Expression of the isiA and isiB genes was analysed in the cyanobacterium Synechocystis sp. PCC 6803 grown in high salt or in iron-deficient medium. The detection of a 2.3-knt transcript in Northern blot experiments indicated cotranscription of isiAB in an operon, which was confirmed by reverse transcriptase PCR. The abundance of a monocistronic 1.25-knt isiA-specific mRNA was about 10-fold higher than the dicistronic message. The isiAB-specific transcripts were most abundant in cells adapted to 342 mM NaCl and under iron deficiency. The promoter of the operon was mapped to 211 bp upstream of the translational start. A putative Fur binding site was detected immediately upstream of the GTG start codon. A preliminary transcript of about 0.2 knt was detected in cells grown in conditions in which the isiAB operon was not transcribed. This indicates that a repressor binds to the identified Fur binding site and thus inhibits isiAB transcription under low salt and iron replete conditions. z

The iron deficiency-dependent regulation of isiAB transcription in Synechocystis sp. PCC 6803 was analyzed by fusion of modified isiAB promoter fragments to gfp and in vivo quantification of Gfp fluorescence. For the putative Fur box only a slight repressing impact on promoter activity could be shown. In a heteroallelic fur mutant a corresponding incomplete repression of isiAB transcription under iron-replete conditions confirmed the role of Fur in isiAB regulation. However, a 90 bp region upstream of the putative 335 box of the isiAB promoter was essential for full promoter activity under iron-deplete conditions. This pattern indicates a dual promoter regulation by both a repressing mechanism exhibited via the Fur system and an unknown activating mechanism.

To identify genes transcribed preferentially under salt stress, a subtractive RNA hybridization procedure was applied to the cyanobacterium Synechocystis sp. PCC 6803. The screening of a genomic library led to the identification of several RNA species that were more abundant in salt-stressed cells than in control cells. Salt-dependent transcription of the identified genes was verified in Northern blot experiments. In addition to the previously characterized genes cpn60 (encoding GroEL; a molecular chaperone) and isiA (encoding a chlorophyll-binding protein), genes encoding a protein of unknown function (slr0082) and a putative RNA helicase (slr0083) were identified as salt-regulated genes in Synechocystis. Genes slr0082 and slr0083, located at sites adjacent to each other on the Synechocystis chromosome, were transcribed from separate promoters and showed the most significant induction 1-3 h after salt shock. The salt-regulated promoters of these two genes were mapped. Genes cpn60, slr0082, and slr0083 were also found to be induced by a cold shock. The possible role of the identified gene products for salt adaptation of Synechocystis is discussed.

In order to investigate the metabolic importance of glycine decarboxylase (GDC) in cyanobacteria, mutants were generated defective in the genes encoding GDC subunits and the serine hydroxymethyl-transferase (SHMT). It was possible to mutate the genes for GDC subunits P, T, or H protein in the cyanobacterial model strain Synechocystis sp. PCC 6803, indicating that GDC is not necessary for cell viability under standard conditions. In contrast, the SHMT coding gene was found to be essential. Almost no changes in growth, pigmentation, or photosynthesis were detected in the GDC subunit mutants, regardless of whether or not they were cultivated at ambient or high CO2 concentrations. The mutation of GDC led to an increased glycine/serine ratio in the mutant cells. Furthermore, supplementation of the medium with low glycine concentrations was toxic for the mutants but not for wild type cells. Conditions stimulating photorespiration in plants, such as low CO2 concentrations, did not induce but decrease the expression of the GDC and SHMT genes in Synechocystis. It appears that, in contrast to heterotrophic bacteria and plants, GDC is dispensable for Synechocystis and possibly other cyanobacteria.