Isolation of Regulated Genes of the Cyanobacterium Synechocystis sp. Strain PCC 6803 by Differential Display

Abstract

Global identification of differentially regulated genes in prokaryotes is constrained because the mRNA does not have a 3 polyadenylation extension; this precludes specific separation of mRNA from rRNA and tRNA and synthesis of cDNAs from the entire mRNA population. Knowledge of the entire genome sequence of Synechocystis sp. strain PCC 6803 has enabled us to develop a differential display procedure that takes advantage of a short palindromic sequence that is dispersed throughout the Synechocystis sp. strain PCC 6803 genome. This sequence, designated the HIP (highly iterated palindrome) element, occurs in approximately half of the Synechocystis sp. strain PCC 6803 genes but is absent in rRNA and tRNA genes. To determine the feasibility of exploiting the HIP element, alone or in combination with specific primer subsets, for analyzing differential gene expression, we used HIP-based primers to identify light intensity-regulated genes. Several gene fragments, including those encoding ribosomal proteins and phycobiliprotein subunits, were differentially amplified from RNA templates derived from cells grown in low light or exposed to high light for 3 h. One novel finding was that expression of certain genes of the pho regulon, which are under the control of environmental phosphate levels, were markedly elevated in high light. High-light activation of pho regulon genes correlated with elevated growth rates that occur when the cells are transferred from low to high light. These results suggest that in high light, the rate of growth of Synechocystis sp. strain PCC 6803 exceeds its capacity to assimilate phosphate, which, in turn, may trigger a phosphate starvation response and activation of the pho regulon.

Consequences of a Deletion in dspA on Transcript Accumulation in Synechocystis sp. Strain PCC6803

“…Surprisingly, most of the phosphate starvation-induced genes (pho), extensively studied in E. coli (78), were in group 2; this group includes pstA (slr1249), pstB (sll0684, sll0683), and pstS (slr1247). Some transcripts for pho regulon polypeptides exhibited a transient increase in HL, as is clearly observed for pstC and pstS (Tables 2 and 3 previously (4). Interestingly, even in LL, the dspA strain showed very high levels (relative to wild-type cells) of pstS (both sll0680 and slr1247), pstC (sll0681 and slr1248, integral membrane protein), and pstB (sll0683 and slr1250, nucleotide binding protein) transcripts, as well as of transcripts encoding an alkaline phosphatase (sll0654) and the extracellular NucH nuclease (sll0656) ( Table 3).…”

“…Transcripts from other genes such as those belonging to the pho regulon, and especially those involved in phosphate transport, were shown to increase following the exposure of cells to HL. This was first observed in studies in which differential display was used to identify genes responsive to HL conditions (4). Differences in values and precise sets of genes modulated by HL conditions are likely to reflect differences in conditions (and strains) used for the experiments.…”

Effects of high light on transcripts of stress-associated genes for the cyanobacteria Synechocystis sp. PCC 6803 and Prochlorococcus MED4 and MIT9313

“…RNA was isolated from pelleted cells that had been frozen at 280 uC, using a modification of the method of de Saizieu et al (1998), as described by Bhaya et al (2000). Briefly, 500 ml acidified phenol and 500 ml NAES (50 mM sodium acetate pH 5?1, 10 mM EDTA, 1 % SDS) were added to pelleted cells from 150 ml cultures; 100 mg glass beads (0?1 mm mean diameter, Bio-Rad) was placed in the suspension, which was then vortexed three times for 20 s each.…”

Ecological Genomics of Marine Picocyanobacteria

“…Such multiplicity may reflect functionally distinct copies, e.g., with differences in P affinity which allow transport over an extended range of P concentration, or variability in their capacity to act as part of the P sensor, or regulation by different environmental variables. For instance, light is known to induce one of the PstS copies in the freshwater cyanobacterium Synechocystis (17). Variability of copy number between strains is doubtless also a function of the local P environment occupied by individual strains.…”