Genetic and Phenotypic Comparison of Facultative Methylotrophy between Methylobacterium extorquens Strains PA1 and AM1

Abstract

Methylobacterium extorquens AM1, a strain serendipitously isolated half a century ago, has become the best-characterized model system for the study of aerobic methylotrophy (the ability to grow on reduced single-carbon compounds). However, with 5 replicons and 174 insertion sequence (IS) elements in the genome as well as a long history of domestication in the laboratory, genetic and genomic analysis of M. extorquens AM1 face several challenges. On the contrary, a recently isolated strain - M. extorquens PA1- is closely related to M. extorquens AM1 (100% 16S rRNA identity) and contains a streamlined genome with a single replicon and only 20 IS elements. With the exception of the methylamine dehydrogenase encoding gene cluster (mau), genes known to be involved in methylotrophy are well conserved between M. extorquens AM1 and M. extorquens PA1. In this paper we report four primary findings regarding methylotrophy in PA1. First, with a few notable exceptions, the repertoire of methylotrophy genes between PA1 and AM1 is extremely similar. Second, PA1 grows faster with higher yields compared to AM1 on C1 and multi-C substrates in minimal media, but AM1 grows faster in rich medium. Third, deletion mutants in PA1 throughout methylotrophy modules have the same C1 growth phenotypes observed in AM1. Finally, the precision of our growth assays revealed several unexpected growth phenotypes for various knockout mutants that serve as leads for future work in understanding their basis and generality across Methylobacterium strains.

Methylamine Utilization via the N -Methylglutamate Pathway in Methylobacterium extorquens PA1 Involves a Novel Flow of Carbon through C 1 Assimilation and Dissimilation Pathways

“…better-characterized AM1 strain (25), here we find that it lacks methylamine dehydrogenase and possesses only the NMG pathway for MA use. By creating mutant strains with lesions in one or more genes of the NMG pathway and assaying the growth patterns of each of these strains on MA, we uncovered that the NMG pathway has a linear topology in PA1 akin to that very recently suggested for DM4 (17) and M. silvestris BL2 (3).…”

“…A strain lacking ftfL (encoding formyl-tetrahydrofolate ligase, which catalyzes the reduction of formate and free H 4 F to formyl-H 4 F in an ATP-dependent manner [29]) cannot grow on C 1 compounds like methanol and formate (25) because of an early block in assimilation (29). We recently reported that a ⌬ftfL mutant of PA1 has the same phenotype as the ⌬ftfL mutant of AM1 (25). Contrary to the findings for other C 1 compounds, the ⌬ftfL mutant could grow on MA at a rate indistinguishable from that of the WT (P ϭ 0.8887).…”

Transposon Sequencing Uncovers an Essential Regulatory Function of Phosphoribulokinase for Methylotrophy

“…Strain PA1 is phylogenetically closely related to strain AM1 (100% 16 S rRNA gene identity) and was isolated from the phyllosphere of Arabidopsis thaliana [22]. Due to several advantages over AM1, including faster growth on methanol, a simpler genome structure [23,24], and better suitability for transposon mutagenesis [25], it has been proposed as a new model strain for methylotrophy [26]. Furthermore, it is a competitive plant colonizer [22], making it a suitable model to study methylotrophy in its natural environment.…”

Methylobacterium extorquens: methylotrophy and biotechnological applications

“…The slow growth of M. extorquens AM1 on ethanol is a drawback for competitive industrial applications. In this respect, strain PA1 might represent an attractive alternative candidate with higher growth rates on methanol, ethanol, and multi-carbon substrates (Nayak and Marx 2014a).…”