Balanced cell division is secured by two different regulatory sites in OxyS RNA.

The hydrogen peroxide-induced small RNA OxyS has been proposed to originate from the 3' UTR of a peroxide mRNA. Unexpectedly, phylogenetic OxyS targetome predictions indicate that most OxyS targets belong to the category of "cell cycle," including cell division and cell elongation. Previously, we reported that Escherichia coli OxyS inhibits cell division by repressing expression of the essential transcription termination factor nusG, thereby leading to the expression of the KilR protein, which interferes with the function of the major cell division protein, FtsZ. By interfering with cell division, OxyS brings about cell-cycle arrest, thus allowing DNA damage repair. Cell division and cell elongation are opposing functions to the extent that inhibition of cell division requires a parallel inhibition of cell elongation for the cells to survive. In this study, we report that in addition to cell division, OxyS inhibits mepS, which encodes an essential peptidoglycan endopeptidase that is responsible for cell elongation. Our study indicates that cell-cycle arrest and balancing between cell division and cell elongation are important and conserved functions of the oxidative stress-induced sRNA OxyS.

Expression of the Cyanobacterial FoF1 ATP Synthase Regulator AtpΘ Depends on Small DNA-Binding Proteins and Differential mRNA Stability.

FoF1 ATP synthases produce ATP, the universal biological energy source. ATP synthase complexes on cyanobacterial thylakoid membranes use proton gradients generated either by photosynthesis or respiration. AtpΘ is an ATP synthase regulator in cyanobacteria which is encoded by the gene atpT. AtpΘ prevents the hydrolysis of ATP (reverse reaction) that otherwise would occur under unfavorable conditions. In the cyanobacterium Synechocystis sp. PCC 6803, AtpΘ is expressed maximum in darkness but at very low levels under optimum phototrophic growth conditions or in the presence of glucose. DNA coimmunoprecipitation experiments followed by mass spectrometry identified the binding of the two transcriptional regulators cyAbrB1 and cyAbrB2 to the promoter and the histone-like protein HU to the 5'UTR of atpT. Analyses of nucleotide substitutions in the promoter and GFP reporter assays identified a functionally relevant sequence motif resembling the HLR1 element bound by the RpaB transcription factor. Electrophoretic mobility shift assays confirmed interaction of cyAbrB1, cyAbrB2, and RpaB with the promoter DNA. However, overall the effect of transcriptional regulation was comparatively low. In contrast, atpT transcript stabilities differed dramatically, half-lives were 1.6 min in the light, 33 min in the dark and substantial changes were observed if glucose or DCMU were added. These findings show that transcriptional control of atpT involves nucleoid-associated DNA-binding proteins, positive regulation through RpaB, while the major effect on the condition-dependent regulation of atpT expression is mediated by controlling mRNA stability, which is related to the cellular redox and energy status. IMPORTANCE FoF1 ATP synthases produce ATP, the universal biological energy source. Under unfavorable conditions, ATP synthases can operate in a futile reverse reaction, pumping protons while ATP is used up. Cyanobacteria perform plant-like photosynthesis, but they cannot use the same mechanism as plant chloroplasts to inhibit ATP synthases during the night because respiratory and photosynthetic complexes are both located in the same membrane system. AtpΘ is a small protein encoded by the gene atpT in cyanobacteria that can prevent the ATP synthase reverse reaction (ATPase activity). Here we found that three transcription factors contribute to the regulation of atpT expression. However, the control of mRNA stability was identified as the major regulatory process governing atpT expression. Thus, it is the interplay between transcriptional and posttranscriptional regulation that position the AtpΘ-based regulatory mechanism within the context of the cellular redox and energy balance.

Resequencing of a mutant bearing an iron starvation recovery phenotype defines Slr1658 as a new player in the regulatory network of a model cyanobacterium.

Photosynthetic microorganisms encounter an erratic nutrient environment characterized by periods of iron limitation and sufficiency. Surviving in such an environment requires mechanisms for handling these transitions. Our study identified a regulatory system involved in the process of recovery from iron limitation in cyanobacteria. We set out to study the role of bacterioferritin co-migratory proteins during transitions in iron bioavailability in the cyanobacterium Synechocystis sp. PCC 6803 using knockout strains coupled with physiological and biochemical measurements. One of the mutants displayed slow recovery from iron limitation. However, we discovered that the cause of the phenotype was not the intended knockout but rather the serendipitous selection of a mutation in an unrelated locus, slr1658. Bioinformatics analysis suggested similarities to two-component systems and a possible regulatory role. Transcriptomic analysis of the recovery from iron limitation showed that the slr1658 mutation had an extensive effect on the expression of genes encoding regulatory proteins, proteins involved in the remodeling and degradation of the photosynthetic apparatus and proteins modulating electron transport. Most significantly, expression of the cyanobacterial homologue of the cyclic electron transport protein PGR5 was upregulated 1000-fold in slr1658 disruption mutants. pgr5 transcripts in the Δslr1658 mutant retained these high levels under a range of stress and recovery conditions. The results suggest that slr1658 is part of a regulatory operon that, among other aspects, affects the regulation of alternative electron flow. Disruption of its function has deleterious results under oxidative stress promoting conditions.

Acclimation of Oxygenic Photosynthesis to Iron Starvation Is Controlled by the sRNA IsaR1.

Oxygenic photosynthesis crucially depends on proteins that possess Fe2+ or Fe/S complexes as co-factors or prosthetic groups. Here, we show that the small regulatory RNA (sRNA) IsaR1 (Iron-Stress-Activated RNA 1) plays a pivotal role in acclimation to low-iron conditions. The IsaR1 regulon consists of more than 15 direct targets, including Fe2+-containing proteins involved in photosynthetic electron transfer, detoxification of anion radicals, citrate cycle, and tetrapyrrole biogenesis. IsaR1 is essential for maintaining physiological levels of Fe/S cluster biogenesis proteins during iron deprivation. Consequently, IsaR1 affects the acclimation of the photosynthetic apparatus to iron starvation at three levels: (1) directly, via posttranscriptional repression of gene expression; (2) indirectly, via suppression of pigment; and (3) Fe/S cluster biosynthesis. Homologs of IsaR1 are widely conserved throughout the cyanobacterial phylum. We conclude that IsaR1 is a critically important riboregulator. These findings provide a new perspective for understanding the regulation of iron homeostasis in photosynthetic organisms.

Awakening of a Dormant Cyanobacterium from Nitrogen Chlorosis Reveals a Genetically Determined Program.

The molecular and physiological mechanisms involved in the transition of microbial cells from a resting state to the active vegetative state are critically relevant for solving problems in fields ranging from microbial ecology to infection microbiology. Cyanobacteria that cannot fix nitrogen are able to survive prolonged periods of nitrogen starvation as chlorotic cells in a dormant state. When provided with a usable nitrogen source, these cells re-green within 48 hr and return to vegetative growth. Here we investigated the resuscitation of chlorotic Synechocystis sp. PCC 6803 cells at the physiological and molecular levels with the aim of understanding the awakening process of a dormant bacterium. Almost immediately upon nitrate addition, the cells initiated a highly organized resuscitation program. In the first phase, they suppressed any residual photosynthetic activity and activated respiration to gain energy from glycogen catabolism. Concomitantly, they restored the entire translational apparatus, ATP synthesis, and nitrate assimilation. After only 12-16 hr, the cells re-activated the synthesis of the photosynthetic apparatus and prepared for metabolic re-wiring toward photosynthesis. When the cells reached full photosynthetic capacity after ∼48 hr, they resumed cell division and entered the vegetative cell cycle. An analysis of the transcriptional dynamics during the resuscitation process revealed a perfect match to the observed physiological processes, and it suggested that non-coding RNAs play a major regulatory role during the lifestyle switch in awakening cells. This genetically encoded program ensures rapid colonization of habitats in which nitrogen starvation imposes a recurring growth limitation.

The hierarchy of transition metal homeostasis: iron controls manganese accumulation in a unicellular cyanobacterium.

Iron and manganese are part of a small group of transition metals required for photosynthetic electron transport. Here, we present evidence for a functional link between iron and manganese homeostasis. In the unicellular cyanobacterium, Synechocystis sp. PCC 6803, Fe and Mn deprivation resulted in distinct modifications of the physiological status. The effect on growth and photosynthetic activity under Fe limitation were more severe than those observed under Mn limitation. Moreover, the intracellular elemental quotas of Fe and Mn were found to be linked. Fe limitation reduced the intracellular Mn quota. Mn limitation did not exert a reciprocal effect on Fe quotas. Microarray analysis comparing Mn and Fe limitation revealed a stark difference in the extent of the transcriptional response to the two limiting conditions, reflective of the physiological responses. The effects of Fe limitation on the transcriptional network are widespread while the effects on Mn limitation are highly specific. Our analysis also revealed an overlap in the transcriptional response of specific Fe and Mn transporters. This overlap provides a framework for explaining Fe limitation induced changes in Mn quotas.

Iron deprivation in Synechocystis: inference of pathways, non-coding RNAs, and regulatory elements from comprehensive expression profiling.

Iron is an essential cofactor in many metabolic reactions. Mechanisms controlling iron homeostasis need to respond rapidly to changes in extracellular conditions, but they must also keep the concentration of intracellular iron under strict control to avoid the generation of damaging reactive oxygen species. Due to its role as a redox carrier in photosynthesis, the iron quota in cyanobacteria is about 10 times higher than in model enterobacteria. The molecular details of how such a high quota is regulated are obscure. Here we present experiments that shed light on the iron regulatory system in cyanobacteria. We measured time-resolved changes in gene expression after iron depletion in the cyanobacterium Synechocystis sp. PCC 6803 using a comprehensive microarray platform, monitoring both protein-coding and non-coding transcripts. In total, less than a fifth of all protein-coding genes were differentially expressed during the first 72 hr. Many of these proteins are associated with iron transport, photosynthesis, or ATP synthesis. Comparing our data with three previous studies, we identified a core set of 28 genes involved in iron stress response. Among them were genes important for assimilation of inorganic carbon, suggesting a link between the carbon and iron regulatory networks. Nine of the 28 genes have unknown functions and constitute key targets for further functional analysis. Statistical and clustering analyses identified 10 small RNAs, 62 antisense RNAs, four 5'UTRs, and seven intragenic elements as potential novel components of the iron regulatory network in Synechocystis. Hence, our genome-wide expression profiling indicates an unprecedented complexity in the iron regulatory network of cyanobacteria.