Fig 1.
SaPIs promote the survival of transductants.
S. aureus RN4220 derivative strains either devoid of (red) or harboring either SaPIbov1 (blue), SaPINY940 (green) or SaPI1 (purple) were infected with an MOI of 1:10 (phage:bacteria) of the indicated phages and acquisition of a chromosomal cadmium resistance marker (lateral transduction) (top row panels) or a plasmid-borne chloramphenicol resistance cassette (generalized transduction) (bottom row panels) was determined 18 h post-infection. Bold horizontal lines in each boxplot represent the median and lower and upper hinges the first and third quartiles, respectively (n = 7 biological replicates for strains infected with either Φ11 or Φ11 Δdut or n = 3 biological replicates for strains infected with either 80α or 80α Δsri). Assessment of statistically significant differences between groups was performed using a two-sided Student’s t-test on log10 transformed data. p-values are indicated above the respective comparison, ns not significant. Limit of detection (LOD) for this assay is 10 transductants per ml.
Fig 2.
SaPIs reduce phage reproduction and lysogenization but increase cell viability.
S. aureus RN4220 derivative strains either devoid of (red) or harboring either SaPIbov1 (blue), SaPINY940 (green) or SaPI1 (purple) were infected with an MOI of 1:10 (phage:bacteria) of the indicated phages. Phage titers (A), viable cells (B) and the relative number of lysogens per viable cell (C) for each recipient were determined. Note that the number of lysogens could not be determined for SaPINY940 due to an incompatibility of selection markers. Bold horizontal lines in each boxplot represent the median and lower and upper hinges the first and third quartiles, respectively (n = 7 biological replicates for strains infected with either Φ11 or Φ11 Δdut or n = 3 biological replicates for strains infected with either 80α or 80α Δsri). Assessment of statistically significant differences between groups was performed using a two-sided Student’s t-test on log10 transformed data. p-values are indicated above the respective comparison, ns not significant. Limits of detection (LOD) for this assay are 100 plaques per ml (A), 100 colonies per ml (B).
Fig 3.
Assessment of strain viability, phage titers and transduced markers at different time points following infection.
The indicated strains either devoid of (red) or harboring either SaPIbov1 (blue) or SaPINY940 (green) were infected with an MOI of 1:10 (phage:bacteria) with the indicated phage donor lysates. Samples for viable cell count, phage titer and number of cells transduced with either antibiotic resistance marker were taken at the indicated timepoints. Note that at 0 h, viable cells correspond to the number of infected cells used, phage titers correspond to the number phages from the donor lysate added for the infection and resistance marker titers are 0 as the recipient cells are sensitive at this stage. Error bars are s.d. from the mean. Assessment of statistically significant differences between groups was performed using a two-sided Student’s t-test on log10 transformed data. p-values are as follows: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns not significant. Limits of detection (LOD) are 100 colonies per ml for viable cells counts, 100 plaques per ml for phage titers and 10 colonies per ml for transductions.
Fig 4.
Impact of MOI on horizontal gene transfer, phage titers and viability.
S. aureus RN4220 derivative strains either devoid of (red) or harboring either SaPIbov1 (blue), SaPINY940 (green) or SaPI1 (purple) were infected with an MOI of either 1:1 or 1:10 (phage:bacteria) of the indicated phages and acquisition of a chromosomal cadmium resistance marker (lateral transduction) (A), a plasmid-borne chloramphenicol resistance cassette (generalized transduction) (B), phage titers (C) and cell viability (D) was determined 18 h post-infection. Bold horizontal lines in each boxplot represent the median and lower and upper hinges the first and third quartiles, respectively (n = 3–4 biological replicates as indicated). Assessment of statistically significant differences between groups was performed using a two-sided Student’s t-test on log10 transformed data. p-values are indicated above the respective comparison, ns not significant. Limit of detection (LOD) of transductants (A&B) is 10 transductants per ml, 100 plaques per ml for phage titers (C), and 100 colonies per ml for viable cells (D).
Fig 5.
SaPIs increase population heterogeneity.
S. aureus RN4220 derivative strains either devoid of (RN4220) or harboring SaPIbov1 were infected with an MOI of 1:10 (phage:bacteria) of a Φ11 or Φ11 Δdut mutant lysate derived from a donor strains containing a chromosomal cadmium and plasmid-borne chloramphenicol resistance marker. Number of viable cells, transductants and lysogenization frequencies of the defined populations and their relative frequencies to the total number of viable cells were determined. Population frequencies of (A) cells acquiring the chromosomal cadmium resistance marker or (B) the plasmid-encoded chloramphenicol resistance marker in the defined recipient strains. Bold horizontal lines in each boxplot represent the median and lower and upper hinges the first and third quartiles, respectively (n = 4 biological replicates for all samples). Assessment of statistically significant differences between groups was performed using a two-sided Student’s t-test on log10 transformed data. p-values are indicated above the respective comparison, ns not significant. Note that 10−9 as relative population frequency in these data indicates populations that were completely absent. (C) Schematic representation of the presence of different cell populations in either a recipient lacking a SaPI (left) or containing a SaPI (right).
Fig 6.
Model of the impact of SaPI protection from phage predation on horizontal gene transfer and population heterogeneity.
In SaPI-free recipients (grey cells), infecting phages can reproduce without restrictions and lyse their host cell (indicated dashed lines and skull and crossbones symbol). Only lysogenization (cells shaded blue) can protect the host cell from future phage infection and killing. Any cells with horizontally acquired genes (red and green shaded cells) that do not undergo lysogenization are also subject to subsequent rounds of phage infection increasing the likelihood of their loss. In the presence of SaPIs, recipient strains can successfully block phage replication and can survive without the need for lysogenization. Consequently, transductants are also more likely to survive leading to an overall increase in population heterogeneity. We propose that this occurs mainly via a general reduction of phage reproduction and burst size protects at population level. However, SaPIs could also act at the individual cell level and protect the infected cell from lysis. The mechanistic details of protection are likely to be diverse and different SaPIs could employ replication-dependent and -independent mechanisms to achieve this.
Table 1.
Strains and plasmids used in this study.