Fig. S1. Unrooted phylogenetic consensus trees constructed after Bayesian inference of phylogeny from the ClustalX alignments of 127 PxmA, PmoA and AmoA and 51 PxmB, PmoB and AmoB protein sequences subjected to a Bayesian inference of phylogeny using the BEAST package [BEAUti v1.5.3, BEAST v1.5.3, TreeAnnotator v1.5.3, FigTree v.1.3 (Drummond and Rambaut, 2007)] as described in Fig. 1 (Monte-Carlo Markov Chain with 10 000 000 generations, WAG substitution model). Unrooted 50% majority rule consensus phylograms (panel A, A protein alignments; panel B, B protein alignments) were constructed and mean branch lengths (posterior probability values of all observed clades < 1.0 are indicated) are characterized by a scale bar indicating the evolutionary distance (changes per amino acid position). Protein sequence accession numbers for all protein sequences in the alignments were included into the branch labels together with information of the source organism. The trifurcation point identified by the star topology of the tree shown in Fig. 1 is indicated.

Fig. S2. Operon structure of pxmABC genes in Methylomonas sp. strain LW13. A. Retrieved contiguous sequence contains pxm genes in the non-canonical order A-B-C as indicated. An open reading frame (orf1) encoding a protein related to LysR type transcription regulators is divergently transcribed upstream of pxm coding sequence. Dashed lines indicate partial ORFs. Approximate locations of PCR primers (given in Table S1) are indicated by arrows. B. The putative pxm initial transcribed region is predicted to contain two alternative hairpins. The longer hairpin contains features suggesting a transcriptional regulatory role.

Fig. S3. Endpoint analysis of Reverse-Transcription real-time PCR for detection of pxmA and pmoA transcripts in creek sediment samples. PCR-amplification was performed using three serial dilution samples (lanes 2, 3 and 4) of cDNA generated from DNase-treated RNA obtained from creek sediments (‘RT’) as described in Fig. 3 and three serial dilution samples (lanes 5, 6 and 7) of the DNase-treated RNA incubated with reverse transcription buffer omitting reverse transcriptase enzyme (‘no-RT’). ‘no-RT’ samples were used as controls to demonstrate absence of contamination by genomic DNA in the cDNA. Lane 1 contains 4 ul of the size standard (Low range ladder, Fisher Scientific, Pittsburgh, PA) to estimate size and intensity of the obtained amplicons. Cycle threshold determined during real time PCR is indicated. The melting temperature (Tm) indicates that the pxmA and pmoA amplicons are distinct species.

Table S1. Primers and conditions used in this study.

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