Objectif
The objective on the T-Project on lactic acid bacteria (LAB) is the development and advancement of the knowledge of genetics, molecular biology, physiology and biochemistry of LAB and the identification and/or construction of improved starter culture strains which meet the requirements of relevant industries that are of major economic importance in Europe.
Lactococcin B from Lactococcus lactis has been subjected to detailed study with advanced genetic analysis down to the deoxyribonucleic acid (DNA) sequence level. Recent work on the mode of action has shown it to depend on the proton motive force and to involve a collapse of the cytoplasmic membrane potential. Lactococcin M has been purified and shown to contain lanthionine residues but is distinct from the classic lactococcal lantibiotic nisin. Genetic analysis of it is well advanced.
A number of Lactobacillus isolates have been shown to produce distinct antimicrobials. One of them, lactocin S, was also shown to be a novel lantibiotic that is plasmid encoded and currently the subject of advanced genetic analysis. 2 pediocins, PA 1 and A, have been studied in detail with a complete analysis of the former down the DNA sequence level and ongoing gene cloning of the latter from characterized plasmids.
Some work has been done on the genetic element responsible for nisin biosynthesis genes and is relevant to the characterization of the novel lantibiotics produced by other strains. The range of bacteriocins currently under study includes novel lantibiotics from Lactococcus lactic, Lactobacillus sake, Lactobacillus plantarum and Carnobacterium piscicola.
Large numbers of lactic acid bacteria have been tested for a variety of key industrially relevant properties. Some 600 isolates have been screened for production of bacteriocins, proteolytic activities and exo-polysaccharides. This yielded 8 nisin like bacteriocin producers, 30 exo-polysaccharide producing strains and a classification of strains into groups on the basis of proteolytic activity. Distinct studies analyzed 30 strains from the port manufacturing environment, 127 strains from neutral fermentations, 226 strains from homemade cheeses, 23 strains from fresh sheep's milk and a large collection of isolates from traditional Greek dairy products. This screening activity is backed up by taxonomic studies including the analysis of protein profiles using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and the use of ribosomal ribonucleic acid (RNA) probes.
Metabolic activity analysis includes the study of malate and citrate fermentation in Leuconostoc oenos, particularly the malolactic fermentation that is important in wine fermentation. Lactobacilli involved in the sourdough fermentation are under study with emphasis on the fermentation of maltose. The citrate fermentation pathway in Lactococcus lactis subs lactis biovariety diacetylactis and Leuconostoc species is under study in order to determine the route to and from the key flavour compound diacetyl. The enzyme genes in the citrate metabolic pathway are being cloned and analysed which will facilitate a metabolic engineering approach to the elevation of a diacetyl yield in lactic fermentations.
The enzyme, lactate dehydrogenase, which controls the final conversion of pyruvate to lactic acid in the catabolism of lactose pathway has been cloned and sequenced.
Efforts directed towards characterizing the specific mechanisms and components involved in proteolysis have provided much valuable information on these systems in lactic acid bacteria, particularly the lactococci. There are 3 essential elements involved in proteolysis. Firstly, proteinases which are cell membrane anchored serine proteases of which 3 types have been indentified, PI, PIII and PI/PIII, based on their cleavage specificity on, and preference for, a and/or b casein. Secondly, peptidases which degrade peptides released from casein by the action of starter proteinase and other protease enzymes (eg chymosin). These include endopeptidases, general aminopeptidases, x-prolyl-di-peptidyl aminopeptidase, di- and tri-peptidases, prolidase and iminopeptidase. Thirdly, transport systems for amino acids, di- and tri-peptidases and oligopeptides.
A number of the enzyme components involved in proteolysis have been characterized at an advanced level. The genes encoding lactococcal proteinases have been cloned, sequenced and subjected to comparative analysis and the sites determining catalytic and specificity functions have been identified. Protein engineering technology has been applied to generate enzymes with hybrid or novel caseinolytic activities. Studies relating to proteinase expression and posttranslational processing are also ongoing. A range of peptidases form Lactococcus and Lactobacillus have been isolated and characterized biochemically and the genetic determinants for a number of these have been analysed at sequence level. In addition, genes encoding peptide transport proteins from Lactococcus and Lactobacillus have been cloned and sequenced. Lactococcal hosts in which components of their proteolytic system have been modified through genetic manipulation (ie with altered proteinase or peptidase activities) have been used to manufacture cheddar cheese on a laboratory scale. Organoleptic and chemical analyses of the cheeses is ongoing.
Phages of lactic acid bacteria are under study to elucidate the molecular basis for their interaction with their hosts. A genetic basis has been established for classifying phages of Lactococcus, Lactobacillus and Streptococcus thermophilus based on hybridization analysis and homology studies which have supported analyses based on morphological and structural protein criteria. The structural organisation of Lactobacillus and lactococcal phage genomes is being determined and a number of phage genes have now been cloned and sequenced. These include genes for phage lysin and phage structural proteins. Specific loci involved in integration of temperate phage genomes (attachment sites), the cohesive ends (cos) involved in phage genome circularization and the packaging sites (pac) of circularly permuted phages have been localized and, in many cases, characterized at a molecular level.
Analysis of phage host interaction in lactic acid bacteria has led to the identification of a number of mechanisms which mediate a high level of phage resistance in these hosts. Those encountered in Lactococcus strains have been studied at the most advanced level and include:
adsorption blocking (Ads) mechanisms which interfere with the initial attachment of the phage particle to the host cell surface;
restriction modification (RM) systems which involve a set of complementary enzyme activities which complete to either degrade (restriction endonuclease) or protect (modification methylase) infecting phage deoxyribonucleic acid (DNA);
abortive infection (Abi) mechanisms which prevent the progression of intracellular phage development.
In lactococci these mechanisms are usually, but in the case of RM not always, plasmid encoded and the genes encoding a range of RM and Abi mechanisms have been cloned and sequenced.
Phage resistance plasmids have now been applied to construct lactococcal strains with increased resistance properties. The strategy used is a food grade one and involves the transfer of the plasmids by conjunction to the appropriate recipient strains. Strains constructed in this manner are currently used in commercial cheese making.
Studies on various aspects of risk assessment have been initiated using genetically modified lactic acid bacteria. These include the survival and gene transfer in the laboratory, under practical conditions, and in axenic mice. In addition, lactococci with autolytic phenotypes have been created using well characterized genes for phage lysins. Molecular monitoring techniques are being developed for various lactic acid bacteria.
Several dominant homologous marker genes have been developed based on lactose metabolism and nisin resistance. The versatile food grade selection system based on lactose fermentation has proved its utility by selecting and stably maintaining recombinant deoxyribonucleic acid (DNA) in lactococci. For instance it has been used to construct strains that overproduce the debittering aminopeptidase N, PepN. The genetic modification of the latter strains involved DNA molecules that were entirely derived from the host organisms, and have been designated selfcloning.
More than 400 lactic acid bacteria (LAB) strains have been isolated from traditional food fermentation processes such as sour dough, semidry sausages (salami, chourico), cheeses, other dairy products and olives. Another 200 LAB strains have come from culture collections. These strains have been screened for some properties relevant to the food and feed industry: production of bacteriocins; proteolytic activity and production of exopolysaccharides (EPS). The screening resulted in: 8 bacteriocin producing strains (all of them nisin like); the subdivision of all tested strains into 4 groups with increasing proteolytic activity; and 30 EPS producing strains. A taxonomical characterization of the most relevant strains has been carried out using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) protein pattern analysis.
800 lactic acid bacteria (LAB) strains were investigated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) of whole cell proteins. Representative culture collections strains from Bifidobacterium, Carnobacterium, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus and Vagococcus (including 80 type strains) as well as a large number of wild strains were fingerprinted. The densitometric traces of the protein profiles were stored in database format and are available for identification of unknown isolates.
A very good agreement was found with the results of published deoxyribonucleic acid (DNA): DNA hybridizations. Reproducibility over a 3 year period was found to be greater than or equal to 92%. The influence of growth temperature (30 C or 37 C), oxygen concentration (aerobic or anaerobic), and growth medium was always found to be within the limits of the reproducibility.
Comparative sequence analysis of 16S and 23S ribosomal ribonucleic acid (rRNA) genes was used to design rRNA targeted oligonucleotide probes specific for various lactic acid bacteria that play an important role in the production of fermented foods. Species specific probes for Streptococcus thermophilus, Lactobacillus sake, Lactobacillus curvatus, Lactobacillus pentosus and Lactobacillus plantarum, Lactobacillus delbruecckii, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus gasseri and Lactobacillus sanfrancisco were successfully applied using dot blot and colony hybridization assays combined with isotopic or nonisotopic detection procedures. Because of the high copy number of ribosomes and the difference extent of conservation within the target molecule rRNA targeted probes exhibit a greater sensitivity and a wider specificity than other deoxyribonucleic acid (DNA) probes.
A genetic and physical map of the chromosome of currently used lactococcal laboratory strains is under construction. During this work, pulsed field gel electrophoresis (PFGE) has been developed and the data obtained of restriction digests of genomic deoxyribonucleic acid (DNA) establish this technique as a simple and reliable method for identification of lactococcal strains and study of their genetic history.
An immunoenzymatic detection test of bacteriophages of lactococcal strains of first generation, based on a classical sandwich enzyme linked immunosorbent assay (ELISA) test in 2 steps, was found to be not sensitive enough. Preliminary data has been obtained for the development of an immunoenzymatic test of second generation, using as the main component hybrid proteins containing common phage antigens fused to the beta galactosidase.
An endolysin encoding gene, lsyA, of Lactobacillus bulgaricus bacteriophage mv1 has been characterized at a molecular level.
The antimicrobial (bacteriocin) activity of lactic acid bacteria from meat origin has been tested against a number of selected indicator microorganisms to identify suitable bacteriocin producers on which further biochemical, immunological and molecular biology work can be performed. A number of lactobacilli and pediococci have been identified as bacteriocin producers that effectively inhibit growth of undesirable and pathogenic bacteria potentially present in foods. From 2 of the isolates, Lactobacillus sake 148 and Pediococcus species number 347, information has been gained on growth and production of the activity on several media at various temperatures, as well as on the partial purification of their antagonistic activity by lyophilisation and gel filtration. The antimicrobial activities are destroyed by proteolytic enzymes but they are resistant to heat. Experiments are in progress to further purify both antagonistic activities to homogeneity, as well as to obtain adequate immunosera for immunological studies and to perform molecular biology studies about synthesis and regulation of the bacteriocins at the genetic level.
Organisms from olive fermentations have been screened for antimicrobial production, and suitable candidates chosen for more detailed studies. One strain of Lactobacillus plantarum, LPCO-10, which produces a proteinaceous bacteriocin, has been shown to be active against a range of lactic acid bacteria. This bacteriocin, plantaricin S, has been found to be active against a number of indicator strains including members of the genera Lactobacillus, Pediococcus, Propionibacterium and Clostridium. The last 2 are particularly important, since they are the principal agents of spoilage in olive fermentations.
The possibility that plantaricin S and nisin were the same molecule has been discounted by challenging total deoxyribonucleic acid (DNA) from a nisin producing strain of Lactococcus lactis and Lb plantarum LPCO-10 with oligonucleotides specific to nisin in polymerase chain reaction (PCR) experiments. The results suggest that a PCR product results only from the Lc lactis strain, suggesting that LPCO-10 does not possess a nisin gene. Purification of the active antimicrobial compound of plantaricin S is in progress.
The metabolism of olive fermenting strains of Lb plantarum has been examined under different physiological conditions, such as varying salt concentrations and sugar substrates. The effect of medium components such as citrate on metabolism have also been investigated. Comparative studies of the growth characteristics of a plantaricin S producing strain and a nonplantaricin S producing strain under identical laboratory fermentation conditions have also been performed. The performance of strain LPCO-10 and an isogenic nonplantaricin S producing mutant have been examined in real olive fermentations, to determine their effectiveness and examine whether the antimicrobial compound is produced under field conditions.
Gene transfer, antimicrobial production and phage resistance were investigated in strains of Lactobacillus, Pediococcus and Streptococcus salivarius subspecies thermophilus. Lactobacillus plantarum 4B2 strain exhibits a constitutive cell aggregation and efficient conjugal transfer ability. Analysis of the aggregation process revealed a secreted protein (32 kda) to be involved in the cell clumping and demonstrated that lipoteichoich acids act as receptors of this protein. The use of purified preparations of the secreted protein was shown to dramatically improve conjugal transfer efficiency in matings involving strains belonging to Lactobacillus plantarum, Lactobacillus reuteri and Enterococcus faecalis.
Naturally occurring phage resistant mutants were characterized in one strain of Streptococcus salivarius subspecies thermophilus, currently used as dairy starter. The resistance mechanism was shown to be a plasmid encoded restriction and modification (R/M). The plasmid was transferred, by means of conjugal comobilization, in isogenic, phage sensitive clones. Tranconjugants showed the same level of phage resistance as the donor strain. Technologically relevant properties of phage resistant mutants were characterized and clones with unaltered traits were selected.
Mutants resistant to the lytic phage ATCC 15807 B1 were also isolated from sensitive cells of Lactobacillus helveticus ATCC 15807. The resistance mechanism seems to be a block in the phage adsorption process. Resistance mutants exhibit different levels of caseinase activity.
Preliminary procedures were carried out allowing isolation of active fractions of the bacteriocin, Pediocin A. The plasmid coding for Pediocin production and immunity was subcloned in Escherichia coli, Bacillus subtilis and Lactobacillus reuteri. Expression of the antimicrobial activity was not detected in heterologous hosts.
Research into lactic acid bacteria (LAB) is being undertaken in the following areas:
isolation and characterization of peptidases and proteinases responsible for development of flavour peptides in foods;
cloning, analysis and overexpression of peptidase genes in LAB;
analysis and exploitation of in vivo gene transfer mechanisms;
development of genetic engineering technologies in food grade selection, chromosomal integration, gene expression and secretion;
molecular genetics and protein engineering of nisins (antimicrobial polypeptides produced by LAB);
characterization and exploitation of bacteriophage lysins;
analysis and metabolic engineering of the citrate pathway;
development of rapid ribosomal ribonucleic acid (rRNA) (recombinant deoxyribonucleic acid) (rDNA) sequencing strategies and construction of a large (23s) subunit rRNA sequence database.
It has been shown that a lysin gene from a bacteriophage active against Listeria can be expressed in Lactococcus lactis from the lacA promoter. Furthermore the incorporation of this lysin gene together with a lactococcal phage lysin gene on the same plasmid can result in the release of the LM-4 lysin into the culture supernatant.
13 restriction and modification (R/M) systems of phage resistance were characterised. 8 were isolated from Lactococcus lactis, 2 from Streptococcus thermophilus and 3 from Lactobacillus delbrueckii subspecies bulgaricus. 7 of the lactoccal R/M systems were compared and 6 has different specificities. Several phage abortive infection mechanisms were also identified. 3 different abortive infection (Abi) encoding determinants were cloned and 2 of them were sequenced. The results indicate that different lactococcal genes can encode the phage Abi phenotype which might be the result of different processes at the molecular level. The method of interaction of these mechanisms with phage development is under study.
Lactococcal bacteriophages have been isolated and characterized. The physical and genetic characterization involved structural protein analysis, deoxyribonucleic acid (DNA)-DNA hybridizations, electron microscopy, and host range studies. The phages can be divided into 3 major morphological types: small isometric headed; large isometric headed; and prolate headed. The small isometric type is the most common. Extensive molecular analysis of a representative of this type is under way.
Research has been done on the detection of host associated phage insensitivity in lactococci. Various plasmids have been identified that confer resistance to bacteriophages on the strain harbouring the plasmid. These constitute a reservoir of phage resistance determinants with potential application in the improvement of industrial starter cultures. The introduction of a number of these plasmids into commerical strain has resulted in elevated levels of phage resistance. This has been achieved in a food grade manner acceptable to the food industries.
Starter cultures of Lactococcus lactis can be made more resistant to phages by the introduction of genes encoding different restriction and modification (R/M) systems. For this reason different R/M systems are being identified, isolated and characterised. The endonucleases will be purified and characterized. The endonucleases (also called restriction enzymes) can probably be used commercially in biotechnology. Construction of probes specific for the different R/M systems can be used as an easy method to screen Lactococcus lactis strains for their content of R/M system. This will facilitate the combination of strains containing different R/M systems in multiple starter culture.
Non dairy lactic acid fermentations (sausage, sourdough, sauerkraut, fruit mashes, olives and malolactic fermentation of wine) are under study. Research is underway in the areas of: taxonomy of bacteria; development of starter cultures; physiological characterisation; biogenic amines; bacteriophages; genetical characterisation; applied genetics; risk assessment and deoxyribonucleic acid (DNA) probes.
Screening was carried out in lactic acid bacteria isolated from Portuguese wines originating from different geographical regions. Strains were selected according to their capabilities to perform malolactic fermentation under wine conditions. Spoilage bacteria have also been isolated from port wines. Initial screening of bacteria suggests that one species is predominantly responsible for the spoilage.
The effects of aeration and the presence of malic acid on the metabolism of glucose were monitored both in growing and nongrowing cells of Leuconostoc oenos. Considerable alterations on the pattern of end products were observed when anaerobic conditions were compared.
In vivo carbon-13 nuclear magnetic resonance (NMR) was used to investigate the metabolism of glucose, fructose and ribose in nongrowing cells under a gas phase of oxygen, air, nitrogen or carbon dioxide. Erythritol and glycerol were detected as 2 major products of glucose metabolism and the ratio of the concentrations of these 2 metabolites was strongly influenced by the aeration conditions. The metabolites detected and the metabolic shift observed in the presence of oxygen indicated that glucose was catabolized heterofermentatively as well as via a novel erythritol forming pathway. This pathway was elucidated by using carbon-13 nuclear magnetic resonance labelled glucose on carbon 1, carbon 2 or carbon 6. The carbon flux in the 2 pathways was determined by proton NMR analysis.
3 homemade cheeses were screened for lactic acid bacteria. Samples were taken at different stages of ripening, their physicochemical characteristics determined and the microorganismspresent quantified and isolated. Those belonging to any of the lactic acid bacteria genera, as well as others isolated from cheese and wine, were tested for activities such as growth in milk, rate of acidification, presence of bacteriophages, antimicrobial production, etc, in order to select strains to be used as starters.
Among the strains tested, 2 lactobacilli showed production of bacteriocins which remained active in culture supernatants and had a spectrum of activity that comprised some gram positive spoilage bacteria. One of the bacteriocins has been purified in order to study its characteristics and achieve cloning of the corresponding genes. Bacteriocin like antagonism activity has been also detected in 2 lactococcal strains.
Bacteriophages active on lactobacilli have been isolated and one chosen for further studies. Its structural characteristics are being elucidated as a step towards the knowledge of its intracellular development.
The isolation and sequence determination of the lysine-aminopeptidase gene (lap, pepN) from Lactococcus lactis subspecies cremoris Wg2 is reported. The gene has been cloned from genomic libraries of size fractionated lactococcal deoxyribonucleic acid (DNA). The complete nucleotide (nt) sequence of the lap gene has been determined. A large open reading frame (ORF) of 2538 nt is predicted to encode a polypeptide of 846 amino acids (aa). A recombinant plasmid containing the lap gene with its flanking sequences was shown to direct in vivo synthesis of LAP activity in Escherichia coli, indicating that the cloned DNA fragment is the lap gene. The lap gene seems to be present in most strains of L lactis and L cremoris.
Inactivation studies of the lap gene in Lactococcus were performed by integration of a truncated part of the gene into the chromosomal genome by homologous additive integration. The resulting lactococcal transformants demonstrated a substantially reduced LAP activity when compared to the untransformed strain.
Comparison of the deduced aa sequence indicates that the LAP has extensive homology to mammalian aminopeptidases and belongs to the super family of zinc (2+) ion metallohydrolases and show identity in the core decapeptide consensus sequence for the zinc (2+) ion binding motif of these enzymes.
To characterize the pattern of peptidolytic activities of mesophilic lactobacilli used in manufacturing Swiss type cheese, cell free extracts of Lactobacillus (Lb) delbrueckii subspecies (ssp) lactis WS87 were fractionated and incubated with selected peptide substrates. Of 8 peptidases detected, one (peptidase A) has been purified and biochemically characterized. In order to isolate some of the corresponding peptidase genes, plasmid libraries of the Lb delbrueckii ssp lactis WS87 genome were constructed. The libraries were screened by complementation of peptidase deficiencies in the heterologous Escherichia (E) coli system by using enzyme specific chromogenic substrates. This allowed the identification, sucbcloning and sequencing of genes encoding an X-prolyl-dipeptidyl-aminopeptidase (pepA) and an aminopeptidase (pepB). In E coli, both genes are heavily overexpressed from their authentic promoters.
The deduced amino acid sequences of the pepA and pepB products exhibits extensive homologies to enzymes with similar substrate specificities from other lactic acid bacteria. Using a newly constructed Lactobacillus vector, the pepB gene could be stably maintained in Lb casei.
E coli strain UK61, being deficient in the uptake of x-pro and pro-x dipeptides, was transformed with a plasmid library of the Lb delbrueckii ssp lactis chromosome. Complementation experiments resulted in the isolation of a transport gene which is distinct from the E coli dppA and oppA genes. This gene shows significant homology to genes involved in the transport of branched amino acids in Pseudomonas.
Lactococcal starter cultures have been used with manipulated proteolytic systems to produce cheddar cheese in a closed laboratory scale environment. Cheeses were made using lactococcal strains with the following characteristics: those defective in protease activity; those harbouring cloned protease genes which produced an elevated level of protease activity; a strain harbouring a neutral protease from Bacillus (B) subtilis; a strain harbouring a cloned lysyl-aminopeptidase which is expressed at elevated levels; and a parental strain with an unaltered proteolytic system.
Based on limited organoleptic analysis and detailed chemical analysis, the following conclusions can be drawn from the cheese making trials. Although starter proteases are required for accumulation of small peptides and free amino acids in cheddar cheese, increasing the concentration of the cell wall associated protease approximately 3 fold did not enhance proteolysis. The strain that produced the cell wall associated protease appeared to contribute more to proteolysis than that which secreted the enzyme. Cheeses made with strains deficient in protease activity lacked flavour. Cheeses made with those strains harbouring the neutral protease from B subtilis exhibited a greatly accelerated rate of ripening as well as elevated levels of nitrogen in water soluble fractions.
Screening programmes are executed in order to select and improve starter cultures. Desirable traits screened for are probiotic properties. Claims concerning health beneficial activities (antitumour activities, competitive exclusion, anticholesteremic activities, etc) of some strains are investigated. Enzyme treatment of agroindustrial waste products and conservation by ensilage are under investigation. Research is also focused on antimicrobial activities produced by some lactobacilli as this could serve as an alternative to chemical food perservatives.
Undesirable properties, for instance decarboxylating activities resulting in the formation of biogenic amines, are screened for, in order to circumvent fermentation products which are causing serious health problems. The role of lactobacilli in food spoilage and shelf life extension is under investigation and the subject of predictive modelling studies.
Molecular genetic tools (transformation protocols, plasmid vectors, chromosomal integration vectors, expression vectors, isolated genes, etc) are being developed to improve Lactobacillus strains. Novel oral Lactobacillus based vaccines are under construction by equipping selected strains with the property to synthesize specific antigens. Lactobacillus strains are also being constructed that will affect fat resorption in animals and humans. Improvement of antimicrobial compounds in lactobacilli is under study. Recombinant deoxyribonucleic acid (DNA) technology is applied in studies concerning the rapid detection and typing of Lactobacillus strains.
An in vitro model simulating the gastrointestinal tract was developed to predict physiological and nutritional properties of new (fermented) foods, food additives and pharmaceuticals. Assessment of the environmental impact related to the oral intake of genetically engineered microorganisms is studied in this model system. It also provides a model for studying the interactive processes between food and gastrointestina l components and the microecology of the intestine.
Lactobacillus (Lb) helveticus is used as a starter culture along with Streptococcus salivarius subspecies (ssp) thermophilus and Lb delbrueckii ssp bulgaricus in the fermentation of milk to produce cheese, yoghurt and other dairy products. Studies have been performed to manipulate the key enzyme alpha galactosidase in order to stabilize and improve expression of this gene in Lb helveticus. Cloning of the beta galactosidase gene has been attempted and hybridisation studies of mutant strains performed to determine the nature of the mutation events leading to the loss of the lac+ phenotype.
Further studies have been performed to develop an efficient, reproducible method of transformation of Lb helveticus thereby allowing the introduction of foreign genes into certain strains. This has involved the use of electroporation of the Lb helveticus cultures.
An attempt is being made to modify the bacterial strains which are already used for fermentation. The types of genes which may be interesting to establish in these bacteria include the bacteriocins (to combat toxic bacterial contaminants), bacteriophage resistance genes, and novel proteases (for taste). For this purpose some genetic tools have been developed which allow transfer of foreign deoxyribonucleic acid (DNA) into different organisms, and stable integration of this DNA in chromosome. A temperature sensitive plasmid has been combined with an origin of conjugational transfer to make a cloning vector. This vector can be readily transferred between lactic strains. If a fragment of chromosomal DNA is present on this plasmid, it is possible to select for stable integration in the chromosome of the information carried by the vector.
The use of lactic bacteria as vaccine and drug carriers is under study and relies on the development of DNA transfer systems such as the one described.
The regulatory mechanisms and circuits which are operating in lactococci are under study. The study was initiated on 3 models, namely the biosynthesis of: tryptophan; branched chain amino acids (isoleucine, leucine and valine); and histidine. The biosynthetic genes involved in these pathways were characterised by cloning and sequence analysis. The main conclusions, based on analysis of over 30 kb of sequence, are that: most if
This proposal involves the transnational collaboration of eight research.teams that wish to constitute the CORE Group in the T project on the Biotechnology of Lactic Acid Bacteria (LAB). A critical research mass, With multi-disciplinary skills including physiology, biochemistry and molecular biology, has been assembled to advance the fundamental knowledge of IAB. Lactococcus will be used as a model system for studies on gene expression and regulation, chromosome structure, conjugation, and the development of food grade cloning and integration vectors but attention will also be focused on other industrially important LAB e.g. Lactobacillus, Leuconostoc and thermophilic Streptococcus. The participating groups have targeted key industrially important traits of LAB such as proteolysis, phage and phage resistance, antimicrobials and metabolism of lactose, maltose and citrate for specific research attention. screening for LAB strains with improved industrial traits is an inherent part of this proposal.
Since these are LEAF activities in the T-project, the groups in this proposal also wish to participate in specific LEAFS so that the maximum progress can be ensured by the two-way interaction of CORE and LEAF Groups, including industry partners.
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CORDIS classe les projets avec EuroSciVoc, une taxonomie multilingue des domaines scientifiques, grâce à un processus semi-automatique basé sur des techniques TLN. Voir: Le vocabulaire scientifique européen.
- sciences médicales et de la santé biotechnologie médicale génie génétique thérapie génique
- sciences naturelles sciences chimiques chimie organique acides organiques
- sciences naturelles sciences biologiques microbiologie virologie
- sciences naturelles sciences biologiques génétique ADN
- sciences médicales et de la santé médecine fondamentale pharmacologie et pharmacie produit pharmaceutique vaccins
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