Comparative genome analysis of the closely related Synechocystis strains PCC 6714 and PCC 6803

Abstract

Synechocystis sp. PCC 6803 is the most popular cyanobacterial model for prokaryotic photosynthesis and for metabolic engineering to produce biofuels. Genomic and transcriptomic comparisons between closely related bacteria are powerful approaches to infer insights into their metabolic potentials and regulatory networks. To enable a comparative approach, we generated the draft genome sequence of Synechocystis sp. PCC 6714, a closely related strain of 6803 (16S rDNA identity 99.4%) that also is amenable to genetic manipulation. Both strains share 2838 protein-coding genes, leaving 845 unique genes in Synechocystis sp. PCC 6803 and 895 genes in Synechocystis sp. PCC 6714. The genetic differences include a prophage in the genome of strain 6714, a different composition of the pool of transposable elements, and a ∼ 40 kb genomic island encoding several glycosyltransferases and transport proteins. We verified several physiological differences that were predicted on the basis of the respective genome sequence. Strain 6714 exhibited a lower tolerance to Zn(2+) ions, associated with the lack of a corresponding export system and a lowered potential of salt acclimation due to the absence of a transport system for the re-uptake of the compatible solute glucosylglycerol. These new data will support the detailed comparative analyses of this important cyanobacterial group than has been possible thus far. Genome information for Synechocystis sp. PCC 6714 has been deposited in Genbank (accession no AMZV01000000).

Keywords: comparative genomics; cyanophages; genome sequence; prophage; salt acclimation.

Figure 1.

Genome coverage based on circular genome plots of the Synechocystis 6803 chromosome and its four large plasmids pSYSA, pSYSG, pSYSM ,and pSYSX. Tracks from the outside show (1) regions with BLASTN hit in Synechocystis 6714 and identity between 50% (grey) and 100% (red); (2) CDS features from forward and reverse strand in Synechocystis 6803; (3) GC content.

Figure 2.

A likely genomic island in two Synechocystis strains. A genomic segment of ∼40 kb from Synechocystis 6803 is shown with some genes annotated for orientation (EPS, exopolysaccharide export protein; CmcI, Cephalosporin hydroxylase protein; GT1, GT1 family of glycosyltransferases; GTA-GTB, fusion protein joining a glycosyltransferase family A with a glycosyltransferase family B domain; Norf2 is a 68 amino acid peptide-encoding gene originally predicted on basis of transcriptome data indicating the presence of an mRNA for this conserved reading frame). Adjacent genes to this region are in the two strains of the gene pairs slr0976/slr0977 and sly1015510/sly1015500 encoding a DUF820 protein and an ABC transporter permease component; left side in 6803) and slr1084/slr1085 and sly1015040/sly1015030 (encoding a WcaF-type acyl transferase and a glycosyltransferase; right side in 6803). The GC % content, indicated by the green bars (each representing 1000 nt), drops considerably within this region. Thus, this region has features of a genomic island. The nucleotide identity to matching segments in the Synechocystis 6714 genome is colour coded (red >90%, light red >70%). The corresponding stretch in the Synechocystis 6714 genome encompasses genes sly1015490– sly1015020, almost entirely belonging to the list of unique genes in that strain (Supplementary Table S6). The proteins encoded by these genes are annotated as hypothetical proteins, UDP-glucose 4-epimerase, several different glycosyltransferases, rhamnogalacturonides degradation protein RhiN, dTDP-glucose 4′6′-dehydratase, methylase/methyltransferase, ABC transporter, GDP-mannose 4′6′dehydratase and as NAD-dependent epimerase/dehydratase.

Figure 3.

Prophage Psy1 inserted into the trnF^GAA gene (labelled by the green stars) of Synechocystis 6714. Genome position is drawn along the x-axis. Protein coding genes are shown in red if conserved in Synechocystis 6803 and in blue if not; trnF^GAA is shown in green. Transcriptome read counts per 100 million for the forward strand are plotted above and for the reverse strand below the CDS features. The GC % content is indicated by the green bars (each representing 1000 nt). The following genes were annotated as coding for bacteriophage-related proteins and are mentioned in the text: sly1027640, HK97 family phage portal protein; sly1027490, bacteriophage lysozyme-like protein; sly1027670 and sly1027680, remotely similar to bacteriophage Cro repressor; sly1027700, D5 N terminal like domain-containing protein of phage D5 proteins and bacteriophage P4 DNA primases; sly1027750, phage integrase.

Figure 4.

Verification of physiological and genetic differences predicted upon draft genome analysis of Synechocystis 6714. (A) Phototactic motility of Synechocystis. Cells from liquid cultures (OD₇₅₀ = 0.2) were dropped onto a BG11-agar plate, pre-cultivated under standard conditions for 3 days and afterwards exposed to a gradient of incident light with intensity 50 µE. The photograph was taken before and after further 5 days. (B) Drop dilution assay showing the growth on solid media in the presence of increasing concentrations of Zn²⁺ ions. The photograph was taken after 10 days of standard cultivation.

Figure 5.

Comparative genome analysis reveals the absence of the genes encoding the glucosylglycerol transport system (Ggt). (A) Genomic region encompassing the ggtA gene in Synechocystis 6803 and of the corresponding region in Synechocystis 6714. (B) Genomic region encompassing the ggtBCD operon. Apart from Ggt, both loci are well conserved (protein identity scores of ca. 90%). (C) Occurrence of mRNAs for ggtA, ggtBCD, and ggpS in salt-treated cells of both strains. For ggpS, salt-dependent expression was observed in both strains, whereas mRNAs for ggtA and ggtBCD were not detected in strain 6714.

Figure 6.

Effects of the absence or presence of the ggtABCD system. (A) Measurement of intracellular and extracellular GG content in the two Synechocystis strains. (B) Long-term growth of Synechocystis 6803 and 6714 in liquid cultures under salt stress. Cells were pre-cultivated without salt for 2 days before salt was added to a final concentration of 2, 4, and 6% (w/v), respectively (time point is marked by a red arrow). After further 4 days, samples for RNA extraction and GG measurements were taken (marked by a black arrow). The data are representatives of two independent experiments. (C) Drop dilution assay illustrating the growth on solid media in the presence of increasing NaCl concentrations. Cell material of exponentially growing cultures was diluted to an OD₇₅₀ of 0.3 and 20 µl of this suspension as well as a dilution series were dropped on NaCl-containing, agar-solidified BG11 medium. The photograph was taken after 10 days under constant illumination of 50–60 µE.