Binding of the IclR-type regulator HutR in the histidine utilization (hut) gene cluster of the human pathogen Corynebacterium resistens DSM 45100.
ABSTRACT The genome of the human pathogen Corynebacterium resistens DSM 45100 is equipped with a histidine utilization (hut) gene cluster encoding a four-step pathway for the catabolism of l-histidine and a transcriptional regulator of the IclR superfamily, now named HutR. The utilization of l-histidine might be relevant for the growth of C. resistens in its natural habitat, probably the histidine-rich inguinal and perineal areas of the human body. The ability of C. resistens to utilize l-histidine as a sole source of nitrogen was demonstrated by growth assays in synthetic minimal media. Reverse transcriptase PCRs revealed enhanced transcript levels of the hut genes in C. resistens cells grown in the presence of l-histidine. Promoter-probe assays showed that the hut genes are organized in three transcription units: hutHUI, hutR, and hutG. The respective transcriptional start sites were mapped by 5' RACE-PCR to detected putative promoter regions. DNA band shift assays with purified HutR protein identified the 14-bp DNA sequence TCTGwwATwCCAGA located upstream of the mapped promoters. This DNA motif includes a 4-bp terminal palindrome, which turned out to be essential for HutR binding in vitro. These data add a new physiological function to the large IclR family of transcriptional regulators.
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ABSTRACT: Novel shuttle promoter-probe vectors replicating in Escherichia coli, Corynebacterium glutamicum, and Rhodococcus erythropolis were constructed on the basis of the C. glutamicum plasmid pCG1. The vectors carry reporter genes coding for fluorescent proteins, which allow the measurement of promoter activities in vivo. The promoter-probe vector pPRE11 contains the rsgfp reporter gene, coding for a variant of green fluorescent protein (GFP) with a red-shifted excitation maximum. To ensure efficient expression of the gfp gene in R. erythropolis from the tested promoters, the promoterless gene gfpuv, with 5' end fusion with the initial six codons of the aph gene and upstream insertion of the aph Shine-Dalgarno sequence, was used as a reporter gene in the promoter-probe vector pEPR1. Insertion of the rfp reference gene, coding for a variant of the red fluorescent protein DsRed.T4 and cloned under the strong constitutive C. glutamicum promoter P-45, into the vector pEPR1 resulted in a new-generation promoter-probe vector (pRAG5). All vectors were tested using a set of mutant P-dapA promoters displaying various transcriptional activities. The vector pRAG5 is suitable for normalized measurements of promoter activities during the growth of bacterial batch cultures because estimation of the GFP-to-red fluorescent protein fluorescence ratio in strains carrying the plasmid pRAG5 with the tested promoters upstream of gfpuv avoids the influence of plasmid copy number variations on the promoter activity assay.Current Microbiology 10/2007; 55(3):234-9. · 1.52 Impact Factor
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ABSTRACT: The hutC gene in Pseudomonas putida encodes a repressor protein that negatively regulates the expression of all hut genes. We have overexpressed this cloned hutC gene in Escherichia coli to identify P. putida hut regions that could specifically bind the repressor. Ten restriction fragments, some of which were partially overlapping and spanned the coding portions of the P. putida hut region, were labeled and tested for their ability to recognize repressor in a filter binding assay. This procedure identified three binding sites, thus supporting previous indications that there were multiple operons. A 1.0-kilobase-pair SalI restriction fragment contained the operator region for the hutUHIG operon, whereas a 1.9-kilobase-pair SmaI fragment contained the hutF operator. A 2.9-kilobase-pair XhoI segment appeared to contain the third operator, corresponding to a separate and perhaps little used control region for hutG expression only. The addition of urocanate, the normal inducer, caused dissociation of all operator-repressor complexes, whereas N-formylglutamate, capable of specifically inducing expression of the hutG gene, inhibited binding only of repressor to fragments containing that gene. Formylglutamate did not affect the action of urocanate on the repressor-hutUHIG operator complex, indicating that it binds to a site separate from urocanate on the repressor. DNA footprinting and gel retardation analyses were used to locate more precisely the operator for the hutUHIG operon. A roughly 40-base-pair portion was identified which contained a 16-base-pair region of dyad symmetry located near the transcription initiation site for this operon.Journal of Bacteriology 09/1989; 171(8):4189-95. · 3.19 Impact Factor
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ABSTRACT: DNA-binding transcription factors regulate the expression of genes near to where they bind. These factors can be activators or repressors of transcription, or both. Thus, a fundamental question is what determines whether a transcription factor acts as an activator or a repressor? Previous research into this question found that a protein's regulatory function is determined by one or more of the following factors: protein-protein contacts, position of the DNA-binding domain in the protein primary sequence, altered DNA structure, and the position of its binding site on the DNA relative to the transcription start site. Although there are many aspects specific to different transcription factors, in this work we demonstrate that, in general, in the prokaryote Escherichia coli, a transcription factor's protein family is not indicative of its regulatory function, but the position of its binding site on the DNA is.Trends in Genetics 03/2003; 19(2):75-9. · 9.77 Impact Factor