Light regulation of type IV pilus-dependent motility by chemosensor-like elements in Synechocystis PCC6803

Abstract

To optimize photosynthesis, cyanobacteria move toward or away from a light source by a process known as phototaxis. Phototactic movement of the cyanobacterium Synechocystis PCC6803 is a surface-dependent phenomenon that requires type IV pili, cellular appendages implicated in twitching and social motility in a range of bacteria. To elucidate regulation of cyanobacterial motility, we generated transposon-tagged mutants with aberrant phototaxis; mutants were either nonmotile or exhibited an "inverted motility response" (negative phototaxis) relative to wild-type cells. Several mutants contained transposons in genes similar to those involved in bacterial chemotaxis. Synechocystis PCC6803 has three loci with chemotaxis-like genes, of which two, Tax1 and Tax3, are involved in phototaxis. Transposons interrupting the Tax1 locus yielded mutants that exhibited an inverted motility response, suggesting that this locus is involved in controlling positive phototaxis. However, a strain null for taxAY1 was nonmotile and hyperpiliated. Interestingly, whereas the C-terminal region of the TaxD1 polypeptide is similar to the signaling domain of enteric methyl-accepting chemoreceptor proteins, the N terminus has two domains resembling chromophore-binding domains of phytochrome, a photoreceptor in plants. Hence, TaxD1 may play a role in perceiving the light stimulus. Mutants in the Tax3 locus are nonmotile and do not make type IV pili. These findings establish links between chemotaxis-like regulatory elements and type IV pilus-mediated phototaxis.

Figure 1

(A) Physical map of Tax loci showing three Tax loci (Tax1, Tax 2, and Tax3) and taxAY3. Arrowheads indicate the position of transposons that map to specific tax genes. (Bar, 200 amino acids or 600 nucleotides.) (B) Directional motility assays. Cells were spotted (shown by dotted circle) onto 0.4% agar plates and placed in a directional light source (arrowhead) for 3 days. 1, wild-type; 2, taxAY1-5′; 3, taxAY1-3′; 4, taxD1; 5, taxAY3; and 6, the taxD3 transposon mutant.

Figure 2

(A) Diagram of TaxAY histidine kinases showing the different domains (P1–P5) and the CheY-like domains (dotted boxes). CheA from E. coli is shown in line 1; numbers in parentheses represent protein size in amino acids. Black bar in P1 represents the H box. (B) Diagram of TaxD chemosensor-like polypeptides showing putative transmembrane helices (black bars), phytochrome-like domains in TaxD1 (gray boxes), and signaling domains (white boxes). The HAMP domain of TaxD2 is shown as a dark gray box. Numbers in parentheses represent protein size in amino acids.

Figure 3

Comparison of phytochrome-like domains of TaxD1 (phy1 and phy2) with chromophore-binding domain of phytochrome E of Arabidopsis thaliana (PhyE, 181–358) and RcaE (89) of Fremyella diplosiphon. phy1 (210) and phy2 (376) are shown on lines 1 and 2, respectively. Arrows indicate conserved cysteine and histidine residues. Black boxes indicate identical or conserved residues in all four sequences, whereas dark gray and light gray boxes represent identical or conserved residues in two or three sequences, respectively.

Figure 4

Transmission electron micrographs of wild-type (A), taxAY1-3′ (B), taxAY1-5′ (C), and taxAY3 (D) null mutants. Cells were stained with 1% uranyl acetate, and cell edge with pili is shown at a final magnification of 112,500.

Figure 5

Northern blot hybridizations. (Upper) RNA from wild-type (lane 1), taxAY1-3′ (lane 2), taxAY1-5′ (lane 3), taxD1 (lane 4), taxAY3 (lane 5), and taxD3 (lane 6) mutants was probed with pilA1 gene-specific probe. Ten micrograms total RNA were loaded in each lane. (Lower) 16S RNA shown as loading control.

Figure 6

Regulation of Tfp-mediated motility through chemosensor-like elements. Gray boxes represent mutants that show the inverted response, circles represent nonmotile mutants that have Tfp, and white boxes represent nonmotile mutants lacking Tfp. The black box with a question mark represents a putative photoreceptor that has not yet been identified (see text for details). The circles and boxes with dotted outlines represent elements whose functions have not been tested.