Outer Membrane Porin F in E. coli Is Critical for Effective Predation by Bdellovibrio

ABSTRACT Bdellovibrio and like organisms (BALOs) are a unique bacterial group that live by predating on other bacteria, consuming them from within to grow and replicate before the progeny come out to complete the life cycle. The mechanisms by which these predators recognize their prey and differentiate them from nonprey bacteria, however, are still not clear. Through genetic knockout and complementation studies in different Escherichia coli strains, we found that Bdellovibrio bacteriovorus strain 109J recognizes outer membrane porin F (OmpF) on the E. coli surface and that the activity of the E. coli EnvZ-OmpR regulatory system significantly impacts predation kinetics. OmpF is not the only signal by which BALOs recognize their prey, however, as B. bacteriovorus could eventually predate on the E. coli ΔompF mutant after prolonged incubation. Furthermore, recognizing OmpF as a prey surface structure was dependent on the prey strain, as knocking out OmpF protein homologues in other prey species, including Escherichia fergusonii, Klebsiella pneumoniae, and Salmonella enterica, did not always reduce the predation rate. Consequently, although OmpF was found to be an important surface component used by Bdellovibrio to efficiently recognize and attack E. coli, future work is needed to determine what other prey surface structures are recognized by these predators. IMPORTANCE Bdellovibrio bacteriovorus and like organisms (BALOs) are Gram-negative predatory bacteria that attack other Gram-negative bacteria by penetrating their periplasm and consuming them from within to obtain the nutrients necessary for the predator’s growth and replication. How these predators recognize their prey, however, has remained a mystery. Here, we show that the outer membrane porin F (OmpF) in E. coli is recognized by B. bacteriovorus strain 109J and that the loss of this protein leads to severely delayed predation. However, predation of several other prey species was not dependent on the recognition of this protein or its homologues, indicating that there are other structures recognized by the predators on the prey surface that are yet to be discovered.


Supplementary methods
Bacterial strains and culturing conditions 30 All the bacterial strains used in this study are listed in Table S1. Each of the prey and their isogenic mutants were routinely propagated on lysogen broth (LB) agar plates. Fresh single colonies were cultured in LB broth, incubated overnight in a shaking incubator at 37 ºC, centrifuged (5000 x g, 15 min) and the pellet was re-suspended in the predation media. All the predatory strains were routinely grown as described previously (1, 2) using E. coli 35 MG1655/pUCDK as the prey.

Bioluminescence assay to monitor the predation kinetics
The E. coli prey strains were rendered bioluminescent by transforming them with pGEN-luxCDABE (3), a gift from Harry Mobley (Addgene plasmid # 44918; 40 http://n2t.net/addgene:44918; RRID: Addgene_44918). Overnight cultures of these prey strains were grown in LB broth supplemented with 100 µg/ml ampicillin at 37 ºC before being diluted to an optical density (OD600nm) of 0.05 in dilute nutrient broth (DNB; 1/10 NB) containing 3 mM MgCl2 and 2 mM CaCl2. The predator was grown as described above, filtered, and diluted two-fold in 25 mM HEPES (with 3 mM MgCl2 and 2 mM CaCl2, pH 7.2). 45 The predator and prey cell densities in each sample were determined using top agar plates and colony counts, respectively, as described previously (4), and were used to calculate the predator-prey ratio (PPR). The predator and prey preparations were mixed 1:1 (v:v; 100 µl each) in the wells of a 96-well plate (white, Greiner, USA) and the bioluminescence was measured every ten minutes as described previously (5).

Prey viability assessment
For these experiments, the prey was diluted to OD 0.05 in DNB. B. bacteriovorus 109J was grown as above and diluted in HEPES buffer (with 3 mM MgCl2 and 2 mM CaCl2, pH 7.2).
The predator dilutions and prey suspensions were mixed (1:1 (v:v)) so that the predator-to-55 prey ratio was 6.25, 25 or 100. Each sample was then incubated with shaking incubator (250 rpm) at 30 ºC for 1 hour, after which the viability of the prey was determined using plate counts on LB agar plates.

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The pCA24N plasmid was linearized for In-Fusion® cloning using primers pCA ir and pCA if primers (Table S2). The ompF gene was amplified from E. coli MG1655 with its flanking regions using primers pCA-Omp F and pCA-Omp R primers (Table S2). After purifying both the vector and insert, they were recombined using the In-Fusion® manufacturer's suggested protocol, generating the complementation plasmid, pCA-ompF. This plasmid was 65 transformed into E. coli DH5α cells, which were then grown on LB agar plates containing chloramphenicol (35 µg/ml). The plasmid from an individual colony was purified and sequenced using the pCA24 seq F and pCA24 seq R primer set (Table S2). Once the sequence was verified, the plasmid (pCA-ompF) was transformed into the E. coli BW25113 strains.

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The same was used to also construct the pCA-ompC plasmid. For this, the ompC gene was amplified from wild-type E. coli BW25113 with additional flanking region using primers pCA-ompC F and pCA-ompC R (Table S2). After construction, transformation and purification of the plasmid as above, its sequence was confirmed using primers pCA24 seq F 75 and pCA24 seq R (Table S2).

Microscopic analyses of predation
Using E. coli S17 λ-pir as the donor, plasmid pMQ414, which expresses the tdTomato fluorescence protein (6), was transferred into B. bacteriovorus 109J through conjugation. The 80 predation tests were conducted using this fluorescent predatory strain and a synchronized attack, to ensure many of the prey were attacked at the same time, as described previously (7).
Briefly. after growth of the fluorescent predator using the same protocol as described above, it was concentrated 10-fold by centrifugation (7000 x g, 15 min) and resuspended in fresh HEPES buffer (with 3 mM MgCl2 and 2 mM CaCl2, pH 7.2). For these predation assays, 85 wild-type E. coli BW25113 and three isogenic mutants (i.e., ΔompF, ΔompR and ΔenvZ) were all used as prey. Each was grown overnight as above, centrifuged (7000 x g, 15 min) and resuspended in HEPES buffer to an OD600nm of 4.0. These samples (i.e., predator and prey solutions) were stored at 30 ºC for 10 minutes before they were mixed 1:1 (v:v). The mixed cultures were incubated in a shaking incubator at 30 ºC and samples were taken at set 90 times (i.e., 0, 20 and 60 min) and fixed with an equal volume of 8% (w/v) paraformaldehyde (PFA) prepared in the same HEPES buffer. The fixed samples were stored at 4 ºC until being observed by confocal microscopy. The number of each prey cell type (i.e., free prey, prey with a predator attached or bdelloplast) were counted and analyzed at the indicated time points.

Growth of E. coli at Higher Osmolalities
To study the impact of the medium osmolality on predation rates, E. coli BW25113 and E. coli JW0912 (ΔompF) were grown in LB medium prepared without NaCl addition. Before autoclaving, NaCl was added to a final concentration of 0, 0.25, 0.5 and 1% (w:v), generating 100 osmolalities of 78, 162, 256 and 427 mOsm/kg, respectively, for each medium. Growth of the prey was conducted as described above. After growth overnight, the prey cells were pelleted (5000 x g, 15 min), washed in sterile HEPES to remove the salts (1, 8) and resuspended in fresh DNB to an OD of 0.05, as described previously (9). Deletion of the ompF gene in each E. coli strain was confirmed by PCR using the primers listed in Table S2.
Constructing ompF knockout mutants in the non-E. coli prey 115 Deletion of the ompF gene in E. fergusonii and ompK35 (ompF homologue (68% identity based on amino acid sequence)) in K. pneumoniae was achieved using a suicide plasmid as described previously (12). Briefly, a suicide plasmid harboring a sacB gene cassette, kanamycin resistance gene cassette, the R6K replication origin and a RP4-oriT was constructed. Sets of primers (Table S2) were used to amplify approximate 1 kb homologous 120 recombination arms flanking the genes in K. pneumoniae and E. fergusonii. These homologous recombination arms included the first and the last 20~30 amino acids of the gene in each case. Each was then fused through a third PCR reaction and ligated to the suicide plasmid using the In-Fusion® HD Cloning kit (Clontech). The ligated plasmid was transformed into E. coli S17 λ-pir through chemical transformation and transferred via 125 conjugation to the corresponding recipient strain (E. fergusonii or K. pneumoniae). E.
fergusonii ATCC 35473 and both K. pneumoniae WGLW1 and WGLW2 are naturally resistant to ampicillin, allowing us to screen the conjugants on LB agar plates containing 100 µg/mL ampicillin (to select against the E. coli S17 λ-pir donor cells) and 50 µg/mL kanamycin (to select for merodiploids). One merodiploid mutant in each case was selected and grown on an LSW-sucrose agar plate (tryptone 10 g/L, yeast extract 5 g/L, glycerol 5 ml/L, NaCl 0.4 g/l, sucrose 100 g/L and agar 20 g/L) (12) to generate a double crossover mutant. One colony was then selected and grown on LB agar with no antibiotics. Loss of the conjugated plasmid in this strain was confirmed by PCR using the primers listed in Table S2, as well as phenotypically as the mutant was unable to grow in presence of kanamycin.

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To generate an ompF deletion in S. enterica LT2, lambda red recombineering was employed as previously described (10,13). The resulting ompF knockout clone had its gene replaced with the kanamycin resistance cassette, leaving only the first 30 and last 18 amino acids of the host ompF gene. plates were incubated at 30 °C until clear plaques were visualized. Individual plaques were then collected and sub-cultured with freshly prepared prey to isolate the predators. Each predatory strain was identified as being Bdellovibrio based on their 16S rDNA sequence, which had high homology to that of B. bacteriovorus 109J (Table S3).

Reproducibility and statistical analysis
Unless specified, each experiment was performed in triplicate and the standard deviations are presented on the graphs as error bars. Normal distribution of each dataset was verified using the Shapiro-Wilk test. None of the samples showed a substantial departure from the normality (p > 0.05) and, thus, the student t-test was used to evaluate statistical significance between 160 two sets of data. Significance is indicated within the graphs using: a -p < 0.05; b -p < 0.01; c -p < 0.001.   The alignment was performed using the PROMALS3D server using the default parameters 230 (17,18). The structure of OmpF of E. coli MH225 was retrieved from RCSB Protein Data Bank (https://www.rcsb.org) and used as the reference structure (Sequence "s001"). The first line in each block shows conservation indices for positions with a conservation index above 5.
Each representative sequence has a magenta name and is colored according to PSIPRED (19)