Detection of the isiA gene across cyanobacterial strains: potential for probing iron deficiency

Abstract

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.

FIG. 1

Growth rate and isiA expression of Synechocystis strain MpIGisi and dependence on the starting and stopping of iron-depleted growth in batch cultures with CO₂-enriched aeration. +Fe, 19.2 μM Fe(NH₄) citrate; −Fe, 0 μM Fe(NH₄) citrate. (A) Specific growth rate; (B) Northern blot hybridization signals after application of an isiA-specific probe; (C) Northern blot hybridization signals after application of a 16S rRNA-specific probe; (D) quantitative estimation of isiA mRNA normalized to the 16S rRNA content (highest content corresponds to 100%). d, days.

FIG. 2

GFP fluorescence in cells of Synechocystis strain MpIGisi after 9 days of cultivation in media containing different concentrations of Fe(NH₄) citrate. The fluorescence level of precultivated cells [0.7 μM Fe(NH₄) citrate] at the beginning of the experiment corresponds to 100%. Error bars denote standard deviations of triplicate samples.

FIG. 3

Separation of PCR fragments obtained with DNAs from various organisms (Table 1) using degenerate isiA-specific primers. In all cases, the 840-bp fragment could be verified as an isiA gene fragment by sequencing. Lanes: 1, marker (λ DNA EcoRI/HindIII digested); 2, Synechococcus sp. strain SAG 14.02-1; 3, Synechocystis sp. strain PCC 6803; 4, Synechococcus sp. strain PCC 7942; 5, Synechococcus sp. strain PCC 7002; 6, Anabaena sp. strain PCC 7120; 7, Fischerella muscicola PCC 73103; 8, Anabaena torulosa SAG 26.79; 9, Lyngbya lagerheimii SAG 24.99; 10, Microcystis aeruginosa BM Mi/5; 11, Oscillatoriales sp. strain 99-2/6.2.2 (isolate from Baltic Sea shoreline, Zingst); 12 to 15, BB 1, BB 2, BB 3, and BB 7 (enrichment cultures of estuarine field samples); 16, Oscillatoriales sp. strain 99-3/5.3.1 (isolate from Lake Kummerow); 17, Chlorogloeopsis fritschii SAG 1411-1; 18, Anabaenopsis elenkinii SAG 252.80.

FIG. 4

Rooted cladogram (TreeView version 1.5; R. D. M. Page, 1998) after phylogenetic analysis of translated IsiA protein sequences (PAUP software package, neighbor-joining method). The isiA sequences (830-bp internal fragment) come from databases (marked by ) and from the present study of different cyanobacterial strains, clones from field samples, and enrichment cultures. Bootstrap values were calculated after 1,000 replications; only branchings with values above 50% are shown.

FIG. 5

Separation of internal isiA gene fragments obtained by RT-PCR using RNAs isolated from different cyanobacterial samples cultivated for 12 days under restricted iron availability (−Fe) in contrast to conditions for control cells (+Fe). Lanes BB, enrichment cultures of estuarine field samples (numbers signify the different cultures); lanes 6803, Synechocystis sp. strain PCC 6803; lanes 1, marker (λ DNA EcoRI and HindIII digested).