The cyanobacterial diversity in the pelagic of a shallow estuary at the Southern Baltic Sea has been investigated by a combination of classical morphological data and a polymerase chain reaction (PCR)-based molecular approach. The aim of the study was to investigate possible changes in the composition of the cyanobacterial community along the salinity and nutrient gradients. For this purpose partial gene sequences of cyanobacterial 16S rDNA and of two functional genes (ggpS- salinity tolerance marker, isiA- iron starvation marker) were amplified and compared with total community DNA. Random distribution of ggpS genotypes along the salinity gradient suggests that synthesis of the osmolyte glucosylglycerol is not restricted to higher salinity sampling sites. Most of the isiA sequences formed a new homogenous cluster in a phylogenetic tree, which indicates that the indigenous cyanobacterial community comprises a group of unknown species. Minimum iron concentrations, which can activate isiA transcription in model cyanobacteria, occurred at a few sampling sites with high phytoplankton biomass and moderate salinity. Nevertheless, isiA expression could be detected at all sampling sites, which indicated restricted iron supply to cyanobacterial phytoplankton in summer.

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 M. 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 ironregulated isiA expression can serve to indicate iron deficiency in cyanobacterial samples of differing origins from the field.Iron as an important micronutrient of phytoplankton had been neglected for a long period before Martin and coworkers (28-30) revived its consideration in their approach to defining the basic pattern of plankton productivity in the open oceans. Subsequent research dealt with iron as the crucial nutrient in high-nitrate, low-chlorophyll regions of the open oceans and its ecological importance (1,12,16,22,43). Some evidence of iron-limited phytoplankton development also exists for coastal upwelling regions (13). Owing to the complex chemical behavior of iron, the bioavailable iron concentration cannot easily be manipulated in oxygen-rich waters by iron enrichment and iron depletion procedures. Trace-metal precipitation and adsorption to particles as well as ion-exchange processes (for a review, see reference 39) are only a few of the potential effects of these procedures that can hamper the desirable effects of fertilization and depletion experiments. Although these are methods that provide support for the characterization of the concentration of bioavailable iron in water (42), the great number and variety of variously efficient mechanisms of organismic iron sequestration (6, 14, 43) limit the interpretation of the corresponding analyses. Thus, the use of molecular markers to detect the physiological state of iron limitation in microalgae without the use of artificial manipulations of water chemistry would represent an important achievement in proving iron limitation in parts of phytoplankton.Besides producing results of general interest, the use of molecular approaches became necessary to go beyond the limits of conventional methodology in aquatic ecology. Scanlan et al. (36) introduced the phosphate-binding protein PstS as a potential diagnostic marker for investigating phosphate stress in photosynthetic picoplankton. For iron limitation, flavodoxin accumulation was used as a biochemical marker and its detection in single cells pr...

Prochlorothrix hollandica is the only filamentous chlorophyll b (Chlb)-containing oxyphotobacterium that has been found in freshwater habitats to date. Chlb serves as a light-harvesting pigment which is bound to special binding proteins (Pcb). Even though Prochlorothrix was initially characterized as a highly salt-sensitive species, we detected it in a brackish water environment that is characterized by salinities of up to 12 practical salinity units. Using PCR and reverse transcription, we amplified pcb gene fragments of phytoplankton samples taken along a salinity gradient in the eutrophic Darss-Zingst estuary (southern Baltic Sea). After sequencing, high levels of homology to the pcbB and pcbC genes of P. hollandica were found. Furthermore, autofluorescence of Prochlorothrix-like filaments that indicated that Chlb was present was detected in enrichment cultures prepared from the estuarine phytoplankton. The detection of Chlb-containing filaments, as well as the pcb and 16S ribosomal DNA sequences, suggests that Prochlorothrix is an indigenous genus in the Darss-Zingst estuary and may also inhabit many other brackish water environments. The potential of using pcb gene detection to differentiate Prochlorothrix from morphologically indistinguishable species belonging to the genera Pseudanabaena and Planktothrix (Oscillatoria) in phytoplankton analyses is discussed.

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 10fold 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. ß