The role of core and accessory type IV pilus genes in natural transformation and twitching motility in the bacterium Acinetobacter baylyi.

Here we present an examination of type IV pilus genes associated with competence and twitching in the bacterium Acinetobacter baylyi (strain ADP1, BD413). We used bioinformatics to identify potential competence and twitching genes and their operons. We measured the competence and twitching phenotypes of the bioinformatically-identified genes. These results demonstrate that competence and twitching in A. baylyi both rely upon a core of the same type IV pilus proteins. The core includes the inner membrane assembly platform (PilC), a periplasmic assemblage connecting the inner membrane assembly platform to the secretin (ComM), a secretin (ComQ) and its associated pilotin (PilF) that assists with secretin assembly and localization, both cytoplasmic pilus retraction ATPases (PilU, PilT), and pilins (ComP, ComB, PilX). Proteins not needed for both competence and twitching are instead found to specialize in either of the two traits. The pilins are varied in their specialization with some required for either competence (FimT) and others for twitching (ComE). The protein that transports DNA across the inner membrane (ComA) specializes in competence, while signal transduction proteins (PilG, PilS, and PilR) specialize in twitching. Taken together our results suggest that the function of accessory proteins should not be based on homology alone. In addition the results suggest that in A. baylyi the mechanisms of natural transformation and twitching are mediated by the same set of core Type IV pilus proteins with distinct specialized proteins required for each phenotype. Finally, since competence requires multiple pilins as well as both pilus retraction motors PilU and PilT, this suggests that A. baylyi employs a pilus in natural transformation

Reactive design patterns

<p><b>Twitching zones on (a) soft vs. (b) hard agar for multiple twitching phenotypes.</b> On soft agar (0.5%) in panel (a), mutants <i>fimU</i> and <i>comA</i> twitch a comparable amount to the wildtype, while <i>comP</i> and <i>comE</i> are substantially impaired in agreement with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182139#pone.0182139.g003" target="_blank">Fig 3</a>. Panel (b) shows the same mutants but now on hard agar (1.5%). In this case all diameters are comparable.</p

Model of the ADP1 type IV pilus with associated competence and signal transduction proteins.

<p>Components of the model are predicted by homology from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182139#pone.0182139.t001" target="_blank">Table 1</a> and are supported by data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182139#pone.0182139.g003" target="_blank">Fig 3</a>.</p

Alignment of FimU and FimT pilins from ADP1.

<p>A. The length of the primary sequence is indicated above with Pfam motifs found in both proteins diagrammed below. N = PF07963.11; GspH = PF12019.7. Such motifs are identified using multiple sequence alignments. B. Pairwise alignment of FimU and FimT. Identical amino acids are indicated by a vertical line connecting them while: and. represent higher and lower degrees of chemical similarity between the two amino acids, respectively. Boldface amino acids are part of the Pfam motifs diagrammed in A.</p

Protein motif diagrams of the type IV pilin proteins encoded by ADP1.

<p>Length in amino acids is indicated along the top, with pilins diagrammed below. Pilins absolutely required for both twitching and competence are in boldface. Pfam motifs are indicated by filled, labeled boxes. The Pfam motif names are: N = PF07963.11 Prokaryotic N-terminal methylation motif; Pilin = PF00114.18 Pilin (bacterial filament); GspH = PF12019.7 Type II transport protein GspH; PilW = PF16074.4 Type IV Pilus-assembly protein W; PilX_N = PF14341.5 PilX N-terminal; ComP_DUS = PF16732.4 Type IV minor pilin ComP, DNA uptake sequence receptor.</p

Competence and twitching phenotypes of null mutations.

<p>All data in this figure were taken using complex media and incubating at 37°C. Both competence and twitching assays were performed on agar: 1.5% for competence and 0.5% for twitching. Color scheme is the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182139#pone.0182139.g001" target="_blank">Fig 1</a>. Like symbols indicate that genes are part of the same operon. All data points contain multiple measurements for both competence and twitching. Error bars represent the standard deviation of multiple measurements. For the x-axis standard deviation is given by where x = log and ΔTE is defined as the standard deviation from the mean transformation efficiency, . The detection limit for competence is 10⁻⁹ and for twitching is 0.4. Data points that fall below either detection limit appear on the graph in a “below detection limit” region. Their position within this region has no physical interpretation beyond indicating that they fall below this limit.</p

Type IV pilus protein homologs in ADP1.

<p>Type IV pilus protein homologs in ADP1.</p

Full-length homologs of competence and twitching proteins encoded by the ADP1 genome.

<p>Full-length homologs of competence and twitching proteins encoded by the ADP1 genome.</p

Alignment of PilT and PilU retraction motors from ADP1.

<p>A. The length of the primary sequence is indicated above with Pfam motifs found in both proteins diagrammed below. T2SSE = Type II/IV secretion system protein; AAA_30 = AAA_proteins. AAA_30 domains contain a P-loop motif with Walker A and Walker B motifs. Such motifs are identified using multiple sequence alignments. B. Pairwise alignment of PilT and PilU. Identical amino acids are indicated by a vertical line connecting them while: and. represent higher and lower degrees of chemical similarity between the two amino acids, respectively. Boldface amino acids are part of the Pfam motifs diagrammed in A while the blue text indicates more specifically the AAA_30 motifs.</p