Markerless Mutagenesis inMethanococcus maripaludisDemonstrates Roles for Alanine Dehydrogenase, Alanine Racemase, and Alanine Permease

Abstract

Among the archaea, Methanococcus maripaludis has the unusual ability to use L-or D-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method for markerless mutagenesis that builds on previously established genetic tools for M. maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of the M. maripaludis hpt gene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition, we developed a negative selection method to stably incorporate constructs into the genome at the site of the upt gene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation in the gene for alanine racemase lost only the ability to grow on D-alanine. The wild-type gene for alanine dehydrogenase, incorporated into the upt site, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer, the dehydrogenase is specific for the L isomer, and the racemase converts the D isomer to the L isomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent G؉C gram-positive bacteria.

Essential anaplerotic role for the energy-converting hydrogenase Eha in hydrogenotrophic methanogenesis

“…In each case, the resulting plasmid contained an in-frame deletion consisting of a start codon and a stop codon with intervening codons contained within an AscI site. Plasmid DNA was used to make deletions using a markerless mutagenesis method with neomycin for positive selection and 6-azauracil for negative selection as described (7,20). When formate was present as electron donor, neomycin was increased to 5 mg/mL on liquid or solid medium (7).…”

Protein complexing in a methanogen suggests electron bifurcation and electron delivery from formate to heterodisulfide reductase

“…This vector was transformed into M. maripaludis S2 as described in ref. 4 and selected in McCas medium (4) containing 1 mg/mL neomycin followed by selection for a mutant containing the in-frame deletion of upt on medium S2 with an in frame deletion of the uracil phosphoribosyltransferase gene (Mmp0680) MM1262 MM901 with an in frame deletion of fdhA2B2 (Mmp0138 and Mmp0139) MM1263…”

“…Cultures were grown until steady state was reached and OD 660 remained stable at ∼0.6 for >48 h. Cultures were then collected as described below. For growth with formate as the sole electron donor, cultures were grown in 400-mL batch culture at 37°C with agitation at 100 rpm (Jeio Tech SK-600 shaker) with described medium (2) with 20 mM NH 4 Cl in place of casamino acids (formate medium). Cultures were grown for ∼24 h to a final OD 660 of ∼0.4 to 0.5 and collected as described below.…”

“…To this end, we performed experiments with the hydrogenotrophic methanogen, Methanococcus maripaludis. M. maripaludis is ideal for such an undertaking because of its rapid growth under laboratory conditions, a well-developed set of genetic tools (3,4), the ability to grow in continuous culture under conditions of defined nutrient limitation (5), and the availability of an exhaustive dataset from quantitative measurements of the proteome (6,7).…”

Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase

“…A M. maripaludis strain (Mm1036) with an in-frame deletion in nifH (⌬4-268) was constructed from strain Mm900 for expression of the His-tagged NifH. Regions flanking nifH were PCR amplified and cloned into ApaI-XbaI digested pCRPrtNeo, and marker replacement was performed as described (36). All constructs were confirmed by sequencing.…”