Spo0M exhibits a scattered distribution pattern in cells, appearing near the cell poles and the septum region.
We generated a Spo0M:DsRed fusion protein via allelic replacement using the pSGGS construct (A) (see S1 File). R1, 500 bp upstream of the 5’ intergenic region of spo0M; SpR, spectinomycin resistance cassette; KnR, 500 bp of the CT region of a kanamycin resistance cassette. B. We analyzed the phenotypes of these strains using light microscopy and observed that the wild type phenotype was restored. The absence of elongated cells in the Spo0M:DsRed strains indicated that Spo0M functionality was complemented by the fusion protein C. Spore quantification of the wild type, mutant and Spo0M:DsRed strains. All of the strains were cultured in Spizizen sporulation medium for 40 h. The total number of spores in each sample was quantified manually using a bright field microscope. The number of spores generated by the Spo0M:DsRed strain was restored to wild type levels, validating that the Spo0M phenotype was complemented with the fusion protein. Data was analyzed through an ANOVA test and a Turkey multiple analysis of means, with a p value of 0.001, blue bar corresponds to the mean value and red bar represents the standard deviation. We used the fluorescent fusion protein to analyze the localization pattern of Spo0M during bacterial growth in rich and sporulation media. D. In rich medium (exponential phase of growth, 6–8 h of culture), Spo0M formed a scattered distribution near the cell poles (orange arrows) and in the central region of the cell, where a septum would be expected to form (white arrow). This distribution pattern remained until the appearance of the first spores (20–24 h). E. In sporulation medium (16–20 h of culture), Spo0M was localized to the forespores (arrow head), and it remained contained in these structures even after their release as mature spores (after 18–20 h). Scale bar inside the box is 1μm.