Fusions at residues Gly109, Gly133, Lys157, and Tyr177 yielded alternating low and high PhoA activities (Fig. 1c), indicating that these regions have corresponding alternate cytoplasmic and periplasmic locations; this location was confirmed by fusions Gly109, Gly133, and Lys157 also yielding alternate high and low LacZ activities (Fig. 1c). The topology of this region, which spans the last four TMSs of Chr3C, was in complete agreement with prediction models (Fig. S1b). Together, these results suggested a topology of five TMSs for Chr3C, with the N-terminal end in the cytoplasm and the C-terminal end in the periplasm (Fig. 1d). In conclusion, membrane topology of the B. subtilis Chr3N/Chr3C

homologous pair, as determined by translational fusions, consists of five TMSs in antiparallel orientation, with the N-terminal end of Chr3N located in the periplasm and the N terminus of Chr3C located in the cytoplasm (Fig. 1b and d). Eighty-two amino acid Tipifarnib clinical trial sequences, retrieved LDK378 nmr during Blastp

searches at the UniProt site, were identified as members of the short-chain CHR3 subfamily (orthologous Chr3N/Chr3C) by phylogenetic analyses with the mega5 software. All chr3N/chr3C genes found are organized as tandem pairs and belong mainly to bacteria from the phylum Firmicutes (Bacillales; 76 protein sequences) and the γ-proteobacteria (Oceanospirillales; six protein sequences) group. Table S2 shows all Chr3N/Chr3C amino acid sequences studied in this work. A multiple protein sequence alignment was constructed with the 82 orthologous Chr3N/Chr3C sequences. Kyte-Doolittle hydropathic profiles, von Heijne transmembrane profiles, and free energy (ΔGapp) for membrane insertion of potential transmembrane helices were

calculated for each sequence and are shown in Fig. S1a. Profiles for Chr3N and Chr3C are very similar, suggesting that both types of proteins possess the same number of TMSs. Figure S1a shows five evident local minima of calculated Bcl-w ΔGapp values that represent candidate TMSs (shaded areas). Additional local minima weakly supported are indicated by empty areas. As expected, these local minima corresponded with local maxima of hydrophobicity, supporting the existence of the abovementioned putative TMSs. ΔG prediction server v1.0 (Hessa et al., 2007) recognized a range from three to six TMSs for each identified Chr3N/Chr3C protein sequences. Thus, TMS3 and TMS4 were recognized, with no exceptions, in all short-chain CHR3 subfamily members; TMS5 and TMS6 were predicted in the majority of analyzed Chr3N/Chr3C sequences, and TMS1 was recognized in all of Chr3C sequences and in the majority of Chr3N sequences (Table 1). In contrast, TMS2 (indicated by empty areas in Fig. S1a) was recognized only in one Chr3N and in none Chr3C sequences (Table 1). These data agree with calculated values of average ΔGapp for membrane insertion of each of the six potential TM helices for Chr3N and Chr3C proteins (Table 1).