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RNA degradation is crucial for many processes in pro- and eukaryotic organisms. In bacteria, the preference of the central ribonucleases RNase E, RNase J and RNase Y towards 5’-monophosphorylated RNAs is considered important for RNA degradation. For RNase E, the underlying mechanism is termed 5’ sensing. Cyanobacteria, such asSynechocystissp. PCC 6803 (Synechocystis), encode RNase E and RNase J homologs. Here, we constructed aSynechocystisstrain lacking the 5’ sensing function of RNase E and mapped on a transcriptome-wide level 292 5’-sensing-dependent cleavage sites. These included so far unknown targets such as the 5’ untranslated region of the response regulator genelsiR;trxA, apcEandatpImRNAs, encoding proteins related to energy metabolism; as well assbtBandrbcLXSencoding proteins relevant for carbon fixation. Cyanobacterial 5’ sensing is important for the maturation of rRNA and several tRNAs, including tRNAGluUUC. This tRNA activates glutamate for tetrapyrrole biosynthesis in plant chloroplasts and most prokaryotes. We found that increased RNase activities leads to a higher copy number of the majorSynechocystisplasmids pSYSA and pSYSM. The results provide a first step towards understanding the relative importance of different target mechanisms of RNase E outsideEscherichia coli.

Synthetic biology approaches toward the development of cyanobacterial producer strains require the availability of appropriate sets of plasmid vectors. A factor for the industrial usefulness of such strains is their robustness against pathogens, such as bacteriophages infecting cyanobacteria. Therefore, it is of great interest to understand the native plasmid replication systems and the CRISPR-Cas based defense mechanisms already present in cyanobacteria. In the model cyanobacteriumSynechocystissp. PCC 6803, four large and three smaller plasmids exist. The ∼100 kb plasmid pSYSA is specialized in defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy number in the cell. The pSYSA copy number is positively correlated with the expression level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the pSYSA-encodedssr7036transcript. Together with a cis-located abundant antisense RNA (asRNA1), this mechanism resembles the control of ColE1-type plasmid replication by two overlapping RNAs, RNA I and II. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains. Deletion ofslr7037led to the integration of pSYSA into the chromosome or the other large plasmid pSYSX. Moreover, the presence ofslr7037was required for successful replication of a pSYSA-derived vector in another model cyanobacterium,Synechococcus elongatusPCC 7942. Therefore, we annotated the protein encoded byslr7037as Cyanobacterial Rep protein A1 (CyRepA1). Our findings open new perspectives on the development of shuttle vectors for genetic engineering of cyanobacteria and of modulating the activity of the entire CRISPR-Cas apparatus inSynechocystissp. PCC 6803.

Small regulatory RNAs and antisense RNAs play important roles in the regulation of gene expression in bacteria but are underexplored, especially in natural populations. While environmentally relevant microbes often are not amenable to genetic manipulation or cannot be cultivated in the laboratory, extensive metagenomic and metatranscriptomic datasets for these organisms might be available. Hence, dedicated workflows for specific analyses are needed to fully benefit from this information. Here, we identified abundant sRNAs from oceanic environmental populations of the ecologically important primary producerProchlorococcusstarting from a metatranscriptomic differential RNA-Seq (mdRNA-Seq) dataset. We tracked their homologs in laboratory isolates, and we provide a framework for their further detailed characterization. Several of the experimentally validated sRNAs responded to ecologically relevant changes in cultivation conditions. The expression of the here newly discovered sRNA Yfr28 was highly stimulated in low-nitrogen conditions. Its predicted top targets include mRNAs encoding cell division proteins, a sigma factor, and several enzymes and transporters, suggesting a pivotal role of Yfr28 in the coordination of primary metabolism and cell division. Acis-encoded antisense RNA was identified as a possible positive regulator ofatpFencoding subunit b’ of the ATP synthase complex. The presented workflow will also be useful for other environmentally relevant microorganisms for which experimental validation abilities are frequently limiting although there is wealth of sequence information available.

Marine cyanobacteria of the genus Prochlorococcus thrive in high cell numbers throughout the euphotic zones of the world's subtropical and tropical oligotrophic oceans, making them some of the most ecologically relevant photosynthetic microorganisms on Earth. The ecological success of these free-living phototrophs suggests that they are equipped with a regulatory system competent to address many different stress situations. However, Prochlorococcus genomes are compact and streamlined, with the majority encoding only five different sigma factors, five to six two-component systems and eight types of other transcriptional regulators. Here, we summarize the existing information about the functions of these protein regulators, about transcriptomic responses to defined stress conditions, and discuss the current knowledge about riboswitches, RNA-based regulation and the roles of certain metabolites as co-regulators. We focus on the best-studied isolate, Prochlorococcus MED4, but extend to other strains and ecotypes when appropriate, and we include some information gained from metagenomic and metatranscriptomic analyses.

Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression. A key endoribonuclease in many bacteria is RNase E. To ensure an appropriate supply of RNase E, some bacteria, such as E. coli, have evolved tightly functioning feedback regulation of RNase E that is mediated in cis by the rne 5′-untranslated region (5′ UTR); however, the mechanisms involved in the control of RNase E in other bacteria largely remain unknown. Cyanobacteria rely on solar light as an energy source for photosynthesis, despite the inherent ultraviolet (UV) irradiation. Here, we investigated the global gene expression response in the cyanobacterium Synechocystis sp. PCC 6803 after exposure to UV light and discovered a unique response of RNase E: a rapidly increasing enzymatic activity, although the stability of the protein was decreased. In parallel, we observed an increased accumulation of full-length rne mRNA that was caused by the stabilization of its 5′ UTR and suppression of premature transcriptional termination but not by an increased transcription rate. Mapping of RNA 3′ ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5′ UTR, indicating autoregulation via its own 5′ UTR. These observations imply that RNase E in cyanobacteria contributes substantially to reshaping the transcriptome during the UV stress response and that its required activity level is maintained despite enhanced turnover of the protein by posttranscriptional feedback regulation.

Ribonucleases are crucial enzymes in RNA metabolism and post-transcriptional regulatory processes in bacteria. Cyanobacteria encode the two essential ribonucleases RNase E and RNase J. Cyanobacterial RNase E is shorter than homologues in other groups of bacteria and lacks both the chloroplast-specific N-terminal extension as well as the C-terminal domain typical for RNase E of enterobacteria. In order to investigate the function of RNase E in the model cyanobacterium Synechocystis sp. PCC 6803, we engineered a temperature-sensitive RNase E mutant by introducing two site-specific mutations, I65F and spontaneously occurring V94A. This enabled us to perform RNA-seq after the transient inactivation of RNase E by a temperature shift (TIER-seq) and to map 1,472 RNase-E-dependent cleavage sites. We inferred a dominating cleavage signature consisting of an adenine at the -3 and a uridine at the +2 position within a single-stranded segment of the RNA. The data identified putative RNase-E-dependent instances of operon discoordination, mRNAs likely regulated jointly by RNase E and an sRNA, potential 3’ end-derived sRNAs and a dual-acting mechanism for the glutamine riboswitch. Our findings substantiate the pivotal role of RNase E in post-transcriptional regulation and suggest the redundant or concerted action of RNase E and RNase J in cyanobacteria.

RNA helicases play crucial functions in RNA biology. In plants, RNA helicases are encoded by large gene families, performing roles in abiotic stress responses, development, the post-transcriptional regulation of gene expression as well as house-keeping functions. Several of these RNA helicases are targeted to the organelles, mitochondria and chloroplasts. Cyanobacteria are the direct evolutionary ancestors of plant chloroplasts. The cyanobacterium Synechocystis 6803 encodes a single DEAD-box RNA helicase, CrhR, that is induced by a range of abiotic stresses, including low temperature. Though the ΔcrhR mutant exhibits a severe cold-sensitive phenotype, the physiological function(s) performed by CrhR have not been described. To identify transcripts interacting with CrhR, we performed RNA co-immunoprecipitation with extracts from a Synechocystis crhR deletion mutant expressing the FLAG-tagged native CrhR or a K57A mutated version with an anticipated enhanced RNA binding. The composition of the interactome was strikingly biased towards photosynthesis-associated and redox-controlled transcripts. A transcript highly enriched in all experiments was the crhR mRNA, suggesting an auto-regulatory molecular mechanism. The identified interactome explains the described physiological role of CrhR in response to the redox poise of the photosynthetic electron transport chain and characterizes CrhR as an enzyme with a diverse range of transcripts as molecular targets.HighlightThe cyanobacterial DEAD-box RNA helicase CrhR binds mainly photosynthesis-associated and redox-controlled transcripts connecting its regulation, localization and phenotypes of mutants for the first time with a set of potential RNA targets.

Significance During genome analysis, genes encoding small proteins are frequently neglected. Accordingly, small proteins have remained underinvestigated in all domains of life. Based on a previous systematic search for such genes, we present the functional analysis of the 66 amino acids protein NblD in a photosynthetic cyanobacterium. We show that NblD plays a crucial role during the coordinated dismantling of phycobilisome light-harvesting complexes. This disassembly is triggered when the cells become starved for nitrogen, a condition that frequently occurs in nature. Similar to NblA that tags phycobiliproteins for proteolysis, NblD binds to phycocyanin polypeptides but has a different function. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration., Phycobilisomes are the major pigment–protein antenna complexes that perform photosynthetic light harvesting in cyanobacteria, rhodophyte, and glaucophyte algae. Up to 50% of the cellular nitrogen can be stored in their giant structures. Accordingly, upon nitrogen depletion, phycobilisomes are rapidly degraded following an intricate genetic program. Here, we describe the role of NblD, a cysteine-rich, small protein in this process in cyanobacteria. Deletion of the nblD gene in the cyanobacterium Synechocystis sp. PCC 6803 prevented the degradation of phycobilisomes, leading to a nonbleaching (nbl) phenotype, which could be complemented by a plasmid-localized gene copy. Competitive growth experiments between the ΔnblD and the wild-type strain provided direct evidence for the physiological importance of NblD under nitrogen-limited conditions. Ectopic expression of NblD under nitrogen-replete conditions showed no effect, in contrast to the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation. Transcriptome analysis indicated increased nblA1/2 transcript levels in the ΔnblD strain during nitrogen starvation, implying that NblD does not act as a transcriptional (co)regulator. However, immunoprecipitation and far-western experiments identified the chromophorylated (holo form) of the phycocyanin β-subunit (CpcB) as its target, while apo-CpcB was not bound. The addition of recombinant NblD to isolated phycobilisomes caused a reduction in phycocyanin absorbance and a broadening and shifting of the peak to lower wavelengths, indicating the occurrence of structural changes. These data demonstrate that NblD plays a crucial role in the coordinated dismantling of phycobilisomes and add it as a factor to the genetically programmed response to nitrogen starvation.

Phycobilisomes (PBSs), the principal cyanobacterial antenna, are among the most efficient macromolecular structures in nature, and are used for both light harvesting and directed energy transfer to the photosynthetic reaction center. However, under unfavorable conditions, excess excitation energy needs to be rapidly dissipated to avoid photodamage. The orange carotenoid protein (OCP) senses light intensity and induces thermal energy dissipation under stress conditions. Hence, its expression must be tightly controlled; however, the molecular mechanism of this regulation remains to be elucidated. Here, we describe the discovery of a posttranscriptional regulatory mechanism in Synechocystis sp. PCC 6803 in which the expression of the operon encoding the allophycocyanin subunits of the PBS is directly and in an inverse fashion linked to the expression of OCP. This regulation is mediated by ApcZ, a small regulatory RNA that is derived from the 3′-end of the tetracistronic apcABC–apcZ operon. ApcZ inhibits ocp translation under stress-free conditions. Under most stress conditions, apc operon transcription decreases and ocp translation increases. Thus, a key operon involved in the collection of light energy is functionally connected to the expression of a protein involved in energy dissipation. Our findings support the view that regulatory RNA networks in bacteria evolve through the functionalization of mRNA 3′-UTRs.

Phycobilisomes are the major pigment-protein antenna complexes that perform photosynthetic light harvesting in cyanobacteria, rhodophyte and glaucophyte algae. Up to 50% of the cellular nitrogen can be stored in their giant structures. Accordingly, upon nitrogen depletion, phycobilisomes are rapidly degraded. This degradation is tightly coordinated, follows a genetic program and involves small proteins serving as proteolysis adaptors. Here, we describe the role of NblD, a novel factor in this process in cyanobacteria. NblD is a cysteine-rich, 66-amino acid small protein that becomes rapidly induced upon nitrogen starvation. Deletion of thenblDgene in the cyanobacteriumSynechocystisprevents the degradation of phycobilisomes, leading to a nonbleaching (nbl) phenotype. Competition experiments provided direct evidence for the physiological importance of NblD. Complementation by a plasmid-localized gene copy fully restored the phenotype of the wild type. Overexpression of NblD under nitrogen-replete conditions showed no effect, in contrast to the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation ectopically. Transcriptome analysis revealed that nitrogen starvation correctly inducesnblA1/2transcription in the ΔnblDstrain implying that NblD does not act as a transcriptional (co-)regulator. However, fractionation and coimmunoprecipitation experiments indicated the presence of NblD in the phycobilisome fraction and identified the β-phycocyanin subunit as its target. These data add NblD as a new factor to the genetically programmed response to nitrogen starvation and demonstrate that it plays a crucial role in the coordinated dismantling of phycobilisomes when nitrogen becomes limiting.Significance StatementDuring genome analysis, genes encoding small proteins are frequently neglected. Accordingly, small proteins have remained underinvestigated in all domains of life. Based on a previous systematic search for such genes, we present the functional analysis of the small protein NblD in a photosynthetic cyanobacterium. We show that NblD plays a crucial role during the coordinated dismantling of phycobilisome light-harvesting complexes. This disassembly is triggered when the cells run low in nitrogen, a condition that frequently occurs in nature. Similar to the NblA proteins that label phycobiliproteins for proteolysis, NblD binds to phycocyanin polypeptides but has a different function. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration.

The sRNA Yfr1 and members of the Yfr2 sRNA family are almost universally present within cyanobacteria. The conserved motifs of these sRNAs are nearly complementary to each other, suggesting their ability to participate in crosstalk. The conserved motif of Yfr1 is shared by members of the Yfr10 sRNA family, members of which are otherwise less conserved in sequence, structure, and synteny compared to Yfr1. The different structural properties enable the discrimination of unique targets of Yfr1 and Yfr10. Unlike most studied regulatory sRNAs, Yfr1 gene expression only slightly changes under the tested stress conditions and is present at high levels at all times. In contrast, cellular levels of Yfr10 increase during the course of acclimation to darkness, and levels of Yfr2 increase when cells are shifted to high light or nitrogen limitation conditions. In this study, we investigated the targetomes of Yfr2, Yfr1, and Yfr10 in Prochlorococcus MED4, establishing CRAFD-Seq as a new method for identifying direct targets of these sRNAs that is applicable to all bacteria, including those that are not amenable to genetic modification. The results suggest that these sRNAs are integrated within a regulatory network of unprecedented complexity in the adjustment of carbon and nitrogen-related primary metabolism.

We evaluated the seasonal contribution of heterotrophic and autotrophic diazotrophy to the total dinitrogen (N2) fixation in a representative pelagic station in the northern Gulf of Aqaba in early spring when the water column was mixed and during summer under full thermal stratification. N2 fixation rates were low during the mixed period (∼ 0.1 nmol N L−1 d−1) and were significantly coupled with both primary and bacterial productivity. During the stratified period N2 fixation rates were four-fold higher (∼ 0.4 nmol N L−1 d−1) and were significantly correlated solely with bacterial productivity. Furthermore, while experimental enrichment of seawater by phosphorus (P) enhanced bacterial productivity and N2 fixation rates during both seasons primary productivity was stimulated by P only in the early spring. Metatranscriptomic analyses from the stratified period identified the major diazotrophic contributors as related to heterotrophic prokaryotes from the Euryarchaeota and Desulfobacterales (Deltaproteobacteria) or Chlorobiales (Chlorobia). Moreover, during this season, experimental amendments to seawater applying a combination of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and a mixture of amino acids increased both bacterial productivity and N2 fixation rates. Our findings from the northern Gulf of Aqaba indicate a~shift in the diazotrophic community from phototrophic and heterotrophic populations, including small blooms of the cyanobacterium Trichodesmium, in winter/early spring, to predominantly heterotrophic diazotrophs in summer that may be both P and carbon limited as the additions of P and amino acids illustrated.

Prochlorococcus is a marine picocyanobacterium with a streamlined genome that is adapted to different ecological niches in the oligotrophic oceans. There are currently >20 regulatory small RNAs (sRNAs) that have been identified in the model strain Prochlorococcus MED4. While most of these sRNAs are ecotype-specific, sRNA homologs of Yfr1 and of the Yfr2 family are widely found throughout the cyanobacterial phylum. Although they were identified 13 yrs ago, the functions of Yfr1 and Yfr2 have remained unknown. We observed a strong induction of two Yfr2 sRNA homologs of Prochlorococcus MED4 during high light stress and nitrogen starvation. Several Prochlorococcus and marine Synechococcus yfr2 promoter regions contain a conserved motif we named CGRE1 (cyanobacterial GntR family transcriptional regulator responsive element 1). Using the conserved promoter region as bait in a DNA affinity pull-down assay we identified the GntR family transcriptional regulator PMM1637 as a binding partner. Similar to Yfr2, homologs of PMM1637 are universally and exclusively found in cyanobacteria. We suggest that PMM1637 governs the induction of gene expression of Yfr2 homologs containing CGRE1 in their promoters under nitrogen-depleted and high-light stress conditions.

Previous studies have shown that infection of Prochlorococcus MED4 by the cyanophage P-SSP7 leads to increased transcript levels of host endoribonuclease (RNase) E. However, it has remained enigmatic whether this is part of a host defense mechanism to degrade phage messenger RNA (mRNA) or whether this single-strand RNA-specific RNase is utilized by the phage. Here we describe a hitherto unknown means through which this cyanophage increases expression of RNase E during phage infection and concomitantly protects its own RNA from degradation. We identified two functionally different RNase E mRNA variants, one of which is significantly induced during phage infection. This transcript lacks the 5′ UTR, is considerably more stable than the other transcript, and is likely responsible for increased RNase E protein levels during infection. Furthermore, selective enrichment and in vivo analysis of double-stranded RNA (dsRNA) during infection revealed that phage antisense RNAs (asRNAs) sequester complementary mRNAs to form dsRNAs, such that the phage protein-coding transcriptome is nearly completely covered by asRNAs. In contrast, the host protein-coding transcriptome is only partially covered by asRNAs. These data suggest that P-SSP7 orchestrates degradation of host RNA by increasing RNase E expression while masking its own transcriptome from RNase E degradation in dsRNA complexes. We propose that this combination of strategies contributes significantly to phage progeny production.

The ecologically important cyanobacterium Prochlorococcus possesses the smallest genome among oxyphototrophs, with a reduced suite of protein regulators and a disproportionately high number of regulatory RNAs. Many of these are asRNAs, raising the question whether they modulate gene expression through the protection of mRNA from RNase E degradation. To address this question, we produced recombinant RNase E from Prochlorococcus sp. MED4, which functions optimally at 12 mM Mg(2+), pH 9 and 35°C. RNase E cleavage assays were performed with this recombinant protein to assess enzyme activity in the presence of single- or double-stranded RNA substrates. We found that extraordinarily long asRNAs of 3.5 and 7 kb protect a set of mRNAs from RNase E degradation that accumulate during phage infection. These asRNA-mRNA duplex formations mask single-stranded recognition sites of RNase E, leading to increased stability of the mRNAs. Such interactions directly modulate RNA stability and provide an explanation for enhanced transcript abundance of certain mRNAs during phage infection. Protection from RNase E-triggered RNA decay may constitute a hitherto unknown regulatory function of bacterial cis-asRNAs, impacting gene expression.

Motivation: Prochlorococcus possesses the smallest genome of all sequenced photoautotrophs. Although the number of regulatory proteins in the genome is very small, the relative number of small regulatory RNAs is comparable with that of other bacteria. The compact genome size of Prochlorococcus offers an ideal system to search for targets of small RNAs (sRNAs) and to refine existing target prediction algorithms. Results: Target predictions for the cyanobacterial sRNA Yfr1 were carried out with INTARNA in Prochlorococcus MED4. The ultraconserved Yfr1 sequence motif was defined as the putative interaction seed. To study the impact of Yfr1 on its predicted mRNA targets, a reporter system based on green fluorescent protein (GFP) was applied. We show that Yfr1 inhibits the translation of two predicted targets. We used mutation analysis to confirm that Yfr1 directly regulates its targets by an antisense interaction sequestering the ribosome binding site, and to assess the importance of interaction site accessibility. Contact: backofen@informatik.uni-freiburg.de; claudia.steglich@biologie.uni-freiburg.de Supplementary information: Supplementary data are available at Bioinformatics online.

The visualization of massive datasets, such as those resulting from comparative metatranscriptome analyses or the analysis of microbial population structures using ribosomal RNA sequences, is a challenging task. We developed a new method called CoVennTree (Comparative weighted Venn Tree) that simultaneously compares up to three multifarious datasets by aggregating and propagating information from the bottom to the top level and produces a graphical output in Cytoscape. With the introduction of weighted Venn structures, the contents and relationships of various datasets can be correlated and simultaneously aggregated without losing information. We demonstrate the suitability of this approach using a dataset of 16S rDNA sequences obtained from microbial populations at three different depths of the Gulf of Aqaba in the Red Sea. CoVennTree has been integrated into the Galaxy ToolShed and can be directly downloaded and integrated into the user instance.

ProchlorococcusMED4 has, with a total of only 1,716 annotated protein-coding genes, the most compact genome of a free-living photoautotroph. Although light quality and quantity play an important role in regulating the growth rate of this organism in its natural habitat, the majority of known light-sensing proteins are absent from its genome. To explore the potential for light sensing in this phototroph, we measured its global gene expression pattern in response to different light qualities and quantities by using high-density Affymetrix microarrays. Though seven different conditions were tested, only blue light elicited a strong response. In addition, hierarchical clustering revealed that the responses to high white light and blue light were very similar and different from that of the lower-intensity white light, suggesting that the actual sensing of high light is mediated via a blue-light receptor. Bacterial cryptochromes seem to be good candidates for the blue-light sensors. The existence of a signaling pathway for the redox state of the photosynthetic electron transport chain was suggested by the presence of genes that responded similarly to red and blue light as well as genes that responded to the addition of DCMU [3-(3,4-dichlorophenyl)-1,1-N-N′-dimethylurea], a specific inhibitor of photosystem II-mediated electron transport.

The marine unicellular cyanobacterium Prochlorococcus is the smallest-known oxygen-evolving autotroph1. It numerically dominates the phytoplankton in the tropical and subtropical oceans2,3, and is responsible for a significant fraction of global photosynthesis. Here we compare the genomes of two Prochlorococcus strains that span the largest evolutionary distance within the Prochlorococcus lineage4 and that have different minimum, maximum and optimal light intensities for growth5. The high-light-adapted ecotype has the smallest genome (1,657,990 base pairs, 1,716 genes) of any known oxygenic phototroph, whereas the genome of its low-light-adapted counterpart is significantly larger, at 2,410,873 base pairs (2,275 genes). The comparative architectures of these two strains reveal dynamic genomes that are constantly changing in response to myriad selection pressures. Although the two strains have 1,350 genes in common, a significant number are not shared, and these have been differentially retained from the common ancestor, or acquired through duplication or lateral transfer. Some of these genes have obvious roles in determining the relative fitness of the ecotypes in response to key environmental variables, and hence in regulating their distribution and abundance in the oceans.

Summary Marine cyanobacteria of the genus Prochlorococcus belong to one of two ecotypes that are specifically adapted to either low light (LL) or high light (HL) conditions. Previous analyses of the differences in pigmentation and gene complement revealed that LL-adapted ecotypes carry a gene cluster to produce a functional phycoerythrin, whereas in the fully sequenced genome of the HL-adapted strain MED4, only a single and free-standing cpeB gene occurs. This gene encodes a derived form of β-phycoerythrin, the function of which has remained enigmatic so far. Here, an analysis of HL-adapted Prochlorococcus strains from different ocean provinces revealed the presence of a cpeB gene highly similar to that of MED4. To investigate whether the presence of particular phycoerythrin genes is a common characteristic of the LL- and HL-adapted ecotypes, primer sets targeting specific motifs in LL-cpeB and HL-cpeB were designed for polymerase chain reaction (PCR) analysis of Red Sea phytoplankton. A major PCR product for Prochlorococcus HL-cpeB was obtained from samples taken at 5–70 m depth and for LL-cpeB from 70–125 m. The high sensitivity of this approach allowed the detection of HL-cpeB down to 100 m and LL-cpeB as deep as 175 m. DNA sequence and phylogenetic analysis of 70 individual clones for HL-cpeB and of 68 clones for LL-cpeB revealed a monophyletic origin for the HL and LL sequences respectively. This study shows that cpeB sequences are suitable as very sensitive molecular markers for the study of natural populations of Prochlorococcus. The low sequence divergence of HL-cpeB among Prochlorococcus strains, which have been isolated from the Mediterranean Sea, the Arabian Sea and the Southern Pacific Ocean as well as in populations from the Red Sea, suggests the HL-cpeB gene to be conserved and its product to be functional in Prochlorococcus.

Regulatory RNAs play a central role in the regulation of gene expression and can act on several regulatory levels from transcriptional initiation and RNA processing to the control of initiation of translation and RNA stability. One class of these molecules is non-coding (nc)RNAs in bacteria that typically lack protein-coding potential, range in size between 50 and 500nt and originate from intergenic regions. Common methods for the identification of these RNAs are either based on computational predictions, or on transcriptomic analyses of laboratory cultures, whereas very little is known about ncRNAs in environmental microbial populations. Here, we have combined a metatranscriptomics approach with a selective enrichment protocol for ncRNAs. The primary objective of this study was the identification of novel, environmentally relevant ncRNAs focusing on the cyanobacterium Prochlorococcus, which was one of the dominant microorganisms of the marine community of the Gulf of Aqaba when samples were taken.

Prochlorococcus is a genus of abundant and ecologically important marine cyanobacteria. Here, we present a comprehensive comparison of the structure and composition of the transcriptomes of two Prochlorococcus strains, which, despite their similarities, have adapted their gene pool to specific environmental constraints. We present genome-wide maps of transcriptional start sites (TSS) for both organisms, which are representatives of the two most diverse clades within the two major ecotypes adapted to high- and low-light conditions, respectively. Our data suggest antisense transcription for three-quarters of all genes, which is substantially more than that observed in other bacteria. We discovered hundreds of TSS within genes, most notably within 16 of the 29 prochlorosin genes, in strain MIT9313. A direct comparison revealed very little conservation in the location of TSS and the nature of non-coding transcripts between both strains. We detected extremely short 5′ untranslated regions with a median length of only 27 and 29 nt for MED4 and MIT9313, respectively, and for 8% of all protein-coding genes the median distance to the start codon is only 10 nt or even shorter. These findings and the absence of an obvious Shine–Dalgarno motif suggest that leaderless translation and ribosomal protein S1-dependent translation constitute alternative mechanisms for translation initiation in Prochlorococcus. We conclude that genome-wide antisense transcription is a major component of the transcriptional output from these relatively small genomes and that a hitherto unrecognized high degree of complexity and variability of gene expression exists in their transcriptional architecture.

An intrinsic divinyl-chlorophyll a/b antenna and a particular form of phycobiliprotein, phycoerythrin (PE) III, coexist in the marine oxyphotobacterium Prochlorococcus marinusCCMP 1375. The genomic region including the cpeB/A operon of P. marinus was analysed. It encompasses 10 153 nucleotides that encode three structural phycobiliproteins and at least three (possibly five) different polypeptides analogous to cyanobacterial or red algal proteins involved either in the linkage of subunits or the synthesis and attachment of chromophoric groups. This gene cluster is part of the chromosome and is located within a distance of less than 110 kb from a previously characterized region containing the genes aspA-psbA-aroC. Whereas the Prochlorococcus phycobiliproteins are characterized by distinct deletions and amino acid replacements with regard to analogous proteins from other organisms, the gene arrangement resembles the organization of phycobiliprotein genes in some other cyanobacteria, in particular marine Synechococcusstrains. The expression of two of the Prochlorococcuspolypeptides as recombinant proteins in Escherichia coliallowed the production of individual homologous antisera to the Prochlorococcus and PE subunits. Experiments using these sera show that the ProchlorococcusPEs are specifically associated to the thylakoid membrane and that the protein level does not significantly vary as a function of light irradiance or growth phase. Abbreviations: Chl, chlorophyll; IPTG, iso-propyl-thiogalactoside; PC, phycocyanin; PE, phycoerythrin; PBS, phycobilisome; PUB, phycourobilin

This paper discusses the effects of intercropping and tree pruning on root distribution and soil water depletion in an alley cropping system with Acacia saligna and Sorghum bicolor in northern Kenya. Root distribution was determined by destructive sampling, and the soil water suction was measured with tensiometers and gypsum blocks, both up to 150 cm depth. The root systems of the intercropped trees and crops were distinguished using the natural 13 C discrimination between C3 and C4 plants. The root carbohydrate contents were used to estimate plant water stress integrated over time. The highest root length density was always measured in the topsoil, regardless of season or cropping system. In the dry season, the proportion of roots under the tree row compared to the alley was higher than during the wet season; the same was found for the proportion of roots in the subsoil compared to the topsoil. Pruning decreased the total root length density of sole cropped trees by 47%. The highest root length density was found when the pruned trees were intercropped with Sorghum. If the trees were not pruned, combining trees and crops did not increase root length density. Intercropping resulted in a spatial separation of the root systems of trees and crops between the hedgerows, Sorghum having more roots in the topsoil and the trees having more roots in the subsoil under alley cropping than in monoculture. At the hedgerow of the agroforestry system, however, the root systems of trees and crop overlapped and more roots were found than the sum of roots of sole cropped trees and crops. Soil water depletion was higher under the tree row than in the alley and higher in alley cropping than in monocultural systems. Water competition between tree and crop was confirmed by the carbohydrate analyses showing lower sugar contents of roots in agroforestry than in monoculture. The agroforestry combination used the soil water between the hedgerows more efficiently than the sole cropped trees or crops, as water uptake of the trees reached deeper and started earlier after the flood irrigation than of the Sorghum, whereas the crop could better utilize topsoil water. Under the experimental conditions, the root system of the alley cropped Acacia and Sorghum exploited a larger soil volume utilizing soil resources more efficiently than the respective monocultures.

Prochlorococcus ecotypes are a useful system for exploring the origin and function of diversity among closely related microbes. The genetic variability between phenotypically distinct strains that differ by less that 1% in 16 S ribosomal RNA sequences occurs mostly in genomic islands. Island genes appear to have been acquired in part by phage-mediated lateral gene transfer, and some are differentially expressed under light and nutrient stress. Furthermore, genome fragments directly recovered from ocean ecosystems indicate that these islands are variable among cooccurring Prochlorococcus cells. Genomic islands in this free-living photoautotroph share features with pathogenicity islands of parasitic bacteria, suggesting a general mechanism for niche differentiation in microbial species.

There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS were located upstream of an annotated gene; another third were on the reverse complementary strand of 866 genes, suggesting massive antisense transcription. Orphan TSS located in intergenic regions led us to predict 314 noncoding RNAs (ncRNAs). Complementary microarray-based RNA profiling verified a high number of noncoding transcripts and identified strong ncRNA regulations. Thus, ∼64% of all TSS give rise to antisense or ncRNAs in a genome that is to 87% protein coding. Our data enhance the information on promoters by a factor of 40, suggest the existence of additional small peptide-encoding mRNAs, and provide corrected 5′ annotations for many genes of this cyanobacterium. The global TSS map will facilitate the use of Synechocystis sp. PCC6803 as a model organism for further research on photosynthesis and energy research.

Summary The synthesis and accumulation of compatible solutes represent an essential part of the salt acclimation strategy of microorganisms. Glucosylglycerol is considered to be the typical compatible solute among marine cyanobacteria. However, genes that encode enzymes for the synthesis of glucosylglycerol were not detected in the genome sequences of marine picoplanktonic Prochlorococcus strains. Instead, we noticed the presence of genes that putatively encode for glucosylglycerate (GGA) synthesis among Prochlorococcus and most other closely related marine picocyanobacteria. Recombinant proteins from Prochlorococcus marinus SS120 and Synechococcus sp. PCC 7002 exhibited glucosyl-phosphoglycerate synthase (GpgS) activity, and GpgS is a key enzyme of GGA synthesis. GGA accumulation was found to be salt- as well as nitrogen-regulated in the coastal strain Synechococcus sp. PCC 7002. Moreover, GGA was also detected in all picoplanktonic Prochlorococcus and Synechococcus strains harbouring gpgS genes, especially under N-limiting conditions. These results suggest that marine picocyanobacteria acquired the capacity to synthesize the negatively charged compound GGA during their evolution. Our results establish GGA as the fifth most widespread compatible solute among cyanobacteria. Additionally, GGA appears to replace glutamate as an anion to counter monovalent cations in marine picocyanobacteria from N-poor environments.

The marine cyanobacterium Prochlorococcus MED4 has the smallest genome and cell size of all known photosynthetic organisms. Like all phototrophs at temperate latitudes, it experiences predictable daily variation in available light energy which leads to temporal regulation and partitioning of key cellular processes. To better understand the tempo and choreography of this minimal phototroph, we studied the entire transcriptome of the cell over a simulated daily light-dark cycle, and placed it in the context of diagnostic physiological and cell cycle parameters. All cells in the culture progressed through their cell cycles in synchrony, thus ensuring that our measurements reflected the behavior of individual cells. Ninety percent of the annotated genes were expressed, and 80% had cyclic expression over the diel cycle. For most genes, expression peaked near sunrise or sunset, although more subtle phasing of gene expression was also evident. Periodicities of the transcripts of genes involved in physiological processes such as in cell cycle progression, photosynthesis, and phosphorus metabolism tracked the timing of these activities relative to the light-dark cycle. Furthermore, the transitions between photosynthesis during the day and catabolic consumption of energy reserves at night— metabolic processes that share some of the same enzymes — appear to be tightly choreographed at the level of RNA expression. In-depth investigation of these patterns identified potential regulatory proteins involved in balancing these opposing pathways. Finally, while this analysis has not helped resolve how a cell with so little regulatory capacity, and a ‘deficient’ circadian mechanism, aligns its cell cycle and metabolism so tightly to a light-dark cycle, it does provide us with a valuable framework upon which to build when the Prochlorococcus proteome and metabolome become available.

In the high-light-adapted unicellular marine cyanobacterium Prochlorococcus sp. MED4 the cpeB gene is the only gene coding for a structural phycobiliprotein. The absence of any other phycoerythrin gene in the fully sequenced genome of this organism, the previous inability to detect a gene product, and the mutation of two out of four cysteine residues, normally involved in binding chromophores, suggested that MED4-cpeB might not code for a functional protein. Here, transcription of MED4-cpeB at a low level was detected and the transcriptional start site was mapped. Enrichment of the protein identified phycoerythrobilin as its sole chromophore in vivo, which was confirmed by chromophorylation assays in vitro using the recombinant protein. Phycourobilin is the major chromophore in low-light-adapted Prochlorococcus ecotypes such as strain SS120. Therefore, spectrally tuned phycoerythrins are a characteristic feature of distinct Prochlorococcus ecotypes. Further in vitro mutagenesis experiments replacing one or both cysteines C61R/C82S by arginine or serine, respectively, revealed that only Cys82 is required for chromophore binding. Thus, an unusual green light-absorbing phycoerythrin evolved in the high-light-adapted ecotypes of Prochlorococcus, which potentially serves as a photoreceptor.

International audience; Effects of nitrogen limitation on Photosystem II (PSII) activities and on phycoerythrin were studied in batch cultures of the marine oxyphotobacterium Prochlorococcus marinus. Dramatic decreases in photochemical quantum yields (F-V/F-M), the amplitude of thermoluminescence (TL) B-band, and the rate of QA reoxidation were observed within 12 h of growth in nitrogen-limited conditions. The decline in F-V/F-M paralleled changes in the TL B-band amplitude, indicative of losses in PSII activities and formation of non-functional PSII centers. These changes were accompanied by a continuous reduction in D1 protein content. In contrast, nitrogen deprivation did not cause any significant reduction in phycoerythrin content. Our results refute phycoerythrin as a nitrogen storage complex in Prochlorococcus. Regulation of phycoerythrin gene expression in Prochlorococcus is different from that in typical phycobilisome-containing cyanobacteria and eukaryotic algae investigated so far. (C) 2001 Elsevier Science B.V. All rights reserved.

The D1 protein of photosystem II in the thylakoid membrane of photosynthetic organisms is encoded by psbA genes, which in cyanobacteria occur in the form of a small gene family. Light-dependent up-regulation of psbA gene expression is crucial to ensure the proper replacement of the D1 protein. To gain a high level of gene expression, psbA transcription can be enhanced by several orders of magnitude. Recent transcriptome analyses demonstrated a high number of cis-encoded antisense RNAs (asRNAs) in bacteria, but very little is known about their possible functions. Here, we show the presence of two cis-encoded asRNAs (PsbA2R and PsbA3R) of psbA2 and psbA3 from Synechocystis sp. PCC 6803. These asRNAs are located in the 5′ untranslated region of psbA2 and psbA3 genes. Their expression becomes up-regulated by light and down-regulated by darkness, similar to their target mRNAs. In the PsbA2R-suppressing strain [PsbA2R(−)], the amount of psbA2 mRNA was only about 50% compared with the control strain. Likewise, we identified a 15% lowered activity of photosystem II and a reduced amount of the D1 protein in PsbA2R(−) compared with the control strain. The function of PsbA2R in the stabilization of psbA2 mRNA was shown from in vitro RNase E assay when the AU box and the ribosome-binding site in the 5′ untranslated region of psbA2 mRNA were both covered by PsbA2R. These results add another layer of complexity to the mechanisms that contribute to psbA gene expression and show PsbA2R as a positively acting factor to achieve a maximum level of D1 synthesis.

Prochlorococcus marinus strain CCMP 1375 is the sole prokaryote to possess phycoerythrin in addition to (divinyl-)chlorophyll a/b binding antenna complexes. Here we demonstrate, employing a spectrofluorimetric assay, that phycoerythrin serves a light-harvesting antenna function (transfers energy to chlorophylls).

Prochlorococcus is a marine cyanobacterium that numerically dominates the mid-latitude oceans and is the smallest known oxygenic phototroph. Numerous isolates from diverse areas of the world's oceans have been studied and shown to be physiologically and genetically distinct. All isolates described thus far can be assigned to either a tightly clustered high-light (HL)-adapted clade, or a more divergent low-light (LL)-adapted group. The 16S rRNA sequences of the entire Prochlorococcus group differ by at most 3%, and the four initially published genomes revealed patterns of genetic differentiation that help explain physiological differences among the isolates. Here we describe the genomes of eight newly sequenced isolates and combine them with the first four genomes for a comprehensive analysis of the core (shared by all isolates) and flexible genes of the Prochlorococcus group, and the patterns of loss and gain of the flexible genes over the course of evolution. There are 1,273 genes that represent the core shared by all 12 genomes. They are apparently sufficient, according to metabolic reconstruction, to encode a functional cell. We describe a phylogeny for all 12 isolates by subjecting their complete proteomes to three different phylogenetic analyses. For each non-core gene, we used a maximum parsimony method to estimate which ancestor likely first acquired or lost each gene. Many of the genetic differences among isolates, especially for genes involved in outer membrane synthesis and nutrient transport, are found within the same clade. Nevertheless, we identified some genes defining HL and LL ecotypes, and clades within these broad ecotypes, helping to demonstrate the basis of HL and LL adaptations in Prochlorococcus. Furthermore, our estimates of gene gain events allow us to identify highly variable genomic islands that are not apparent through simple pairwise comparisons. These results emphasize the functional roles, especially those connected to outer membrane synthesis and transport that dominate the flexible genome and set it apart from the core. Besides identifying islands and demonstrating their role throughout the history of Prochlorococcus, reconstruction of past gene gains and losses shows that much of the variability exists at the “leaves of the tree,” between the most closely related strains. Finally, the identification of core and flexible genes from this 12-genome comparison is largely consistent with the relative frequency of Prochlorococcus genes found in global ocean metagenomic databases, further closing the gap between our understanding of these organisms in the lab and the wild., Author Summary Prochlorococcus—the most abundant photosynthetic microbe living in the vast, nutrient-poor areas of the ocean—is a major contributor to the global carbon cycle. Prochlorococcus is composed of closely related, physiologically distinct lineages whose differences enable the group as a whole to proliferate over a broad range of environmental conditions. We compare the genomes of 12 strains of Prochlorococcus representing its major lineages in order to identify genetic differences affecting the ecology of different lineages and their evolutionary origin. First, we identify the core genome: the 1,273 genes shared among all strains. This core set of genes encodes the essentials of a functional cell, enabling it to make living matter out of sunlight and carbon dioxide. We then create a genomic tree that maps the gain and loss of non-core genes in individual strains, showing that a striking number of genes are gained or lost even among the most closely related strains. We find that lost and gained genes commonly cluster in highly variable regions called genomic islands. The level of diversity among the non-core genes, and the number of new genes added with each new genome sequenced, suggest far more diversity to be discovered.

Prochlorococcus marinus SS120 is an ecologically important and biochemically intriguing marine cyanobacterium. In addition to divinyl chlorophylls (DV-Chls) a and b it possesses a particular form of phycoerythrin (PE), but no other phycobilins and therefore no complete phycobilisomes. Here, a spectroscopic characterisation of these DV-Chls and PE is provided. Comparison of fluorescence quantum yields, excited state lifetimes and absorption characteristics indicate similar light-harvesting properties of the DV-Chls as their monovinyl counterparts. PE, which is present only in tiny amounts, was purified and considerably enriched. A phycourobilin to phycoerythrobilin ratio of 3:1 chromophores per (alphabeta) PE monomer is suggested. The in vitro fluorescence lifetime of PE is 1.74 ns. In vivo time-resolved fluorescence measurements with synchrotron radiation were used to investigate the possible role of PE in light-harvesting. The fluorescence decay time for PE is about 550 ps, indicating an unusually slow excitation energy transfer. The decay time slowed to 1 ns after addition of glycerol to cell cultures. The contribution of PE to total light-harvesting capacity was estimated to be about one (alphabeta) PE monomer per 330 DV-Chl b molecules. Thus, the capacity of PE to function primarily as a photosynthetic light-harvesting pigment in P. marinus SS120 is low.