CO2 Mediated Interaction in Yeast Stimulates Budding and Growth on Minimal Media

Here we show that carbon dioxide (CO2) stimulates budding and shortens the lag-period of Saccharomyces cerevisiae cultures, grown on specific weak media. CO2 can be both exogenous and secreted by another growing yeast culture. We also show that this effect can be observed only in the lag-period, and demonstrate minimal doses and duration of culture exposition to CO2. Opposite to the effects of CO2 sensitivity, previously shown for pathogens, where increased concentration of CO2 suppressed mitosis and stimulated cell differentiation and invasion, here it stimulates budding and culture growth.

The ''mitogenetic effect'' consists in a distant stimulation of mitosis in prokaryotic and eukaryotic cells by optical contact with other well growing cultures. The effect was shown for bacterial cultures [10], yeast [11], etc., and ultraweak ultraviolet luminescence was stated to be the mediator of these cell-cell interactions [12,13]. Altogether several hundred articles and monographies appeared in this area, mostly in 1920-1950s, both verifying [14][15][16] and refuting [17,18] original results. Still, the problem of mitogenetic effect remains unsolved till nowadays.
The effects of chemical cell-cell interactions in microbial cultures, most of them denoted by the notion ''quorum sensing'', are proved much more unequivocally. The ''quorum sensing'' phenomenon (the name given in [19]) consists in cooperative ''behavior'' of microbial cultures depending on the population density and composition, and including gene expression, cell differentiation, antibiotic secretion, and various virulence-dealing processes, such as hyphae, biofilm and spore formation, and substrate invasion. The mechanism lies in simultaneous secretion and reception of certain species-specific or more or less universal chemicals (small peptides, alcohols, ethers etc.), which accumulate in the medium and switch on certain intracellular signaling pathways when reaching a certain concentrational threshold (for reviews see [3,4,20,21]). Besides these specific signaling factors, cell interaction can be mediated by such a ''simple'' molecule as NH 3 [5], which is ''used'' to synchronize cell differentiation and general morphology of neighboring colonies [22] and prevent them from spreading too close to each other [5].
Can CO 2 also be a factor of cell-cell interaction? CO 2 sensitivity of mammalian cells has been known for nearly 50 years [23] and investigated in detail [24]. It has also been shown for cyanobacteria [7], and pathogenic fungi [25], in which it plays the role of ''host tissue sensor''. But there are practically no works dealing with CO 2 -mediated cell-cell interaction [9], especially in nonpathogens (discussion of some doubtful data [26] see below). In this work a new case of yeast cell-cell interaction was shown, and the mediator of this was proved to be CO 2 . Thus the observed effect turned out a new case of CO 2 sensitivity in microorganisms, and a new type of CO 2 -mediated processes, where cell cycle is stimulated rather than suppressed ''in favor'' of cell differentiation, as it had been well shown for pathogens before.

Measurement
Culture density and budding were measured during experiments.
To evaluate density of agar culture, it was carefully washed off the plate with three portions of water, and optical density of the resulting suspension was measured with nephelometer at 650 nm (OD 650 ).
Culture budding was characterized with budding index (BI)total number of buds divided by the total number of cells counted, in %. To calculate BI of the culture, agar sections of 4 cm 2 in area were cut off the plates, fixed with formalin, and microphotographed with a phase contrast microscope with 406 objective and digital 5 MP camera. The photographs were digitally processed with specially created original software [27], automatically recognizing cells and buds in digital pictures ( fig. 1). No less than 1500 cells were counted for each BI calculation.
Both culture density and budding were measured periodically, to obtain growth and budding dynamics of the culture ( fig. 2).

Experiment
The ''induction'' experiment. Two open plates with yeast cultures were fixed together, their cell layers directed towards each other, and left for 10-150 min ( fig. 3A). After that, one of the plates (called ''inductor'') was removed, and the other one (called ''recipient'') -closed, and left at 30uC for further growth. The recipient density and budding were periodically gauged, every 15-30 min for 3-5 hours, and compared to budding and culture density in separated single control plates with identical medium and culture.
In fig. 3C a modification of the standard induction experiment is shown. A small Petri dish with 1M NaOH was fixed inside the recipient plate to partially absorb CO 2 from the atmosphere inside.
CO 2 inducing experiments. To check the inducing capacity of CO 2 , recipient cultures were put into hermetically closed containers (2062064 cm), and atmosphere with various concentrations of CO 2 (0,1-4%) was created inside by injecting the needed volume of 99,99% CO 2 into the container, through an airtight rubber stopper.

Reproducibility
Altogether more than 500 budding curves were registered at various conditions: media content, age of the recipient and inductor cultures, and duration of induction. Each point on the budding curve was obtained by automatic counting of 1500-2000 cells in microphotographs. Each particular experiment was repeated no less than 7 times; some experiments were repeated up to 20 times.
The main experimental data obtained in our work, were budding curves of yeast cultures, which (although looking like standard S-shaped functions), could not be correctly approximated by functions of a single family. Thus we preferred to compare values of budding index in individual time points on the curves. According to our experimental scheme, each experiment had its own control, and thus criteria for dependent samples could be used. As not all the data were always distributed normally, we preferred to use nonparametric Wilcoxon T-test for dependent samples to calculate the data confidence. Intervals given in tables are effective 99% confident intervals calculated from normal approximation.

Distant Stimulation of Budding and Growth
The ''induction'' experiment, as shown in fig. 3A, was performed under various conditions -age of the inductor and the recipient cultures, their medium content, and the induction length. Under particular conditions (see below) the experiment led to stimulation of budding and growth in the recipient culture (compared to adequate control).
The main conditions for the induction effect are listed below.
The ''recipient'' cultures can be stimulated only on weak media. Plated onto rich growth media (YPD or 2-18u beer worth), the recipient cultures didn't react to induction, i.e. both  Plated onto minimal medium with 0,1% glucose, yeast showed a slower (suppressed) dynamics of budding (compare control lines in fig. 4A and B). On this medium induction led to budding stimulation, the culture achieving maximal BI ,1 hour earlier than in control ( fig. 4B). Plated onto minimal acetate-containing medium, control culture showed practically no budding (BI,10%) up to 270 min after inoculation ( fig. 4C), and the induced culture achieved maximal BI of ,50%, which was 5-10 times higher than in control at the same time (see 210-270 min period in fig. 4C, P,10 25 ). Budding-stimulation on minimal media led also to growth stimulation ( fig. 5). Still, the budding stimulation effect could also be observed even on extremely weak media lacking nitrogen, where subsequent growth was impossible ( fig. 4D, growth not shown).
When on similar media, with malate, succinate or fumarate as the only substrate (instead of glucose or acetate), or with no substrate at all, the recipient culture showed no budding either in control or after induction (data not shown).
The recipient cultures can be stimulated only during the lag period of budding, with the induction lasting from 15 to 150 min. The recipient cultures could be stimulated only during the first ,2 hours after inoculation, and the earlier the recipient was subjected to induction, the higher was the observed effect ( fig. 6). Notice that the effect of induction exhibited ,2 hours after the beginning of induction, and 1-1,5 hour after its end.
The minimal length of induction that produced any buddingstimulation effect was found to be 10-15 min. The effect was increasing to maximum with the induction length rising up to 60-90 min, and remained constant for longer inductions (table 1).
Yeast cultures used as inductors must be alive and growing on rich media. Cultures grown on minimal media didn't produce reliable budding stimulation effect (on any recipient cultures) with induction lasting either 30 or 90 min (data not shown). Yeast of the same strain grown on rich growth media (YPD or beer worth) produced budding stimulation effect (on proper recipient cultures) from exponential phase to the beginning of stationary phase (4-30 hours old -see sect. 4

The Induction Effect is Caused by a Volatile Chemical Factor that can be Absorbed by Alkaline Solution
To test whether the inducing factor was a volatile chemical, we separated the inductor and the recipient with metal, glass and quartz plates, and the budding-stimulating effect disappeared in any case (data not shown). The effect was also missing if the atmosphere between the inductor and the recipient was being constantly renewed during the experiment (data not shown). Two identical recipient plates fixed inside a big container with inductor, equally accessible to volatile chemicals, but oppositely located Table 1. Budding index of agar cultures of S.cerevisiae, 270 min after inoculation -in control and after induction of various length (see fig. 4D for the whole budding curve on this medium). The experiment scheme is given in fig. 3A. Inductor -S.cerevisiae culture on YPD-agar in early stationary phase (20 hour old), recipient -S.cerevisiae culture 15 min after inoculation. Medium content: CH3COONa 0,1%+KH2PO4 0,1%+MgSO4 0,05%+CaCl2 0,01%+NaCl 0,01%+agar 3%, pH5,5. doi:10.1371/journal.pone.0062808.t001 Table 2. Budding index of agar cultures of S.cerevisiae, 210 min after inoculation -in control and after 120 min induction with various inductors (see fig. 4D for the whole budding curve on this medium).

Length of induction, min
Inductor Budding index, % (age of recipient -210 min)
Thus the budding stimulation effect was caused by a volatile chemical factor, secreted by yeast cultures from early exponential phase to early stationary phase, and absorbed by alkaline solutions.

Exogenous Carbon dioxide Mimics the Induction Effect
The only known chemical secreted by yeast and corresponding to all the data obtained, is CO 2 . NH 3 and the known quorum sensing factors -tryptophol and phenilethanol -are not absorbed by alkaline solutions. Besides, the quorum sensing factors are not volatile, and NH 3 is not secreted by yeast colonies till rather late stationary phase (4-10 days old [5]).
Measured with infrared CO 2 sensor, the rate of CO 2 production by our inductor cultures was found ,0,1 micromole/sec from 1 cm 2 of agar medium (V. Ptushenko, unpublished data). This rate of CO 2 production leads to accumulation of ,1% CO 2 in 10-20 min, in the atmosphere between the recipient and the inductor. To check the inducing capacity of CO 2 , the recipient cultures were put into hermetically closed containers, and atmosphere with various concentrations of CO 2 (0,1-4%) was created inside. Exogenous CO 2 stimulated budding in the recipient at least in the concentrations from 0,1% to 4% (table 3), and at induction length more than 10 min (table 4). These conditions generally corresponded to the amount of CO 2 secreted by the inductor culture.
Thus the effect of budding stimulation, observed in S.cerevisiae cultures on specific poor media, when in contact with another actively growing yeast culture, was caused by CO 2 , secreted by the latter, and exerting the stimulating influence in concentrations 0,1-4% in the atmosphere, and at the induction length $10 min.
To test the first opportunity, we performed the main budding stimulation experiments on media with different pH. Budding stimulation, both by inductor yeast cultures, and exogenous CO 2 , was equally observed (on appropriate minimal media -see section 4.1.1) at pH from 4,5 to 6 (data not shown). The medium pH in the recipient culture after the end of induction was equal to pH in the control culture (and not changed comparing to initial pH of the medium). Thus, the stimulation effect was not connected to any CO 2 -induced change of the medium pH.
Metabolic CO 2 fixation is for the greatest part taking place in reactions of phoshptryose carboxylation, generating oxaloacetate (OA) and ''supporting'' the Krebs cycle [30]. This way is important on media with glucose, and practically useless on media with acetate, as all OA is generated through glyoxylate bypass   fig. 3C). Inductor -S.cerevisiae culture on YPD-agar in early stationary phase (20 hour old). Medium content: CH3COONa 0,1%+KH2PO4 0,1%+MgSO4 0,05%+CaCl2 0,01%+NaCl 0,01%, pH5,5. doi:10.1371/journal.pone.0062808.g008 Table 3. Budding index of agar cultures of S.cerevisiae, 270 min after inoculation -in control and after induction with CO 2 of various concentration (see fig. 4D for the whole budding curve on this medium).   [30]. To test whether the budding stimulation effect was connected to metabolic CO 2 fixation, we performed our main experiments on glucose and acetate containing minimal media (see fig. 1B and C), with addition of 0,1% oxaloacetate. This led to increase of both control and CO 2 -stimulated budding dynamics on both media (comparing to identical media without OA -see table 5), but didn't decrease the culture sensitivity to CO 2 . Absolute increase of budding, caused by CO 2 , was equal or even slightly higher on media with OA than on identical media without OA (table 5, column DBI). Thus, OA was used as additional substrate, important for the culture budding (table 5) and growth (data not shown), but didn't ''substitute'' exogenous CO 2 . Besides, CO 2 action on yeast was equally high on glucose and acetate containing media, with or without additional OA. Thus, the CO 2 -induced budding stimulation effect in our experiments was not connected to non-specific stimulation of metabolism through CO 2 fixation, and remained as high (or even higher) on media containing excessive amount of oxaloacetate, the key product of CO 2 fixation.

Discussion
In the last decade a number of works appeared, showing CO 2 sensitivity for a vast number of microorganisms [9]. Two general mechanisms of CO 2 action on the cell are known: (1) metabolicheterotrophic fixation, and (2) regulatory -participation in signaling pathways. Heterotrophic fixation of CO 2 , long known for S.cerevisiae [30], Schizosacharomyces pombe [29], and other species, is essential for culture growth on minimal media, mainly by supporting Krebs cycle through phosphotriose to oxaloacetate carboxylation. This way is not active when the culture is grown on rich media, or on minimal acetate-containing media, as all the needed oxaloacetate is produced in Krebs cycle (rich media) or glyoxylate bypass (acetate-containing media).
Regulatory action of CO 2 goes through class IIIb (soluble or cytoplasmic) adenylyl cyclases by direct binding with their catalytic domain. This way is shown for mammals, cyanobacteria [7], and pathogenic fungi [25], in which it stimulates cell differentiation and virulence [31]. Regulatory pathway of CO 2 sensitivity was also supposed for S. pombe [29], and argued for S.cerevisiae [26], but disproved by the same authors in [32], where they showed that the effect of HCO 3 stimulated spore formation, observed in their work, was caused by alkalization of the medium [33].
The present work is the first to show significant effects of CO 2mediated interaction of cells on S.cerevisiae. We cannot make any direct statements concerning mechanisms of our effect yet. Still we can conclude that (1) it is not connected to the medium pH shift, and (2) it is not connected to heterotrophic fixation and metabolic use of CO 2 . This allows us to suppose the budding-stimulation effect going through regulatory, rather than metabolic pathways. Table 4. Budding index of agar cultures of S.cerevisiae, 270 min after inoculation -in control and after induction with S.cerevisiae culture, or with 1% exogenous CO 2 (see fig. 4D for the whole budding curve on this medium).  Medium content: A -Minimal medium with glucose (glucose 0,1%+(NH 4 ) 2 SO 4 0,1%+KH 2 PO 4 0,1%+MgSO 4 0,05%+CaCl 2 0,01%+NaCl 0,01%, pH5,5), B -Minimal medium with acetate, without nitrogen (CH 3 COONa 0,1%+KH 2 PO 4 0,1%+MgSO 4 0,05%+CaCl 2 0,01%+NaCl 0,01%, pH5,5). Inductor -4% CO 2 , length of induction -120 min. Recipient -S.cerevisiae culture 15 min after inoculation. doi:10.1371/journal.pone.0062808.t005 Besides, the effect is observed 1,5-2 hour later than the interaction is finished. The main difference of our results from the effects of CO 2 action, known for pathogenic fungi, is that here CO 2 increase stimulates cell division, rather than mitosis block and cell differentiation [9,31].
Anyway, the effect of distant CO 2 -mediated interaction of S. cerevisiae cultures, shown in this work, can be interpreted as cell-cell interaction, regulating cell behavior according to the culture density, i.e. a quorum sensing effect.