Third, the colR-deficient strain possessed slightly less OprB1 in its OM than the wild-type (Figure 6C), indicating that the membrane of the colR mutant is probably sensitive to accumulation of OprB1. Thus, our data suggest that ColRS is necessary for P. putida to maintain the cell membrane homeostasis and this becomes particularly important during up-regulation of certain OMPs such as OprB1. We detected the glucose-specific cell lysis of the colR-deficient strain only on solid and not in liquid medium (Figure 1).

Bacterial population growing on solid medium is highly heterogeneous and it is obvious that bacteria located at the edge of the growth area experience different conditions compared to the cells in the centre of the population. Gradient fields of carbon source as well as of excreted metabolites develop during the growth, putting the cells in the centre of the population under more restrictive conditions than those at the periphery. It has BTK inhibitor been shown that such gradient fields govern cellular responses of multicellular solid medium populations and regulate development of gene expression patterns in space and time [11]. Our previous results revealed a spatial aspect NVP-BKM120 cell line of ColR-dependent lysis. Colonies of the colR-deficient strain developed central concavities when growing on the glucose medium which we interpreted as an elevated lysis of central population [25].

Here, we proved that the degree of lysis of the colR mutant is spatially different. However, contrary to our expectations the lysis of peripheral cells was significantly higher than that of the central cells. Yet, it is important to point out that in Org 27569 the current study we analyzed the bacteria grown on a sector (1/6 of the Petri plate), the area of which is more than 100 times bigger than that of a single colony. Therefore, the nutrient gradients building up in the medium under central cells of a sector and under the central part of a colony are not really

comparable. We suggest that lysis occurs at a certain glucose concentration range and whether this develops in the centre or in the periphery of a population depends on the size of the cells’ growth area. This study indicated that the glucose-specific lysis of the colR-deficient P. putida occurs among a subpopulation of cells adapting to nutrient limitation. This was most strongly evidenced by the fact that the degree of lysis depended both on time and glucose concentration. We suggest that the continuous increase of the colR mutant lysis during the first 48 hours of growth on 0.2% glucose solid medium (Figure 1 and Figure 5) is caused by a gradual decrease of glucose concentration. Given that significantly less lysis was observed on 0.4% glucose and that no lysis was detected on 0.8% glucose medium (Figure 5), it is possible to conclude that the ColR-dependent cell lysis occurs only when the amount of glucose decreases below a certain threshold level.