Identification of the cglC, cglD, cglE, and cglF genes and their role in cell contact-dependent gliding motility in Myxococcus xanthus

Abstract

Within Myxococcus xanthus biofilms, cells actively move and exchange their outer membrane (OM) lipoproteins and lipids. Between genetically distinct strains, OM exchange can regulate recipient cell behaviors, including gliding motility and development. Although many different proteins are thought to be exchanged, to date, only two endogenous OM lipoproteins, CglB and Tgl, are known to be transferred. Protein exchange requires the TraAB proteins in recipient and donor cells, where they are hypothesized to facilitate OM fusion for transfer. To better understand the types of proteins exchanged, we identified the genes for the remaining set of cgl gliding motility mutants. These mutants are unique because their motility defect can be transiently restored by physical contact with donor cells that encode the corresponding wild-type protein, a process called stimulation. Similar to CglB and Tgl, the cglC and cglD genes encode type II signal sequences, suggesting that they are also lipoproteins. Surprisingly, the cglE and cglF genes instead encode type I signal sequences, suggesting that nonlipoproteins are also exchanged. Consistent with this idea, the addition of exogenous synthetic CglF protein (71 amino acids) to a cglF mutant rescued its motility defect. In contrast to a live donor cell, stimulation with purified CglF protein occurred independently of TraA. These results also indicate that CglF may localize to the cell surface. The implications of our findings on OM exchange are discussed.

Fig 1

Genetic maps of cglC and cglD regions. The locations of Tn5 transposon insertions are indicated by triangles and omega numbers. The precise insertion site of Ω1903 in the DK1622 genome was at bp 2,958,528, and that of Ω1904 was at bp 1,117,552. The calculated physical distances between the cgl mutations and Tn5 insertions derived from phage Mx8 cotransduction frequencies are shown. The ability of plasmids containing the indicated genes to rescue cgl mutations are listed. Locus tags are shown as MXAN numbers. (A) cglC gene cluster. Plasmid rescue was conducted against strain DK1633. (B) cglD gene cluster. Plasmid rescue was conducted against strain DK391. The images are not to scale.

Fig 2

Genetic map of cglE-and-cglF region. See Fig. 1 legend for details. The precise insertion site of Ω1905 in the DK1622 genome was at bp 6,077,315, that of Ω1919 was at bp 6,107,659, and that of Ω1931 was at bp 6,116,470. Plasmid rescue experiments were conducted against strains DK360 (cglE1) and DW704 (cglF1). The image is not to scale.

Fig 3

Motility and stimulation phenotypes of cgl mutants. The top panels show the colony edge swarming properties of the indicated isogenic cgl mutants. These strains contain a ΔpilQ mutation that abolishes S motility. The strains used were DW1438 (cglC2 mutant), DW1440 (cglD1 mutant), DW1443 (cglE1 mutant), and DW1445 (cglF1 mutant). The nonmotile donor (DK6204 [ΔmglBA mutant]) and reference A⁺ S⁻ strain (DK8615; bottom right panel) colony edges are shown. Indicated bottom panels were 1:1 mixtures of the donor strain mixed with the indicated cgl mutants. Phase-contrast micrographs (10× objective) were taken after 2 days of incubation at 33°C.

Fig 4

Single-cell motility analysis of cgl mutants. Time-lapse microscopy was conducted for 30 min with micrographs taken every 30 s. For each strain at least 50 individual cells were visually tracked. A single discernible cell movement over the duration of the 30 min movie was sufficient for a positive score. Isogenic strain sets contain a ΔpilQ mutation and strain numbers are listed in Fig. 3 legend.

Fig 5

CglD domain structure and phenotypes. (A) Relevant features include type II signal sequence (SS), repeat elements 1 and 2, VWA domain (VWA) and C-terminal cysteine-rich domain. CglD amino acid substitutions and the ΔcglD deleted region (black bar) are shown for indicated mutants. (B) Colony edge morphologies of cglD strains in isogenic ΔpilQ background. Strains used were DK8615 (cglD⁺), DW1440 (cglD1), DW1461 (cglD2) and DW1433 (ΔcglD). Micrographs (10× objective) were taken after 1 day incubation on [1/2] CTT 1.5% agar supplemented with 2 mM CaCl₂, except as noted.

Fig 6

Addition of synthetic CglF peptide stimulates a cglF mutant in a TraA-independent manner. CglF synthetic peptide (20 μg) was mixed with nonmotile (A⁻ S⁻) cglF mutants and incubated as shown. Strains used were DW1460 (ΔcglF), DW1405 (cglF1 traA::km) and the nonmotile donor strain was DK6204.