Showing papers on "Lactococcus lactis published in 1995"

A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes.

Abstract: A system for generating chromosomal insertions in lactococci is described. It is based on the conditional replication of lactococcal pWV01-derived Ori+ RepA- vector pORI19, containing lacZ alpha and the multiple cloning site of pUC19. Chromosomal AluI fragments of Lactococcus lactis were cloned in pORI19 in RepA+ helper strain Escherichia coli EC101. The frequency of Campbell-type recombinants, following introduction of this plasmid bank into L. lactis (RepA-), was increased by combining the system with temperature-sensitive pWV01 derivative pVE6007. Transformation of L. lactis MG1363 (pVE6007) with the pORI19 bank of lactococcal chromosomal fragments at the permissive temperature allowed replication of several copies of a recombinant plasmid from the bank within a cell because of the provision in trans of RepA-Ts from pVE6007. A temperature shift to 37 degrees C resulted in loss of pVE6007 and integration of the pORI19 derivatives at high frequencies. A bank of lactococcal mutants was made in this way and successfully screened for the presence of two mutations: one in the monocistronic 1.3-kb peptidoglycan hydrolase gene (acmA) and one in the hitherto uncharacterized maltose fermentation pathway. Reintroduction of pVE6007 into the Mal- mutant at 30 degrees C resulted in excision of the integrated plasmid and restoration of the ability of ferment maltose. The integration plasmid (pMAL) was rescued by using the isolated plasmid content of a restored Mal+ colony to transform E. coli EC101. Nucleotide sequencing of the 564-bp chromosomal fragment in pMAL revealed an internal part of an open reading frame of which the translated product showed significant homology with ATP-binding proteins MalK of E. coli, Salmonella typhimurium, and Enterobacter aerogenes and MsmK of Streptococcus mutans. This combined use of two types of conditional replicating pWV01-derived vectors represents a novel, powerful tool for chromosomal gene inactivation, targeting, cloning, and sequencing of the labelled gene.

Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation.

Abstract: A gene of Lactococcus lactis subsp. cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells. In cell extracts of L. lactis MG1363 and several halo-producing E. coli transformants, lytic bands of similar sizes were identified by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing L. lactis or M. lysodeikticus cell walls. Of these clearing bands, corresponding to the presence of lytic enzymes with sizes of 46 and 41 kDa, the 41-kDa band was also present in the supernatant of an L. lactis culture. Deletion analysis of one of the recombinant plasmids showed that the information specifying lytic activity was contained within a 2,428-bp EcoRV-Sau3A fragment. Sequencing of part of this fragment revealed a gene (acmA) that could encode a polypeptide of 437 amino acid residues. The calculated molecular mass of AcmA (46,564 Da) corresponded to that of one of the lytic activities detected. Presumably, the enzyme is synthesized as a precursor protein which is processed by cleavage after the Ala at position 57, thus producing a mature protein with a size of 40,264 Da, which would correspond to the size of the enzyme whose lytic activity was present in culture supernatants of L. lactis. The N-terminal region of the mature protein showed 60% identity with the N-terminal region of the mature muramidase-2 of Enterococcus hirae and the autolysin of Streptococcus faecalis. Like the latter two enzymes, AcmA contains C-terminal repeated regions. In AcmA, these three repeats are separated by nonhomologous intervening sequences highly enriched in serine, threonine, and asparagine. Genes specifying identical activities were detected in various strains of L. lactis subsp. lactis and L. lactis subsp. cremoris by the SDS-polyacrylamide gel electrophoresis detection assay and PCR experiments. By replacement recombination, an acmA deletion mutant which grew as long chains was constructed, indicating that AcmA is required for cell separation.

Genes involved in immunity to the lantibiotic nisin produced by Lactococcus lactis 6F3.

Abstract: The lantibiotic nisin is produced by several strains of Lactococcus lactis. The complete gene cluster for nisin biosynthesis in L. lactis 6F3 comprises 15 kb of DNA. As described previously, the structural gene nisA is followed by the genes nisB, nisT, nisC, nisI, nisP, nisR, and nisK. Further analysis revealed three additional open reading frames, nisF, nisE, and nisG, adjacent to nisK. Approximately 1 kb downstream of the nisG gene, three open reading frames in the opposite orientation have been identified. One of the reading frames, sacR, belongs to the sucrose operon, indicating that all genes belonging to the nisin gene cluster of L. lactis 6F3 have now been identified. Proteins NisF and NisE show strong homology to members of the family of ATP-binding cassette (ABC) transporters, and nisG encodes a hydrophobic protein which might act similarly to the immunity proteins described for several colicins. Gene disruption mutants carrying mutations in the genes nisF, nisE, and nisG were still able to produce nisin. However, in comparison with the wild-type strain, these mutants were more sensitive to nisin. This indicates that besides nisI the newly identified genes are also involved in immunity to nisin. The NisF-NisE ABC transporter is homologous to an ABC transporter of Bacillus subtilis and the MbcF-MbcE transporter of Escherichia coli, which are involved in immunity to subtilin and microcin B17, respectively.

Genetic marking of Lactococcus lactis shows its survival in the human gastrointestinal tract.

Abstract: A human feeding study was performed with Lactococcus lactis TC165.5, which is genetically marked by insertion of the sucrose-nisin conjugative transposon Tn5276 and chromosomal resistance to rifampin and streptomycin. The fate of strain TC165.5 and its nucleic acids was monitored by conventional plating methods and by molecular detection techniques based on specific PCR amplification of the nisin (nisA) gene from DNA extracted from human feces. A method was developed for the efficient extraction of microbial DNA from human feces. The results show that a fraction of viable cells of L. lactis TC165.5 survived passage through the human gastrointestinal tract. Only cells that passed within 3 days of ingestion could be recovered from the feces of the volunteers, and they accounted for approximately 1% of the total number of cells consumed. The presence of nisA in DNA extracted from feces could be detected up to 4 days, when viable cells were no longer present.

Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80.

Abstract: Transposon Tn917-LTV1 was used to produce a collection of Lactococcus lactis strains with fusion of a promoterless lacZ gene to chromosomal loci. Screening 2,500 Tn917-LTV1 integrants revealed 222 that express beta-galactosidase on plates at 30 degrees C. Pulsed-field gel electrophoresis revealed Tn917-LTV1 insertions in at least 13 loci in 15 strains analyzed. Integrants in which beta-galactosidase expression was regulated by temperature or pH and/or arginine concentration were isolated. In most cases, the regulation observed on plates was reproducible in liquid medium. One integrant, PA170, produces beta-galactosidase at pH 5.2 but not at pH 7.0, produces more beta-galactosidase at 15 degrees C than at 30 degrees C, and has increased beta-galactosidase activity in the stationary phase. DNA fragments potentially carrying promoters from selected Lactococcus lactis integrants were cloned in Escherichia coli. A new promoter probe vector, pAK80, containing promoterless beta-galactosidase genes from Leuconostoc mesenteroides subsp. cremoris and the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid replication region was constructed, and the lactococcal fragments were inserted. Plasmid pAK80 was capable of detecting and discriminating even weak promoters in Lactococcus lactis. When inserted in pAK80, the promoter cloned from PA170 displayed a regulated expression of beta-galactosidase analogous to the regulation observed in PA170.

Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicrobial peptides exported by gram-positive bacteria.

Abstract: Lantibiotics form a family of highly modified peptides which are secreted by several Gram-positive bacteria. They exhibit antimicrobial activity, mainly against other Gram-positive bacteria, by forming pores in the cellular membrane. These antimicrobial peptides are ribosomally synthesized and contain leader peptides which do not show the characteristics of signal sequences. Several amino acid residues of the precursor lantibiotic are enzymatically modified, whereafter secretion and processing of the leader peptide takes place, yielding the active antimicrobial substance. For several lantibiotics the gene clusters encoding biosynthetic enzymes, translocator proteins, self-protection proteins, processing enzymes and regulatory proteins have been identified. This MicroReview describes the current knowledge about the biosynthetic, immunity and regulatory processes leading to lantibiotic production. Most of the attention is focused on the lantibiotic nisin, which is produced by the food-grade bacterium Lactococcus lactis and is widely used as a preservative in the food industry.

Oligopeptides are the main source of nitrogen for Lactococcus lactis during growth in milk.

Abstract: The consumption of amino acids and peptides was monitored during growth in milk of proteinase-positive (Prt+) and -negative (Prt-) strains of Lactococcus lactis. The Prt- strains showed monophasic exponential growth, while the Prt+ strains grew in two phases. The first growth phases of the Prt+ and Prt- strains were in same, and no hydrolysis of casein was observed. Also, the levels of consumption of amino acids and peptides in the Prt+ and Prt- strains were similar. At the end of this growth phase, not all free amino acids and peptides were used, indicating that the remaining free amino acids and peptides were unable to sustain growth. The consumption of free amino acids was very low (about 5 mg/liter), suggesting that these nitrogen sources play only a minor role in growth. Oligopeptide transport-deficient strains (Opp-) of L. lactis were unable to utilize oligopeptides and grew poorly in milk. However, a di- and tripeptide transport-deficient strain (DtpT-) grew exactly like the wild type (Opp+ Dtpt+) did. These observations indicate that oligopeptides represent the main nitrogen source for growth in milk during the first growth phase. In the second phase of growth of Prt+ strains, milk proteins are hydrolyzed to peptides by the proteinase. Several of the oligopeptides formed are taken up and hydrolyzed internally by peptidases to amino acids, several of which are subsequently released into the medium (see also E.R.S. Kunji, A. Hagting, C.J. De Vries, V. Juillard, A.J. Haandrikman, B. Poolman, and W.N. Konings, J. Biol. Chem. 270:1569-1574, 1995).(ABSTRACT TRUNCATED AT 250 WORDS)

The genus Lactococcus

Abstract: Lactococci are coccoid Gram-positive, anaerobic bacteria which produce l(+)-lactic acid from lactose in spontaneously fermented raw milk which is left at ambient temperatures around 20–30°C for 10–20 h. They are commonly called ‘mesophilic lactic streptococci’. It is tempting to suggest that the first isolation, identification and description of the chemical entity lactic acid by Carl Wilhelm Scheele from sour milk in Sweden in the year 1780, was actually l (+)-lactic acid produced by lactococci. The microbial nature of lactic fermentation was recognized in 1857 by Louis Pasteur. The first bacterial pure culture on earth, obtained and scientifically described by Joseph Lister (1873) was Lactococcus lactis, at that time called: ‘Bacterium lactis’. Admitting then that we had here to deal with only one bacterium, it presents such peculiarities both morphologically and physiologically as to justify us, I think, in regarding it a definite and recognizable species for which I venture to suggest the name Bacterium lactis. This I do with diffidence, believing that up to this time no bacterium has been defined by reliable characters. Whether this is the only bacterium that can occasion the lactic acid fermentation, I am not prepared to say.

Detection and characterization of lactose-utilizing Lactococcus spp. in natural ecosystems.

Abstract: The presence of lactose-utilizing Lactococcus species in nondairy environments was studied by using identification methods based on PCR amplification and (sub)species-specific probes derived from 16S rRNA sequences. Environmental isolates from samples taken on cattle farms and in the waste flow of a cheese production plant were first identified to the genus level, using a Lactococcus genus-specific probe. Isolates which showed a positive signal with this probe were further identified to the (sub)species level. Lactococcus lactis isolates were also characterized at the phenotypic level for the ability to hydrolyze arginine, to ferment citrate, and to produce proteases and bacteriocins. With specific PCR amplifications, the presence of sequences related to citP, coding for citrate permease; prtP, coding for protease; and nisA or nisZ, the structural genes for production of nisin A or nisin Z, respectively, was verified. By these methods, it was possible to isolate lactococci from various environmental sources, such as soil, effluent water, and the skin of cattle. The strains of L. lactis isolated differed in a number of properties, such as the ability to hydrolyze arginine or the absence of citP-related sequences, from those found in industrial starter cultures. The results indicate that the majority of the industrially produced lactococci do not survive outside the dairy environment, although natural niches are available. However, from those niches strains with the potential to be developed into novel starter cultures may be isolated.

The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides.

Abstract: The peptides released from beta-casein by the action of PI-type proteinase (PrtP) from Lactococcus lactis subsp. cremoris Wg2 have been identified by on-line coupling of liquid chromatography to mass spectrometry. After 24 h of incubation of beta-casein with purified PrtP, a stable mixture of peptides was obtained. The trifluoroacetic acid-soluble peptides of this beta-casein hydrolysate were fractionated by high-performance liquid chromatography and introduced into the liquid chromatography-ion spray mass spectrometry interface. Multiply charged ions were generated from trifluoroacetic acid-soluble peptides under low nozzle voltage conditions, yielding the MH+ mass of each eluted peptide. All peptides corresponding to each of the MH+ calculated masses were determined. In those cases in which different peptides were possible, further identification was achieved by collision-induced dissociation under higher nozzle voltage conditions. Hydrolysis of beta-casein by PrtP was observed to proceed much further than reported previously. More than 40% of the peptide bonds are cleaved by PrtP, resulting in the formation of more than 100 different oligopeptides. With the exception of Phe, significant release of amino acids or di- and tripeptides could not be observed. Interestingly, one-fifth of the identified oligopeptides are small enough to be taken up by the oligopeptide transport system. Uptake of these peptides could supply L. lactis with all amino acids, including the essential ones, indicating that growth of L. lactis might be possible on peptides released from beta-casein by proteinase only.

Stress response in Lactococcus lactis: cloning, expression analysis, and mutation of the lactococcal superoxide dismutase gene.

Abstract: In an analysis of the stress response of Lactococcus lactis, three proteins that were induced under low pH culture conditions were detected. One of these was identified as the lactococcal superoxide dismutase (SodA) by N-terminal amino acid sequence analysis. The gene encoding this protein, designated sodA, was cloned by the complementation of a sodA sodB Escherichia coli strain. The deduced amino acid sequence of L. lactis SodA showed the highest degree of similarity to the manganese-containing Sod (MnSod) of Bacillus stearothermophilus. A promoter upstream of the sodA gene was identified by primer extension analysis, and an inverted repeat surrounding the -35 hexanucleotide of this promoter is possibly involved in the regulation of the expression of sodA. The expression of sodA was analyzed by transcriptional fusions with a promoterless lacZ gene. The induction of beta-galactosidase activity occurred in aerated cultures. Deletion experiments revealed that a DNA fragment of more than 130 bp surrounding the promoter was needed for the induction of lacZ expression by aeration. The growth rate of an insertion mutant of sodA did not differ from that of the wild type in standing cultures but was decreased in aerated cultures.

Rational development of a simple synthetic medium for the sustained growth of Lactococcus lactis

Abstract: The growth of two strains of Lactococcus lactis subsp. lactis from vegetable (NCDO 2118) and dairy origin (IL 1403) were compared on various culture media. Both strains grew more rapidly on a complex organic medium than on a defined synthetic medium. The best growth was obtained under nitrogen gas phase. The single omission technique was applied to each component of a non-optimized synthetic medium in order to determine the true nutritional requirements. Requirements for macro-elements, oligo-elements, bases and vitamins were identical for the two strains. As expected, the dairy strain (IL 1403) was seen to be auxotrophic for some amino acids, whereas the vegetable strain (NCDO 2118) was seen to be prototrophic for all amino acids when using the single omission technique. Growth was then characterized on progressively simplified media and the composition of the absolute minimal media for the growth of both strains was defined. Sustained growth of the vegetable strain was only possible in minimal media supplemented with six amino acids (Glu, Met, Ile, Leu, Val, Ser), indicating that the definition of prototrophy/auxotrophy is partly dependent upon the medium composition. The dairy strain showed a requirement for Arg, His and Thr in addition to the six amino acids necessary for growth of the vegetable strain. The removal of ammonium salt from the medium did not affect the growth, illustrating that the amino acids may satisfy the totality of the nitrogen requirement for biomass synthesis.

Microbiological profile in Serra ewes' cheese during ripening

Abstract: The microflora of Serra cheese was monitored during a 35 d ripening period at three different periods within the ewe's lactation season. After 7 d ripening, the numbers of micro-organisms reached their maximum, and lactic acid bacteria (LAB) and coliforms were the predominant groups. Pseudomonads were not detected after 1 week of ripening. At all stages of ripening, cheeses manufactured in spring exhibited the lowest numbers of LAB and yeasts, whereas cheeses manufactured in winter showed the lowest numbers of coliforms and staphylococci. Leuconostoc lactis was the most abundant LAB found in Serra cheese whereas Enterococcus faecium and Lactococcus lactis spp. lactis exhibited the highest decrease in percentage composition. Numbers of both Leuc. mesenteroides and Lactobacillus paracasei tended to increase throughout ripening. The most abundant coliform was Hafnia alvei. Klebsiella oxytoca was found in curd but declined in number during ripening. Staphylococcal flora of curd was mainly composed of Staphylococcus xylosus, Staph. aureus and Staph. epidermidis. Staphylococcus xylosus was the major species found at the end of ripening. Pseudomonas fluorescens, was the only Pseudomonas species isolated from the curd. Although a broad spectrum of yeasts were found in Serra cheese, Sporobolomyces roseus was the most abundant yeast isolated.

Purification and Characterization of Cystathionine (beta)-Lyase from Lactococcus lactis subsp. cremoris B78 and Its Possible Role in Flavor Development in Cheese.

Abstract: An enzyme that degrades sulfur-containing amino acids was purified from Lactococcus lactis subsp. cremoris B78; this strain was isolated from a mixed-strain, mesophilic starter culture used for the production of Gouda cheese. The enzyme has features of a cystathionine (beta)-lyase (EC 4.4.1.8), a pyridoxal-5(prm1)-phosphate-dependent enzyme involved in the biosynthesis of methionine and catalyzing an (alpha),(beta)-elimination reaction. It is able to catalyze an (alpha),(gamma)-elimination reaction as well, which in the case of methionine, results in the production of methanethiol, a putative precursor of important flavor compounds in cheese. The native enzyme has a molecular mass of approximately 130 to 165 kDa and consists of four identical subunits of 35 to 40 kDa. The enzyme is relatively thermostable and has a pH optimum for activity around 8.0; it is still active under cheese-ripening conditions, viz., pH 5.2 to 5.4 and 4% (wt/vol) NaCl. A possible essential role of the enzyme in flavor development in cheese is suggested.

Cloning and DNA sequence analysis of two abortive infection phage resistance determinants from the lactococcal plasmid pNP40.

Abstract: The lactococcal plasmid pNP40, from Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, confers complete resistance to the prolate-headed phage phi c2 and the small isometric-headed phage phi 712 in L. lactis subsp. lactis MG1614. A 6.0-kb NcoI fragment of pNP40 cloned in the lactococcal Escherichia coli shuttle vector pAM401 was found to confer partial resistance to phi 712. Subcloning and deletion analysis of the recombinant plasmid pPG01 defined a 2.5-kb ScaIHpaI fragment as conferring phage insensitivity. Sequence analysis of this region confirmed the presence of two overlapping open reading frames (ORFs). Further subcloning of pNP40 to characterize the resistance determinant active against phi c2 identified a 5.6-kb EcoRV fragment of pNP40 which, when cloned in pAM401, conferred partial resistance to both phi c2 and phi 712. Subcloning and deletion analysis of the recombinant plasmid pCG1 defined a 3.7-kb EcoRV-XbaI fragment as encoding phage insensitivity. DNA sequence analysis of this region revealed the presence of a single complete ORF. The introduction of a frameshift mutation at the unique BglII site within this ORF disrupted the phage resistance phenotype, confirming that this ORF is responsible for the observed phage insensitivity. The mechanisms encoded by pPG01 and pCG1 in L. lactis subsp. lactis MG1614 conformed to the criteria defining abortive infection and were designated AbiE and AbiF, respectively. Analysis of the phage DNA content of phi 712-infected hosts containing AbiF demonstrated that it inhibited the rate of phage DNA replication, while AbiE had little effect on phage DNA replication, suggesting a later target of inhibition. The predicted protein product of abiF shows significant homology to the products of two other lactococcal abortive infection genes, abiD and abiD1.

Comparison of proteolytic activities in various lactobacilli.

Abstract: A total of 169 Lactobacillus strains from 12 species (Lb. acidophilus, Lb. brevis, Lb. buchneri, Lb. casei, Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. delbrueckii, Lb. delbrueckii subsp. lactis, Lb. fermentum, Lb. helveticus, Lb. paracasei subsp. paracasei, Lb. plantarum and Lb. rhamnosus), isolated from raw milk and various milk products, and 9 Lactococcus lactis strains were evaluated for peptidase activities with five chromogenic substrates and a tryptic digest of casein. Within each species, the peptidase activity of the cell-free extracts of the strains varied. Furthermore, differences were observed between the Lactobacillus species and Lc. lactis. Lb. helveticus had by far the highest hydrolysing activities towards all substrates, indicating the presence of powerful aminopeptidases, X-prolyl-dipeptidyl aminopeptidases and proline iminopeptidases. Lb. delbrueckii subsp. bulgaricus possessed high hydrolysing activities towards substrates containing proline, alanyl-prolyl-p-nitroanilide and prolyl-p-nitroanilide. On the other hand, Lb. fermentum and Lb. brevis could be considered as weakly proteolytic species. A more detailed study with highly proteolytic Lactobacillus strains indicated that at least three different proteinases or endopeptidases were present. Compared with Lc. lactis, the Lactobacillus strains had a much lower hydrolytic action on glutamyl-glutamic acid, suggesting that glutamyl aminopeptidase was absent in lactobacilli.

The recA gene of Lactococcus lactis: characterization and involvement in oxidative and thermal stress

Abstract: The role of recA in Lactococcus lactis, a microaerophilic fermenting organism, was examined by constructing a recA-disrupted strain. This single alteration had a surprisingly pleiotropic effect. In addition to its roles in homologous recombination and DNA repair, recA is also involved in responses to oxygen and heat stresses. We found that oxygen stress induced by aeration causes reductions in growth and stationary-phase survival of the recA strain. Toxicity is a consequence of hydroxyl radical production via the Fenton Reaction and is alleviated by catalase or Ferrozine addition. These results suggest that oxygen radicals are not efficiently eliminated and accumulate in lactococcal cultures, and that RecA is needed to deal with the damage they incur. Unexpectedly, thermal stress arrested growth of the recA strain. Immunological data indicate that the recA mutant is deficient in heat-shock proteins DnaK, GroEL, and GrpE. Poor growth at elevated temperature is therefore due to a diminished heat-shock response in the recA strain. In contrast, levels of a novel heat-shock protein, HfIB, are elevated. In Escherichia coli, HfIB downregulates the heat-shock response by promoting degradation of the transcription factor sigma 32. We propose that recA regulates the heat-shock response via HfIB. This work provides the first evidence showing that two major pathways of stress response, induced by heat shock and DNA damage, are interactive.

Isolation of nisin‐producing Lactococcus lactis strains from dry fermented sausages

Abstract: A total of 4608 lactic acid bacteria (LAB) were isolated from 24 Spanish fermented sausages and screened for bacteriocin production. Two strains, BB24 and G18, produced bacteriocins that inhibited a broad spectrum of Gram-positive bacteria. BB24 and G18 were tentatively identified as Lactococcus lactis by carbohydrate fermentation patterns and other biochemical characteristics. The characterization of their bacteriocins suggested that both could be the well-known lantibiotic nisin. This was confirmed by PCR analysis of their genomic DNA. Nucleotide sequencing revealed that they produced nisin A. The fact that BB24 and G18 were isolated from sausages produced in two different regions of Spain suggests that nisin-producing L. lactis strains may be more widespread in meat products than previously thought. Nisin produced by L. lactis BB24 has been purified to homogeneity by a procedure that included ammonium sulphate precipitation and cation-exchange, hydrophobic-interaction and reverse-phase chromatography. The purification procedure was simple, rapid and reproducible.

Nutritional factors affecting nisin production by Lactococcus lactis subsp. lactis NIZO 22186 in a synthetic medium.

Abstract: L. DE VUYST. 1995 A minimal synthetic medium (SM8) for nisin-producing Lactococcus lactis subsp. lactis strains has been designed; it consists of eight growth-stimulating amino acids (glutamic acid, methionine, valine, leucine, threonine, arginine, isoleucine and histidine), five vitamins (biotin, calcium pantothenate, nicotinic acid, pyridoxine and riboflavin) and the mineral salts dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, magnesium sulphate and trisodium citrate. Nisin biosynthesis is strongly dependent on the presence of a sulphur source, either an inorganic salt (magnesium sulphate or sodium thiosulphate) or the amino acids methionine, cysteine or cystathionine. The amino acids serine, threonine and cysteine highly stimulate nisin production without affecting the final cell yield, indicating their precursor role during nisin biosynthesis.

Analysis and production of two exopolysaccharides from Lactococcus lactis subsp. cremoris LC330

Abstract: Two polysaccharides produced concurrently by Lactococcus lactis subsp. cremoris strain LC330 have been identified. One had a high molecular mass (> 1 × 106 Da) and was neutral. The second was smaller (∼ 10000 Da), charged and had a high phosphorus content. Sugar composition also differed. In chemostat culture the neutral polysaccharide was influenced by temperature and by nitrogen limitation. This polysaccharide was branched with terminal galactose moieties and contained galactose, glucose and glucosamine. The phosphopolysaccharide was more complex with glucose, rhamnose, galactose and glucosamine in an approximate ratio of 6:5:4:1.

Isolation of Lactococcus lactis nonsense suppressors and construction of a food‐grade cloning vector

Abstract: Nonsense suppressor strains of Lactococcus lactis were isolated using plasmids containing nonsense mutations or as revertants of a nonsense auxotrophic mutant. The nonsense suppressor gene was cloned from two suppressor strains and the DNA sequence determined. One suppressor is an ochre suppressor with an altered tRNA(gln) and the other an amber suppressor with an altered tRNA(ser). The nonsense suppressors allowed isolation of nonsense mutants of a lytic bacteriophage and suppressible auxotrophic mutants of L. lactis MG1363. A food-grade cloning vector based totally on DNA from Lactococcus and a synthetic polylinker with 11 unique restriction sites was constructed using the ochre suppressor as a selectable marker. Selection, following electroporation of a suppressible purine auxotroph, can be done on purine-free medium. The pepN gene from L. lactis Wg2 was subcloned resulting in a food-grade plasmid giving a four- to fivefold increase in lysine aminopeptidase activity.

Metabolic engineering of Lactococcus lactis: influence of the overproduction of alpha-acetolactate synthase in strains deficient in lactate dehydrogenase as a function of culture conditions.

Abstract: The als gene for alpha-acetolactate synthase of Lactococcus lactis MG1363 was cloned on a multicopy plasmid under the control of the inducible L. lactis lacA promoter. More than a hundredfold overproduction of alpha-acetolactate synthase was obtained in L. lactis under inducing conditions as compared with that of the host strain, which contained a single chromosomal copy of the als gene. The effect of alpha-acetolactate synthase overproduction on the formation of end products in various L. lactis strains was studied under different fermentation conditions. Under aerobic conditions and with an initial pH of 6.0, overexpression of the als gene resulted in significant acetoin production that amounted to more than one-third of the pyruvate converted. However, the effect of the alpha-acetolactate synthase overproduction was even more pronounced in the lactate dehydrogenase-deficient strain L. lactis NZ2700. Anaerobic cultivation of this strain resulted in a doubling of the butanediol formation of up to 40% of the converted pyruvate. When cultivated aerobically at an initial pH of 6.8, overexpression of the als gene in L. lactis NZ2700 resulted in the conversion of more than 60% of the pyruvate into acetoin, while no butanediol was formed. Moreover, at an initial pH of 6.0, similar amounts of acetoin were obtained, but in addition approximately 20% of the pyruvate was converted into butanediol. These metabolic engineering studies indicate that more than 80% of the lactose can be converted via the activity of the overproduced alpha-acetolactate synthase in L. lactis.

Secretion of biologically-active murine interleukin-2 by lactococcus-lactis subsp lactis.

Abstract: Secretion of functional recombinant murine interleukin-2 (mIL2) by Lactococcus lactis was achieved by fusion of the sequence encoding mature mIL2 to the secretion signal leader of the lactococcal usp45 gene placed under transcriptional control of the phage T7 promoter-T7 RNA polymerase expression system. The recombinant mature mIL2 was one of only a few proteins which accumulated in the growth medium. Sequence analysis revealed correct processing at the first amino acid of the mature protein. A T-cell proliferation assay showed that the recombinant protein has the same specific biological activity as mIL2 obtained from a natural source.

Medium-dependent regulation of proteinase gene expression in Lactococcus lactis: control of transcription initiation by specific dipeptides.

Abstract: Transcriptional gene fusions with the Escherichia coli beta-glucuronidase gene (gusA) were used to study the medium- and growth-dependent expression of the divergently transcribed genes involved in proteinase production (prtP and prtM) of Lactococcus lactis SK11. The results show that both the prtP and prtM genes are controlled at the transcriptional level by the peptide content of the medium and, to a lesser extent, by the growth rate. A more than 10-fold regulation in beta-glucuronidase activity was observed for both prtP and prtM promoters in batch and continuous cultures. The level of expression of the prtP and prtM promoters was high in whey permeate medium with relatively low concentrations of peptides, whereas at increased concentrations the expression of the promoters was repressed. The lowest level of expression was observed in peptide- and amino acid-rich laboratory media, such as glucose-M17 and MRS. The addition of specific dipeptides, such as leucylproline and prolylleucine, to the growth medium negatively affected the expression of the prtP-gusA fusions. The repression by dipeptides was not observed in mutants defective in the uptake of di-tripeptides, indicating that the internal concentration of dipeptides or derivatives is important in the regulation of proteinase production.

Characterization of the lactococcal abiD1 gene coding for phage abortive infection.

Abstract: Lactococcal phage abortive infection (AbiD1) determined by plasmid pIL105 is active on both prolate- and small-isometric-head phages of the C6A and 936 phage groups, respectively, which are considered two different species. The Abi phenotype was found to be encoded by a single gene, designated abiD1. The abiD1-encoded protein (351 amino acids) does not show homology with any known protein and has a deduced isoelectric point of 10. It also possesses two helix-turn-helix structures and an unusually high content of asparagine, isoleucine, and lysine. A consensual promoter with a TGy extension to the -10 box was mapped 76 bp upstream of the start codon. Transcription initiated at this strong promoter stops at a terminator located 48 bp downstream from the promoter. The termination process is very efficient, and transcripts corresponding to the abiD1 gene were not visible in our experimental conditions with or without phage infection. Expression of abiD1 under the control of a T7 promoter induced a lag phase in Lactococcus lactis cell growth, suggesting that overproduction of AbiD1 could be toxic for the cells. AbiD1 protein was visualized in Escherichia coli by using a tightly controlled expression system.