Bacterial strains and culture
B. bacteriovorus HD100, and fluorescently tagged or gene-deletion strains, were grown predatorily on stationary-phase E. coli S17-1 prey (16 h, 29 °C), in Ca–HEPES buffer (5.94 g l−1 HEPES free acid, 0.284 g l−1 calcium chloride dihydrate, pH 7.6), or on YPSC (Yeast Extract Peptone Sodium Acetate Calcium) overlay platesas plaques within a lawn of E. coli S17-1 prey, as described previously37. To provide selection for fluorescent-tag or gene-deletion constructs, 50 mg ml−1 of kanamycin sulphate was added to growth media where appropriate. E. coli S17-1 cells, for B. bacteriovorus prey or fluorescent-tagging and gene-deletion manipulations, were grown for 16 h in YT broth (5 g NaCl, 5 g Difco Yeast extract, 8 g Difco Bacto Tryptone per litre, pH adjusted to 7.5 with NaOH) at 37 °C with shaking at 200 rpm.
Plasmid and strain construction
The primers used to generate fluorescently tagged or gene-deletion strains of B. bacteriovorus are documented in Supplementary Table 5, and the plasmid constructs used are documented in Supplementary Table 6. PCR amplification was performed with Phusion polymerase (New England Biolabs) according to the manufacturer’s instructions, using primers listed in Supplementary Table 5.
Generation of markerless deletion mutants
To construct markerless gene deletions of bd1334, bd2133, bd2439, bd2734, bd2740 and bd3182, between 750 bp and 1,000 bp of DNA upstream and downstream of the gene of interest were cloned into the suicide vector pK18mobsacB by Gibson assembly38 using the NEBuilder HiFi DNA assembly cloning kit (New England BioLabs). Gene-deletion vectors were introduced into B. bacteriovorus by conjugation (using donor E. coli S17-1 strains) and subsequently cured of the donor plasmid by sucrose suicide counter-selection, resulting in the integration of the gene knockout constructs via double-crossover homologous recombination. This process is further detailed and was described previously9,39. All gene deletions were verified by Sanger sequencing. To visualize any effect of the single-gene deletions bd2734 and bd2740, during the initial stages of the predatory cycle, a C-terminal mCherry fusion to the constitutively expressed PilZ protein Bd0064 was introduced via single-crossover recombination, illuminating the B. bacteriovorus cell body, as described previously40,41,42.
Generation of Bd3182 mutation of catalytic serine S782 to alanine
The S782A mutation of Bd3182 was introduced by amplifying the gene on either side of the S74 codon and introducing the mutation in the primers (Supplementary Table 4), followed by Gibson assembly as above.
Fluorescence tagging of B. bacteriovorus gene products
Protein fluorophore tags (mCherry, mNeonGreen, mTFP or mCerulean3) were fused to the C-terminus of target genes by PCR amplification of the target gene, without its stop codon, and amplification of the fluorophore gene, followed by Gibson cloning using the NEBuilder HiFi or GeneArt assembly kits (New England Biolabs) according to the manufacturer’s instructions, into the mobilizable broad-host-range vector pK18mobsacB43. Each construct was conjugated into B. bacteriovorus HD100 as described previously37. In some cases, a particular fluorescent tag, such as mCherry for Bd2133, was not tolerated by a gene under study for reasons unknown. In such cases, a different colour combination of strains was used in experiments. Single-crossover constructs of Bd0635–mCherry and Bd0635–mTeal were obtained by standard cloning methods rather than by Gibson assembly (details in Supplementary Table 4).
For multiple fluorophore combinations, either the Bd0064 or the CpoBBd0635 tagging constructs were made with 1,000 bp of flanking DNA and ex-conjugants of these were subjected to sucrose suicide selection to generate a double-crossover event, replacing the genomic copy of the gene with the tagged version, via the same methodology used to generate single-gene-deletion strains (above). The second fluorophore-tagged gene was then introduced, where required, as above by single crossover into the B. bacteriovorus genome, as before.
Labelling of cell wall muropeptides with fluorescent d-amino acids and imaging
Pulse labelling of predator-modified prey peptidoglycan during early predation events with the ‘blue’ fluorescent d-amino acid HADA to label predatory porthole formation was carried out as described previously1. B. bacteriovorus HD100 cells were grown predatorily for 16 h at 29 °C on stationary-phase E. coli S17-1 prey, until the prey culture was lysed. The B. bacteriovorus were then filtered through a 0.45 µm filter (yielding ~2 × 108 plaque-forming units (pfu) ml−1) and concentrated 30 times by centrifugation at 12,000 × g for 5 min. The resulting pellet was resuspended in Ca–HEPES buffer (2 mM CaCl2, 25 mM HEPES, pH 7.6). E. coli S17-1 cells were grown for 16 h in Luria–Bertani medium at 37°C with shaking at 200 rpm and were back diluted to an optical density at 600 nm (OD600) of 1.0 in Ca–HEPES buffer (yielding ~1 × 109 colony-forming units (cfu) ml−1). A total of 50 μl of this B. bacteriovorus culture was mixed with 40 µl of the pre-labelled E. coli and 30 µl of Ca–HEPES buffer and incubated at 30 °C. For pulse labelling, 1.2 µl of a 50 mM stock of HADA in DMSO was added 10 min before the sampling time point for microscopy and returned to 30 °C incubation. At each time point, all of the 120 µl predator–prey sample was transferred to 175 µl ice-cold ethanol and incubated at −20 °C for at least 15 min to fix the cells. The cells were pelleted by centrifugation at 17,000 × g for 5 min, washed with 500 µl PBS and resuspended in 5 µl Slowfade (Molecular Probes), and stored at −20 °C before imaging. Samples (2 µl) were imaged using a Nikon Ti-E inverted fluorescence microscope equipped with a Plan Apo 100× 1.45 Ph3 objective, a DAPI filter cube and an Andor Neo sCMOS camera using DAPI settings for detection of HADA (emission maximum 450 nm).
B. bacteriovorus predation on E. coli in liquid culture
Assays were based on, and modified from, those detailed in a previous study11. In summary, B. bacteriovorus gene-deletion mutants were grown predatorily (as above). The pfu inputs for each gene-deletion strain were matched using the SYBR Green DNA stain, to ensure that equal titres of B. bacteriovorus for each strain were used as starting inputs. Briefly, B. bacteriovorus were incubated with SYBR Green dye for 90 min (300 rpm double orbital, in darkness, in triplicate), before fluorescence was measured using a FLUOstar Omega plate reader (BMG Labtech; excitation 485 nm, emission 520 nm, gain 800), with fluorescence values being interpolated into relative pfu per ml counts using a pfu–SYBR Green fluorescence correlation curve. E. coli S17-1 cells were grown as above and subsequently back diluted to OD600 1.0 (approximately 1 × 109 cfu ml−1) in dilute nutrient broth. Predatory cultures (containing approximately 1 × 109 cfu ml−1 E. coli prey and 1 × 108 pfu ml−1 B. bacteriovorus WT or mutant) were inoculated in triplicate into a black OptiPlate (Corning), along with media-only, B. bacteriovorus-only (no prey) and prey-only (no B. bacteriovorus) controls. The OD600 of the predatory culture was measured every 20 min, for 18 h (200 rpm, double orbital, in triplicate) to give a prey survival curve, in which a drop in OD600 is indicative of successful predation and prey lysis, because prey cells, but not predators, are large enough to produce an optical density at 600 nm.
OD600 data were exported to Excel 2016 and then analysed using CurveR, according to the method documented previously11 to analyse prey cell lysis and predation dynamics. Rmax indicates the maximum rate of prey cell lysis. S indicates the inflection point, the time at which the maximum rate of prey cell lysis (Rmax) occurs, that is, the steepest point on the curve.
To further assess the effect of deletion in early stages of the predatory cycle, synchronous predatory infections of B. bacteriovorus HD100:Bd0064mCherry:Δbd2734 and HD100: Bd0064mCherry:Δbd2740 on E. coli S17-1 PZMR100 were set up as described above, with aliquots removed at 30 min after the mixing of predator and prey. Cells were immobilized on a thin 1% Ca–HEPES buffer agarose pad and imaged as above. Images were minimally processed using the sharpen and smooth tools (ImageJ), with adjustments to brightness and contrast to ensure clarity. Full fields of view were visually scored for complete B. bacteriovorus entry as described previously44. The percentage of bdelloplasts with a fully entered B. bacteriovorus at 30 min was derived from manual inspection of the total number of bdelloplasts with B. bacteriovorus fully entered at 30 min divided by the total number of bdelloplasts visually characterized. Images were analysed from three biological repeats. Each deletion mutant strain was imaged and analysed alongside B. bacteriovorus HD100 (control) within the same experiment.
Phase-contrast and epifluorescence microscopy
The in vivo fluorescence of MAT protein–XFP tags (XFP meaning any of the different fluorescent protein tags) during the predatory life cycle was examined. Approximately synchronous predation of E. coli S17-1 PZMR100 by B. bacteriovorus strains was prepared by combining a 10-times-concentrated B. bacteriovorus predatory culture with E. coli S17-1 PZMR100 (standardized to an OD600 of 1) and Ca–HEPES at a ratio of 5:4:3, respectively, as described previously32,45. Progress through the predatory life cycle (and position of the fluorescently tagged protein under investigation) was visualized via fluorescence microscopy (10 s exposure) at the following time points (in minutes): 0, 15, 30, 45, 60, 120, 180 and 240, by withdrawing 10 μl of the culture and immobilizing on a thin 1% Ca–HEPES buffer agarose pad. Cells were visualized with a Nikon Ti-E inverted epifluorescence microscope equipped with a Plan Apo 100× Ph3 oil objective lens (Numerical Aperture 1.45) and the following filters: mCherry (excitation 550–600 nm, emission 610–665 nm), GFP, green fluorescent protein, (for mNeonGreen; excitation 460–500 nm, emission 515–530 nm), CFP, cerulean fluorescent protein, (mCerulean; excitation 420–450 nm, emission 470–490 nm) and TFP, teal fluorescent protein, (mTeal; excitation 420–450 nm, emission 515–530 nm).
Images were acquired using an Andor Neo sCMOS camera with Nikon NIS software and analysed using ImageJ (Fiji). Images were minimally processed using the sharpen and smooth tools, with adjustments to brightness and contrast. Where stated, false colour was used in channels for display purposes.
Image analysis
Images were manipulated with ImageJ (Fiji distribution) software using the sharpen and smooth tools, and by duplication of the region of interest for presentation. Images were analysed using the MicrobeJ plug-in for ImageJ46, which automates the detection of bacteria within an image. Attack-phase B. bacteriovorus cells were detected with parameters as default, with fluorescence detected by the foci method with default parameters and associated with parent bacteria with a tolerance of 0.1 µm.
For the analysis of CpoB–mCherry foci with Bd0064–mCerulean cytoplasmically labelled B. bacteriovorus, prey E. coli were detected as bacteria, B. bacteriovorus cells were detected using the medial axis method in the mCerulean channel and associated with bacteria with a tolerance of 0.1 µm, and CpoB–mCherry foci were detected using the fit shape as circle method in the mCherry channels with a maximum area of 0.25 µm2 and associated with B. bacteriovorus cells with a tolerance of 1 µm.
Manual inspection of the analysed images confirmed that the vast majority of cells and foci were correctly assigned. The shape measurements including the angularity, area, aspect ratio, circularity, curvature, length, roundness, sinuosity, solidity and width were measured for each type of cell.
Co-localization analysis
Images were analysed using ImageJ software (Fiji), using the cell counter plug-in. Adjustments to brightness and contrast for whole images were made until CpoBBd0635 (mCherry: Bd2133–mNeonGreen or mTeal (Bd2439–mCherry or Bd2740–mCherry)) and Bd2133–mNeonGreen, Bd2439–mCherry or Bd2740–mCherry foci, if present, were visible. Bdelloplasts were then manually (visually) scored for the presence of a CpoBBd0635 focus. Of the bdelloplasts that contained a CpoBBd0635 focus, the number of bdelloplasts for which a Bd2133, Bd2439 or Bd2740 focus was coincident was scored for approximately 60 bdelloplasts, originating from two biological replicates.
Visualization of external mCherry-tagged MAT protein expression
Semi-synchronous predation experiments were set up as above. Samples of 1.4 ml were pre-fixed in 0.25% paraformaldehyde and recovered by centrifugation at 17,000 × g for 2 min, then fixed in 2.5% paraformaldehyde in Dulbecco’s PBS for 10 min at 37 °C. Cells were recovered by centrifugation at 17,000 × g for 2 min, washed twice in 100 µl blocking solution (2% bovine serum albumin, BSA, in Dulbecco’s phosphate buffered saline, PBS) and then incubated in blocking solution for 45 min. Samples were further incubated in anti-mCherry antibody (Invitrogen PA5-34974) diluted 1:1,000 in blocking solution for 1 h. Cells were recovered by centrifugation at 17,000 × g for 2 min, then washed twice in blocking solution before a final incubation with secondary antibody of goat anti-rabbit IgG with Alexa Fluor plus 488 (Invitrogen A32731). Cells were then washed twice with blocking solution before imaging with a Nikon Ti-E microscope as described above. Some samples were further stained with FM46-4 by resuspension in 10 mM stain in water. Images were analysed using ImageJ software (Fiji). Adjustments to brightness and contrast for whole images were made until FM46-4-stained flagella and anti-mCherry foci were visible. Cells were then manually (visually) scored for the presence of flagella (FM46-4 red channel) and the presence and positioning of anti-mCherry foci (GFP channel).
Microscopic statistical analysis
Statistical analysis was performed in Prism 8.2.0 (GraphPad). Data were first tested for normality and then analysed using the appropriate statistical test. The number of biological repeats, n values for cell numbers and the statistical test applied are described within each figure legend.
Cloning, expression and purification
Bd213321–1,031 was synthesized and inserted into pET29a between NdeI and XhoI (Twist Bioscience), producing a construct with an N-terminal PelB leader sequence and a hexa-His tag. Bd2133662–1,031 and Bd3182668–922 were cloned into pCold1 using NdeI and XhoI restriction sites. The final constructs contained an N-terminal 24-amino-acid tag containing a transcription-enhancing element, a hexa-His tag, a factor Xa cleavage site and a Tobacco Etch Virus (TEV) cleavage site. Bd2133910–1,031 was produced by Q5 (New England Biolabs) deletion using the Bd2133662–1,031 plasmid as a template. Bd3182668–922_S782A was produced by Q5 mutagenesis using Bd3182632–922 as a template. Bd1334818–1,031 and Bd1334914–1,151 were cloned into pCold1 using XhoI and HindIII restriction sites. The final constructs contained an N-terminal 30-amino-acid tag containing a transcription-enhancing element, a hexa-His tag, a factor Xa cleavage site and a TEV cleavage site. Bd2734691–843 was cloned into pET26b using NcoI and XhoI restriction sites. The final construct contained a pelB leader sequence, Met-Ala and a hexa-His tag. Bd2439837–1,107 was cloned into pET26b using BamHI and XhoI restriction sites. The final construct contained a pelB leader sequence, Met-Asp-Ile-Phe-Ile-Asn-Ser-Asp-Pro and a hexa-His tag. Bd2740518–627 was synthesized and inserted into pET29a between NdeI and XhoI (Twist Bioscience), producing a construct with an N-terminal PelB leader sequence, a hexa-His tag and an Ala-Ser linker.
Constructs were expressed in E. coli BL21 RIPL (DE3). Cells were grown to an OD600 of 0.5–0.7 and induced with 0.5 mM IPTG (isopropyl ß-D-1-thiogalactopyranoside) at 18 °C for 16 h. Cells were harvested by centrifugation and resuspended in 50 mM HEPES, 500 mM NaCl and 20 mM imidazole, pH 7.5. The cells were lysed by sonication on ice with the exception of Bd213321–1,031, which was lysed by three passages through an EmulsiFlex C3 high-pressure homogenizer (Avestin). The lysates were loaded onto a 5 ml Ni-NTA column (GE Healthcare) and washed with 10 column volumes of lysis buffer. The protein was eluted with a gradient of 20–500 mM imidazole. TEV was added to constructs containing cleavage sites that were subsequently dialyzed into 150 mM NaCl, 20 mM HEPES and 20 mM imidazole, pH 8.0, overnight at 4 °C. The protein was then passed through a 1 ml Ni-NTA column to remove TEV and uncleaved protein. The proteins were finally passed through a Superdex 200 26/60 column (GE Healthcare), which was pre-equilibrated with 20 mM HEPES and 150 mM NaCl, pH 7.5.
To express selenomethionine-labelled Bd2133662–922, a 60 ml overnight culture of BL21 RIPL (DE3) cells in LB was centrifuged and resuspended in minimal media supplemented with kanamycin and chloramphenicol to a final volume of 1 l. At OD600 0.4, 0.1 g of lysine, 0.1 g of threonine, 0.1 g of phenylalanine, 0.05 g leucine, 0.05 g of isoleucine, 0.05 g of valine and 0.06 g of selenomethionine were added to the culture. After 15 min, IPTG was added to 1 mM. Cells were harvested by centrifugation after 16 h at 18 °C and stored at −20 °C. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels were imaged using Quantity One v4.6.8.
Crystallization and data collection
For crystallization, proteins were concentrated to 3–10 mg ml−1 using a spin concentrator. Protein was mixed with precipitant in a 1:1 ratio, with drop sizes of 0.8–4 μl, and crystallized using vapour diffusion at 18 °C.
Crystals of Bd3182632–922 formed in Morpheus screen C1 (0.03 M sodium nitrate, 0.03 M sodium phosphate dibasic, 0.03 M ammonium sulphate, 0.1 M imidazole and MES (2-ethanesulfonic acid), pH 6.5, 20% v/v PEG (polyethylene glycol) 500 MME (monomethylether), 10% w/v PEG 20000) in spacegroup P21, Morpheus screen G12 (1.0 M sodium citrate tribasic dihydrate, 0.1 M HEPES, pH 7.0) with spacegroup I2 and Proplex screen E3 (0.1 M magnesium acetate tetrahydrate, 0.1 M MES, pH 6.5, 10% w/v PEG 10000) in a different P21 spacegroup. Crystals were subject to diffraction at Diamond Light Source at 100 K at wavelength 0.970–0.976 Å on beamline IO3. The phases were solved using Phenix MR47 with a low sequence homology (<10% sequence ID) ensemble of pruned T5 phage-tail fibre (PDB code 4UW8 (ref. 14)) and GP12 (PDB code 3GW6 (ref. 10)).
Crystals of Bd2734691–843 grew in JCSG+ screen A3 (0.2 M ammonium citrate dibasic, 20% w/v PEG 3350) in spacegroup P21. Crystals were subject to diffraction at Diamond Light Source at 100 K at wavelength 0.98 Å on beamline IO4. The phases were solved using Phenix MR47 with a homology model generated by ColabFold13.
Crystals of Bd1334914–1,151 formed in PACT screen B9 (0.2 M lithium chloride, 0.1 M MES, pH 6.0, 20% w/v PEG 6000) in spacegroup R3. Crystals were subject to diffraction at Diamond Light Source at 100 K at wavelength 0.98 Å on beamline IO4. Crystals of Bd1334818–1,151 grew in Morpheus screen C1 (0.03 M sodium nitrate, 0.03 M sodium phosphate dibasic, 0.03 M ammonium sulphate, 0.1 M imidazole and MES, pH 6.5, 20% v/v PEG 500 MME, 10% w/v PEG 20000) in spacegroup C2. Crystals were subject to diffraction at Diamond Light Source at 100 K at wavelength 1 Å on beamline IO4. The phases of the Bd1334818–1,151 crystals were solved using Phenix MR47 with a homology model generated by ColabFold13. The phases of the Bd1334914–1,151 crystals were solved using Phenix MR47 with a fragment of the Bd1334818–1,151 structure.
Crystals of Bd2439837–1,107 grew in multiple conditions: PACT H3 (0.2 M sodium iodide, 0.1 M Bis–Tris propane, pH 8.5, 20% w/v PEG 3350) in spacegroup P21221 and MIDASplus E9 (0.1 M lithium sulphate, 0.1 M tris, pH 8.0, 25% v/v Jeffamine ED-2003) in spacegroup P4. Crystals were cryocooled in the mother liquor with an addition of 25% ethylene glycol. Crystals were subject to diffraction at the European Synchrotron Radiation Facility at 100 K at wavelength 0.976 Å on beamlines id30a and id30b. The phases were solved using Phenix MR47 with a homology model generated by ColabFold13. Bd2439837–1,107–GlcNAc–MurNAc crystals were grown in PACT H3 as above, supplemented with 100 mM GlcNAc–MurNAc. To prevent ethylene glycol entering the binding pocket, these crystals were cryoprotected with 25% PEG 400.
Crystals of native and selenomethionine-labelled Bd2133662–1,031 grew in Morpheus screen B3 (0.03 M sodium fluoride, 0.03 M sodium bromide, 0.03 M sodium iodide, 0.1 M imidazole and MES, pH 6.5, 20% v/v glycerol, 10% w/v PEG 4000) in spacegroup P6322. Crystals were subject to diffraction at Diamond Light Source at 100 K at wavelength 0.98 Å (native) on beamline IO4-1 and 0.89 Å (selenomethionine protein) on beamline IO3. The phases of the Bd2133662–1,031 crystals were solved using experimental phasing. Selenium sites of the selenomethione-labelled protein crystals were identified via SAD by SHELX48, and Phenix47 Autosol was used to phase the data via SIRAS using the selenomethionine and native datasets. For model building, the symmetry was dropped to p63 to allow asymmetric modelling of the N-terminal beta strands. Molecular replacement of the selenomethioine-labelled protein structure was used to solve the phases of the native dataset. Crystals of Bd2133910–1,031 grew in Morpheus screen A4 (0.03 M magnesium chloride, 0.03 M calcium chloride, 0.1 M imidazole and MES, pH 6.5, 12.5% v/v MPD (2-methyl-2,4-pentanediol), 12.5% PEG 1000, 12.5% w/v PEG 3350) in spacegroup P212121. Crystals were subject to diffraction at Diamond Light Source at 100 K at wavelength 0.75 Å on beamline IO4 and solved using a fragment of the larger model.
Bd2740518–627 crystallized in MIDAS screen F1 (0.1 M HEPES, pH 6.5, 40% polypropylene glycol bisaminopropylether 2000) in space group P212121. Crystals were subject to diffraction at Diamond Light Source at 100 K at wavelength 0.979 Å on beamline IO4 and solved using molecular replacement with a ColabFold13 model.
All structures were manually completed and altered in CCP4i2 v1.1.0 and COOT 0.9.8.1 (ref. 49), and refined using Phenix 1.20.1-4487 (ref. 47). Statistics for data collection and refinement are presented in Supplementary Table 2.
Glycan arrays
Glycan arrays were performed as a service by Z Biotech. The array was blocked for 30 min using glycan array blocking buffer (Z Biotech item number 10106). Alexafluor-555-labelled samples were diluted in glycan array assay buffer (Z Biotech item number 10107) to the desired concentrations and then applied directly to the array. The array was covered with an adhesive film and shaken at 80 rpm for 1 h at room temperature. The array was then washed three times with glycan array assay buffer and two times with MilliQ water. The array was read using an Innopsys InnoScan 710 Microarray Scanner with a high-power laser at 1× photomultiplier gain. Proprietary software was used to detect each spot on the array and calculate the relative fluorescence unit (RFU) intensity for each spot. Background RFU was subtracted from each spot’s RFU value. The median of four repeat spots was determined for each glycan.
Electron microscopy
To visualize a full-length fibre, Bd213321–1,031 was subjected to negative-stain electron microscopy. Formvar and carbon grids (EMResolutions) were glow discharged with an Elmo glow discharge system (Cordouan) for 1 min at 3 mA. The protein was diluted to 1 μg ml−1 in 150 mM NaCl and 20 mM HEPES, pH 7.5, and 5 μl of protein was added followed by 5 μl of 2% uranyl acetate. Micrographs were taken using a 200 kV LaB6 Jeol 2100Plus 200 kV with a Gatan OneView IS at 50,000× magnification.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
