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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Physiological and molecular adaptations of Lactococcus lactis to near-zero growth conditions
    Ercan, O. - \ 2014
    Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): Eddy Smid. - Wageningen : Wageningen University - ISBN 9789462570719 - 206
    lactococcus lactis - adaptatiefysiologie - voedselmicrobiologie - groeitempo - groeispanning - transcriptomica - metabolomica - lactococcus lactis - adaptation physiology - food microbiology - growth rate - growth stress - transcriptomics - metabolomics

    Lactococcus lactis is an important lactic acid bacteria (LAB) species that is used for the manufacture of dairy products, such as cheese, buttermilk, and other fermented products. The predominant function of this bacterium in dairy fermentation is the production of lactic acid, as its major fermentation end-product that contributes to preservation and microbial safety of the product. Moreover, L. lactis is frequently encountered in natural ecosystems such as in (rotting) plant material.

    Due to restricted energy source availability, natural microbial communities commonly live in a situation that can be characterized as ‘hunger’, which is different from strict nutrient-starvation. As a consequence, environmental microbes commonly grow at very low-growth rates as compared to laboratory cultures. Analogously, microorganisms can experience such nutrient-poor conditions in diverse industrial fermentation applications. For example, LAB encounter extreme low or no energy source availability during the extended ripening process of cheeses or dry sausages, which can take months. Despite these harsh environmental conditions, many LAB are able to remain viable in these processes for months and sustain a low-level metabolic activity, which plays an important role in their contribution to flavor and aroma formation in the product matrix.

    In this thesis, the quantitative physiology of L. lactis at near-zero specific growth rates was studies, employing both metabolic and genome-wide transcriptome studies in an experimental set-up of carbon-limited retentostat cultivation. Chapter 2 describes how retentostat cultivation enables uncoupling of growth and non-growth related processes in L. lactis, allowing the quantitative analysis of the physiological adaptations of this bacterium to near-zero growth rates. In chapter 3, transcriptome and metabolome analyses were integrated to understand the molecular adaptation of L. lactis to near-zero specific growth rate, and expand the studies in chapter 2 towards gene regulations patterns that play a profound role in zero-growth adaptation. Chapter 4 describes the enhanced robustness to several stress conditions of L. lactis after its adaptation to extremely low-specific growth rate by carbon-limited retentostat cultivation. In this chapter correlations were modelled that quantitatively and accurately describe the relationships between growth-rate, stress-robustness, and stress-gene expression levels, revealing correlation coefficients for each of the varieties involved. Chapter 5 evaluates the distinction between the transcriptome responses to extended carbon-limited growth and severe starvation conditions, where the latter condition was elicited by switching off the medium supply of the retentostat cultures described in chapter 1. Chapter 6 highlights the comparison of the physiological and molecular adaptations of industrially important microorganisms towards carbon-limited retentostat conditions. In conclusion, this thesis describes the quantitative physiological, metabolic, and genome-wide transcriptional adaptations of L. lactis at near-zero specific growth rates induced by carbon source limited retentostat cultivation, and compares these molecular adaptations to those elicited by strict carbon-starvation conditions.

    Community dynamics of complex starter cultures for Gouda-type cheeses and its functional consequences
    Erkus, O. - \ 2014
    Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): Eddy Smid. - Wageningen UR : Wageningen - ISBN 9789462570108 - 215
    goudse kaas - lactococcus lactis - microbiële diversiteit - gouda cheese - lactococcus lactis - microbial diversity

    Lactic acid bacteria (LAB) are used as starter and adjunct cultures for the production of artisanal and industrial fermented milk products such as yoghurt and cheese. Artisanal fermentations is propagated with the transfer of an inoculum from old batch of fermented food to the new batch (back-slopping) to initiate the fermentation with the activity of the indigenous microbiota present in the inoculum. In industrial production, these inocula with indigenous microbiota are replaced with the starter cultures that contain lower numbers of LAB species for better controlled fermentation process and consistent final product quality. Cheese manufacturing is still performed in both artisanal ways and with the use of starter cultures. Gouda cheese starter cultures constitute several strains from the subspecies of Lactococcus lactisand Leuconostocs mesenteroidesin different combinations. The mixed and undefined type of starter culture may harbour variable number of strains that contribute unique functionalities to the cheese manufacturing process. Therefore, understanding, controlling and predicting the cheese manufacturing processes require the determination of strain level diversity in the starter culture, their collective and specific metabolic complement, and their activity throughout the cheese manufacturing process, including the interactions between the strains. The first two studies that are covered in this thesis describes the development of a high resolution AFLP fingerprinting tool allowing the discrimination of closely related strains in the starter culture and the subsequent analysis of the microbial community of Gouda cheese starter with this implemented technique and with metagenomics. Furthermore, the thesis includes the development of another tool to selectively amplify DNA only from live fraction of the microbial community in cheese using propidium monoazide (PMA), which is required to study community dynamics with culture independent approaches. The last study in the thesis describes the effects of the variation in propagation regime on the community composition of a mixed starter culture and connects the composition change to the functionalities that impact on flavour development during cheese manufacturing. Overall, the approaches presented in this thesis are intended to eventually enable accurate prediction and control of the cheese manufacturing process using (un)defined starter cultures, but may also allow rational design and development of new starter cultures.

    Metabolic shifts in microorganisms: the case of Lactococcus lactis
    Goel, A. - \ 2013
    Wageningen University. Promotor(en): Willem de Vos; B. Teusink, co-promotor(en): D. Molenaar. - S.l. : s.n. - ISBN 9789461737342 - 184
    lactococcus lactis - metabolisme - enzymactiviteit - enzymen - systeembiologie - metabolische studies - lactococcus lactis - metabolism - enzyme activity - enzymes - systems biology - metabolic studies

    A commonly observed organismal response to changing growth rate is a metabolic shift from one mode of metabolism to another. This phenomenon is potentially interesting from a fundamental and industrial perspective because it can influence cellular choices and can limit the capacity of industrial microorganisms to channel nutrients to desired products. The mechanistic cause of the metabolic shift may vary between species, but the presence of such shifts from bacteria to man suggests functional relevance, which may be understood through an evolutionary perspective. One of the many existing hypotheses (reviewed in Chapter 2) states that protein investment costs affect the metabolic strategy employed, and that the implemented strategy is the result of a cost-benefit analysis. To test this experimentally, we performed a global multi-level analysis using the model lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363, which shows a distinct, anaerobic version of the bacterial Crabtree/Warburg effect: at low growth rates it produces “mixed-acids” (acetate, formate and ethanol) and at high growth rates it produces predominantly lactate from glucose.

    We first standardized growth conditions and established an in vivo–like enzyme assay medium mimicking the intracellular environment for enzyme activity measurements of growing cells of L. lactis (Chapter 3). With standardized experimental procedures we characterized at multiple cellular levels, glucose-limited chemostat cultures of L. lactis at various growth rates. More than a threefold change in growth rate was accompanied by metabolic rerouting with, surprisingly, hardly any change in transcription, protein ratios, and enzyme activities (Chapter 4). Even ribosomal proteins, constituting a major investment of cellular machinery, scarcely changed. Thus, contrary to the original hypothesis, L. lactis displays a strategy where its central metabolism appears always prepared for high growth rate and it primarily employs the regulation of enzyme activity rather than alteration of gene expression. Only at the highest growth rate and during batch growth – conditions associated with glucose excess – we observed down-regulated stress protein levels and up-regulated glycolytic protein levels. We conclude from this that for glucose, transcription and protein expression largely follow a binary feast / famine logic in L. lactis.

    To delve deeper into the mechanism of regulation of the shift in L. lactis, we tested a mixed-acid fermentative lactose-utilizing L. lactis MG1363 derivative and showed that there is a strong positive correlation between glycolytic flux and the extent of homolactic fermentation: a correlation caused by metabolic regulation (Chapter 5). We subsequently provided new evidence for a causal relationship between the concentration of the glycolytic intermediate, fructose-1,6-bisphosphate (FBP) and the metabolic shift. We showed that 2,5-anhydromannitol, which converts to a non-metabolizable FBP analogue in vivo, almost doubles the flux towards lactate when taken up by the cells. In vitro the activating effect of the analogue on lactate dehydrogenase is similar to native FBP, whereas it had no effect on the enzyme phosphotransacetylase (part of the mixed-acid pathway). The activation concentration of the analogue, however, is much lower than normal intracellular FBP concentrations. This may imply that the activation of lactate dehydrogenase in vivo requires a much higher concentration of FBP, but this remains to be resolved. We subsequently put the regulatory relationships of glycolytic flux, FBP, the redox potential and allosteric effectors on enzymes of the glycolytic and downstream pathways together in a mathematical model to test and investigate whether these interactions can explain the metabolic shift (Chapter 6). Although the model was not able to consistently fit combined data from the chemostats at various dilution rates, and in vivo–NMR data of glucose pulsed non-growing cells, we found for the best fitted model that the parameters most influencing the metabolic shift were those involved in regulation by FBP and inorganic phosphate.

    In conclusion, L. lactis seems to be always prepared for high growth rate as it carries a high overcapacity of enzymes, a property retained even after evolving for 800 generations under constant environmental conditions. Moreover, its growth rate-related metabolic shift does not appear to be an outcome of growth-rate optimization with protein cost as a major driver. At the mechanistic level, the choice of the strategy is regulated via alterations in metabolite levels, with FBP (and probably phosphate) exerting a central role.

    Regulatory and adaptive responses of Lactococcus lactis in situ
    Bachmann, H. - \ 2009
    Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): J. van Hylckama. - [S.l.] : s.n. - ISBN 9789085854265 - 192
    lactococcus lactis - kaasbereiding - kazen - adaptatie - genetische regulatie - lactococcus lactis - cheesemaking - cheeses - adaptation - genetic regulation - cum laude
    cum laude graduation (with distinction)
    Electron transport chains of lactic acid bacteria
    Brooijmans, R.J.W. - \ 2008
    Wageningen University. Promotor(en): Willem de Vos, co-promotor(en): J. Hugenholtz. - S.l. : S.n. - ISBN 9789085853022 - 228
    elektronenoverdracht - melkzuurbacteriën - lactococcus lactis - lactobacillus plantarum - haem - menaquinonen - electron transfer - lactic acid bacteria - lactococcus lactis - lactobacillus plantarum - haem - menaquinones
    Lactic acid bacteria are generally considered facultative anaerobic obligate fermentative bacteria. They are unable to synthesize heme. Some lactic acid bacteria are unable to form menaquinone as well. Both these components are cofactors of respiratory (electron transport) chains of prokaryotic bacteria.
    Lactococcus lactis, and several other lactic acid bacteria, however respond to the addition of heme in aerobic growth conditions. This response includes increased biomass and robustness. In this study we demonstrate that heme-grown Lactococcus lactis in fact do have a functional electron transport chain that is capable of generating a proton motive force in the presence of oxygen. In other words, heme addition induces respiration in Lactococcus lactis. This aerobic electron transport chain contains a NADH-dehydrogenase, a menaquinone-pool and a bd-type cytochrome.
    A phenotypic and genotypic screening revealed a similar response, induced by heme (and menaquinone) supplementation, in other lactic acid bacteria.
    The genome of Lactobacillus plantarum WCFS1 was predicted to encode a nitrate reductase A complex. We have found that Lactobacillus plantarum is capable of using nitrate as terminal electron acceptor, when heme and menaquinone are provided. Nitrate can be used by Lactobacillus plantarum as effective electron sink and allows growth on a extended range of substrates. The impact of both the aerobic and anaerobic electron transport chain, on the metabolism and global transcriptome of Lactobacillus plantarum were studied in detail.
    This work has resulted in the discovery of novel electron transport chains and respiratory capabilities of lactic acid bacteria. The potential respiratory capabilities of other, previously considered (strictly) anaerobic prokaryotic bacteria, were reviewed.









    Metabolic engineering of mannitol production in Lactococcus lactis
    Wisselink, H.W. - \ 2004
    Wageningen University. Promotor(en): Willem de Vos, co-promotor(en): J. Hugenholtz. - [S.l.] : S.n. - ISBN 9789085041108 - 149
    metabolisme - mannitol - lactococcus lactis - genetische modificatie - metabolism - mannitol - lactococcus lactis - genetic engineering
    Mannitol is a sugar alcohol that is produced by a wide variety of (micro) organisms. It is assumed to have several beneficial effects as a food additive. It can serve as an antioxidant and as a low-calorie sweetener which can replace high-calorie sugars such as sucrose, lactose, glucose and fructose. In addition, it has been shown that mannitol has a protective effect for lactic acid bacteria, such as Lactococcus lactis , when they are subjected to drying and/or freezing. The viability of starter cultures containing these lactic acid bacteria may thus be enhanced by inducing mannitol production in these strains. In addition the use of a mannitol producing L. lactis may result in fermented products with extra nutritional value.

    The metabolic engineering of mannitol production in L. lactis, using a combined approach of predicting the control points for mannitol production in L. lactis by a kinetic glycolysis model, and experimental metabolic engineering steps, are described in this thesis. Based on the model predictions, we combined engineering steps such as knocking out lactate dehydrogenase, reduction of phosphofructokinase and fructokinase activity, and overexpression of genes involved in mannitol biosynthesis, such as mannitol 1-phosphate dehydrogenase, mannitol 1-phosphatase, and mannitol dehydrogenase. This resulted in different levels of mannitol production in several L. lactis strains. Especially the combination of overexpression of genes encoding mannitol 1-phosphate dehydrogenase and a mannitol-1-phosphatase resulted in high mannitol production by L. lactis . Moreover, it was found that mannitol 1-phosphatase has a high control on the mannitol production, and a clear correlation between the activity of this enzym and the mannitol production was shown.

    The research described in his thesis is a text book example of metabolic engineering. It contributes to the understanding of mannitol biosynthesis in L. lactis , and how the mannitol production can be improved by metabolic engineering. Moreover, it provides suggestions for food-grade applications, especially in the field of the production of low-calorie sweeteners.
    Metabolic engineering of exopolysaccharide production in Lactococcus lactis
    Boels, I.C. - \ 2002
    Wageningen University. Promotor(en): W.M. de Vos; M. Kleerebezem. - S.l. : S.n. - ISBN 9789058086938 - 144
    polysacchariden - melkzuurbacteriën - lactococcus lactis - productie - biosynthese - metabolisme - polysaccharides - lactic acid bacteria - lactococcus lactis - production - biosynthesis - metabolism

    Exopolysaccharides (EPS) produced by lactic acid bacteria are important structural components in fermented foods. In addition, they may confer health benefits to the consumer, as mouse model studies have indicated that EPS may have immunostimulatory, anti-tumoral, or cholesterol-lowering activity. Lactococcus lactis strain NIZO B40 produces a phosphorylated EPS with a branched repeating unit containing glucose, galactose and rhamnose. The biosynthesis of this polymer depends on both the specific eps genes organized in a plasmid-located gene cluster and on several household genes that are involved in biosynthesis of the primary EPS precursors, the nucleotide-sugars. This work focused on the household genes to induce overproduction and/or structural modification of EPS. Therefore, we cloned, characterized, and controlled expression of the genes that encode enzymes involved (i) in primary sugar metabolism ( glk , pfk , fbp , pgm ), (ii) the biosynthetic pathway from glucose-1P to the EPS precursors UDP-glucose ( galU ), UDP-galactose ( galU and galE ) and dTDP-rhamnose ( rfbACBD) , and (iii) in the specific pathway ( epsA-K ) for the assembly of the repeating unit, export and polymerization of the NIZO B40 EPS. We provide evidence for metabolic control of the gal and rfb genes in EPS precursor and EPS production. Overexpression of the galU , pgm or the rfb genes resulted in a significant increase of EPS-precursors. Moreover, overexpression of the eps genes led to four-fold increased NIZO B40 EPS production. In addition, reduction of the UDP-galactose level by galE disruption abolished EPS production while a rfb conditional knock out yielded an EPS with altered sugar composition and different physical characteristics.

    The research described in this thesis contributes to the understanding of exopolysaccharide biosynthesis in lactic acid bacteria and provides a starting point for applications in the dairy industry, especially with respect to the texture and health benefits of fermented products.

    Molecular characterization of a family of cold-shock proteins of Lactococcus lactis
    Wouters, J.A. - \ 2000
    Agricultural University. Promotor(en): F.M. Rombouts; W.M. de Vos; O.P. Kuipers; T. Abee. - S.l. : S.n. - ISBN 9789058082084 - 126
    melkzuurbacteriën - lactococcus lactis - koudeshock - eiwitten - cryobeschermingsmiddelen - lactic acid bacteria - lactococcus lactis - cold shock - proteins - cryoprotectants

    Lactic acid bacteria (LAB) are widely used as starter cultures in fermentation processes. The stress response of LAB during different industrial processes, and during low-temperature conditions in particular, requires a better understanding. For that reason a research project on the cold adaptation of Lactococcus lactis MG1363, a model LAB strain, was initiated. Research focused on the identification and characterization of a family of five csp genes, named cspA, cspB, cspC, cspD and cspE , encoding highly similar cold-shock proteins (CSPs; 65-85% identity). On the L. lactis MG1363 chromosome two tandem groups of csp genes ( cspA/cspB and cspC/cspD ) were identified, whereas cspE was found as a single gene.

    Transcription analysis showed that cspE is the only non-cold-induced csp gene, whereas the other csp genes are induced 10- to 40-fold at different times after cold shock. The 7-kDa CSPs, corresponding to the csp genes of L. lactis MG1363, were the highest induced proteins upon cold shock to 10°C as was shown by two-dimensional gel electrophoresis. Using the nisin-inducible expression system CspB, CspD and CspE could be overproduced to high levels. For CspA and CspC limited overproduction was obtained, that could be explained by low stability of cspC mRNA and by low stability of CspA. For L. lactis NZ9000ΔAB (deleted in cspAB ) and NZ9000ΔABE (deleted in cspABE ) no differences in growth at normal and at low temperature were observed, compared to that of the wild-type strain L. lactis NZ9000. The deletion of csp genes was compensated by increased expression of the remaining csp genes. These data indicate that the expression of csp genes in L. lactis is regulated by a tightly controlled transcription network.

    When L. lactis cells were shocked to 10°C for 4 h the survival to freezing increased approximately 100-fold compared to mid-exponential phase cells grown at 30°C. L. lactis cells overproducing CspB, CspD or CspE at 30°C show a 2-10 fold increased survival after freezing compared to control cells. The adaptive response to freezing conditions by prior exposure to 10°C was significantly delayed in strain NZ9000ΔABE compared to strains NZ9000 and NZ9000ΔAB.

    In combination, these data indicate that 7-kDa CSPs of L. lactis enhance the survival capacity after freezing. CSPs either have a direct protective effect during freezing, e.g. by RNA stabilization, and/or induce other factors involved in the freeze-adaptive response. A group of strongly cold-induced 7-kDa proteins was also identified for Streptococcus thermophilus and, indeed, enhanced production of these proteins also coincided with increased survival to freezing of this bacterium.

    Using two-dimensional gel electrophoresis, induction of several (non-7 kDa) cold-induced proteins (CIPs) of L. lactis was observed upon overproduction of CSPs. Furthermore, several CIPs were no longer cold induced in the csp -deleted strains, which indicates that CSPs might activate the expression of certain CIPs. A selection of CIPs of L. lactis was identified and appears to be implicated in a variety of cellular processes, e.g. transcriptional and translational control, sugar metabolism and signal sensing. Furthermore, it was shown that the maximal glycolytic activity measured at 30°C increases (approximately 2.5-fold) upon incubation at 10°C for two to four h, a process for which protein synthesis is required. Based on their cold induction and involvement in cold adaptation of glycolysis, it is proposed that the CcpA/HPr control circuit regulates a (unidentified) factor involved in the increased glycolytic activity.

    The research described in this thesis contributes to the understanding of the response of lactic acid bacteria to low temperatures and might yield applications for dairy industry, especially with respect to fermentation performance and the survival of starter bacteria during freezing.

    Structural characterisation and enzymic modification of exopolysaccharides from Lactococcus lactis
    Casteren, W. van - \ 2000
    Agricultural University. Promotor(en): A.G.J. Voragen; H.A. Schols. - S.l. : S.n. - ISBN 9789058082206 - 114
    lactococcus lactis - polysacchariden - lactococcus lactis - polysaccharides

    Since ancient times, lactic acid bacteria have been used for the preservation of food. Some of these bacteria are able to produce exopolysaccharides (EPSs), which may contribute to the peculiar rheology and texture of, for example, milk-derived products. Insight into the relationship between the chemical structure of EPSs and their physical properties can lead to tailor-made polysaccharides, which meet particular requirements in terms of structure and function. In this thesis, the elucidation of the chemical structures of three exopolysaccharides from Lactococcus lactis subsp. cremoris is described. Enzymes are used as a tool during the structural characterisation and modification of EPS and the action of three enzymes having activity towards (chemically modified) EPSs is investigated as well. Finally, a start has been made within this project to study the effect of structural changes of EPSs on the physical properties.

    In chapter 1, a brief general introduction into the research subject is given. Besides background information about the use of bacterial EPSs in food and the biosynthesis of EPS, attention is paid to common features in EPS structures from lactic acid bacteria. Different ways to obtain structurally related EPSs are presented and the use of enzymes in polysaccharide research is outlined.

    Chapter 2 describes the study of the chemical structure of EPS B40, explaining earlier reported analytical discrepancies.The EPS contains rhamnose:galactose:glucose:phosphate in a molar ratio of 1:1.3:2:1.1. 31P NMR indicated that a single phosphate group is present as a phosphodiester. EPS B40 has chemically been modified using 0.3 M H 2 SO 4 , 28 M HF or 2 M NaOH. From these modifications it is concluded that during the hydrolysis step prior to sugar composition analysis the galactose 3-phosphate linkages are split only partially and that, consequently, the amount of galactose is underestimated. The backbone of HF-modified EPS B40 can be degraded by a crude cellulase preparation from Trichoderma viride . Purification and characterisation of the obtained oligomers (chapter 3), together with the characterisation of the polymer (chapter 2), has resulted in a chemical structure for EPS B40 identical to the repeating unit already described for EPS SBT 0495:

    Inline Image Figure 01

    In chapter 3, the enzyme activity responsible for the degradation of HF-modified EPS B40 is identified as an endoglucanase (endoV). Thus, after complete removal of galactosyl residues and phosphate and partial removal of rhamnosyl residues, endo glucanase is able to cleave the backbone consisting of glucosyl and galactosyl residues. Characterisation of the resulting homologous series of oligomers by MS and NMR unequivocally demonstrated that endoV is able to cleave theβ-(1→4) linkage between two glucopyranosyl residues when the galactopyranosyl residue towards the nonreducing end is unsubstituted. The mode of action of endoV on HF-modified EPS B40 is discussed on the basis of the subsite model for endoV, described in literature. The crude cellulase preparation from T. viride has also been shown to contain a phosphatase able to act on EPS B40 after removal of rhamnosyl and galactosyl residues by mild CF 3 CO 2 H treatment.

    In chapter 4, the structural elucidation of EPS B39 is outlined. This novel exopolysaccharide structure contains l-Rha, d-Gal and d-Glc in a molar ratio of 2:3:2. Enzymic modification, methylation analysis and 1D/2D NMR experiments (both 1H- 1H and 1H- 13C) revealed that EPS B39 consists of a branched heptasaccharide repeating unit with the following structure:

    Inline Image Figure 02

    Chapter 5 describes the chemical structure of EPS B891, which contains d-Gal and d-Glc in a molar ratio of 2:3. The polysaccharide is partially O -acetylated. By means of HF solvolysis, O -deacetylation, enzymic modification, methylation analysis and 1D/2D NMR studies the novel exopolysaccharide is shown to be composed of repeating units with the following structure:

    Inline Image Figure 03

    EPS B39 and O -deacetylated EPS B891 both contain lactosyl side chains and it is demonstrated that the terminally linked galactosyl residues can be removed by using a crude commercial enzyme preparation from Aspergillus aculeatus . The purification and characterisation of theβ-galactosidase responsible for this modification is described in chapter 6. The enzyme has a molecular mass of approximately 120 kDa, a pI between 5.3-5.7 and is optimally active at pH 5.4 and 55-60 oC. Based on the N-terminal amino acid sequence, the enzyme probably belongs to family 35 of the glycosyl hydrolases. The catalytic mechanism is shown to be retaining and transglycosylation products are demonstrated using lactose as a substrate. Theβ-galactosidase is able to release terminally linked galactosyl residues from EPS B891 in presence of acetyl groups, but the hydrolysing rate after O -deacetylation is higher. Furthermore, O -deacetylated EPS B891 is degalactosylated faster than EPS B39.

    In chapter 7, the results of this thesis are discussed. Emphasis is placed on the approach of using enzymes in structure (-function) studies of exopolysaccharides. Furthermore, the use of (modified) exopolysaccharides for characterising enzyme activities is outlined. Finally, the influence of various structural modifications on the physical properties of EPSs is briefly discussed.

    Physiology of exopolysaccharide biosynthesis by Lactococcus lactis
    Looijesteijn, E. - \ 2000
    Agricultural University. Promotor(en): J.A.M. de Bont; J. Hugenholtz. - S.l. : S.n. - ISBN 9789058082862 - 197
    lactococcus lactis - industriële microbiologie - biosynthese - biochemie - fysiologie - oligosacchariden - koolhydraten - melkzuurbacteriën - lactococcus lactis - industrial microbiology - biosynthesis - biochemistry - physiology - oligosaccharides - carbohydrates - lactic acid bacteria

    Several lactic acid bacteria (LAB) produce exopolysaccharides (EPS). EPSs produced by LAB are a potential source of natural additives and because LAB are food grade organisms, these EPSs can also be produced in situ . The amount of EPS in milk fermented with strain NIZO B40, which produces an anionic EPS composed of glucose, rhamnose, galactose and phosphate, is very low. This relatively low concentration could be increased by optimising the culture conditions and medium composition. Using pH-controlled fermentations and a chemically defined medium, the total EPS production was highest at pH 5.8 and 25 °C. Glucose was demonstrated to be the most efficient sugar source for EPS production by L. lactis NIZO B40. With fructose as the sugar source only a minor amount of EPS was produced. The intracellular levels of sugar nucleotides, the EPS precursors, were much lower in fructose- than in glucose-grown cultures. The activity of the enzymes involved in the biosynthesis of the sugar nucleotides were however unaffected by the source of sugar but the activity of fructose-1,6-bisphosphatase (FBPase) was very low. FBPase catalyses the conversion of fructose-1,6-diphosphate into fructose-6-phosphate, an essential step for the biosynthesis of sugar nucleotides from fructose but not from glucose. Overexpression of the fbp gene resulted in increased EPS synthesis on fructose.

    Most culture conditions influenced growth as well as EPS formation and EPS synthesis itself was also influenced by the growth rate. EPS production by strain NIZO B40 starts at the exponential growth phase but continues during the stationary phase in batch cultures, indicating that EPS biosynthesis and growth are not strictly coupled. Indeed we found that non-growing cultures were still able to produce EPS, making it possible to study the influence of different culture conditions on EPS biosynthesis independent of growth.

    The amounts of EPS produced by L. lactis NIZO B40 and NIZO B891 were comparable under glucose and leucine limitation. The efficiency of EPS production, the quantity of EPS produced per quantity of glucose consumed, was however much higher under conditions of glucose limitation. The production of phosphorylated B40 EPS and of unphosphorylated B891 EPS was strongly reduced under conditions of phosphate limitation. The sugar composition of both B40 and B891 EPS and the phosphate content of B40 EPS were unaffected by the type of limitation but surprisingly, glucose limitation resulted in the production of EPSs with strongly reduced molecular masses.

    Anionic B40 EPS in suspension and a cell-associated layer of this EPS protected the bacteria against toxic copper ions and nisin, probably due to charge interactions. Furthermore, cell-associated EPS resulted in a decrease in the sensitivity of the bacteria to bacteriophages and lysozyme, most likely by masking the targets for the phages and the enzyme. The protection of EPS against nisin and bacteriophages could be a competitive advantage in mixed strain dairy starter cultures. Unfortunately, the EPS yields were not increased in the presence of copper, bacteriophages, nisin or lysozyme.

    Exopolysaccharide biosynthesis in Lactococcus lactis : a molecular characterisation
    Kranenburg, R. van - \ 1999
    Agricultural University. Promotor(en): W.M. de Vos. - S.l. : S.n. - ISBN 9789058081353 - 122
    lactococcus lactis - biosynthese - polysacchariden - lactococcus lactis - biosynthesis - polysaccharides

    Lactic acid bacteria are Gram-positive bacteria which are used for industrial food fermentation processes. Some have the ability to form exopolysaccharides (EPSs) and these bacteria or the produced EPSs can be used to enhance the structural properties of food products. Furthermore, these EPSs are claimed to be health beneficial. This thesis describes the results of a study on the biosynthesis of these polymers in Lactococcus lactis strains.

    Chapter 1 provides an overview of the current knowledge of cell-surface polysaccharide biosynthesis, the glycosyltransferases involved, and export and polymerisation processes. Special attention is paid to genetics, regulation, and EPSs produced by LAB.

    Chapter 2 describes the characterisation of EPS production by L. lactis NIZO B40. The strain produces an extracellular phosphopolysaccharide containing galactose, glucose, and rhamnose. The EPS production is encoded on a 40-kb plasmid, which was isolated after conjugation and subsequent plasmid curing. On this plasmid, a 12-kb region containing 14 genes with the order epsRXABCDEFGHIJKL was identified encoding putative gene products which shared sequence homologies with gene products involved in cell-surface polysaccharide biosynthesis of other bacteria. Based on these homologies, predicted functions as regulation ( epsR ), polymerisation and export ( epsA , epsB , epsI , epsK ), or biosynthesis of the repeating unit ( epsD , epsE / epsF , epsG , epsH ) could be assigned. The eps genes are co-ordinately expressed and transcribed as a single 12-kb mRNA from a promoter upstream of epsR . Heterologous expression of epsD in Escherichia coli showed that its gene product is the so-called priming glucosyltransferase, linking the first sugar of the repeating unit to the lipid carrier.

    Chapter 3 describes the functional analysis of the glycosyltransferase genes of the NIZO B40 eps gene cluster. The genes were cloned and expressed in E. coli and L. lactis to determine their function and the sugar-specificity of the encoded enzymes. The EPS consists of repeating units containing a trisaccharide backbone of two glucose and one galactose moieties. The epsDEFG gene products are involved in the synthesis of this trisaccharide, linking glucose to a lipid carrier in the membrane (EpsD), glucose to lipid-linked glucose (EpsE/EpsF), and galactose to lipid-linked cellobiose (EpsG), respectively. The epsJ gene product was found to be involved in the biosynthesis of EPS and is likely to act either as a galactosyl phosphotransferase or as an enzyme which releases the backbone oligosaccharide from the lipid carrier.

    Chapter 4 describes the variety of EPS production by L. lactis . Sixteen EPS-producing L. lactis strains were analysed and based on the chemical composition of the EPSs they formed and the genotype of their eps genes, they were grouped in three major groups and two unique strains. Representatives of the three major groups were studied in detail. Group I comprises strain NIZO B40 which was characterised in the previous chapters. Fragments of the eps gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating unit synthesis.

    First successful attempts for genetic engineering of the EPS production were achieved by replacing the NIZO B40 priming glucosyltransferase gene, epsD , by an erythromycin resistance gene which resulted in the loss of EPS production and the complementation of the EPS-producing phenotype by controlled expression of priming glycosyltransferase genes from Gram-positive organisms with known function and substrate specificity.

    In Chapter 5 the regions involved in replication and mobilisation of the NIZO B40 EPS-plasmid pNZ4000 were characterised. The plasmid contains four highly conserved replication regions that belong to the lactococcal theta replicon family and all are functional and compatible in L. lactis . Plasmid pNZ4000 was shown to be a mobilisation plasmid and two regions involved in mobilisation were identified. Both regions contained a functional origin of transfer ( oriT ). One oriT sequence was followed by a mobA gene, coding for a trans -acting protein involved in conjugative transfer and likely to be the relaxase nicking the nic sites of the oriT sequences.

    Chapter 6 describes the complete nucleotide sequence of the EPS-plasmid pNZ4000, which amounts to 42810 bp and represents one of the largest sequenced plasmids in LAB to date. Apart from the regions involved in EPS biosynthesis, replication, and mobilisation, described in Chapters 2 and 5, two regions potentially involved in transport of divalent cations were localised on pNZ4000.

    In Chapter 7 the results of the previous chapters are discussed and their implications on practical applications and in particular the perspectives for polysaccharide engineering are described.

    Carbon catabolite repression and global control of the carbohydrate metabolism in Lactococcus lactis
    Luesink, E.J. - \ 1998
    Agricultural University. Promotor(en): W.M. de Vos; O.P. Kuipers. - S.l. : Luesink - ISBN 9789054859314 - 136
    koolhydraatmetabolisme - bestrijdingsmethoden - lactococcus lactis - bacillus megaterium - carbohydrate metabolism - control methods - lactococcus lactis - bacillus megaterium

    In view of the economic importance of fermented dairy products considerable scientific attention has been given to various steps of fermentation processes, including the L-lactate formation of lactic acid bacteria (de Vos, 1996). In particular, the carbohydrate metabolism of L. lactis has been the subject of extensive research and several genes encoding proteins involved in the central carbohydrate metabolism have been described (Llanos et al., 1992; Llanos et al., 1993; Cancilla et al., 1995a; Cancilla et al., 1995b; Qian et al., 1997). Although several findings have established that the carbohydrate metabolism is subject to several forms of regulation, detailed information concerning this regulation and, in particular, the transcriptional control of the central carbohydrate metabolism is lacking (Collins and Thomas, 1974; Fordyce et al., 1982; Hardman et al., 1985; Garrigues et al., 1997).

    A better understanding of the regulatory mechanisms involved would be an advantage for metabolic pathway engineering. Metabolic engineering is mainly aimed at the optimization of the metabolism and the diversion from L-lactate to other desired metabolites. The metabolite formation depends on the activity of enzymes of the central metabolic pathway and is therefore also subject to regulatory mechanisms in response to the carbon source provided. The research reported in this thesis has focussed on carbon catabolite repression (CCR), a global control system which regulates the transcription of genes involved in the carbohydrate metabolism depending on the carbon source availability (Hueck and Hillen, 1995). An overview of the present state of the art on CCR in Gram-positive bacteria is presented in Chapter 1.

    The aim of the work presented in this thesis was to investigate the elements involved in CCR in L. lactis and their effects on the carbohydrate metabolism. Several cis- and trans-acting elements involved in specific and global control systems were identified and their role in the transcriptional and allosteric control of carbohydrate metabolism was characterized. The salient features of their sequences are summarized in the Appendix.

    Chapter 2 describes the transcriptional and functional analysis of Tn5276-located genes involved in sucrose metabolism in L. lactis. The observation that the transcription of the previously cloned sacA gene was subject to glucose repression and the identification of a cre element in the promoter region of the sucrose genes lead to the choice of the sucrose genes as a model system to study the effects of CCR. (Rauch and de Vos, 1992). In addition to the sacA gene, encoding a sucrose-6-phosphate hydrolase, three new complete genes were identified. The sacB gene encodes a sucrose-specific EII protein of the phosphotransferase system (PTS) and its disruption resulted in the inability of the strain to utilize sucrose as carbon and energy source, thereby confirming the functionality of the sacB gene in the sucrose metabolism of L. lactis.. Downstream of the sacB gene the sacK gene was identified encoding a fructokinase. Partially overlapping sacA, the sacR gene was identified, the deduced protein sequence of which showed high homology to regulatory proteins of the LacI/GalR family. The L. lactis sucrose gene is the only gene cluster reported so far containing all three structural genes necessary for the complete catabolism of sucrose as well as a specific regulatory gene.

    Transcriptional analysis of the sucrose gene cluster lead to the identification of three sucrose-inducible transcripts. One of 3.2 kb containing sacB and sacK which initiates from the sacB promoter and is likely to terminate at the inverted repeat located downstream of the sacK gene. Another transcript of 3.4 kb, was shown to contain the sacA and sacR genes and initiates from the sacA promoter. Furthermore, a third sucrose-inducible transcript of 1.8 kb was identified, which contains the only the sacR gene and initiates from a promoter which was mapped upstream of the sacR gene.

    Disruption of the sacR gene resulted in the constitutive transcription of the sacBK and sacAR transcripts suggesting that SacR acts as a negative regulator of transcription. Under non-induced circumstances SacR most likely binds to the putative operator sites that were identified in the three promoters of the sucrose operon, resulting in repression of transcription. The presence of an inducer molecule (most likely sucrose-6-P) may result in the dissociation of SacR from the operator leading to transcription of the sucrose genes. The sacR gene is subject to a negative autoregulatory mechanism that results in higher levels of the repressor protein under induced circumstances compared to the non-induced situation. This control system allows for a very tight control of the expression of all the sucrose genes and to fast adaptation to environmental changes and resembles the system identified for the transcriptional control of the galactose genes in E. coli (Weickert and Adhya. 1993).

    The transcription of the sacA and sacB promoters in the wild-type strain is subject to glucose repression. The disruption of the sacR gene resulted in the complete absence of the glucose repression observed in the wild-type. This suggested that the glucose repression is dependent on SacR and is most likely due to a reduced induction resulting from lower concentrations of inducer molecules rather than the activity of a general regulatory mechanism like CcpA-mediated CCR. The concentration of inducer molecules may be affected by inducer control mechanisms like inducer exclusion and inducer expulsion (see below). The tight regulation of the expression of the sac genes by the operon-specific regulator SacR and the apparent independency of the chromosomally encoded CcpA-mediated CCR, may be a consequence of their location on a conjugative transposon of non-lactococcal origin that may be transferred to a variety of hosts.

    Chapter 3 deals with the detection of CcpA-like proteins in different Gram-positive bacteria including L. lactis. Polyclonal antibodies raised against purified Bacillus megaterium CcpA were used to screen protein extracts of several Gram-positive bacteria of high and low GC content. The results indicate that cross-reacting proteins were present in all Gram-positive bacteria tested, and suggest that a CcpA-mediated regulatory mechanism, like CCR, is a wide-spread phenomenon.

    In Chapter 4 the cloning and analysis of the L. lactis ccpA gene is described. An L. lactis expression library was constructed and screened with the CcpA antiserum resulting in the isolation of the L. lactis ccpA gene. In contrast to the Staphylococcus xylosus and Lactobacillus casei ccpA genes, the expression level of the L. lactis ccpA gene does not vary significantly in response to the carbon source provided (Egeter and Brückner, 1996; Monedero et al., 1997). The observed negative autoregulation of ccpA in Staphylococcus xylosus and Lactobacillus casei probably provides the cell with a mechanism controlling CCR by varying the level of CcpA protein. The observed regulation of the expression level of the L. lactis ptsH gene (see below) might allow a similar regulation of CCR activity in L. lactis because it affects the concentration of HPr(Ser-P), which functions as a coregulator. Inactivation of the L. lactis ccpA gene resulted in a reduced growth rate on all sugars tested, suggesting an involvement of CcpA in the regulation of a key metabolic pathway.

    Because the sucrose gene cluster, despite the presence of a cre element, appeared to be independent of CcpA-mediated CCR, a new model system to study CCR was required. The recently identified galactose gene cluster, containing the genes involved in the catabolism of galactose via the Leloir pathway, contains a cre element in the promoter region and was therefore a likely candidate for CcpA-mediated CCR (Grossiord et al., 1998). Disruption of the ccpA gene confirmed this involvement because the transcription of the gal operon in the resulting strain was partly relieved from CCR. However, the transcription of the gal operon was not completely relieved from CCR, suggesting that another regulatory mechanism was functional. Possible mechanisms for mediating the residual glucose repression are inducer exclusion and inducer expulsion.

    The expression of the genes encoding the glycolytic enzymes pyruvate kinase and L-lactate dehydrogenase is subject to carbon source dependent regulation since higher activities of both enzymes were measured in cells grown on glucose compared to cells grown on galactose. The genes encoding pyruvate kinase and L-lactate dehydrogenase are located in an operon structure together with the gene encoding the glycolytic enzyme phosphofructokinase (Llanos et al., 1992; Llanos et al., 1993). This operon, designated las for lactic acid synthesis, contains a cre site in the promoter region and is therefore a likely candidate for CcpA-mediated regulation. The inactivation of the ccpA gene resulted in a four-fold reduction of the transcription of the las operon genes indicating that CcpA acts as a transcriptional activator. CcpA has been reported to act as a transcriptional activator of the Bacillus subtilis alsS and ackA genes encoding a-acetolactate synthase and acetate kinase, respectively (Grundy et al., 1993; Renna et al., 1993). However, the CcpA-mediated transcriptional activation of the L. lactis las operon is the first report of transcriptional control of genes encoding enzymes involved in the central carbohydrate metabolism in Gram-positive bacteria.

    The lower transcription level of the las operon was reflected in reduced activities of pyruvate kinase and L-lactate dehydrogenase, resulting in a lower production of L-lactate. Furthermore, the fermentation pattern after growth on glucose had changed from almost homolactic, in case of the wild-type strain, to a more mixed-acid pattern in the ccpA knock out strain. The observation that the B. subtilis ccpA was capable of complementing the transcriptional activation, combined with the presence of cre sites in the promoter regions of glycolytic genes of different Gram-positive bacteria, strongly suggests that the observed transcriptional activation of glycolytic genes is not limited to L. lactis.

    The analysis of the L. lactis ptsHI genes is described in Chapter 5. The ptsHI operon is transcribed as a 2.0-kb transcript from a single promoter mapped upstream of the ptsH gene. Furthermore, a 0.3-kb transcript was detected that contained only the ptsH gene. This transcript originates from the ptsH promoter and terminates at a stem-loop structure located downstream of the ptsH gene. This transcriptional organization most likely results in a higher expression of the ptsH gene, explaining the higher amount of HPr protein compared to enzyme I as observed in several bacteria, including Staphylococcus carnosus (Kohlbrecher et al., 1992).

    The expression of the ptsHI genes appeared to be regulated since lower transcription levels were observed when the cells were grown on the non-PTS sugar galactose compared to the PTS sugar glucose. Induction of the ptsHI expression by glucose has also been observed in Bacillus subtilis and allows the cell to control the activity of the PTS in response to the carbon source availability (Stülke et al., 1997). The glucose induction of the Bacillus subtilis ptsHI genes is mediated via an antitermination mechanism and is dependent of a characteristic terminator structure located upstream of the ptsH gene. Because no obvious recognition sites for transcriptional regulators could be identified at relevant positions in the L. lactis ptsHI operon, the mechanism by which the transcriptional control of this operon operates remains to be clarified.

    The disruption of both the ptsH and the ptsI genes resulted in the absence of growth on sucrose and fructose, indicating that these sugars are exclusively taken up by the PTS. The growth rate on glucose was severely reduced, suggesting that in addition to the PTS, another glucose uptake system is present. This finding is in agreement with the results of Thompson and coworkers who presented biochemical evidence that L. lactis uses the PTS and a non-PTS permease for the uptake of glucose (Thompson et al., 1985). Complementation of the ptsH and ptsI genes with the appropriate L. lactis genes under the control of an inducible promoter confirmed the functionality of both genes. Furthermore, the growth rate on galactose and maltose, two sugars that are most likely taken up via a non-PTS system was reduced two-fold. This observation suggests an involvement of the PTS with either protein activities involved in the galactose and maltose catabolism or the regulation of the expression of the encoding genes. In Gram-positive bacteria, the PTS has been reported to control catabolic pathways, like the Bacillus subtilis levanase or glycerol pathways (Stülke et al., 1995; Charrier et al., 1997) or the lactose uptake in Streptococcus thermophilus (Poolman et al., 1995), by HPr(His-P)-dependent phosphorylation of either enzymes or regulatory proteins resulting in enhanced or reduced activities.

    In order to analyze the regulatory role of HPr(Ser-P) in the sugar metabolism of L. lactis, mutant HPr proteins were constructed that were affected in the phosphorylation of residue Ser-46. Overproduction in a wild-type strain of HPr(S46D) where residue Ser-46 has been changed into an aspartic acid, that mimics a phosphorylated serine, resulted in a reduction of the growth rate on galactose, whereas the growth rate on glucose was not affected. These results suggested that HPr(Ser-P) is involved in the CCR of the galactose metabolism. Whether this regulation occurs in combination with the inducer control mechanisms or at the transcriptional level in combination with CcpA remains to be determined.

    In addition to its role in the CCR of the genes involved in the galactose metabolism, HPr(Ser-P) is also involved in the positive regulation of the enzymes encoded by the las operon. Expression of the gene encoding S46D HPr in wild-type cells grown on galactose resulted in increased activities of both pyruvate kinase and L-lactate dehydrogenase. Since the positive effect of the production of S46D HPr on the activities of pyruvate kinase and L-lactate dehydrogenase depends on the presence of the ccpA gene, it is feasible that the regulation occurs at the transcriptional level.

    These findings established the function of HPr(Ser-P) as a signal molecule in several allosteric and transcriptional metabolic control systems in L. lactis . The occurrence of HPr(Ser-P) results in a reduced entry of new sugar phosphates into the glycolysis due to the inducer control mechanisms and CCR. In addition, the catabolite activation of the las operon results in an increased flux through the glycolysis. Consequently, the inducer control systems as well as the CcpA-mediated catabolite control can be seen as mechanisms to prevent the wasteful and possibly toxic accumulation of early glycolytic intermediates.

    The studies described in this thesis have resulted in the characterization of different regulatory mechanisms involved in the control of the carbohydrate metabolism in L. lactis. The regulation of the expression of the Tn5276-located sucrose genes appeared to be dependent on the operon-specific regulator, SacR. This apparent independence of chromosomally encoded global regulation might be a result of the fact that these genes are located on transposons, which can be conjugally transferred to other species and therefore require a host-independent transcriptional control system. The analysis of the L. lactis ccpA gene lead to the identification of two CcpA-dependent regulatory systems i.e. the CcpA-mediated CCR of the galactose operon and the transcriptional activation of the glycolytic las operon. The CCR of the galactose operon mediated by CcpA confirmed previous reports on the role of CcpA in other Gram-positive bacteria. So far, CcpA-mediated transcriptional activation of gene expression was only identified in B. subtilis.

    However, the observation that the L. lactis CcpA mediates the expression of genes encoding key enzymes of the glycolysis suggests that CcpA is involved in the global transcriptional control of the metabolic activity, in response to carbon source availability. The observation that the seryl-phosphorylated form of HPr is involved as coregulator in CCR as well as the CcpA-mediated transcriptional activation of gene expression established its important role as signal molecule reflecting the energy state of the cell.

    The new information concerning the elements involved in the CcpA-mediated catabolite activation of the central carbohydrate metabolism can be used to accelerate the L-lactate formation in certains strains. The disruption of the ccpA gene most likely results in an increased intracellular concentration of early glycolytic intermediates like FDP, which might lead to an increased biosynthesis of e.g. extracellular polysaccharides since precursors thereof are derived from these metabolites. Data emerging from the L. lactis sequencing project (Bolotin et al., 1998) in combination with new technologies like the microarray technology (de Saizieu et al., 1998), allowing genome-wide monitoring of gene expression, and the knowledge of the global regulatory mechanisms presented in this thesis will facilitate the design of metabolic engineering strategies.

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