Residues that influence in vivo and in vitro CbbR function in Rhodobacter sphaeroides and identification of a specific region critical for co‐inducer recognition

Abstract

SummaryCbbR is a LysR-type transcriptional regulator (LTTR) that is required to activate transcription of the cbb operons, responsible for CO 2 fixation, in Rhodobacter sphaeroides . LTTR proteins often require a coinducer to regulate transcription. Previous studies suggested that ribulose 1,5-bisphosphate (RuBP) is a positive effector for CbbR function in this organism. In the current study, RuBP was found to increase the electrophoretic mobility of the CbbR/ cbb I promoter complex. To define and analyse the co-inducer recognition region of CbbR, constitutively active mutant CbbR proteins were isolated. Under growth conditions that normally maintain transcriptionally inactive cbb operons, the mutant CbbR proteins activated transcription. Fourteen of the constitutively active mutants resulted from a single amino acid substitution. One mutant was derived from amino acid substitutions at two separate residues that appeared to act synergistically. Different mutant proteins showed both sensitivity and insensitivity to RuBP and residues that conferred constitutive transcriptional activity could be highlighted on a three-dimensional model, with several residues unique to CbbR shown to be at locations critical to LTTR function. Many of the constitutive residues clustered in or near two specific loops in the LTTR tertiary structure, corresponding to a proposed site of co-inducer binding.

CbbR, the Master Regulator for Microbial Carbon Dioxide Fixation

“…Additionally, in vitro, the CbbR from R. sphaeroides was shown to alter the angle at which it bends the cbb I promoter DNA in the presence of RuBP, as illustrated by a change in the mobility of the CbbR/DNA complex in gel mobility shift assays (76). DNase I footprint analysis also demonstrates that RuBP improves protection of the cbb I promoter by CbbR (76).…”

“…Effector binding occurs in a small cleft formed between RD-I and RD-II (Fig. 1) and is a hallmark of the LTTR family (22,(70)(71)(72)(73)(74)(75)(76)(77)(78)(79)(80). A recent study using nondenaturing mass spectrometry has illustrated for the first time that an LTTR tetramer binds four molecules of its effector in a stepwise pattern while bound to DNA (81).…”

“…Effector binding to the LTTR can also inhibit transcription (19,82). Studies illustrating DNA bend angle modification or initiation of transcription brought about by effector binding have been reported for some CbbRs (8,76,82,83). The effector molecules may be different for each CbbR, depending on the organism.…”

“…The suggestion was based on a study employing a strain deleted for form I and form II RubisCO, leading to an accumulation of RuBP and a subsequent increase in transcription for both the cbb I and cbb II operons (84). Additionally, in vitro, the CbbR from R. sphaeroides was shown to alter the angle at which it bends the cbb I promoter DNA in the presence of RuBP, as illustrated by a change in the mobility of the CbbR/DNA complex in gel mobility shift assays (76). DNase I footprint analysis also demonstrates that RuBP improves protection of the cbb I promoter by CbbR (76).…”

“…The effector molecules may be different for each CbbR, depending on the organism. Figure 4 illustrates the relaxation of the cbb promoter bend angle imposed by CbbR subsequent to conformational changes elicited by effector (RuBP) binding after the switch to autotrophic growth for R. sphaeroides, R. capsulatus, Xanthobacter flavus, and H. thermoluteolus (8,76,82,83).…”