Fractionation of cells from the rim and hub of the mat.
Growth on low-agar plates produced a clear visual differentiation between the cells at the rim and those at the hub of a wild-type mat (Fig. ). In order to analyze the two populations of cells, we overlaid the mat with plastic wrap. When the plastic wrap was withdrawn, the nonadherent cells at the rim were drawn up with the wrap (Fig. , rim), whereas the agar-adherent cells in the hub remained behind (Fig. , hub).
This analysis revealed a new aspect of mat formation: the growing mat could be partitioned into two distinct subpopulations based on agar adherence properties. The point on the growing mat at which the appearance changed from smooth (rim) to channel-ridden (hub) (Fig. ) is also the point at which the adherence phenotype changed. By contrast, the flo11Δ strain, which is uniformly smooth, failed to adhere to the agar and was completely removed from the agar by the plastic wrap (Fig. ).
A glucose gradient is present in the mat and influences its development.
Since a low glucose concentration promotes adhesion of Saccharomyces
cells to polystyrene (24
), we determined whether there was a gradient of glucose that correlated with the different cell populations revealed by the overlay adhesion assay. This analysis revealed that the uniform glucose level present prior to growth of the mat (2%) was replaced during growth by a concentration gradient that decreased from the rim toward the hub (Fig. ). Thus, there is a gradient of glucose in the growing mat such that the concentration is higher at the rim and lower at the hub.
Glucose limitation appeared to be an important signal for regulating mat formation, but other nutrients could be depleted in the hub and might play a role as well. If glucose is the key nutrient in regulating mat formation, then medium containing increased levels of glucose, but identical levels of all other components, should slow mat formation. Plates were poured that contained 1% yeast extract, 2% peptone, and 2%, 4%, or 6% glucose. It was found that, in comparison to 2% glucose plates, hub formation was delayed by 1 day or 2 days in plates containing 4% or 6% glucose, respectively (Fig. ).
Increased glucose concentrations delay mat formation. The wild-type (TBR1) strain was inoculated onto the center of YPD plates with 0.3% agar containing 2%, 4%, or 6% glucose and grown at 23°C for 7 days.
Although a glucose gradient is formed in the growing mat (Fig. ), it was unclear if the gradient itself was required for mat formation. In order to test this, the gradient was perturbed in a mat by the addition of glucose to the medium outside of the growing mat. If a glucose gradient were required for maturation of the mat, then the addition of glucose to one side of the mat should slow maturation of the mat on the side to which glucose was added. After a wild-type mat had grown for 3 days, a small pit was dug in the agar outside of the mat with a pipette tip, and 50 μl of 40% glucose or water was added to the pit (Fig. , ). After an additional 2 days of growth, the mat growing near the pit containing 40% glucose underwent mat formation normally except for a large smooth region of the mat immediately adjacent to the pit. This region lacked the channels typical of the developing mat (Fig. ). Conversely, the mat facing the pit containing water underwent mat formation normally on all sides (Fig. ).
FIG. 3. Disruption of the glucose gradient perturbs mat formation. The glucose gradient set up within the growing mat was perturbed by adding 50 μl of 40% glucose or water as a negative control to a pit dug in the agar at day 3 (3d). After two (more ...)
The fact that a gradient of glucose drives mat formation and that the hub is depleted of glucose compared to the rim suggested that cells in the rim were actively growing whereas those in the hub had stopped growing. This was tested by a “healing” assay. The mat was damaged by using a pipette tip to tear a hole in both the rim and the hub on day 5 (Fig. ). Two days later (day 7), the hole made in the rim was detectable only as a depression in the agar beneath the mat as cells had proceeded to grow over it. Conversely, the hole in the hub looked much like it had at day 5 with only slight changes in appearance (Fig. ). This result indicated that the cells in the hub exhibited little to no growth, whereas the cells in the rim continued to divide and spread.
FIG. 4. Cells in the rim (R) are actively growing whereas cells in the hub (H) exhibit very little growth. Wild-type (TBR1) yeast was grown on YPD-0.3% agar plates, and at day 5 a pipette tip was used to gouge a hole in the hub and rim. The mat was then (more ...) Glucose-sensing genes control mat formation and FLO11 expression.
Several signaling pathways are associated with sensing glucose levels and regulating filamentous growth in S. cerevisiae
. These pathways include the Ras/cyclic AMP, Snf1p kinase, and Yak1p kinase pathways. Genes representing these pathways include SNF1
), and RAS2
has been shown to be required for mat formation on low agar (21
), biofilm formation on plastic, FLO11
), and filamentous growth on solid 2% agar (7
). Isogenic mutants for these genes were generated in the strain TBR1 (24
) and tested for mat formation. Disruptions of SNF1
, or RAS2
all caused defects in mat formation (Fig. ). Although they differed in the extent of their adhesion defects (Fig. ), all three mutants formed few or no discernible channels and spread poorly.
FIG. 5. Strains carrying mutations that interfere with glucose signaling exhibit defects in mat formation. Strains were inoculated onto the center of YPD plates containing 0.3% agar, grown at 23°C for 5 days (Mat), and subjected to the overlay (more ...)
The three glucose-sensing mutants, yak1Δ, ras2Δ, and snf1Δ, were tested for their effects on FLO11 expression. It was found that all three mutants caused a decrease in FLO11 expression compared to the wild-type strain when grown to exponential phase in liquid YPD medium containing 2% glucose (Fig. ). The ras2Δ mutant did not cause a strong decrease in FLO11 expression compared to the yak1Δ or snf1Δ mutants. However, when the three mutants were compared to wild type in liquid YPD medium during glucose stress (growth to post-exponential phase), the ras2Δ mutant, like the yak1Δ and snf1Δ mutants, showed a significant decrease in FLO11 expression (Fig. ).
FIG. 6. Mutants in glucose-sensing pathways have variable effects on FLO11 expression. Wild-type and mutant strains were grown in YPD medium to logarithmic phase (OD600 of 0.5 to 1.0) (A) or post-logarithmic phase (OD600 of ~4.0) (B) where the cells are (more ...) Flo11p is expressed on the surface of cells in the rim and hub.
Since the glucose-sensing genes affect the expression of FLO11
and glucose levels are lower in the hub, the morphological differences between the rim and the hub could be a consequence of FLO11
mRNA levels were measured in the rim, the intermediate region where the rim and hub meet (middle), and the center of the hub by real-time RT-PCR. If ACT1
is used to normalize the FLO11
levels, there is no significant difference in FLO11
mRNA levels between the rim and hub (Fig. ). By contrast, if SNR190
is used for normalization, FLO11
mRNA levels appear highest in the rim and lowest in the hub (Fig. ). This difference in expression is also observed in the ACT1
mRNA levels, which appear to be higher in the rim than the hub when SNR190
is used for normalization (Fig. ). ACT1
levels are affected by the growth state of cells (5
), but SNR190
levels are stable between cells in logarithmic and stationary phase (7a
). The indeterminate growth state of cells in the hub makes an accurate measurement of FLO11
mRNA levels difficult, but it is clear that FLO11
mRNA is expressed in both parts of the mat.
FIG. 7. FLO11 is expressed in the rim, hub, and intermediate (middle) regions of the mat, but the other FLO genes are not expressed. (A) Real-time RT-PCR was used to measure the expression of FLO11 and the other FLO genes, and the levels of expression were normalized (more ...)
The availability of a strain expressing a functional version of Flo11p with an HA tag (10
) permitted a direct assessment of the levels of Flo11p on the surface of cells in the rim and hub. This strain, containing the FLO11
construct inserted into the chromosome at the FLO11
locus, forms mats like the wild-type strain. The percentage of cells expressing Flo11p on their surfaces was assessed by indirect immunofluorescence and was found to be the same in the rim and hub (38% ± 2% and 38% ± 5%, respectively) (Fig. ). As has been previously reported (10
), there was heterogeneity of FLO11
expression among the cells, but there did not appear to be any difference in the distribution of the cells expressing Flo11p in the two populations.
FIG. 8. Flo11-HA was expressed at similar levels in both the rim and hub of the mat. Cells were collected from both the rim (A and C) and hub (B and D). Cells were stained with an anti-HA monoclonal antibody and viewed by differential interference contrast (A (more ...)
The same RNAs from the rim, hub, and middle that were used to assess the FLO11
levels were used to ascertain the levels of the other FLO genes (FLO1
, and FLO10
), which are known to affect aggregation and adhesion (30
). Although strains from the ∑1278b background do not express these other FLO genes in liquid, they might be expressed on the low-agar petri dishes. Analysis of these transcript levels by real-time RT-PCR using primers specific for these genes showed no significant expression in any portion of the mat using either ACT1
as a normalization standard (Fig. , respectively).
Mat formation is controlled by a pH gradient.
Analysis of the mat revealed that in addition to the glucose gradient (Fig. ), the mat also established a pH gradient. The pH of the medium was originally 5.8 but was altered by the yeast, and after growth of the mat, a pH gradient formed between the sites of the hub (4.7) and rim (5.0) (Fig. ). Moreover, the cells at the edge of the rim were juxtaposed to medium at pH 5.8. To test whether the pH gradient was an important component in the differentiation of the mat, the pH of the medium was buffered with 20 mM citrate buffer to a final pH of 4.9, 5.4, or 5.8, and mat formation was initiated on these plates (Fig. ). Wild-type mats growing on plates buffered with citrate buffer exhibited generally decreased spreading compared to mats growing on plates without citrate buffer (Fig. ). A slight rim and hub region could be detected in mats grown on plates buffered to pH 5.4, based on appearance (Fig. ). However, at pH 5.8, the channels that characterize the hub did not form, and the surface of the whole mat was smooth. In the cases of both types of plates (pH 5.4 and 5.8), the surfaces of the whole mats were removed by the overlay adhesion assay (Fig. ). In contrast, the entire surfaces of mats formed on pH 4.9 plates resembled hubs, and no material could be removed from the surface of the plate using the overlay adhesion assay (Fig. ). This strong adherence at pH 4.9 is dependent on FLO11 as the flo11Δ mutant was completely removed from pH 4.9 plates during the overlay adhesion assay (Fig. ).
Mat formation correlates with a gradient of pH. pH strips were used to measure the pH of medium outside the mat and inside the rim and the hub. The pH values are indicated next to the strips. The lines indicate the sampling points.
FIG. 10. pH controls mat formation. Wild-type or flo11Δ strains were inoculated onto low-agar YPD plates that were buffered to pH 4.9, 5.4, and 5.8 with 20 mM Na citrate buffer; cultures were grown for 5 days at 23°C and photographed. The mat in (more ...)