The N Terminus of Myxococcus xanthus CarA Repressor Is an Autonomously Folding Domain That Mediates Physical and Functional Interactions with Both Operator DNA and Antirepressor Protein

Abstract

Expression of the Myxococcus xanthus carB operon, which encodes the majority of the enzymes involved in light-induced carotenogenesis, is down-regulated in the dark by the CarA repressor binding to its bipartite operator. CarS, produced on illumination, relieves repression of carB by physically interacting with CarA to dismantle CarA-DNA complexes. Here, we demonstrate that the N-and C-terminal portions of CarA are organized as distinct structural and functional domains. Specifically, we show that the 78 N-terminal residues of CarA, CarA(Nter), form a monomeric, highly helical, autonomously folding unit with significant structural stability. Significantly, CarA(Nter) houses both the operator and CarS binding specificity determinants of CarA. CarA(Nter) binds operator with a lower affinity than whole CarA, and the CarA(Nter)-CarS complex has a 1:1 stoichiometry. In vitro, sufficiently high concentrations of CarA(Nter) block M. xanthus RNA polymerase-promoter binding, and this is relieved by CarS. In vivo, substitution of the gene carA by that for CarA(Nter) results in constitutive expression of carB just as in a carA-deleted background. However, re-engineering the latter strain to overexpress CarA(Nter) restores repression of carB. Thus, the 78-residue N-terminal portion of CarA is an autonomously folded, dual function domain that orchestrates specific DNA-protein and protein-protein interactions and, when overexpressed, can be functionally competent in vivo.

Light-dependent gene regulation by a coenzyme B 12 -based photoreceptor

“…S1C). A plasmid construct with the gene for C1 or C2 (or CarH, as a control) expressed from a constitutive promoter (12,20) was introduced into the M. xanthus strain lacking endogenous CarA and CarH, where it integrates at an unlinked site in the chromosome. Unlike the Car C recipient strain, the resulting transformants were Car (+) : yellow in the dark only in the presence of B 12 and red in the light, just like the control strain with CarH (Fig.…”

“…S1B). Besides their autonomously stable, N-terminal DNA-binding domains (CarANt and CarHNt, respectively) that recognize the same operator (12,19,20), CarA and CarH have C-terminal domains (CarACt and CatHCt, respectively) with a B 12 -binding motif of the type found in enzymes like methionine synthase (6,12,(21)(22)(23). However, only CarH requires B 12 for its repressor activity (12).…”

Operator Design and Mechanism for CarA Repressor-mediated Down-regulation of the Photoinducible carB Operon in Myxococcus xanthus

“…Oligomerization could also play an important role in driving the binding of CarA to the low affinity site pII. Indeed, deletion of its oligomerization domain leads to a constitutive light-independent expression of P B in vivo (55). The particular spatial arrangement and the differential repressor binding affinities of pI and pII in the design of the CarA operator may have evolved to achieve the proper finetuning in the control of carB expression, as discussed next.…”

The CarA/LitR-Family Transcriptional Regulator: Its Possible Role as a Photosensor and Wide Distribution in Non-Phototrophic Bacteria

“…CarA represses the transcription of carotenoid biosynthesis genes in the dark, and light-induced sequesterization of CarA induces expression of the biosynthesis genes, 4,5) but there is no evidence regarding the direct response of CarA to an illumination signal. The relation between LitR function and illumination was also unclear in S. coelicolor A3(2), since our attempts to obtain true knockout mutants for litR and litQ in this organism failed.…”

“…In M. xanthus, light-induced carotenogenesis is clearly observed in media supplied with vitamin B12. 14) Recently, PerezMarin et al 5) suggested that CarA binds vitamin B12 in vitro. The property of absorbing blue light, which is a characteristic of vitamin B12 proteins such as methionine synthase, 15) implies that the cofactor plays a significant role in the light-dependent function of the family of transcriptional regulators.…”