Computational protocol: Experimental Horizontal Gene Transfer of Methylamine Dehydrogenase Mimics Prevalent Exchange in Nature and Overcomes the Methylamine Growth Constraints Posed by the Sub-Optimal N-Methylglutamate Pathway

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Protocol publication

[…] Amino acid similarity to mauA and mauB (which encode the small and large subunit of MaDH) from M. extorquens AM1 [] and gmaS, mgsC and mgdC (which encode γ-glutamylmethylamide synthetase, and subunits of N-methylglutamate synthase and N-methylglutamate dehydrogenase respectively) from M. extorquens PA1 [] were used to detect the presence of MaDH and/or the N-methylglutamate pathway, respectively, in sequenced methylotrophic Proteobacterial genomes deposited in NCBI. The criterion for detecting genes representing each of these MA oxidation pathways was based upon a sequence similarity and sequence coverage cutoff. The cutoff was determined based on either: (a) parameter values below which a significant, sharp rise in the e-value of the BLAST search results was observed (mauA and mauB) or (b) parameter values below which functionally distinct homologs were observed (gmaS, mgsC, mgdC). Sequence coverage of >96% and >85%, sequence identity of >54% and >43% (corresponding to an e-value of 4e-55 and 2e-51) was used as the criterion for detecting mauA and mauB like sequences, respectively, in the genomes of sequenced methylotrophs within the Proteobacteria. Sequence coverage of >96%, >96% and >87% and sequence identity of >40%, >81%, and >35% was used as the threshold for the detection of gmaS, mgsC and mgdC like sequences in the genomes of sequenced methylotrophs within the Proteobacteria. Sequence alignment was conducted using the MUSCLE alignment software [] with a maximum of 127 iterations. ML (Maximum Likelihood) phylogenetic analysis was performed with the PhyML package built in Geneious version 5.4 [] using the HKY85 substitution model [] and the Dayhoff substitution model [] for nucleotide and amino acid sequences, respectively, and employed 500 bootstrap resamplings. The proportion of invariable sites, gamma distribution parameters, and transition/transversion ratios (only for nucleotide sequences) were estimated; four substitution rate categories were used, and the topology, branch length, as well as rate parameters were optimized. […]

Pipeline specifications

Software tools MUSCLE, PhyML, Geneious
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Methylobacterium extorquens, Methylobacterium extorquens AM1
Chemicals Carbon, Nitrogen