Streptococcus mutans is a gram-positive bacterium found in the oral cavity, and is considered a leading cause of dental caries. S. mutans has a glnK gene which codes for a PII-like protein possibly involved in the integration of carbon, nitrogen and energy metabolisms. To characterize the S. mutans GlnK protein, the glnK gene was amplified by PCR using synthetic oligonucleotides, cloned into the expression vectors pET29b(+) and pET28b(+). The resulting plasmids were named pMEGP2 and pMEGP3, respectively. The pMEGP2 plasmid expresses the S. mutans GlnK protein in its native form (GlnK-Sm) and the pMEGP3 plasmid expresses S. mutans GlnK protein fused to an N-terminal histidine-tag (GlnK-His-Sm). The native GlnK-Sm was purified through anionic exchange (Q-Sepharose) and affinity (Hi-Trap Heparin) chromatography. The GlnK-His-Sm protein was purified using a Hi-Trap Chelating-Ni²⁺ column. Gel filtration experiments indicated that this protein is a homohexamer in solution. The GlnK-His-Sm is not uridylylated by the Escherichia coli GlnD protein. The activities of the GlnK-Sm and GlnK-His-Sm proteins were assayed in E. coli backgrounds expressing the Klebsiella pneumoniae nifLA operon constitutively. In K. pneumoniae, NifL inhibits NifA activity under high levels of ammonium but the GlnK protein is required to relieve the inhibition of NifL under low ammonium levels. The GlnK-Sm protein was unable to relieve NifL inhibition of NifA protein. Furthermore, in the presence of the GlnK-Sm protein NifA was constitutively inhibited by NifL, indicating that this protein antagonizes the relieving activity of the E. coli GlnB and GlnK proteins on the NifL-NifA complex, under nitrogen limiting conditions. Surprisingly, the GlnK-His-Sm protein was capable to partially relieve NifL inhibition of the NifA protein under nitrogen limitation conditions, in a manner similar to the E. coli GlnK protein.