Predicted domain structure and purification of the PcrA-like proteins from <i>M. pneumoniae</i> and <i>M. genitalium</i>.

<p>(A) Schematic representation of the predicted (sub)domains (1A, 1B, 2A and 2B) and motifs (I, Ia and II to VI) of the PcrA-like proteins from <i>M. pneumoniae</i> and <i>M. genitalium</i>. The predictions are based on the multiple alignment shown in supporting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070870#pone.0070870.s001" target="_blank">Fig. S1</a>, and on the crystal structures of PcrA/UvrD/Rep homologs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070870#pone.0070870-Subramanya1" target="_blank">[35]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070870#pone.0070870-Bird2" target="_blank">[60]</a>. Each subdomain is indicated by a separate color. Counterparts of the predicted 2B domains from PcrA<i>_Mpn</i> and PcrA<i>_Mge</i> (in blue) are absent from the PcrA^s_M129 and PcrA^s_FH proteins. (B) ORF structure of the genomes of <i>M. pneumoniae</i> (<i>Mpn</i>, at the top) and <i>M. genitalium</i> (<i>Mge</i>, at the bottom) in the region surrounding the ORFs encoding the PcrA-like proteins. All ORFs in these regions have the same orientation, i.e. 5′→3′ from left to right. The ORFs encoding the PcrA-like proteins are presented with the same color scheme as that of their encoded proteins in (A). Neighboring ORFs that are conserved between <i>M. pneumoniae</i> and <i>M. genitalium</i> are indicated in dark grey. The cluster of genes that is unique to <i>M. pneumoniae</i> (consisting of ORFs MPN342 to MPN347) is indicated at the top. (C) Purification of MBP, PcrA^s_M129, K29A, K29R, PcrA^s_FH, PcrA<i>_Mpn</i> and PcrA<i>_Mge</i>. K29A and K29R are point mutants of protein PcrA^s_M129. All proteins were purified as fusions to MBP by using the same protocol. Some of the protein preparations, including those of MBP and PcrA<i>_Mge</i>, contained minor protein species that are smaller than the full-length proteins. These species are not contaminants, but breakdown products of the full-length proteins; this was demonstrated by MALDI-TOF analysis of the two protein species that are indicated with asterisks (*). Samples of the purified proteins (as indicated above the lanes) were analyzed by SDS-PAGE (10%) and Coomassie brilliant blue (CBB)-staining. The sizes of protein markers (lane 1; PageRulerTM Prestained Protein Ladder [Fermentas]) are shown on the left-hand side of the figure in kDa.