Physiological role for the GlnK protein of enteric bacteria: relief of NifL inhibition under nitrogen-limiting conditions

Abstract

In Klebsiella pneumoniae, NifA-dependent transcription of nitrogen fixation (nif) genes is inhibited by a flavoprotein, NifL, in the presence of molecular oxygen and/or combined nitrogen. We recently demonstrated that the general nitrogen regulator NtrC is required to relieve NifL inhibition under nitrogen (N)-limiting conditions. We provide evidence that the sole basis for the NtrC requirement is its role as an activator of transcription for glnK, which encodes a PII-like allosteric effector. Relief of NifL inhibition is a unique physiological function for GlnK in that the structurally related GlnB protein of enteric bacteria-apparently a paralogue of GlnK-cannot substitute. Unexpectedly, although covalent modification of GlnK by uridylylation normally occurs under N-limiting conditions, several lines of evidence indicate that uridylylation is not required for relief of NifL inhibition. When GlnK was synthesized constitutively from non-NtrC-dependent promoters, it was able to relieve NifL inhibition in the absence of uridylyltransferase, the product of the glnD gene, and under N excess conditions. Moreover, an altered form of GlnK, GlnKY51N, which cannot be uridylylated due to the absence of the requisite tyrosine, was still able to relieve NifL inhibition.

FIG. 1

Amounts of NifL and NifA in wild-type (wt) and glnK mutant strains of E. coli. Amounts of proteins in crude cell extracts were determined by Western blotting with polyclonal antisera against NifL (lanes 1 to 4) or NifA (lanes 5 to 8). All strains carried plasmid pNH3 (Ptac-nifLA). Cultures were grown in K medium under derepressing conditions, and expression of NifL and NifA was induced with 10 μM IPTG (+) or was not induced (−). Lanes: 1, 2, 5, and 6, strain NCM1528 (wild type); 3, 4, 7, and 8, strain NCM1974 (glnK mutant).

FIG. 2

Uridylylation (A) and different levels of accumulation (B) of GlnK (Y) and GlnK^Y51N (N). (A) Cultures were grown in K medium under anaerobic conditions with (+) or without (−) NH₄Cl (see Materials and Methods). Proteins were separated by SDS-PAGE in the Tris-Tricine system (see Materials and Methods) and detected with anti-GlnB serum. Lanes: 1 to 4, glnD⁺ background (NCM1851); 5 to 8, glnD mutant background (NCM1687). The strains used for lanes 1, 3, 5, and 7 contained pJES1104 (Ptet-glnK in vector pACYC184); the strains used for lanes 2, 4, 6, and 8 contained plasmid pJES1106 (Ptet-glnK5; GlnK^Y51N in vector pACYC184). When cell extracts were treated with snake venom phosphodiesterase to remove uridylyl groups prior to electrophoresis, only the lower band was detected (data not shown). Treatment with alkaline phosphatase had no effect. (B) Cultures were grown in K medium under derepressing conditions (see Materials and Methods). Proteins were separated by SDS-PAGE in the 2× Tris-glycine system (see Materials and Methods) and detected with anti-GlnK serum. Lanes: 1, strain NCM1528 (wild type); 2, strain NCM1974 (glnK mutant); 3, strain NCM2069 (glnK/pJES1161 [PglnK-glnK⁺ in mini-F vector]); 4, strain NCM2087 (glnK/pJES1104 [Ptet-glnK⁺ in vector pACYC184]); 5, strain NCM2081 (glnK/pJES1162 [PglnK-glnK5; GlnK^Y51N in mini-F vector]); 6, strain NCM2088 (glnK/pJES1106 [Ptet-glnK5; GlnK^Y51N in vector pACYC184]). In this panel, bands were not resolved well enough for assessment of uridylylation.

FIG. 2

Uridylylation (A) and different levels of accumulation (B) of GlnK (Y) and GlnK^Y51N (N). (A) Cultures were grown in K medium under anaerobic conditions with (+) or without (−) NH₄Cl (see Materials and Methods). Proteins were separated by SDS-PAGE in the Tris-Tricine system (see Materials and Methods) and detected with anti-GlnB serum. Lanes: 1 to 4, glnD⁺ background (NCM1851); 5 to 8, glnD mutant background (NCM1687). The strains used for lanes 1, 3, 5, and 7 contained pJES1104 (Ptet-glnK in vector pACYC184); the strains used for lanes 2, 4, 6, and 8 contained plasmid pJES1106 (Ptet-glnK5; GlnK^Y51N in vector pACYC184). When cell extracts were treated with snake venom phosphodiesterase to remove uridylyl groups prior to electrophoresis, only the lower band was detected (data not shown). Treatment with alkaline phosphatase had no effect. (B) Cultures were grown in K medium under derepressing conditions (see Materials and Methods). Proteins were separated by SDS-PAGE in the 2× Tris-glycine system (see Materials and Methods) and detected with anti-GlnK serum. Lanes: 1, strain NCM1528 (wild type); 2, strain NCM1974 (glnK mutant); 3, strain NCM2069 (glnK/pJES1161 [PglnK-glnK⁺ in mini-F vector]); 4, strain NCM2087 (glnK/pJES1104 [Ptet-glnK⁺ in vector pACYC184]); 5, strain NCM2081 (glnK/pJES1162 [PglnK-glnK5; GlnK^Y51N in mini-F vector]); 6, strain NCM2088 (glnK/pJES1106 [Ptet-glnK5; GlnK^Y51N in vector pACYC184]). In this panel, bands were not resolved well enough for assessment of uridylylation.