Nitrogen control in Mycobacterium smegmatis: nitrogen-dependent expression of ammonium transport and assimilation proteins depends on the OmpR-type regulator GlnR

Abstract

The effect of nitrogen regulation on the level of transcriptional control has been investigated in a variety of bacteria, such as Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, and Streptomyces coelicolor; however, until now there have been no data for mycobacteria. In this study, we found that the OmpR-type regulator protein GlnR controls nitrogen-dependent transcription regulation in Mycobacterium smegmatis. Based on RNA hybridization experiments with a wild-type strain and a corresponding mutant strain, real-time reverse transcription-PCR analyses, and DNA binding studies using cell extract and purified protein, the glnA (msmeg_4290) gene, which codes for glutamine synthetase, and the amtB (msmeg_2425) and amt1 (msmeg_6259) genes, which encode ammonium permeases, are controlled by GlnR. Furthermore, since glnK (msmeg_2426), encoding a PII-type signal transduction protein, and glnD (msmeg_2427), coding for a putative uridylyltransferase, are in an operon together with amtB, these genes are part of the GlnR regulon as well. The GlnR protein binds specifically to the corresponding promoter sequences and functions as an activator of transcription when cells are subjected to nitrogen starvation.

FIG. 1.

Alignment of GlnR proteins from various actinomycetes. Abbreviations used for GlnR homologs with annotation numbers: MSMEG, Mycobacterium smegmatis; MAP, Mycobacterium avium subsp. paratuberculosis; Mb, Mycobacterium bovis; Rv, Mycobacterium tuberculosis H37rv; SCO, Streptomyces coelicolor; SAV, Streptomyces avermitilis; nfa, Nocardia farcinica; RHA1, Rhodococcus sp. strain RHA1; FRAAL, Frankia alni ACN14a; Francci3, Frankia sp. strain Cci3; Tfu, Thermobifida fusca; BL, Bifidobacterium longum; AAur, Arthrobacter aurescens; Lxx, Leifsonia xylii; ANA, Actinomyces naeslundii; AAN77733, Amycolatopsis mediterranei. Identical amino acid residues are indicated by a black background, and similar amino acids are indicated by a gray background. M. smegmatis GlnR contains a putative phosphorylation pocket, located at amino acid residues 43 to 53, and a C-terminal DNA binding domain (helix turn helix), located at amino acid residues 144 to 218 (indicated by a bar above the sequence). *, identical residues; :, conserved substitutions/residues; ., semiconserved substitutions.

FIG. 2.

GlnR binding motif in mycobacteria. Sequence logos of putative GlnR cis elements were identified upstream of the M. smegmatis (msmeg), M. tuberculosis (H37rv), M. bovis (bovis), and M. avium (avium) glnA, amtB, and amt1 genes (amt1 is present only in the M. smegmatis genome). The standard code of the Weblogo server is shown in gray scale at the top.

FIG. 3.

DNA affinity purification of GlnR. Magnetic beads coated with glnA, amtB, and amt1 promoter DNA were incubated with M. smegmatis cytoplasmic proteins. After washing steps, proteins bound to the DNA fragments were eluted using buffers containing different NaCl concentrations. Eluted proteins were separated by SDS-PAGE and subjected to tryptic in-gel digestion, MALDI-TOF MS, and peptide mass fingerprint analyses. The numbers indicate proteins identified by this approach, as shown in Table 1.

FIG. 4.

Transcriptional responses of the M. smegmatis wild-type strain and glnR deletion strain MH1 to nitrogen deprivation: hybridization of RNA isolated from M. smegmatis cells before and after induction of nitrogen starvation by incubation in nitrogen-free medium. (A) Transcription of the glutamine synthetase-encoding glnA gene, amtB and amt1 coding for ammonium uptake systems, and glnR in the wild type. The time points indicated (t₀, t₃₀, t₉₀, and t₁₅₀) are sampling times (in min) after resuspension of centrifuged and washed cells. One microgram of total RNA per spot was applied. (B) Transcription of genes in M. smegmatis glnR deletion strain MH1.

FIG. 5.

Real-time RT-PCR of the M. smegmatis wild-type strain. Cells were grown until the exponential growth phase was reached (OD₆₀₀, approximately 0.6 to 0.8). To induce nitrogen starvation, cells were harvested, washed, and resuspended in prewarmed Middlebrook 7H9 medium without a nitrogen source (−N); prewarmed standard Middlebrook 7H9 medium was used as a control (+N). RNA was isolated at the indicated time points, and transcription of amtB (open bars), amt1 (gray bars), and glnA (black bars) was monitored by real-time RT-PCR. For each gene the control value at zero time was defined as 1.

FIG. 6.

Induction of the nitrogen starvation response by MSX: hybridization of RNA isolated from M. smegmatis cells before (t₀) and after addition of MSX. t₁₅, 15 min after addition of MSX; t₃₀, 30 min after addition of MSX; t₆₀, 60 min after addition of MSX. (A) Transcription of the glutamine synthetase-encoding glnA gene, as well as amtB and amt1 coding for ammonium uptake systems. (B) Transcription of M. smegmatis glnR.

FIG. 7.

Complementation of M. smegmatis glnR deletion strain MH1: hybridization of RNA isolated from M. smegmatis cells carrying control plasmid pMN016 or glnR delivery plasmid pMN016-glnR before (t₀) and after induction of nitrogen starvation by addition of MSX. t₁₅, 15 min after addition of MSX; t₃₀, 30 min after addition of MSX; t₆₀, 60 min after addition of MSX. (A) Transcription control for M. smegmatis glnR. (B) Transcription of glnA, amtB, and amt1.

FIG. 8.

Gel retardation assay. amtB (220 bp) (A), glnA (219 bp) (B), and glnR (249 bp) (C) apparent promoter fragments were incubated with different amounts of purified GlnR protein. Lanes 0, 1, 2, 3, 4, and 5 contained 0, 1, 2, 3, 4, and 5 μl of a 0.3-μg μl⁻¹ solution, respectively.