Abstract
Some cyanobacteria couple oxygenic photosynthesis in vegetative cells with O2-sensitive N2 fixation in differentiated cells called heterocysts. Heterocyst differentiation involves extensive biochemical and structural changes that collectively permit heterocysts to assimilate N2 aerobically and supply the products of N2 fixation to vegetative cells. HepK and DevR are required for the development of functional heterocysts in Anabaena and Nostoc, respectively. We show that HepK is an autokinase and that Anabaena DevRA is its cognate response regulator, together comprising part or all of a two-component system that mediates developmental regulation of biosynthesis of a heterocyst envelope polysaccharide. Recombinant N-hexahistidine-tagged HepK (H6HepK), the cytoplasmic portion H6'HepK of H6HepK, H6DevR, and H6DevRA were overexpressed in Escherichia coli and purified to homogeneity. H6'HepK, but not H6HepK, autophosphorylates with [gamma-32P]ATP. ADP, specifically, elicits dephosphorylation of phosphorylated H6'HepK. The phosphoryl group of H6'HepK is transferred rapidly and efficiently to both H6DevR and H6DevRA but not to His-tagged OmpR, whose cognate sensor kinase is EnvZ. Sequence comparisons, the results of site-specific mutagenesis, and tests of chemical stability support identification of HepK-His348 and DevR-Asp53 as the phosphorylated residues. The mutation HepK-H348A abolishes both in vitro autokinase activity and in vivo functionality of HepK. Heterocysts of both hepK Anabaena and devRA Anabaena lack an envelope polysaccharide layer and are nonfunctional. Consistent with the normal site of deposition of that polysaccharide, a hepK::gfp transcriptional fusion is expressed principally in proheterocysts. HepK/DevRA is the first two-component system identified that regulates the biosynthesis of a polysaccharide as part of a patterned differentiation process.
Highlights
When combined nitrogen becomes limiting, Anabaena species and their close relatives respond by initiating a developmental program that results in the production of terminally differentiated, nitrogen-fixing cells called heterocysts
We show that HepK is an autokinase and that Anabaena DevRA is its cognate response regulator, together comprising part or all of a two-component system that mediates developmental regulation of biosynthesis of a heterocyst envelope polysaccharide
HepK/DevRA is the first two-component system identified that regulates the biosynthesis of a polysaccharide as part of a patterned differentiation process
Summary
Culture and Preparation for Electron Microscopy—Anabaena sp. strain PCC 7120 and derivatives of it were grown at 30 °C in the light at approximately 140 microeinsteins mϪ2 sϪ1 (Li-Cor Quantum Radiometer/Photometer model LI-185A; Lincoln, NE) on AA ϩ N agar [47] in the presence of appropriate antibiotics or on a rotary shaker in medium AA/8 or AA/8 ϩ N [47] plus appropriate antibiotics. The PCR product was first cloned into the EcoRV site of pBluescript SKϩ (Stratagene, La Jolla, CA) to produce pRL2483 for DNA sequencing; an NdeIBamHI fragment containing ompR was transferred between the NdeI and BamHI sites of pET-14b, yielding pRL2484. At the indicated reaction times, 15-l portions of samples were rapidly transferred to an equal volume of 2ϫ concentrated SDS-PAGE loading buffer. The mixtures were incubated for 60 min in a water bath at 42 °C Another sample, to which 2 l of water was added, was kept on ice. The samples were separated by SDS-PAGE and analyzed for radioactivity. ADP, CDP, GDP, or TDP (sodium salts; Sigma; sodium ADP was purchased from TCI America for confirmation) at a final concentration of 0.5 mM was incubated for 5 min in a 15-l phosphorylation reaction mixture containing 2 g of H6ЈHepK-32P. Fluorescence microscopy and photography of the hepK::gfp strain was performed as described [53]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
