Ras GTPase-like protein MglA, a controller of bacterial social-motility in Myxobacteria, has evolved to control bacterial predation by Bdellovibrio

Abstract

Bdellovibrio bacteriovorus invade Gram-negative bacteria in a predatory process requiring Type IV pili (T4P) at a single invasive pole, and also glide on surfaces to locate prey. Ras-like G-protein MglA, working with MglB and RomR in the deltaproteobacterium Myxococcus xanthus, regulates adventurous gliding and T4P-mediated social motility at both M. xanthus cell poles. Our bioinformatic analyses suggested that the GTPase activating protein (GAP)-encoding gene mglB was lost in Bdellovibrio, but critical residues for MglA(Bd) GTP-binding are conserved. Deletion of mglA(Bd) abolished prey-invasion, but not gliding, and reduced T4P formation. MglA(Bd) interacted with a previously uncharacterised tetratricopeptide repeat (TPR) domain protein Bd2492, which we show localises at the single invasive pole and is required for predation. Bd2492 and RomR also interacted with cyclic-di-GMP-binding receptor CdgA, required for rapid prey-invasion. Bd2492, RomR(Bd) and CdgA localize to the invasive pole and may facilitate MglA-docking. Bd2492 was encoded from an operon encoding a TamAB-like secretion system. The TamA protein and RomR were found, by gene deletion tests, to be essential for viability in both predatory and non-predatory modes. Control proteins, which regulate bipolar T4P-mediated social motility in swarming groups of deltaproteobacteria, have adapted in evolution to regulate the anti-social process of unipolar prey-invasion in the "lone-hunter" Bdellovibrio. Thus GTP-binding proteins and cyclic-di-GMP inputs combine at a regulatory hub, turning on prey-invasion and allowing invasion and killing of bacterial pathogens and consequent predatory growth of Bdellovibrio.

Figure 1. Predation and in cis complementation of B. bacteriovorus ΔmglA HI strains, on E. coli prey.

(A) Predation efficiency of the ΔmglA HI strain was assayed against predatory and non-predatory controls by the reduction of E. coli numbers over 48 hours. Three wild-type HI strains (HID13, HID26 and HID50) reduced E. coli numbers in liquid cultures by up to four logs (grey region shows known natural variation in predation rate between different wild-type HI isolates). The ΔmglA HI strain showed no reduction in E. coli numbers, comparable to a previously-studied, non-predatory ΔpilA HI strain, and to E. coli with no added B. bacteriovorus. (B) Reintroduction of the mglA ORF in cis to the ΔmglA HI strain in plasmid pK18::mglA restored predatory growth. Error bars represent 1 SD from the mean (for predation-testing of Δ bd2492 strain see Figure S5).

Figure 2. Host-independent invasion and attachment assays of ΔmglA strain and wild-type controls.

(A) Attachment assay: After 1 hour, 21.0% of E. coli cells were attached to and invaded by wild-type Bdellovibrio HI strain HID26 cells. A further 22.5% of E. coli cells were attached to, but not invaded by HID26 cells. 23.0% and 17.4% of E. coli cells were attached to by ΔmglA HI and ΔpilA HI, respectively. The ΔmglA HI and ΔpilA HI strains never invaded to form bdelloplasts. The attachment assay has the following variability: Percentage points (pp): WT 43.5%±15.5 pp; ΔmglA 23.0%±4.2 pp; ΔpilA 17.4%±7.3 pp. (B) Invasion assay: HI wild-type Bdellovibrio control HID50 was able to infect E. coli prey cells (26.3% of E. coli cells invaded to form bdelloplasts after 22 hours, 45/171 E. coli cells). Bdellovibrio ΔmglA HI strain could not invade E. coli prey (0.0% of E. coli cells invaded to form bdelloplasts after 22 hours, 0/319 E. coli cells). Fluorescent images show representative fluorescent E.coli S17-1::pMAL_p2-mCherry cells, either uninfected or rounded to form bdelloplasts.

Figure 3. B. bacteriovorus ΔmglA HI cells show sustained gliding motility; MglA-His₈ HD cells show hyper-reversals.

(A) B. bacteriovorus ΔmglA HI cells showed gliding motility on 1% agarose/CaHEPES. Gliding was sustained and progressive (cells were not hyper-reversing), as in the arrowed cell which moved at 9.49 µm hr⁻¹. Each panel is a 15 minute timepoint, starting from 540 minutes after the cells were added to the agarose surface (three “bystander” cells that have not yet commenced gliding represent a stationary marker). (B) B. bacteriovorus MglA-His₈ HD cells showed a high incidence of reversals during gliding motility on 1% agarose/CaHEPES compared to wild-type cells. Each larger panel shows a “trail-montage” of 60 minutes of gliding motility (150 second per frame): MglA-His₈ cells show no progressive gliding motility (reversing rapidly), whilst wild-type cells show sustained runs of gliding (seen as curving trails with direction changes). (C) Smaller panels show individual wild-type and MglA-His₈ HD cells gliding from an original start point (indicated by white dashed region), starting at 180 minutes after addition to the agarose surface and at 150 second intervals; arrow indicates direction of movement.

Figure 4. Protein alignment of MglA_Mx and MglA_Bd.

MglA_Mx (MXAN1925) alignment with MglA_Bd (Bd3734) shows significant sequence similarity between the two proteins. (A) The P-loop is conserved in B. bacteriovorus, although a serine is present in place of a glycine residue at position 21 (signified by arrow). The PM1/G1 threonine residue (B) and PM3 (C); and G2 (D) motifs are all conserved between the two proteins (for mglA genes, encoding MglA G21, co-occurring with mglB genes see Figure S1).

Figure 5. Gene synteny of bd2492–bd2495 homologues is conserved in B. bacteriovorus, M. xanthus and B. marinus.

Genes encoding a TPR domain protein are followed by genes encoding a DUF490 domain protein and an Omp85 superfamily protein in all three bacterial species. In M. xanthus, the three genes are interrupted by a gene encoding a putative Sec system ATPase, MXAN_5765. Percentage protein sequence identities and similarities with the B. bacteriovorus protein (NEEDLE global alignment) are shown underneath (for operon-confirmation of bd2492-2495 see Figure S6).

Figure 6. B. bacteriovorus RomR-mCherry and Bd2492-mCherry localised at the prey-interaction pole; MglA-mCherry showed variable diffuse foci.

B. bacteriovorus cells were incubated with E. coli S17-1 prey cells for 5 minutes, allowing sufficient time for some of the Bdellovibrio cells to attach to prey. Panels- A: The lower prey-cell shows a typical attached Bdellovibrio cell, with a RomR-mCherry focus at the anterior (attached) pole of the Bdellovibrio. B: The rightmost prey-cell shows a typical attached Bdellovibrio cell, with a Bd2492-mCherry focus at the anterior (attached) pole of the Bdellovibrio. C: MglA-mCherry Bdellovibrio cells had variable foci, including diffuse and unipolar localisations. From left to right, all panels show brightfield, fluorescent, and merged images and a graphical representation. Fluorescent exposure = 2 seconds.

Figure 7. Model for B. bacteriovorus predatory-pole regulation during prey-invasion and its relationship to M. xanthus bipolar motility-control proteins.

During prey-invasion; TamA_Bd, RomR_Bd and CdgA protein interactions occur (see Figure S2, S3 S4) at the single B. bacteriovorus pole. This could control localization of the TamA_Bd-like OMP at the prey-interaction pole or activate it to receive, (via its POTRA domains), and secrete predatory outer membrane or autotransporter proteins. The action of this secretion via Bd2495 TamA is essential to both predatory and HI lifestyles, and RomR_Bd, (which is also essential), may regulate or report the activity of the TamAB transport system, at the single predatory pole. Additional regulation of this activity could be influenced by c-di-GMP for which CdgA, (another hub protein that binds RomR and TPR Bd2492), is a receptor in Bdellovibrio. MglA_Bd interacts with Bd2492 at the predatory pole but also is found more diffusely in the cell. MglA interactions may regulate prey entry via TPR Bd2492, as deletion of MglA or TPR Bd2492 abolishes prey-invasion but not prey attachment. MglA deletion in Bdellovibrio greatly reduces the level of Type IV pilus formation at the single anterior pole. In contrast Keilberg and co-workers showed that M. xanthus RomR and MglB localise bipolarly asymmetrically, while MglA typically localises at the leading cell pole, during surface movement, to regulate both A- and S- motility (M. xanthus after [23]).