NMR in Microbiology: Theory and Applications Chapter Abstracts
CHAPTER 1: NMR Basic Principles
Jean-Michel Franconi, Dominique Cailleu, Jean-Charles Portais and Paul Canioni
Abstract
The Nuclear Magnetic Resonance (NMR) applications are growing exponentially during the last 20 years. In order to understand and use the last NMR sequences, it is necessary to have an initial knowledge of the NMR physics. In the first chapter we will develop the basic principles of NMR spectroscopy and imaging. The phenomenon will be first described by the classical theory. The phenomenon will be dissociated in three 'major' steps: polarisation, resonance and relaxation. Then we will describe the basic principles NMR spectroscopy and the NMR spectral parameters. The final part of the chapter will deal with the description of some more sophisticated issues like polarisation transfer and the rules of NMR sequence build-up.
CHAPTER 2: NMR and Microorganisms
Jean-Philippe Grivet
Abstract
This chapter is an introduction to the use of NMR for the investigation of microbial physiology and metabolism. NMR parameters which determine the sensitivity and resolving power of the method are reviewed. A broad survey of current applications follows. Qualitative uses are described first; they include compound identification and localisation. Quantitative aspects, such as pH, concentration and flux measurements are then examined, as well as the corresponding experimental constraints. The chapter ends with suggestions of possible future developments in instrument capabilities aimed at improving sensitivity: higher fields, spectroscopic and imaging microprobes.
CHAPTER 3: In situ NMR Systems
Jacqueline V. Shanks
Abstract
In situ NMR is becoming an established technology for applications in bioprocessing and metabolic engineering. The in situ NMR biosensor acts as a noninvasive pH, ion, and concentration meter, with 31P and 13C as the two main isotopes of study. A substantial data base now exists for phosphorus and carbon spectra of bacteria and yeast. In situ NMR can provide many of the state variables needed for modeling glycolytic pathway function. NMR micro-reactor technology has improved significantly in the last decade. Several designs for immobilized cell reactors have been tested, and in particular, considerable gains have been made in the feasibility of studying aerobic, chemostat cultures with in situ NMR. Acquisition of 31P spectra from cell suspensions of 3-5% v/v under controlled conditions can be made in 3 7 minute time resolution in several systems.
CHAPTER 4: Use of 13C labelling and NMR Spectroscopy in Metabolic Flux Analysis
Albert A. de Graaf
Abstract
NMR spectroscopy in combination with stable isotope labelling plays an increasingly important role in the identification and quantitation of metabolic reactions in living systems. Several key studies have demonstrated the potential of 13C labelling and NMR spectroscopy for the determination of metabolic fluxes in the pathways of primary metabolism. Isotopic measurement data may be obtained from excreted products or cytoplasmic metabolites, but also from macromolecular cell constituents after prolonged incubation with the 13C-labelled substrate. Modern methods, especially so-called inverse proton NMR techniques, allow the highly precise and accurate determination of positional 13C enrichments as well as of 13C isotopomer distributions in biogenic compounds. Using sophisticated, matrix-based explicit computation methods, complete metabolic networks can now be analysed from a comprehensive 13C NMR isotopomer data set in a few minutes time on a modern PC. Optimal results are obtained when the NMR data are treated in an integrated analysis together with extracellular measurement data of substrate uptake and product excretion rates, and when a detailed account is kept of the amounts of precursor metabolites that are needed to supply the anabolic reactions.
CHAPTER 5: Data Analysis And Modelisation
Tran-Dinh Son
Abstract
In this chapter, we focus our attention mainly on the applications of 13C-NMR spectroscopy in investigating the metabolism in cells. We show how the various isotopomers and the isotopomer distribution in randomly (or non-randomly) and uniformly (or non-uniformly) labelled compounds can be determined from one-dimensional 13C-NMR spectra. A formalism using matrix operations is presented for studying the transfer of carbons and the change in the isotopomer distribution between metabolites along a linear or cyclic pathway. Very simple models containing only one general equation are constructed on the basis of 13C-NMR data of specific carbon labelling of glutamate and aspartate. These models are then used to determine the population of all glutamate or aspartate isotopomers and to evaluate, by numerical or analytical methods, the metabolic flux in the citric acid cycle associated, or not, with another pathway such as the glyoxylate cycle or the malate-aspartate shuttle.
CHAPTER 6: Two-Dimensional NMR Spectroscopy
Jack Skalicky and Thomas Szyperski
Abstract
Two-dimensional (2D) NMR spectroscopy is nowadays routinely used in biological research. When compared with its one-dimensional congener, it offers increased resolution and allows indirect detection of otherwise non-observable nuclear spin quantum states. This chapter outlines basic concepts of 2D NMR spectroscopy.
CHAPTER 7: Structure Determination of Microbial Polysaccharides by High Resolution NMR Spectroscopy
Dusan Uhrín and Jean-Robert Brisson
Abstract
A general strategy for de novo structure elucidation of an unknown polysaccharide by NMR is outlined. The main emphasis is on the determination of the primary structure of polysaccharides, although some attention is given to the investigation of their three-dimensional structures. Selected NMR techniques are demonstrated using the specific capsular polysaccharide of Streptococcus pneumoniae type 7F. It is argued that the judicial combination of 2D homonuclear NMR, 1D selective excitation methods and 2D heteronuclear methods can now lead to the determination of even the most complex polysaccharide structures by NMR. Two-dimensional homonuclear methods remain the most sensitive and routine methods. They provide most, but usually not all, the information needed to determine the structure of a polysaccharide. One-dimensional selective methods require more experimental set-up but with the advent of more sophisticated software these can be conducted in a routine fashion. Concatenation of several polarization transfer step in 1D double-selective experiments is introduced as a valuable addition to the arsenal of 2D homonuclear techniques already established in this field. Two-dimensional heteronuclear experiments can now be performed on sub milligram quantities of compounds when using special probes. All the techniques discussed here use pulsed field gradients which significantly reduce artifacts and enhance the performance of all classes of NMR experiments.
CHAPTER 8: NMR Studies of Bacterial Sugar Ttransport System
Gerd Gemmecker, Horst Kessler and Bernhard Erni
Abstract
The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) links sugar uptake with regulatory processes. It consists of a cascade of several phosphoproteins transfering phosphoryl groups from phosphoenolpyruvate to the different sugars. Enzyme I and HPr are two general PTS proteins at the top of the cascade, while IIA and IIB are phosphorylated subunits of the different sugar-specific transport complexes.
The possibilities of protein NMR studies are exemplarily demonstrated on the IIB domain of the E.coli glucose transporter. With triple-resonance NMR techniques applied to stabile isotope labeled protein samples, a high resolution structure of IIBGlc was obtained, showing a unique new protein fold for this family of enzymes. With the help of an in situ phosphorylation system of the other PTS proteins, IIBGlc could be kept in the phosphorylated state for several days, allowing NMR measurements of the effect of protein phosphorylation upon protein structure. Complexation studies with differentially isotope-labeled IIAGlc and IIBGlc showed that both proteins maintain their tertiary structure upon mutual binding. From an analysis of the signal shifts induced by complexation, the binding sites of both proteins could be mapped, and a model for the complex between IIIAGlcand IIBGlc derived.
CHAPTER 9: Use of NMR in Protein-Water Exchange Studies
Marie-Claire Parker and Catherine Sarazin
Abstract
It is important to control enzyme hydration as the catalytic performance will depend on the water content of the organic medium in which it is suspended. Therefore solution-state NMR has been used to measure protein hydration and to determine water activity in situ. A very sensitive 2H NMR method has been developed for measuring deuterated water bound to proteins suspended in nonpolar solvents. This has been used to determine the amount of bound water as a function of water activity for subtilisin Carlsberg suspended in n-hexane, benzene and toluene. Comparison of the degree of enzyme hydration in the three solvents are very similar showing that water activity can be usefully employed to predict the amount of water bound to protiens in nonpolar organic media. The isotherms of proteins in nonpolar solvents increasingly diverge from the conventional isotherm measured through the vapour phase, at higher aw levels and is as a result of preferential hydration of the hydrophobic surface patches on the protein by the nonpolar solvent. A 1H NMR method has been developed to monitor on-line lipase catalysed esterification reactions without the need to sample the reaction medium. This method is used to determine simultaneously the water activity and the time course of reaction. As chemical shift signal of hydroxylic hydrogens in fast exchange varies with water activity and ester content, one relationship has been established between them. This has been used to evaluate water partitioning during esterification.
CHAPTER 10: In Situ Measurements of Enzyme Activities by NMR
Zheng-Yu Wang and Tsunenori Nozawa
Abstract
The capability of high-resolution solution NMR spectroscopy to monitor, in situ, the enzyme activities for two selected reactions, light-driven phosphorylation of chromatophores and CO2 fixation of Rubisco from photosynthetic bacteria, is demonstrated. Depending on the purpose of investigation, 1H-, 31P- and 13C spectra, or combinations thereof can be employed at different levels for measuring the progress of the reactions. It is possible to conduct 1H-NMR experiments in H2O by using field gradient to suppress the H2O signal. Measurement by 1H-NMR is quick and provides real-time information on distribution of both substrates and products. For reactions containing phosphorus compounds, 31P-NMR is most convenient and has the advantages of showing degree of the reaction at any time during the reaction and simultaneous detection of pH value in the reaction solution. 13C-NMR is particularly useful for identifying the incorporated carbon atoms and for assessment of metabolic reactions with use of specific 13C-labeling. Results obtained from the NMR methods and those from other analytical techniques are compared.
CHAPTER 11: Biological Membrane Structure by Solid-State NMR
Michèle Auger
Abstract
Nuclear magnetic resonance (NMR) spectroscopy, and particularly solid-state NMR spectroscopy, is a method of choice to study the structure and dynamics of both the lipid and the protein components of model and biological membranes. Different approaches have been developed to study these systems in which the restricted molecular motions result in broad NMR spectra. This contribution will first present an overview of the different techniques used to study lipid bilayers, namely 31P, 2H and 13C solid-state NMR spectroscopy. On the other hand, the study of the structure of membrane peptides and proteins is a rapidly growing field and several methods developed in the last two decades will be presented. These methods allow the investigation of protein systems for which structural information is often difficult to obtain by techniques such as X-ray diffraction and multidimensional solution NMR.
CHAPTER 12: Pathways with NMR: Metabolism of Amino Acids in Corynebacteria
Stéphane Guillouet, Philip A. Lessard and Anthony J. Sinskey
Abstract
Amino acids produced by Corynebacteria in fermentations provide the basis of a global food and animal feed industry. Commercial production of amino acids is critically dependent on the efficiency of the pathways bacteria use to synthesize amino acids. Direction of carbon flux through the different cellular metabolic pathways can have profound implications for cellular energy requirements, yield on the basis of the growth substrate and, ultimately, productivity. NMR and 13C labeling experiments have made it possible to monitor how bacteria control the distribution of carbon resources during growth and amino acid production, and how this distribution affects energy requirements and substrate utilization for both growth and amino acid productivity. Studies to date have focused on three problems: (i) flux distribution through the split pathway of lysine synthesis, (ii) carbon flux distribution between the pentose phosphate pathway and glycolysis, and (iii) central carbon metabolism. These insights have made it possible to design new strategies to improve amino acid production in corynebacteria.
CHAPTER 13: Propionic Acid Bacteria
Patrick Boyaval and Catherine Deborde
Abstract
Lactic acid bacteria metabolism has been widely investigated. By comparison, NMR spectroscopy applied to bacterial metabolism is only in its infancy. Only a few works have been realized either to explore uncommon pathways (erythritol anabolism), to examine the energetic states of the cell through 31P NMR or to discriminate between several proposed pathways (diacetyl synthesis, methionine catabolism). Compared with the lactic acid bacteria used as starter cultures in cheesemaking and winemaking, relatively little is known about propionibacteria. Nevertheless, some investigations have been done on metabolic behavior of propionibacteria by NMR. These metabolic studies of propionibacteria by NMR should be divided in two groups, not based on the differences between classical and clinical species, but on primary metabolism and secondary metabolism, i.e. propionic acid production and energy metabolism versus vitamin B12 production. In addition, some structural and stereochemical aspects of specific enzymes (transcarboxylase, polyphosphate dependent phosphofructokinase and superoxyde dismutase) are also introduced.
CHAPTER 14: NMR Investigations of Yeast Metabolism: Biochemistry and Biotechnology Applications
Hans J. Vogel and Elke M. Lohmeier-Vogel
Abstract
Metabolism in suspensions of yeast cells can be studied by noninvasive NMR methods. In particular phosphorus-31 NMR and carbon 13 NMR have been used to study glycolysis and tricarboxylic acid cycle activity, energy metabolism and pH control in Saccharomyces cerevisiae and a host of other yeast strains. In addition much has been learned about metabolites that are prevalent in yeast, such as polyphosphates which act as a vacuolar phosphate store, and trehalose, which protects cells (and its proteins) from stresses such as dessication and heat shock. Yeast is an important organism in biotechnology, and the fermentation of waste biomass hexose and pentose sugars to ethanol, has been studied in detail. NMR studies have also provided novel insights into the physiology of immobilized gel-entrapped cells. Finally NMR methods have been used to study protein motions and the possibility of metabolon formation in living cells. Overall, NMR studies of yeast have provided unique insight in the versatility of this simple eukaryote.
CHAPTER 15: NMR Investigations of Polymer Biosynthesis. The Case of a MultiproductiveBbacterium, Sinorhizobium meliloti
Isabelle Gosselin, Jean-Noël Barbotin and Jean-Charles Portais
Abstract
NMR is increasingly used for the elucidation of the biosynthesis of microbial polymers, both in vitro and in vivo. It offers a suitable tool for probing both the dynamics of polymer synthesis (or degradation) and the assessment of the central pathways involved. 13C-NMR has been the main employed approach for such a purpose. After a short - and non-exhaustive - survey of some NMR works dealing with the biosynthesis of microbial polymers, the present chapter states a more detailed illustration by taking example of Sinorhizobium meliloti, a bacterium that produces many polymers at the same time.
CHAPTER 16: Sugar Transport and Metabolism in Fermentative Bacteria
O.M.M. Bouvet and M-N. Rager
Abstract
NMR spectroscopy has become a particulary attractive tool for the study of metabolic diversity. The ability of 13C and 31P NMR spectroscopies to monitor complex metabolic pathways in four Gram-negative bacteria (Aeromonas hydrophila, Escherichia coli, Pasteurella multocida, Plesiomonas shigelloides) is demonstrated in this report. The central pathway for glucose catabolism was investigated in resting cell in vivo using 13C NMR spectroscopy and in cell-free extracts in vitro using 31P NMR spectroscopy. Metabolism via the Embden-Meyerhof-Parnas and pentose phosphate pathways were separately quantitated by using glucose substrates with 13C label at different carbon atoms.
31P NMR has become a very useful method for enzyme kinetics studies in vitro. Using a spectrophotometric method, a very weak activity of the 6-phosphofructokinase was detected in cell-free extracts of P. multocida. Evidence for the presence of this enzyme was obtained using 31P NMR. Furthemore the presence of a constitutive fructose-6-phosphate reductase, an unusual enzyme converting fructose-6-phosphate to mannitol-1-phosphate, and a mannitol-1-phosphatase which converted mannitol-1-phosphate to mannitol were also detected in cell-free extracts of P. multocida.
CHAPTER 17: NMR on Bacterial Biofilms and Model Systems
Christian Mayer
Abstract
The mechanical stability of biofilms and other microbial aggregates is of great importance for both the maintenance of biofilm processes and the removal of undesired biofilms. The potential binding forces are weak interactions such as London dispersion forces, electrostatic interactions and hydrogen bonds. In a first attempt to elucidate their contribution, extracellular polymeric substances (EPS) from a mucoid strain of Pseudomonas aeruginosa are studied by solid state NMR and rheological measurements. Polyacrylic acid is identified as a suitable model system for EPS when the influence of electrolytes on molecular interactions is observed. Effects on biofilm solutions and model systems are monitored by solid state NMR techniques, viscosimetry and dynamic light scattering. Based on the results, electrostatic interactions and hydrogen bonds are identified as the dominating forces among macromolecules within the biofilm.
CHAPTER 18: Comparative NMR Analysis of Stable Isotope Labeling Patterns. Biosynthesis of Gallic Acid
Wolfgang Eisenreich, Ingo Werner and Adelbert Bacher
Abstract
The biosynthesis of gallic acid was studied in cultures of the fungus Phycomyces blakesleeanus and in leaves of the tree Rhus typhina by labeling experiments using [1-13C]glucose and a mixture of [U-13C6]glucose and unlabeled glucose (1:25, w/w). A detailed quantitative NMR analysis of gallic acid and amino acids isolated from the cells showed that the bulk amount of gallic acid is formed directly via 5-dehydroshikimate and not via phenylalanine. The retrobiosynthetic approach used in this study is described in detail and compared with classical approaches of in vivo incorporation experiments.
CHAPTER 19: Microbial Degradation of Xenobiotics
Anne-Marie Delort and Bruno Combourieu
Abstract
Nuclear magnetic resonance (NMR) offers a powerful tool to study the degradation of xenobiotics by microorganisms. However, most studies have been carried out classically on large amounts of purified metabolites (mg). More recently, some microbial degradation pathways of drugs or pollutants were investigated by in vivo or in situ NMR, directly on whole cells or on culture media containing small amounts of metabolites (mg). Although the numerous advantages of 19F NMR studies are limited by the narrow range of xenobiotics bearing 19F, fluorouracil and fluorophenol derivatives can be studied. The use of 13C and 2H NMR has been greatly limited in xenobiotic degradation as 13C or 2H labeled xenobiotics are not commercially available. Consequently very few studies are reported; they include formamide, methylsulfate and acenaphthene metabolisms. To avoid the problems caused by the presence of water and intrinsic metabolite signals, the culture medium was completely deuterated to study benzoate derivative metabolism, or protons linked to the 13C-15N enriched pattern were specifically detected during glyphosate degradation. In situ 1H NMR was applied successfully to study the metabolism of heterocyclic compounds, such as morpholine. Also, 1H HPLC-NMR was used to study non-labeled warfarin metabolites.
CHAPTER 20: Application of Solid-State NMR to Investigate Organic Biogeochemistry in Soils
D.W. Hopkins, J.A. Chudek, S.F.I. Haslam and E.A. Webster
Abstract
Solid-state 13C nuclear magnetic resonance (using cross polarization, magic angle spinning NMR) has been widely used to characterize chemically C and investigate the processes of decomposition of organic materials in soils. Solid-state 13C NMR is used because semi-quantitative information is provided about a range of functional groups indicative of some of the main classes of biochemicals involved in decomposing organic materials. Characterization of soil C usually relies on the naturally abundant 13C, the low concentration of which together with the presence of Fe in soils, make spectroscopy difficult. Addition to and recovery from soil of C-rich or 13C-enriched materials allows NMR to be used to follow the decomposition processes.
CHAPTER 21: Metabolic Engineering: A Framework for the Integration of Genomic and Physiological Data
Maria I. Klapa and Gregory Stephanopoulos
Abstract
Metabolic engineering strives to systematically induce biological changes that will produce desired cellular properties. As such it favors the analysis of integrated metabolic networks and the use of fluxes to obtain a detailed picture of cellular physiology. The development of technologies for the measurement of genome-wide gene expression (DNA microarrays) and cell-wide protein production (2-dimensional gels) data will introduce a new dimension in biological and biotechnological research. For the first time, physiological data will be complemented to such large extent by information from both the genomic and proteomic level. The integration of such diverse information is required for the determination of gene regulation and cellular physiology. Metabolic engineering can play a key role towards this direction by providing the framework for the systematic and combined application of the available methodologies in the elucidation of biological systems in their entirety. In this chapter, we present the two main computational methodologies of the metabolic engineering toolbox in the post-genomic era. How flux quantification and gene expression analysis, along with sophisticated experimental techniques, can be combined to upgrade the content of information in the physiological and genomic/proteomic data towards the unraveling of cellular function and regulation, is discussed at the end of the chapter.
CHAPTER 22: Future Trends in Complex Microbial Reaction Studies
Uwe Sauer, Thomas Szyperski, and James E. Bailey
Abstract
The complexity of biological systems poses formidable scientific challenges to a comprehensive, integrated understanding of how components contribute to overall function. Recent advances in global mRNA and protein analyses provide access to certain aspects of this complexity, but there is currently no direct link between such compositional data and the dynamic metabolic and physiological aspects of cellular systems. NMR spectroscopy is expected to play a major role in global investigations of microbial metabolism, and we provide here a general overview of current and future NMR methods in this field.
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