Regulation of proline utilization in Salmonella typhimurium: a membrane-associated dehydrogenase binds DNA in vitro

Abstract

The PutA protein is a membrane-associated enzyme that catalyzes the degradation of proline to glutamate. Genetic evidence suggests that in the absence of proline, the PutA protein also represses transcription of the put4 and putP genes. To directly determine whether PutA protein binds to the put control region, we analyzed gel retardation of put control region DNA by purified PutA protein in vitro. The put control region is 420 bp. Purified PutA protein bound specifically to several nonoverlapping fragments of control region DNA, indicating the presence of multiple binding sites in the control region. Electrophoretic abnormalities and behavior of circularly permuted fragments of control region DNA indicate that it contains a region of intrinsically curved DNA. To determine whether the multiple binding sites or the DNA curvature are important in vivo, two types of deletions were constructed: (i) deletions that removed sequences predicted to contribute to DNA curvature as well as potential operator sites and (ii) deletions that removed only potential operator sites. Both types of deletions increased expression of the put genes but were still induced by proline, indicating that multiple cis elements are involved in repression. These data suggest a model for put repression that invokes the formation of a complex between PutA protein molecules bound at different sites in the control region, brought into proximity by a loop of curved DNA.

Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme

“…Formation of the PutA47-DNA complex seems to involve cooperativity consistent with properties of some RHH DNA-binding proteins (17,29). In previous studies with PutA, cooperative DNA binding has not been described, although multiple PutA binding sites exist in the put control DNA (7,12,14). Detailed analysis of PutA-DNA interactions will be required to determine whether the observed cooperative DNA binding of PutA47 is relevant to the function of PutA as a transcriptional repressor or is unique to PutA47.…”

“…3. PutA is known to have transcriptional repressor activity from Salmonella typhimurium, Klebsiella aerogenes, and Pseudomonas putida (7,22,23). In the remaining bacteria, such as Yersinia pestis, Ralstonia solanacearum, and Burkholderia fungorum, the regulation of the put regulon has not been described; based on the alignment in Fig.…”

“…PutP encodes a high affinity sodium-proline transporter (9). PutA represses the expression of the put genes, which are transcribed in opposite directions, by binding to the put intergenic DNA region (1,7,8). The transcriptional repression of the put genes is relieved in the presence of proline by the translocation of PutA to a peripheral position on the membrane, where proline is efficiently converted to glutamate.…”

Flavin Redox Switching of Protein Functions

“…PutA represses the put genes by binding to five operator sites in the 419 bp intergenic control region from which the putP and putA genes are transcribed in opposite directions (7,51,52,85) (Fig. 6B).…”

“…In the absence of proline, EcPutA resides in the cytoplasm and acts as a transcriptional repressor of the put genes (7,52). PutA represses the put genes by binding to five operator sites in the 419 bp intergenic control region from which the putP and putA genes are transcribed in opposite directions (7,51,52,85) (Fig.…”