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Metabolic Interactions Between Dynamic Bacterial Subpopulations
Adam Z. Rosenthal
1
, Yutao Qi
1
, Sahand Hormoz
1
, Jin Park
1
, Sophia Hsin-Jung Li
2
, and Michael Elowitz
1
1)
Division of Biology and Biological Engineering and Department of Applied Physics, California
Institute of Technology, Pasadena, CA 91125, USA
2)
Department of Molecular Biology, Princeton University, Princeton, United States
3) Howard Hughes Medical Institute, USA
Corresponding author: Michael B Elowitz (melowitz@caltech.edu)
Abstract
Within multi-species microbial communities, individual species are known to occupy distinct
metabolic niches. By contrast, it has remained largely unclear whether and how metabolic
specialization occurs within clonal bacterial populations. The possibility of such metabolic
specialization in clonal populations raises several questions: Does specialization occur, and if it
does, which metabolic processes are involved? How is specialization coordinated? How rapidly do
cells switch between states? And finally, what functions might metabolic specialization provide?
One potential function of metabolic specialization could be to manage overflow metabolites such
as acetate, which presents a toxic challenge due to low pH, and protective pH-neutral overflow
metabolites. Here we show that exponentially dividing
Bacillus subtilis
cultures divide into distinct
interacting metabolic subpopulations including one population that produces acetate, and
another population that differentially expresses metabolic genes for the production of acetoin, a
pH-neutral storage molecule. These subpopulations grew at distinct rates, and cells switched
dynamically between states, with acetate influencing the relative sizes of the different
subpopulations. These results show that clonal populations can use metabolic specialization to
control the environment through a process of dynamic, environmentally-sensitive state-
switching.
Introduction
The Gram-positive bacterium
Bacillus subtilis
has two preferred carbon sources: glucose and
malate (Kleijn et al. 2010). When both of these carbon sources are available they are consumed
simultaneously, generating growth rates that surpass those achieved with either substrate alone
(Kleijn et al. 2010). Under conditions of rapid growth, co-consumption of glucose and malate
leads to the accumulation of high levels of acetate (Kleijn et al. 2010). As a weak organic acid,
acetate can be harmful to cells even in buffered medium (Rosenthal, Kim, and Gralla 2008).
Acetate and related short-chain fatty acids enter the cell passively in the neutral form and then
dissociate intracellularly, releasing a proton and transiently acidifying the cytoplasm (Russell and
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Diez-Gonzalez 1997; A. J. Roe et al. 1998). The intracellular dissociation of acetate also disrupts
the cellular anion balance, with negative effects on metabolism (A. J. Roe et al. 1998; Andrew J.
Roe et al. 2002) and transcription (Rosenthal, Kim, and Gralla 2008). When extracellular acetate
levels rise to toxic levels the growing
Bacillus subtilis
culture consumes the acetate and produces
acetoin, a non-toxic pH-neutral overflow metabolite that can be used as a carbon source in later
growth stages (Speck and Freese 1973) (Fig. 1A).
A biphasic growth strategy, in which acetate is produced to a toxic level and then reabsorbed and
replaced by a non-toxic metabolite (Wolfe 2005), is common to many bacterial species and is
important both for understanding the basic biology of bacterial growth in culture, and for
applications in metabolic engineering (Papagianni 2012). However, it has generally been studied
only at the population level, implicitly assuming a homogeneous progression of the entire culture
from acetate producing to acetate detoxifying states. However, recent work at the single cell level
suggests that bacterial systems can exhibit enormous heterogeneity in functional and gene
expression states across diverse systems (Eldar et al. 2009a; Locke et al. 2011a; Süel et al. 2006;
Levine et al. 2012). This prompts the questions of whether microbial cells differentiate into
metabolically distinct subpopulations, and more specifically, whether acetate production and
detoxification might occur in distinct cells specializing in acetate production or detoxification,
respectively.
Results
To address these questions we constructed a library of strains with reporters for key genes
involved in central carbon metabolism, acetate production, and organic acid detoxification (Fig.
1A). We introduced a fluorescent protein (YFP) under the control of promoters for 13 different
metabolic genes and stably incorporated them into the commonly used
sacA
site within the
genome (Table S1), (Eldar et al. 2009a; Locke et al. 2011a). Using quantitative single-cell
fluorescence microscopy, we analyzed the distribution of expression levels of these genes in
individual cells at different times along the growth curve in buffered culture medium containing
0.4% glucose and 50 mM malate. To eliminate oxygen gradients, 10 mL cultures were grown in
250 mL flasks with rapid shaking (250 RPM). Most genes showed unimodal and relatively tight
distributions (Figure S1) with coefficients of variation (CV)<25%). By contrast, expression of
sucC
and
alsS
, genes that encode succinate co-A ligase and acetolactate synthase, respectively, was
more heterogeneous (Fig. 1B). We observed CVs greater than 30%, with 3.8% of P
sucC
-YFP and
8.1% of P
alsS
-YFP cells exhibiting high expression levels (≥2 standard deviations above the mean)
at OD
600
~
0.8 (
sucC
) and OD
600
~
2 (
alsS
). Thus, at least two metabolic genes are heterogeneously
expressed under these conditions.
To better understand when this heterogeneity emerges in batch culture, we performed a time
course analysis of the fraction of
sucC
and
alsS
positive cells (≥2 standard deviations above the
mean denoted
sucC+
and
alsS+
, Fig. 1C). We observed that the subpopulation of cells expressing
sucC
only exists transiently, in mid- to late-exponential phase (Fig. 1C). Furthermore, this dynamic
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pulse of
sucC
+ cells coincided with the time and culture optical density at which acetate
production was observed (
~
180-300 min, Fig. 1D). This observation suggested that
sucC
expression could be involved in acetate production. A parallel analysis of
alsS
expression revealed
the opposite behavior, with
alsS
expression dynamics coinciding with a decrease in acetate and a
concomitant increase in acetoin levels (Fig. 1C, D). This behavior is generally consistent with the
known role of
alsS
in acetoin production in response to acetate toxicity (Speck and Freese 1973).
Together, these results show that a dynamic change in acetate and acetoin levels in the culture
overlaps with changes in the population fraction of
sucC
and
alsS
expressing cells.
A role for SucC in acetate production has not been studied previously. To understand the
relationship between the
sucC+
subpopulation and acetate production, we used fluorescence
activated cell sorting (FACS) of the P
sucC
YFP reporter strain to sort cells expressing YFP from a
SucC
promoter at the time of peak acetate levels, and performed RNAseq to compare gene expression
profiles (Fig. 2A, Fig. S2). As expected,
sucC
expression was elevated 2-fold in the
sucC+
sorted
subpopulation (blue dot, Fig 2A). For most genes, we observed a broad correlation in gene
expression between the two populations. However, RNAseq analysis with cuffdiff (Trapnell et al.
2010) and gene set enrichment analysis with GSEA (Subramanian et al. 2005) showed that genetic
competence genes (Berka et al. 2002) were significantly enriched in the
~
300 upregulated genes
in the
sucC
+ subpopulation (red dots and inset, Fig. 2A and Table S2; GSEA p <e-16). The
sucC+
population also exhibited increased expression of the phosphate acetyltransferase gene,
pta
(green dot, Fig. 2A), the enzyme that catalyzes the final step in overflow acetate production.
sucC
+
cells apparently represent a distinct gene expression state that could be involved in acetate
production.
Based on the strong correlation between
sucC
expression and competence gene expression in the
RNAseq results (Fig. 2A), we next asked whether the
sucC+
subpopulation represented the
competent state. To analyze the relationship between
sucC
expression and genetic competence
in single cells, we constructed four dual reporter strains, expressing CFP from the
sucC
promoter
and YFP from one of four competence promoters:
comG
,
comK
,
nucA
and
rapH
(Berka et al. 2002).
Imaging revealed a clear positive correlation between
sucC
and the competence genes (Fig. 2B,
Fig S3). This positive correlation was not general to all metabolic genes, as
sucC
expression was
anti-correlated with
pckA
(fig 2C, Fig S4), a gene involved in phosphoenolpyruvate synthesis
(Meyer and Stülke 2013). We note that
pta
and
sucC
were previously observed to be up-regulated
in the competent state (Berka et al. 2002). Together, these results suggest that individual cells can
exist in at least two distinct metabolic states, one of which represents genetically competent cells
and involves increased expression of
sucC
and
pta
, among other genes.
We next assessed how competence might be linked to elevated acetate production. The
competence system is controlled by a noise-excitable gene circuit that stochastically initiates
transient episodes of differentiation in individual cells (Suel et al. 2007; Süel et al. 2006; Cağatay
et al. 2009; Maamar, Raj, and Dubnau 2007; J. Hahn, Kong, and Dubnau 1994) To better
understand the relationship between competence and acetate metabolism, we next asked
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whether activation of the competence system is necessary for increased
sucC
expression. Strains
in which the competence master transcription factor
comK
is deleted (Table S1) exhibited reduced
acetate production (Fig. 2D) and a loss of
sucC
as well as
comG
expression (Fig. S5). Although the
competent state has been suggested to be involved in other functions, such as attachment,
motility, antibiotic resistance, and DNA metabolism (Redfield 1993; Jeanette Hahn et al. 2015;
Bakkali 2013; Finkel and Kolter 2001), a role in central carbon metabolism has not been
appreciated. These results indicate that the
sucC
subpopulation is controlled by the competence
system, linking competence both to an alternative metabolic state and to the control of acetate
levels in culture.
To better understand the dynamics with which cell switch into the competence and later into the
alsS+
, we used the “Mother Machine” microfluidic device (Fig 4A), to conduct long-term analysis
of individual cells over tens of cell generations under chemostatic conditions (Wang et al. 2010;
Norman et al. 2013). We set up the Mother Machine as described previously (Norman et al. 2013),
but cultured cells with conditioned media obtained from batch growth of
B. subtilis
cultures at
different final optical densities. Specifically, we used media from cultures at OD
600
0.8 and OD
600
2.0, points during the peak of
sucC+
or
alsS+
expression, respectively. This approach provides the
simplicity of long-term chemostatic analysis with the ability to compare cellular behavior at
different culture time-points.
Using the Mother Machine, we analyzed cell lineages for up to 4 days (approximately 60
generations) for a total of 1,400 cell generations. With
sucC
-inducing media (conditioned at OD
0.8), we observed rare episodes of
sucC
activation in some cells, lasting for approximately four
hours each (252 ± 89 minutes). Consistent with previous analysis of competence dynamics (Süel
et al. 2006), these cells divided infrequently, and grew more slowly than other cells in the same
movies (elongation rates of 47.4 ±
2.7 %/hr and 67.7 ±
2.3 %/hr, respectively). Cells in the
activated state could switch out of the
sucC+
state and resume normal growth rates (Fig. S6,
Movie S1). Under these conditions, we did not observe activation of
alsS
expression. By contrast,
in the OD 2.0 conditioned media we did not observe activation of
sucC
expression, but we did
observe frequent pulses of
alsS
gene expression.
alsS+
cells grew at a slightly reduced elongation
rate (63% ±
2.6 %/hr increase compared to 74% ±
2.3 %/hr for alsS- cells, Fig. 3B, right panel,
Movie S2). Together, these results provide the rates of transitions into the
sucC+
(competent) and
alsS+
gene expression states, and show that these states have altered growth rates and respond
to medium composition.
These results suggested the possibility that acetate predominantly produced by
sucC+
cells early
in the growth could be inducing cell switching to the
alsS+
state in later growth stages, when it
accumulates to toxic levels. However, many components could differ between the OD
600
0.8 and
OD
600
2.0 medium. To determine whether acetate was sufficient to affect
alsS
expression, we
cultured reporter cells in varying levels of acetate, in unconditioned liquid medium, and quantified
the fraction of
alsS+
cells. We observed both a systematic increase in the distribution of alsS
expression levels, and in the fraction of cells in the high expressing “tail” of the distribution (Fig.
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peer-reviewed) is the author/funder. It is made available under a
The copyright holder for this preprint (which was not
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http://dx.doi.org/10.1101/208686
doi:
bioRxiv preprint first posted online Oct. 25, 2017;