A photosensory two-component system regulates bacterial cell attachment

Abstract

Flavin-binding LOV domains are blue-light photosensory modules that are conserved in a number of developmental and circadian regulatory proteins in plants, algae, and fungi. LOV domains are also present in bacterial genomes, and are commonly located at the amino termini of sensor histidine kinases. Genes predicted to encode LOV-histidine kinases are conserved across a broad range of bacterial taxa, from aquatic oligotrophs to plant and mammalian pathogens. However, the function of these putative prokaryotic photoreceptors remains largely undefined. The differentiating bacterium, Caulobacter crescentus, contains an operon encoding a two-component signaling system consisting of a LOV-histidine kinase, LovK, and a single-domain response regulator, LovR. LovK binds a flavin cofactor, undergoes a reversible photocycle, and displays increased ATPase and autophosphorylation activity in response to visible light. Deletion of the response regulator gene, lovR, results in severe attenuation of cell attachment to a glass surface under laminar flow, whereas coordinate, low-level overexpression of lovK and lovR results in a light-independent increase in cell-cell attachment, a response that requires both the conserved histidine phosphorylation site in LovK and aspartate phosphorylation site in LovR. Growing C. crescentus in the presence of blue light dramatically enhances cell-cell attachment in the lovK-lovR overexpression background. A conserved cysteine residue in the LOV domain of LovK, which forms a covalent adduct with the flavin cofactor upon absorption of visible light, is necessary for the light-dependent regulation of LovK enzyme activity and is required for the light-dependent enhancement of intercellular attachment.

Fig. 1.

lovK and lovR are organized in an operon whose transcription is temporally regulated across the cell cycle. (A) The gene structure of the lovKR operon (top line) with the cc7 regulatory motif GGAACC-N15-CGTT (18) upstream of lovK indicated with a triangle. The domain structure of the LovK protein is schematized below. (B) Asymmetric division and developmental progression of C. crescentus. The swarmer cell is characterized by a single polar flagellum and polar pili, which are lost during the swarmer-to-stalked transition and replaced by a membranous stalk; at the tip of the stalk is an adhesive holdfast. The G1, S, and G2 phases of the cell cycle are indicated. The chromosome is represented by an oval inside the cell; chromosome replication is indicated by the theta structure. (C) Transcription of lovK (dotted line) and lovR (solid line) are correlated (Pearson R = 0.92) across the cell cycle. Levels of both mRNAs peak during the swarmer-to-stalked transition, fall during DNA replication in the S phase, and rise again during cytokinesis. The time scale is shared for B and C.

Fig. 2.

LovK exhibits a canonical light-dependent LOV absorption spectrum and light regulated kinase activity. (A) Light-minus-dark difference absorption spectrum of LovK is qualitatively identical to that of the oat phototropin-1 LOV2 domain. Light-dependent spectral changes are abolished in the LovK (C70A) mutant. (B) LovK photorecovery in vitro, monitored by the reappearance of the 447-nm peak as the flavin–cysteine adduct ruptures. LovK dark decay is approximated by fitting to the sum of two exponential decay events, indicating that multiple rate constants govern photorecovery. (C) Autophosphorylation of the LovK histidine kinase, detected by autoradiography of an SDS/PAGE gel, is up-regulated by white light. (D) ATPase activity of LovK is up-regulated by white light as measured by monitoring the hydrolysis of [γ-³²P]ATP via thin layer chromatography.

Fig. 3.

Surface attachment is severely attenuated in a lovR deletion background. (A) Schematic showing attachment of C. crescentus cells inside a microfluidic channel. Cells attach to the glass coverslip via the stalk holdfast. When a flow is applied to the system, cells experience flow forces that are sufficient to lay them flat against the coverslip, permitting individual cell attachment to be quantified via phase contrast microscopy. (B) The flow of a water-based medium in a microfluidic channel of these dimensions is laminar; thus flow velocity approaches zero at the sides of the channel. The arrows inside the parabolic flow profile are a cartoon representation of the decreasing medium flow velocity from the center of the channel to the sides in a two-dimensional cross-section. (C) Bar graph of the number of cells that attached to the glass surface of the channel coverslip and remained attached after 12 ml/min medium flow was applied to the system. Tested strains are labeled on the horizontal axis and the average number of cells attached in a field of view is on the vertical axis with error bars showing 1 standard error of the mean.

Fig. 4.

Overexpression of LovK and LovR increases cell-cell adhesion in a light modulated manner. (A) Coordinate, low-level overexpression of both LovK and LovR with intact phosphorylation sites leads to elevated levels of cell-cell adhesion in dark-grown cultures. The proteins overexpressed in each strain are indicated below the plot. LovK overexpression variants or an empty plasmid were integrated into the vanR locus and LovR variants or empty plasmid were integrated into the xylX locus. Total numbers of rosettes observed for each strain are indicated in parentheses. Illustrative examples of the three rosette types are shown on the right. (B) Light-dependent enhancement of cell-cell attachment in a LovK/LovR overexpression background requires the C70 residue of LovK. Average number of large rosettes binned by approximate size; error bars indicate standard error of the mean. Representative images of rosettes in each size class are shown above each bin. (Scale bar, 5 μm, except for the largest rosette where it represents 2.5 μm.) (C) A proposed model of the LovK/LovR signaling pathway. Gene deletion and gene overexpression data demonstrate that LovK and LovR are regulators of cell attachment. Deletion of lovR nearly abolishes attachment to a glass surface, whereas deletion of lovK only modestly attenuates attachment, suggesting there may be an additional regulator of LovR (shown as a question mark in this panel). Mutagenesis of the conserved phosphorylation sites in this two-component system provides evidence that LovK and LovR phosphorylation is necessary for the large increase in intercellular attachment during coordinate LovK/LovR overexpression. Light acts to up-regulate both the kinase activity of LovK in vitro, and cell–cell attachment in vivo; both of these responses require cysteinyl-flavin adduct formation in the LOV domain of LovK. Filled arrows indicate phosphotransfer events. Double-headed arrows indicate signal input and cellular output.