Sequence-dependent cleavage site selection by RNase Z from the cyanobacterium synechocystis sp. PCC 6803

Biosynthesis of transfer RNA requires processing from longer precursors at the 5′- and 3′-ends. In eukaryotes, in archaea, and in those bacteria where the 3′-terminal CCA sequence is not encoded, 3′ processing is carried out by the endonuclease RNase Z, which cleaves after the discriminator nucleotide to generate a mature 3′-end ready for the addition of the CCA sequence. We have identified and cloned the gene coding for RNase Z in the cyanobacterium Synechocystis sp. PCC 6803. The gene has been expressed in Escherichia coli, and the recombinant protein was purified. The enzymatic activity of RNase Z from Synechocystis has been studied in vitro with a variety of substrates. The presence of C or CC after the discriminator nucleotide modifies the cleavage site of RNase Z so that it is displaced by one and two nucleotides to the 3′-side, respectively. The presence of the complete 3′-terminal CCA sequence in the precursor of the tRNA completely inhibits RNase Z activity. The inactive CCA-containing precursor binds to Synechocystis RNase Z with similar affinity than the mature tRNA. The properties of the enzyme described here could be related with the mechanism by which CCA is added in this organism, with the participation of two separate nucleotidyl transferases, one specific for the addition of C and another for the addition of A. This work is the first characterization of RNase Z from a cyanobacterium, and the first from an organism with two separate nucleotidyl transferases.Ministerio de Ciencia y Tecnología BMC2001–3789, BFU2004 – 00076/BMCJunta de Andalucía CVI21

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

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.España Ministerio de Educación, Cultura y Deporte (FPU014/05123 and EST16-00088)España Ministerio de Economía y Competitividad BFU2013-48282-C2-1España Agencia Estatal de Investigación (AEI), Ministerio de Economía, Industria y Competitividad, both cofinanced by the Fondo Europeo de Desarrollo Regional (FEDER) BFU2016-74943-C2-1-

NsrR1, a Nitrogen stress-repressed sRNA, contributes to the regulation of nblA in Nostoc sp. PCC 7120

Small regulatory RNAs (sRNAs) are currently considered as major post-transcriptional regulators of gene expression in bacteria. The interplay between sRNAs and transcription factors leads to complex regulatory networks in which both transcription factors and sRNAs may appear as nodes. In cyanobacteria, the responses to nitrogen availability are controlled at the transcriptional level by NtcA, a CRP/FNR family regulator. In this study, we describe an NtcA-regulated sRNA in the cyanobacterium Nostoc sp. PCC 7120, that we have named NsrR1 (nitrogen stress repressed RNA1). We show sequence specific binding of NtcA to the promoter of NsrR1. Prediction of possible mRNA targets regulated by NsrR1 allowed the identification of nblA, encoding a protein adaptor for phycobilisome degradation under several stress conditions, including nitrogen deficiency. We demonstrate specific interaction between NsrR1 and the 5′-UTR of the nblA mRNA, that leads to decreased expression of nblA. Because both NsrR1 and NblA are under transcriptional control of NtcA, this regulatory circuit constitutes a coherent feed-forward loop, involving a transcription factor and an sRNA.Agencia Estatal de Investigación (AEI) BFU2016-74943- C2-1-PMinisterio de Economía y Competitividad BFU2013-48282-C2-1-P, BES-2014- 06848

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

Heterocystous cyanobacteria such as Nostoc sp. are filamentous photosynthetic organisms that, in response to nitrogen deficiency, undergo a differentiation process transforming certain, semi-regularly spaced cells into heterocysts, devoted to nitrogen fixation. During transition to a nitrogen-fixing regime, growth of most vegetative cells in the filament is temporarily arrested due to nutritional deprivation, but developing heterocysts require intense transcriptional activity. Therefore, the coexistence of arrested vegetative cells and actively developing prospective heterocysts relies on the simultaneous operation of somewhat opposite transcriptional programs. We have identified genes with multiple nitrogen-responsive transcriptional starts appearing in seemingly paradoxical combinations. For instance, sigA, encoding the RNA polymerase housekeeping sigma factor, is transcribed from one major nitrogen stress-repressed promoter and from a second, nitrogen stress-induced promoter. Here, we show that both promoters are expressed with complementary temporal dynamics. Using a gfp reporter we also show that transcription from the inducible promoter takes place exclusively in differentiating heterocysts and is already detected before any morphological or fluorescence signature of differentiation is observed. Tandem promoters with opposite dynamics could operate a compensatory mechanism in which repression of transcription from the major promoter operative in vegetative cells is offset by transcription from a new promoter only in developing heterocyst.Ministerio de Economía y Competitividad BFU2013-48282-C2-1Agencia Estatal de Investigación (AEI) BFU2016-74943-C2-1-PMinisterio de Educación, Cultura y Deportes FPU014/0512

A New Type of Asymmetrically Acting β-Carotene Ketolase Is Required for the Synthesis of Echinenone in the Cyanobacterium Synechocystis sp. PCC 6803

We have isolated, based on the knowledge of the complete genomic sequence of the cyanobacterium Synechocystis sp. PCC 6803, an open reading frame (slr0088) similar to known bacterial carotene desaturases and have analyzed the function of the encoded protein. Surprisingly, this protein has no detectable desaturase activity with phytoene, hydroxyneurosporene, or ζ-carotene as substrates, but is rather a β-carotene ketolase that acts asymmetrically introducing a keto group on only one of the two β-ionone rings of β-carotene to generate echinenone. This is in contrast to the so far characterized β-carotene ketolases that act symmetrically, producing the di-keto carotenoid canthaxanthin from β-carotene without significant accumulation of echinenone. We have designated this new gene crtO The function of the crtO gene product has been demonstrated by 1) the biosynthesis of echinenone when the crtO gene is expressed in an Escherichia coli strain able to accumulate β-carotene, 2) the in vitro biosynthesis of echinenone from β-carotene with cell free extracts from E. coli cells that express the crtO gene, and 3) the absence of echinenone in a Synechocystis strain in which the crtO gene has been insertionally inactivated. The primary structure of the Synechocystis asymmetric ketolase bears no similarity with the known β-carotene ketolases. crtO is not required for normal growth under standard or high light conditions, neither is the photosynthetic activity of the crtO-deficient strain affected

Conditional Expression of RNase P in the CyanobacteriumSynechocystis sp. PCC6803 Allows Detection of Precursor RNAs

We have constructed a strain (CT1) that expresses RNase P conditionally with the aim to analyze the in vivotRNA processing pathway and the biological role that RNase P plays inSynechocystis 6803. In this strain, the rnpBgene, coding for the RNA subunit of RNase P, has been placed under the control of the petJ gene promoter (PpetJ), which is repressed by copper, cell growth, and accumulation of RNase P RNA is inhibited in CT1 after the addition of copper, indicating that the regulation by copper is maintained in the chimerical PpetJ -rnpB gene and that RNase P is essential for growth in Synechocystis. We have analyzed several RNAs by Northern blot and primer extension in CT1. Upon addition of copper to the culture medium, precursors of the mature tRNAs are detected. Furthermore, our results indicate that there is a preferred order in the action of RNase P when it processes a dimeric tRNA precursor. The precursors detected are 3′-processed, indicating that 3′ processing can occur before 5′ processing by RNase P. The size of the precursors suggests that the terminal CCA sequence is already present before RNase P processing. We have also analyzed other potential RNase P substrates, such as the precursors of tmRNA and 4.5 S RNA. In both cases, accumulation of larger than mature size RNAs is observed after transferring the cells to a copper-containing medium

Unexpected diversity of RNase P, an ancient tRNA processing enzyme: challenges and prospects

For an enzyme functioning predominantly in a seemingly housekeeping role of 5′ tRNA maturation, RNase P displays a remarkable diversity in subunit make-up across the three domains of life. Despite the protein complexity of this ribonucleoprotein enzyme increasing dramatically from bacteria to eukarya, the catalytic function rests with the RNA subunit during evolution. However, the recent demonstration of a protein-only human mitochondrial RNase P has added further intrigue to the compositional variability of this enzyme. In this review, we discuss some possible reasons underlying the structural diversity of the active sites, and use them as thematic bases for elaborating new directions to understand how functional variations might have contributed to the complex evolution of RNase P

A functional RNase P protein subunit of bacterial origin in some eukaryotes

RNase P catalyzes 5′-maturation of tRNAs. While bacterial RNase P comprises an RNA catalyst and a protein cofactor, the eukaryotic (nuclear) variant contains an RNA and up to ten proteins, all unrelated to the bacterial protein. Unexpectedly, a nuclear-encoded bacterial RNase P protein (RPP) homolog is found in several prasinophyte algae including Ostreococcus tauri. We demonstrate that recombinant O. tauri RPP can functionally reconstitute with bacterial RNase P RNAs (RPRs) but not with O. tauri organellar RPRs, despite the latter’s presumed bacterial origins. We also show that O. tauri PRORP, a homolog of Arabidopsis PRORP-1, displays tRNA 5′-processing activity in vitro. We discuss the implications of the striking diversity of RNase P in O. tauri, the smallest known free-living eukaryote.Ministerio de Ciencia e Innovación European Regional Fund BFU2007-60651Junta de Andalucía P06-CVI-01692National Science Foundation MCB-0238233 MCB-0843543European Union ASSEMBLE 22779

Identification of conserved and potentially regulatory small RNAs in heterocystous cyanobacteria

Small RNAs (sRNAs) are a growing class of non-protein-coding transcripts that participate in the regulation of virtually every aspect of bacterial physiology. Heterocystous cyanobacteria are a group of photosynthetic organisms that exhibit multicellular behavior and developmental alternatives involving specific transcriptomes exclusive of a given physiological condition or even a cell type. In the context of our ongoing effort to understand developmental decisions in these organisms we have undertaken an approach to the global identification of sRNAs. Using differential RNA-Seq we have previously identified transcriptional start sites for the model heterocystous cyanobacterium Nostoc sp. PCC 7120. Here we combine this dataset with a prediction of Rho-independent transcriptional terminators and an analysis of phylogenetic conservation of potential sRNAs among 89 available cyanobacterial genomes. In contrast to predictive genome-wide approaches, the use of an experimental dataset comprising all active transcriptional start sites (differential RNA-Seq) facilitates the identification of bona fide sRNAs. The output of our approach is a dataset of predicted potential sRNAs in Nostoc sp. PCC 7120, with different degrees of phylogenetic conservation across the 89 cyanobacterial genomes analyzed. Previously described sRNAs appear among the predicted sRNAs, demonstrating the performance of the algorithm. In addition, new predicted sRNAs are now identified that can be involved in regulation of different aspects of cyanobacterial physiology, including adaptation to nitrogen stress, the condition that triggers differentiation of heterocysts (specialized nitrogen-fixing cells). Transcription of several predicted sRNAs that appear exclusively in the genomes of heterocystous cyanobacteria is experimentally verified by Northern blot. Cell-specific transcription of one of these sRNAs, NsiR8 (nitrogen stress-induced RNA 8), in developing heterocysts is also demonstrate