Role and Function of Class III LitR, a Photosensor Homolog from Burkholderia multivorans

Abstract

The LitR/CarH protein family is an adenosyl B12 (AdoB12)-dependent photoreceptor family with DNA-binding activity, and its homologs are widely distributed in the genomes of diverse bacterial genera. In this investigation, we studied the role and functions of a LitR homolog from a Gram-negative soil bacterium, Burkholderia multivorans, which does not possess an AdoB12-binding domain. Transcriptome analysis indicated the existence of 19 light-induced genes, including folE2, cfaB, litS, photolyase gene phrB2, and cryB, located in the region flanking litR. Disruption of litR caused constitutive expression of all the light-inducible genes, while mutation in the light-induced sigma factor gene, litS, abolished the transcription of the phrB2 operon and the cfa operon, indicating that LitR and LitS play a central role in light-inducible transcription. A gel shift assay showed that recombinant protein LitR specifically binds to the promoter regions of litR and the folE2 operon, and its binding was weakened by UV-A illumination. LitR absorbs light at maximally near 340 nm and exhibited a photocyclic response and light-dependent dissociation of multimer into tetramer. The litR mutant produced a 20-fold-higher intracellular level of folate than that of the wild-type strain. Thus, the evidence suggests that LitR light-dependently regulates the transcription of litR itself and the folE2 operon, resulting in the production of folate, and then the expressed RNA polymerase complex containing σLitS directs the transcription of the phrB2 operon and the cfa operon. These light-dependent characteristics suggest that class III LitR, in complex with a UV-A-absorbing molecule, follows a novel light-sensing mechanism. IMPORTANCE Members of the LitR/CarH family are adenosyl B12-based photosensory transcriptional regulator involved in light-inducible carotenoid production in nonphototrophic bacteria. Our study provides the first evidence of the involvement of a class III LitR, which lacks an adenosyl B12-binding domain in the light response of Burkholderia multivorans belonging to betaproteobacteria. Our biochemical analysis suggests that class III LitR protein exhibits features as a photosensor including absorption of light at the UV-A region (λmax = ca. 340 nm), photocyclic response, and light-dependent dissociation. This suggests that class III LitR associates with a UV-A-absorbing molecule, and it has a photosensing mechanism distinguishable from that of the B12-based type.

Light Response of Pseudomonas putida KT2440 Mediated by Class II LitR, a Photosensor Homolog

“…The light-inducible expression of phrB (PP_0739) and the photosensitivity of the folate derivative lead to our assumption that the upregulation of folE2 in response to illumination is needed to supply the second chromophore to Phr for its efficient DNA repair activity. Recently, we showed that the class III LitR of Burkholderia multivorans induces the expression of folE2 in a lightdependent manner (23). The involvement of PhrR, a class I LitR of Halomonas spp., in folate biosynthesis was also reported (39).…”

“…Quantitative RT-PCR analysis. cDNA was synthesized using 2 g of total RNA with SuperScript VILO cDNA synthesis kit (Thermo Fisher Scientific) and quantified by using PowerUp SYBR green master mix (Thermo Fisher Scientific) and an Applied Biosystems 7500 real-time PCR system (Thermo Fisher Scientific) according to manufacturer's manual and our previous study (23). The cDNAs of phrB, pplR1, PP_742, PP_743, folE2, PP_2727, PP_2729, ufaM, PP_2736, PP_3161, PP_3276, PP_3282, PP_3657, PP_4656, and rpoD were detected by the primer pairs summarized in Table S2 in the supplemental material.…”

“…Recently, we reported the results of phylogenetic analysis demonstrating that LitR/CarH family proteins are divided into at least five classes (I to V) based on the primary structure of their C-terminal photosensing domain (23). Of these classes, class I LitR is the above-mentioned AdoB 12 -dependent type widely distributed among Gram-negative and Gram-positive nonphototrophic bacteria.…”

“…Class III LitR, a group lacking B 12 -binding domain, is mainly found in betaproteobacteria, including Burkholderia spp. Recently, we discovered that the class III LitR recombinant protein of B. multivorans prepared in E. coli exhibits some expected characteristics of photosensors, such as a photocyclic response and absorption of light in the UV-A region, which suggests that its photosensing mechanism is distinct from that of the B 12 -dependent type (23).…”

Bioluminescence and Photoreception in Unicellular Organisms: Light-Signalling in a Bio-Communication Perspective

“…However, it has recently been shown that other types of receptors such as the chemoreceptors Aer, Tar, Tsr, Tap and Trg also mediate bacterial responses to blue-light [ 184 ]. In addition, a new photosensor family uses coenzyme B 12 for sensing blue and green-light and regulating gene expression [ 185 , 186 , 187 , 188 , 189 , 190 , 191 , 192 ]. A bacteriochlorophyll usually involved in photosynthesis with bacteria has been found as a photosensor in the retina of dragon-fish Malacosteus niger living in the deep-sea [ 193 ].…”