Occurrence of cyclic di-GMP-modulating output domains in cyanobacteria: an illuminating perspective

Abstract

Microorganisms use a variety of metabolites to respond to external stimuli, including second messengers that amplify primary signals and elicit biochemical changes in a cell. Levels of the second messenger cyclic dimeric GMP (c-di-GMP) are regulated by a variety of environmental stimuli and play a critical role in regulating cellular processes such as biofilm formation and cellular motility. Cyclic di-GMP signaling systems have been largely characterized in pathogenic bacteria; however, proteins that can impact the synthesis or degradation of c-di-GMP are prominent in cyanobacterial species and yet remain largely underexplored. In cyanobacteria, many putative c-di-GMP synthesis or degradation domains are found in genes that also harbor light-responsive signal input domains, suggesting that light is an important signal for altering c-di-GMP homeostasis. Indeed, c-di-GMP-associated domains are often the second most common output domain in photoreceptors-outnumbered only by a histidine kinase output domain. Cyanobacteria differ from other bacteria regarding the number and types of photoreceptor domains associated with c-di-GMP domains. Due to the widespread distribution of c-di-GMP domains in cyanobacteria, we investigated the evolutionary origin of a subset of genes. Phylogenetic analyses showed that c-di-GMP signaling systems were present early in cyanobacteria and c-di-GMP genes were both vertically and horizontally inherited during their evolution. Finally, we compared intracellular levels of c-di-GMP in two cyanobacterial species under different light qualities, confirming that light is an important factor for regulating this second messenger in vivo.

Importance: This study shows that many proteins containing cyclic dimeric GMP (c-di-GMP)-regulatory domains in cyanobacteria are associated with photoreceptor domains. Although the functional roles of c-di-GMP domain-containing proteins in cyanobacteria are only beginning to emerge, the abundance of these multidomain proteins in cyanobacteria that occupy diverse habitats ranging from freshwater to marine to soil environments suggests an important role for the regulation of c-di-GMP in these organisms. Indeed, we showed that light distinctly regulates c-di-GMP levels in Fremyella diplosiphon and Synechocystis sp. strain PCC6803. Our findings are consistent with the occurrence of c-di-GMP domains based on evolutionary origin and as an adaptation to specific habitat characteristics. Phylogenetic analyses of these domains clearly separate two distinctive clades, one composed of domains belonging predominantly to cyanobacteria and the other belonging to a mix of cyanobacteria and other bacteria. We further demonstrate that in cyanobacteria the acquisition of c-di-GMP signaling domains occurred both vertically and horizontally.

FIG 1

Partner domain occurrence for c-di-GMP domains, in percentages. The most common domain associated with c-di-GMP domains was PAS (28%), followed by photoreceptors (26%) and CheY-like domains (23%). PAS, Per-Arnt-Sim domain; CheY-like, receiver domain; FHA, forkhead associated; CHASE, cyclases/histidine kinases, associated sensory extracellular; HAMP, histidine kinases, adenylate cyclases, methyl-accepting proteins, and phosphatases.

FIG 2

Correlation between genome size (A), total number of domains (B), and total number of photoreceptor domains (C) associated with c-di-GMP modulating domains in publically available sequenced cyanobacteria. Each dot represents every species present in Table 1, for a total of 37 species. Colored dots represent individual species: red, Trichodesmium erythraeum; green, Microcystis aeruginosa; orange, Synechocystis sp. strain PCC6803; and blue, Cyanothece sp. strain ATCC 51142.

FIG 3

Phylogenetic analysis based on multiple presumably functionally conserved EAL-only sequences (left). (Right) Phylogenetic analysis based on 16S genes of the species representing the EAL-only genes. Dotted lines link species to their genes that were presumably acquired vertically. Continuous lines link species to their genes that were presumably acquired horizontally. Blue, cyanobacterial species; red, bacterial species.

FIG 4

Alignment of EAL-only domains showing insertion and deletion regions. Gene names starting with a “C” denote cyanobacterial genes. Genes starting with a “B” denote bacterial genes. Blue backgrounds show deletion regions present in the genes cya_1130, cocor_05649, mxam_2424, and staur_3026 that are not present in other EAL domains. Gray backgrounds show insertion and deletion regions present in the genes pcc8801_0177 and pstab_3153 that are not present in other EAL domains. Green backgrounds show conserved regions between a28ld_0392 and slr6110 genes. Amino acid residues critical for activity are indicated with black backgrounds (70).

FIG 5

Effect of light quality on intracellular concentration of c-di-GMP normalized to total proteins. Cyclic di-GMP concentration (in nmol c-di-GMP μg protein⁻¹) in F. diplosiphon (A) and Synechocystis (B). The colors of the bars indicate color of light under which cells were grown. WL, white light; RL, red light; GL, green light; BL, blue light. Means with different letters are significantly different (P < 0.05). Vertical bars represent standard errors.