Molecular Identification of the Urea Uptake System and Transcriptional Analysis of Urea Transporter- and Urease-Encoding Genes in Corynebacterium glutamicum

Abstract

The molecular identification of the Corynebacterium glutamicum urea uptake system is described. This ABC-type transporter is encoded by the urtABCDE operon, which is transcribed in response to nitrogen limitation. Expression of the urt genes is regulated by the global nitrogen regulator AmtR, and an amtR deletion strain showed constitutive expression of the urtABCDE genes. The AmtR repressor protein also controls transcription of the urease-encoding ureABCEFGD genes in C. glutamicum. The ure gene cluster forms an operon which is mainly transcribed in response to nitrogen starvation. To confirm the increased synthesis of urease subunits under nitrogen limitation, proteome analyses of cytoplasmic protein extracts from cells grown under nitrogen surplus and nitrogen limitation were carried out, and five of the seven urease subunits were identified.Urea is a readily available nitrogen source, since it is excreted by a variety of organisms into the environment. In bacteria able to metabolize this solute, urea is hydrolyzed by the cytoplasmic urease enzyme complex, leading to one CO 2 and two ammonium molecules (for review, see reference 20). Although urea is small and uncharged and can therefore easily pass the bacterial membrane, many prokaryotes, including Corynebacterium glutamicum, synthesize energy-dependent transport systems for its uptake. When present in high concentrations, sufficient urea crosses the C. glutamicum cytoplasmic membrane by passive diffusion, and only under conditions of nitrogen starvation is an energy-dependent urea uptake system synthesized (27). With a K m of 8 M for urea, the affinity of this uptake system is much higher than the affinity of urease for its substrate (K m of approximately 55 mM). The maximum urea uptake rate depends on the level of expression and is relatively low at 2.0 to 3.5 nmol mg (dry weight) Ϫ1 min Ϫ1 (27). Genes coding for the C. glutamicum urea uptake system have not been previously identified.The genes encoding the urease enzyme complex were isolated and sequenced (21, 24). While ureA, ureB, and ureC encode the urease structural subunits, the ureE, ureF, ureG, and ureD genes code for accessory proteins. As shown by mutant analyses, at least the ureC and the ureD products are essential for a functional urease (21). Depending on the growth conditions, urease activity observed in the wild type varies between 0.9 and 2.2 U mg of protein Ϫ1 for cells grown in ammonium-rich minimal medium (21, 24), between 1.0 and 1.6 U mg of protein Ϫ1 when glutamine is used as nitrogen source (24), and between 2.0 and 6.1 U mg of protein Ϫ1 when different concentrations of urea were added to the medium as sole nitrogen source (21, 24). These medium-dependent changes in urease activity indicate that the enzyme is moderately regulated, either on the level of activity or on the level of gene expression.Here, we describe the identification of genes encoding the C. glutamicum urea uptake system and their transcriptional organization and regulation, as well as the organization and control ...

Functional characterization ofActinobacillus pleuropneumoniaeurea transport protein, ApUT

“…Under conditions of nitrogen starvation, in Methylophilus methylotropus and Corynebacterium glutamicaum, an ATP-dependent ABC type transporter is responsible for urea uptake into the cell (2,24). Passive or facilitated movement of urea is seen in Helicobacter pylori where urea uptake is mediated by an acid-activated urea channel, UreI, which was shown to be essential for survival in vivo (33).…”

Proteomics analysis of the response of the marine bacterium Marinobacter adhaerens HP15 to the diatom Thalassiosira weissflogii

“…We further observed the down-regulation of the periplasmic substrate-binding compound UrtA of the urea uptake system (Beckers et al 2004). Urea serves as a nitrogen source for a wide range of bacteria (Mobley & Hausinger 1989).…”

“…Amino acids other than glutamate might serve as possible additional carbon and nitrogen sources when diatoms are present, suggesting that several of such amino acids might be secreted by the diatom cells. This finding is supported by the down-regulation of a urea uptake transporter element during co-cultivation with the diatom, a process usually associated with growth under nitrogen-rich conditions (Beckers et al 2004). Only 1 protein of a whole transporter unit, which comprises several proteins, was found to be altered (Beckers et al 2004).…”

“…This finding is supported by the down-regulation of a urea uptake transporter element during co-cultivation with the diatom, a process usually associated with growth under nitrogen-rich conditions (Beckers et al 2004). Only 1 protein of a whole transporter unit, which comprises several proteins, was found to be altered (Beckers et al 2004). This observation may be partly explained by our experimental approach that did not isolate and identify membrane-associated, hydrophobic proteins (e.g.…”

“…Urea serves as a nitrogen source for a wide range of bacteria (Mobley & Hausinger 1989). The operon encoding for the urea uptake system (made up of 5 genes, urt -ABCDE) is induced by nitrogen-limiting conditions in Corynebacterium glutamicum (Beckers et al 2004). Due to the down-regulation of 1 protein of the urea uptake system, the nitrogen supply seems to be more optimal in the treatment protocol as compared to the reference protocol in which the amino acid glutamate is the only nitrogen source provided.…”