Genetic analysis reveals the identity of the photoreceptor for phototaxis in hormogonium filaments of Nostoc punctiforme

Abstract

In cyanobacterial Nostoc species, substratum-dependent gliding motility is confined to specialized nongrowing filaments called hormogonia, which differentiate from vegetative filaments as part of a conditional life cycle and function as dispersal units. Here we confirm that Nostoc punctiforme hormogonia are positively phototactic to white light over a wide range of intensities. N. punctiforme contains two gene clusters (clusters 2 and 2i), each of which encodes modular cyanobacteriochrome-methyl-accepting chemotaxis proteins (MCPs) and other proteins that putatively constitute a basic chemotaxis-like signal transduction complex. Transcriptional analysis established that all genes in clusters 2 and 2i, plus two additional clusters (clusters 1 and 3) with genes encoding MCPs lacking cyanobacteriochrome sensory domains, are upregulated during the differentiation of hormogonia. Mutational analysis determined that only genes in cluster 2i are essential for positive phototaxis in N. punctiforme hormogonia; here these genes are designated ptx (for phototaxis) genes. The cluster is unusual in containing complete or partial duplicates of genes encoding proteins homologous to the well-described chemotaxis elements CheY, CheW, MCP, and CheA. The cyanobacteriochrome-MCP gene (ptxD) lacks transmembrane domains and has 7 potential binding sites for bilins. The transcriptional start site of the ptx genes does not resemble a sigma 70 consensus recognition sequence; moreover, it is upstream of two genes encoding gas vesicle proteins (gvpA and gvpC), which also are expressed only in the hormogonium filaments of N. punctiforme.

FIG 1

(A) Images of hormogonium filaments of wild-type N. punctiforme (strain UCD 154) migrating on representative phototaxis plates after 24 h of directed-light incubation using white-light LEDs. The light was applied from the direction of the bottom of the photograph. The intensity of the light used is given below each panel. (B) Quantification (see supplemental information) of each of the images in panel A. Cells moving downward in the images are scored as moving toward light, and cells moving upward in the images are scored as moving away from light. Integrated density values were derived from the ImageJ analysis software. (C) Images of phototaxis plates incubated under tungsten lamps, except for the first picture on the left, of a plate incubated with a white LED light, which is included here as a control for purposes of comparison. The intensity of the light applied is given below each image. The direction of light was from the bottom of the photograph.

FIG 2

The 5 chemotaxis-like loci. Numerical designations for each locus (to the left of each plot) are based on the system of Wuichet and Zhulin (21). (Left) Graphs of microarray data. The x axis values are hours after HIF induction, and the y axis values are expression levels (log base 2) of genes at each time point relative to expression at time zero. (Right) Open reading frame maps. The relative sizes of genes in each cluster are drawn to scale, but not all maps reflect the same size relative to each other. The approximate size of each locus is given on the right. The following abbreviations are used for the domains: HAMP, histidine kinase, adenylate cyclase, methyl-accepting protein, phosphatase; Sig, signaling domain; Hpt, histidine-containing phosphotransfer domain; Hase, histidine kinase; W, CheW binding domain; RR, response regulator receiver domain; G, GAF (cGMP-specific phosphodiesterase, adenylyl cyclase, FhlA); B, CheB methylesterase domain.

FIG 3

(Top) Representative phototaxis images of the wild-type strain (wt) and mutant strains with alterations in each of the 5 chemotaxis-like loci, incubated with white-light LEDs at 0.130 W/m² for 24 h. (Bottom) Quantification of each of the images.

FIG 4

(Top) Locus 2i is drawn to scale, showing the approximate locations of the transcriptional start site (TSS) and the 5′ processed end of the mRNAs. (Bottom) (Left and right) Gels show the results of 5′ RACE. TAP, tobacco acid pyrophosphatase. (Center) Sequence of the putative promoter region.

FIG 5

(Top) Locus 2i is drawn to scale, with upside-down triangles indicating the approximate locations of insertion mutations and right-side-up triangles indicating those of in-frame deletion mutations. (Bottom) Representative images of mutant strains after 24 h of incubation using white-light LEDs at 0.130 W/m². An image of the wild-type strain is shown for comparison.