Selected gene classes with differential expression in the two nutrient treatments.

<p>The “direction” column indicates whether genes showed increased or decreased expression in the frog skin treatment (relative to the tryptone treatment). Both log2 fold change and absolute-value fold change are given. The <i>p</i>-values are Benjamini and Hochberg corrected for multiple testing. The “Bd-specific” column indicates with asterisks those genes that were found to be unique to Bd <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049924#pone.0049924-Rosenblum3" target="_blank">[31]</a>. The “life stage” column indicates those genes that were found to have significantly increased expression in the zoospore (Z) or sporangia (S) life stage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049924#pone.0049924-DaSilva1" target="_blank">[30]</a>.</p

The proportion of Bd genes with significantly increased or decreased expression in the frog skin treatment (relative to the tryptone treatment) shown for all genes, Bd-specific genes, and several families of putative pathogenicity genes.

<p>Genes with significant differences in expression were identified using a correction for multiple testing. Abbreviations include M36 = fungalysin metalloprotease, S41 = serine protease, Asp = aspartyl protease, CRN = Crinkler-like effectors.</p

MasABK Proteins Interact with Proteins of the Type IV Pilin System to Affect Social Motility of <em>Myxococcus xanthus</em>

<div><p>Gliding motility is critical for normal development of spore-filled fruiting bodies in the soil bacterium <em>Myxococcus xanthus</em>. Mutations in <em>mgl</em> block motility and development but one <em>mgl</em> allele can be suppressed by a mutation in <em>masK</em>, the last gene in an operon adjacent to the <em>mgl</em> operon. Deletion of the entire 5.5 kb <em>masABK</em> operon crippled gliding and fruiting body development and decreased sporulation. Expression of <em>pilAGHI</em>, which encodes type IV pili (TFP) components essential for social (S) gliding, several cryptic <em>pil</em> genes, and a LuxR family protein were reduced significantly in the Δ<em>mas</em> mutant while expression of the myxalamide operon was increased significantly. Localization and two-hybrid analysis suggest that the three Mas proteins form a membrane complex. MasA-PhoA fusions confirmed that MasA is an integral cytoplasmic membrane protein with a ≈100 amino acid periplasmic domain. Results from yeast two-hybrid assays showed that MasA interacts with the lipoprotein MasB and MasK, a protein kinase and that MasB and MasK interact with one another. Additionally, yeast two-hybrid analysis revealed a physical interaction between two gene products of the <em>mas</em> operon, MasA and MasB, and PilA. Deletion of <em>mas</em> may be accompanied by compensatory mutations since complementation of the Δ<em>mas</em> social gliding and developmental defects required addition of both <em>pilA</em> and <em>masABK</em>.</p> </div

<i>masABK</i> and <i>pilA</i> are both required for fruiting body formation.

<p><i>M. xanthus</i> strains were concentrated to a density of 5×10⁹ cells/ml and 20 µl aliquots were plated on starvation (TPM) agar as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054557#s4" target="_blank">Methods</a>. After 96 hours incubation, spots were photographed under using the 10× objective as seen above. The black bar in panel A denotes 100 µm. A: DK1622 (WT), B: DK10407, C: MxH2604, D: MxH2622, E: MxH2609, F: MxH2624.</p

<i>pilA</i> and <i>masABK</i> are required for motility in 0.5% methylcellulose.

<p>Cells were grown overnight in CTPM with appropriate antibiotics and overlaid with methylcellulose to a final concentration of 0.5% w/v. Velocities were determined using Metamorph (n = 50). Cells above a threshold velocity of 1.5 µm/min were used to calculate the reversal frequencies.</p

Phase Variation in <i>Myxococcus xanthus</i> Yields Cells Specialized for Iron Sequestration

<div><p><i>Myxococcus xanthus</i> undergoes phase variation during growth to produce predominantly two colony phenotypes. The majority are yellow colonies containing swarm-proficient cells and a minority are tan colonies containing swarm-deficient cells. Comparison of the transcriptomes of a yellow variant, a tan variant, and three tan mutants led to the identification of differentially-regulated genes that define key segments of the phase variation pathway. For example, expression of genes for the yellow pigment DKxanthene and the antibiotic myxovirescin was increased significantly in yellow variants. In contrast, expression of the siderophore myxochelin, hemin binding proteins, and iron transport proteins was increased specifically in tan strains. Thus, a consequence of phase variation is that yellow cells shift from producing antibiotic and pigment to producing components involved in acquisition of iron, which may increase fitness during periods of iron limitation. Multiple protein kinases and HTH-Xre DNA-binding proteins identified in this study may be involved in the regulatory hierarchy that governs phase variation.</p></div

Deletion of the <i>masABK</i> operon affects swarming.

<p>A: WT and the Δ<i>mas</i> strain were grown in CTPM to a concentration of 5×10⁸ cells/ml. After 10-fold concentration, 3 µl were plated on 1.5% and 0.3% agar plates as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054557#pone.0054557-Shi1" target="_blank">[1]</a>. Swarm diameters was measured after 96 hours. Data represent an average of nine samples plated in three separate assays. B: Photomicrographs of the colony edge after 4 days on CTPM 1.5% agar reveal isolated cells indicative of A-motility. White arrows designate the leading edge of the colony.</p

qRT-PCR confirms <i>pilA</i> expression decline is specific.

<p>A: Relative quantity of S-motility gene expression in <i>M xanthus</i> WT and Δ<i>mas</i> strains. Gene expression in the Δ<i>mas</i> strain was normalized to the WT reference (set to 1) and relative quantitation are represented as a ratio of strain/WT, such as Δ<i>mas</i>/WT. Values represent the average of three PCR reactions. <i>pilA</i> is shown in red (panels A and B), <i>pilS</i> is shown in orange and <i>pilT</i> is in lighter blue. B: Genomic context of the <i>pilA</i> gene within the 20 kb <i>pil</i> gene cluster. Arrows denote known promoter regions for <i>pilSR, pilAGHI</i>, <i>gspO</i> and <i>pilS2/R2</i> transcription units <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054557#pone.0054557-Wu3" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054557#pone.0054557-Wu5" target="_blank">[57]</a>.</p

The <i>mas</i> operon is predicted to encode three proteins, MXAN_1927 (MasA), MXAN_1928 (MasB) and MXAN_1929 (MasK).

<p>A: MasA possesses two transmembrane helices, and a periplasmic domain of 100 amino acid residues (green). MasB is a membrane bound lipoprotein (purple), anchored to the inner membrane at residue 25 (black dot). MasK is a membrane-associated protein kinase (red) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054557#pone.0054557-Thomasson1" target="_blank">[15]</a>. B: Genomic context of the <i>masABK</i> operon. The <i>mas</i> operon is 327 bp upstream of the <i>mglBA</i> operon (blue arrows) and is transcribed from the opposite strand. The <i>mas</i> operon coordinates are indicated below the ORFs for reference. The spacing of the three predicted open reading frames (ORF) suggests that the genes are cotranscribed. C: Primer extension analysis of cDNA transcript. cDNA transcript was generated using a primer specific for MasK, 810, and the template was amplified using primers 801 and 810 (illustrated using inward arrows). The resulting amplicon was 5.4 kb and confirms expression of the <i>masABK</i> operon involves the formation of a polycistronic mRNA transcript.</p

MasA and MasK are membrane proteins.

<p>A. MasA has two predicted membrane spanning regions (TM  =  transmembrane). B. PhoA fusions demonstrate that MasA has a periplasmic domain. MasA was cloned into pCR Blunt II TOPO under a <i>lac</i> promoter to form pSF8. pSF20 is a derivative of pSF8 with an in-frame insertion of the <i>E. coli phoA</i> gene at the <i>Pst</i>I site (*), while pACW1 has an in-frame fusion at the <i>Mlu</i>I site (†) shown in panel A. Overnight cultures were adjusted to an OD₆₀₀ of 1.0 and serially diluted. 5 μl of each dilution was incubated on LB agar with 35 µg/ml X-P at 37°C for 16 hours. C. Fluorescence from a MasK-mcherry fusion expressed in an otherwise WT strain was concentrated at one pole of the cell. Yellow arrows indicate the direction of movement of cells on 1.5% agar surface.</p