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.

FIG. 1.

Urea consumption in C. glutamicum wild type (open squares) and urtA, urtC, and urtE mutant strains (open triangles, open circles, and inverted closed triangles, respectively) grown in minimal medium with urea as single nitrogen source. Under the experimental conditions used (urea concentration, 4 μM), diffusion of urea across the cytoplasmic membrane was negligible.

FIG. 2.

Transcription of the genes encoding the C. glutamicum urea uptake system. (A) RT-PCR using primers annealing to urtA and urtE. Total RNA isolated from wild-type strain ATCC 13032 after 30 min of nitrogen starvation (lane 1) and from amtR deletion strain MJ6-18 grown under nitrogen surplus (lane 3) was used as template. The RT-PCR products (indicated by the arrow) had an expected size of 4.0 kb. Control reactions without the addition of RT (lanes 2 and 4) gave no PCR product, validating that the RNA preparations used were DNA free (lane 5). Lane M, marker DNA (1-kb DNA ladder; New England Biolabs). (B) RNA hybridization experiments with an urtA probe and total RNA (1 μg per slot) prepared from the wild type and from the amtR deletion strain MJ6-18 grown in nitrogen-rich minimal medium and after 5, 15, and 30 min of nitrogen limitation (lanes 1 to 4). (C) Gel retardation experiment. Increasing amounts of AmtR-containing E. coli cell extracts (lanes 1 to 5: 0, 5, 10, 20, and 30 μg of protein, respectively) were added to a digoxigenin-labeled 122-bp DNA fragment from the urtA promoter region spanning the two identified putative AmtR binding sites. As a control, cell extract lacking AmtR was used (lane 6; 30 μg). DNA fragments with shifted mobilities depending on AmtR are indicated by arrows.

FIG. 3.

Transcription of the urease-encoding genes in C. glutamicum. (A) RT-PCR with primers annealing to ureA and ureD cluster. As template, total RNA isolated from amtR deletion strain MJ6-18 was used. The RT-PCR product (lane 1; indicated by an arrow) had an expected size of 5.1 kb. A PCR control without addition of RT (lane 2) showed no signal, indicating DNA-free RNA preparation; lane 3, marker DNA (1-kb DNA ladder; New England Biolabs). (B) RNA hybridization experiments with a ureA probe and total RNA (1 μg per slot) prepared from the wild type, from amtR deletion strain MJ6-18, and from the ureR gene disruption mutant Res167::pDRIVE-ureR under nitrogen surplus (lanes 1 and 5); after 5, 15, and 30 min of incubation with no nitrogen source starvation (lanes 2 to 4); and after 5, 15, and 30 min of incubation in the presence of 15 mM urea (lanes 6 to 8). (C) Gel retardation experiment using increasing amounts of AmtR-containing E. coli cell extracts (lanes 1 to 5: 0, 5, 10, 20, and 30 μg of protein, respectively) and a digoxigenin-labeled 216-bp DNA fragment from the ure promoter region spanning the identified putative AmtR binding site. As a control, cell extract lacking AmtR was used (lane 6; 30 μg). The DNA fragment that shifted in its electrophoretic mobility due to the presence of AmtR is indicated by an arrow.

FIG. 4.

Identification of urease subunits in the protein extract of nitrogen-starved cells. Cytoplasmic proteins (120 μg) of C. glutamicum ATCC 13032 grown in standard minimal medium and incubated for 3 h in nitrogen-free minimal medium were separated by two-dimensional gel electrophoresis and stained with colloidal Coomassie brilliant blue. Marked protein spots, which were not detectable in extracts from cells grown in nitrogen-rich medium (data not shown), were identified by MALDI-TOF MS and peptide mass fingerprint analysis. Experiments were carried out independently in duplicate; molecular mass markers and pH values of the gel section are indicated.