Cell Wall Amidase AmiC1 Is Required for Cellular Communication and Heterocyst Development in the Cyanobacterium Anabaena PCC 7120 but Not for Filament Integrity

Abstract

Filamentous cyanobacteria of the order Nostocales display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N 2 takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the Gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue amiC2 gene in Nostoc punctiforme ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two amiC paralogues from Anabaena sp. strain PCC 7120. The amiC1 ( alr0092 ) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the amiC2 ( alr0093 ) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.

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

“…Mutation of the amiC2 locus in Nostoc punctiforme also resulted in a strain that could not differentiate heterocysts, however, in contrast to the normal morphology of the murC and murB mutants following the removal of combined nitrogen to induce differentiation, the amiC2 mutant produced an aberrant phenotype consisting of groups of fused cells with incomplete septal cleavage (24). Mutation of the amiC1 and alr5045 genes from Anabaena resulted in a reduced percentage of heterocysts that formed after a time delay (25,54). Finally, mutation of the pbp6 and hcwA genes in Anabaena yielded strains that produced a normal pattern of heterocysts that could not fix nitrogen under aerobic conditions (22,23).…”

“…Similarly, transposon inactivation of the gene encoding the penicillin-binding protein Pbp6, a member of a class of proteins known to be involved in peptidoglycan formation and maintenance, resulted in a normal pattern of heterocysts that could fix nitrogen only under anoxic conditions (23). The amidase-encoding genes amiC1 and amiC2 were both found to be required for proper heterocyst differentiation in Anabaena and in the closely related strain Nostoc punctiforme ATCC 29133, respectively (24,25). Mutation of amiC1 in Anabaena resulted in the greatly reduced formation of heterocysts, and the mutant was impaired in the movement of the fluorescent dye calcein-AM between cells (25).…”

“…The amidase-encoding genes amiC1 and amiC2 were both found to be required for proper heterocyst differentiation in Anabaena and in the closely related strain Nostoc punctiforme ATCC 29133, respectively (24,25). Mutation of amiC1 in Anabaena resulted in the greatly reduced formation of heterocysts, and the mutant was impaired in the movement of the fluorescent dye calcein-AM between cells (25). Although also impaired in intercellular dye movement, an N. punctiforme amiC2 mutant was entirely incapable of forming heterocysts and, instead, created cellular aggregates of fragmented filaments due to incomplete septal cleavage between dividing cells (24).…”

Cell Envelope Components Influencing Filament Length in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

“…Two other class A PBPs, All2981 and Alr4579, are also specifically required for aerobic N 2 fixation, and their inactivation results in an altered polysaccharide layer of the heterocyst envelope (31). In addition, an N-acetylmuroamoyl-L-alanine amidase is required specifically for heterocyst maturation (38,39). Therefore, PG metabolism-related enzymes appear to be important during the differentiation of heterocysts: they may be needed for the proper assembly of the glycolipid and polysaccharide layers of the heterocyst envelope or for remodeling of the vegetative-cell-heterocyst intercellular septa.…”

An amidase is required for proper intercellular communication in the filamentous cyanobacteriumAnabaenasp. PCC 7120

“…The nanopores are located in the central areas of the septa. Formation of the nanopores on the septa between the cells requires amidases in both Anabaena 7120 and Nostoc punctiforme (29,31,32).…”