Analysis of the role of methyl-accepting chemotaxis proteins and the GGDEF motif in the signal transduction chain of photoactive yellow protein
Abstract
Many bacterial genomes encode photoreceptors which detect illumination and send signal cascades to response-regulated proteins to illicit physiological responses. The Photoactive yellow protein (PYP) is a known photoreceptor which has been characterized to illicit a range of biological responses in different bacteria upon blue light illumination indicating diversity in its downstream signal transduction chain. Such responses include photoregulation of photo-protective pigment biosynthesis, photoregulation of biofilm formation, and negative phototaxis. We performed bioinformatics analyses of PYP homologs to identify both recurring genes within the same predicted operon as PYP and protein domains occurring in multidomain PYPs to further understand possible biological functions of PYP and its signal transduction pathways. Two identified genes of interest include the PYP-MCP fusion protein and the GGDEF motif as it relates to the signal transduction chain of PYP. The domain structure of the PYP-MCP fusion protein suggests that it is sensitive to blue light illumination via its PYP domain and that it controls motility via its methyl-accepting chemotaxis protein (MCP) moiety, leading to the hypothesis that this protein triggers negative phototaxis. To further investigate the PYP-MCP fusion protein and its signal transduction chain, we experimentally analyzed the Nitrincola alkalilacustris sp., which contains a multidomain PYP, to test the prediction that N. alkalilacustris sp. containing the MCP-PYP fusion protein will respond to a stimulus of blue-light and undergo negative phototactic movement due to the downstream signaling from the PYP to the methyl accepting chemotaxis protein which controls the MCP moiety. Using prior studies as well as bioinformatics research the GGDEF domain is suggested to be a key player in regulating biofilm formation upon light illumination triggered by the pyp gene. The GGDEF proteins have been characterized to convert GTP into cyclic-di-GMP which is needed to synthesize exopolysaccharides in biofilm formation. Therefore, the domain structure of tis GGDEF motif coupled with the PYP suggests that it is sensitive to light illumination via its PYP and it controls the regulation of biofilm formation via its GGDEF protein, leading to the hypothesis that this multi-domain protein triggers the regulation of biofilm formation. To investigate this GGDEF motif as a multi-domain PYP we experimentally analyzed Massilia albidiflava and Massilia plicata species containing the GGDEF motif in a multi-domain PYP using Idiomarina loihiensis as an experimental control as it has been described to have biofilm formation regulation via they PYP to test the prediction that, M. albidiflava and M. pilcata containing the PYP-GGDEF motif will respond to the blue-light stimulus and undergo photo-regulation of biofilm formation due to downstream signaling from the photoactive yellow protein to the GGDEF motif in the multidomain PYP. Since PYP functional photocycle is initiated by photoisomerization of its p-coumaric acid (pCA) chromophore, we used the addition of pCA to indicate the blue-light illumination response to be triggered by the PYP signaling due to PYP being the only photoreceptor to use pCA as its key component in photoisomerization of its chromophore to initiate an active response. Upon the addition of pCA within the media used to test phototaxis within N. alkalilacustris sp, there appears to be a restoration in positive phototaxis, which needs further biological replicates to be a conclusive result. Furthermore, for the analysis of biofilm formation there were inconclusive results for M. albidiflava while the addition of pCA did not appear to have an effect on M. plicata. Upon further investigation of M. plicata there does however appear to be a light effect which causes a decrease in biofilm formation upon light illumination, again further biological replicates are needed to confirm these results. The addition of a trans-locked pCA chromophore is also needed to determine this result is due to the PYP.