2.5.7.1 DNA extraction and pooling
DNA was extracted from all 40 isolates within each population, normalised and pooled, and the pooled DNA from each population was sequenced, allowing an estimation of the frequency of variants in the population. DNA was extracted from overnight cultures using the Wizard genomic DNA extraction kit (Promega) and eluted in Qiagen elution buffer. DNA for each isolate was quantified in triplicate (2.3.5) and normalised to the lowest concentration in the pool, resulting in equal amounts of DNA for isolates across the population.
2.5.7.2 DNA sequencing
Library preparation (500 bp paired-end) was done by the Liverpool Centre for Genomic Research (CGR). Samples were sequenced on a HiSeq 2000 (Illumina), with multiplexing of 3 samples per flow cell lane.
2.5.7.3 Bioinformatics analysis
Bioinformatics analysis was performed by Dr. Sam Haldenby, CGR. Briefly, single- nucleotide variants (SNVs) were called by mapping to the reference strain PAO1- UW. Pre-existing variants in our PAO1 strain were filtered out after resequencing of this strain, as PAO1 is known to demonstrate substantial diversity between
laboratories (Klockgether et al., 2010). Prophage positions in the PAO1 genome were determined by examination of contig ends.
57
2.5.7.4 PCR confirmation of single nucleotide polymorphisms (SNPs) in the
lasR gene
An 854 bp region, encompassing the entire lasR gene (719 bp) and a small region of the adjacent rsaL gene was amplified using standard PCR, using the LasRF and LasRR primer pair (Table 2.2). Amplified DNA was purified using the QIAquick PCR purification kit (Qiagen), according to the manufacturer’s instructions, and Sanger sequenced (Source Bioscience) using the reverse primer. Sequence quality was assessed by inspection of the sequence chromatogram using Finch TV
(Geospiza). Good quality sequence was similarity searched using the nucleotide basic local alignment search tool (BLASTn), against P. aeruginosa PAO1 sequences in the non-redundant nucleotide database. All other parameters were set to the default. Alignments were inspected to identify variants.
2.5.7.5 PCR confirmation of phage integration sites
PCR amplification was conducted to confirm phage integration sites, and quantify the exact frequency at which a specific integration site occurred in a population. Dual PCR amplification was used to amplify both the left and right phage-bacterial
chromosomal junctions, and the principle is summarised in Figure 2.3. Primer pair 1 targeted the left bacterial-phage junction, with the forward primer designed to bind to the bacterial gene and the reverse primer to the 3ʹ end of the phage. Primer pair 2 was designed to target the right junction, with the forward primer designed to bind to the 5ʹ end of the phage and the reverse primer to the bacterial gene. Positive
amplification yielding two products confirmed occupation of the locus by LESφ4. Positive amplification between the forward and reverse primer of primer pair 1 and 2, respectively, yielding a single product, indicated an intact integration site. If an isolate was PCR positive for prophage, but had an intact integration site, the phage was assumed to have integrated elsewhere in the PAO1 genome. Primers were designed to span the fimU integration locus in population 7, and the pilV integration locus in population 11. All 40 endpoint isolates were PCR screened to obtain each integration frequencies, in addition to 20 isolates per time point, per population, at transfers 1, 5 and 15.
58
Figure 2.3 Diagram to illustrate the concept of PCR confirmation of LES φ4 integration sites in the PAO1 genome.
2.5.7.6 Inverse PCR to determine LES φ4 phage integration site
Inverse PCR (IPCR) was used to determine LESφ4 integration sites in the single- phage control treatment (as sequence data was not available), allowing a comparison of phage integration sites in the presence and absence of other LES phages. The principle of IPCR is summarised in Figure 2.4
59 .
Figure 2.4 Diagram to illustrate the concept of inverse PCR to determine LESφ4 integration sites in the PAO1 genome.
2.5.7.6.1 Restriction endonuclease digestion
Genomic DNA was extracted (2.5.7.1) and digested with NarIrestriction enzyme (New England Biolabs), selected as it was known to cut 775 bp into the 5̍ end of LESφ4, and also at numerous points throughout the PAO1 genome. Digestion was performed according to manufacturer’s recommendations. The enzyme was
inactivated by heating to 65 ˚C for 20 minutes, and the digested DNA purified using the QIAquick PCR purification kit (Qiagen), and quantified.
60
2.5.7.6.2 Recircularisation of DNA fragments
Ligation is favoured at low DNA concentrations and large reaction volumes. To this end, DNA was diluted to <1 ng µl-1 and 45 µl diluted DNA mixed with 5 µl 10X ligase buffer and 3 µl T4 DNA ligase (Stratagene). The mix was incubated at 18 ˚C overnight, then heated to 100 ˚C for 3 minutes, to introduce nicks in the circularised DNA. The DNA was purified again, as before, and eluted in a small (30 µl) volume to ensure the DNA was as concentrated as possible.
2.5.7.6.3 PCR amplification of DNA
Primers were designed using the free software FastPCR, which has a tool to allow design of primers for IPCR. Primers were designed to face outwards (as opposed to conventional primers which face inwards) at the 5̍ end of the phage, in the region between the end of the phage and the first restriction enzyme cut site. Each PCR reaction contained: 500 nM each primer, 10 µM each dNTP (Bioline), 10 µl Q5 reaction buffer, 10 µl high GC enhancer, 1 U Q5 high-fidelity DNA polymerase (all New England Biolabs), 5 µl ligated DNA and DEPC treated water, to a final volume of 50 µl. Q5 DNA polymerase was selected due to its low error rate and its improved ability to amplify long and difficult sequences, relative to other polymerases. Primers and cycling conditions are described in (Table 2.2).
2.5.7.6.4 Sanger sequencing of DNA amplicons to determine phage integration
site
The entire PCR reaction was subjected to agarose gel electrophoresis. The most visible band was selected and the DNA extracted (2.3.4). The amplicon was Sanger sequenced (Source Bioscience) using the reverse primer. BLASTn analysis revealed a small portion of sequence with high similarity to the end of the phage (c repressor gene), and the remainder of the sequence matched to the phage integration locus in the PAO1 genome.
2.6 Direct competition of evolved populations with ancestor in ASM
Endpoint evolved populations were competed against antibiotic labelled PAO1 strains, so that the competitors could be easily separated. Whole-populations were competed, rather than individual isolates, as it is known that in genetically diverse
61 populations, the fitness of individual isolates are not representative of population level-fitness (Wong et al., 2012). Populations were competed against their “ancestor”, PAO1::GmR, and to control for the competitive effect of phages,
populations were also competed against a strain that does not produce the receptor required for phage infection, PAO1 pilA-::TetR. To confirm the absence of any fitness
costs of the antibiotic gene cassette, wild-type PAO1 was competed against both antibiotic marked strains in LB.
Prior to initiation of the competition, both competitors were acclimatised to the competition medium (ASM). Biofilm homogenate from endpoint populations, and stocks of the ancestral competitor strains, stored at -80 ˚C, were defrosted.
Approximately 5 x 105 bacterial cells were inoculated into 1 ml of ASM in a sterile 24 well tissue culture plate and incubated for 24 hours. Cultures were homogenised with Sputasol and diluted 5-fold. The OD600 for each competitor was measured and
adjusted to 0.4. Competitions were initiated with a 50:50 starting ratio of each competitor; 50 µl of each competitor was inoculated into 5 ml ASM and incubated under conditions identical to 1 cycle of the coevolution experiment (2.5.1). The exact input cell density of each competitor was quantified by plating onto Columbia agar. After 4 days, biofilms were homogenized and diluted cultures plated onto both non- selective media (LB agar) and the appropriate antibiotic media (LB agar containing 10 µg ml-1 gentamicin or 50 µg ml-1 tetracycline), in order to quantify the density of each competitor. The density of the evolved PAO1 population was calculated by subtracting the density on antibiotic media from the density on non-selective media. Five independent biological replicates were performed for each competition.