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

Abstract

Yfr1 is a strictly conserved small RNA in cyanobacteria. A bioinformatic prediction to identify possible interactions of Yfr1 with mRNAs was carried out by using the sequences of Yfr1 from several heterocyst-forming strains, including Nostoc sp. strain PCC 7120. The results of the prediction were enriched in genes encoding outer membrane proteins and enzymes related to peptidoglycan biosynthesis and turnover. Heterologous expression assays with Escherichia coli demonstrated direct interactions of Yfr1 with mRNAs of 11 of the candidate genes. The expression of 10 of them (alr2458, alr4550, murC, all4829, all2158, mraY, alr2269, alr0834, conR, patN) was repressed by interaction with Yfr1, whereas the expression of amiC2, encoding an amidase, was increased. The interactions between Yfr1 and the 11 mRNAs were confirmed by site-directed mutagenesis of Yfr1. Furthermore, a Nostoc strain with reduced levels of Yfr1 had larger amounts of mraY and murC mRNAs, supporting a role for Yfr1 in the regulation of those genes. Nostoc strains with either reduced or increased expression of Yfr1 showed anomalies in cell wall completion and were more sensitive to vancomycin than the wild-type strain. Furthermore, growth in the absence of combined nitrogen, which involves the differentiation of heterocysts, was compromised in the strain overexpressing Yfr1, and filaments were broken at the connections between vegetative cells and heterocysts. These results indicate that Yfr1 is an important regulator of cell wall homeostasis and correct cell wall remodeling during heterocyst differentiation. IMPORTANCE Bacterial small RNAs (sRNAs) are important players affecting the regulation of essentially every aspect of bacterial physiology. The cell wall is a highly dynamic structure that protects bacteria from their fluctuating environment. Cell envelope remodeling is particularly critical for bacteria that undergo differentiation processes, such as spore formation or differentiation of heterocysts. Heterocyst development involves the deposition of additional layers of glycolipids and polysaccharides outside the outer membrane. Here, we show that a cyanobacterial phylogenetically conserved small regulatory RNA, Yfr1, coordinates the expression of proteins involved in cell wall-related processes, including peptidoglycan metabolism and transport of different molecules, as well as expression of several proteins involved in heterocyst differentiation.

NsiR1, a small RNA with multiple copies, modulates heterocyst differentiation in the cyanobacterium Nostoc sp. PCC 7120

“…As an alternative strategy to deplete NsiR1 without altering the genomic region upstream of hetF , we have followed a strategy similar to that in Brenes‐Álvarez et al . (2020) and generated a Nostoc strain (OE_as_NsiR1) that constitutively expresses the sequence complementary to nsiR1 . 4 from the trc promoter (Fig.…”

Regulatory RNA at the crossroads of carbon and nitrogen metabolism in photosynthetic cyanobacteria

“…homeostasis and correct cell wall remodeling during heterocysts differentiation [58]. These observations are reminiscent of the competing endogenous RNA (ceRNA) hypothesis in eukaryotic cells, which suggests that competition for a limited pool of regulatory RNAs mediates cross-regulation of mRNAs [61,62].…”

“…Moreover, in predictions of secondary structures, these likely interacting sequences are located in single-stranded regions. Interaction in vitro has been shown for Yfr1 and Yfr2 from Nostoc 7120 [58].…”

“…B. In more complex cyanobacteria, such as Nostoc 7120, Yfr1 targets genes encoding outer membrane proteins and enzymes related to peptidoglycan biosynthesis and turnover [58].…”

Molecular circuit of heterocyst differentiation in cyanobacteria

“…Furthermore, it is reported that the expression of gene amiC2 is increased due to this small RNA. The overexpression of Yfr1 resulted in a filament broken at connection points between vegetative cells and heterocyst, revealing its role in remodeling in the cell wall during heterocyst differentiation [30].…”