The accumulation of glutamate is necessary for optimal growth of Salmonella typhimurium in media of high osmolality but not induction of the proU operon

Abstract

Synthesis of glutamate can be limited in bacterial strains carrying mutations to loss of function of glutamate synthase (2-oxoglutarate.glutamine aminotransferase) by using low concentrations of NH4' in the growth medium. By using such gtBID mutant strains of Sabnonella typhimnurum, we demonstrated that: (i) a large glutamate pool, previously observed to correlate with growth at high external osmolality, is actually required for optimal growth under these conditions; (ii) the osmoprotectant glycine betaine (NNN-trimethylglycine) apparently cannot substitute for glutamate; and (iii) accumulation of glutamate is not necessary for high levels of induction of the proU operon in vivo. Expression of the proU operon, which encodes a transport system for the osmoprotectants proline and glycine betaine, is induced >100-fold in the wild-type strain under conditions of high external osmolality. Intracellular osmolality of organisms is determined by a few species of solutes known as compatible solutes, whose concentrations vary in parallel with the osmolality of the environment (3). Glutamate, trehalose, K+, glycine betaine, and proline are some of the important compatible solutes in members of the family Enterobacteriaceae (reviewed in reference 7). In these organisms, glutamate and trehalose are synthesized internally, but the other three are taken up from the medium under conditions of high external osmolality. It is well documented that K+ is an essential nutrient in all environments (12,13), that trehalose is required for growth in media of high osmolality (15), and that exogenous proline and glycine betaine can alleviate the inhibitory effects of high osmotic stress (7). However, a requirement for high internal concentrations of glutamate to counter high external osmolality has not been demonstrated.In bacteria, glutamate is synthesized by two pathways: biosynthetic glutamate dehydrogenase (GDH) and glutamate synthase (2-oxoglutarate:glutamine aminotransferase [GOGATI) in conjunction with glutamine sythetase (Fig. 1) (49). Since GDH has a relatively poor affinity for NH4' (Kin, -1 to 5 mM), glutamate is synthesized primarily via the glutanine synthetase-GOGAT cycle in media containing low concentrations of NH4' (2,34,49). In gitB or gltD mutant strains, which lack a functional GOGAT, glutamate can be synthesized only by GDH, and therefore it is possible to limit glutamate synthesis in such strains by growing them in media containing suboptimal concentrations of NH4+. We exploited this property of glutamate synthase * Corresponding author. Phone: (317) 496-1496. mutants of Salnonella typhimnuium to study the role of glutamate in osmotic adaptation and found that high concentrations of this solute are necessary for optimal growth in media of high osmolality.In S. typhimurium and Escherichia coli, proline and glycine betaine are accumulated to high concentrations under conditions of high osmolality by two permeases: the ProP system, which is synthesized constitutively, and the ProU system, which is induced >100-f...

Identification of genetic and phenotypic differences associated with prevalent and non-prevalent Salmonella Enteritidis phage types: analysis of variation in amino acid transport

“…The proU operon (encoding a transport system for proline and glycine betaine) promoter is induced by low water potential in Escherichia coli and Salmonella (Wright & Beattie, 2004). Similarly, glutamate has also been shown to be necessary for S. Typhimurium to grow optimally in a highosmolarity environment (Csonka et al, 1994). We insertionally inactivated the amino acid transporter genes (SEN1417-1420) in a PT4 strain and confirmed the mutation by thorough PCR and CGH analysis.…”

Lack of Protective Osmolytes Limits Final Cell Density and Volumetric Productivity of Ethanologenic Escherichia coli KO11 during Xylose Fermentation

“…Osmolyte pools were estimated using a standard curve for each compound with acetone as the internal standard. Intracellular pools are reported in nanomoles per milliliter per optical density at 550 nm [OD 550 ] unit as described by Csonka et al (14).…”

“…Glutamate is the most abundant free amino acid in the cytoplasm of E. coli and related organisms during aerobic growth in mineral salts media (14,27,33). In addition to essential roles in nitrogen assimilation and biosynthesis, glutamate also serves as a protective intracellular osmolyte during adaptation to osmotic stress (13-15, 32, 50).…”

Roles of SpoT and FNR in NH4+ assimilation and osmoregulation in GOGAT (glutamate synthase)-deficient mutants of Escherichia coli

“…Csonka et al (13) 4 ϩ with L-aspartate or L-asparagine abolishes its osmosensitivity; and (iii) a multicopy plasmid which suppresses the gltBD defect in the mutant also confers osmotolerance. These data implicate a defect in N assimilation as the basis for the osmosensitive phenotype of GJ193 and provide additional evidence that increased L-glutamate synthesis and accumulation are important in E. coli osmoregulation (2,13,37).…”

“…Botsford et al (2) and McLaggan et al (37) have argued that the increase in L-glutamate pools in cultures grown at elevated osmolarity represents a very small, and possibly insignificant, load in comparison with the total biosynthetic flux through the amino acid. On the other hand, Csonka et al (13) have recently shown that, in GOGAT-defective mutants of S. typhimurium grown under ammonia-limiting conditions, there is an inverse relationship between growth rate and the osmolarity of the culture medium. The latter result provides indirect genetic evidence that increased L-glutamate synthesis is necessary for optimal growth under hyperosmotic stress.…”