Materials and methods
Bacterial strains and strain construction. Bacterial strains were
selected from replicate populations started with strains REL606 and
REL607, which are isogenic except for a neutral arabinose marker and a
mutation in recD that also appears to be neutral (Tenaillon et
al. 2016). Populations were evolved in Davis-Mingioli (DM) minimal media
supplemented with different presentations of glucose (175 μg/ml) and
lactose (210 μg/ml) (Cooper and Lenski 2010). The evolution environments
included DM supplemented with lactose (Lac), daily fluctuations of
glucose and lactose (G/L), or long-term switching from glucose to
lactose (G_L) or from lactose to glucose (L_G) every 2,000
generations. Each population was evolved for 8,000 generations, except
for one G/L population, which was evolved for 7,000 generations (G/L2).
Six replicate populations were evolved in each environment, the
replicate number is indicated by the number following the evolution
environment designation. Clones with the lacI- mutation were
selected from populations based on their growing as a blue colony on
indicator plates that contained X-gal
(5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) and glucose (TGX
plates) (Fig. 1; Quan et al. 2012). On TGX plates, the blue phenotype is
indicative of a strain constitutively expressing the lac operon.
A total of nine clones were selected, one from each of three lactose
only populations, a G_L population, and five G/L populations. In all
clones the lacI gene was amplified and sequenced to verify thatlac operon constitutive expression was caused by a mutant LacI
repressor (Fig.1). Amplification was carried out using the primers:
5’-GCGGAGCTGAATTACATTCC-3’ (11-F) and 5’-GGGTGCCTAATGAGTGAGCT-3’ (12-R).
To determine the effect of substituted lacI- mutations, we first
constructed lacI + derivatives of our focal evolved strains. To do
this, we PCR amplified the ancestral allele using primers 11-F and 12-R,
ligated this product into pCR2.1 using a TA cloning kit (Invitrogen, CA
USA). The plasmid was used to transform TOP10F’ cells, which were
blue/white screened to identify transformants having a plasmid insert.
pCR2.1::lacI + plasmids were purified, and the lacI +
fragment excised and cloned into pDS132 using enzymes SacI and XbaI. The
resulting plasmid, pDS132::lacI+ was used to transform
MFDpir cells (Ferrières et al. 2010). MFDpir(pDS132::lacI +) was separately conjugated with
each target evolved strain by mixing donor and recipient at a 1:2 ratio,
respectively. Conjugation was carried out on LB agar plates supplemented
with 2,6-diaminopimelic acid (30 μM), which MFDpir needs for
growth, for 3-4 hours at 37 ° C. The conjugation mixture was
resuspended in 200 μL DM medium and plated onto minimal glucose agar
(MG) supplemented with chloramphenicol (Cm; 20 μg/mL) to select for
recipient strains with the pDS132 plasmid successfully integrated into
the chromosome (Philippe et al. 2004). After overnight
incubation, six colonies were restreaked onto MG + Cm agar, again
incubated overnight, and then restreaked a second time to the same
medium. A colony descended from each of the original six was resuspended
in 500 μL DM base liquid media and plated onto sucrose plates
supplemented with X-gal to select for excision of the pDS132 plasmid
(Philippe et al. 2004) and screen for clones that had retained the
introduced lacI + allele. One blue colony and one white colony
were selected from each plate and restreaked twice onto sucrose + X-gal
plates. White colonies indicate clones that successfully integrated thelacI + allele. Blue colonies, which retained the originallacI- allele were used as negative controls to test for the
presence of additional mutations that occurred during the allele
replacement process. All clones were tested for proper pDS132 plasmid
excision by checking for chloramphenicol sensitivity. The lacI +
insertion was verified by sequencing.
To evaluate the possibility of secondary mutations being inadvertently
added during construction of lacI + strains, we measured the
fitness of control strains that went through the conjugation process but
did not retain the introduced lacI+ allele in competition with an
otherwise isogenic strain with a distinct neutral ara marker. Theara marker strains were constructed either by pairwise
conjugations between the evolved strain and MFDpir (pDS132::ara -)
(same method as above except X-gal was not used in the media), or by
plating 100 μL of overnight LB cultures onto minimal arabinose agar (MA)
and selecting for spontaneous ara + mutants. Indistinguishable
fitness between competing strains was interpreted as indicating the
absence of fitness-effecting secondary mutations.
Fitness assays. Fitness effect of the lacI - mutation in
each isolated strain was measured in glucose (175 μg/ml) and lactose
(210 μg/ml) only environments. Cells were transferred from freezer
stocks initially to LB medium, then, after overnight growth, to minimal
medium supplemented with glucose or lactose as used in the particular
competition assay. Strains were initially acclimated to the assay
environment over two 24 hr transfer cycles with a 1:100 dilution
occurring between each cycle. Pre-conditioned competitors were mixed at
a 1:1 ratio by diluting each competing strain 1:200 directly into the
assay environment. Competitions were carried out over two (lactose
competitions) or four (glucose competitions) transfer cycles. On the
initial and final day of competitions, cells were plated onto TGX plates
and incubated at 37 ° C for 18-20 hours in order to distinguish
competing genotypes. Relative fitness effect of the lacI -
mutation was determined based on the change in density of blue
(lacI -) and white (lacI +) colonies on TGX plates using the
formula: ln(blue2 × 100t /
blue0)/ ln(white2 ×
100t / white0), where
subscripts indicate the time at which competitor density was estimated,
and t accounts for transfer cycles during the competition (Lenski
et al. 1991). Test competitions checking for additional mutations
occurring during allele replacement procedures or selection of
spontaneous ara + mutants were performed as described above,
except competitors were distinguished by plating onto tetrazolium
arabinose agar (TA).
Expression assays. Expression of the lac operon was
measured using a GFP reporter construct controlled by the
Plac promoter region including the O1 and O3 LacI
operator sequences, and native primary CRP binding site (Quan et al.
2012). This reporter was previously cloned into a mini-Tn7cassette in a suicide-vector (Quan et al. 2012) and was introduced into
target strains by tri-parental conjugations between a target recipient
evolved strain, a donor strain (MFDpir(pUC18R6KT::Plac -GFP, kanr)),
and a helper strain (MFDpir (pTSN2)) (Choi et al. 2005;
Ferrières, et al. 2010; Quan et al. 2012). Strains were mixed at
a 3:1:1 ratio (recipient : donor : helper) on LB + DAP (30 μM) agar and
incubated at 37 ° C for 3 hours. The conjugation mix was
resuspended in DM medium and plated onto LB + kanamycin (Km; 60 μg/mL) +
isopropyl β-D-1-thiogalactopyranoside (IPTG; 1 mM) agar plates.
Kanamycin selects for clones that successfully obtained the
Plac -GFP reporter while IPTG induces expression
of the reporter allowing identification of clones that stably expressed
GFP. After 24-36 hours of growth, six fluorescent colonies were
restreaked onto LB + Km (60 μg/mL) plates. Restreaked colonies were
streaked a second time on LB + Km + IPTG plates and then tested for
absence of the delivery plasmid by spotting colonies on LB + Ap (100
μg/mL) agar. Insertion of the Plac -GFP reporter into theatt Tn7 site was confirmed by PCR (primers:
5’-TAACAGCCAGCACCACGCCG-3’ (120-F) and 5’-CGCGAATCCGATCTGGCGCT-3’
(121-R)). Transposition of the reporter into each recipient strain’satt Tn7 site allows consistent insertion of the reporter
into the same region of the genome, minimizing divergent effects on
reporter fitness and expression costs.
To measure Plac -GFP reporter expression, reporter
strains were grown overnight in LB broth from freezer stocks, diluted
1:10,000 in the assay environment, and allowed to grow for 24 hours. The
following day cultures were diluted again 1:10,000 in the assay
environment and grown in a VersaMax spectrophotometer (Molecular
Dynamics, CA) until mid-log phase (OD450~0.1-0.15) to allow measurement of a steady-state level
of Plac -GFP reporter expression.
Plac -GFP expression was measured in an Accuri C6
flow cytometer (Becton Dickinson, NJ). Assay environments included
DM+175 μg/mL glucose, DM+210 μg/mL lactose.
Statistical analyses. Data was analyzed using R (version 3.5.0)
(R Core Team, 2018). Dunnett’s tests for one against many comparisons
were performed using the glht function in the multcomp package. Where
appropriate, experimental block was included in analyses as a random
effect in mixed-model ANOVA performed using the lmer function in the
lme4 package. Fixed effects were tested for significance by comparing
models fitted with and without the factor of interest using a χ2 test .