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

Immunoblot analysis shows that expression of PilA is abolished in a <i>mas</i> deletion strain and is only rescued by addition of a <i>pilA</i> construct.

<p>Lane 1 represents the WT level of PilA expression, whereas deletion of the <i>pilA</i> gene is sufficient to abolish all detectible expression (lane 2). Addition of <i>pilA</i> at the chromosomal site rescues the defect as shown in lane 3. Lane 4 represents a strain deficient in <i>pilA</i> but merodiploid for the <i>masABK</i> locus, and does not rescue the <i>pilA</i> defect. Δ<i>masABK</i> mutants lack <i>pilA</i> expression as seen in lane 5. Addition of <i>masABK</i> at the <i>att</i> site does not rescue the phenotype, as shown in lane 7, but addition of a WT copy of the <i>pilA</i> gene and necessary promoter region rescues expression of PilA in <i>M. xanthus</i> (lanes 6 and 8). Immunoblot was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054557#s4" target="_blank">materials and methods</a>, with a polyclonal anti-pilin antibody diluted to 1∶1000.</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

MasAB-PilA interaction drives transcription from the GAL promoter in a Y2H assay.

<p>Diploid yeast strains containing both a pGAD-C1 and pGBD-C1 derived vectors. Dilutions of overnight culture were plated on a low stringency medium (SC-LTH) Panel A, and medium stringency medium (SC-LTA), Panel B as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054557#s4" target="_blank">Materials and Methods</a>. Row 1: <i>FOS/JUN</i> (+) control, Row 2: Empty vector (pGAD, pGBD negative control), Row 3: pGAD<i>masA</i>, pGBD<i>pilA</i>, Row 4, pGAD<i>masB</i>, pGBD<i>pilA</i>.</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

Sporulation is decreased in a Δ<i>mas</i> mutant.

<p>Spores were harvested from a TPM plate after 5 days of growth at 32°C and resuspended in 100 µl of TPM buffer. After 2 h incubation at 50°C, spore aliquots were diluted and plated on CTPM agar. After 3 days incubation at 32°C, CFU's from heat resistant spores were counted. All values are given as a percentage of the WT level, normalized to 100%.</p

Deletion of <i>pilA</i> does not affect <i>masB</i> transcription.

<p>RNA was harvested from 1×10⁸ cells and mRNA was used to generate cDNA using a random hexanucleotide primer. Gene specific primers for <i>masB</i> and the <i>16S</i> gene (endogenous control) were used in the amplification reaction with cDNA. <i>masB</i> expression was amplified from cDNA using 50, 5 and 0.5 ng template, while <i>16 S</i> expression was amplified using 200, 20 and 2 pg cDNA. Both the WT and <i>pilA</i> strain express <i>masB</i> (black band in lanes 1 and 4 respectively). Hence, deletion of <i>pilA</i> does not adversely affect the expression of <i>masB</i> in <i>M. xanthus</i>. Deletion of <i>masABK</i> abolished <i>masB</i> expression (lanes 7–9).</p