Appropriate control of the rate of cell growth is central to the long-term survival of species, particularly microorganisms. A fast growth rate is a competitive advantage when environmental conditions are favourable, while slow growth or even quiescence may allow survival under stress conditions such as rapid changes in the physicochemical environment, starvation, or exposure to toxins. Thus most organisms have evolved stringent controls over the rate of cell growth, and any disruption of these controls has severe consequences; an obvious example being the development of cancer as a result of uncontrolled cell division.
Our genome-scale approach to the investigation of growth rate control in the model eukaryote cell, S. cerevisiae
, is based on the concepts of Metabolic Control Analysis (MCA; [1
]). MCA defines the extent of control exerted over the flux through a pathway by its components. In our context, flux is the growth rate of cells, pathway components are the gene products, and control coefficients are the ratios of the fractional changes in growth rate to the fractional changes in the concentrations of gene products. Thus the term "control" has a special meaning in the context of MCA. Those cell components that exert control over flux are not necessarily regulatory molecules. Rather, they exert control because the flux through the pathway is sensitive
to changes in the concentration (or activity) of the component. In this paper, we shall routinely use the term 'growth rate', rather than 'flux', since we have measured differences in growth rate consequent on reducing the copy number of individual genes from two to one in diploid yeast.
As part of our overall approach, we have designed experiments in two categories to elucidate the control coefficients of gene products for growth rate. In Category 1 experiments, we measured the change in the concentration of gene products as a response to a change in flux ([2
]). We report no new Category 1 experiments in the present study, but we shall refer to our earlier experiments in this Category. In Category 2 experiments, the concentration of gene products is altered and any impact on growth rate measured [4
]; all new data reported in this paper relates to Category 2 experiments.
In our earlier Category 1 experiments, we grew a reference yeast strain in carbon-, nitrogen-, phosphorus-, or sulphur-limited conditions in chemostat culture at dilution rates, D = 0.07, 0.1 and 0.2 h-1
(equivalent to doubling times ~ 10, 7, and 3.5 h, respectively), and collected samples at each of the different steady states. We analysed our samples for changes in the levels of mRNAs, proteins, and metabolites with respect to dilution rate and identified a set of growth-rate-regulated (GR) genes; i.e.
a set of genes that significantly changed their expression levels in response to the change in growth rate, irrespective of the specific nutrient whose rate of supply determined the rate of growth [2
]. There is also a nutrient-regulated (NR) gene set, whose expression levels change according to the nutrient whose rate of supply is determining the growth rate; again, we have reported on these previously [3
]. The level of expression of NR genes may vary in either a growth-rate-dependent or growth-rate-independent manner.
In the set of Category 2 experiments that we reported previously, we established a growth-rate competition between yeast deletion mutants (each hemizygous for just one of the organisms 5,800 protein-encoding genes [5
]) under carbon, nitrogen, or phosphorus limitation in chemostats at D = 0.1 h-1
]. We classified all the mutants according to the sign of their relative growth rate, the mutants with negative relative growth rate (i.e.
whose proportion in the population fell significantly overtime) were classified as haploinsufficient (HI) and the mutants with positive relative growth rate (i.e.
whose proportion in the population significantly increased with time) were classified as haploproficient (HP). The set of haploinsufficient and haploproficient genes together form the set of high flux control (HFC) genes, i.e.
those genes for which a reduction of their copy number in diploid cells from 2 to 1 results in a significant change in growth rate (flux).
Our earlier results showed that, under all three nutrient limitations and at a dilution rate of 0.1 h-1
, there was little overlap between HFC genes from Category 2 experiments and the GR genes defined in Category 1 experiments; i.e.
the genes regulated by growth rate are not, themselves, regulators of growth. At the time [4
], we noted that this result might, in the jargon of Systems Biology, represent a 'design rule' for the eukaryotic cell. However, since cells are the products of evolution rather than design, such rules may change according to the selection pressures to which an organism is exposed. Thus, while this rule about HFC genes holds for nutrient-limited environments, further studies are required to determine whether it has any greater generality. This paper reports these additional studies.
Since our previous set of Category 2 experiments was carried out at a low growth rate and under nutrient limitation, we decided to determine whether the lack of overlap between HFC and GR gene sets still held at high growth rates or under nutrient-unconstrained conditions. Chemostats tend to be unstable at dilution rates close to the cells' maximum specific growth rate (μmax
) and so, to establish a steady state at μmax
, we used turbidostats to allow us to monitor competition between the pool of hemizygous yeast deletants growing in a complex synthetic medium (FPM [6
]; see Methods section for modifications). Turbidostats are continuous cultures in which the cells, rather than the experimenter, control the growth rate [7
] since the nutrient supply is determined by the biomass concentration in the growth vessel. Accordingly, we established a turbidostat in which the biomass concentration was held at a value equivalent to that of a mid-exponential phase batch culture. The turbidostat is unconstrained by the supply of nutrients and so equilibrates at the maximum specific growth rate of the yeast strain used. However, if the culture is a pool of hemizygous mutants of S. cerevisiae
, and if the different mutants in the pool can have different maximum growth rates, those mutants that can achieve a μmax
greater than the population average will increase in the population over time, while those with a μmax
less than the population average will decrease in the population. (Those readers unfamiliar with chemostats and turbidostats will find a fuller explanation in Additional File 1
, Additional Text; also see reviews by Bull [8
] and Pirt [7
Turbidostats, just like chemostats, represent a sensitive way of identifying haploinsufficient and haploproficient phenotypes, with the difference that (in a turbidostat) haploproficient mutants must be capable of growing at a rate greater than the previously recorded μmax. Thus, in this study, we have searched for sets of genes that exhibit either haploinsufficient or haploproficient phenotypes (these are both sub-sets of the class of HFC genes) in rapidly growing yeast cultures, either in nutrient-limited chemostats operated at D-values close to μmax or in the nutrient-unconstrained conditions of turbidostat culture. Further, we investigated subsets of HFC genes that have closely related cellular functions in order to elucidate the gene and protein features that mediate their high degree of control over growth rate. Finally, we propose an extended approach to growth rate (or flux) control, without limiting its definition to enzymes or haploinsufficiency.