Genetic screen for regulatory mutations in Methanococcus maripaludis and its use in identification of induction-deficient mutants of the euryarchaeal repressor NrpR

Abstract

NrpR is an euryarchaeal transcriptional repressor of nitrogen assimilation genes. Previous studies with Methanococcus maripaludis demonstrated that NrpR binds to palindromic operator sequences, blocking transcription initiation. The metabolite 2-oxoglutarate, an indicator of cellular nitrogen deficiency, induces transcription by lowering the affinity of NrpR for operator DNA. In this report we build on existing genetic tools for M. maripaludis to develop a screen for change-of-function mutations in a transcriptional regulator and demonstrate the use of an X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) screen for strict anaerobes. We use the approach to address the primary structural requirements for the response of NrpR to 2-oxoglutarate. nrpR genes from the mesophilic M. maripaludis and the hyperthermophilic Methanopyrus kandleri were targeted for mutagenesis. M. maripaludis nrpR encodes a protein with two homologous NrpR domains while the M. kandleri nrpR homolog encodes a single NrpR domain. Random point mutagenesis and alanine replacement mutagenesis identified two amino acid residues of M. kandleri NrpR involved in induction of gene expression under nitrogen-deficient conditions and thus in the response to 2-oxoglutarate. Mutagenesis of the corresponding regions in either domain of M. maripaludis NrpR resulted in a similar effect, demonstrating a conserved structure-function relationship between the two repressors. The results indicate that in M. maripaludis, both NrpR domains participate in the 2-oxoglutarate response. The approach used here has wide adaptability to other regulatory systems in methanogenic Archaea and other strict anaerobes.

FIG. 1.

Alignment of NrpR domains of M. kandleri (Mk), M. maripaludis (Mm), and M. acetivorans (Ma). D1 and D2 differentiate between individual NrpR domains of M. maripaludis and M. acetivorans. Numbers at ends of rows indicate the amino acid positions based on the full-length NrpR sequences. Amino acids shown to be involved in 2OG sensing are underlined, and their mutations are indicated in brackets. Black, grey, and light grey represent 100%, 80%, and 60% conservation, respectively.

FIG. 2.

Genetic arrangement for screening mutant nrpR alleles. M. maripaludis strain Mm1043 and the pWLG40-based replicative plasmid containing the nrpR gene are described in the text. Arrows represent genes or operons. P_nif represents wild-type nif promoter and P_nif* represents a mutated version to allow constitutive nif gene expression. The in-frame deletion of nrpR is indicated (×). The inverted triangle represents insertion of the P_nif-lacZ fusion into the chromosome at the upt site (12).

FIG. 3.

(A) M. maripaludis colonies expressing M. kandleri nrpR (strain Mm1058) or His-tagged M. maripaludis nrpR (strain Mm1048). Colonies were grown with ammonia or N₂ as a nitrogen source. Numbers below images are β-galactosidase activities (Miller units) from the same strains grown in liquid medium with the same nitrogen sources. (B) Screen for random mutants of M. kandleri NrpR. Lighter-colored mutant colonies are indicated by arrows.

FIG. 4.

nif regulation by wild-type M. kandleri NrpR and alanine replacement mutants. Vector control, strain Mm1171 (no NrpR); wild type, strain Mm1058 (M. kandleri NrpR). Amino acid changes are indicated on the x axis. Black bars, ammonia-grown cultures; white bars, dinitrogen-grown cultures.

FIG. 5.

(A) nif regulation by mutants of M. kandleri NrpR resulting from random and site-specific mutagenesis. Vector and wild-type controls are as described in the legend of Fig. 4. Mm1119, Mm1120, and Mm1124 are mutant strains isolated after random mutagenesis. C167A and S168A are M. kandleri NrpR site-specific mutants. Black bars, ammonia-grown cultures; white bars, dinitrogen-grown cultures. (B) nif regulation by wild-type M. maripaludis NrpR and site-specific mutants. Vector control, strain Mm1171 (no NrpR); wild type, strain Mm1048 (His-tagged M. maripaludis NrpR); black bars, ammonia-grown cultures; white bars, dinitrogen-grown cultures.

FIG. 6.

Electrophoretic mobility shift assay of binding of wild-type and mutant M. maripaludis NrpR in cell extracts to nif operator DNA. Vector control, Mm1171 (no NrpR); wild type, strain Mm1048 (His-tagged M. maripaludis NrpR).