Graphical output from SIMBAL computation and post-processing.

<p>Fig 2A shows the triangle heat map obtained for query sequence NP_718091.1, a rhombosortase from <i>Shewanella oneidensis</i> MR-1, obtained as described in the text. Each colored pixel in the heat map conveys three pieces of information: a SIMBAL score (color, where red indicates greater statistical significance), the length of the subsequence being scores (height on the Y-axis), and the location of the middle of the subsequence along the length of the complete protein (position on the X-axis). Subsequences are evaluated from a minimum length of 9 (bottom of the heat map) to a maximum of 204, the full length of the protein, at the top of the heatmap. Scores are computed as the negative log₁₀ of the odds against encountering, purely by chance, at least as great a preponderance of YES set-derived sequences among the top BLAST hits. Note that nearby pixels may differ sharply in score, and that the deepest red colors appear in a “plume” of pixels whose corresponding subsequences all contain the same key small region. Fig 2<b>B</b> shows a smoothed heatmap that results from re-processing SIMBAL scores so that each pixel represents a blend of its own score and those of longer sequences that contain it, performed iteratively starting with the longest sequences. The heritability parameter used was 0.93. Fig 2<b>C</b> shows data in numerical form corresponding to a line passing through the heatmap of Fig 2<b>A</b> very near its base, at a subsequence length of 9, with height rather than color showing the score. Fig 2<b>D</b> shows the corresponding slice through the rescored heatmap of Fig 2B, with a greatly reduced jitter in scores from one pixel to the next, and a clear indication of which short subsequences most likely contain key sites that discriminate rhombosortases from other rhomboid family proteases.</p

Training Set Construction.

<p>Fig 1A illustrates the simplest method for training set construction. Each genome (gray circles) is treated as a “bag of genes”; distance relationships between genes are ignored. One hidden Markov model (HMM) identifies target family proteins (orange squares) in the corresponding proteome. A second HMM finds proteins from a second family (yellow stars) whose presence or absence in the proteome is the attribute that controls how target family proteins are sorted. If an attribute family protein is found, members of the target family get sorted to the YES set (green container). If not, then target family proteins go to the NO set (red container). The training set builder (TSB) always works on one target protein family at a time, but more complicated rules may require multiple attributes to be jointly present for the YES set, and multiple attributes to be jointly absent for the NO set. Fig 1B shows training set construction using a distance rule. The S-shaped curved represents a long segment of genomic DNA. A target protein is sorted to the YES set if and only if its gene lies within a user-specified distance from the attribute protein’s gene. Target proteins from genomes that lack the attribute completely go to the NO set. A target protein goes to the FAR set if and only its gene sufficiently far from the nearest attribute gene, and the genome has already sent a target protein to the YES set. If a genome encodes an attribute family protein, but no target family protein qualifies for the YES set, then target family proteins are not sorted to any bin.</p

Sequential steps in a SIMBAL analysis.

<p>Sequential steps in a SIMBAL analysis.</p

Stimulation of gliding motility depends on TraA.

<p>(A) Indicated motility phenotypes shown for reference. To easily observe stimulation the donor strain (DK6204) was nonmotile (A<b>⁻</b>S<b>⁻</b>), i.e. produces sharp colony edges, and nonstimulatable, while the (B) <i>cgl</i> and <i>tgl</i> recipients were phenotypically A<b>⁻</b>S<b>⁻</b> but stimulatable. (C) Indicated mutants mixed 1∶1 with the donor exhibiting various degrees of A- or S-motility stimulation. (D) Same as C, except strains mixed with isogenic <i>traA</i>::km donor. (E) Same as C, except isogenic recipient strains contain the <i>traA</i>::km mutation. Cells were incubated for 22 hrs at 33°C and observed with 10× objective. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002626#pgen.1002626.s011" target="_blank">Table S2</a> lists strain genotypes.</p

Lipophilic fluorescent dye (DiD) transfer depends on TraA and cell motility.

<p>Column headers list relevant properties of isogenic recipients. The same nonmotile (DK8601) donor was used in all mixtures and recipients were DW1414 (A⁺ <i>traA⁺</i>), DW1416 (A⁺ <i>traA⁻</i>) and DK8606 (A⁻ <i>traA⁺</i>). The ability of the DiD dye (red fluorescence) to be transferred to GFP recipients was assessed in merged panels (100× objective). Here yellow color indicates DiD transfer to GFP recipients and representative cells are noted by arrows.</p

SS_OM-mCherry transfer requires TraA.

<p>A 1∶1 mixture of donor and recipient cells were mixed and spotted on ½ CTT 1% agar and incubated for 4 hrs. Swarms were harvested and single cells were microscopically examined on glass slides to test whether GFP recipients became red by obtaining SS_OM-mCherry. White arrows highlight two cells where transfer occurred. Column micrographs (100× objective) were of identical fields. Rod shaped <i>M. xanthus</i> cells were ∼0.5×6.0 microns. Strains were as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002626#pgen-1002626-g002" target="_blank">Figure 2</a> and were <i>traA⁺</i> unless indicated otherwise in column headers.</p

Genetic and modular structure of TraAB.

<p>(A) Operon structure depicting genes that translationally overlap. (B) Domain and motif architecture and the DK396 amino acid substitution indicated. (C) Modeled three-dimensional structure and electrostatic surface potential of TraA PA14 domain. Features shown in green in the ribbon diagram (left) could serve to recognize glycans through potential side-chain coordination of a calcium ion by Asp183, Asp184, Glu237 (only Cα-Cβ shown), and the location of an insertion important for carbohydrate-binding specificity in FLO5 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002626#pgen.1002626-Veelders1" target="_blank">[24]</a>. Graphics produced with PyMOL (Molecular Graphics System, Version 1.3, Schrödinger, LLC) and APBS Tools2 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002626#pgen.1002626-Lerner1" target="_blank">[61]</a>. (D) consensus sequence LOGO <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002626#pgen.1002626-Crooks1" target="_blank">[62]</a> for Cys-repeats found in TraA and myxobacteria family members designated TIGR04201.</p

Transfer of heterologous SS_OM-mCherry reporter in <i>M. xanthus</i> swarm requires TraA.

<p>Nonmotile SS_OM-mCherry donors (DW1411 or DW1412) were mixed 1∶3 with A-motile GFP labeled recipients (DW1414 or DW1416). After 1 day incubation on TPM agarose pad A-motile recipients readily swarm out from the inoculum spot. Column micrographs were of identical fields taken under phase contrast and GFP or mCherry fluorescence (20× objective). Indicated isogenic strains contain <i>traA⁺</i> or <i>traA</i>::km alleles. In panels F and I arrows indicate nonmotile donor cells that were pushed or dragged to the swarm edge <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002626#pgen.1002626-Wei1" target="_blank">[6]</a>. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002626#pgen.1002626.s011" target="_blank">Table S2</a> lists strain genotypes.</p

Working model for OM and lipoprotein transfer between <i>M. xanthus</i> biofilm cells.

<p>See text for details.</p

Consensus sequence LOGO of indicated bacterial C-terminal protein sorting motifs.

<p>Subcellular membrane topology predictions are shown. Black arrows indicate predicted or known proteolytic processing sites for MYXO-CTERM (TIGR03901) and LPXTG <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002626#pgen.1002626-Paterson1" target="_blank">[28]</a>, respectively. Note the N-terminal conserved sequences vary between motifs, while C-terminal sequences are all enriched for arginine and lysine residues.</p