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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.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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A global survey of urban water tariffs: are they sustainable, efficient and fair?
Zetland, D.J. ; Gasson, C. - \ 2013
International Journal of Water Resources Development 29 (2013)3. - ISSN 0790-0627 - p. 327 - 342.
This paper examines the relations between tariffs and sustainability, efficiency and equity, using a unique data-set for 308 cities in 102 countries. Higher water tariffs are correlated with lower per capita consumption, smaller local populations, lower water availability, higher demand and a lower risk of shortage. Aggregating to the national level, higher tariffs are correlated with higher GDP and better governance. A different country-level analysis shows that a higher percentage of the population with water service is correlated with better governance, higher GDP and a greater risk of water shortage. The relation between water prices and service coverage is statistically inconsistent.
Discovering lactic acid bacteria by genomics
Klaenhammer, T. ; Altermann, E. ; Arigoni, F. ; Bolotin, A. ; Breidt, F. ; Broadbent, J. ; Cano, R. ; Chaillou, S. ; Deutscher, J. ; Gasson, M. ; Guchte, M. van de; Guzzo, J. ; Hartke, A. ; Hawkins, T. ; Hols, P. ; Hutkins, R. ; Kleerebezem, M. ; Kok, J. ; Kuipers, O. ; Lubbers, M. ; Maguin, E. ; McKay, L. ; Mills, D. ; Nauta, A. ; Overbeek, R. ; Pel, H. ; Pridmore, D. ; Saier, M. ; Sinderen, D. van; Sorokin, A. ; Steele, J. ; O'Sullivan, D. ; Vos, W. de; Weimer, B. ; Zagorec, M. ; Siezen, R. - \ 2002
Antonie van Leeuwenhoek: : Nederlandsch tijdschrift voor hygiëne, microbiologie en serologie 82 (2002)1-4. - ISSN 0003-6072 - p. 29 - 58.
This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, nvironmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram–positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.
Improved applicability of nisin in novel combinations with other food preservation factors
Pol, I.E. - \ 2001
University. Promotor(en): F.M. Rombouts; Eddy Smid. - S.l. : S.n. - ISBN 9789058083821 - 95
voedselbewaring - nisine - listeria monocytogenes - bacillus cereus - food preservation - nisin
<h3>General discussion</h3><p>Modern consumers nowadays, have a preference for more natural, mildly preserved food products with a fresh appearance over traditionally preserved products. Mild preservation techniques applied singly are usually not sufficient to control microbial outgrowth and combinations of measures are needed to ensure complete safe products (16). Bacteriocins, produced by lactic acid bacteria have been successfully used as biopreservatives in a number of food products to inhibit the growth of pathogenic and spoilage organisms (27). Up till now, nisin is the only bacteriocin that has been approved by the WHO to be used as a food preservative. Due to its restricted inhibition spectrum and the decreased solubility and heat sensitivity at neutral pH, application is still limited (10). The study described in this thesis aimed to increase the practical application of nisin by combinations with other biopreservatives or mild preservation techniques.</p><h3>Nisin and essential oils</h3><p>Essential oils, derived from plants, are known for their flavor characteristics. Many of the compounds found in essential oils possess antimicrobial activity (4, 9, 14, 22), and therefore are suitable candidates for mild food preservation in combination with nisin. The essential oils dramatically enhance the bactericidal activity of nisin at concentrations, which alone do not affect the bacterial cell counts of the foodborne pathogens <em>Listeria monocytogenes</em> and <em>Bacillus cereus</em> (chapter 2). Adaptation of these cells to lower temperatures resulted in an increased sensitivity towards nisin, possibly due to an altered membrane composition leading to a change in membrane fluidity or to an increased electrostatic interaction of nisin with phospholipids in the membrane caused by an increase in negative charges (8, 18 - 21, 31). Alternatively a decrease in lipid II content as a result of changes in the membrane composition might explain the decreased activity of nisin (5). Lowering the temperature had a negative influence on the synergy between nisin and the essential oils, which might result from the lower sensitivity of the cells towards essential oils at lower temperatures (28).</p><p>The exact mechanism underlying this synergy is not exactly known. Both nisin and carvacrol cause a dissipation of the proton motive force as well as depletion of the internal ATP pool (6, 12, 23, 26, 30, 32, chapter 3). In combination, carvacrol enhances the membrane potential dissipating effect of nisin, at concentrations which do not affect the viable count of <em>B. cereus</em> . Apparently cells are able to cope with low concentrations of nisin and carvacrol. When concentrations increase, cells are no longer able to compensate for loss of membrane integrity and a synergistic reduction of the pH gradient and depletion of the intracellular ATP pool were observed. The reduction in internal ATP is not proportional to the increase in external ATP and no additional increase in external ATP was observed upon simultaneous exposure to nisin and carvacrol. This observation excludes increased leakage of ATP as an explanation for the synergistic depletion of the intracellular ATP pool. Consequently, the underlying mechanism of the synergistic inactivation of <em>B. cereus</em> is most likely not the increased poreforming ability of nisin by carvacrol. Presumably, the rate of ATP hydrolysis is increased upon simultaneous addition of nisin and carvacrol or the internal ATP pool is exhausted in an attempt to reenergize the membrane (1, 23, 29). Alternatively, the disturbance of the membrane permeability by carvacrol and nisin might lead to impairment of membrane bound enzymes like ATPase, resulting in a decreased ATP synthesis (15, 26).</p><h3>Nisin and PEF treatment</h3><p>In addition to essential oils, Pulsed Electric Field treatment was also found to improve the antimicrobial action of nisin against <em>B. cereus.</em> Synergy was only found when PEF treatment was spread over a period of 10 minutes to match the relevant inactivation time scale of nisin's action. <em></em> The additional stress imposed by PEF treatment possibly facilitates the incorporation of nisin into the cytoplasmic membrane resulting in more or larger pores or pores with a longer lifetime (chapter 4). Further reduction of the intensities of the treatments was achieved by adding carvacrol as a third hurdle to the combination of nisin and PEF treatment (chapter 5).</p><p>The fact that synergy was found between the three treatments renders the combination very interesting for mild food preservation. However, extrapolation of the results from labscale experiments in buffer systems to food model matrices is usually difficult and the influence of food ingredients on the efficiency of preservation techniques are not fully understood. The efficiency of PEF treatment against vegetative cells of <em>B. cereus</em> is not affected by proteins in skimmed milk (20 %). However, the proteins do have a negative influence on the nisin activity, either as a result of a decreased bioavailability of nisin due to binding of the molecule to proteins or because of protection of the microorganisms by the proteins. As a consequence, the synergy between nisin and PEF treatment is less pronounced in skimmed milk (20 %).</p><p>In sharp contrast to the improved bactericidal activity found in HEPES buffer, carvacrol is not able to enhance the synergy between nisin and PEF treatment in diluted milk (only in high concentrations (1.2 mM)). Possibly, carvacrol binds to the proteins, reducing the availability of the molecule. However, this is not consistent with the fact that carvacrol increases the antimicrobial activity of PEF treatment in milk. Therefore, the absence of synergy between nisin, PEF treatment and carvacrol is more likely explained by the decreased bioavailability of nisin, thereby decreasing the extent of synergy between nisin and carvacrol and consequently between all three treatments. The influence of PEF treatment on the behavior of proteins is not exactly known. Proteins can carry electric charges and might behave as dipoles when subjected to PEF treatment, which cause the macromolecules to reorient or deform (such as protein unfolding and denaturation), and possibly some breakdown of covalent bonds or casein micelles may occur (3). These PEF induced changes in the structure of proteins may play a role in the existence of synergy between carvacrol and PEF. Dilution of the milk to 5 % still provides enough proteins to stimulate synergy between carvacrol and PEF treatment (chapter 5).</p><p>Before such novel techniques can replace currently used thermal processes, more insight into spore inactivation is needed (chapter 6). Nisin and PEF treatment do not directly inactivate or damage spores of <em>B. cereus</em> , however germinated spores can be inactivated by nisin or PEF treatment to a certain extent. The PEF resistance of the germinated spores is lost 50 minutes after the onset of germination. Nisin resistance was lost immediately in parallel to heat resistance, suggesting that loss of nisin resistance might be ascribed to changes in the dehydrated state of the core. Sulfhydryl groups in the membrane, not available in ungerminated spores, were suggested to be the natural target for nisin and therefore access to the membrane is a prerequisite for inactivation (17, 24, 25). In addition, the increase in availability of the membrane-anchored cell wall precursor Lipid II upon germination could also play a role in the loss of nisin resistance (5). Apparently, nisin has gained access to the membrane by penetrating the coat, which was made more permeable upon germination or alternatively, the protective coat was degraded by spore lytic enzymes, allowing nisin to reach the cytoplasmic membrane. The late loss of PEF resistance can be explained by its dependence on the degradation of the spore coat. To exert antimicrobial inactivation by PEF treatment, free migration of ions is needed to increase the transmembrane potential of the spores. Formation of pores occurs after compression of the membrane and reorientation of the phospholipids in the membrane. In spores the ions are immobilized by proteins or DPA, restricting their mobility (7, 13) and subsequently the build up of an increased transmembrane potential is prevented. Secondly, the spore core is surrounded by several rigid protecting layers limiting the compression and reorientation of the phospholipids (2).</p><p>Combining nisin and PEF treatment did not result in additional inactivation of the germinating spores. Since loss of PEF resistance occurs only after 50 minutes of germination and loss of nisin resistance seems to be an early event in spore germination, synergy would therefore be less likely due to different time scales of action. Furthermore, the incomplete germination of the spores reduces the margins to observe synergy. Ideally, complete and synchronized germination is needed to quantify the inactivation by nisin or PEF treatment and determine precisely the onset of loss of nisin or PEF resistance.</p><p>One of the main problems associated with the use of antimicrobial compounds is the development of tolerance or resistance to certain compounds. Adaptation of cells to carvacrol was correlated to a decrease in membrane fluidity as demonstrated by Ultee <em>et al.</em> (30). In addition, they observed a change in phospholipid composition of the membrane. Cells adapted to carvacrol exhibited an increased sensitivity towards nisin compared to control cells (chapter 6). A decrease in the membrane fluidity is not expected to increase nisin's action, but a change in the head group composition, with an increase in negatively charged lipids, might stimulate the electrostatic binding of nisin and in this way enhance nisin's action (8, 18 - 21, 31). Alternatively an increase in lipid II content in carvacrol-adapted cells as a result of changes in the membrane composition might explain the increased activity of nisin (5). A decrease in the membrane fluidity did not change the susceptibility towards a PEF treatment. A more rigid membrane is less likely to be compressed by accumulating charges as a result of applied field strength and the ordered state of the phospholipids in the membrane decreases the chance of reorientation, which would reasonably lead to a decreased inactivation by PEF treatment. Although the bactericidal activity of nisin was increased by adaptation to carvacrol, the synergy between nisin and PEF treatment was not influenced by a change in membrane fluidity and membrane composition. Attemps to change the membrane composition of spores by adaptation of vegetative cells to carvacrol prior to and during sporulation did not lead to inactivation of spores by either nisin or PEF treatment.</p><h3>Application</h3><p>Combinations of nisin with essential oils or PEF treatment have been successful in overcoming the restrictions in practical application of nisin. For instance, the inhibition spectrum of nisin can be widened by combination with other preservation technologies like PEF treatment. In addition, the limited activity of nisin at higher temperatures can be complemented by the increased synergy between nisin and essential oils.</p><p>The application of multiple hurdles has great potential to be used as a mild food preservation technology. The occurrence of synergy between nisin and essential oils or PEF technology allows for a reduction in the intensities of the treatments demonstrating the suitability for mild preservation. Increasing the number of hurdles (lysozyme) improves the observed synergy and further increases the mildness of the preservation technology (chapter 1).</p><p>Consumer's acceptation of these combination techniques in case of the essential oils is not expected to meet difficulties. This combination meets with present preference for more natural and mild preservation methods. Herbs and spices, of which essential oils are the active components, are already used for centuries as flavoring agents and in homeopathic products and medicines. Currently, carvacrol is Generally Recognized As Safe (GRAS) and has been approved by the Code of Federal Regulation (CFR) to be used as a flavoring agent (11). However, when the essential oils are used for their antimicrobial activity, they will be regarded as new food additives and subsequently require a non-toxicity report (27). To circumvent these problems, the original herbs and spices can be used as food flavoring agents, while at the same time advantage can be taken of their antimicrobial activity. However, the producer has to take into account the low concentration of the active compound in herbs and spices. Furthermore, the essential oils have a strong and specific flavor and can only be applied in products where this aroma is appreciated.</p><p>Acceptance of PEF technology is expected to give more problems and introduction of this technology has to be handled carefully. Consumers might associate PEF treated foods with residual electromagnetic raditation, just like radiated foods are associated with radioactivity. Only when PEF technology is introduced carefully and the consumers are supplied with the right information, they will accept this technology as mild preservation.</p><p>At the moment, not enough information is known about PEF technology and its mechanism of action. Evidently, more research needs to be done to verify the influence of other food ingredients including fat particles on the antimicrobial activity. Furthermore the influence of PEF treatment on the product quality needs to be investigated. The fresh-like appearance, color and the vitamin content are seemingly unaffected however, the influence of PEF treatment on proteins, polysaccharides macromolecules, or lipids is not exactly known.</p><p>The development of tolerance or resistance to the PEF treatment or the combination treatments is not clear and should receive more attention, since microorganisms generally adapt to environmental stress factors. Increased tolerance towards nisin and carvacrol has been studied in more detail (8, 18 - 21, 31) however, no such research has been conducted concerning PEF technology. Combining preservation technologies in which the microorganism is attacked from different sides should reduce the development of tolerance to a minimum. Inactivation of spores is another challenge to be overcome before such combination technologies can be implemented in current preservation strategies.</p><p>In conclusion, these combination techniques are a welcome alternative to currently used pasteurization methods. The current limitations in the application of nisin can be complemented by the inhibition spectrum of the combination treatment. In addition, the synergy observed between the different preservation techniques allows for a reduction of the used intensities increasing the suitability for mild preservation.</p><h3>References</h3><ol><li>Abee, T., F. M. Rombouts, J. Hugenholtz, G. Guihard, and L. Letellier. 1994. Mode of action of Nisin Z against <em>Listeria monocytogenes</em> Scott A grown at high and low temperatures. Applied and Environmental Microbiology 60(6):1962-1968.</li><li>Barbosa-Cánovas, G. V., M. Marcela Góngora-Nieto, U. R. Pothakamury, and G. S. Barry. 1999. Preservation of foods with pulsed electric fields. Academic Press, San Diego.</li><li>Barsotti, L., P. Merle, and J. C. Cheftel. 1999. Food Processing by pulsed electric fields. I. Physical aspects. Food Review International 15(2):163-180.</li><li>Beuchat, L. R., M. R. S. Clavero, and C. B. Jaquette. 1997. Effects of nisin and temperature on survival, growth and enterotoxin production characteristics of psychrotrophic <em>Bacillus cereus</em> in beef gravy. Applied and Environmental Microbiology 63(5):1953-1958.</li><li>Breukink, E., I. Wiedemann, C. van Kraaij, O. P. Kuipers, H-G. Sahl, and B. de Kruijff. 1999. Use of the cell wall precursor lipid II by the pore-forming peptide antibiotic. Science 286:2361-2364.</li><li>Bruno, M. E. C., A. Kaiser, and T. J. Montville. 1992. Depletion of proton motive force by nisin in <em>Listeria monocytogenes</em> cells. Applied and Environmental Microbiology 58(7):2255-2259.</li><li>Carstensen, E. L., and R. E. Marquis. 1974. Dielectric and electrochemical properties of bacterial cells. <em>In</em> Spores VI. Michigan, 10-13 October 1974.</li><li>Crandall, A. D., and T. J. Montville. 1998. Nisin resistance in <em>Listeria monocytogenes</em> ATCC 700302 is a complex phenotype. Applied and Environmental Microbiology 64(1):231-237.</li><li>Deans, S. G., and G. Ritchie. 1987. Antibacterial properties of plant essential oils. International Journal of Food Microbiology 5 <strong>:</strong> 165-180.</li><li>Delves-Broughton, J., and M. J. Gasson. 1994. Nisin, p. 99-131. <em>In</em> V. M. Dillon and R. G. Board (ed.), Natural antimicrobial systems and food preservation, vol. 328p. Cab international, Oxon, UK.</li><li>Fenaroli, G. 1995. Fenaroli's handbook of flavor ingredients, third ed. CRC Press, Boca Rotan.</li><li>Garcerá, M. J. G., M. G. L. Elferink, A. J. M. Driessen, and W. N. Konings. 1993. In vitro pore-forming activity of the lantibiotic nisin. Role of protonmotive force and lipid composition. European Journal of Biochemistry 212:417-422.</li><li>Gould, G. W., and G. J. Dring. 1971. Biochemical mechanism of spore germination. <em>In</em> Spores V. Fontana, Wisconsin, 8-10 October.</li><li>Kim, J. M., J. A. Marshall, J. A. Cornell, J. F. Preston III, and C. I. Wei. 1995. Antibacterial activity of carvacrol, citral and geraniol against <em>Salmonella typhimurium</em> in culture medium and on fish cubes. Journal of Food Science 60(6):1364-1368.</li><li>Knobloch, K., H. Weigand, N. Weis, H-M. Schwarm, and H. Vigenschow. 1986. Action of terpenoids on energy metabolism, p. 429-445. <em>In</em> E. J. Brunke (ed.), Progress in essential oil research. de Gruyter, Berlin.</li><li>Leistner, L., and L. G. M. Gorris. 1995. Food preservation by hurdle technology. Trends in Food Science and Technology 6(2):41-46.</li><li>Lui, W., and J. N. Hansen. 1990. Some chemical and physical properties of nisin, a small protein antibiotic produces by <em>Lactococcus lactis.</em> Applied and Environmental Microbiology 56(8):2551-2558.</li><li>Mazzotta, A. S., and T. J. Montville. 1999. Characterization of fatty acid composition, spore germination and thermal resistance in a nisin-resistant mutant of <em>Clostridium botulinum</em> 169B and in the wild-type strain. Applied and Environmental Microbiology 65(2):659-664.</li><li>Mazzotta, A. S., A. D. Crandall, and T. J. Montville. 1997. Nisin resistance in <em>Clostridium botulinum</em> spores and vegetative cells. Applied and Environmental Microbiology 63(7):2654-2659.</li><li>Ming, X., and M. A. Daeschel. 1993. Nisin resistance of foodborne bacteria and the specific resistance responses of <em>Listeria monocytogenes</em> Scott-A. Journal of Food Protection 56:944-948.</li><li>Ming. X., and Daeschel, M. A. 1995. Correlation of cellular phospholipid content with nisin resistance of <em>Listeria monocytogenes</em> Scott A. Journal of Food Protection 58:416-420.</li><li>Moleyar, V., and P. Narasimham. 1986. Antifungal activity of some essential oil components. Food Microbiology 3:331-336.</li><li>Montville, T. J., and Y. Chen. 1998. Mechanistic action of pediocin and nisin: recent progress and unresolved questions. Applied Microbiology and Biotechnology 50:511-519.</li><li>Morris, S. L., and J. N. Hansen. 1981. Inhibition of Bacillus cereus spore outgrowth by covalent modification of a sulfhydryl group by nitrosothiol and iodoacetate. Journal of Bacteriology 148(2):465-471.</li><li>Morris, S. L., R.C. Walsh, and J. N. Hansen. 1984. Identification and characterization of some bacterial membrane sulfhydryl groups which are target of bacteriostatic and antibiotic action. The Journal of Biological Chemistry 259(21) <strong>:</strong> 13590-13591.</li><li>Okereke, A., and T. J. Montville. 1992. Nisin dissipates the proton motive force of the obligate anaerobe <em>Clostridium sporogenes</em> PA 3679. Applied and Environmental Microbiology 58(8):2463-2467.</li><li>Smid, E. J., and L. G. M. Gorris. 1999. Natural antimicrobials for food preservation, p. 285-308. <em>In</em> M. Shafiurr Rahman (ed.), Handbook of food preservation. Marcel Dekker, Inc., New York.</li><li>Ultee, A., L. M. G. Gorris, and E. J. Smid. 1998. Bactericidal activity of carvacrol towards the food-borne pathogen <em>Bacillus cereus</em> . Journal of Applied Microbiology 85:211-218.</li><li>Ultee, A., E. P. W. Kets, and E. J. Smid. 1999. Mechanisms of action of carvacrol on the foodborne pathogen <em>Bacillus cereus</em> . Applied and Environmental Microbiology 65:4606-4610.</li><li>Ultee, A., E. P. W. Kets, M. Alberda, F. A. Hoekstra, and E. J. Smid. 2000. Adaptation of the foodborne pathogen <em>Bacillus cereus</em> to carvacrol. Archives of microbiology 174:233-238.</li><li>Verheul, A., N. Russel, J., R. Van 't Hof, F. M. Rombouts, and T. Abee. 1997. Modifications of membrane phospholipid composition in nisin-resistant <em>Listeria monocytogenes</em> Scott A. Applied and Environmental Microbiology 63(9):3451-3457.</li><li>Winkowski, K., M. E. C. Bruno, and T. J. Montville. 1994. Correlation of bioenergetic parameters with cell death in <em>Listeria monocytogenes</em> cells exposed to nisin. Applied and Environmental Microbiology 60:4186-4188.</li></ol>
Colophospermum reduced to Hardwickia (Leguminosae-Caesalpinioideae).
Breteler, F.J. ; Ferguson, I.K. ; Gasson, P.E. ; Welle, B.J.H. ter - \ 1997
Adansonia 19 (1997)2. - ISSN 1280-8571 - p. 279 - 291.
Protein engineering of lantibiotics.
Kuipers, O.P. ; Bierbaum, G. ; Ottenwälder, G. ; Dodd, H.M. ; Horn, N. ; Metzger, J. ; Kupke, T. ; Gnau, V. ; Bongers, R. ; Boogaard, P. van den; Kosters, H. ; Rollema, H.S. ; Vos, W.M. de; Siezen, R.J. ; Jung, G. ; Götz, F. ; Sahl, H.G. ; Gasson, M.J. - \ 1996
Antonie van Leeuwenhoek: : Nederlandsch tijdschrift voor hygiëne, microbiologie en serologie 69 (1996). - ISSN 0003-6072 - p. 161 - 169.
Gene cloning and expression systems in Lactococci.
Vos, W.M. de; Simons, G.F.M. - \ 1994
In: Genetics and biotechnology of lactic acid bacteria / Gasson, M.J., de Vos, W.M., London : Chapman & Hall - p. 52 - 105.
Genetics and biotechnology of lactic acid bacteria.
Gasson, M.J. ; Vos, W.M. de - \ 1994
London : Chapman and Hall - 310 p.
The proteolytic system of lactic acid bacteria.
Kok, J. de; Vos, W.M. de - \ 1994
In: Genetics and biotechnology of lactic acid bacteria / Gasson, M.J., de Vos, W.M., London : Chapman & Hall - p. 169 - 210.
Characterization of the Lactococcus lactis lactose genes and regulation of their expression
Rooijen, R.J. van - \ 1993
Agricultural University. Promotor(en): W.M. de Vos. - S.l. : Van Rooijen - ISBN 9789090059853 - 185
lactobacillus - melkzuurbacteriën - micro-organismen - genetica - heritability - lactose - biochemie - metabolisme - synthese - moleculaire biologie - genexpressie - lactic acid bacteria - microorganisms - genetics - biochemistry - metabolism - synthesis - molecular biology - gene expression
<p>An important trait of the lactic acid bacterium <em>Lactococcus lactis</em> , that is used in industrial dairy fermentations, is the conversion of lactose into lactic acid. The enzymatic steps involved in the breakdown of lactose, that is transported into the cell via a phosphoenolpyruvate-dependent lactose phosphotransferase system (PEP-PTS <sup>lac</SUP>), have been well established (Fig. 1). However, except for the molecular cloning and characterization of the plasmid-located phospho-B-galactosidase gene (Boizet <em>et al.</em> , 1988; De Vos and Gasson, 1989), relatively little data have emerged concerning the genetic information for the lactose catabolic enzymes. A solid genetic basis of this key metabolic route is essential for the development of food-grade selection markers and pathway engineering strategies for <em>L. lactis.</em> In addition, since high lactose-specific enzyme activities are observed during growth on lactose, which are repressed during growth on glucose, expression of the <em>lac</em> genes is probably under control of a strong and inducible promoter. Such a promoter would be applicable as a 'genetic switch' in the controlled overexpression of homologous and heterologous genes in <em>Lactococci</em> . Isolation and elucidation of the mechanism of control of the <em>lac</em> promoter would be beneficial for the development of such strains. This thesis describes the characterization and organization of the genes involved in the lactose metabolism of <em>L. lactis</em> subsp. <em>lactis.</em> In addition, several <em>cis</em> - and <em>trans</em> -acting factors that are involved in the regulation of their expression were identified.<p>In <strong>Chapter 1</strong> some background information is given about the enzymology and genetics of lactose metabolism in lactic acid bacteria. In addition, this Chapter provides a brief overview of the various mechanisms that may be involved in the regulation of gene expression in bacteria, and presents the state-of-the-art concerning gene regulation in lactic acid bacteria.<p>The characterization of the genetic determinants for lactose metabolism, including the PEP-PTS <sup>lac</SUP>(LacEF), phospho-β-galactosidase (LacG) and tagatose-6-phosphate pathway enzymes (LacABCD), is presented in <strong>Chapters 2</strong> and <strong>3</strong> . The <em>lac</em> genes of the <em>L. lactis</em> subsp. <em>lactis</em> strain MG1820, that are located on the 23.7-kb plasmid pMG820, appeared to be organized in a 7.8-kb operon-structure with the gene order <em>lacABCDFEGX</em> (Fig. 1). The <em>lacE</em> and <em>lacF</em> genes encode the PEP-PTS <sup>lac</SUP>proteins Enzyme II <sup>lac</SUP>(62 kDa) and Enzyme III <sup>lac</SUP>(11 kDa), that are involved in the translocation across the cell membrane and subsequent phosphorylation of lactose (Chapter 2). Crosslinking studies with purified enzyme showed that Enzyme III <sup>lac</SUP>is active as a trimer. The identity of the <em>lacF</em> gene was confirmed by complementation of <em>lacF</em> deficiency in <em>L. lactis</em> strain YP2-5, that appeared to contain a G 18E mutation in the deduced LacF protein. Homology was observed between the deduced amino acid sequences of the <em>L.</em> lactis lacE and lacF genes and those of <em>Lactobacillus c</em> a <em>sei</em> and <em>Staphylococcus aureus</em> . In addition, the deduced <em>L. lactis</em> LacE and LacF amino acid sequences were homologous to those of CelA, CelB and CelC that are involved in the cellobiose PTS of <em>Escherichia coli</em> (Reizer <em>et al.,</em> 1990). The <em>lacG</em> gene codes for the phospho-β-galactosidase enzyme (54 kDa) that catalyzes the hydrolysis of lactose-6-phosphate into galactose-6-phosphate and glucose (De Vos and Gasson, 1989). The <em>L. lactis</em> phospho-β-galactosidase has been purified from an overexpressing <em>E.coli</em> strain (De Vos and Simons, 1988) and belongs to the superfamily of β-glycohydrolases (Hassouni <em>et al.</em> 1992). The tagatose-6-phosphate pathway enzymes were shown to be encoded by the <em>lacABCD</em> genes (Chapter 3). The first enzyme of the tagatose-6-phosphate pathway, the galactose-6-phosphate isomerase (LacAB), is encoded by the first two genes of the <em>lac</em> operon, the <em>lacAB</em> genes. Galactose-6- phosphate activities were only observed in <em>E.coli</em> cells overexpressing both the <em>lacA</em> and <em>lacB</em> genes, whereas no activity was found in cells expressing solely LacA (15 kDa) or LacB (19 kDa). The <em>lacC</em> and <em>lacD</em> genes encode the tagatose-6-phosphate kinase (33 kDa) and tagatose-1,6-diphosphate aldolase (36 kDa), respectively, as was evident form their enzyme activities in overexpressing <em>E.coli</em> cells. The deduced amino acid sequences of the <em>lacABCD</em> genes appeared to be strongly homologous to those of <em>S. aureus</em> and <em>S</em> . <em>mutans</em> (Jagusztyn- Krynicka <em>et</em><em>al.</em> 1992). In addition, the <em>L. lactis</em> LacC sequence is homologous to the <em>E. coli</em> enzyme phosphofructosekinase B, that catalyzes the phosphorylation of tagatose-6- phosphate in the galactitol catabolic pathway. The function of the distal <em>lacX</em> gene, encoding a 34-kDa protein, is still unclear. No significant homology was found with other sequences in DNA or protein databases. However, the <em>lacX</em> gene seems not to be essential for lactose catabolism, since <em>L. lactis</em> strains in which transcription of <em>lacX</em> was prevented did not show significantly altered growth characteristics or phospho-β-galactosidase activities during growth on lactose (Simons <em>et al.,</em> 1993). Northern-blot analysis showed that the <em>lac</em> genes are transcribed as two 6.0- and 8.0-kb polycistronic transcripts, of the <em>lacABCDFE</em> and <em>lacABCDFEGX</em> genes, respectively. An inverted repeat which is located between the <em>lacE</em> and <em>lacG</em> genes could function as the transcription termination site for the 6.0-kb transcript. In cells shifted from glucose to lactose, <em>lac</em> operon transcription was induced similarly as lactose enzyme activities (approximately 5-10 fold), indicating that the expression of the <em>lac</em> operon is regulated at the transcriptional level. The 3' end of the <em>lacABCDFEGX</em> operon appeared to be followed by an <em>iso</em> -IS <em>S1</em> element ( <strong>Chapter 4</strong> ). This element is flanked by 16-bp inverted repeats and contains a divergently transcribed gene ( <em>orf1</em> ) encoding a putative transposase that is highly homologous to that of other <em>iso</em> -IS <em>S1</em> elements. It remains to be determined whether this IS-element, or one of the other IS-elements that have been located on pMG820 (Fig. 1; Van Rooijen, unpublished results), are involved in the conjugal transfer of this or related lactose plasmids.<p>Transcription of the <em>lacABCDFEGX</em> operon was found to be regulated by the product of the divergently transcribed 0.8-kb <em>lacR</em> gene ( <strong>Chapter 5</strong> ). The <em>lacR</em> gene was characterized by overexpression in <em>E.coli</em> and DNA sequencing and found to encode a 28- kDa protein. Northern-blot analysis showed that, in contrast to the <em>lacABCDFEGX</em> genes, the <em>lacR</em> gene is induced during growth on glucose. The deduced amino acid sequence of LacR appeared to be homologous to those of the <em>E. coli</em> DeoR, GutR, and FucR, <em>S.aureus</em> and <em>S.mutans</em> LacR, and <em>Agrobacterium tumefaciens</em> AccR repressors. None of these repressors belongs to one of the known LacI/GaIR or LysR repressor families. Since the DeoR repressor was the first repressor to be identified, this group of repressors was designated the <em>E. coli</em> DeoR family of repressors. Common characteristics of the members of the DeoR family are the presence of a helix-turn-helix motif near their N-termini and a conserved region near their C-termini, that for the <em>L.lactis</em> LacR repressor appeared to be involved in DNA and inducer binding, respectively (see below). In addition, all members have in common that expression of the catabolic operon they control is induced by a phosphorylated sugar, or a derivative thereof. The functionality of the <em>lacR</em> gene product as a repressor was demonstrated after introduction of multiple copies of the <em>lacR</em> gene in <em>L. lactis</em> strain MG5267, that contains a single chromosomal copy of the pMG820 <em>lac</em> operon. Whereas no effects were observed during growth on glucose, significant decreased growth rates and <em>lac</em> operon activities were observed during growth on lactose, indicating that <em>lacR</em> specifically represses expression of the <em>lac</em> operon.<p>Characterization of the <em>lac</em> promoter and modulation of promoter activity by the <em>lacR</em> gene product is presented in <strong>Chapter 6</strong> . The transcription initiation site of the <em>lac</em> promoter was determined by primer extension mapping. The <em>lac</em> promoter canonical -35 and -10 sequences correspond closely to those described for gram-positive bacteria and are located in a back-to-back configuration with those of the divergently orientated <em>lacR</em> promoter (Fig. 1). The effects on <em>lac</em> promoter activity of flanking sequences and the <em>lacR</em> gene were studied in <em>L. lactis</em> and <em>E. coli</em> by using transcriptional fusions with a promoterless chloramphenicol acetyltransferase ( <em>cat</em> -86) gene. In the presence of the <em>lacR</em> gene both in <em>L. lactis</em> and <em>E. coli,</em> significantly decreased CAT activities were observed, indicating that the <em>lacR</em> gene product represses <em>lac</em> promoter activity. In addition, to obtain inducible CAT-activities a <em>lac</em> promoter fragment of at least 0.5 kb was required, suggesting that regions flanking the promoter are involved in regulation. These studies also showed that sequences flanking the <em>lac</em> promoter significantly contribute to the promoter efficiency in <em>L. lactis.</em> Enhancement of promoter activity in <em>L. lactis</em> of up to 38-fold was observed.<p><img src="/wda/abstracts/i1628_1.gif" height="757" width="600"/><p>The interaction between the LacR repressor and the <em>lac</em> promoter region is described in <strong>Chapter 7</strong> . For this purpose, LacR was overexpressed in <em>E. coli</em> and purified in a three-step procedure. Cross-linking studies with glutaraldehyde showed the ability of LacR to generate dimers. Gel-mobility shift assays and DNase I footprinting studies demonstrated the presence of two LacR-binding sites, <em>lacO1</em> and <em>lacO2,</em> in the intercistronic region between the <em>lacA</em> and <em>lacR</em> genes (Fig. 1). The <em>lacO1</em> operator is located at positions -31 to +6 and -96 to -59 relative to the transcription initiation sites of the <em>lac</em> operon and <em>lacR</em> gene, respectively. The distances between <em>lacO1</em> and transcription initiation sites of the <em>lac</em> operon and <em>lacR</em> gene are comparable to those often observed for repressor and activator binding sites, respectively, as is illustrated in Fig. 2. The <em>lacO2</em> operator is located at positions -313 to -278 and +188 to +223 relative to the transcription initiation sites of the <em>lac</em> operon and the <em>lacR</em> gene, respectively. Since a TGTTT sequence is present as an inverted repeat in <em>lacO1</em> and as a direct repeat in <em>lacO2,</em> we proposed that the TGTTT box comprises the LacR recognition sequence. Titration experiments with purified LacR and DNA-fragments containing <em>lacO1, lac02,</em> or both <em>(lacO1O2)</em> showed that <em>lacO1</em> and <em>(lacO1O2)</em> have a three-fold higher affinity than <em>lacO2,</em> for LacR binding. This indicated that the presence of <em>lac02 in cis</em> does not significantly enhance binding <em>of</em> LacR to <em>lacO1. To</em> identify the metabolite that induces <em>lac</em> operon expression during growth on lactose, gel mobility shift assays were carried out with the LacR repressor and <em>(lacO1O2)</em> in the presence <em>of</em> various phosphorylated monosaccharide intermediates from the tagatose-6-phosphate and glycolytic pathways. Dissociation of the LacR <em>-lacO1O2</em> complex was observed only in the presence of tagatose-6- phosphate, which is an intermediate of <em></em> the tagatose-6-phosphate pathway. No dissociation was observed with galactose-6-phosphate, tagatose-1,6-diphosphate, glucose-6- phosphate, fructose-6-phosphate and fructose- 1,6-diphosphate. Therefore, it was concluded that tagatose-6-phosphate is the physiological inducer of lac operon expression. This is supported by the observation that <em>lac</em> operon expression is also induced during growth on galactose, that is transported via a galactose-PTS and is metabolized through the tagatose-6-phosphate pathway.<p>In order to study whether the LacR repressor is the only determinant in the control <em>of lac</em> operon expression and to develop an expression system in <em>L.lactis</em> that allowed screening <em>of</em> mutated <em>lacR</em> genes, the chromosomally located <em>lacR</em> gene <em>of</em> strain MG5267 was deleted by replacement recombination ( <strong>Chapter 8</strong> ). The resulting strain was designated <em>L.lactis</em> NZ3015. As expected, determination <em>of</em> phospho-β-galactosidase (LacG) and lactose phosphotransferase (LacEF) activities, and <em>lac</em> mRNA levels of <em></em> lactose- and glucose-grown NZ3015 cells showed that expression of <em></em> the <em>lac</em> operon was significantly derepressed in the glucose-grown cells. However, approximately one fifth <em>of</em> the wild-type regulation level remained, as was demonstrated by the 1.6-fold (average) higher <em>lac</em> operon activities during growth on lactose than on glucose. This indicates that an additional control circuit is involved in the regulation of the <em>lac</em> operon. Since the RNA- studies demonstrated that this regulatory circuit mediates <em>lac</em> operon expression at the transcriptional level, we searched for DNA sequences in the <em>lac</em> promoter region that were homologous to a putative glucose-responsive-element (GRE) from <em>Bacillus.</em> Five basepairs downstream of the <em>lacO1</em> operator a sequence was detected that showed strong homology to the <em>Bacillus</em> GRE sequence. The <em>L. lactis</em> GRE sequence was also found in the promoter region of the <em>S.aureus lac</em> operon, that is strongly homologous to that <em>of L. lactis.</em><p><img src="/wda/abstracts/i1628_2.gif" height="553" width="600"/><p>The last two <strong>Chapters 9</strong> and <strong>10</strong> present the identification <em>of</em> amino acids in the <em>L. lactis</em> LacR repressor that are involved in the inducer response and binding to DNA, respectively. This was realized by studying the effects on the regulation of <em>lac</em> operon expression in the LacR-deficient strain NZ3015 and wild-type strain MG5267, after introduction of mutated <em>lacR</em> genes. Since LacR belongs to the <em>E.coli</em> DeoR family of repressors, in which all members have in common that their inducer is a phosphorylated sugar, it was anticipated that within this family there will be conserved amino acid residues that are involved the response to the inducer tagatose-6-phosphate. Various amino acid residues in LacR that are conserved in other DeoR family members and located outside the DNA-binding motif, were replaced by alanine or arginine. Cells of strain NZ3015 containing K72A-, K80A-, D210A-, or K213A-LacR, were unable to derepress phospho-β-galactosidase activities during growth on lactose. These low phospho-β-galactosidase activities resulted in significantly decreased growth rates on lactose, and strongly suggested that these LacR mutant proteins had lost their ability to respond to inducer. This hypothesis was verified by carrying out gel mobility shift assays with <em>lacO1O2</em> operator and purified K72A-, K80A-, D210A-, and K213A-LacR proteins in the presence or absence of the inducer tagatose-6-phosphate. None of the complexes between the <em>lacO1O2</em> and the mutant proteins was affected by tagatose-6-phosphate, whereas the complex between <em>lacO1O2</em> and wild-type LacR dissociated in the presence of tagatose-6-phosphate. From these experiments it was concluded that Lys-72, Lys-80, Asp- 210, and Lys-213 are involved in the inducer response of the LacR repressor. It is not yet clear whether these residues are involved in the actual binding of tagatose-6-phosphate or, upon binding, the allosteric transition of LacR into a molecule with a decreased affinity for lacO1O2. In addition, these results confirm that <em>in vivo</em> tagatose-6-phosphate is the inducer of the L.lactis lac operon.<p>To identify the residues in LacR involved in DNA-binding, amino acid residues in the putative N-terminal DNA-binding domain, that contains a helix-turn-helix motif, were replaced by alanine. The LacR mutants M34A and R38A showed a 10- and 25-fold decrease of the <em>in</em> vivo DNA-binding constant, indicating that Met-34 and Arg-38 are involved in DNA-binding. Two LacR mutants, D30A and D33A, were constructed with a 4-fold increased DNA-binding constant, indicating that it is possible to improve the relatively weak binding of LacR to its operator. Based on the similarities between the LacR repressor and the <em>lacO1</em> operator and the <em>E.coli</em> LacI repressor variant 44 and its corresponding operator, a model for the binding of LacR to the <em>lacO1</em> operator was presented.<p>Based on the studies presented in this thesis a model for the action of the LacR repressor in the regulation of the <em>L.lactis lac</em> operon is proposed. Below, three stages of the model will be discussed. <strong></strong><p><strong>1. Binding of LacR repressor to operator <em>lacO1</em> during growth on glucose results in autoactivation of <em>lacR</em> gene expression</strong> . The induction of <em>lacR</em> on glucose and the high affinity of the LacR repressor for <em>lacO1</em> are evident from the Northern-studies (Chapter 5) and gel mobility shift titration experiments (Chapter 7), respectively. The distance between location of <em>lacO1</em> and the <em>lacR</em> transcription initiation site coincides with the distance that is commonly observed for an activator (Fig. 2). The involvement of <em>lacO1</em> in the regulation of <em>lacR</em> is supported by the observation that partial deletion of <em>lacO1</em> resulted in the loss of <em>lacR</em> regulation (Chapter 9, Fig. 3). However, no experimental data have been generated to establish the <u>direct</u> involvement of LacR in activating expression of its own gene. Since the transcriptional fusion studies (Chapter 6) showed that <em>lacO1</em> alone is incapable of regulating CAT-expression, we presume that no repression of <em>lac</em> operon expression occurs at this stage.<p><strong>2. Binding of <em></em> LacR repressor to <em>lacO2</em> at increasing LacR concentrations during growth on glucose results in repression of <em>lacR</em> gene and lac operon expression</strong> . Since it has been shown that <em>lacO2</em> has a lower affinity for LacR than <em>lacO1</em> (Chapter 7), <em>lacO2</em> will only be bound at increasing LacR concentrations. The CAT-reporter studies showed that both <em>lacO1</em> and <em>lacO2</em> are required for repression of CAT-activity during growth on glucose (Chapter 6). Therefore, repression of transcription initiation of lac operon occurs when LacR is bound to both <em>lacO1</em> and <em>lacO2</em> . The exact repression mechanism has not been elucidated, but might include the formation of a DNA loop between <em>lacO1</em> and <em>lacO2</em> , as has been described for other regulatory systems (Matthews, 1992). The postulated repression of <em>lacR</em> expression upon binding of LacR to <em>lacO2</em> , would prevent the cell from overproduction of LacR due to continuous activation by <em>lacO1</em> and results in a certain steady state concentration of LacR. However, no experiments have been carried out to establish the role of <em>lacO2</em> , in the putative autoregulation of <em>lacR</em> .<p><strong>3. Binding of LacR repressor to tagatose-6-phosphate during growth on lactose results in dissociation of the LacR-operator complex concomitant with the induction of <em>lac</em> operon expression</strong> . From the gel mobility shift studies in Chapter 7 it is evident that the LacR- <em>lacO1O2</em> complex dissociates in the presence of tagatose-6-phosphate, that is an intermediate of the tagatose-6-phosphate pathway. In addition, LacR mutants were constructed, the presence of which in <em>L. lactis</em> resulted in an inability to induce lac operon activity on lactose, that had lost their sensitivity to tagatose-6-phosphate. Therefore, the complex between LacR and tagatose-6-phosphate that is formed during growth on lactose does not bind to the <em>lac</em> operators, resulting in the restoration of transcription initiation from the <em>lac</em> promoter. As a result of the absence of LacR bound to <em>lacO1</em> the <em>lacR</em> gene is probably no longer (auto)activated resulting in a decreased level of <em>lacR</em> expression. The presence of multiple copies of constitutively expressed lacR results in an additional repression of <em>lac</em> promoter activity during growth on both glucose and lactose (Chapters 5 and 6), suggesting that due to the overproduction of LacR relatively more <em>lacO2</em> , is bound by LacR. Due to the limited amount of inducer (Chapter 8), it would under these conditions then be theoretically possible that, in contrast to the situation in wild-type cells, <em>lacR</em> expression is induced during growth on lactose. This might be a consequence of the dissociation of only the <em>lacO2</em> -LacR complex in these cells during growth on lactose. In contrast, in wild-type cells, where the LacR concentration is lower, LacR dissociates from both operators during growth on lactose.<p>The studies described in this thesis have provided more insight in the genetic basis and regulation of lactose catabolism in <em>L. lactis</em> . Parts of this knowledge have already been used for the development of a food-grade selection system for <em>L. lactis</em> based on the <em>lacF</em> gene (De Vos, 1988). In addition, the <em>lac</em> promoter has already been successfully used for the expression of mutated <em>nisZ</em> genes in <em>L. lactis</em> (Kuipers <em>et al.</em> , 1992). Since <em>L. lactis</em> preferentially metabolizes glucose it should be possible, by the starting the fermentation with a certain amount of glucose, to overexpress genes of interest under control of the <em>lac</em> promoter at a defined stage in a dairy fermentation.
Boekbespreking: The economics of parttime farming, R. Gasson.
Vries, W.M. de - \ 1990
Sociologia Ruralis 30 (1990). - ISSN 0038-0199 - p. 361 - 363.
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