Showing papers on "Lactococcus lactis published in 1996"
TL;DR: The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes.
Abstract: The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes. In the nisin-producing L. lactis strain NZ9700, the specific beta-glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached. Expression of the gusA gene was also studied in L. lactis NZ9800, an NZ9700 derivative carrying a deletion in the structural nisA gene that abolishes nisin production, and in L. lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome. In both strains, beta-glucuronidase activity was linearly dependent on the amount of nisin added to the medium. Without nisin, no beta-glucuronidase production was observed. To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of the nisA gene. Use of the translational fusion vector yielded up to six times more beta-glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin. In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold. The beta-glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800. This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein. These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins.
866 citations
TL;DR: The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria, suggesting that these starters provide a means of controlling developing microflora in ripened fermented products.
Abstract: Lactococcus lactis DPC3147, a strain isolated from an Irish kefir grain, produces a bacteriocin with a broad spectrum of inhibition. The bacteriocin produced is heat stable, particularly at a low pH, and inhibits nisin-producing (Nip+) lactococci. On the basis of the observation that the nisin structural gene (nisA) does not hybridize to DPC3147 genomic DNA, the bacteriocin produced was considered novel and designated lacticin 3147. The genetic determinants which encode lacticin 3147 are contained on a 63-kb plasmid, which was conjugally mobilized to a commercial cheese starter, L. lactis subsp. cremoris DPC4268. The resultant transconjugant, DPC4275, both produces and is immune to lacticin 3147. The ability of lacticin 3147-producing lactococci to perform as cheddar cheese starters was subsequently investigated in cheesemaking trials. Bacteriocin-producing starters (which included the transconjugant strain DPC4275) produced acid at rates similar to those of commercial strains. The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria. Such results suggest that these starters provide a means of controlling developing microflora in ripened fermented products.
398 citations
TL;DR: Results show that the nisF and nisA promoter sequences have significant similarities and contain a conserved region that could be important for transcriptional control.
Abstract: The promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoterless beta-glucuronidase gene (gusA). Three promoters preceding the nisA, nisR, and nisF genes, which all give rise to gusA expression in the nisin-producing strain L. lactis NZ9700, were identified. The transcriptional autoregulation of nisA by signal transduction involving the sensor histidine kinase NisK and the response regulator NisR has been demonstrated previously (0. P. Kuipers, M. M. Beerthuyzen, P. G. G. A. de Ruyter, E. J. Luesink, and W. M. de Vos, J. Biol. Chem. 270: 27299-27304, 1995), and therefore the possible nisin-dependent expression of gusA under control of the nisR and nisF promoters was also investigated. The nisR promoter was shown to direct nisin-independent gusA expression in L. lactis MG 1363, which is a nisin-transposon- and plasmid-free strain. L. lactis NZ9800, which does not produce nisin because of a deletion in the nisA gene, containing the nisF-gusA fusion plasmid, gave rise to beta-glucuronidase production only after induction by nisin. A similar regulation was found in L. lactis NZ3900, which contains a single copy of the nisR and nisK genes but no other genes of the nisin gene cluster. In contrast, when the nisK gene was disrupted, no beta-glucuronidase activity directed by the nisF promoter could be detected even after induction with nisin. These results show that, like the nisA promoter, the nisF promoter is nisin inducible. The nisF and nisA promoter sequences have significant similarities and contain a conserved region that could be important for transcriptional control.
341 citations
01 Jan 1996
TL;DR: The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes, illustrating the potential of the nisi-inducible expression system for overproduction of desired proteins.
Abstract: The kinetics, control, and efficiency of nisin-induced expression directed by thenisApromoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter gene. In the nisin-producing L. lactis strain NZ9700, the specific b-glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached. Expression ofthegusAgenewasalsostudiedinL.lactisNZ9800,anNZ9700derivativecarryingadeletioninthestructural nisA gene that abolishes nisin production, and in L. lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome. In both strains, b-glucuronidase activity was linearly dependent on the amount of nisin added to the medium. Without nisin, no b-glucuronidase production was observed. To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of thenisAgene. Use of the translational fusion vector yielded up to six times more b-glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin. In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold. The b-glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800. This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein. These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins.
245 citations
TL;DR: The results show that L. lactis possesses an inducible response to acid stress in exponential phase, and suggest that the stringent response pathway, which is involved in starvation and stationary phase survival, may also be implicated in acid pH tolerance.
Abstract: It is now generally recognized that cell growth conditions in nature are often suboptimal compared to controlled conditions provided in the laboratory. Natural stresses like starvation and acidity are generated by cell growth itself. Other stresses like temperature or osmotic shock, or oxygen, are imposed by the environment. It is now clear that defense mechanisms to withstand different stresses must be present in all organisms. The exploration of stress responses in lactic acid bacteria has just begun. Several stress response genes have been revealed through homologies with known genes in other organisms. While stress response genes appear to be highly conserved, however, their regulation may not be. Thus, search of the regulation of stress response in lactic acid bacteria may reveal new regulatory circuits. The first part of this report addresses the available information on stress response in Lactococcus lactis.
166 citations
TL;DR: The addition of Ca2+ to the medium showed a stimulating effect on the production of nisin Z and a maximum activity of 3150 IU/ml was obtained during pH-controlled batch fermentation in the medium supplemented with 0.1 M CaCl2.
Abstract: The influence of several parameters on the fermentative production of nisin Z by Lactococcus lactis IO-1 was studied. Considerable attention has been focused on the relationship between the primary metabolite production of bacteriocin and lactate and cell growth, which has so far not been clarified in detail. Production of nisin Z was optimal at 30°C and in the pH range 5.0–5.5. The addition of Ca2+ to the medium showed a stimulating effect on the production of nisin Z. A maximum activity of 3150 IU/ml was obtained during pH-controlled batch fermentation in the medium supplemented with 0.1 M CaCl2. It was about three times higher than that obtained under the optimal conditions for cell growth and lactic acid production.
162 citations
TL;DR: It will be demonstrated that the metabolic transformation of pyruvate can be predicted if the growth-limiting constraints are adequately established and the normal pathways can no longer maintain balanced carbon flux.
Abstract: Lactococcus lactis, a homofermentative lactic acid bacterium, has been studied extensively over several decades to obtain sometimes conflicting concepts relating to the growth behaviour. In this review some of the data will be examined with respect to pyruvate metabolism. It will be demonstrated that the metabolic transformation of pyruvate can be predicted if the growth-limiting constraints are adequately established. In general lactate remains the major product under conditions in which sugar metabolism via a homolactic fermentation can satisfy the energy requirements necessary to assimilate anabolic substrates from the medium. In contrast, alternative pathways are involved when this energy supply becomes limiting or when the normal pathways can no longer maintain balanced carbon flux. Pyruvate occupies an important position within the metabolic network of L. lactis and the control of pyruvate distribution within the various pathways is subject to co-ordinated regulation by both gene expression mechanisms and allosteric modulation of enzyme activity.
155 citations
TL;DR: A commonly used agar diffusion bioassay technique for quantification of nisin in food samples was modified to increase its sensitivity, accuracy and precision.
Abstract: Nisin, a bacteriocin produced by Lactococcus lactis subsp. lactis, is used in some types of food preservation due to its inhibitory action on Gram-positive bacteria and their spores. A commonly used agar diffusion bioassay technique for quantification of nisin in food samples was modified to increase its sensitivity, accuracy and precision. Several variables were evaluated. Results showed Micrococcus luteus as the most sensitive organism tested, a lower agar concentration (0 x 75% compared 1 x 5%) increased the sensitivity of the assay (21% improvement over standard method), and incorporation of 1% Na2HPO4 buffer into the bioassay agar made it possible to prevent false inhibitory zones from developing due to the low pH of the test solutions. This resulted in a 57% improvement in accuracy and a 12% improvement in precision compared to the standard method.
145 citations
TL;DR: The results establish that L. lactis can adapt to lactic acid exposure in two different ways: a logarithmic phase LATR, which may be activated by protons, and a stationary-phase LATR which needs no activation by protONS.
Abstract: The lactic acid tolerance response (LATR) of the lactic acid bacterium Lactococcus lactis subsp. lactis has been studied. A dramatic increase in survival to a severe acid stress (pH 3.9) was obtained by preexposing the cells for 30 min to a mildly acid shock at pH 5.5. Whole-cell protein extract analysis revealed that during the acid tolerance response 33 polypeptides are induced over the level of naive cells. Among these are the major heat shock proteins DnaK and GroEL. In conjunction with a previous report (Hartke et al. 1994), the results establish that L. lactis can adapt to lactic acid exposure in two different ways: a logarithmic phase LATR, which may be activated by protons, and a stationary-phase LATR, which needs no activation by protons. Both systems are independent of de novo protein synthesis.
144 citations
TL;DR: In this paper, the effect of gelatin, xanthan gum or maltodextrins on the survival during freeze-drying and stability during storage of four lactic acid bacteria were studied.
141 citations
TL;DR: A versatile set of cloning and expression vectors has been developed for application in self-cloning and other genetic modifications of Lactococcus lactis, equipped with the controlled and strong lacA promoter of the lactococcal lactose operon.
Abstract: A versatile set of cloning and expression vectors has been developed for application in self-cloning and other genetic modifications of Lactococcus lactis. The expression vectors were equipped with the controlled and strong lacA promoter of the lactococcal lactose operon. In addition, the transcriptional terminator of the aminopeptidase N gene, pepN, was inserted, which in some cases increased the genetic stabilities of the vectors and the cloned DNA. The small, 0.3-kb lacF gene encoding the soluble carrier enzyme IIALac was used as a dominant selection marker in the plasmid-free L. lactis strain NZ3000 carrying an in-frame deletion of the chromosomal lacF gene. Lactose-utilizing transformants were easily selected on lactose indicator plates at high frequencies and showed a copy number of approximately 50 plasmids per cell. All vectors were stably maintained in the lacF strain NZ3000 when grown on lactose, and only the high-level expression vectors showed some instability when their host was grown on glucose-containing medium. The application potentials of the expression vectors carrying the lacF marker were determined by cloning of the promoterless Escherichia coli gusA reporter gene under control of the lacA promoter followed by analysis of its expression. While in one of the vectors this resulted in a promoter-down mutation in the -10 region of the lacA promoter, in other vectors high-level and controlled expression of the gusA gene was observed.
TL;DR: This review will discuss past and present examples and future possibilities of metabolic engineering of lactic acid bacteria for the production of important compounds, including lactic and other acids, flavor compounds, and exopolysaccharides.
Abstract: Lactic acid bacteria are characterized by a relatively simple sugar fermentation pathway that, by definition, results in the formation of lactic acid. The extensive knowledge of traditional pathways and the accumulating genetic information on these and novel ones, allows for the rerouting of metabolic processes in lactic acid bacteria by physiological approaches, genetic methods, or a combination of these two. This review will discuss past and present examples and future possibilities of metabolic engineering of lactic acid bacteria for the production of important compounds, including lactic and other acids, flavor compounds, and exopolysaccharides.
TL;DR: To assess the frequency of extracellular superoxide (O-2) production by enterococci, multiple species were surveyed in a whole organism assay for their ability to reduce ferricytochrome c in a superoxide dismutase-inhibitable fashion to suggest an association between invasiveness andextracellular O-2 production.
Abstract: To assess the frequency of extracellular superoxide (O 2 - ) production by enterococci, multiple species were surveyed in a whole organism assay for their ability to reduce ferricytochrome c in a superoxide dismutase-inhibitable fashion. For stool and clinical enterococcal isolates and 12 type strains, only Enterococcus faecalis (87/91 isolates and ATCC 19443), Enterococcus faecium (5/13 isolates), Enterococcus casseliflavus (ATCC 25788), and Enterococcus gallinarum (ATCC 35038) produced extracellular O 2 - . Among 106 strains comprising 13 species of enteric gram-negative bacilli and gram-positive cocci, only Lactococcus lactis subspecies lactis produced extracellular O 2 - . Mean (±SE) rates of extracellular O 2 - production in vitro by E. faecalis for isolates associated with bacteremia and endocarditis and for isolates from stool were 2.4 ± 0.2, 1.9 ± 0.2, and 1.5 ± 0.3 nmol of O 2 - min -1 10 9 cfu -1 , respectively (P =.025, analysis of variance), suggesting an association between invasiveness and extracellular O 2 - production.
TL;DR: The overall kinetics of retting, a spontaneous fermentation of cassava roots performed in central Africa, was investigated in terms of microbial-population evolution and biochemical and physicochemical parameters.
Abstract: The overall kinetics of retting, a spontaneous fermentation of cassava roots performed in central Africa, was investigated in terms of microbial-population evolution and biochemical and physicochemical parameters. During the traditional process, endogenous cyanogens were almost totally degraded, plant cell walls were lysed by the simultaneous action of pectin methylesterase and pectate lyase, and organic acids (C(inf2) to C(inf4)) were produced. Most microorganisms identified were found to be facultative anaerobes which used the sugars (sucrose, glucose, and fructose) present in the roots as carbon sources. After 24 h of retting, the fermentation reached an equilibrium that was reproducible in all the spontaneous fermentations studied. Lactic acid bacteria were largely predominant (over 99% of the total flora after 48 h) and governed the fermentation. The epiphytic flora was first replaced by Lactococcus lactis, then by Leuconostoc mesenteroides, and finally, at the end of the process, by Lactobacillus plantarum. These organisms produced ethanol and high concentrations of lactate, which strongly acidified the retting juice. In addition, the rapid decrease in partial oxygen pressure rendered the process anaerobic. Strict anaerobes, such as Clostridium spp., developed and produced the volatile fatty acids (mainly butyrate) responsible, together with lactate, for the typical flavor of retted cassava. Yeasts (mostly Candida spp.) did not seem to play a significant role in the process, but their increasing numbers in the last stage of the process might influence the flavor and the preservation of the end products.
TL;DR: The results supply the first direct evidence for the functioning of lactococcal peptidases in the degradation of milk proteins, and provides critical information about the relative importance of the peptidase for growth in milk, the order of events in the proteolytic pathway, and the regulation of its individual components.
Abstract: To examine the contribution of peptidases to the growth of lactococcus lactis in milk, 16 single- and multiple-deletion mutants were constructed. In successive rounds of chromosomal gene replacement mutagenesis, up to all five of the following peptidase genes were inactivated (fivefold mutant): pepX, pepO, pepT, pepC, and pepN. Multiple mutations led to slower growth rates in milk, the general trend being that growth rates decreased when more peptidases were inactivated. The fivefold mutant grew more than 10 times more slowly in milk than the wild-type strain. In one of the fourfold mutants and in the fivefold mutant, the intracellular pools of amino acids were lower than those of the wild type, whereas peptides had accumulated inside the cell. No significant differences in the activities of the cell envelope-associated proteinase and of the oligopeptide transport system were observed. Also, the expression of the peptidases still present in the various mutants was not detectably affected. Thus, the lower growth rates can directly be attributed to the inability of the mutants to degrade casein-derived peptides. These results supply the first direct evidence for the functioning of lactococcal peptidases in the degradation of milk proteins. Furthermore, the study provides critical information about the relative importance of the peptidases for growth in milk, the order of events in the proteolytic pathway, and the regulation of its individual components.
TL;DR: Research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual with the availability of this new technology.
Abstract: Current knowledge of the genomes of the lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, and members of the genera Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium is reviewed The genomes contain a chromosome within the size range of 18 to 34 Mbp Plasmids are common in Lactococcus lactis (most strains carry 4–7 different plasmids), some of the lactobacilli and pediococci, but they are not frequently present in S thermophilus, Lactobacillus delbrueckii subsp bulgaricus or the intestinal lactobacilli Five IS elements have been found in L lactis and most strains carry multiple copies of at least two of them; some strains also carry a 68-kbp conjugative transposon IS elements have been found in the genera Lactobacillus and Leuconostoc, but not in S thermophilus Prophages are also a normal component of the L lactis genome and lysogeny is common in the lactobacilli, however it appears to be rare in S thermophilus Physical and genetic maps for two L lactis subsp lactis strains, two L lactis subsp cremoris strains and S thermophilus A054 have been constructed and each reveals the presence of six rrn operons clustered in less than 40% of the chromosome The L lactis subsp cremoris MG1363 map contains 115 genetic loci and the S thermophilus map has 35 The maps indicate significant plasticity in the L lactis subsp cremoris chromosome in the form of a number of inversions and translocations The cause(s) of these rearrangements is (are) not known A number of potentially powerful genetic tools designed to analyse the L lactis genome have been constructed in recent years These tools enable gene inactivation, gene replacement and gene recovery experiments to be readily carried out with this organism, and potentially with other lactic acid bacteria and Gram-positive bacteria Integration vectors based on temperate phage attB sites and the random insertion of IS elements have also been developed for L lactis and the intestinal lactobacilli In addition, a L lactis sex factor that mobilizes the chromosome in a manner reminiscent to that seen with Escherichia coli Hfr strains has been discovered and characterized With the availability of this new technology, research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual
TL;DR: It is proposed that the aerobic class I ribonucleotide reductases contain two subclasses, one coded by nrdAB, active in E. coli and eukaryotes and other microorganisms present in various microorganisms (class Ib); the NrdEF enzymes use NrdH proteins as electron transporter in place of thioredoxin or glutaredoxin used by NrdAB enzymes.
TL;DR: The MICs of antimicrobial agents contained in the SCEPTOR Streptococcus MIC panels (Becton Dickinson Microbiology Systems) were determined for Lactococcus lactis, L. garvieae, and unknown Lactitis species, indicating that knowledge of the L lactococcus species causing an infection might influence the choice of antim antibiotics.
Abstract: The MICs of antimicrobial agents contained in the SCEPTOR Streptococcus MIC panels (Becton Dickinson Microbiology Systems) were determined for Lactococcus lactis, L. garvieae, and unknown Lactococcus species. Several isolates had reduced susceptibilities to many of the antimicrobial agents contained in the panel. For L. garvieae, the MICs of penicillin and, possibly, cephalothin were higher than for L. lactis, and unlike L. lactis, L. garvieae was resistant to clindamycin, indicating that knowledge of the Lactococcus species causing an infection might influence the choice of antimicrobial therapy. Susceptibility to clindamycin can also be used to differentiate between L. lactis and L. garvieae.
TL;DR: Reversed-phase fast protein liquid chromatography of gliadin digests showed a more-complex peptide pattern produced by the proteinase of Lactobacillus sanfrancisco CB1.
Abstract: A cell envelope 57-kDa proteinase, a cytoplasmic 65-kDa dipeptidase, and a 75-kDa aminopeptidase were purified from Lactobacillus sanfrancisco CB1 sourdough lactic acid bacterium by sequential fast protein liquid chromatography steps. All of the enzymes are monomers. The proteinase was most active at pH 7.0 and 40 degrees C, while aminopeptidase and dipeptidase had optima at pH 7.5 and 30 to 35 degrees C. Relatively high activities were observed at the pH and temperature of the sourdough fermentation. The proteinase is a serine enzyme. Urea-polyacrylamide gel electrophoresis of digest of alpha s1- and beta-caseins showed differences in the pattern of peptides released by the purified proteinase and those produced by crude preparations of the cell envelope proteinases of Lactobacillus delbrueckii subsp. bulgaricus B397 and Lactococcus lactis subsp. lactis SK11. Reversed-phase fast protein liquid chromatography of gliadin digests showed a more-complex peptide pattern produced by the proteinase of Lactobacillus sanfrancisco CB1. The dipeptidase is a metalloenzyme with high affinity for dipeptides containing hydrophobic amino acids but had no activity on tripeptides or larger peptides. The aminopeptidase was also inhibited by metal-chelating agents, and showed a broad N-terminal hydrolytic activity including di- and tripeptides. Km values of 0.70 and 0.44 mM were determined for the dipeptidase on Leu-Leu and the aminopeptidase on Leu-p-nitroanilide, respectively.
TL;DR: Six of the 2100 colonies of lactic acid bacteria isolated from 4 month old Cheddar cheese and raw buffalo milk showed antifungal activity against Aspergillus flavus IARI when tested by the well agar diffusion assay on Potato Dextrose Agar containing 0.1% Triton X-100.
TL;DR: The bactericidal activity of the bacteriocin was more potent when sensitive strains were in the logarithmic growth phase, inducing cell lysis, as observed by decreases in optical density and release of intracellular marker enzymes.
Abstract: A total of 203 lactic acid bacteria isolated from raw goat's milk and artisanal cheese were tested for antibacterial activity. Only two strains of Lactococcus lactis, one strain of Enterococcus faecalis and one strain of Lactobacillus curvatus were shown to produce a bacteriocin-like substance. Lactobacillus curvatus IFPL105 produced a heat-stable bacteriocin, which was hydrolysed by α-chymotrypsin, proteinase K and pancreatin and exhibited a broad spectrum of inhibitory activity. The bactericidal activity of the bacteriocin was more potent when sensitive strains were in the logarithmic growth phase, inducing cell lysis, as observed by decreases in optical density and release of intracellular marker enzymes. Curing experiments resulted in variants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of the parental strain and the bacteriocin-negative variants indicated that a plasmid of about 46 kbp may be involved in bacteriocin production and immunity to this antibacterial compound.
TL;DR: The C-terminal fragment of cow's κ-casein (residues: 106–169) is the main factor stabilizing casein micelles and glycosidic moieties connected to this fragment enhance the ability of λ- casein to stabilize micells and also the resistance of this protein to the action of proteolytic enzymes and high temperature in simple model systems.
TL;DR: Results showed that the nisin promoters are under positive control in an autoregulatory manner and that antimicrobial peptides can also function as signal molecules.
Abstract: Nisin is a small post-translationally modified lanthionine-containing peptide (lantibiotic) produced by certain Lactococcus lactis strains which has a high antimicrobial activity against several pathogenic Gram-positive bacteria. Northern blots and RT/PCR analyses of the nisin-producing strain N8 revealed that the nisZBTCIPRKFEG gene cluster, responsible for nisin biosynthesis, immunity and regulation, consists of two operons, nisZBTCIPRK and nisFEG. The promoter of the nisFEG operon was mapped. The −35 to −1 region upstream of the transcription start of the nisFEG promoter showed 73% identity with the corresponding region upstream of the nisA and nisZ gene. In contrast to earlier reports, nisin was found to be secreted during the early stages of growth as well as later in the growth cycle. The secreted nisin was adsorbed on the surface of the cells and was released to the medium during mid-exponential growth, when the pH in the medium fell below 5.5. In nisZB antisense and nisT deletion mutant strains constructed in this study the transcription of the nisin operons, nisin production and immunity were lost. Provision of external nisin restored the transcription of both operons in the mutant strains, showing that the operons are coordinately regulated by mature nisin. Nisin induction of the mutant strains also resulted in an increased amount of the Nisl protein and an increase in the level of immunity. Induction using higher concentrations of nisin yielded a higher level of immunity. These results showed that the nisin promoters are under positive control in an autoregulatory manner and that antimicrobial peptides can also function as signal molecules.
TL;DR: The HPLC method developed proved to be a precise tool to monitor the organic acid content of milk and can be used to follow the fermentation ability of starter cultures, providing information about the type of fermentation.
Abstract: The major function of lactic starter cultures in cheese making is to produce lactic and other organic acids from the carbohydrates present in milk. The activity of six starter cultures consisting of two Lactococcus lactis ssp. lactis, two Lactococcus lactis ssp. lactis biovar. diacetylactis and two Leuconostoc strains, was tested by monitoring the evolution of the organic acid composition of milk by a modified HPLC method. In addition, their performance as cheese starters was also tested. The HPLC method developed proved to be a precise tool to monitor the organic acid content. Thus, it can be used to follow the fermentation ability of starter cultures, providing information about the type of fermentation. The use of any of the six starters assayed is suggested for manufacturing Afuega'l Pitu cheese.
TL;DR: It is shown for the first time that phosphopolysaccharide derived from a dairy lactic acid bacterium can induce IFN-gamma and IL-1 alpha production in macrophages and this findings strongly suggest that the phosphopoly Saccharide is a type of 'biological response modifier' and the fermented dairy foods containing Lactococcus lactis ssp.
TL;DR: A regulatory region of the temperate Lactococcus lactis bacteriophage r1t chromosome that encompasses the two divergently oriented genes rro, encoding the phage repressor, and tec has been cloned and characterized.
Abstract: A regulatory region of the temperate Lactococcus lactis bacteriophage r1t chromosome has been cloned and characterized, It encompasses the two divergently oriented genes rro, encoding the phage repressor, and tee. Both genes, of which the transcription start sites have been mapped, are preceded by consensus -35 and -10 promoter sequences, The region contains three 21 bp direct repeats with internal dyad symmetry which probably act as operators, Two of these repeats partially overlap the two promoter sequences, The distant third repeat is located within the tec coding sequence, Gel mobility shift assays demonstrated that Rro specifically binds to this sequence, To study possible transcriptional regulation of the region, a lacZ translational fusion with an open reading frame following tec was constructed, Under conditions that favour the lysogenic life cycle of r1t, beta-galactosidase activity was very low, Expression of the lacZ fusion could be induced 70-fold by the addition of mitomycin C at a concentration which promotes the switch of r1t from the lysogenic to the lytic life cycle, In non-induced cells, promoter activity was repressed by Rro, as a frameshift mutation in rro resulted in constitutive expression of the lacZ gene fusion.
TL;DR: A mutant of the peptide antibiotic nisin in which the dehydroalanine residue at position 5 has been replaced by an alanine has been produced and structurally characterized and has activity very similar to that of wild-type nisin but is very much less active than nisin as an inhibitor of the outgrowth of spores of Bacillus subtilis.
Abstract: A mutant of the peptide antibiotic nisin in which the dehydroalanine residue at position 5 has been replaced by an alanine has been produced and structurally characterized. It is shown to have activity very similar to that of wild-type nisin in inhibiting growth of Lactococcus lactis and Micrococcus luteus but is very much less active than nisin as an inhibitor of the outgrowth of spores of Bacillus subtilis. These observations, which parallel those of W. Liu and J. N. Hansen (Appl. Environ. Microbiol. 59:648-651, 1993) on the corresponding mutant of the related antibiotic subtilin, are discussed in terms of the mechanism(s) of action of these antibiotics.
TL;DR: It appears that pNP40 encodes a novel phage resistance mechanism which blocks DNA penetration specifically for (phi)c2.
Abstract: The lactococcal plasmid pNP40 mediates insensitivity to (phi)c2 by an early-acting phage resistance mechanism in addition to the previously identified abortive infection system, AbiF, in the Lactococcus lactis subsp. lactis MG1614 background. A second abortive infection determinant on pNP40, AbiE, does not confer resistance to (phi)c2. The early-acting mechanism on pNP40 does not prevent phage adsorption nor does it appear to operate by restriction/modification. Phage DNA was not detected in pNP40-containing cells until 30 min following exposure to (phi)c2 compared with 5 min in a sensitive host; however, electroporation of phage DNA into resistant hosts resulted in the release of phage progeny from a dramatically elevated number of cells compared with conventionally infected hosts. It appears therefore that pNP40 encodes a novel phage resistance mechanism which blocks DNA penetration specifically for (phi)c2.
TL;DR: An ABC-transport system consisting of two membrane proteins and a cytoplasmic protein play a role in immunity of nisin, subtilin and epidermin by import, export or inhibition of pore formation by the membrane components of the transport systems.
Abstract: Bacteria producing bacteriocins have to be protected from being killed by themselves. This mechanism of self-protection or immunity is especially important if the bacteriocin does not need a specific receptor for its action, as is the case for the type A lantibiotics forming pores in the cytoplasmic membrane. At least two different systems of immunity have evolved in this group of bacteriocins containing modified amino acids as a result of posttranslational modification. The immunity mechanism of Pep5 in Staphylococcus epidermidis is based on inhibition of pore formation by a small 69-amino acid protein weakly associated with the outer surface of the cytoplasmic membrane. In Lactococcus lactis and Bacillus subtilis the putative immunity lipoproteins NisI and SpaI, respectively, are also located at the outer surface of the cytoplasmic membrane, suggesting that a similar mechanism might be utilized by the producers of nisin and subtilin. In addition an ABC-transport system consisting of two membrane proteins, (NisEG, SpaG and the hydrophobic domain of SpaF, and EpiEG) and a cytoplasmic protein (NisF, the cytoplasmic domain of SpaF, and EpiF) play a role in immunity of nisin, subtilin and epidermin by import, export or inhibition of pore formation by the membrane components of the transport systems. Almost nothing is known of the immunity determinants of newly described and other type of lantibiotics.
TL;DR: The results suggest that in Leuconostoc oenos the genes that encode the malolactic enzyme and a malate carrier protein are organized in a cluster.
Abstract: Using degenerated primers from conserved regions of the protein sequences of malic enzymes, we amplified a 324-bp DNA fragment by PCR from Leuconostoc oenos and used this fragment as a probe for screening a Leuconostoc oenos genomic bank. Of the 2,990 clones in the genomic bank examined, 7 with overlapping fragments were isolated by performing colony hybridization experiments. Sequencing 3,453 bp from overlapping fragments revealed two open reading frames that were 1,623 and 942 nucleotides long and were followed by a putative terminator structure. The first deduced protein (molecular weight, 59,118) is very similar (level of similarity, 66%) to the malolactic enzyme of Lactococcus lactis; as in several malic enzymes, highly conserved protein regions are present. The synthesis of a protein with an apparent molecular mass of 60 kDa was highlighted by the results of labelling experiments performed with Escherichia coli minicells. The gene was expressed in E. coli and Saccharomyces cerevisiae and conferred "malolactic activity" to these species. The second open reading frame encodes a putative 34,190-Da protein which has the characteristics of a carrier protein and may have 10 membrane-spanning segments organized around a central hydrophilic core. Energy-dependent L-[14C]malate transport was observed with E. coli dicarboxylic acid transport-deficient mutants carrying the malate permease-expressing vector. Our results suggest that in Leuconostoc oenos the genes that encode the malolactic enzyme and a malate carrier protein are organized in a cluster.