Inactivation of Three RG(S/T)GR Pentapeptide-Containing Negative Regulators of HetR Results in Lethal Differentiation of Anabaena PCC 7120

The filamentous cyanobacterium Anabaena sp. PCC 7120 produces, during the differentiation of heterocysts, a short peptide PatS and a protein HetN, both containing an RGSGR pentapeptide essential for activity. Both act on the master regulator HetR to guide heterocyst pattern formation by controlling the binding of HetR to DNA and its turnover. A third small protein, PatX, with an RG(S/T)GR motif is present in all HetR-containing cyanobacteria. In a nitrogen-depleted medium, inactivation of patX does not produce a discernible change in phenotype, but its overexpression blocks heterocyst formation. Mutational analysis revealed that PatX is not required for normal intercellular signaling, but it nonetheless is required when PatS is absent to prevent rapid ectopic differentiation. Deprivation of all three negative regulators—PatS, PatX, and HetN—resulted in synchronous differentiation. However, in a nitrogen-containing medium, such deprivation leads to extensive fragmentation, cell lysis, and aberrant differentiation, while either PatX or PatS as the sole HetR regulator can establish and maintain a semiregular heterocyst pattern. These results suggest that tight control over HetR by PatS and PatX is needed to sustain vegetative growth and regulated development. The mutational analysis has been interpreted in light of the opposing roles of negative regulators of HetR and the positive regulator HetL.

The Proposed Neurotoxin β-N-Methylamino-l-Alanine (BMAA) Is Taken up through Amino-Acid Transport Systems in the Cyanobacterium Anabaena PCC 7120

Produced by cyanobacteria and some plants, BMAA is considered as an important environmental factor in the occurrence of some neurodegenerative diseases. Neither the underlying mechanism of its toxicity, nor its biosynthetic or metabolic pathway in cyanobacteria is understood. Interestingly, BMAA is found to be toxic to some cyanobacteria, making it possible to dissect the mechanism of BMAA metabolism by genetic approaches using these organisms. In this study, we used the cyanobacterium Anabaena PCC 7120 to isolate BMAA-resistant mutants. Following genomic sequencing, several mutations were mapped to two genes involved in amino acids transport, suggesting that BMAA was taken up through amino acid transporters. This conclusion was supported by the protective effect of several amino acids against BMAA toxicity. Furthermore, targeted inactivation of genes encoding different amino acid transport pathways conferred various levels of resistance to BMAA. One mutant inactivating all three major amino acid transport systems could no longer take up BMAA and gained full resistance to BMAA toxicity. Therefore, BMAA is a substrate of amino acid transporters, and cyanobacteria are interesting models for genetic analysis of BMAA transport and metabolism.

RNase II binds to RNase E and modulates its endoribonucleolytic activity in the cyanobacterium Anabaena PCC 7120

In Escherichia coli, the endoribonuclease E (RNase E) can recruit several other ribonucleases and regulatory proteins via its noncatalytic domain to form an RNA degradosome that controls cellular RNA turnover. Similar RNA degradation complexes have been found in other bacteria; however, their compositions are varied among different bacterial species. In cyanobacteria, only the exoribonuclease PNPase was shown to bind to the noncatalytic domain of RNase E. Here, we showed that Alr1240, a member of the RNB family of exoribonucleases, could be co-isolated with RNase E from the lysate of the cyanobacterium Anabaena PCC 7120. Enzymatic analysis revealed that Alr1240 is an exoribonuclease II (RNase II), as it only degrades non-structured single-stranded RNA substrates. In contrast to known RNase E-interacting ribonucleases, which bind to the noncatalytic domain of RNase E, the Anabaena RNase II was shown to associate with the catalytic domain of RNase E. Using a strain in which RNase E and RNase II were tagged in situ with GFP and BFP, respectively, we showed that RNase E and RNase II form a compact complex in vivo by a fluorescence resonance energy transfer (FRET) assay. RNase E activity on several synthetic substrates was boosted in the presence of RNase II, suggesting that the activity of RNase E could be regulated by RNase II-RNase E interaction. To our knowledge, Anabaena RNase II is an unusual ribonuclease that interacts with the catalytic domain of RNase E, and it may represent a new type of RNA degradosome and a novel mechanism for regulating the activity of the RNA degradosome. As Anabaena RNase E interacts with RNase II and PNPase via different regions, it is very likely that the three ribonucleases form a large complex and cooperatively regulate RNA metabolism in the cell.