Corynebacteria: Genomics and Molecular Biology
Caister Academic Press
Friedrich-Alexander Universitaet Erlangen-Nuernberg, 91058 Erlangen, Germany
viii + 340
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Corynebacteria are a diverse group Gram-positive bacteria found in a range of different ecological niches such as soil, vegetables, sewage, skin, and cheese smear. Some, such as Corynebacterium diphtheriae, are important pathogens while others, such as Corynebacterium glutamicum, are of immense industrial importance. In fact C. glutamicum is one of the biotechnologically most important bacterial species in use today with an annual production of more than two million tons of amino acids, mainly L-glutamate and L-lysine. Due to its industrial importance, C. glutamicum has been studied extensively over the years, and the publication of the C. glutamicum genome sequence in 2003 provided renewed impetus to these studies. To date, the complete genome sequences of four different species have been published, and sequencing of at least two more species is ongoing. These genomic data have enabled a dramatic improvement in our understanding of the corynebacterial genome architecture, metabolic processes, species-specific traits, industrial capabilities, and potential roles in pathogenicity of humans and animals. In addition these genome sequences, allied with newly developed genetic tools will consolidate C. glutamicum as a model organism for systems biology. Research in this area has never been more exciting.
This volume brings together the expertise and enthusiasm of today's leading experts to provide a description of the current state-of-the-art molecular biology toolbox available for Corynebacterium research, including global analyses techniques such as comparative genomics, transcriptome, proteome and metabolome analysis as well as the most recent knowledge on Corynebacterium promoter structures and vector systems. Furthermore, topics such as regulatory networks controlling carbon, nitrogen, phosphorus, sulphur and iron metabolism, cell wall structure, proteolysis and environmental stress response are covered. Essential reading for all Corynebacterium scientists and everyone with an interest in global analysis techniques, global regulation and systems biology.
"it is not surprising that this book has followed the Handbook of Corynebacterium glutamicum (2005, Eggling and Bott, eds). Obviously there is some overlap in these volumes; however, this one is updated and brings in information relevant to other Corynebacteria ... There is a chapter on plasmids and promoters and their applications that researchers will find very useful in a practical sense ... researchers in the field will find this a useful and up to date addition to their library." from Microbiology Today (2008)
"This solid book provides a generous amount of important information on the Corynebacterium species. It will serve as a primary resource for scientists researching and using these bacteria." from Doodys (2008)
Table of contents
1. The Discovery of Corynebacterium glutamicum and Birth of Amino Acid Fermentation Industry in Japan
Amino acids were once prepared by decomposition of proteins and considered as precious natural products of limited availability. Nowadays, amino acids such as L-glutamate and L-lysine are produced in scale of million of tons by fermentation. The prosperity of amino acid fermentation industry started about half a century ago with my discovery of a glutamic acid-producing bacterium. This essay summarizes the story of the isolation of Corynebacterium glutamicum, the first approaches, the successful screening procedure and the prerequisites that were crucial for the discovery of this amino acid-producing bacterium.
2. Genomics of Industrially and Medically Relevant Corynebacteria
The genus Corynebacterium is a diverse collection of Gram-positive microorganisms with a DNA of high G+C content. Many species of this genus were originally isolated from human clinical samples, whereas others were detected in animals and diverse environmental habitats. The apparent heterogeneity of the genus Corynebacterium poses challenges and opportunities when characterizing individual species at the genomic level. To date, the complete genome sequences of four species have been published, and sequencing of at least two more species is ongoing. These genomic data dramatically improved our understanding of the corynebacterial genome architecture, metabolic processes, species-specific traits, industrial capabilities, and potential roles in pathogenicity of humans and animals. This article describes the current status of Corynebacterium genome projects and summarizes the major findings of functional and comparative genomics studies.
3. DNA Microarray-based Transcriptome Analysis in C. glutamicum
Volker F. Wendisch
Since the availability of DNA microarray technology for Corynebacterium glutamicum and completion of the genome sequence, transcriptome analyses have immensely contributed to further our understanding of this bacterium, a non-pathogenic actinomycete of biotechnological importance. This chapter describes the use of DNA microarray-based transcriptome analyses to elucidate global gene expression changes in response to environmental changes (stimulons), to determine targets of transcriptional regulators (regulons), to map undirected mutations and to improve amino acid production by C. glutamicum.
4. Proteomics of Corynebacterium glutamicum and Other Corynebacteria
Jörn Kalinowski, Dirk Wolters and Ansgar Poetsch
Corynebacteria were subject to several proteomics studies in the past, either aiming to describe comprehensive cytosolic, extracellular and membrane proteomes or comparing proteomes of different species, wildtype and mutant strains, respectively. In these surveys, a variety of techniques were applied, one- and two-dimensional gel electrophoresis as well as liquid chromatography (LC) separations. Identification of proteins was done mostly by protease digestion and mass-spectrometry, employing MALDI or ESI mass spectrometers and subsequent comparison of acquired mass spectra with databases. In the next years, automated separation and online identification by LC-MALDI or LC-ESI will progressively replace the gel-based methods, ultimately leading to real quantitative data. While mostly hydrophilic proteins can be analyzed comprehensively with routine techniques, separation and identification of membrane proteins is more complex and was investigated in detail in a number of recent studies. This chapter will first review the initial work on hydrophilic proteins and then focus on membrane proteomics as the novel and fast proceeding field of research.
5. Metabolic Network Analysis and Design in Corynebacterium glutamicum
Christoph Wittmann and Elmar Heinzle
Methods of metabolic flux analysis and design of metabolic networks for the overproduction of metabolites are reviewed and discussed with respect to their importance for Corynebacterium glutamicum. These methods are relevant for an in-depth understanding of the in vivo function of metabolism and its directed engineering. The methods discussed here are based on solid material balances and well established genomic and biochemical knowledge about metabolic reactions. Monitoring of metabolic fluxes yields very reliable and quantitative genome wide data reflecting the genomic background and environmental influences on cells studied. Particularly flux split ratios between glycolysis and pentose phosphate pathway and activities around the pyruvate node are elucidated that are most relevant for the supply of precursors and NADPH. The paper outlines also methods for design of metabolic pathways optimal for the overproduction of metabolites.
6. Plasmids and Promoters in Corynebacteria and Their Applications
Jan Nešvera and Miroslav Pátek
The discovery of small plasmids in corynebacteria initiated genetic analysis and molecular breeding of industrially important Corynebacterium strains. The constructed plasmid vectors for corynebacteria include cloning vectors, expression vectors and promoter-probe vectors. They were particularly used for the isolation of various genes and their overexpression and inactivation, which allowed their metabolic role to be deduced. Using promoter-probe vectors, many promoters of corynebacteria were localized and characterized. A systematic promoter sequence analysis and mutational studies resulted in the definition of promoter consensus sequences. In addition to the promoters of housekeeping genes, promoters recognized by alternative sigma factors of RNA polymerase have recently been characterized. Plasmid vectors for corynebacteria are important tools, not only for the detailed analysis of gene functions and their regulation, but also for the improvement of industrial strains. The gene manipulations within the chromosome using integrative plasmid vectors and graded expression from altered promoters, both of which result in stable recombinant strains, are considered a promising approach to strain breeding.
7. Regulation of Carbon Metabolism in Corynebacterium glutamicum
Annette Arndt and Bernhard J. Eikmanns
The amino acid producing Corynebacterium glutamicum grows aerobically on a variety of carbohydrates, organic acids and alcohols as single or combined sources of carbon and energy. Among the substrates metabolized are glucose, L-lactate, acetate and ethanol which all four can also serve as substrates for amino acid production. Based on biochemical and genetic studies, on quantitative determination of metabolic fluxes and/or on genome-based methods such as DNA microarray analyses, this chapter mainly summarizes the present knowledge on the different steps and the regulation of the fundamental pathways in C. glutamicum during growth on glucose, L-lactate, acetate and/or ethanol. It becomes evident that carbon metabolism in this organism is subject to complex regulation at the transcriptional and at the post-transcriptional level. So far, eight transcriptional regulators, i.e., SugR, GntR1, GntR2, AcnR, LldR, RamA, RamB and GlxR and the serine/threonine (Ser/Thr) protein kinase G, phospho-Ser/Thr protein phosphatase and their target protein OdhI have been identified to be involved in the regulation of key enzymes in carbon metabolism, i.e., in the regulation of substrate uptake and activation, glycolysis, tricarboxylic acid (TCA) cycle, glyoxylate cycle and/or gluconeogenesis. Although the molecular mechanisms of transcriptional and post-transcriptional regulation of the enzymes involved in carbon metabolism is not yet completely understood, the recent findings led to a much better understanding of the adaptation of C. glutamicum to a given carbon source at the molecular level.
8. Molecular Mechanisms of Nitrogen Control in Corynebacteria
Eva Hänßler and Andreas Burkovski
Nitrogen is one of the macro-elements necessary for cell maintenance and growth. As part of amino acids, nucleotides, and amino sugars nitrogen is an essential component of almost all macromolecules in a bacterial cell, e.g. proteins, nucleic acids, and the murein sacculus determining its shape. Besides for the standard model bacteria for Gram-negatives, Escherichia coli, and Gram-positives, Bacillus subtilis, nitrogen metabolism and nitrogen regulation is best understood in the mycolic acids-containing actinomycete Corynebacterium glutamicum. In this review, uptake and assimilation of nitrogen sources in C. glutamicum are shortly summarized, before the current knowledge on regulatory mechanisms including transcriptional control and post-transcriptional regulation is described. Furthermore, the available data on nitrogen metabolism and regulation in closely related actinomycetes are summarized.
9. Phosphorus Metabolism and its Regulation
Volker F. Wendisch and Michael Bott
The phosphorus metabolism of Corynebacterium glutamicum and its regulation have been studied to some detail using a combination of genetic, biochemical and transcriptome analyses. C. glutamicum utilizes a number of inorganic and organic phosphates as sources of phosphorus. When orthophosphate (Pi), its preferred phosphorus source, is abundant, C. glutamicum accumulates polyphosphate. When Pi becomes scarce, a complex genetic response is elicited enabling C. glutamicum to scavenge low Pi concentrations, to take up transportable organophosphates and to access Pi from nontransportable Pi esters. The two-component signal transduction system PhoS-PhoR serves a role in the transcriptional regulation of the Pi starvation-inducible genes.
10. Sulfur Metabolism in Corynebacterium glutamicum
Christian Rückert and Jörn Kalinowski
Sulfur is an important element for life as it is a constituent of a number of essential organic molecules like cysteine, methionine, Coenzyme A, or iron sulfur clusters. In turn, these compounds are involved in a number of essential cellular processes like protein biosynthesis or the transfer of electrons and acyl groups. Yet, an external supply of these compounds is essential for many eucaryotes and, due to their scarcity in many foods and feeds, their biosynthesis is of great industrial interest. Therefore, the metabolism of sulfur in Corynebacterium glutamicum has been studied in more and more detail in recent years. Besides the pathways to obtain and utilize sulfur from the environment especially the reactions leading to and from the sulfur-containing amino acids cysteine and methionine have been analyzed in great detail, revealing a number of so far unique metabolic routes. In addition, the regulation of sulfur metabolism has been analyzed on the transcriptional as well as on the enzymatic level, revealing the presence of at least three transcriptional regulators and a high number of feed-back inhibitions of key enzymes. In this chapter, the current state of knowledge concerning the central pathways of sulfur metabolism (i.e. those consuming most of the cellular sulfur) is summarized and a short outlook on the perspectives to produce methionine and cysteine is given.
11. Regulation of Iron Homeostasis in Corynebacterium glutamicum
Julia Frunzke und Michael Bott
The relevance of iron to corynebacteria was first noticed in the 1930s, when the synthesis of diphtheria toxin by Corynebacterium diphtheriae was shown to be dependent on the iron supply. Meanwhile, the DtxR protein responsible for this regulation has been intensively characterized biochemically. It functions as an intracellular Fe2+ sensor and, when its low-affinity iron-binding site is occupied, as a transcriptional regulator. In C. diphtheriae, several DtxR targets have been identified and thoroughly characterized, but no genome-wide studies were performed yet. In the non-pathogenic Corynebacterium glutamicum, on the other hand, the DtxR regulon has been determined by genome-wide studies including transcriptome comparisons of wild type and dtxR deletion mutants using DNA microarrays. By this approach, more than 50 genes were found to be repressed by DtxR under iron excess, most of which encode proteins involved in iron acquisition, notably several ABC transporters for siderophores. Moreover, genes which are presumably activated by DtxR have been identified, such as iron storage proteins and proteins responsible for iron-sulfur cluster assembly. Three of the target genes repressed by DtxR in C. glutamicum (ripA, cgtR11, cg0527) encode themselves transcriptional regulators. The AraC-type regulator RipA has been shown to repress a set of genes which encode prominent iron proteins of the cell, such as aconitase, succinate dehydrogenase or catalase. In this way, the iron demand of the cell is reduced, possibly allowing prolonged survival under iron limitation. In the case of the response regulator CgtR11, evidence for its involvement in heme utilization has been obtained, whereas the function of Cg0527 is not yet known. In summary, key players of a complex regulatory network controlling iron homeostasis in C. glutamicum have been elucidated in recent years.
12. Structure and Synthesis of the Cell Wall
Lothar Eggeling, Gurdyal S. Besra and Luke Alderwick
Apart from a general interest in understanding the living world, there are at least two other reasons for investigating the cell wall of Corynebacterium glutamicum. First of all, there is the influence of the cell-wall structure and its regulation on the amino acid efflux and, secondly, this knowledge would facilitate the understanding of the complex cell-wall structure of Mycobacterium tuberculosis. We summarize here what is known on the cell wall synthesis of C. glutamicum and describe how studies with this organism improved the understanding on the structure and synthesis of the polysaccharides arabinogalactan, lipomannan, and lipoarabinomannan in Corynebacterianeae encluding M. tuberculosis. While general reviews on cell wall synthesis and cell wall structure of Corynebacterium are available (Eggeling et al., 2001; Dover et al., 2004; Daffe, 2005), the present review focusses on the latest discoveries encluding the key functions of glycosyltransferases in building the cell wall of C. glutamicum.
13. General and Regulatory Proteolysis in Corynebacteria
Johannes Amon, Alja Lüdke, Fritz Titgemeyer, and Andreas Burkovski
Proteases comprise a broad variety of functions in cells. They are involved in the degradation of polypeptides to supply amino acids for the synthesis of new proteins or for catabolism as carbon and energy source. Others remove misfolded or denatured proteins or are involved in the maturation of preproteins, the removal of signal peptides, and in the regulatory proteolysis of signal transduction proteins or transcriptional regulators. Furthermore, in pathogens, proteases are also important virulence factors. Reflecting this multiplicity of functions, in bacteria typically a broad set of proteases can be found. The composition of this set of proteolytic enzymes is specific for a single bacterium and depends on the ecological niche inhabited. This review focuses on the role of proteases in corynebacteria. The proteolytic equipment of the sequenced Corynebacterium species is compared to the number of proteases in other important groups of Gram-positive bacteria, namely bacilli, streptomycetes, and mycobacteria. Recent data on the Corynebacterium glutamicum Clp and FtsH protease complexes are summarized and the putative role of secreted and surface-anchored proteases in pathogenic corynebacteria, especially in Corynebacterium diphtheriae, is discussed.
14. Environmental Stress Response of Corynebacterium glutamicum
Susanne Morbach and Reinhard Krämer
Bacteria, like Corynebacterium glutamicum, are confronted with numerous stress situations. These can be caused by two principally different conditions, namely (i) by limitations in the supply of various nutrients, or (ii) by influences from the environment such as variations in temperature, osmolality, pH, and oxygen content, as well as increased heavy metal concentrations. In this chapter we will not describe adaptation processes related to nutrient limitation because this type of stress will be described elsewhere in this issue. Instead we will focus on external stress factors. So far only data are available dealing with the response of C. glutamicum to influences of osmotic, heat and chill stress. These changes are often accompanied by the formation of reactive oxygen species, thus leading also to oxidative stress. In order to adapt to these unfavourable conditions, C. glutamicum has developed several adaptation strategies, which are acting at different levels of the regulatory network as for example by the transcriptional response of a whole sigma factor-dependent regulon or directly at the level of activity of an enzyme or a carrier. In this review we will summarize the knowledge about adaptation strategies of C. glutamicum to heat, oxidative, osmotic, and chill stress.
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(EAN: 9781904455301 9781913652098 Subjects: [bacteriology] [microbiology] [medical microbiology] [molecular microbiology] [genomics] [environmental microbiology] )