An ancient bacterial zinc acquisition system identified from a cyanobacterial exoproteome.

Bacteria have developed fine-tuned responses to cope with potential zinc limitation. The Zur protein is a key player in coordinating this response in most species. Comparative proteomics conducted on the cyanobacterium Anabaena highlighted the more abundant proteins in a zur mutant compared to the wild type. Experimental evidence showed that the exoprotein ZepA mediates zinc uptake. Genomic context of the zepA gene and protein structure prediction provided additional insights on the regulation and putative function of ZepA homologs. Phylogenetic analysis suggests that ZepA represents a primordial system for zinc acquisition that has been conserved for billions of years in a handful of species from distant bacterial lineages. Furthermore, these results show that Zur may have been one of the first regulators of the FUR family to evolve, consistent with the scarcity of zinc in the ecosystems of the Archean eon.

Role of a cryptic tRNA gene operon in survival under translational stress.

As compared to eukaryotes, bacteria have a reduced tRNA gene set encoding between 30 and 220 tRNAs. Although in most bacterial phyla tRNA genes are dispersed in the genome, many species from distinct phyla also show genes forming arrays. Here, we show that two types of arrays with distinct evolutionary origins exist. This work focuses on long tRNA gene arrays (L-arrays) that encompass up to 43 genes, which disseminate by horizontal gene transfer and contribute supernumerary tRNA genes to the host. Although in the few cases previously studied these arrays were reported to be poorly transcribed, here we show that the L-array of the model cyanobacterium Anabaena sp. PCC 7120, encoding 23 functional tRNAs, is largely induced upon impairment of the translation machinery. The cellular response to this challenge involves a global reprogramming of the transcriptome in two phases. tRNAs encoded in the array are induced in the second phase of the response, directly contributing to cell survival. Results presented here show that in some bacteria the tRNA gene set may be partitioned between a housekeeping subset, which constantly sustains translation, and an inducible subset that is generally silent but can provide functionality under particular conditions.

Trans-oligomerization of duplicated aminoacyl-tRNA synthetases maintains genetic code fidelity under stress.

Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNA(Thr) synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNA(Thr). Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs.

Membrane anchoring of aminoacyl-tRNA synthetases by convergent acquisition of a novel protein domain.

Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl-, and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process also dependent on the CAAD domain.

Intraphylum diversity and complex evolution of cyanobacterial aminoacyl-tRNA synthetases.

A comparative genomic analysis of 35 cyanobacterial strains has revealed that the gene complement of aminoacyl-tRNA synthetases (AARSs) and routes for aminoacyl-tRNA synthesis may differ among the species of this phylum. Several genes encoding AARS paralogues were identified in some genomes. In-depth phylogenetic analysis was done for each of these proteins to gain insight into their evolutionary history. GluRS, HisRS, ArgRS, ThrRS, CysRS, and Glu-Q-RS showed evidence of a complex evolutionary course as indicated by a number of inconsistencies with our reference tree for cyanobacterial phylogeny. In addition to sequence data, support for evolutionary hypotheses involving horizontal gene transfer or gene duplication events was obtained from other observations including biased sequence conservation, the presence of indels (insertions or deletions), or vestigial traces of ancestral redundant genes. We present evidences for a novel protein domain with two putative transmembrane helices recruited independently by distinct AARS in particular cyanobacteria.

FtsZ of Filamentous, Heterocyst-Forming Cyanobacteria Has a Conserved N-Terminal Peptide Required for Normal FtsZ Polymerization and Cell Division.

Filamentous cyanobacteria grow by intercalary cell division, which should involve distinct steps compared to those producing separate daughter cells. The N-terminal region of FtsZ is highly conserved in the clade of filamentous cyanobacteria capable of cell differentiation. A derivative of the model strain Anabaena sp. PCC 7120 expressing only an FtsZ lacking the amino acids 2-51 of the N-terminal peptide (ΔN-FtsZ) could not be segregated. Strain CSL110 expresses both ΔN-FtsZ, from the endogenous ftsZ gene promoter, and the native FtsZ from a synthetic regulated promoter. Under conditions of ΔN-FtsZ predominance, cells of strain CSL110 progressively enlarge, reflecting reduced cell division, and show instances of asymmetric cell division and aberrant Z-structures notably differing from the Z-ring formed by FtsZ in the wild type. In bacterial 2-hybrid assays FtsZ interacted with ΔN-FtsZ. However, ΔN-FtsZ-GFP appeared impaired for incorporation into Z-rings when expressed together with FtsZ. FtsZ, but not ΔN-FtsZ, interacted with the essential protein SepF. Both FtsZ and ΔN-FtsZ polymerize in vitro exhibiting comparable GTPase activities. However, filaments of FtsZ show a distinct curling forming toroids, whereas ΔN-FtsZ form thick bundles of straight filaments. Thus, the N-terminal FtsZ sequence appears to contribute to a distinct FtsZ polymerization mode that is essential for cell division and division plane location in Anabaena.

Sub-Cellular Localization and Complex Formation by Aminoacyl-tRNA Synthetases in Cyanobacteria: Evidence for Interaction of Membrane-Anchored ValRS with ATP Synthase.

tRNAs are charged with cognate amino acids by aminoacyl-tRNA synthetases (aaRSs) and subsequently delivered to the ribosome to be used as substrates for gene translation. Whether aminoacyl-tRNAs are channeled to the ribosome by transit within translational complexes that avoid their diffusion in the cytoplasm is a matter of intense investigation in organisms of the three domains of life. In the cyanobacterium Anabaena sp. PCC 7120, the valyl-tRNA synthetase (ValRS) is anchored to thylakoid membranes by means of the CAAD domain. We have investigated whether in this organism ValRS could act as a hub for the nucleation of a translational complex by attracting other aaRSs to the membranes. Out of the 20 aaRSs, only ValRS was found to localize in thylakoid membranes whereas the other enzymes occupied the soluble portion of the cytoplasm. To investigate the basis for this asymmetric distribution of aaRSs, a global search for proteins interacting with the 20 aaRSs was conducted. The interaction between ValRS and the FoF1 ATP synthase complex here reported is of utmost interest and suggests a functional link between elements of the gene translation and energy production machineries.

CURT1,CAAD-containing aaRSs, thylakoid curvature and gene translation.

CURT1 proteins induce membrane curvature to grana margins in Arabidopsis (Arabidopsis thaliana) thylakoids. A domain sharing sequence and structural features with CURT1 is found in some cyanobacterial aminoacyl-tRNA synthetases (aaRSs) that show an unusual localization to the thylakoid membranes. Evolutionary scenarios and functional implications are discussed in this article.

The plastid ancestor originated among one of the major cyanobacterial lineages.

The primary endosymbiotic origin of chloroplasts is now well established but the identification of the present cyanobacteria most closely related to the plastid ancestor remains debated. We analyse the evolutionary trajectory of a subset of highly conserved cyanobacterial proteins (core) along the plastid lineage, those which were not lost after the endosymbiosis. We concatenate the sequences of 33 cyanobacterial core proteins that share a congruent evolutionary history, with their eukaryotic counterparts to reconstruct their phylogeny using sophisticated evolutionary models. We perform an independent reconstruction using concatenated 16S and 23S rRNA sequences. These complementary approaches converge to a plastid origin occurring during the divergence of one of the major cyanobacterial lineages that include N2-fixing filamentous cyanobacteria and species able to differentiate heterocysts.

Genomic survey of cAMP and cGMP signalling components in the cyanobacterium Synechocystis PCC 6803.

Cyanobacteria modulate intracellular levels of cAMP and cGMP in response to environmental conditions (light, nutrients and pH). In an attempt to identify components of the cAMP and cGMP signalling pathways in Synechocystis PCC 6803, the authors screened its complete genome sequence by using bioinformatic tools and data from sequence-function studies performed on both eukaryotic and prokaryotic cAMP/cGMP-dependent proteins. Sll1624 and Slr2100 were tentatively assigned as being two putative cyclic nucleotide phosphodiesterases. Five proteins were identified as having all the determinants required to be cyclic nucleotide receptors, two of them being probably more specific for cGMP (an element of two-component regulatory systems - Slr2104 - and a putative cyclic-nucleotide-gated cation channel - Slr1575), the three others being probably more specific for cAMP: (i) a protein of unidentified function (Slr0842); (ii) a putative cyclic-nucleotide-modulated permease (Slr0593), previously annotated as a kinase A regulatory subunit; and (iii) a putative transcription factor (CRP-SYN: =Sll1371), which possesses cAMP- and DNA-binding determinants homologous to those of the cAMP receptor protein of Escherichia coli (CRP-EC:). This homology, together with the presence in Synechocystis of CRP-EC:-like binding sites upstream of crp, cya1, slr1575, and several genes encoding enzymes involved in transport and metabolism, strongly suggests that CRP-SYN: is a global regulator.

The NblAI protein from the filamentous cyanobacterium Tolypothrix PCC 7601: regulation of its expression and interactions with phycobilisome components.

Cyanobacteria respond to changes in light or nutrient availability by modifications in their photosynthetic light harvesting antenna. In unicellular cyanobacteria a small polypeptide (NblA) is required for phycobilisome degradation following environmental stresses. In the filamentous strain Tolypothrix sp. PCC 7601 the nblAI gene, encoding a NblA homologue, is located upstream of the operon coding for phycoerythrin (cpeBA). The nblAI transcripts all originate from a single transcription start point; their intracellular levels vary according to nitrogen regimes but not with light spectral quality. Using recombinant His-tagged NblAI protein, we found that in vitro NblAI has affinity for both phycocyanin and phycoerythrin subunits from Tolypothrix sp. PCC 7601, but not for allophycocyanin from this cyanobacterium or for phycobiliproteins from other cyanobacterial species. We also observed that although nblAI is mainly expressed under nitrogen starvation, NblAI polypeptides are always present in the cell; a significant portion of them co-purify with phycobilisome preparations but only if cells were grown under red light. Our data indicate that NblAI attaches to the phycobilisomes even under non-inducing conditions and suggest a preferential affinity of NblAI for phycocyanin.

Co-ordinated expression of phycobiliprotein operons in the chromatically adapting cyanobacterium Calothrix PCC 7601: a role for RcaD and RcaG.

In the cyanobacterium Calothrix sp. PCC 7601 the cpc2 operon encoding phycocyanin 2 (PC2) is expressed if red radiations are available. RcaD was previously identified in extracts from red-light-grown cells as an alkaline phosphatase-sensitive protein that binds upstream of the transcription start point (TSP) of the cpc2 operon. In this work, RcaD was purified, and the corresponding gene cloned with a PCR probe obtained using degenerated primers based on RcaD peptide sequences (accession no. AJ319541). Purified RcaD binds to the cpc2 promoter region and also to those of the constitutive cpc1 and apc1 operons that encode phycocyanin 1 and allophycocyanin. Escherichia coli-overexpressed RcaD can bind to the cpc2 promoter region. The rcaD gene is upstream of an open reading frame (ORF) termed rcaG. Co-transcription of both genes was demonstrated by reverse transcription (RT)-PCR experiments, and found to be independent of the light wavelengths. A single TSP was mapped. Sequence features of RcaD and RcaG led us to propose a functional relationship between these two proteins. A rcaD mutant generated by allelic exchange exhibited altered expression of the cpc2, cpeBA, apc1 and cpc1 operons upon green to red-light shifts. RcaD seems to be a co-activator co-ordinating the transcription of the phycobiliprotein operons upon changes in light spectral quality.