The delayed outgrowth of ∆ybeX is heterogeneous at the
individual cell level
When streaking out mutant strains from glycerol stocks and overnight
grown stationary phase cultures, we noticed that while the ∆ybeZstrain produces wild-type-like colonies and ∆ybeY strain has
uniformly small colonies, the ∆ybeX strain produces heterogeneous
colonies. Re-streaking of small and large ∆ybeX colonies resulted
in well-grown large second-generation colonies, indicating that the
heterogeneous phenotype is not caused by a genetic mutation (data not
shown). We also tested whether the colony heterogeneity is caused by
freeze-thawing in the glycerol mix by growing the ∆ybeX and
wild-type cells in liquid media into stationary phase and then plating
the cells directly onto LB agar plates. Again, while wild-type cells
exhibited uniform colonies, the ∆ybeX strain gave heterogeneous
colony growth (Fig. 2A ). Thus, it is likely that the growth
heterogeneity of ∆ybeX depends on the heterogeneity of the
initial physiological states of individual stationary cells.
We quantified colony radiuses of wild-type and ∆ybeX strains
grown in LB and MOPS minimal media supplemented with 0.3% glucose using
AutocellSeg (Khan et al. , 2018). ∆ybeX cells tend to form
smaller colonies than wild-type cells when grown in LB and MOPS media
(Fig. 2A ). ∆ybeX colonies are heterogeneous when grown
in LB medium (Fig. 2B ), while the colony radiuses are
homogeneous when ∆ybeX cells are grown in MOPS liquid medium
(Fig. 2C ).
We then asked how heat shock affects cell growth and heterogeneity.
Overnight cultures were diluted and plated on LB agar plates following
16-18 hours of incubation at 37°C or 42°C (Fig. S3a ). We
observed fewer ∆ybeX colonies at 42°C (p < 0.0001 and p
= 0.02 for LB and MOPS, respectively; Fig. 2D ).
We inspected the colony growth of ∆ybeX::kan (ybeX single
deletion strain in BW25113 background constructed via lambda red
recombination) and ∆ybeX/kan- (the kanamycin cassette removed
from the inhouse constructed ∆ybeX::kan ) cells at 37°C for 24 and
48 hours (Fig. S3b, c ). No significant differences were
observed for ∆ybeX::kan and ∆ybeX/kan- . Furthermore,
although the observed tiny colonies of ∆ybeX were increasing in
size over time, they consistently remained smaller than WT-like∆ybeX colonies (Fig. S3b, c ).
To better understand the nature of the observed lag phase phenotype at
the individual cell level, we quantified colony radiuses of the∆ybeX and the WT cells at 37°C and 42°C using cells pre-grown for
16-18 hours in liquid LB or MOPs minimal media prior to plating.
Inspection of four independent stationary phase outgrowth experiments
showed, in accordance with our previous observations, that at both
temperatures WT cells tend to form larger colonies, while the colony
radiuses of ∆ybeX cells are heterogeneous and possibly dimorphic.
These intuitions were formalized by jointly modelling means and standard
deviations of colony radiuses and, in a separate model, the colony
radiuses as mixtures of two normal distributions (see Materials and
Methods for details). The estimated mean colony radiuses are smaller in
the ybeX strain by about 1/3 (the difference, in arbitrary units, at
37°C is 3.58 [95% CI 2.89, 4.24] and at 42°C is 3.72 [3.01,
4.42]), and the ∆ybeX colony radiuses have a larger standard
deviation (the difference in 37°C is 0.67 [0.36, 1.04] and at 42°C
is 0.91 [0.53, 1.34]). Interestingly, modelling the colony radiuses
as emanating from two distinct gaussian populations resulted in a
superior out-of-sample fit, as assessed by leave-one-out-cross
validation (data not shown), supporting the conjecture that ∆ybeXcells grow in at least two distinct regimes, one of which is similar to
WT growth, while another results in up to two-fold smaller colonies (WT
vs. ∆ybeX difference in 37°C: µ1 (estimate for
the mean of the first gaussian): 1.73 [0.47, 2.93],
µ2: 5.58 [4.71, 6.39], and in 42°C:
µ1:1.39 [0.06, 2.81], µ2: 5.92
[4.99, 6.79]).