Parallel Evolution and Horizontal Gene Transfer of the pst Operon in Firmicutes from Oligotrophic Environments

Abstract

The high affinity phosphate transport system (pst) is crucial for phosphate uptake in oligotrophic environments. Cuatro Cienegas Basin (CCB) has extremely low P levels and its endemic Bacillus are closely related to oligotrophic marine Firmicutes. Thus, we expected the pst operon of CCB to share the same evolutionary history and protein similarity to marine Firmicutes. Orthologs of the pst operon were searched in 55 genomes of Firmicutes and 13 outgroups. Phylogenetic reconstructions were performed for the pst operon and 14 concatenated housekeeping genes using maximum likelihood methods. Conserved domains and 3D structures of the phosphate-binding protein (PstS) were also analyzed. The pst operon of Firmicutes shows two highly divergent clades with no correlation to the type of habitat nor a phylogenetic congruence, suggesting horizontal gene transfer. Despite sequence divergence, the PstS protein had a similar 3D structure, which could be due to parallel evolution after horizontal gene transfer events.

Figure 1

Gene architecture of the pst operon in different groups of bacteria. A: Bacillus cereus group, marine Bacillus and Clostridium; B: Listeria and Streptococcus; C: Bacillus subtilis group, Bacillus marisflavi, and Bacillus sp. CH108; D: Brevibacillus, Oceanobacillus, Desulfitobacterium, Acaryochloris; E: Synechococcus, Geobacillus kaustophilus, Sebaldella termitidis, B. cereus group. In C–E, the regulatory gene phoU is found elsewhere in the genome, not as part of the operon or entirely missing.

Figure 2

Maximum likelihood phylogenetic reconstruction of the concatenated PstC, PstA, and PstB (PstBB) protein sequences encoded by the pst operon. Branch colors indicate the two divergent clades: subtilis-like and cereus-like. Tag colors indicate the type of habitat where each species is found. Bootstrap values above 70% are indicated with an asterisk. The phylogeny of the individual proteins has a very similar topology (data not shown).

Figure 3

Maximum likelihood phylogeny of Firmicutes based on the concatenated amino acid sequence of 14 housekeeping genes and dated with a penalized likelihood method. The branch colors indicate the type of operon present in each taxon. Tag colors refer to the type of habitat. Clade I corresponds to aerobic Firmicutes and clade II includes CCB and marine Bacillus. Clade I had a fixed age of 2300 my and clades III and IV had a fixed minimum age of 35 my. Bootstrap values above 70% are denoted with an asterisk. Clades with branch lengths of 0 were collapsed (D. hafniense DCB-2-D. hafniense Y51 and G. thermodenitrificans NG80-2-G.sp. G11MC16).

Figure 4

Conserved motifs of the PstS protein for both cereus-like and subtilis-like operons. Red stars on top of residues on motif 2 indicate the binding site of phosphate. Motifs 4 and 5 are aligned (black lines) to show homologous amino acid positions. The height of the blocks is proportional to the e-value of each motif. Motif 3 for subtilis-like PstS has an e-value <10⁻¹⁰.

Figure 5

Comparison of 3D structures with TOPOFIT of PstS from B. subtilis (red), with (a) B. sp. NRRL-14911 (cyan; r.m.s.d. = 1.02, z-score = 39.89), (b) Bacillus sp. m3-13 (green; r.m.s.d. = 1.53, z-score = 20.24) and (c) B. coahuilensis m4-4 (yellow; r.m.s.d. = 1.49, z-score = 9.65). Residues involved in phosphate binding are highlighted in purple. (d) shows an alignment of the active site of PstS and the different amino acids involved in phosphate binding are highlighted in gray.