Figure 5 Comparison of lysis of peripheral and central subpopulations of P. putida PaW85 wild-type (wt) and colR -deficient (colR) strains grown on solid glucose medium. A. Representation
of a Petri plate with three growth sectors of bacteria and subpopulations sampled for β-galactosidase analysis. Unmasked β-galactosidase activity was assayed from the cells of peripheral subpopulation (area encircled by the dotted line and indicated by the white arrow) and from central one (indicated by the black arrow). Black circles indicate the areas sampled for the measurement of residual glucose concentration in the medium (data is presented in Table 3). The check details degree of lysis is presented as unmasked β-galactosidase activity which was measured from bacteria Selleck BI 6727 grown either 24, 48 or 72 hours on solid media with 0.2% (B), 0.4% (C) or 0.8% (D) of glucose see more (glc) as the carbon source. Due to the spatiotemporal character of the lysis of the colR mutant we hypothesized that nutrient limitation could be involved in cell death. During the active growth of bacteria
on agar plate the concentration of glucose in the growth area decreases, yet, it is obvious that compared to the central population the peripheral cells are nutritionally less limited due to diffusion of glucose from the adjacent medium. To evaluate the glucose consumption dynamics during 72 hours of bacterial growth on 0.2% (9 mM) glucose solid medium, we measured the glucose concentration in the growth
agar by sampling the regions underneath the cell lawn and adjacent to the bacterial growth area (sampling regions are indicated in Figure 5A). Already at 24 hours of growth, the amount of glucose in the medium underneath the bacterial lawn had dropped below the detection level of the assay (0.1 mM). Concentration of glucose in the medium adjacent to the growth area continuously dropped down to 1.6 mM by 72 hours of growth (Table 3). These results show that bacteria constantly consume glucose that is diffusing from adjacent region of agar plate and that peripheral population of bacteria has to adapt to gradient of glucose. Notably, glucose consumption Rebamipide dynamics for the wild-type and the colR mutant were similar. Table 3 Glucose concentration in the bacteria-free agar medium adjacent to the growth area of the cells Glucose concentration (mM) Initially After 24 hours After 48 hours After 72 hours 9 (0.2%) 6.9 ± 0.3 2.9 ± 0.6 1.6 ± 0.2 18 (0.4%) 14.0 ± 1.0 5.9 ± 0.4 3.5 ± 0.4 36 (0.8%) 29.2 ± 0.3 13.0 ± 1.3 6.8 ± 0.9 Accumulating evidence indicates that bacteria growing under subsaturating nutrient levels express a transient response called hunger response, which helps them to cope with limiting conditions [48]. The most obvious feature of hunger response is up-regulation of nutrient uptake systems, including several OM porins [3, 5]. This lead us to hypothesize that elevated lysis of peripheral cells on 0.