nM for each component peptide (in combination), suggesting a peptide stoichiometry of 1:1. Interestingly, the LtnA1 peptide demonstrated independent inhibitory activity, with an MIC50 of 200 nM against L. lactis HP. In parallel studies, the single peptide bacteriocin nisin exhibited an MIC50 of 50 nM against the same target strain. Sequential peptide addition (with an intermediate washing step) demonstrated that LtnA1 must be added before LtnA2 rather than vice versa to observe inhibitory activity. The nanomolar activity of the lacticin peptides suggests the involvement of a docking molecule, speculated to be lipid II. Taken together with the recently determined structure of lacticin 3147 (N. I. Martin, T. Sprules, M. R. Carpenter, P. D. Cotter, C. Hill, R. P. Ross, and J. C. Vederas, Biochemistry, 43:3049-3056, 2004), these data support the hypothesis that the mode of action for lacticin 3147 involves a lipid II binding step (by the mersacidin-like LtnA1 peptide, which would explain its independent inhibitory activity), followed by insertion of the more linear LtnA2 peptide into the target membrane, resulting in pore formation and ultimate cell death.
TL;DR: Results showed that inactivation of both the lactate and acetate pathways with the coexpression of PEPC and PYC was most effective in improving succinate production.
Abstract: Sorghum vulgare phosphoenolpyruvate carboxylase (PEPC) and Lactococcus lactis pyruvate carboxylase (PYC) were overexpressed in Escherichia coli concurrently to improve the production of succinate, a valuable industrial specialty chemical. This coexpression system was also applied to E. coli mutant strains strategically designed by inactivating the competing pathways of succinate formation. The highest level of succinate production was observed in E. coli strains coexpressing both PEPC and PYC when compared with E. coli strains individually overexpressing either PEPC or PYC. Lactate production was also significantly reduced with PEPC and PYC coexpression. Lactate and acetate pathways were inactivated to eliminate the competing pathways of succinate formation. Results showed that inactivation of both the lactate and acetate pathways with the coexpression of PEPC and PYC was most effective in improving succinate production. Inactivating the lactate or acetate pathway alone only caused a majority of the carbon flux to shift to other metabolites rather than succinate. Coexpression of PEPC and PYC was also applied to an E. coli mutant strain deficient in lactate dehydrogenase and pyruvate:formate lyase that accumulated a substantial amount of the intermediate metabolite pyruvate during growth. Results showed that PEPC and PYC coexpression was effective in depleting pyruvate accumulation and increasing the production of metabolites.
TL;DR: It was concluded that the use of different starter bacteria caused significant differences in the quality of the cheese, and that each starter culture contributed to proteolysis to a different degree.
TL;DR: It is demonstrated that Smb expression is induced by the competence-stimulating peptide (CSP) and that a com box-like sequence is located in the smb promoter region, suggesting that SMB belongs to the class I bacteriocin family, and its expression is dependent on CSP-induced quorum sensing.
Abstract: A dipeptide lantibiotic, named Smb, in Streptococcus mutans GS5 was characterized by molecular genetic approaches. The Smb biosynthesis gene locus is encoded by a 9.5-kb region of chromosomal DNA and consists of seven genes in the order smbM1, -T, -F, -M2, -G, -A, -B. This operon is not present in some other strains of S. mutans, including strain UA159. The genes encoding Smb were identified as smbA and smbB. Inactivation of smbM1, smbA, or smbB attenuated the inhibition of the growth of the indicator strain RP66, confirming an essential role for these genes in Smb expression. Mature Smb likely consists of the 30-amino-acid SmbA together with the 32-amino-acid SmbB. SmbA exhibited similarity with the mature lantibiotic lacticinA2 from Lactococcus lactis, while SmbB was similar to the mersacidin-like peptides from Bacillus halodurans and L. lactis. We also demonstrated that Smb expression is induced by the competence-stimulating peptide (CSP) and that a com box-like sequence is located in the smb promoter region. These results suggest that Smb belongs to the class I bacteriocin family, and its expression is dependent on CSP-induced quorum sensing.
TL;DR: The genome of Lactococcus lactis has a multicistronic folate synthesis operon that includes an open reading frame (ylgG) specifying a putative Nudix hydrolase, and it is proposed that ylG be redesignated as folQ, confirming that YlgG functions in folate biosynthesis.
TL;DR: The possibility of continuous production of high concentration of bacteriocins by lactic acid bacteria for use as food biopreservatives is illustrated using a packed-bed bioreactor.
TL;DR: D-Ala depletion of the cell wall causes lysis by two distinct mechanisms, which results in an altered peptidoglycan that is more susceptible to lyses by AcmA and also by other factors, e.g., one or more of the other cell wall hydrolases expressed by L. lactis.
Abstract: Mutations in the genes encoding enzymes responsible for the incorporation of D-Ala into the cell wall of Lactococcus lactis affect autolysis An L lactis alanine racemase (alr) mutant is strictly dependent on an external supply of D-Ala to be able to synthesize peptidoglycan and to incorporate D-Ala in the lipoteichoic acids (LTA) The mutant lyses rapidly when D-Ala is removed at mid-exponential growth AcmA, the major lactococcal autolysin, is partially involved in the increased lysis since an alr acmA double mutant still lyses, albeit to a lesser extent To investigate the role of D-Ala on LTA in the increased cell lysis, a dltD mutant of L lactis was investigated, since this mutant is only affected in the D-alanylation of LTA and not the synthesis of peptidoglycan Mutation of dltD results in increased lysis, showing that D-alanylation of LTA also influences autolysis Since a dltD acmA double mutant does not lyse, the lysis of the dltD mutant is totally AcmA dependent Zymographic analysis shows that no degradation of AcmA takes place in the dltD mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain In L lactis, LTA has been shown to be involved in controlled (directed) binding of AcmA LTA lacking D-Ala has been reported in other bacterial species to have an improved capacity for autolysin binding Mutation of dltD in L lactis, however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered In conclusion, D-Ala depletion of the cell wall causes lysis by two distinct mechanisms First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, eg, one or more of the other (putative) cell wall hydrolases expressed by L lactis Second, reduced amounts of D-Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity
TL;DR: Physiological and proteomic characterization of this adaptive response indicated that L. lactis reorganizes its metabolism in response to acid stress to a great extent and quite differently in the two media.
Abstract: Exponentially growing cells of Lactococcus lactis MG1363 are able to develop an Acid Tolerance Response (ATR) when incubated at pH 5, in both rich (M17) – and chemically defined (SA) – culture media. Physiological and proteomic characterization of this adaptive response indicated that L. lactis reorganizes its metabolism in response to acid stress to a great extent and quite differently in the two media. The development of ATR was fully dependent on protein de novo synthesis in SA and only partly dependent in M17. 2D gel electrophoresis revealed a total of 90 spots induced by acidity, 80 of which were identified by mass spectrometry. Only 10 proteins (BglA, PycA, GlmS, HasC, ArgS, GatA, AtpA, ArcB, Cfa, and SodA) were overproduced in the two media. A transcriptional analysis of the corresponding genes suggested that for half of them the mode of regulation may differ in the two media. Among the protein spots upregulated during the ATR in SA but not in M17, 13 already displayed an elevated rate of synthesis in M17 at neutral pH. These proteins could play an important role in the development of the protein de novo synthesis-independent ATR observed in M17.