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.

FIG. 1

Positions of HIP elements in the Synechocystis sp. strain PCC 6803 genome. The position of each HIP element is marked with a cross (+). The sequence begins at the lower left (nucleotide 1 of the sequence) and ends at the top right (nucleotide 3573470). Positions of the rRNA genes which lack HIP elements are shown by thick lines.

FIG. 2

PCR from genomic DNA using different HIP primers. HIP primers were used for PCR with 5 ng of genomic DNA template. Primers used were HIPA (lane 2), HIPC (lane 3), W2-HIPA (lane 4), W2-HIPC (lane 5), W2-HIPAC (lane 6), and HIPACC (lane 7). Molecular weight markers, in kilobase pairs, are shown in lane 1. PCR conditions are described in Materials and Methods.

FIG. 3

Differential display of W2-HIPG-primed RNA from cells grown in LL and exposed to HL. RT-PCR was performed as described in Materials and Methods with 100 ng of RNA from cells either grown in LL (lane 4) or exposed to HL for 3 h (lane 5). In control reactions (in which the RT step was omitted [-RT]), RNA from LL-grown (lane 2) and HL-exposed (3 h; lane 3) cells was used. Filled arrows mark products that differentially accumulate; the open arrow marks a product that does not differentially accumulate. Molecular weight markers, in kilobase pairs, are shown in lane 1.

FIG. 4

Map and expression of the cpcBACHD operon. (A) The map of the cpcBACHD operon shows the relative positions and sizes of the five cpc genes. The positions of the W2-HIPG-like elements are shown as rectangles below the map. The thin line below the map depicts the position and size of the differentially displayed product, while the thick line shows the size and position of the cpcA gene-specific probe. Thin arrows indicate positions of primers used to generate the cpcA-specific probe. (B) Northern blot hybridization using the cpcA-specific probe. Each lane contained 5 μg of RNA. Lanes 1 and 2 show signals from RNA isolated from LL- and HL-grown cells, respectively, hybridized to the cpcA probe. The major transcripts in lane 1 are 3.5, 2.6, 2.0, and 1.4 kb. RNA size markers are given on the left. (C) RT-PCR was performed with RNA from LL- and HL-grown cells using cpcA-specific primers; 100 ng of RNA was used for the reaction, as described in Materials and Methods. Lanes 2 and 3 show the no-RT control; Lanes 4 and 5 show the RT-PCR product (397 bp) generated from LL- and HL-grown cells, respectively.

FIG. 5

Map and expression of the rpl11-rpl1 operon. (A) The map of the rpl11-rpl1 operon shows the relative positions and sizes of nusG, rpl11, and rpl1. The positions of the W2-HIPG elements are shown as black rectangles below the map. The thin line below the map represents the size and position of the differentially displayed product. (B) Probes specific for rpl11 and rpl1 were made as described in Materials and Methods and used for RPA. In the RPA for rpl11 (top), the undigested probe migrates at 162 bp and is indicated with a filled arrow. A strongly protected fragment of 102 bp, indicated by an open arrow, is seen in lane 2 (HL), with a much fainter protected fragment in lane 1 (LL). Similar results are observed in the lower panel, in which a rpl1-specific probe was used (sizes of the unprotected and protected fragments are 304 and 223 bp, respectively). Lanes 3 in both panels show the control (C) (total RNA replaced with yeast RNA), in which no protected fragment is seen.

FIG. 6

Use of HIP primer combinations for differential display. RT-PCR conditions as described in Materials and Methods were used. W2-HIPAC was used as the RT primer in lanes 2 to 5, while W2-HIPGC was used as the RT primer in lanes 6 to 9. This was followed by PCR using either W2-HIPAC (lanes 2, 3, 6, and 7) or W2-HIPGC (lanes 4, 5, 8, and 9). Alternate rows are from LL (lanes 2, 4, 6, and 8) or HL (lanes 3, 5, 7, and 9). Molecular weight markers are shown in kilobase pairs in lane 1. Several putative differentially displayed products are visible in all lanes.

FIG. 7

Differential expression of genes in the pho regulon with respect to light intensity. (A) Differential display shows in the induction of the phoA gene (encoding alkaline phosphatase) in HL. LL- or 18-h HL-grown cells were used for RNA isolation. RT-PCR was performed using HIPAAA and conditions described in Materials and Methods. Lanes 1 and 2 show products from LL- HL-grown cells, respectively. A band at 1 kbp is visible only in lane 2 (marked with an asterisk), while bands at 800 and 750 bp appear in both lanes (including in the no-RT control lanes [data not shown]). (B) Gene-specific probes were used for hybridizations to phoA (top panel) and pstS (middle panel) transcripts. RNA was isolated from LL-grown cells (lane 1) or cells transferred to HL for 1 h (lane 2), 3 h (lane 3), or 8 h (lane 4). Blots were also probed with rDNA-specific probes as a loading control (lower panel).