Functions of the Essential Gene mraY in Cellular Morphogenesis and Development of the Filamentous Cyanobacterium Anabaena PCC 7120

Bacterial cell shape is determined by the peptidoglycan (PG) layer. The cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a filamentous strain with ovoid-shaped cells connected together with incomplete cell constriction. When deprived of combined nitrogen in the growth medium, about 5–10% of the cells differentiate into heterocysts, cells devoted to nitrogen fixation. It has been shown that PG synthesis is modulated during heterocyst development and some penicillin-binding proteins (PBPs) participating in PG synthesis are required for heterocyst morphogenesis or functioning. Anabaena has multiple PBPs with functional redundancy. In this study, in order to examine the function of PG synthesis and its relationship with heterocyst development, we created a conditional mutant of mraY, a gene necessary for the synthesis of the PG precursor, lipid I. We show that mraY is required for cell and filament integrity. Furthermore, when mraY expression was being limited, persistent septal PG synthetic activity was observed, resulting in increase in cell width. Under non-permissive conditions, filaments and cells were rapidly lysed, and no sign of heterocyst development within the time window allowed was detected after nitrogen starvation. When mraY expression was being limited, a high percentage of heterocyst doublets were found. These doublets are formed likely as a consequence of delayed cell division and persistent septal PG synthesis. MraY interacts with components of both the elongasome and the divisome, in particular those directly involved in PG synthesis, including HetF, which is required for both cell division and heterocyst formation.

Heterocyst Development and Diazotrophic Growth of Anabaena variabilis under Different Nitrogen Availability

Nitrogen is globally limiting primary production in the ocean, but some species of cyanobacteria can carry out nitrogen (N) fixation using specialized cells known as heterocysts. However, the effect of N sources and their availability on heterocyst development is not yet fully understood. This study aimed to evaluate the effect of various inorganic N sources on the heterocyst development and cellular growth in an N-fixing cyanobacterium, Anabaena variabilis. Growth rate, heterocyst development, and cellular N content of the cyanobacteria were examined under varying nitrate and ammonium concentrations. A. variabilis exhibited high growth rate both in the presence and absence of N sources regardless of their concentration. Ammonium was the primary source of N in A. variabilis. Even the highest concentrations of both nitrate (1.5 g L−1 as NaNO3) and ammonium (0.006 g L−1 as Fe-NH4-citrate) did not exhibit an inhibitory effect on heterocyst development. Heterocyst production positively correlated with the cell N quota and negatively correlated with vegetative cell growth, indicating that both of the processes were interdependent. Taken together, N deprivation triggers heterocyst production for N fixation. This study outlines the difference in heterocyst development and growth in A. variabilis under different N sources.

Preventing Accidental Heterocyst Development in Cyanobacteria

ABSTRACT The filamentous cyanobacterium Anabaena can form heterocysts specialized in N2 fixation, mostly through a cascade of transcriptional activation in response to the nitrogen starvation signal 2-oxoglutarate. It is reported now that a transcription repressor, CalA, acts as a safety device to prevent heterocyst development under certain conditions where the 2-oxoglutarate level may touch the threshold to trigger unnecessary initiation of heterocyst development. Such a control may increase the fitness of Anabaena under a constantly changing environment.

A Heterocyst-Specific Antisense RNA Contributes to Metabolic Reprogramming in Nostoc sp. PCC 7120

Upon nitrogen deficiency, some filamentous cyanobacteria differentiate specialized cells, called heterocysts, devoted to N2 fixation. Heterocysts appear regularly spaced along the filaments and exhibit structural and metabolic adaptations, such as loss of photosynthetic CO2 fixation or increased respiration, to provide a proper microaerobic environment for its specialized function. Heterocyst development is under transcriptional control of the global nitrogen regulator NtcA and the specific regulator HetR. Transcription of a large number of genes is induced or repressed upon nitrogen deficiency specifically in cells undergoing differentiation. In recent years, the HetR regulon has been described to include heterocyst-specific trans-acting small RNAs and antisense RNAs (asRNAs), suggesting that there is an additional layer of post-transcriptional regulation involved in heterocyst development. Here, we characterize in the cyanobacterium Nostoc (Anabaena) sp. PCC 7120 an asRNA, that we call as_glpX, transcribed within the glpX gene encoding the Calvin cycle bifunctional enzyme sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (SBPase). Transcription of as_glpX is restricted to heterocysts and is induced very early during the process of differentiation. Expression of as_glpX RNA promotes the cleavage of the glpX mRNA by RNase III, resulting in a reduced amount of SBPase. Therefore, the early expression of this asRNA could contribute to the quick shut-down of CO2 fixation in those cells in the filament that are undergoing differentiation into heterocysts. In summary, as_glpX is the first naturally occurring asRNA shown to rapidly and dynamically regulate metabolic transformation in Nostoc heterocysts. The use of antisense transcripts to manipulate gene expression specifically in heterocysts could became a useful tool for metabolic engineering in cyanobacteria.

Three Substrains of the CyanobacteriumAnabaenasp. Strain PCC 7120 Display Divergence in Genomic Sequences andhetCFunction

ABSTRACTAnabaenasp. strain PCC 7120 is a model strain for molecular studies of cell differentiation and patterning in heterocyst-forming cyanobacteria. Subtle differences in heterocyst development have been noticed in different laboratories working on the same organism. In this study, 360 mutations, including single nucleotide polymorphisms (SNPs), small insertion/deletions (indels; 1 to 3 bp), fragment deletions, and transpositions, were identified in the genomes of three substrains. Heterogeneous/heterozygous bases were also identified due to the polyploidy nature of the genome and the multicellular morphology but could be completely segregated when plated after filament fragmentation by sonication.hetCis a gene upregulated in developing cells during heterocyst formation inAnabaenasp. strain PCC 7120 and found in approximately half of other heterocyst-forming cyanobacteria. Inactivation ofhetCin 3 substrains ofAnabaenasp. PCC 7120 led to different phenotypes: the formation of heterocysts, differentiating cells that keep dividing, or the presence of both heterocysts and dividing differentiating cells. The expression of PhetZ-gfpin thesehetCmutants also showed different patterns of green fluorescent protein (GFP) fluorescence. Thus, the function ofhetCis influenced by the genomic background and epistasis and constitutes an example of evolution under way.IMPORTANCEOur knowledge about the molecular genetics of heterocyst formation, an important cell differentiation process for global N2fixation, is mostly based on studies withAnabaenasp. strain PCC 7120. Here, we show that rapid microevolution is under way in this strain, leading to phenotypic variations for certain genes related to heterocyst development, such ashetC. This study provides an example for ongoing microevolution, marked by multiple heterogeneous/heterozygous single nucleotide polymorphisms (SNPs), in a multicellular multicopy-genome microorganism.

Mutation of themurCandmurBGenes Impairs Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

ABSTRACTTo stabilize cellular integrity in the face of environmental perturbations, most bacteria, including cyanobacteria, synthesize and maintain a strong, flexible, three-dimensional peptidoglycan lattice.Anabaenasp. 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. ThemurCandmurBgenes 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 colistrains deficient for these enzymes. Cells depleted of eithermurCormurBexpression failed to differentiate heterocysts under normally inducing conditions and displayed decreased filament integrity. To identify the stage(s) of development affected bymurCormurBdepletion, the spatial distribution of expression of the patterning marker gene,patS, was examined. WhereasmurBdepletion did not affect the pattern ofpatSexpression,murCdepletion led to aberrant expression ofpatSin all cells of the filament. Finally, expression ofgfpcontrolled by the region of DNA immediately upstream ofmurCwas 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 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.