Cell-to-Cell Transfer of Bacterial Outer Membrane Lipoproteins

Eric Nudleman,^* Daniel Wall, Dale Kaiser

^* Present address: Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

Present address: Anadys Pharmaceuticals, 3115 Merryfield Row, San Diego, CA 92121, USA.

To whom correspondence should be addressed. E-mail: kaiser{at}cmgm.stanford.edu

Myxobacteria move by gliding on surfaces—an effective mode of translocation in their soil habitat (1). Myxococcus xanthus moves smoothly in the direction of its long axis, then reverses and moves in the opposite direction. It has no flagella and cannot swim; its gliding is propelled by two polar engines that are encoded by the A and the S genes (2). The A engines appear to extrude polymer chains from the trailing ends of cells, which gelate, pushing the cells forward (3). Slime is extruded from the trailing end only (4). The S genes encode hairlike appendages projecting from the leading end of the cell—the type IV pili (5, 6)—and they pull a cell forward by retracting (7). Many proteobacterial pathogens have type IV pili (8). M. xanthus type IV pili are polar (6), and S motility is limited to cells within pilus-contact distance of each other (9).

A common set of 10 proteins is necessary for the assembly and retraction of type IV pili in M. xanthus, Pseudomonas aeruginosa, and Neisseria gonorrhoeae (8). One of those proteins, known as PilA in M. xanthus, constitutes the pilus filament, a helical array of PilA monomers (10). The filament is thought to pass through the outer membrane within a gated channel formed by the PilQ secretin protein (11). Secretins are multimeric channels large enough to pass folded proteins (12). Secretins often have cognate lipoproteins that facilitate their assembly in the outer membrane (13, 14).

The Tgl lipoprotein is necessary to assemble a detergent-resistant PilQ secretin in M. xanthus (15). Nevertheless, tgl mutants can be stimulated to assemble pili and to glide after transient contact with tgl⁺ donor cells (16, 17). Cells with a tgl mutation lack S motility but have normal A motility. Tgl stimulation specifically restores their S motility (2). To render stimulation visible by its effect on motility, we transferred a mutant tgl locus into an A^– background, creating a nonmotile strain. Strain DK8602 (table S1) lacked both A and S motility and formed a smooth-edged colony (Fig. 1A). When DK8602 was mixed with a nonmotile tgl⁺ donor strain (pilA, aglB1), the mixed population initially had a smooth colony edge reflecting both strains' lack of A and S motility (Fig. 1A). Recipient cells swarm beyond the original colony edge as flares when they are stimulated (Fig. 1B), which shows that the cells had assembled retractile pili.

Fig. 1. Tgl stimulation. Stimulated recipient cells became motile and swarmed outward, the donor strain remained nonmotile; it was found inside the original edge of the colony. A tgl mutant carrying an A– mutation (A–S– strain DK8602) was mixed with another nonmotile tgl+ mutant strain (DK8601) and spotted on agar. (A) At 0 hours, the mixed colony had a smooth edge (black arrowhead) because there were no motile cells (Scale bar, 500 µm). (B) After several hours, the tgl+ donor cells activate S motility in the tgl mutant recipient cells by stimulation. The outward swarming of the stimulated recipients after 4 days is indicated by the red arrowhead. This motility was transient; it lasted only a week (fig. S2). (C) Phase-contrast image of DK8601 (GFP– donor cells) mixed 1:1 with a mixture of DK8607 [GFP+ recipient cells (table S1)] and DK8602 (GFP– recipient cells), the two last-named strains at a 1:50 ratio. The original colony edge is indicated by the red arrowhead. (D) Epifluorescent image of the field shown in (C). The original colony edge is indicated by the dashed red line. (E) Phase-contrast image of DK8602 (GFP– recipient cells) mixed 1:1 with a mixture of DK8606 (GFP+ donor cells) and DK8601 (GFP– donor cells) at a 1:50 ratio. (F) Epifluorescent image of (E). [View Larger Version of this Image (128K GIF file)]

Because Tgl is required for the assembly of (detergent-resistant) PilQ multimers (15), the S motility of stimulated cells implies that those cells have assembled PilQ multimers, because they are able to extend and retract pili. To investigate the assembly of PilQ in stimulated recipients, a donor strain was needed that lacked such assemblies. The pilQ tgl⁺ strain is not an efficient donor for Tgl stimulation (15). However, although a pilQ¹²⁴¹ nonsense mutant did not assemble stable PilQ multimers (15), it could nevertheless serve as a Tgl donor. When a tgl⁺ pilQ¹²⁴¹ donor strain was mixed with tgl recipients, assembled PilQ multimers were readily detected by their resistance to dissociation after being boiled in SDS-containing buffer (Fig. 2). Unassembled PilQ monomers were also found in the stimulated recipients, as well as a 50-kD PilQ fragment that always accompanied PilQ assembly (15). The recipients before stimulation had only unassembled PilQ monomers and no 50-kD fragment (Fig. 2). The donor did not have assembled PilQ multimers, 50-kD fragment, or PilQ¹²⁴¹ monomers (Fig. 2). It appears that the mutant monomers whose carboxy ends had been truncated were degraded.

Fig. 2. Stimulated assembly of PilQ multimers. A mutant strain with an in-frame deletion in tgl failed to assemble PilQ multimers (top line to left of gel), although it produced the 98-kD PilQ monomer (other line on left). When the tgl strain was stimulated with the donor strain tgl+ pilQ1241 (a pilQ allele that produces unstable PilQ monomers in lane 2), boiling SDS–resistant PilQ multimers apparently formed at the expense of monomers (lane 3). [View Larger Version of this Image (28K GIF file)]

We tested the possibility that Tgl protein was transferred from donor to recipient cells. As expected, Tgl protein was found in the donor cells, but not in the recipient cells (Fig. 3A). However, after stimulation, large amounts of Tgl protein were also found in the harvested stimulated recipient cells (Fig. 3A). Thus, transient contact between Tgl⁺ donor cells and Tgl^– recipients results in the transfer of Tgl protein from donor to recipient. To challenge the specificity of transfer, we examined a cytoplasmic protein, GFP, in donor cells, recipient cells, and stimulated recipient cells. When only recipients contained GFP, it was detected only in the recipient cells and the harvested stimulated recipient cells (Fig. 3A). Conversely, when only donors contained GFP, it was detected only in the donor cells (Fig. 3A), which demonstrated the absence of contaminating donor cells in the harvested stimulated recipient cells. This experiment also shows that GFP is not transferred.

Fig. 3. Detection of Tgl protein transfer from donor cells to recipient cells. (A) Immunoblots lane 1, donor cells, DK8606; lane 2, recipient cells, DK8602; and lane 3, the harvested stimulated recipient cells (SRs). The GFP(a) row is a fluorogram of GFP– donor strain, DK8601, GFP+ recipient, DK8607, and their harvested SRs. The GFP(b) row shows GFP+ donor, DK8606, the GFP– recipient, DK8602, and their harvested SRs. GroEL is the loading control. (B) Fluorescence profile of a mixture of GFP– donor cells (strain DK8601) with GFP+ recipient cells (strain DK8607), separated by fluorescence-activated cell sorting (FACS) 36 hours after mixing. (C) Tgl Immunoblot of fractions from the FACS separation in (B), 0 hours after mixing and 36 hours after mixing. (D) Transfer of the CglB protein from donor (strain DK6204) to recipient cells (strain ASX1). The two peaks evident in B were harvested separately. [View Larger Version of this Image (16K GIF file)]

The only gene that can be stimulated in the S motility system is Tgl. However, five A motility genes can be stimulated: cglB, cglC, cglD, cglE, and cglF (17). The cglB gene has been cloned and sequenced; it encodes a lipoprotein that has no similarity to tgl, apart from a type II signal sequence (18). To test protein transfer in A motility stimulation, stimulated cglB recipient cells were separated from the donor cells after they had spread beyond the edge of the original spot. Indeed, the harvested, stimulated cglB mutant recipients contained large amounts of the CglB protein (Fig. 3D).

The concentration of Tgl and CglB proteins in stimulated cells was similar to the concentration in donor cells (Fig. 3), as if the donor and recipient cells shared their outer membrane lipoproteins equally. Myxobacteria may have evolved an efficient sharing of outer membrane lipoproteins, because they need to reverse their gliding direction frequently, 20 or more times per division cycle (19). Frequent reversal means frequently reconstructing the A and the S engines (4). Tgl and CglB (and perhaps CglC, D, E, and F) are needed to specify which cell poles have pili and which are active in slime secretion. This sharing of outer membrane lipoproteins among the thousands of cells in a swarm creates a primitive tissue.

References and Notes

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