Light dependent synthesis of a nucleotide second messenger controls the motility of a spirochete bacterium

Nucleotide second messengers are universally crucial factors for the signal transduction of various organisms. In prokaryotes, cyclic nucleotide messengers are involved in the bacterial life cycle and in functions such as virulence and biofilm formation, mainly via gene regulation. Here, we show that the swimming motility of the soil bacterium Leptospira kobayashii is rapidly modulated by light stimulation. Analysis of a loss-of-photoresponsivity mutant obtained by transposon random mutagenesis identified the novel sensory gene, and its expression in Escherichia coli through codon optimization elucidated the light-dependent synthesis of cyclic adenosine monophosphate (cAMP). GFP labeling showed the localization of the photoresponsive enzyme at the cell poles where flagellar motors reside. These findings suggest a new role for cAMP in rapidly controlling the flagella-dependent motility of Leptospira and highlight the global distribution of the newly discovered photoactivated cyclase among diverse microbial species.


Results
Light modulates swimming of soil bacteria. The genus Leptospira possesses two endoflagella (one flagellum per cell end, Supplementary Fig. 1). Leptospira spp. swim by rotating the coiled cell body (swimming mode), and smooth swimming is frequently interrupted by rotation without migration (rotation mode) 13,14 . We found that L. kobayashii exclusively showed rotation in the low light condition (0.2 μmol/m 2 /s, "Light OFF" in Fig. 1), but smooth swimming was triggered by light exposure (26.1 μmol/m 2 /s, "Light ON" in Fig. 1, Movie 1, Supplementary Fig. 2). It is known that the transition between the swimming and rotation modes of Leptospira spp. is affected by viscosity 15 and chemical substrates 16,17 , but this is the first report of light-dependent modulation of Leptospira motility. The frequency of swimming reversal decreased with light intensity, increasing the migration distance per unit time ( Supplementary Fig. 3). Therefore, light-dependent motility was quantified using the swimming velocity of individual cells (i.e., migration distance per second), showing an increase in velocity up to fourfold under light (Fig. 1b, Supplementary Fig. 3). Smooth swimming reached the maximum velocity at ~ 1 s after stimulation (Fig. 1c). The light-responsivity depends on the light intensity, and the duration of unidirectional swimming increased with increasing light intensity ( Supplementary Fig. 3). The response to light was observed even at ~ 1 μmol/m 2 /s, which is less than 1/10 of the light in a conventional experimental room illuminated by 32 W fluorescent lamps (500-600 lx) ( Supplementary Fig. 3). The bacteria can respond to green and blue light but not red light (Fig. 1d). Cumulative cell fractions obtained from the velocity histograms show that green light induces more cells to swim smoothly than blue light (Fig. 1e).
Identification of a photoresponsive gene. To identify the sensor gene responsible for the L. kobayashii photoresponsivity, we explored loss-of-function mutants from a kanamycin-resistant library made by transposon random mutagenesis. One of the ~ 2400 clones retained motility but lacked photoresponsivity (Fig. 2a, Movie 2), and this mutant was named Prd (photoresponsivity-deficient). The Prd mutant carried a transposon insertion in the LPTSP3_g09850 gene (Fig. 2b), but the complementation of the LPTSP3_g09850 gene was not able to restore the photoresponsivity of the mutant (Supplementary Fig. 4). Noting that the LPTSP3_g09840 gene is located immediately downstream of the LPTSP3_g09850 gene and encodes adenylate/guanylate cyclase domain-containing protein, we complemented the Prd mutant with both the LPTSP3_g09850 and LPTSP3_ g09840 genes, resulting in the recovery of photoresponsivity (Fig. 2c,d). Then, the photoresponsivity of the Prd www.nature.com/scientificreports/ mutant was recovered to the wild-type level by complementation of the LPTSP3_g09840 gene (Fig. 2d), indicating that the LPTSP3_g09840 gene is responsible for the photoresponsivity of L. kobayashii. We termed the LPTSP3_g09840 gene lprA (leptospiral photoresponsive protein A).
Light-dependent adenylyl cyclase activity of LprA. Photoactivated adenylyl cyclases (PACs) have been found in both eukaryotes and prokaryotes [18][19][20][21][22][23][24] . The alignment of LprA with known functional PACs showed that the C-terminal region of LprA contains a domain similar to the AC domain of known PACs, whereas the N-terminal putative sensor domain of LprA was distinct from the conventional PAC sensor BLUF or LOV domain ( Supplementary Fig. 5). Since the light-dependent elevation of cAMP concentration was not detected in L. kobayashii cells ( Supplementary Fig. 6a), we measured the enzyme activity using the E. coli overexpression system carrying the codon-optimized lprA (Fig. 3a). The overexpressed LprA showed an increase in the cAMP concentration in E. coli under light exposure (Fig. 3b). Very little cGMP was detected independently of light,   Fig. 6b). These results suggest that LprA is a novel photoactivation-associated adenylyl cyclase. The Prd mutant cells kept rotating in the Light ON condition, but the addition of a membrane-permeable cAMP (8-bromo-cAMP) induced smooth swimming of the mutant as well as the wild-type cells exposed to light (Fig. 3c). An 8-bromo-cAMP-dependent increase in swimming velocity was also induced in the WT and Prd under the low light condition (0.2 μmol/m 2 /s; Supplementary Fig. 7). These results suggest that the observed modulation of flagellar rotation was induced by cAMP synthesized upon light stimulation.
Bipolar localization of LprA. We examined the localization of LprA in the cell body by labeling the protein with AcGFP1 (green fluorescent protein derived from Aequorea coerulescens). AcGFP1 labeling did not affect the photoresponsivity of the bacterium (Supplementary Fig. 8a). Epifluorescence microscopy showed the localization of LprA-AcGFP1 at both ends of the cell body (Fig. 4a). Based on the fluorescence intensity of single  www.nature.com/scientificreports/ AcGFP1 molecules, we estimated that 5.5 ± 3.8 molecules of LprA (n = 46 poles) are localized at one pole ( Fig. 4b and Supplementary Fig. 8b), while those in the membrane pool diffuse along the cell body (Movie 3). Although the increase in cAMP concentration in L. kobayashii was likely to be below the detection limit ( Supplementary  Fig. 6), the bipolar localization of LprA could condense cAMP near the flagellar motor, resulting in a rapid response to light.

Discussion
Organisms react to light through various sensory systems, leading to vision, signaling, and energetic activities. For example, rhodopsins, which are retinal-binding membrane proteins, play a crucial role in vision, ion pumping, and microbial taxis 25,26 , and cryptochromes, which are flavin-binding proteins, are involved in the growth of plants and the circadian clock of animals 27 . Investigating such light-driven proteins deepens our understanding of essential cellular activities. In addition, they have great potential for application as an optogenetic tool, enabling energetic or signaling modulation in live cells under arbitrary spatial and temporal conditions. We showed that L. kobayashii modulates its swimming pattern on a subsecond timescale after increasing illumination intensity and that the adenylyl cyclase discovered in this soil spirochete synthesizes cAMP upon light stimulation. The E. coli expression system experiment showed that light exposure is indispensable for activating LprA (Fig. 3b), suggesting that LprA functions as a photosensor by itself or is activated by interacting with another photosensor.
Since photosensory activity needs a chromophore, the former hypothesis implies that LprA may use a common chromophore with E. coli. Although cAMP signaling has been known to be involved in pilus/flagellar biogenesis and biofilm formation in bacteria, these responses are slow and occur through gene regulation 10,12,28 . The current results suggest that cAMP could mediate rapid motility control, although further studies are required to determine whether cAMP acts directly on the flagellar motor. The genus Leptospira comprises pathogenic and saprophytic species 8 . The species that belong to the saprophytic clade S2 containing L. kobayashii can respond to light, whereas one of the major pathogenic species, L. interrogans, swims even in low light and does not react to light despite carrying the homologous gene (Supplementary Fig. 9). Since the LprA-deficient mutant retains the ability to rotate in one position but cannot migrate by swimming (Movie 2), cAMP-dependent swimming could be important for exploring the environments of free-living species. In contrast, the pathogen motility independent of light is significant for migrating within the host body 29 . Interestingly, a search of the amino acid sequence database showed that many microbial species have a gene homologous to LprA in their genome (Supplementary Fig. 10). Although LprA is not involved in photoresponsive motility in all bacteria, cAMP synthesized by LprA may be used in some signal transductions. Adenylyl cyclases are categorized into six classes, e.g., class I has been found in gamma-proteobacteria, and class II is used by pathogenic bacteria, such as Bacillus anthracis and P. aeruginosa 30 . Since PACs that have functional similarity to LprA belong to a universal class III used by many species of eukaryotes (e.g., fungi and protozoa) and prokaryotes (e.g., eubacteria and archaea), the successful expression of functional LprA in other bacterial species is the crucial first step for optogenetic application. Understanding the molecular basis of the fast photosensory responses of LprA and cAMP signaling will be a future research topic. Screening of a photoresponsivity-deficient mutant by random transposon mutagenesis. Random insertion mutagenesis of L. kobayashii strain E30 using Himar1 transposon was conducted as described previously 31,32 . By conjugation with E. coli β2163 carrying pCjTKS2 32 , about 2400 transconjugant colonies were observed on plates of EMJH agar (1% agar) containing kanamycin at a final concentration of 25 μg/ ml. Each transconjugant was independently inoculated to 150 μl of liquid EMJH containing kanamycin and grown at 30 °C for 4 days. The grown bacteria were observed using a dark-field microscope (BX50, mercury lamp, 10 × objective, dry condenser; Olympus) for screening photoresponsivity-deficient mutants. The transposon insertion site was identified using the semi-random PCR technique 32 .

Methods
For the complementation of photoresponsive-deficient mutant, Prd, the LPTSP3_g09850 and/or lprA genes were expressed under the flgB promoter 33,34 . The flgB promoter region was amplified as previously described 29 , and the LPTSP3_g09850/lprA genes, lprA, LPTSP3_g09850 gene with FLAG tag, and lprA with FLAG tag were amplified from genomic DNA of the L. kobayashii E30, and the amplified products were cloned into the SalIdigested pCjSpLe94 35 by NEBuilder HiFi DNA Assembly cloning (New England BioLabs). For the LprA with GFP joined by a flexible (GGGGS) 3 linker, lprA was amplified as described above, and gfp was amplified from pAcGFP1 (Clontech) using the primer containing the (GGGGS) 3 linker sequence, and the amplified products were cloned into the SalI-digested pCjSpLe94 as described above. Primers used in this study are listed in Supplementary Table 1.
Immunoblotting experiments. About 1.5 × 10 8 leptospiral cells suspended in SDS-PAGE sample buffer were subjected to 5-20% SDS-PAGE and Western blotting. The blot was incubated with antisera raised against the peptide fragment of LprA (NH2-LSWADRTDSIYIWK-COOH) and FlaA2 31 or monoclonal antibody for FLAG tag. Full-size images of immunoblotting data without cropping are shown in Supplementary Fig. 11 The number of LprA-AcGFP1 at the cell pole was estimated by comparing the fluorescence spot intensity at the pole with the fluorescence intensity of a single His-AcGFP1 molecule as previously reported 36 . His-AcGFP1 was purified from E. coli C41(DE3) cells carrying pET19b/ His-AcGFP1 using Ni-NTA agarose (Fujifilm Wako). 10 pg/ml of His-AcGFP1 solution was applied to a coverslip washed by 0.1 M KOH and observed by fluorescence microscopy. In the fluorescent images, a rectangular mask for the fluorescent spot of 30 × 30 pixels was applied to the ROI (region of interest). We defined the spot intensity as the sum of all pixel values within the rectangular mask after subtracting the total background intensity from each pixel value. The number of LprA-AcGFP1 per pole was estimated as the intensity of the cell pole divided by the average intensity of a single His-AcGFP1 molecule.
cAMP ELISA assay. A cAMP ELISA kit (ADI-900-066, Enzo Life Sciences) was used to determine intracellular cAMP levels following the manufacturer's instructions. E. coli C41(DE3) cells carrying pET22b/lprA-His were grown in L-broth containing 100 μg/mL ampicillin with or without 1 mM IPTG at 30 °C for 5 h with shaking. The codon usages of the lprA sequence were optimized to those of E. coli for efficient protein expression. L. kobayashii cells were grown at 30 °C for 4 days in EMJH liquid medium until the late-exponential phase. Cells were photo-stimulated with white LED (1156 μmol/m 2 /s) for 3 min and subsequently lysed. Cell lysates were used to calculate cAMP values. The intracellular cAMP concentrations of E. coli and Leptospira cells were normalized to OD 600 and OD 420 , respectively.

Data availability
The data supporting the findings of this study are available from the corresponding author upon request.