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Transcriptome Response of Lactobacillus Plantarum to Global Regulator Deficiency, Stress and Other Environmental Conditions

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Transcriptome Response of Lactobacillus Plantarum to Global Regulator Deficiency, Stress and Other Environmental Conditions Transcriptome Response of Lactobacillus plantarum to Global Regulator Deficiency, Stress and other Environmental Conditions Marc J.A. Stevens Promotoren: Prof. Dr. W. M. de Vos Hoogleraar Microbiologie Wageningen Universiteit Prof. Dr. M. Kleerebezem Hoogleraar Metagenomics Wageningen Universiteit Co-promotor: Dr. D. Molenaar Projectleider NIZO Food Research Promotiecommisie: Dr. J. Sikkema TI Food and Nutrition Prof. Dr. J. Kok Rijksuniversiteit Groningen Prof. Dr. P. Hols Université Louvain Prof. Dr. T. Abee Wageningen Universiteit Dit onderzoek is uitgevoerd binnen de onderzoeksschool VLAG Transcriptome Response of Lactobacillus plantarum to Global Regulator Deficiency, Stress and other Environmental Conditions Marc J. A. Stevens Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. Dr. M. J. Kropff, In het openbaar te verdedigen op vrijdag 11 januarie 2008 des namiddags om half twee in de aula Marc J. A. Stevens. Transcriptome Response of Lactobacillus plantarum to Global Regulator Deficiency, Stress and other Environmental Conditions. PhD Thesis. Wageningen University and Research Centre, The Netherlands, 2008. With Summary in Dutch ISBN: 978-90-8504-869-5 Abstract Lactobacillus plantarum is a lactic acid bacterium encountered in a variety of food and feed fermentations and as a natural inhabitant of human gastrointestinal tract. To survive in these niches and to maintain its capability, L. plantarum has to respond to numerous changing conditions and the cellular processes involved in these responses are frequently regulated at the transcriptional level. For this reason the transcriptome of a cell is of interest, as it contains information about the transcriptional response of individual genes. Transcriptomic approaches allow elucidation of transcriptional behaviour of all genes at once and are therefore a powerful tool to study all the transcriptional response of a bacterium to differential conditions. The complete genome of L. plantarum was sequenced and putative genes were identified and annotated, allowing design and production of a full genome amplicon based microarray. Using this array, the transcriptional response of L. plantarum to a variety of conditions could be identified. The regulon of sigma factor 54 (σ54) was elucidated and consisted of a single operon encoding a mannose phosphotransferase system (PTS). This transport system was found to be the only functional mannose transporter in L. plantarum. Additionally, a glucose transport function was established for this PTS and deletion of the PTS led to an increase of the intracellular phosphoenolpyruvate (PEP) concentration in resting cells, indicating an important role of this glucose transport system in metabolic control of carbohydrate metabolism. The mannose PTS was also found to play an important role in hydrogen peroxide caused oxidative stress survival due to its robustness to oxidative stress. The impact of mild oxidative stress caused by aerobic growth was elucidated by comparing an aerobic grown culture with an anaerobic grown culture. The results led to the identification of the underlying mechanism of an observed growth stagnation, and subsequent adjustments of fermentation conditions led to overcome the stagnation. The role of the global regulator of catabolite control (CcpA) was determined throughout the whole growth curve, showing a global role for this regulator in catabolite control. Additional studies with different carbon sources confirmed this global role of CcpA and suggested further fine-tuning of regulation by local regulators. Contents: Chapter 1: 7 Introduction and Outline of the Thesis Chapter 2: 31 Sigma 54 Mediated Control of the Mannose PTS in Lactobacillus plantarum Impacts on Carbohydrate Metabolism Chapter 3: 61 Global Transcriptional Analysis of the Peroxide Response in Lactobacillus plantarum and Involvement of the Mannose PTS in Survival Chapter 4: 83 Improvement of Lactobacillus plantarum Aerobic fermentation by Comprehensive Transcriptome Analysis Chapter 5: 115 Comparative Transcriptome and Proteome Analyses of the Growth Phase dependent CcpA-regulon in Lactobacillus plantarum Chapter 6: 141 Regulation of Carbohydrate Transport and Utilization in Lactobacillus plantarum Chapter 7: 169 Summary, General Discussion, and Future perspectives Samenvatting 183 Nawoord 187 Curriculum vitae 189 List of publications 191 Chapter 1 Introduction and Outline of the Thesis Chapter 1 Lactobacillus plantarum is encountered in a range of environmental niches, including industrial food fermentations. The control, improvement and innovation of such fermentations demands detailed knowledge about the responses of the bacterium during fermentation and processing conditions. Transcriptome research is a powerful tool to investigate transcriptional regulation as it offers a near-complete view on relative mRNA abundance in a cell and therefore this technique was chosen to study the behavior of L. plantarum under different conditions. The main pillars of the research described in this thesis are introduced in the following chapter: the organism Lactobacillus plantarum, its characteristics and its place within the group of lactic acid bacteria; the regulation of transcription in bacteria with focus on L. plantarum; and the experimental and theoretical background of genome wide transcriptional analyses. At the end of this chapter an outline of the research in this thesis as described in the following chapters is presented. Lactic acid bacteria Lactic acid bacteria (LAB) are low G + C Gram-positive bacteria functionally related by their ability to produce lactic acid through homo- and/or hetero-fermentative carbohydrate metabolism. Their natural habitats are commonly anaerobic or micro-aerobic and are related to their generally high demand for nutrients and specialized carbohydrate utilization ({Lambert, 200782). LAB are used in a variety of industrial food and feed fermentations with 6 predominant species of beneficial and non-pathogenic bacteria: Lactococcus (milk), Lactobacillus (milk, meat, vegetables, cereals), Leuconostoc (vegetables, milk), Pediococcus, (vegetables, meat, milk), Oenococcus (wine), and Streptococcus (milk) (49). Some LAB, especially of the genera Lactobacillus (e. g. L. plantarum, L. acidophilus, L. casei, and L. gasseri) and Leuconostoc (L. argentinum, L. mesenteroides), are encountered in the human gastrointestinal tract (GI-tract) having a potential beneficial effect on the host (2, 27). The behaviour of LAB in fermentation processes and the GI-tract was extensively studied over the last two decades (85). As this research possibly leads to enhanced performance and functionality of LAB, it is of high relevance for the food industry (49). The dawning of the LAB genomics era started with the annotation and publication of the complete genome sequence of Lactococcus lactis ssp. lactis IL1403 in 2001 (16), followed by the genome of Lactobacillus plantarum WCFS1 in 2003, the first complete genome within the species of Lactobacillus. In the years that followed, genomes of representative species and strains of the genera Lactobacillus, Streptococcus, Lactococcus, Oenococcus, Leuconostoc, 8 Introduction and Outline of the Thesis and Pediococcus were sequenced, representing considerable diversity in ecological habitat (milk, meat, plants, GI-tract) and roles (probiotic, fermentation) (49). The availability of complete genome sequences allowed comparisons between LAB species and revealed major genome decay, lateral gene transfer, sequence conservation, metabolic simplification, and expansion of selected gene families within the group of LAB (reviewed by Makarova (58) and by Claesson (22)). The genome decay, expansions of gene families and metabolic simplification are probably due to adaptation to nutritionally rich environments (57). Moreover, comparison within the genus Lactobacillus revealed significant functional differences between Lactobacilli, presenting difficulties or even necessity for revision in terms of their taxonomic classification (57, 86). The ongoing sequencing projects of LAB genomes will enable advanced comparative genomic analyses, but will also form the basis for extensive functional genomics approaches (ranging from transcriptomic, proteomic, to metabolomic approaches) and the outcome of these efforts will expand our understanding and knowledge of LAB in general and of the genus Lactobacillus in particular (22). Eventually, this should enable the design of improved fermentation processes and enhanced probiotic functionality, which will fuel industrial innovation aiming to provide the consumer with tasty and attractive, but also healthy and safe fermented food products. Lactobacillus plantarum Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium found worldwide in the production of fermented food and feed products. Moreover, L. plantarum is frequently encountered as a natural inhabitant of the human gastrointestinal tract (2) and as spoilage organism in wine (12), meat (17), and orange juice (3). The complete genome sequence of Lactobacillus plantarum strain WCFS1, a single colony isolate of the human saliva isolate NCIMB8826, was determined (50) and was predicted to contain approximately 3,000 protein-encoding genes of which approximately 70% could be assigned with a putative function. The genome encodes all enzymes required for the glycolysis and phosphoketolase pathways, as is expected for a facultative hetero-fermentative
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