Novel DNA-Binding Proteins in the Cyanobacterium Anabaena sp. Strain PCC 7120

Cyanobacteria are microorganisms that require only light as a source of energy and water as a source of reductant. These bacteria have been used to study cell division and differentiation, photosynthesis, nitrogen fixation, hydrogen metabolism, and responses to environmental changes. Anabaena sp. strain PCC 7120, a filamentous cyanobacterium, is simultaneously capable of oxygenic photosynthesis and nitrogen fixation. Nitrogen fixation is closely associated with cellular differentiation and intercellular interactions. In the presence of ammonium or nitrate, filaments of Anabaena sp. are comprised solely of photosynthetic vegetative cells. When deprived of a source of fixed nitrogen, vegetative cells differentiate at semiregular intervals along the filaments into heterocysts, cells specialized for the fixation of N2. The nitrogen-fixing activity of heterocysts depends upon products of photosynthesis that they receive from neighboring vegetative cells, to which, in return, heterocysts supply products of nitrogen fixation. The differentiation of heterocysts involves extensive biochemical and structural changes that are regulated by a cascade-like sequence of transcriptional activations whose coordination is little understood (9, 48). Regulatory DNA-binding proteins that are known to affect heterocyst formation in Anabaena sp. include a histone-like protein, HU (27); transcription factor cyclic AMP receptor protein-related proteins NtcA (16, 45) and DevH (21); and PatB, a ferredoxin-like protein with a helix-turn-helix motif (33). However, RNA polymerase sigma factors SigA, which is expressed in both the presence and absence of combined nitrogen, and SigB and SigC, which are expressed transiently under nitrogen-limiting conditions, and five additional sigma factors (SigD, SigE, SigF, SigG, and SigH) are not involved specifically in the regulation of heterocyst differentiation (6, 7; I. Khudyakov, X. Wu, and J. W. Golden, Abstr. 10th Int. Symp. Phototroph. Prokar., p. 193, 2000.). HU is degraded during heterocyst differentiation, and a new, similar but different protein is synthesized in the heterocyst (37). Mutation of hanA, which encodes HU, results in a highly pleiotropic phenotype that includes slow growth, altered pigmentation, resistance to phage A-4(L), cellular fragility, and the inability of heterocysts to differentiate (27). NtcA binds upstream from xisA, which encodes a site-specific recombinase, but also upstream from glnA, rbcL, nifH, and hetC (10, 22, 36) and is required for heterocyst differentiation in Anabaena sp. (16, 36, 46). Despite this accumulation of known factors, the regulatory mechanisms that underlie the progression of differentiation in cyanobacteria remain largely unknown (48). Earlier, hepB, hepK, and neighboring genes hepC and hepA were shown to be required for the normal formation of a principal structural element of heterocysts, the outer, polysaccharide layer of their envelope (48, 50). A DNA sequence upstream from hepC greatly influences induction of hepA upon nitrogen stepdown (50). We have identified four proteins that bind to a 150-bp sequence, X12, upstream of hepC and have shown that two of these proteins play an important role in the differentiation of heterocysts and in their ability to fix N2 aerobically.