Figure 1.
The five-step competition-based adaptive evolution scheme developed here to generate antimicrobial compounds.
Antibiotic producing microorganisms are depicted in blue and competing pathogens in yellow. An evolution would end when a larger zone of inhibition (ZOI) appears around an evolved replicate of the antibiotic producer than any ZOI that might surround the unevolved wild-type. As a control, an additional set of replicates are evolved according to the same scheme but are not exposed to the pathogen until the end.
Figure 2.
Images of unevolved, wild-type S. clavuligerus and five strains (clavu7, clavu9, clavu10, NL2-c4, and ReIN) adaptively evolved against MRSA N315.
The top half of each panel shows each strain plated against drug-sensitive S. aureus 8325-4 while the bottom half shows the same strain plated against MRSA N315. Top left: wild-type S. clavuligerus. Top middle: clavu7. Top right: clavu9. Bottom left: clavu10. Bottom middle: NL2-c4. Bottom right: ReIN. The images were taken 24 hrs after S. aureus 8325-4 and MRSA N315 were plated against the six strains.
Figure 3.
HPLC-MS data from wild-type S. clavuligerus (black), clavu7 (blue), and clavu7 spiked with holomycin (red).
A. HPLC chromatograms of extracts from the three samples. The arrow indicates the peak corresponding to holomycin, which elutes at approximately 11.8 min. Note that this peak is not detected in extracts from wild-type S. clavuligerus. B. MS total ion monitoring at 11.8 min. There is an intense peak at m/z 388 in both wild-type S. clavuligerus and clavu7 samples, but an ion with m/z 215 ([M+H]+), which is holomycin and indicated by the arrow, can be detected in the clavu7 sample only. Inset. Magnification of the region surrounding m/z 215. C. MS and MS/MS select ion monitoring for m/z 215 (MS), m/z 197 (MS/MS; SRM) and m/z 388 (MS). The m/z 197 fragment in the MS/MS in particular is a sensitive signature for holomycin. D. When holomycin and the m/z 388 compound are separated (Figure S1) and tested individually against MRSA N315, only holomycin shows bioactivity. E. The structure of holomycin. High-resolution MS/MS indicates that the m/z 197 fragment corresponds to loss of H2O ([M−H2O+H]+, Figure S3). Abbreviations: SIM, select ion monitoring; SRM, selected reaction monitoring.
Figure 4.
Alignment of wild-type S. clavuligerus and clavu7 resequencing data to the reference S. clavuligerus ATCC 27064 genome.
A. Alignment of Illumina sequencing data from the wild-type S. clavuligerus strain used in this study to initiate adaptive evolution against MRSA N315. As expected, the data maps to the entire reference sequence. The encircled region indicates the position of the 1.8 Mbp pSCL4 plasmid. B. Alignment of Illumina data from clavu7. There is negligible read coverage for pSCL4. C. PCR amplification of five pSCL4 amplicons and one chromosomal amplicon from both wild-type S. clavuligerus and clavu7. The five pSCL4 amplicons can be amplified using DNA from wild-type S. clavuligerus as a template but not from clavu7 DNA, results which reinforce the Illumina sequencing data. The chromosomal amplicon serves as a positive control and corresponds to the same amplicon used to Sanger sequence the SNP in SSCG_05972 (Table 1).
Table 1.
List of mutations detected in clavu7.