Effect of carbon source availability and growth phase on expression of Corynebacterium glutamicum genes involved in the tricarboxylic acid cycle and glyoxylate bypass

Abstract

The effect of different carbon sources on the expression of tricarboxylic acid (TCA) cycle genes, along with glyoxylate bypass genes, in Corynebacterium glutamicum was determined. All TCA cycle genes were coordinately expressed in medium containing acetate. Growth in the presence of acetate gave rise to abundant expression of most TCA cycle genes, with the level of gltA transcript being the highest. However, when the cells entered the stationary phase triggered by acetate exhaustion, all genes were repressed, except sucCD and mdhB, which were slightly induced. Acetate withdrawal from the growth medium during the exponential phase also led to rapid repression of most TCA cycle genes and a corresponding twofold increase in the expression of sucCD, which were strongly induced by citrate and succinate. In addition, glucose depletion during the stationary phase led to a corresponding 8-20-fold induction of the sucCD, aceA and aceB genes. Addition of glucose to acetate medium resulted in about 10-fold induction of sucCD. The strong dependence of TCA cycle sucCD and glyoxylate bypass aceA and aceB expression on carbon source availability was confirmed and the regulatory system will be studied precisely. INTRODUCTIONCorynebacterium glutamicum, a non-pathogenic, facultative anaerobic Gram-positive soil bacterium, is widely used in the industrial production of numerous metabolites, including amino acids and organic acids (Kinoshita et al., 1957;Liebl, 2005;Nishimura et al., 2007). In contrast to closely related, medically important pathogenic species, such as Corynebacterium diphtheriae and Mycobacterium tuberculosis, C. glutamicum is generally recognized as a nonhazardous organism (Dover et al., 2004;Funke et al., 1997). Furthermore, this organism has gained increasing interest as a suitable model organism for high-G+C-content Grampositive bacteria in general and for Corynebacterineae, a suborder of the Actinomycetes, in particular.One of the central metabolic pathways in C. glutamicum and in other aerobic bacteria is the tricarboxylic acid (TCA) cycle, which is responsible for the complete oxidation of acetyl-CoA derived from various substrates and for the provision of precursors for amino acid biosynthesis. TCA cycle intermediates are commonly used by other metabolic reactions in a wide variety of cell types. Due to the commercial importance of the amino acids and organic acids produced by C. glutamicum, the control of TCA cycle enzyme activities has been the subject of intensive studies, and the phenotypes of mutants affecting TCA cycle genes have been analysed (Eikmanns et al., 1994(Eikmanns et al., , 1995Molenaar et al., 1998Molenaar et al., , 2000Usuda et al., 1996;Wittmann & De Graaf, 2005). However, limited work has been devoted to the regulation of the expression of C. glutamicum TCA cycle genes in response to different growth phases and carbon sources. Recently we have reported the transcription of C. glutamicum genes involved in the TCA cycle and glyoxylate bypass (Han et al., 2008). In fact, aspects of the re...

Complex Regulation of the Phosphoenolpyruvate Carboxykinase Gene pck and Characterization of Its GntR-Type Regulator IolR as a Repressor of myo-Inositol Utilization Genes in Corynebacterium glutamicum

“…Additional studies showed that GlxR is a global regulator with dual functionality that is predicted to directly regulate about 14% of the annotated C. glutamicum genes (66)(67)(68)(69). Binding of GlxR to several of its target promoters was shown to be strictly dependent on the presence of cAMP in vitro (40,41,65,68,(70)(71)(72)(73). The intracellular concentration of cAMP in C. glutamicum seems to vary with the carbon source and the growth phase of the culture, as it is, for instance, high when cells are grown on glucose and low when cells are grown on acetate medium (65,74).…”

Genome-Wide Identification of In Vivo Binding Sites of GlxR, a Cyclic AMP Receptor Protein-Type Regulator in Corynebacterium glutamicum

“…However, whether GlxR acts as a transcriptional activator or repressor of most of these genes is difficult to evaluate, not only because construction of a glxR deletion mutant is difficult but because any mutant that has been successfully constructed shows severe growth defects (41,49,59,79). The role of GlxR has been assessed only by in vitro binding assays and overexpression studies in vivo (14,32,33,40,49), although GlxRdependent repression of genes involved in the glyoxylate pathway and glutamate uptake system was recently confirmed by experiments using a glxR mutant (59). Therefore, the physiological function of and environmental signal(s) sensed by GlxR remain poorly understood.…”

“…First characterized as a factor repressing the promoter activity of a gene coding for a glyoxylate pathway enzyme (41), GlxR is involved in the regulation of several other genes. Like E. coli CRP, it binds to the consensus site 5Ј-TGTGA-N 6 -TCACA-3Ј in a cAMP-dependent manner in vitro (32,33,40,41,49). In silico analyses have detected more than 200 potential binding sites for GlxR in the C. glutamicum genome, and binding to 72 of the sites has been verified by in vitro binding assays, revealing that the regulon includes genes for carbon metabolism, nitrogen metabolism, respiration, resuscitation, cell wall formation, and cell division (42,43).…”

Hyperglycemia impedes lung bacterial clearance in a murine model of cystic fibrosis-related diabetes

“…In the setting of high airway glucose concentrations, excessive glucose may serve as a steady source of both carbon and energy for proliferating infectious bacteria, creating an environment within the lung conducive to bacterial growth and endurance (11). Recent in vivo animal data have demonstrated that airway glucose control is essential to maintaining airway sterility (29).…”