SummaryCysB is a LysR-type transcriptional regulator (LTTR) controlling the expression of numerous genes involved in bacterial sulphur assimilation via cysteine biosynthesis. Our previous mutational analysis of CysB identified several residues within the N-terminal domain crucial for DNA-binding function. Here, we focus on the functional significance of CysB residues localized in the turn between the a a a a 2 and a a a a 3 helices forming an N-terminal helix-turn-helix motif. On the basis of the characteristics of alanine-substituted mutants, we propose that CysB residues Y27, T28 and S29, lying in this turn region, comprise an 'activating region' (AR) that is crucial for positive control of the cysP promoter, but not for DNA binding and inducer response activities of CysB. Using a library of alanine substitutions in the C-terminal domain of the RNAP a a a a subunit ( a a a a -CTD), we identify several residues in a a a a -CTD that are important for CysB-dependent transcription from the cysP promoter. After probing potential protein-protein contacts in vivo with a LexA-based two-hybrid system, we propose that the '273 determinant' on a a a a -CTD, including residues K271 and E273, represents a target for interaction with CysB at the cysP promoter.

CysB is a tetrameric

The genome of Burkholderia cenocepacia contains two genes encoding closely related LysR-type transcriptional regulators, CysB and SsuR, involved in control of sulfur assimilation processes. In this study we show that the function of SsuR is essential for the utilization of a number of organic sulfur sources of either environmental or human origin. Among the genes upregulated by SsuR identified here are the tauABC operon encoding a predicted taurine transporter, three tauD-type genes encoding putative taurine dioxygenases, and atsA encoding a putative arylsulfatase. The role of SsuR in expression of these genes/operons was characterized through (i) construction of transcriptional reporter fusions to candidate promoter regions and analysis of their expression in the presence/absence of SsuR and (ii) testing the ability of SsuR to bind SsuR-responsive promoter regions. We also demonstrate that expression of SsuR-activated genes is not repressed in the presence of inorganic sulfate. A more detailed analysis of four SsuR-responsive promoter regions indicated that ϳ44 bp of the DNA sequence preceding and/or overlapping the predicted ؊35 element of such promoters is sufficient for SsuR binding. The DNA sequence homology among SsuR "recognition motifs" at different responsive promoters appears to be limited.