Genetic Screen for Regulatory Mutations in Methanococcus maripaludis and Its Use in Identification of Induction-Deficient Mutants of the Euryarchaeal Repressor NrpR

Reporter gene-based genetic screens for regulatory mutations have enjoyed wide utility in the Bacteria but have had limited use in the Archaea, due in part to the extreme growth conditions typically required by these organisms. However, effective tools for genetic manipulation are now available in several species of Archaea (21, 23), and the time is ripe for the development of genetic screens for mutations that affect the regulation of gene expression. Methanococcus maripaludis, like all methanogenic archaea, requires strict anaerobic conditions for growth. However, genetic screens involving the use of β-galactosidase and its artificial substrate 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) require oxidized conditions for color development (8). Here, we describe for M. maripaludis a procedure involving appropriate genetic manipulations and an X-Gal screen and its use in the identification of induction-deficient mutant alleles of the nitrogen repressor NrpR. The approach has wide adaptability to other regulatory systems in methanogenic Archaea and other strict anaerobes. Most organisms regulate their nitrogen assimilation functions. Among the Bacteria and Archaea, a variety of nitrogen sensors and regulators are known, and most respond to cellular 2-oxoglutarate (2OG) as an indicator of nitrogen deficiency (11, 20). In the Bacteria and Euryarchaeota the most widespread nitrogen sensors belong to the PII superfamily of 2OG sensor proteins, which consists of the GlnB-GlnK family and the NifI family of nitrogenase regulators. In Proteobacteria, GlnB and GlnK proteins sense 2OG and also indirectly sense glutamine as an indicator of nitrogen sufficiency (5, 19). However, it is 2OG sensing that appears to be nearly universal in nitrogen regulation among the Bacteria and many Euryarchaeota (11). In addition to the PII proteins, many 2OG-responsive nitrogen regulators and enzymes are known. These include the transcriptional activators NifA of Gammaproteobacteria (17) and NtcA of unicellular cyanobacteria (7), the glutamine synthetase modifying adenylyltransferase of Gammaproteobacteria (9), and glutamine synthetase itself in the archaeal genus Methanosarcina (4). A recently characterized 2OG-responsive regulator that operates at the transcriptional level in a wide range of Euryarchaeota is the repressor NrpR (13). Detailed studies carried out in M. maripaludis have shown that NrpR binds as a dimer to palindromic operator sequences in promoter regions (15). 2OG acts as an inducer, directly interacting with NrpR and lowering its affinity for operators, resulting in derepression (induction) under nitrogen-deficient conditions (15). In M. maripaludis the regulation by NrpR of glnA encoding glutamine synthetase, the nif operon containing genes for nitrogen fixation (13), and a glnK-amtB operon (E. L. H. Hendrickson et al., unpublished data) containing genes for a PII protein and an ammonia transporter, has been studied. The presence of consensus NrpR binding sequences suggests that NrpR regulates transcription of other genes related to nitrogen assimilation as well (10). NrpR proteins, defined as containing conserved NrpR domains, are present in all methanogens with known genome sequences as well as Archaeoglobus and a few bacterial species (12). Organisms containing NrpR proteins fall into three groups based on how many NrpR domains they contain and the configuration of the NrpR domains (12). M. maripaludis represents one group, containing two tandem NrpR domains within a single polypeptide with an N-terminal helix-turn-helix (HTH) motif. Methanosarcina species represent another group, where two NrpR domains are contained in two separately encoded polypeptides, one with an N-terminal HTH motif and the other without. Methanopyrus kandleri represents the third group, containing a single NrpR domain that is joined to an N-terminal HTH motif. Furthermore, the NrpR domains fall into five phylogenetic groups that correspond to their context: the two domains of the double-domain proteins make up two groups, the two separately encoded domains of Methanosarcina represent two more groups, and the single domain of M. kandleri represents the fifth group. NrpR domains from each of these five groups are aligned in Fig. ​Fig.1.1. Despite this diversity of the NrpR family, each NrpR protein functions in nitrogen regulation, as evidenced by genetic complementation of an M. maripaludis nrpR null mutation (12). Hence, NrpR and its conserved mechanism of action appear to be widespread. 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 ... Although 2OG modulates a variety of nitrogen sensors and regulators, the structural basis for this response is not understood (11). Crystal structures of GlnB and GlnK-like PII proteins are known (see references 3, 6, 16, and 24), including recently a structure with bound 2OG (25). The tertiary structure of NrpR is unknown. Nevertheless, NrpR offers the opportunity to initiate the identification of structural elements involved in responding to 2OG. Specifically, mutant alleles of NrpR that result in repression of gene expression even under inducing conditions should be deficient in the response to 2OG. To this end, we initiated a genetic screen for such mutations in NrpR in M. maripaludis. We report here the identification of four amino acid residues in M. maripaludis NrpR and two in M. kandleri NrpR that are required for the full response to 2OG. The results identify two homologous positions in the conserved NrpR domain that participate in the induction of gene expression by 2OG.