53NISR Research GRANT
2009 Young Investigator Research Grant

LitR-mediated Photoresponse in Non-phototrophic Bacteria:
Studies on Its Molecular Mechanism and Potential Diversity
Hideaki TAKANO
College of Bioresource Sciences, Nihon University

Research aims

Results

Light is an environmental stimulus that affects many living organisms; such is also the case for prokaryotes1).
Light-inducible transcriptional regulator (LitR), a MerRfamily transcriptional regulatory protein is a common central regulator for light-dependent carotenoid (Crt) production in Streptomyces coelicolor, a gram-positive soil bacterium, and Thermus thermophilus, an extremely thermophilic bacterium3). LitR may also serve as a photosensor
by receiving blue light through cobalamin (Cbl; vitamin
B12), a putative ligand for LitR2). To understand the molecular mechanism underlying LitR-mediated photosensing in
non-phototrophic bacteria, we here examined biochemical
properties of the T. thermophilus LitR and TTP55 proteins,which are putative central regulators for light-dependenttranscription in this non-phototrophic bacterium. We also
performed transcriptome analysis to identify novel light-inducible genes in T. thermophilus. In addition, we newly isolated light-responsive microorganisms from environmental
samples with the aim of discovering proteins with novelfunction.

1. Elucidation of the molecular mechanism for photoresponse in T. thermophilus.
Light-dependent transcriptional regulation of the crt
biosynthesis gene cluster is primarily regulated by two transcriptional regulators: LitR and a CRP homolog (TTP55)
encoded by a gene located downstream of the litR gene(Fig. 1). To observe the effect of LitR and TTP55 on thetranscriptional activity of crtB (phytoene synthase gene), anin vitro transcription analysis was carried out. The in vitrotranscription analysis showed that RNA polymerase holoenzyme and the recombinant TTP55 were sufficient for generation of a specific transcript exhibiting the length corresponding to that for crtB. This activation by TTP55 at least
required the position -56 with respect to the transcriptional
start site of crtB. Furthermore, the assay revealed that recombinant LitR inhibits TTP55-dependent transcription ina dose-response manner. DNase I footprinting assay showedthat the LitR binding site is located at the positions between-55 and -89 on the crtB promoter. Transcriptome analysis
using DNA microarray revealed the existence of twentythree novel light-induced genes, including TTHB89-91,
TTHB94-97 and TTHB112-113, which are located in theneighborhood of the litR and crt genes. The function ofmost of these gene products is unknown. This indicates thatfurther studies may lead to the discovery of proteins withnovel functions.
2. Screening for light-dependent microorganisms andtheir function.
From environmental soil samples, we isolated 80 bacterial strains which produce, in response to illumination, yellow or orange pigments that are most likely carotenoids.
Analysis of 16S rRNA sequences indicated that the light-responsive bacteria isolated in this study belong to the following genera: Group 1 contained Bacillus, Lysinibacillus,Staphylococcus, Halomonas, Pseudomonas, Ralstonia, andSphingomonas; Group 2 contained Arthrobacter, Brevibacterium, and Microbacteria. The bacteria were classified into
two groups based on the presence or absence of the litR homologue (or known photosensor genes) on the genomes ofclosely-related bacteria. Bacteria belonging to the group 1
have litR homologue(s) in their genome. In contrast, bacteria belonging to the group 2 do not retain litR or any otherknown photosensor genes. This result indicated the possible
presence of novel light-sensing proteins in the group 2 bac-Methods
Glutathione S-transferase (GST)-tagged LitR and TTP55
proteins were prepared by an E. coli expression systemusing BL21(DE3) strain and pGEX-6P-2 vector. These
GST fusion proteins were purified using a GST column,and the GST tag was removed by treatment with Precision
protease. Using the LitR and TTP55 proteins thus prepared,
DNase I footprinting analysis and in vitro run-off transcription assay were carried out. DNA microarray analysis oflight-inducible genes in T. thermophilus was performed
using GeneChip (Affymetrix), which contained probe setsof 25-mer oligonucleotides representing 2,238 open reading
frames (ORFs), as well as 1,096 intergenic regions. Total
RNA was isolated from cells cultured under the dark or
light conditions.
We isolated approximately 2,300 bacterial strains fromsoil. Bacteria showing photo-dependent phenotypes wereselected by growing the bacteria in an illuminating incubator (BR-180LF; Taitech). Phylogenetic analysis for bacteria
thus selected was performed on the basis of the DNA sequences encoding 16S rRNA.

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Fig. 1. Light-dependent carotenoid production (A) and its regulatory mechanism (B) in T. thermophilus. LitR protein binds to the promoter region of crtB(C) to repress its transcriptional initiation under dark conditions. Absorption of blue light by the LitR-cobalamin (Cb1) complex may cause its inactivation
followed by a conformational change in protein structure, which in turn allows TTP55, a cAMP receptor-like activator protein, to promote the transcriptionof crtB.

teria.

the physiology and ecology of non-phototrophic bacteria.

Conclusion

References

The lines of evidence obtained in this study indicates that
TTP55 is a positive regulator essential for transcriptional
initiation of the crt gene cluster and that LitR confers
photo-dependency to the TTP55-dependent transcription
via its negative regulatory action. Moreover, novel lightresponsive genes related to LitR and TTP55 were identified
via transcriptome analysis. Our screening for non-phototrophic bacteria exhibiting light-responsive phenotype
showed the diversity of photoresponse in these bacteria andimplied the existence of an as-yet-unidentified photosensor
in Actinobacteria. These findings provide new insights into1.

Purcell, E., and S. Crosson. 2008. Photoregulation in prokaryotes.
Curr Opin Microbiol, 11: 168–178.
2. Takano, H., T. Beppu, and K. Ueda. 2006. The CarA/LitR-familytranscriptional regulator: Its possible role as a photosensor and wide
distribution in non-phototrophic bacteria. Biosci Biotechnol
Biochem, 70: 2320–2324.
3. Takano, H., S. Obitsu, T. Beppu, and K. Ueda. 2005. Light-induced
carotenogenesis in Streptomyces coelicolor A3(2): identification ofan extracytoplasmic function sigma factor that directs photodependent transcription of the carotenoid biosynthesis gene cluster. J Bacteriol, 187: 1825–1832.