Strain list

Strain Genotype description Reference

Pseudomonas fluorescens

SBW25 (PBR340)

Ancestor of all evolved and genetically manipulated P. fluorescens strains in this study. A WT strain isolate with a SM morphology.

Rainey and Bailey 1996

SM Δwsp Δaws Δmws (PBR716)

SBW25 ancestor with ORF of wsp aws mws deleted, used to evolve rare WS types

McDonald et al. 2009 LSWS Ancestor WS evolved from SBW25 resulting from a mutation in

wspF (A901C)

Spiers et al. 2002

LSWS lacZ Ancestor WS evolved from SBW25 resulting from a mutation in wspF (A901C), containing a lacZ gene

Zhang and Rainey 2007a IWSG WS type evolved from SBW25 resulting in a mutation to wspF

(C556T), measured by Bantinaki and colleagues as exhibiting the lowest fitness of the independent WS collection

Bantinaki et al. 2007

Original SWS A WS type evolved from SM Δwsp Δaws Δmws, selected in static microcosms over 5 days

McDonald et al. 2009

SWSTN(1-36)

The collection of 36 independent SM mutants resulting from transposon mutagenesis with IS-Ω-Km/hah of ‘original SWS’ (33 mutant genotypes are identified)

This study

PBR716 fadA-fwsR

SM Δwsp Δaws Δmws genotype in which genes PFLU0184- PFLU0183 have been exchanged with the fadA-fwsR deletion mutation from ‘original SWS’ (using primer pairs BglII-PF184f and BglII-PF183R2)

This study

SBW25 fadA-fwsR

Genotype SBW25 genotype in which genes PFLU0184- PFLU0183 have been exchanged with the fadA-fwsR deletion mutation from ‘original SWS’ (using primer pairs BglII-PF184f and BglII-PF183R2)

This study

SBW25 fadA-fwsR- lacZ

Genotype SBW25 fadA-fwsR that contains a chromosome integrated mini-Tn7T-lacZ gene used for fitness assays

This study

ISWS 1-92 A collection of 92 independent WS types evolved from SM Δ

wsp Δaws Δmws This study

PBR716 ISWS32- fadA-fwsR

SM Δwsp Δaws Δmws genotype in which genes PFLU0184- PFLU0183 have been exchanged with the fadA-fwsR fusion from ‘ISWS32” (using primer pairs PF184f and PF183PROD- 4R)

This study

SBW25 IWS32- fadA-fwsR

SBW25 genotype in which genes PFLU0184-PFLU0183 have been exchanged with the fadA-fwsR fusion from ‘ISWS32” (using primer pairs PF184f and PF183PROD-4R)

This study

PBR716 fadA-fwsR EE255-256AA

SM Δwsp Δaws Δmws genotype featuring the reconstructed fadA-fwsR chimera, in which the GGEEF residue motif is replaced with GGAAF (using primer pairs DGCF1 and GGAAF-R, GGAAF-F and DGCR4)

Strain Genotype description Reference

SBW25 fadA-fwsR EE255-256AA

SBW25 genotype featuring the reconstructed fadA-fwsR, in which the GGEEF residue motif is replaced with GGAAF (using primer pairs DGCF1 and GGAAF-R, GGAAF-F and DGCR4)

This study

PBR716 fadA-fwsR F257L

SM Δwsp Δaws Δmws genotype featuring the reconstructed fadA-fwsR chimera, in which the GGEEF residue motif is replaced with GGEEL (using primer pairs DGCF1 and DGCR2, DGCF3 and DGCR4)

This study

SBW25 fadA-fwsR F257L

SBW25 genotype featuring the reconstructed fadA-fwsR chimera, in which the GGEEF residue motif is replaced with GGEEL (using primer pairs DGCF1 and DGCR2, DGCF3 and DGCR4)

This study

PBR716 PfadA- fwsR

SM Δwsp Δaws Δmws featuring the transcriptional fusion of the promoter of fad to the ORF of fwsR, produced via allelic exchange (using primer pairs PF184ORFD1F and PF184ORFD2R, PF184ORFD3F and PF184ORFD4R)

This study

SBW25 PfadA-fwsR

SBW25 featuring the transcriptional fusion of the promoter of fadA to the ORF of fwsR, produced via allelic exchange (using primer pairs PF184ORFD1F and PF184ORFD2R,

PF184ORFD3F and PF184ORFD4R)

This study

PBR716 fadA-3X- fwsR

SM Δwsp Δaws Δmws featuring a transcriptional fusion of fadA and fwsR, separated by three stop codons in each reading frame (using primer pairs PF184f and NEW183PROD2R, NEW183PROD3F and PF183PROD-4R)

This study

SBW25 fadA-3X- fwsR

SBW25 featuring the a transcriptional fusion of WT fadA and fwsR, separated by three stop codons in each reading frame (using primer pairs PF184f and NEW183PROD2R,

NEW183PROD3F and PF183PROD-4R)

This study

PBR716 fadA-1X- fwsR

SM Δwsp Δaws Δmws featuring a transcriptional fusion of fadA and fwsR, separated by one stop codon in each reading frame (using primer pairs PF184f and PF183PROD-2R,

PF183PROD-3F and PF183PROD-4R)

This study

SBW25 fadA-1X- fwsR

SBW25 featuring a transcriptional fusion of WT fadA and fwsR, separated by one stop codon in each reading frame (using primer pairs PF184f and PF183PROD-2R, PF183PROD-3F and PF183PROD-4R)

This study

PBR716 2w2

SM Δwsp Δaws Δmws in which genes PFLU0184-PFLU0183 have been exchanged with the fadA-fwsR fusion from ‘SREE strain 2w2’ (using primer pairs P184BGLF and PF183PROD- 2R)

This study

SBW25 2w2

SBW25 in which genes PFLU0184-PFLU0183 have been exchanged with the fadA-fwsR deletion mutation from ‘SREE strain 2w2’ (using primer pairs P184BGLF and PF183PROD- 2R)

This study

SMΔmws (PBR711) 2w2’ SBW25 featuring a knockout of mwsR produced via allelic exchange

McDonald et al. 2009

SM Δmws Pfad-

mwsr1218-fwsR

SM Δmws featuring an allelic replacement of fadA with the initial translated 1218 bp of mwsR creating a translation fusion to WT fwsR (using primer pairs TMD_F1 and TMD_MWB_R2, TMD_MWB_F3 and TMD_MWA_R4, TMD_MWB_F5 and TMD_R6)

Strain Genotype description Reference

SBW25 mini-Tn7T- LAC-GFP(-)

SBW25 featuring a chromosome integrated mini-Tn7T-LAC plasmid. The negative control for GFP fluorescence

This study

SBW25 mini-Tn7T- LAC-GFP(+)

SBW25 featuring a chromosome integrated mini-Tn7T-LAC plasmid expressing gfp. The positive control for GFP fluorescence (using primer pairs GFPmut3_Tn7LAC and GFP_KpnI_R)

This study

SBW25 mini-Tn7T- LAC-fadA-gfp

SBW25 featuring a chromosome integrated mini-Tn7T-LAC plasmid expressing a transcriptional fusion of fadA to gfp (using primer pairs 184_FP_SPEIRBSF and 184_FP_SBFI-R, GFP_XhoI_F and GFP_KpnI_R)

This study

SBW25 mini-Tn7T- LAC-fwsR-gfp

SBW25 featuring a chromosome integrated mini-Tn7T-LAC plasmid expressing a transcriptional fusion of WT fwsR to gfp (using primer pairs 183_FP_SPEIRBSF and 183_FP_SBFI_R, GFP_XhoI_F and GFP_KpnI_R)

This study

SBW25 mini-Tn7T- LAC-fwsR-gfp

SBW25 featuring a chromosome integrated mini-Tn7T-LAC plasmid expressing a transcriptional fusion of fadA-fwsR to gfp (using primer pairs 184_FP_SPEIRBSF and 183_FP_SBFI_R, GFP_XhoI_F and GFP_KpnI_R)

This study

SBW25 mini-Tn7T- LAC-fadA::3X-fwsR- gfp

SBW25 featuring a chromosome integrated mini-Tn7T-LAC plasmid expressing a transcriptional fusion of fadA-3X-fwsR (see above) to gfp (using primer pairs 184_FP_SPEIRBSF and 183_FP_SBFI_R, GFP_XhoI_F and GFP_KpnI_R)

This study

SBW25 mini-Tn7T- LAC-fws2w2-gfp

SBW25 featuring a chromosome integrated mini-Tn7T-LAC plasmid expressing a transcriptional fusion of fadA-fwsR (isolated from strain ‘2w2’)to gfp (using primer pairs

184_FP_SPEIRBSF and 183_FP_SBFI_R, GFP_XhoI_F and GFP_KpnI_R)

This study

SREE mutants L1- M1 – L8-M8

64 mutants evolved from an SM Δwsp Δaws Δmws ancestor across 8 lines, each isolate adapted to a given alternating environment

This study

SBW25 nlpD

(C565T) SBW25 featuring a nonsense mutation to nlpD (C565T)

Dr C Kost

SBW25 Δwss SBW25 featuring a knockout of the wss operon (wss a-j), using a pUIC3 plasmid gifted by Dr J. Gallie

This study

SBW25 Δwss nlpD (C565T)

SBW25 Δwss featuring the allelic exchange of WT nlpD with a non-synonymous C565T mutation (using primer pairs

PF1302_bglII_F and PF1302_bglII_R)

This study

SBW25 pUIC3- nlpD(WT)

SBW25 featuring pUIC3 recombined between nlpD and rpoS, resulting in two WT nlpD regions (using primer pairs

PF1302_bglII_F and PF1302_bglII_R)

This study

SBW25 pUIC3- 3’nlpD (C565T)

SBW25 featuring pUIC3 recombined between nlpD and rpoS, resulting in WT nlpD 5’ of pUIC3, and mutant nlpD (C565T) 3’ of pUIC3 (using primer pairs PF1302_bglII_F and

PF1302_bglII_R)

This study

SBW25 pUIC3- 5’nlpD (C565T)

SBW25 featuring pUIC3 recombined between nlpD and rpoS, resulting in mutant nlpD (C565T) 5’ of pUIC3, and WT nlpD 3’ of pUIC3 (using primer pairs PF1302_bglII_F and

PF1302_bglII_R)

Strain Genotype description Reference

SBW25 nlpD (C565T)-pUIC3 (C565T)

SBW25 featuring pUIC3 recombined between nlpD and rpoS, resulting in mutant nlpD (C565T) 5’ of pUIC3, and mutant nlpD (C565T) 3’ of pUIC3 (using primer pairs PF1302_bglII_F and PF1302_bglII_R)

This study

SBW25 nlpD (C565T A566G)

SBW25 featuring a knockout of the wss operon (wssa-j), and the allelic exchange of WT nlpD with a non-synonymous C565T and A566G mutation (using primer pairs

PF1302_bglII_F and PF1302_bglII_R)

This study

SBW25 nlpD C565T A566G (1 -38)

33 nlpD chaining mutants evolved from SBW25 featuring a knockout of the wss operon (wssa-j), and the allelic exchange of WT nlpD with a non-synonymous A566G mutation (using primer pairs PF1302_bglII_F and PF1302_bglII_R)

This study

Escherichia coli

TOP10

F' , mcrA , Δ (mrr-hsd RMS-mcrBC), Φ 80lacZΔ M15,

Δlac X74, deoR , recA1, araD139, Δ(ara-leu) 7697, galU,

galK, rpsL, StrR , endA1, nupG Invitrogen

DH5α (λpir)

supE, ΔlacU169 (φ 80 lac ZΔM15) , hsdR , recA , endA , gyrA,

thi, relA (oriR6K replication) Invitrogen Table 2.1: Strains used, evolved or constructed. WT=wild type, SM=smooth, ORF=open reading frame, IWSG=independent wrinkly spreader G, SWS=slow wrinkly spreader, SREE=slow reverse evolution experiment, GFP=green fluorescent protein.

In document Formalist features determining the tempo and mode of evolution in Pseudomonas fluorescens SBW25 : a thesis submitted in partial fulfilment of the requirements for the degree of Ph D in Evolutionary Genetics at Massey University, Auckland, New Zealand (Page 46-49)