Identification of iron-responsive, differential gene expression in the cyanobacterium Synechocystis sp. strain PCC 6803 with a customized amplification library

Abstract

We describe the use of a method called differential expression using customized amplification library (DECAL) to study the global changes in gene expression in iron-deficient versus iron-reconstituting cells of Synechocystis sp. strain PCC 6803. We identified a number of genes, such as isiA, idiA, psbA, cpcG, and slr0374, whose expression either increased or decreased in response to iron availability. Further analysis led to the identification of additional genes related to those identified by DECAL (e.g., psbC, psbO, psaA, apcABC, cpcBAC1C2D, and nblA) that were differentially regulated by iron availability. Expression of cpcG, psbC, psbO, psaA, apcABC, and cpcBAC1C2D increased, whereas that of isiA, idiA, nblA, psbA, and slr0374 decreased, in iron-reconstituting cells. S1 nuclease protection studies showed that increased transcript levels of psbA in iron-deficient cells was due to the increased expression of both psbA2 and psbA3 genes, although the steady-state level of psbA2 remained higher than that of psbA3.

FIG. 1

Flow chart showing various steps involved in construction of a DECAL for Synechocystis sp. strain PCC 6803. (A) Construction of CAL. A cosmid library carrying 35- to 45-kb genomic fragments of Synechocystis sp. strain PCC 6803 was constructed in pYUB328 and screened for clones containing ribosomal DNA sequences. Nonribosomal cosmids were pooled, restricted with suitable enzymes, and gel purified to generate smaller and similar-sized fragments. These fragments were ligated with adapters and PCR amplified to generate CAL sequences. (B) Identification of differentially regulated genes. Total RNA was isolated from Synechocystis sp. strain PCC 6803 cells that were treated under different conditions, reverse transcribed using biotin-labeled random hexamers, and hybridized to CAL sequences. CAL sequences representing cDNA in total RNA was eluted and amplified to generate PCR probes. These probes were radiolabeled and hybridized to replicate colony arrays of the cosmid library. Colonies showing differences in signal in the two arrays were selected, and differential gene expression was confirmed by Northern blot analysis.

FIG. 2

Absorption spectra of the iron-deficient Synechocystis sp. strain PCC 6803 cultures (····), and those collected at 3 (––––), 12 (—·—·—·—), and 24 (————) h after the addition of iron.

FIG. 3

Cosmid arrays hybridized with radioactively labeled CAL sequences selected after hybridization with single-stranded cDNA prepared from iron-deficient cells (A) and 24 h after the addition of iron (B). The boxed spots represent cosmids that demonstrated major (D21, F11, F12, O14, P13, and P14) or minor (A1) density differences between the two conditions and that were analyzed further.

FIG. 4

Northern blot confirmation of genes that were differentially expressed in iron-deficient versus iron-sufficient conditions. Total RNA was isolated from iron-starved cells (0 h) and from cells harvested at 3, 12, and 24 h after the addition of iron. Total RNA (10 μg/lane) was separated on a 1% denaturing agarose gel, capillary transferred, and hybridized with corresponding radiolabeled probes (see Materials and Methods). (A) isiA; (B) idiA; (C) psbA; (D) cpcG; (E) slr0374.

FIG. 5

Steady-state transcript levels of psbO (A), psaA (B), and psbC (C) genes in iron-starved and iron-reconstituted cells. Experimental conditions are as described in the legend to Fig. 3; sources of gene probes are detailed in Materials and Methods.

FIG. 6

S1 nuclease protection assays of psbA2 (A) and psbA3 (B) genes in iron-starved and iron-reconstituted Synechocystis sp. strain PCC 6803 cells. Primers specific to psbA2 and psbA3 were hybridized (at 51°C for 16 h) to 20 μg of total RNA from iron-starved cells or cells collected at 3, 12, and 24 h after the addition of iron. Nonhybridized primers were digested with S1 nuclease, and remaining sample was fractionated on an 8% polyacrylamide gel in the presence of 8 M urea, vacuum dried, and exposed to X-ray films. Lengths of the gene-specific protected fragments, 78 nucleotides (nt) for psbA3 and 50 nt for psbA2, were obtained using the primer homologous to psbA3. In the case of the primer specific to psbA2, gene-specific protected fragments of 72 nt for psbA2 and 58 nt for psbA3 were obtained.

FIG. 7

Northern blot analysis of total RNA from Synechocystis sp. strain PCC 6803 cells probed with DNA from the phycocyanin operon cpcBAC1C2D (A), the allophycocyanin operon apcABC (B), and the nblA gene (C). Experimental conditions are same as for Fig. 3; sources of gene probes are detailed in Materials and Methods.