Makoto Shirai

Specific recognition of the cyanobacterial psbA promoter by RNA polymerases containing principal sigma factors

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. To clarify the promoter recognition by a sigma factor of RNA polymerase, in vivo and in vitro analyses were performed for the photosynthetic gene. Although the specific transcript from the psbA2 promoter, whose sequence is of Escherichia coli consensus type, was observed in both cyanobacterium K-81 and E. coli cells, the expression was light-dependent in K-81 whereas it was constitutive in E. coli under the conditions of light and darkness (L/D). The specific psbA2-dependent transcripts were also detected in vitro by RNA polymerases containing the principal sigma factors, E. coli sigma70 and K-81 sigmaA1 (constitutively exists in K-81 grown under L/D cycles). Furthermore, a series of promoter fragments were constructed to confirm minimal cis elements for the in vitro psbA2 transcription. A -80 to +6 or -38 to +46 region, the sequences of which consisted of a core promoter (-38 to +6), was identified as the potential minimal cis element using the RNA polymerase fraction (*EsigmaA1) containing sigmaA1 partially purified from K-81. These results suggest that the psbA2 transcription with the minimal sequence was induced by the RNA polymerase (EsigmaA1) containing the principal sigma factor, sigmaA1, under both light and dark conditions in K-81.

Cloning, sequencing and characterization of the gene encoding a principal sigma factor homolog from the cyanobacterium Microcystis aeruginosa K-81.

We cloned and sequenced the rpoD1 gene of Microcystis aeruginosa K-81, a unicellular colony-forming cyanobacterium that can perform photosynthesis involving light-responsive gene expression. The deduced amino acid sequence of RpoD1 exhibited extensive homology to the other eubacterial principal sigma factors. Primer extension and Western blot analyses revealed that the rpoD1 gene, which encodes a principle sigma factor homolog, had two transcription start points, P1 and P2. These transcripts, and the corresponding protein, constitutively appeared in M. aeruginosa, irrespective of light or dark conditions.

A new sigma factor homolog in a cyanobacterium: Cloning, sequencing, and light-responsive transcripts of rpoD2 from Microcystis aeruginosa K-81

We isolated an rpoD2 gene encoding the potential sigma factor of RNA polymerase from the cyanobacterium Microcystis aeruginosa K-81, which can perform photosynthesis. The deduced amino acid sequence of RpoD2 (sigmaA2) exhibits extensive homology to other eubacterial RpoD proteins. This gene possessed multiple 5-end transcripts, expressed specifically under light (P(L)), dark (P(D)), or constitutively light/dark (P(C)) conditions during exponential cell growth.

A novel genetic organization: the leuA-rpoD1 locus in the cyanobacterium Microcystis aeruginosa K-81.

We cloned and sequenced the region upstream of rpoD1, which encodes a principal sigma factor in the cyanobacterium Microcystis aeruginosa K-81. An open reading frame (orf1, 1599 bp) was discovered, the deduced amino-acid sequence of which (533 aa, 58, 016 Da) exhibits homology to another bacterial leuA gene product, 2-isopropylmalate synthase. The leuA (orf1) gene specifically complemented an E. coli leuA mutant. The 5-upstream region of leuA did not contain possible leader peptide or stem-loop structures for attenuation. These findings indicate that the genetic structure of the leuA-rpoD1 locus in M. aeruginosa K-81 significantly differs from those of known leuA and rpoD loci found in other bacteria.