Mutation of the murC and murB Genes Impairs Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

To stabilize cellular integrity in the face of environmental perturbations, most bacteria, including cyanobacteria, synthesize and maintain a strong, flexible, three-dimensional peptidoglycan lattice.Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium capable of differentiating morphologically distinct nitrogen-fixing heterocyst cells in a periodic pattern. While heterocyst development has been shown to require proper peptidoglycan remodeling, the role of peptidoglycan synthesis has remained unclear. Here we report the identification of two peptidoglycan synthesis genes,murC (alr5065 ) andmurB (alr5066 ), as required for heterocyst development. ThemurC andmurB genes are predicted to encode a UDP-N -acetylmuramate:l -alanine ligase and a UDP-N -acetylenolpyruvoylglucosamine reductase, respectively, and we confirm enzymatic function through complementation ofEscherichia coli strains deficient for these enzymes. Cells depleted of eithermurC ormurB expression failed to differentiate heterocysts under normally inducing conditions and displayed decreased filament integrity. To identify the stage(s) of development affected bymurC ormurB depletion, the spatial distribution of expression of the patterning marker gene,patS , was examined. WhereasmurB depletion did not affect the pattern ofpatS expression,murC depletion led to aberrant expression ofpatS in all cells of the filament. Finally, expression ofgfp controlled by the region of DNA immediately upstream ofmurC was enriched in differentiating cells and was repressed by the transcription factor NtcA. Collectively, the data in this work provide evidence for a direct link between peptidoglycan synthesis and the maintenance of a biological pattern in a multicellular organism.IMPORTANCE Multicellular organisms that differentiate specialized cells must regulate morphological changes such that both cellular integrity and the dissemination of developmental signals are preserved. Here we show that the multicellular bacteriumAnabaena , which differentiates a periodic pattern of specialized heterocyst cells, requires peptidoglycan synthesis by the murine ligase genesmurC (alr5065 ) andmurB (alr5066 ) for maintenance of patterned gene expression, filament integrity, and overall development. This work highlights the significant influence that intracellular structure and intercellular connections can have on the execution of a developmental program.