GlobalSoilMap for Soil Organic Carbon Mapping and as a Basis for Global Modeling
Arrouays, D. ; Minasny, B. ; McBratney, A. ; Grundy, Mike ; McKenzie, Neil ; Thompson, James ; Gimona, Alessandro ; Hong, Suk Young ; Smith, Scott ; Hartemink, A.E. ; Chen, Songchao ; Martin, Manuel P. ; Mulder, V.L. ; Richer-de-Forges, A.C. ; Odeh, Inakwu ; Padarian, José ; Lelyk, Glenn ; Poggio, Laura ; Savin, Igor ; Stolbovoy, Vladimir ; Leenaars, J.G.B. ; Heuvelink, G.B.M. ; Montanarella, Luca ; Panagos, P. ; Hempel, Jon - \ 2017
In: Proceedings of the global symposium on soil organic carbon 2017. - FAO - p. 27 - 30.
The demand for information on functional soil properties is high and has increased over time. This is especially true for soil organic carbon (SOC) in the framework of food security and climate change. The GlobalSoilMap consortium was established in response to such a soaring demand for up-to-date and relevant soil information. The majority of the data needed to produce GlobalSoilMap soil property maps will, at least for the first generation, come mainly from archived soil legacy data, which could include polygon soil maps and point pedon data, and from available co-variates such as climatic data, remote sensing information, geological data, and other forms of environmental information.
Several countries have already released products according to the GlobalSoilMap specifications and the project is rejuvenating soil survey and mapping in many parts of the world. Functional soil property maps have been produced using digital soil mapping techniques and existing legacy information and made available to the user community for application. In addition, uncertainty has been provided as a 90% prediction interval based on estimated upper and lower class limits. We believe that GlobalSoilMap constitutes the best available framework and methodology to address global issues about SOC mapping. Main scientific challenges include time related and uncertainties issues.
Soil legacy data rescue via GlobalSoilMap and other international and national initiatives
Arrouays, Dominique ; Leenaars, Johan G.B. ; Richer de Forges, Anne C. ; Adhikari, Kabindra ; Ballabio, Cristiano ; Greve, Mogens H. ; Grundy, Mike ; Guerrero, Eliseo ; Hempel, Jon ; Hengl, Tom ; Heuvelink, Gerard ; Batjes, Niels ; Carvalho Ribeiro, Eloi ; Hartemink, Alfred ; Okx, J.P. - \ 2017
GeoResJ 14 (2017). - ISSN 2214-2428 - p. 1 - 19.
GlobalSoilMap - Legacy data - Soil data rescue
Legacy soil data have been produced over 70 years in nearly all countries of the world. Unfortunately, data, information and knowledge are still currently fragmented and at risk of getting lost if they remain in a paper format. To process this legacy data into consistent, spatially explicit and continuous global soil information, data are being rescued and compiled into databases. Thousands of soil survey reports and maps have been scanned and made available online. The soil profile data reported by these data sources have been captured and compiled into databases. The total number of soil profiles rescued in the selected countries is about 800,000. Currently, data for 117, 000 profiles are compiled and harmonized according to GlobalSoilMap specifications in a world level database (WoSIS). The results presented at the country level are likely to be an underestimate. The majority of soil data is still not rescued and this effort should be pursued. The data have been used to produce soil property maps. We discuss the pro and cons of top-down and bottom-up approaches to produce such maps and we stress their complementarity. We give examples of success stories. The first global soil property maps using rescued data were produced by a top-down approach and were released at a limited resolution of 1 km in 2014, followed by an update at a resolution of 250 m in 2017. By the end of 2020, we aim to deliver the first worldwide product that fully meets the GlobalSoilMap specifications.
Grundy, John ; Mistrik, Ivan ; Ali, Nour ; Soley, Richard M. ; Tekinerdogan, Bedir - \ 2016
In: Software Quality Assurance / Mistrik, I., Ali, N., Tekinerdogan, B., Elsevier Inc. Academic Press - ISBN 9780128023013 - p. xxxiii - xlii.
|Architecture Viewpoint for Modeling Dynamically Configurable Software Systems
Tekinerdogan, B. ; Sozer, H. - \ 2016
In: Managing Trade-offs in Adaptable Software Architectures / Mistrik, I., Ali, N., Kazman, R., Grundy, J., Schmerl, B., Elsevier - ISBN 9780128028551 - p. 79 - 95.
|Architectural Perspective for Design and Analysis of Scalable Software as a Service Architectures
Tekinerdogan, B. ; Ozcan, O. - \ 2016
In: Managing Trade-offs in Adaptable Software Architectures / Mistrik, I., Ali, N., Kazman, R., Grundy, J., Schmerl, B., Elsevier - ISBN 9780128028551 - p. 223 - 243.
|Architectural drift analysis using architecture reflexion viewpoint and design structure reflexion matrices
Tekinerdogan, B. - \ 2015
In: Sofware Quality Assurance in Large Scale and Complex Software-Intensive Systems / Mistrik, I., Soley, R.., Ali, N., Grundy, J., Tekinerdogan, B., Elsevier - ISBN 9780128023013 - p. 221 - 236.
Quality Concerns in Large Scale and Complex Software-intensive Systems
Tekinerdogan, B. ; Ali, N. ; Grundy, J. ; Mistrik, I. ; Soley, R. - \ 2015
In: Sofware Quality Assurance in Large Scale and Complex Software-Intensive Systems / Mistrik, I., Soley, R., Ali, N., Grundy, J., Tekinerdogan, B., Elsevier - ISBN 9780128023013 - p. 1 - 17.
|Software quality assurance: in large scale and complex software-intensive systems
Mistrik, I. ; Soley, R. ; Ali, N. ; Grundy, J. ; Tekinerdogan, B. - \ 2015
Burlington, Massachusetts, USA : Morgan Kaufmann Publishers - ISBN 9780128023013 - 420
Software Quality Assurance in Large Scale and Complex Software-intensive Systems presents novel and high-quality research related approaches that relate the quality of software architecture to system requirements, system architecture and enterprise-architecture, or software testing. Modern software has become complex and adaptable due to the emergence of globalization and new software technologies, devices and networks. These changes challenge both traditional software quality assurance techniques and software engineers to ensure software quality when building today (and tomorrow's) adaptive, context-sensitive, and highly diverse applications. This edited volume presents state of the art techniques, methodologies, tools, best practices and guidelines for software quality assurance and offers guidance for future software engineering research and practice. Each contributed chapter considers the practical application of the topic through case studies, experiments, empirical validation, or systematic comparisons with other approaches already in practice. Topics of interest include, but are not limited, to: quality attributes of system/software architectures; aligning enterprise, system, and software architecture from the point of view of total quality; design decisions and their influence on the quality of system/software architecture; methods and processes for evaluating architecture quality; quality assessment of legacy systems and third party applications; lessons learned and empirical validation of theories and frameworks on architectural quality; empirical validation and testing for assessing architecture quality.
Selected Reaction Monitoring method to determine the species origin of blood-based binding agents in meats: a collaborative study
Grundy, H. ; Read, W.A. ; Macarthur, R. ; Alewijn, M. - \ 2013
Food Chemistry 141 (2013)4. - ISSN 0308-8146 - p. 3531 - 3536.
quadrupole mass-spectrometry - liquid-chromatography - products - validation
Binding products or food ‘glues’ are used throughout the food industry to increase the meat use rate or to augment economic efficiency. Some of these binders contain thrombin from bovine and porcine blood. The European parliament has recently banned thrombin-based additives and labelling legislation governs their use in the US. A mass spectrometry screening method is available to detect the addition of thrombin agents to foods as there is a need to protect consumers and to avoid misleading trade practices. We report the details of an inter-laboratory trial to determine the transferability of this method to operators in various food testing laboratories, each using a different triple quadrupole mass spectrometer design. The trial was successful with the species origin of the binding agent contained in each of the forty three test materials being correctly reported by the participants. This is consistent with a false positive and false negative rate of zero percent. This is the first collaborative study, as far as we are aware, which involves a liquid chromatography mass spectrometry (LC-MS/MS) application to approach a food authenticity issue.
|Proteomic analysis of meat and bone meal and animal feed
Reece, P. ; Chassaigne, H. ; Collins, M.J. ; Buckley, M. ; Bremer, M.G.E.G. ; Grundy, H. - \ 2012
In: Detection, identification and quantification of processed animal proteins in feedingstuffs / Jorgensen, J.S, Baeten, V., Namur : Presses universitaires de Namur - ISBN 9782875510297 - p. 113 - 124.
Weed occurrence on pavements in five North European towns
Melander, B. ; Holst, N. ; Grundy, A. ; Kempenaar, C. ; Riemens, M.M. ; Verschwele, A. ; Hansson, D. - \ 2009
Weed Research 49 (2009)5. - ISSN 0043-1737 - p. 516 - 525.
canadian weeds - biology - areas
Weeds on pavements in urban areas are unwanted mainly because they cause an untidy appearance or sometimes structural damage. Glyphosate has been the principal weed control method for years, but policies in several European towns have changed to lower dependence on herbicides. Instead, less effective and more species-dependent non-chemical methods are used, but little is known about the pavement flora. Consequently, we surveyed the flora on pavements in five North European towns [Braunschweig (DE), Malmö (SE), Næstved (DK), Royal Leamington Spa (UK) and Wageningen (NL)] by recording weed species and their coverage in 56 recording points randomly placed in each town. Weeds were recorded at several dates in 2005 and 2006 and no weed control was applied apart from sweeping. Weed coverage increased during the survey (averaging 1.4% in late 2006) and was highest in the towns having the strictest policies limiting herbicide use. Most coverage (averaging 2%) was found along the pavement edge away from the road. Poa annua was the most frequently recorded species, followed by bryophytes (mainly mosses), Sagina procumbens and perennial grasses. Grasses and some other species frequently found, notably Taraxacum officinale, should receive particular attention when planning a non-chemical weed control campaign on pavements.
A review of pesticide policies and regulations for urban amenity areas in seven European countries
Kristoffersen, P. ; Rask, A.M. ; Grundy, A. ; Franzen, I. ; Kempenaar, C. ; Raisio, J. ; Schroeder, H. ; Spijker, J.H. ; Verschwele, A. ; Zarina, L. - \ 2008
Weed Research 48 (2008). - ISSN 0043-1737 - p. 201 - 214.
nonchemical weed-control - hard surfaces - denmark - water
An analysis of the regulations of herbicide use for weed control in non-agricultural/urban amenity areas, including actual pesticide use, was carried out as a joint survey of seven European countries: Denmark, Finland, Germany, Latvia, the Netherlands, Sweden and United Kingdom. Herbicides constitute the major part of the pesticides used in urban amenity areas. Herbicide use on hard surfaces is the largest in terms of volume and potential contamination of surface and groundwater. The aim of the study was to investigate the differences in political interest and public debate on the 'use of pesticides in public urban amenity areas', regulations within each country at national, regional and local levels, possible use of alternative weed control methods and the amounts of pesticides used on urban amenity areas. A comparative analysis revealed major differences in political interest, regulations and availability of statistics on pesticide use. Denmark, Sweden, the Netherlands and Germany have, or have had, a strong public and political interest for reducing the use of herbicides to control weeds in urban amenity areas and also have very strict regulations. The UK is currently undergoing a period of increasing awareness and strengthening regulation, while Latvia and Finland do not have specific regulations for weed control in urban amenity areas or on hard surfaces. Statistics on pesticide/herbicide use on urban amenity areas were only available in Denmark and the Netherlands. Developing this kind of information base reveals the differences in herbicide use, regulations and policies in European countries and may enhance the transfer of knowledge on sustainable weed control across countries.
Nutritional concerns in old age
Groot, C.P.G.M. de; Staveren, W.A. van - \ 2007
In: Ageing Well. Nutrition, Health, and Social Interventions / Dangour, A.D., Grundy, E.M.D., Fletcher, A.E., CRC Press - ISBN 9780849374746 - p. 5 - 13.
All models are wrong - but some are useful : a report from an EWRS workshop on modelling weed population dynamics
Rasmussen, I.A. ; Bastiaans, L. ; Holst, N. ; Grundy, A. ; Melander, B. - \ 2007
In: Proceedings of the 14th European Weed Research Society (EWRS) Symposium, Hamar, Norway, 17 - 21 june, 2007. - Hamar : EWRS - p. 116 - 116.
Efficacy and safety of plant stanols and sterols in the control of blood cholesterol levels
Katan, M.B. ; Grundy, S.M. ; Jones, P.J.H. ; Law, M.R. ; Miettinen, T. ; Paoletti, R. - \ 2003
Mayo Clinic Proceedings 78 (2003). - ISSN 0025-6196 - p. 965 - 978.
coronary heart-disease - sitostanol-ester-margarine - soluble antioxidant concentrations - density-lipoprotein cholesterol - mildly hypercholesterolemic population - randomized controlled-trial - plasma total-cholesterol - serum total cholesterol - e-deficient mi
Foods with plant stanol or sterol esters lower serum cholesterol levels. We summarize the deliberations of 32 experts on the efficacy and safety of sterols and stanols. A meta-analysis of 41 trials showed that intake of 2 g/d of stanols or sterols reduced low-density lipoprotein (LDL) by 10%; higher intakes added little. Efficacy is similar for sterols and stanols, but the food form may substantially affect LDL reduction. Effects are additive with diet or drug interventions: eating foods low in saturated fat and cholesterol and high in stanols or sterols can reduce LDL by 20%; adding sterols or stanols to statin medication is more effective than doubling the statin dose. A meta-analysis of 10 to 15 trials per vitamin showed that plasma levels of vitamins A and D are not affected by stanols or sterols. Alpha carotene, lycopene, and vitamin E levels remained stable relative to their carrier molecule, LDL. Beta carotene levels declined, but adverse health outcomes were not expected. Sterol-enriched foods increased plasma sterol levels, and workshop participants discussed whether this would increase risk, in view of the marked increase of atherosclerosis in patients with homozygous phytosterolemia. This risk is believed to be largely hypothetical, and any increase due to the small increase in plasma plant sterols may be more than offset by the decrease in plasma LDL. There are insufficient data to suggest that plant stanols or sterols either prevent or promote colon carcinogenesis. Safety of sterols and stanols is being monitored by follow-up of samples from the general population; however, the power of such studies to pick up infrequent increases in common diseases, if any exist, is limited. A trial with clinical outcomes probably would not answer remaining questions about infrequent adverse effects. Trials with surrogate end points such as intima-media thickness might corroborate the expected efficacy in reducing atherosclerosis. However, present evidence is sufficient to promote use of sterols and stanols for lowering LDL cholesterol levels in persons at increased risk for coronary heart disease.
AHA scientific statement: Summary of the scientific conference on dietary fatty acids and cardiovascular health
Kris-Etherton, P. ; Daniels, S.R. ; Eckel, R.H. ; Engler, M. ; Howard, B.V. ; Krauss, R.M. ; Lichtenstein, A.H. ; Sacks, F. ; St. Jeor, S. ; Stampfer, M. ; Grundy, S.M. ; Zock, P.L. - \ 2001
The Journal of Nutrition 131 (2001)4. - ISSN 0022-3166 - p. 1322 - 1326.
Conference Planning and Writing Committee:Penny Kris-Etherton, PhD, Stephen R. Daniels, MD, PhD, Robert H. Eckel, MD, Marguerite Engler, PhD, RN, Barbara V. Howard, PhD, Ronald M. Krauss, MD, Alice H. Lichtenstein, DSc, Frank Sacks, MD, Sachiko St. Jeor, PhD, Meir Stampfer, MD, DrPH, For the American Heart Association Nutrition Committee Speakers and Discussants:, Robert H. Eckel, MD, Scott M. Grundy, MD, PhD, Lawrence J. Appel, MD, MPH, Tim Byers, MD, Hannia Campos, PhD, Greg Cooney, PhD, Margo A. Denke, MD, Barbara V. Howard, PhD, Eileen Kennedy, DSc, Ronald M. Krauss, MD, Penny Kris-Etherton, PhD, Alice H. Lichtenstein, DSc, Peter Marckmann, MD, DSc, Thomas A. Pearson, MD, PhD, Gabriele Riccardi, MD, Lawrence L. Rudel, PhD, Mike Rudrum, PhD, Frank Sacks, MD, Daniel T. Stein, MD, Russell P. Tracy, PhD, Virginia Ursin, PhD, Robert A. Vogel, MD, Peter L. Zock, PhD, AHA Members:, Terry L. Bazzarre, PhD and Julie Clark, AHA Staff
Summary of the scientific conference on dietary fatty acids and cardiovascular health: conference summary from the nutrition committee of the American Heart Association
Kris-Etherton, P. ; Daniels, S.R. ; Eckel, R.H. ; Engler, M. ; Howard, B.V. ; Krauss, R.M. ; Lichtenstein, A.H. ; Sacks, F. ; St. Jeor, S. ; Stampfer, M. ; Grundy, S.M. ; Appel, L.J. - \ 2001
Circulation 103 (2001). - ISSN 0009-7322 - p. 1034 - 1039.
Selection with control of inbreeding in populations with overlapping generations : a comparison of methods
Sonesson, A.K. ; Grundy, B. ; Woolliams, J.A. ; Meuwissen, T.H.E. - \ 2000
Animal Science 70 (2000)1. - ISSN 1357-7298 - p. 1 - 8.
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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Katan, M.B. ; Grundy, S.M. ; Willett, W.C. - \ 1998
Nederlands Tijdschrift voor Geneeskunde 142 (1998). - ISSN 0028-2162 - p. 886 - 889.