Five of the SCO4126-4131 genes encoded membrane proteins, while SCO4127 encoded an ATP/GTP-binding protein. Thus, the SCO4126-4131 gene cluster was designated cmdA-F

(a cluster of genes encoding membrane proteins for differentiation). Figure 2 Phenotype of the null mutants of cmdABCDEF on MS plates. (A) Growth of single and multiple null mutants of the cmdABCDEF genes on MS for three days. The parental strain is M145. (B) A time course of culturing M145 and the null mutants. Strains were inoculated as ~1 cm2 patches on MS medium. Time points of observation are shown on the right. Aberrant branches, defective spore septation and abnormal chromosome segregation in null mutants After harvesting, diluting and plating out spores on medium, the numbers of spores (c. 106/ml) obtained from the ΔcmdB and especially ΔcmdA-F CYC202 molecular weight strains were obviously less than that of wide type M145 (c. 108/ml). To characterise these aerial hyphae and spores, we employed phase-contrast and scanning electron microscopy. Under phase-contrast microscopy, normally PS-341 clinical trial long unbranched aerial hyphae were seen in M145, whereas multiple branching from both aerial and apical

hyphae, giving rise to unusually short spore chains, was observed in the ΔcmdB and ΔcmdA-F strains (Figure TCL 3A). Scanning electron microscopy revealed, in contrast to nearly complete septation of aerial hyphae and formation of abundant long spore Elafibranor chains in M145, most aerial hyphae in null mutants of cmdB and cmdA-F were collapsed and unable to septate to become spores, while some of hyphae could eventually develop into short spore chains (Figure 3B). To further dissect these sporulating aerial hyphae, we employed fluorescence microscopy. Sporulating hyphae were fixed and then their chromosomes

were stained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence microscopy revealed that chromosomes in wide-type M145 were distributed at regularly spaced intervals along spore chains (Figure 3C), and anucleate spores were observed at a low frequency (0.1%, c.1000 spores counted). However, incomplete separation of chromosomes was readily seen in the mutants, shown as unevenly stained chromosomes along spore chains (Figure 3C); and anucleate spores appeared at a frequency of 8% and 6% along spore chains for the ΔcmdB and ΔcmdA-F strains (c.500 spores counted), respectively. Taken together, the ΔcmdB or ΔcmdA-F strains showed aberrant branches, defective chromosome segregation and abnormally spaced spore septation.