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Iron deficiency in cyanobacteria can be assessed by measuring the expression of the isiA gene. In Synechocystis sp. strain PCC 6803, gene expression begins when the concentration of iron citrate decreases and growth begins to decline.

Examines the use of isiA gene expression to monitor the iron status of cyanobacteria. Dependence of isiA expression on an organism response to iron deficiency; Amplification of isiA genes; Detection of expression by means of reverse transcription-polymerase chain reaction.

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 -35 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.

A collection of 17 salt-sensitive mutants of the cyanobacterium Synechocystis sp. strain PCC 6803 was obtained by random cartridge mutagenesis. The genes coding for proteins essential for growth at high salt concentrations were mapped on the completely known genome sequence of this strain. The two genes coding for enzymes involved in biosynthesis of the osmolyte glucosylglycerol were affected in nine mutants. Two mutants defective in a glycoprotease encoding gene gcp showed a reduced salt resistance. Four genes were identified not previously known to be essential for salt tolerance in cyanobacteria. These genes (slr1799, slr1087, sll1061, and sll1062) code for proteins not yet functionally characterized.

The use of isiA expression to monitor the iron status of cyanobacteria was investigated. Studies of laboratory cultures of the cyanobacterium Synechocystis sp. strain PCC 6803 showed that isiA expression is dependent on the organism's response to iron deficiency; isiA expression starts as soon as a decline in the rate of growth begins. isiA expression is switched on at concentrations of iron citrate of less than 0.7 microM. A PCR method was developed for the specific amplification of the iron-regulated isiA gene from a variety of cyanobacteria. After we developed degenerate primers, 15 new internal isiA fragments (840 bp) were amplified, cloned, and sequenced from strains obtained from algal collections, from new isolates, and from enriched field samples. Furthermore, isiA expression could be detected by means of reverse transcription-PCR when enriched field samples were exposed to restricted iron availability. These results imply that determining the level of iron-regulated isiA expression can serve to indicate iron deficiency in cyanobacterial samples of differing origins from the field.

The expression of the chlorophyll a-binding, iron stress-induced protein IsiA is part of the cyanobacterial response to iron deficiency. A new isiA gene from the filamentous heterocystous cyanobacterial strain, Fischerella muscicola PCC 73103, was identified using standard and inverse PCR. While in unicellular cyanobacterial strains isiA is organized in an operon with isiB (encoding flavodoxin), in Fischerella not an isiB gene but another chlorophyll-binding protein encoding gene was identified downstream of isiA, which shows significant similarities to Pcb-like protein encoding genes known from prochlorophytes. The expression of both genes was clearly activated under iron deficiency. Although isiA and pcbC were independently transcribed, the size of the pcbC transcript indicates a large iron-regulated operon. Beside a 10-fold increase of isiA transcript content iron-starved cells of Fischerella showed a blue-shift in the red chlorophyll a absorption peak. In addition, chlorophyll fluorescence at 77 K was dominated by an emission peak at 685 nm. These features are in accordance with the characteristics of IsiA accumulation in iron-starved unicellular cyanobacteria, suggesting identical IsiA function in heterocystous strains in spite of different genetic organization.

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.