Experimental evolution of the megaplasmid pMPPla107 in Pseudomonas stutzeri enables identification of genes contributing to sensitivity to an inhibitory agent

Abstract

Horizontally transferred elements, such as plasmids, can burden host cells with various metabolic and fitness costs and may lead to other potentially detrimental phenotypic effects. Acquisition of the Pseudomonas syringae megaplasmid pMPPla107 by various Pseudomonads causes sensitivity to a growth-inhibiting substance that is produced in cultures by Pseudomonads during growth under standard laboratory conditions. After approximately 500 generations of laboratory passage of Pseudomonas stutzeri populations containing pMPPla107, strains from two out of six independent passage lines displayed resistance to this inhibitory agent. Resistance was transferable and is, therefore, associated with mutations occurring on pMPPla107. Resequencing experiments demonstrated that resistance is likely due to a large deletion on the megaplasmid in one line, and to a nonsynonymous change in an uncharacterized megaplasmid locus in the other strain. We further used allele exchange experiments to confirm that resistance is due to this single amino acid change in a previously uncharacterized megaplasmid protein, which we name SkaA. These results provide further evidence that costs and phenotypic changes associated with horizontal gene transfer can be compensated through single mutational events and emphasize the power of experimental evolution and resequencing to better understand the genetic basis of evolved phenotypes. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Keywords: bacteriostatic; experimental evolution; megaplasmid.

Figure 1.

Testing inhibitory agent on all six evolved lines reveals two resistant lines. Six populations carrying pMPPla107 were passaged under laboratory conditions for 500 generations. Single colonies isolated from these populations were tested for sensitivity against inhibitory agent found in Pseudomonas spp. supernatants. Isolates from lines 4B-500 and 5B-500 revert to a non-pMPPla107 phenotype where a zone of inhibition is not present, indicating resistance to the inhibitory agent. All overlays were plated after 4 h of growth in KB and spotted with 10 µl of P. stutzeri filter-sterilized supernatants. The number (1, 2, 3, …) indicates the individual lines and B indicates the second of two isolates taken at Generation 500. All images are representative of three biological replicates.

Figure 2.

Exconjugants of pMPPla107 from populations 4 and 5 into an ancestral chromosomal background transfers resistance to the inhibitory agent. Given that strains 4B-500 and 5B-500 were known to display resistance against the inhibitory agent, we conjugated megaplasmids from these strains into an unevolved P. stutzeri background DBL388. We additionally used conjugations with the megaplasmid from 3B-500 as a sensitive (positive) control as we knew this evolved strain was still sensitive. While a megaplasmid from 3B-500 maintains sensitivity to the inhibitory agent in the DBL388 background, megaplasmids from 4B-400 and 5B-500 transfer resistance. All overlays were plated after 4 h of growth in KB and spotted with 10 µl of P. stutzeri filter-sterilized supernatants. All images are representative of three biological replicates. Raw image files can be found in electronic supplemental material at Figshare (doi:10.6084/m9.figshare.15072537.v3): DBL618 is electronic supplementary material, figure S1, DBL619 is electronic supplementary material, figure S2, DBL620 is electronic supplementary material, figure S3. (Online version in colour.)

Figure 3.

A large deletion occurs early during the passage of population 4 and provides resistance to the inhibitory agent. (a) SynMap dotplot visualizes the large deletion occurring from 131 to 499 kb in the evolved 4B pMPPla107 as a large shift across the x-axis. The remaining portions of the sequences maintain perfect synteny, indicating that a clean deletion occurred. The x-axis is ancestral pMPPla107 gene order where x_{1, …, N} = gene_{1, …, N} and the y-axis is the line 4B evolved pMPPla107 gene order where y_{1, …, N} = gene_1…N.]. (b) We tested single colony isolates from frozen stocks of the passage line 4 populations at generations 0 and 100 for sensitivity to the inhibitory agent. We found that resistance to the inhibitory agent is present in the generation 0 isolate from population 4. This resistance is maintained in generation 100 (shown) through generation 500 and is the only unique mutation in pMPPla107 other than a synonymous SNP. All overlays were plated after 4 h of growth n KB and spotted with 10 µl of P. stutzeri filter-sterilized supernatants. All images are representative of three biological replicates. Raw image files can be found in electronic supplementary material at Figshare (doi:10.6084/m9.figshare.15072537.v3): DBL408 is electronic supplementary material, figure S4, 4A-0 is electronic supplementary material, figure S5, 4A-100 is electronic supplementary material, figure S6. (Online version in colour.)

Figure 4.

Allelic replacement of skaAE395 K provides resistance to inhibitory compounds in Pseudomonas supernatants. We recreated the skaAE395 K allele in a naive chromosomal and megaplasmid background (strain DBL494). A zone of inhibition can be seen for strain DBL494 but not for strain DBL1802. Strain DBL494 contains a wild-type allele of skaA on the megaplasmid while the skaAE395 K mutation has been recombined into the megaplasmid in strain DBL1802. Overlays were plated after 5 h of growth in KB liquid and spotted with 20 µl of P. stutzeri filter-sterilized supernatants. All images are representative of three biological replicates for these strains. Raw image files can be found in electronic supplementary material at Figshare (doi:10.6084/m9.figshare.15072537.v3): DBL494 is electronic supplementary material, figure S8, DBL1802 is electronic supplementary material, figure S10.

Figure 5.

Predicted protein structure of SkaA. We used Alphafold2 to predict the protein structure of SkaA. The position of the E395 K change in the megaplasmid of 5B-500 is denoted with an arrow. (Online version in colour.)