Natural diversity provides a broad spectrum of cyanobacteriochrome-based diguanylate cyclases

Abstract

Cyanobacteriochromes (CBCRs) are spectrally diverse photosensors from cyanobacteria distantly related to phytochromes that exploit photoisomerization of linear tetrapyrrole (bilin) chromophores to regulate associated signaling output domains. Unlike phytochromes, a single CBCR domain is sufficient for photoperception. CBCR domains that regulate the production or degradation of cyclic nucleotide second messengers are becoming increasingly well characterized. Cyclic di-guanosine monophosphate (c-di-GMP) is a widespread small-molecule regulator of bacterial motility, developmental transitions, and biofilm formation whose biosynthesis is regulated by CBCRs coupled to GGDEF (diguanylate cyclase) output domains. In this study, we compare the properties of diverse CBCR-GGDEF proteins with those of synthetic CBCR-GGDEF chimeras. Our investigation shows that natural diversity generates promising candidates for robust, broad spectrum optogenetic applications in live cells. Since light quality is constantly changing during plant development as upper leaves begin to shade lower leaves-affecting elongation growth, initiation of flowering, and responses to pathogens, these studies presage application of CBCR-GGDEF sensors to regulate orthogonal, c-di-GMP-regulated circuits in agronomically important plants for robust mitigation of such deleterious responses under natural growing conditions in the field.

Figure 1

Fusion of CBCR “GAF” domains with DGC “GGDEF” domain of Tlr0924 (SesA). A, Domain architecture of Tlr0924Δ. B, Alignment of fusion protein constructs, color-coded by coding sequence (Tlr0924, pale green; RcaE, pink; NpR6012g4, cyan; dark orange box, short repeated motif). The alignment starts with the last β strand of the GAF domain (LWGLLIAH in Tlr0924Δ), with the GAF domain ending in the vicinity of VALQQ in the same sequence and with the GGDEF domain in the vicinity of NLQ in the same sequence. Numbering is based on full-length Tlr0924 (GenBank accession BAC08476). C, DGC activity of fusion protein constructs (indicated as in panel B) in 15Z (blue) and 15E (orange) photostates. Error bars are drawn at one standard deviation (n = 3). No construct exhibited significant light-regulated DGC activity (P < 0.05).

Figure 2

Tanglegram view of CBCR (left) and GGDEF (right) maximum-likelihood phylogenies. Bold lettering indicates proteins further described in paper. Color of CBCR lettering matches subfamily seen at top of tanglegram. Connecting lines are colored to facilitate comparison of the two trees; instances of co-evolution should thus yield coherent groups of connecting lines. The cluster of proteins which includes NpR1060 was chosen as an arbitrary outgroup and is placed at the bottom of both phylogenies. Scale bars for each tree indicate 0.2 substitutions per position.

Figure 3

Jellybean-style domain diagram displaying selected CBCR-regulated DGC proteins and CBCR-GGDEF truncations. Domain abbreviations: CBS pairs (cystathionine beta synthase domain repeats); EAL (c-di-GMP-dependent PDE); GAF (cGMP-specific PDE/adenylyl cyclase/FhlA); GGDEF (diguanylate cyclase); PAS (Per-Arnt-Sim); PMT2 (protein-O-mannosyltransferase-2). CBCR photocycles indicated by color.

Figure 4

Spectral analysis of selected CBCR-GGDEF proteins. Absorbance spectra of (A) Os6304_3021Δ; (B) S.hof_0333; (C) NpR1060Δ; (D) Lyn.aest_0230Δ; (E) Lyn8106_0097Δ; (F) Cyan7822_5462Δ; (G) Nod_0172Δ; (H) Nod_0172; and (I) Nod_0172 (GAF1) and Nod_0172Δ. For panels A–G, blue traces represent 15Z states and orange traces represent 15E states. For panel H, the teal trace represents the sample irradiated with saturating teal light (500 ± 10 nm). Subsequent irradiation with violet light (400 ± 35 nm; violet trace) or red light (650 ± 20 nm; black red trace) is shown. Panel I depicts the photochemical difference spectra of GAF1 (black red trace), GAF2 (coral trace; obtained by subtraction of the violet trace from the red trace in panel H), and that of Nod_0172Δ (black trace).

Figure 5

DGC activities of selected CBCR-GGDEF proteins. A, NpR1060Δ, Os6304_3021Δ, and S.hof_0333; B, Lyn.aest_0230Δ, Lyn8106_0097Δ, and Cyan7822_5462Δ; C, Nod_0172 and Nod_0172Δ. Total protein concentration of each protein is 5 µM. For panels A and B, the color of light used for sample irradiation is indicated above each bar column, and those columns are colored blue or orange for samples maintained in 15Z or 15E states, respectively. For panel C, the color of light used for sample irradiation is indicated above/within each bar column. Nod_0172 samples labeled G/B and G/R were first irradiated with saturating green light and then maintained under blue or red, respectively. Nod_0172 bar columns are divided by a line and are colored blue or orange to represent the photostate of GAF1 (upper left) and GAF2 (lower right). Error bars were calculated as standard deviation of all replicates (n = 3); Statistically significant differences between photostates are indicated with asterisks (P ≤ 0.025; Student’s t test). G hν, green light; B hν, blue light; R hν, red light.

Figure 6

Selected CBCR-GGDEF proteins exhibit DGC activity in vivo. Cultures were grown in a shaking incubator under filtered light (see the “Materials and methods” section for filters), and expression of selected CBCR-GGDEF proteins was induced using 0.2% arabinose + 1 mM IPTG (Os6304_3021Δ and ΔNpR1060: 0.05% arabinose + 1 mM IPTG). Settling measured as percent OD loss of cultures in darkness after growth. Color of bars indicates the photostate (blue, 15Z; orange, 15E), with Nod_0172FL colored as in Figure 5. Error bars are drawn at one standard deviation (n = 3). Each CBCR-GGDEF construct was assessed for light-controlled cell settling using Student’s t test for unpaired data, with no assumption of equal variances. Cases exhibiting significant light effects (P < 0.025) are indicated with asterisks.