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

Abstract

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) and murB (alr5066), as required for heterocyst development. The murC and murB 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 of Escherichia coli strains deficient for these enzymes. Cells depleted of either murC or murB expression failed to differentiate heterocysts under normally inducing conditions and displayed decreased filament integrity. To identify the stage(s) of development affected by murC or murB depletion, the spatial distribution of expression of the patterning marker gene, patS, was examined. Whereas murB depletion did not affect the pattern of patS expression, murC depletion led to aberrant expression of patS in all cells of the filament. Finally, expression of gfp controlled by the region of DNA immediately upstream of murC 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. IMPORTANCEMulticellular 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 bacterium Anabaena, which differentiates a periodic pattern of specialized heterocyst cells, requires peptidoglycan synthesis by the murine ligase genes murC (alr5065) and murB (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. Bacteria have evolved diverse strategies to combat environmental changes, including osmotic pressure, oxidative damage, and nutrient deprivation (reviewed in references 1 and 2). To prevent lysis from such environmental stressors, two different cell wall architectures have been adopted that also serve as a basis for classification. Gram-positive bacteria have a characteristically thick peptidoglycan layer (20 to 80 nm) outside an inner membrane, while Gram-negative strains have an inner membrane and outer membrane separated by a thin peptidoglycan layer (1 to 7 nm). Cyanobacteria have a Gram-negative cell wa...

A combinatorial strategy of alternative promoter use during differentiation of a heterocystous cyanobacterium

“…All4316 is annotated as MraY, a protein involved in peptidoglycan synthesis and therefore fulfilling a housekeeping function in the context of cell division. In addition, because deposition of heterocyst‐specific envelopes involves remodelling of the cell wall, peptidoglycan synthesis and remodelling are critical aspects of heterocyst differentiation (Lehner et al ., ; Videau et al ., ). In fact, several genes related to peptidoglycan metabolism are transcribed from HetR‐dependent promoters (Mitschke et al ., ).…”

The Integrity of the Cell Wall and Its Remodeling during Heterocyst Differentiation Are Regulated by Phylogenetically Conserved Small RNA Yfr1 in Nostoc sp. Strain PCC 7120

“…Heterocyst differentiation involves important morphological changes in which synthesis and remodeling of peptidoglycan play an important role. Two of the validated targets of Yfr1 (amiC2 and murC) are necessary for proper diazotrophic growth (11)(12)(13). In addition, mraY (also among the targets of Yfr1 validated in this study) has a complex promoter region in which one transcription start site is specifically upregulated in heterocysts (41), suggesting a role of this enzyme in heterocyst differentiation.…”

“…Mutants with mutations in the murein amidases AmiC1 (alr0092) (10) and AmiC2 (alr0093, hcwA) (11,12) show a significant reduction in the number of nanopores and defects in diazotrophic growth. In addition, strains with mutations in the peptidoglycan synthesis enzymes MurC (alr5065) and MurB (alr5066) (13) and in some penicillin binding proteins (PBPs) (those encoded by all2981, alr4579, and alr5051) (14,15) show alterations in heterocyst differentiation and growth defects in media without combined nitrogen. All this evidence points to the synthesis and remodeling of peptidoglycan being essential for proper heterocyst differentiation and diazotrophic growth.…”

The Peptidoglycan Biosynthesis Gene murC in Frankia: Actinorhizal vs. Plant Type

“…The similarity of Frankia MurC2 to plant MurC proteins might be associated with the presence of regulatory processes, which allow for easier structural changes of the peptidoglycan required for the intracellular lifestyle. This assumption is supported by the fact that in cyanobacteria, murC and murB were found to play a role in the differentiation of nitrogen-fixing heterocysts [7]. However, this would not explain why members of Frankia cluster-4, which do not engage in symbiosis, would have retained the MurC type more similar to that of plants over the ancestral MurC type of Gram-positive bacteria.…”

“…Several reports on the activity of Mur ligases have been published for non-pathogenic bacteria. In the cyanobacterium Anabena sp., murC and murB play an important role in heterocyst differentiation [7]. Although, so far, PG layers have not been identified for the chloroplasts of Spermatophyta, it is known that Arabidopsis thaliana contains a copy of murE, and the mutation of this gene leads to the reduced chlorophyll content of leaves, indicating an essential role of murE for chloroplast development [8].…”