Functional analysis of the Pseudomonas putida regulatory protein CatR: transcriptional studies and determination of the CatR DNA-binding site by hydroxyl-radical footprinting

Abstract

CatR, a LysR family protein, positively regulates the Pseudomonas putida catBC operon, which is required for growth on benzoate as a sole carbon source. Transcriptional studies show that the catR and catBC promoters are divergent and overlapping by 2 bp. A I8-galactosidase promoter probe vector was constructed to analyze expression from the catR and catBC promoters under induced and uninduced conditions. As predicted, the catBC promoter is expressed only under induced conditions, while the catR promoter is constitutive. CatR has been shown to specifically bind the catRBC promoter region, and this property was used to devise a purification protocol for CatR. Linear M13 DNA containing the catRBC control region was covalently bound to cyanogen bromide-activated Sepharose in order to construct a DNA ainity column. Crude extracts containing hyperproduced CatR protein were then incubated with the affinity resin under binding conditions, and the CatR protein was eluted with 1 M NaCl. CatR was also purified by heparin-agarose chromatography. This highly purified protein was used for gel retardation and hydroxyl-radical footprinting studies. From this analysis, it was shown that CatR binds upstream of the catBC promoter within the transcribed region of catR.Pseudomonads utilize many natural and man-made organic compounds. In order to develop strains capable of dissimilating recalcitrant compounds such as chlorinated aromatics, it is important to understand how biodegradative pathways evolve in nature and how they are regulated. Pseudomonads provide a good model for studying how degradative pathways evolve in order to expand the substrate range of a microorganism so that it may degrade more complex and toxic compounds. A simple model used for this analysis has been Pseudomonas putida, which is capable of utilizing benzoate and can also degrade 3-chlorobenzoate when harboring plasmid pAC27 (10). While the structural genes encoding enzymes for the dissimilation of benzoate and 3-chlorobenzoate have been fairly well characterized (1,2,13,15,43), the mechanism for regulation of these genes is only now being investigated.The catBC operon encodes the genes for two enzymes: cis,cis-muconate lactonizing enzyme I (EC 5.5.1.1) and muconolactone isomerase (EC 5.3.3.4), respectively. Both of these genes are required for the dissimilation of benzoate (28). The operon is coordinately regulated and requires the product of a regulatory gene for induction (42). As described previously (32), the regulatory gene catR maps upstream of the catBC operon and is divergently transcribed from the catBC operon (Fig. 1). The catR gene encodes a 32.2-kDa polypeptide that binds to the catBC promoter region in vitro in the presence or absence of the inducer cis,cis-muconate. The inducer, however, is required for in vivo transcriptional activation of the catBC operon. CatR was shown to be a member of a large class of procaryotic regulatory proteins,

Interaction of the Histone-Like Nucleoid Structuring Protein and the General Stress Response Regulator RpoS at Vibrio cholerae Promoters That Regulate Motility and Hemagglutinin/Protease Expression

“…In both cases, the promoter fragments were inserted downstream of the rrnB T 1 T 2 transcription terminator in plasmid pTT3 to generate pTT3flrA and pTT3flaA, respectively. Then, the terminator-promoter fragment was inserted upstream of a promoterless lacZ gene in plasmid pKRZ1 (44) to generate pFlrA-LacZ and pFlaA-LacZ. To construct an rpoN-lacZ promoter fusion, we first constructed plasmid pLacZ by ligating a 4.4-kb BamHI-PstI fragment containing the lacZ gene from pKRZ1 to a 3.2-kb BamHI-PstI fragment from pBR322.…”

Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds

“…The role of the RBS and ABS is not completely clarified. Although ClcR and CbnR contacted both the RBS and ABS in the absence of inducer, CatR did not (215,216). It was concluded that the regulatory proteins have a higher binding affinity to the RBS than to the ABS, since a fragment with only the ABS is not bound by ClcR or CatR (139,181).…”

“…The presence of cis,cis-muconate decreases the affinity of the BenM protein for benzoate (31). All LTTRs repress their own expression, and both autorepression and activation of the catabolic operon promoter are exerted from the same binding site, which is called the regulator or repressor binding site (RBS) (20,216,227). Autorepression was not influenced by the presence of an inducer in the case of BenM (20), but expression of the clcR and tcbR promoters was slightly enhanced in the presence of an inducer (33,263).…”

Purification of the LysR family regulator, ClcR, and its interaction with the Pseudomonas putida clcABD chlorocatechol operon promoter

“…4, the DNase I-protected regions of the catBC promoter have been functionally divided into the repression binding site (RBS) and activation binding site (ABS). In the catR-BC promoter region, the RBS and ABS are implicated in the strong binding and autorepression of CatR on the catR promoter (13) and in the activation of the catBC promoter (11), respectively. In the clcR-ABD promoter region, the DNase I-protected sequences can be divided into two regions with sequence identities at 12 of 26 and 13 of 27 positions compared with the catR-BC RBS and ABS, respectively.…”