Identification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP-rhamnose biosynthesis
Boels, I.C. ; Beerthuyzen, M.M. ; Kosters, M.H. ; Kaauwen, M.P.W. van; Kleerebezem, M. ; Vos, W.M. de - \ 2004
Journal of Bacteriology 186 (2004)5. - ISSN 0021-9193 - p. 1239 - 1248.
controlled gene-expression - gram-positive bacteria - subsp cremoris - capsular polysaccharide - streptococcus-pneumoniae - acid bacteria - cell-wall - o-antigen - exopolysaccharide production - escherichia-coli
dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in Lactococcus lactis. We cloned the rfbACBD operon from L. lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in Escherichia coli, the lactococcal rfbACBD genes could sustain heterologous production of the Shigella flexneri O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in L. lactis resulted in doubled dTDP-rhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate. However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels. A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the rfb genes have an essential role in L. lactis. Limitation of RfbBD activities resulted in the production of altered EPS. The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar. Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties. This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.
Sugar utilisation and conservation of the gal-lac gene cluster in Streptococcus thermophilus
Bogaard, P.T.C. van den; Hols, P. ; Kuipers, O.P. ; Kleerebezem, M. ; Vos, W.M. de - \ 2004
Systematic and Applied Microbiology 27 (2004)1. - ISSN 0723-2020 - p. 10 - 17.
lactose transport-system - 16s ribosomal-rna - beta-galactosidase - exopolysaccharide production - phosphotransferase systems - escherichia-coli - acid bacteria - phosphoenolpyruvate - expression - transcription
The adaptation to utilise lactose as primary carbon and energy source is a characteristic for Streptococcus thermophilus. These organisms, however only utilise the glucose moiety of lactose while the galactose moiety is excreted into the growth medium. In this study we evaluated the diversity of sugar utilisation and the conservation of the gal-lac gene cluster in a collection of 18 S. thermophilus strains isolated from a variety of sources. For this purpose analysis was performed on DNA from these isolates and the results were compared with those obtained with a strain from which the complete genome sequence has been determined. The sequence, organisation and flanking regions of the S. thermophilus gal-lac gene cluster were found to be highly conserved among all strains. The vast majority of the S. thermophilus strains were able to utilize only glucose, lactose, and sucrose as carbon sources, some strains could also utilize fructose and two of these were able to grow on galactose. Molecular characterisation of these naturally occurring Gal+ strains revealed up-mutations in the galKTE promoter that were absent in all other strains. These data support the hypothesis that the loss of the ability to ferment galactose can be attributed to the low activity of the galKTE promoter, probably as a consequence of the adaptation to milk in which the lactose levels are in excess.
Ability of Lactococcus lactis to export viral capsid antigens: a crucial step for development of live vaccines
Dieye, Y. ; Hoekman, A.J.W. ; Clier, F. ; Juillard, V. ; Boot, H.J. ; Piard, J.C. - \ 2003
Applied and Environmental Microbiology 69 (2003)12. - ISSN 0099-2240 - p. 7281 - 7288.
bursal disease virus - toxin fragment-c - acid bacteria - bacillus-subtilis - escherichia-coli - mucosal surfaces - subsp lactis - cell-wall - protein - streptococcus
Thefood grade bacterium Lactococcus lactis is a potential vehicle for protein delivery in the gastrointestinal tract. As a model, we constructed lactococcal strains producing antigens of infectious bursal disease virus (IBDV). IBDV infects chickens and causes depletion of B-lymphoid cells in the bursa of Fabricius and subsequent immunosuppression, morbidity, or acute mortality. The two major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were expressed and targeted to the cytoplasm, the cell wall, or the extracellular compartment of L. lactis. Whereas VP3 was successfully targeted to the three compartments by the use of relevant expression and export vectors, VP2 was recalcitrant to export, thus confirming the difficulty of translocating naturally nonsecreted proteins across the bacterial membrane. This defect could be partly overcome by fusing VP2 to a naturally secreted protein (the staphylococcal nuclease Nuc) that carried VP2 through the membrane. Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial compartments were administered orally to chickens. The chickens did not develop any detectable immune response against VP2 and VP3 but did exhibit an immune response against Nuc when Nuc-VP2 was anchored to the cell wall of lactococci.
Engineering of carbon distribution between glycolysis and sugar nucleobiosynthesis in Lactococcus lactis
Boels, I.C. ; Kleerebezem, M. ; Vos, W.M. de - \ 2003
Applied and Environmental Microbiology 69 (2003). - ISSN 0099-2240 - p. 1129 - 1135.
controlled gene-expression - exopolysaccharide biosynthesis - streptococcus-thermophilus - subsp cremoris - cellular phosphoglucomutase - polysaccharide formation - rheological properties - escherichia-coli - acid bacteria - cloning
We describe the effects of modulating the activities of glucokinase, phosphofructokinase, and phosphoglucomutase on the branching point between sugar degradation and the biosynthesis of sugar nucleotides involved in the production of exopolysaccharide biosynthesis by Lactococcus lactis. This was realized by using a described isogenic L. lactis mutant with reduced enzyme activities or by controlled expression of the well-characterized genes for phosphoglucomutase or glucokinase from Escherichia coli or Bacillus subtilis, respectively. The role of decreased metabolic flux was studied in L. lactis strains with decreased phosphofructokinase activities. The concomitant reduction of the activities of phosphofructokinase and other enzymes encoded by the las operon (lactate dehydrogenase and pyruvate kinase) resulted in significant changes in the concentrations of sugar-phosphates. In contrast, a >25-fold overproduction of glucokinase resulted in 7-fold-increased fructose-6-phosphate levels and 2-fold-reduced glucose-1-phosphate and glucose-6-phosphate levels. However, these increased sugar-phosphate concentrations did not affect the levels of sugar nucleotides. Finally, an similar to100-fold overproduction of phosphoglucomutase resulted in 5-fold-increased levels of both UDP-glucose and UDPgalactose. While the increased concentrations of sugar-phosphates or sugar nucleotides did not significantly affect the production of exopolysaccharides, they demonstrate the metabolic flexibility of L. lactis.
Characterization, expression, and mutation of the Lactococcus lactis galPMKTE genes, involved in galactose utilization via the Leloir pathway
Groossiord, B.P. ; Luesink, E.J. ; Vaughan, E.E. ; Arnaud, A. ; Vos, W.M. de - \ 2003
Journal of Bacteriology 185 (2003). - ISSN 0021-9193 - p. 870 - 878.
tagatose 6-phosphate pathway - streptococcus-lactis - acid bacteria - nucleotide-sequence - escherichia-coli - molecular-cloning - melibiose carrier - exopolysaccharide biosynthesis - lactose metabolism - bacillus-subtilis
A cluster containing five similarly oriented genes involved in the metabolism of galactose via the Leloir pathway in Lactococcus lactis subsp. cremoris MG1363 was cloned and characterized. The order of the genes is galPMKTE, and these genes encode a galactose permease (GalP), an aldose I-epimerase (GaIM), a galactokinase (GalK), a hexose-1-phosphate uridylyltransferase (GalT), and a UDP-glucose 4-epimerase (GaIE), respectively. This genetic organization reflects the order of the metabolic conversions during galactose utilization via the Leloir pathway. The functionality of the galP, galK, galT, and galE genes was shown by complementation studies performed with both Escherichia coli and L. lactis mutants. The GalP permease is a new member of the galactoside-pentose-hexuronide family of transporters. The capacity of GalP to transport galactose was demonstrated by using galP disruption mutant strains of L. lactis MG1363. A galK deletion was constructed by replacement recombination, and the mutant strain was not able to ferment galactose. Disruption of the galE gene resulted in a deficiency in cell separation along with the appearance of a long-chain phenotype when cells were grown on glucose as the sole carbon source. Recovery of the wild-type phenotype for the galE mutant was obtained either by genetic complementation or by addition of galactose to the growth medium.
Glutathione protects Lactococcus lactis against oxidative stress
Li, Y. ; Hugenholtz, J. ; Abee, T. ; Molenaar, D. - \ 2003
Applied and Environmental Microbiology 69 (2003)10. - ISSN 0099-2240 - p. 5739 - 5745.
escherichia-coli - saccharomyces-cerevisiae - oxidized glutathione - growth-conditions - acid bacteria - reductase - thioredoxin - purification - peroxidase - metabolism
Glutathione was found in several dairy Lactococcus lactis strains grown in M17 medium. None of these strains was able to synthesize glutathione. In chemically defined medium, L. lactis subsp. cremoris strain SK11 was able to accumulate up to similar to60 mM glutathione when this compound was added to the medium. Stationary-phase cells of strain SK11 grown in chemically defined medium supplemented with glutathione showed significantly increased resistance (up to fivefold increased resistance) to treatment with H2O2 compared to the resistance of cells without intracellular glutathione. The resistance to H2O2 treatment was found to be dependent on the accumulation of glutathione in 16 strains of L. lactis tested. We propose that by taking up glutathione, L. lactis might activate a glutathione-glutathione peroxidase-glutathione reductase system in stationary-phase cells, which catalyzes the reduction of H2O2. Glutathione reductase, which reduces oxidized glutathione, was detectable in most strains of L. lactis, but the activities of different strains were very variable. In general, the glutathione reductase activities of L. lactis subsp. lactis are higher than those of L. lactis subsp. cremoris, and the activities were much higher when strains were grown aerobically. In addition, glutathione peroxidase is detectable in strain SK11, and the level was fivefold greater when the organism was grown aerobically than when the organism was grown anaerobically. Therefore, the presence of glutathione in L. lactis could result in greater stability under storage conditions and quicker growth upon inoculation, two important attributes of successful starter cultures.