Search PubMed⌕ Search

Biomedical subjects

Stephen C Winans

Publications and source records attributed to Stephen C Winans.

8 recordsLinked to original sources
PubMed2003

A LuxR-type regulator from Agrobacterium tumefaciens elevates Ti plasmid copy number by activating transcription of plasmid replication genes.

TraR, a LuxR-type quorum-sensing transcription factor in Agrobacterium tumefaciens, activates genes required for conjugal transfer of the Ti plasmid and also enhances the copy number of a nopaline-type Ti plasmid. Here, we show that TraR increases the copy number of an octopine-type Ti plasmid up to eightfold and that TraR activates transcription of the repABC operon up to 25-fold. The ability of TraR to increase copy number was strictly dependent on several TraR-activated promoters of this operon, indicating that TraR affects copy number solely at the level of transcription. Promoter resections and mRNA transcript analysis revealed the presence of three TraR-dependent promoters. Two TraR-dependent transcription start sites are located 45.5 and 65.5 nucleotides downstream of a site called tra box II, whereas the third start site lies 42.5 nucleotides downstream of a site called tra box III. Purified TraR bound to both tra boxes with comparable affinities, causing moderate DNA bending. TraR bound and bent these two sites independently rather than synergistically. Alteration of tra box III to match the consensus sequence dramatically increased TraR-dependent expression of repABC and plasmid copy number. TraR-dependent elevation of Ti plasmid copy number caused a three- to fourfold increase in plant tumorigenesis.

Agrobacterium tumefaciens↗
PubMed2003

The RepA and RepB autorepressors and TraR play opposing roles in the regulation of a Ti plasmid repABC operon.

The replicator regions of the Ti plasmids of Agrobacterium tumefaciens belong to the repABC family of replication and partitioning systems, members of which are widely distributed among alpha proteobacteria. In the region upstream of the octopine-type Ti plasmid repABC operon, three promoters were recently shown to be activated by the LuxR-type regulator TraR. Activation of these promoters by TraR led to enhanced rep gene expression and increased Ti plasmid copy number. Here we describe a fourth promoter, designated P4. This promoter lies directly upstream of repA and is not regulated by TraR. The promoter was localized by subcloning and demonstrated to be strongly autorepressed. RepA is the major cis-acting autorepressor of this promoter, though RepB enhanced repression and was essential for RepA-mediated repression in trans. Purified RepA bound to an approximately 70-nucleotide operator site overlapping the P4 promoter and extending well downstream. Binding affinity was increased by adenosine di- and tri-phosphates and also by purified RepB. Activation of P1, P2, and P3 enhanced the activity of P4, suggesting that P4 somehow communicates with the upstream promoters. These findings demonstrate that both autoinduction and autorepression play critical and opposing roles in regulating repABC expression and hence in the replication, stability and copy number of the Ti plasmid.

Agrobacterium tumefaciens↗
PubMed2003

Identification and characterization of the conjugal transfer region of the pCg1 plasmid from naphthalene-degrading Pseudomonas putida Cg1.

Hybridization and restriction fragment length polymorphism data (K. G. Stuart-Keil, A. M. Hohnstock, K. P. Drees, J. B. Herrick, and E. L. Madsen, Appl. Environ. Microbiol. 64:3633-3640, 1998) have shown that pCg1, a naphthalene catabolic plasmid carried by Pseudomonas putida Cg1, is homologous to the archetypal naphthalene catabolic plasmid, pDTG1, in P. putida NCIB 9816-4. Sequencing of the latter plasmid allowed PCR primers to be designed for amplifying and sequencing the conjugal transfer region in pCg1. The mating pair formation (mpf) gene, mpfA encoding the putative precursor of the conjugative pilin subunit from pCg1, was identified along with other trb-like mpf genes. Sequence comparison revealed that the 10 mpf genes in pCg1 and pDTG1 are closely related (61 to 84% identity) in sequence and operon structure to the putative mpf genes of catabolic plasmid pWW0 (TOL plasmid of P. putida) and pM3 (antibiotic resistance plasmid of Pseudomonas. spp). A polar mutation caused by insertional inactivation in mpfA of pCg1 and reverse transcriptase PCR analysis of mRNA showed that this mpf region was involved in conjugation and was transcribed from a promoter located upstream of an open reading frame adjacent to mpfA. lacZ transcriptional fusions revealed that mpf genes of pCg1 were expressed constitutively both in liquid and on solid media. This expression did not respond to host exposure to naphthalene. Conjugation frequency on semisolid media was consistently 10- to 100-fold higher than that in liquid media. Thus, conjugation of pCg1 in P. putida Cg1 was enhanced by expression of genes in the mpf region and by surfaces where conditions fostering stable, high-density cell-to-cell contact are manifest.

Bacterial Proteins↗
PubMed2002

Structure of a bacterial quorum-sensing transcription factor complexed with pheromone and DNA.

Many proteobacteria are able to monitor their population densities through the release of pheromones known as N-acylhomoserine lactones. At high population densities, these pheromones elicit diverse responses that include bioluminescence, biofilm formation, production of antimicrobials, DNA exchange, pathogenesis and symbiosis. Many of these regulatory systems require a pheromone-dependent transcription factor similar to the LuxR protein of Vibrio fischeri. Here we present the structure of a LuxR-type protein. TraR of Agrobacterium tumefaciens was solved at 1.66 A as a complex with the pheromone N-3-oxooctanoyl-L-homoserine lactone (OOHL) and its TraR DNA-binding site. The amino-terminal domain of TraR is an alpha/beta/alpha sandwich that binds OOHL, whereas the carboxy-terminal domain contains a helix turn helix DNA-binding motif. The TraR dimer displays a two-fold symmetry axis in each domain; however, these two axes of symmetry are at an approximately 90 degree angle, resulting in a pronounced overall asymmetry of the complex. The pheromone lies fully embedded within the protein with virtually no solvent contact, and makes numerous hydrophobic contacts with the protein as well as four hydrogen bonds: three direct and one water-mediated.

Amino Acid Sequence↗
PubMed2002

Mutations in the occQ operator that decrease OccR-induced DNA bending do not cause constitutive promoter activity.

OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid. Positive control of the occ genes occurs in response to octopine, a metabolite released from plant tumors. Octopine causes DNA-bound OccR to undergo a conformational change from an inactive to an active state; this change is marked by a decrease in footprint length from 55 to 45 nucleotides as well as a relaxation of a high angle DNA bend. In this study, we first used gel filtration chromatography to show that OccR is dimeric in solution, and we used gel shift assays to show that OccR is tetrameric when bound to DNA. We then created a series of site-directed mutations in the OccR-binding site. Some mutations were designed to lock OccR-DNA complexes into a conformation resembling the inactive conformation, whereas other mutations were designed to lock complexes into the active conformation. These mutations altered the conformation of OccR-DNA complexes and their responses to octopine in ways that we had predicted. As expected, operator mutations that locked complexes into a conformation having a long footprint and a high angle DNA bend blocked activation by octopine in vivo. Surprisingly, however, mutations that lock OccR into a short footprint and low angle DNA bend failed to cause the protein to function constitutively. Furthermore, some of the latter mutations interfered with activation by octopine. We conclude that locking OccR into a conformation having a short footprint is not sufficient to cause constitutive activation, and octopine must cause at least one additional conformational change in the protein.

Agrobacterium tumefaciens↗
PubMed2002

Mob psychology.

Explore the source record for details and available documents.

4-Butyrolactone↗
PubMed2001

Constitutive mutations of the OccR regulatory protein affect DNA bending in response to metabolites released from plant tumors.

OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid and is also an autorepressor. Positive control of the occ genes occurs in response to octopine, a nutrient released from crown gall tumors. OccR binds to a site upstream of the occQ promoter in the presence and absence of octopine. Octopine causes prebound OccR to undergo a conformational change at the DNA binding site that causes changes in footprint length and DNA bending. To determine the roles of these conformational changes in transcriptional activation, we isolated 22 OccR mutants that were able to activate the occQ promoter in the absence of octopine. Thirteen of these mutants contained single amino acid substitutions, and nine contained two base pair changes resulting in two amino acid substitutions, which in most cases acted synergistically. These mutations spanned the entire length of the protein. Most of these mutant proteins in the absence of octopine displayed DNA binding and bending properties characteristic of transcriptionally active OccR-octopine complexes.

Agrobacterium tumefaciens↗