Genetic Dissection of the Light-Inducible carQRS Promoter Region of Myxococcus xanthus

In Myxococcus xanthus photoprotective carotenoids are produced in response to illumination due to regulated expression of carotenoid biosynthesis genes at two loci. Induction of the carotenogenesis regulon is dependent on expression of the carQRS operon. The first gene product of the operon, CarQ, is a sigma factor belonging to the ECF family and is responsible for light-dependent initiation of transcription at the carQRS promoter. We defined the minimal carQRS promoter as a 145-bp fragment of DNA upstream of the carQRS transcriptional start site, which includes the promoter for a divergent gene, gufA. In order to elucidate regions with the promoter required for activity, point mutations were introduced into the carQRS promoter between positions 151 and 6. While most sequence changes abolished carQRS promoter activity, two changes enhanced promoter activity and two changes caused the mutant promoter to become constitutive and independent of CarQ. The promoter-null point mutations and 6-bp deletion mutations implied that the carQRS promoter requires a functional gufA promoter for transcriptional activity and vice versa. By mapping the extent of the promoter region, identifying sequences important for promoter activity, and highlighting potential topological effects, we provide a foundation for further analysis of the carQRS promoter. Illuminating cells of the bacterium Myxococcus xanthus with blue light can cause lysis of the cells due to the light-dependent production of reactive species such as singlet oxygen and triplet-state protoporphyrin IX. As a survival response, M. xanthus produces a variety of photoprotective carotenoids by lightregulated expression of the crt biosynthetic genes (14, 29). Most carotenoid biosynthetic genes are located within the crtEBDC operon; the exception is the monocistronic crtI gene (1, 9). In the dark the crtI gene is transcriptionally silent, while the crtEBDC operon is repressed by CarA, which is encoded by the tenth open reading frame of the operon (40). Three regulatory factors governing carotenogenesis are encoded within the carQRS operon, which is expressed in a light-induced fashion (23). The first gene in the operon encodes an extracytoplasmic-function (ECF)-type sigma factor (18), CarQ, which directs transcription from the crtI and carQRS promoters (2, 23). The second gene product of the operon, CarR, acts as an anti-sigma factor (2, 11) by binding to CarQ and preventing CarQ-dependent initiation of transcription in the dark. The final polypeptide encoded in carQRS is CarS, which relieves repression of the crtEBDC operon by binding to the repressor CarA and eliminating its operator binding activity (40). The ECF-type sigma factors (18) are a large class of sigma factors whose promoter recognition characteristics are poorly understood (12). The carQRS promoter possesses a 35 rec