Tools and Approaches for Dissecting Protein Bacteriocin Import in Gram-Negative Bacteria

Abstract

Bacteriocins of Gram-negative bacteria are typically multi-domain proteins that target and kill bacteria of the same or closely related species. There is increasing interest in protein bacteriocin import; from a fundamental perspective to understand how folded proteins are imported into bacteria and from an applications perspective as species-specific antibiotics to combat multidrug resistant bacteria. In order to translocate across the cell envelope and cause cell death, protein bacteriocins hijack nutrient uptake pathways. Their import is energized by parasitizing intermembrane protein complexes coupled to the proton motive force, which delivers a toxic domain into the cell. A plethora of genetic, structural, biochemical, and biophysical methods have been applied to find cell envelope components involved in bacteriocin import since their discovery almost a century ago. Here, we review the various approaches that now exist for investigating how protein bacteriocins translocate into Gram-negative bacteria and highlight areas of research that will need methodological innovations to fully understand this process. We also highlight recent studies demonstrating how bacteriocins can be used to probe organization and architecture of the Gram-negative cell envelope itself.

Keywords: Gram-negative bacteria; bacteriocin; cell envelope; import; methods.

FIGURE 1

Bacteriocin import pathways. Bacteriocins bind to outer membrane receptors to get imported into the cell. Some bacteriocins (group B) use the receptor protein also as a translocator to cross the outer membrane, while exploiting the TonB system and PMF as an energy source. In case of group A bacteriocins the translocator differs from the receptor protein and the Tol system is used to enter the periplasm. Bacteriocins that degrade lipid II and prevent peptidoglycan recycling remain in the periplasm, while pore forming bacteriocins are inserted in the inner membrane. Nuclease bacteriocins use a distinct protein translocator to cross the inner membrane.

FIGURE 2

Approaches to finding bacteriocin translocon components. Translocon components can be isolated from membranes of sensitive strains by use of affinity tagged bacteriocins as bait. Competitive ligand binding or bacteriocin binding to a purified translocon component can inhibit bacteriocin activity and give indications about proteins engaged in bacteriocin entry. Bacteriocin-resistant mutants can be selected by use of high bacteriocin concentrations, or generated by transposon mutagenesis. Genomes of resistant mutants can further be analyzed for mutations underlying the resistance phenotype, which can give gene candidates for translocon components. A component of bacteriocin entry has to be further confirmed by complementation studies, where bacteriocin sensitivity is re-established once a resistant mutant is transformed with a gene of interest.

FIGURE 3

In vivo cross-linking strategy developed by White et al. for dissecting the import pathway of pyocin S2 through the FpvAI receptor. Photoactivatable crosslinking data were used to establish a three-step model of translocation. In step 1, pyocin S2 binds to FpvAI and mimics its cognate ligand, ferro-pyoverdine. In step 2, a PMF-dependent mechanical force, applied via the ExbB–ExbD–TonB1 complex in the IM (not shown), drives unfolding of the labile half of the receptor plug domain and the N terminus of pyocin S2 enters the receptor lumen. In step 3, pyocin S2 binds to TonB1 and this interaction drives further passage of the pyocin through the receptor lumen. Complete translocation is blocked by the force-resistant GFP, which enables identification of translocation intermediates and mapping of interactions that govern pyocin S2 cell entry [image taken and used with permission from White et al. (2017), CC BY-NC-ND 4.0].

FIGURE 4

FRAP experiments can be used to show that bacteriocin import is a PMF-dependent process. P. aeruginosa PAO1 cells are labeled with pyocin S2 AF488. The bleached region is highlighted (dashed circle). FRAP suggests pyoS2NTD-AF488 has translocated to the periplasm, where it can diffuse laterally. Absence of FRAP observed when cells are treated with 100 μM CCCP indicates that the pyocin remains bound to FpvAI in the OM and that the PMF is necessary for pyocin translocation. Scale bars, 1 μm [image taken and used with permission from White et al. (2017), CC BY-NC-ND 4.0].

FIGURE 5

Fluorescently labeled bacteriocins can be used to track outer and inner membrane protein clusters; used by Rassam et al. (2018) to show that bacteriocin-induced clustering of TolA in the IM mirrors that of OMPs in the OM. 2D-SIM z-slice showing significant co-clustering (yellow fluorescence) of GFP-TolA and ColE9AF594 in the IM and OM, respectively. Scale bars, 1 μm [image taken and used with permission from Rassam et al. (2018), CC BY-NC-ND 4.0].