Transcriptional Organization and Regulatory Elements of a Pseudomonas sp. Strain ADP Operon Encoding a LysR-Type Regulator and a Putative Solute Transport System

Abstract

cluster of the Pseudomonas sp. strain ADP atrazine-degradative plasmid pADP-1, which carries genes for an outer membrane protein and the components of a putative ABC-type solute transporter, is located downstream from atzR, which encodes the LysR-type transcriptional regulator of the cyanuric acid-degradative operon atzDEF. Here we describe the transcriptional organization of these genes. Our results show that all six genes are cotranscribed from the PatzR promoter to form the atzRSTUVW operon. A second, stronger promoter, PatzT, is found within atzS and directs transcription of the four distal genes. PatzT is N dependent, activated by NtrC in response to nitrogen limitation with the aid of IHF, and repressed by AtzR. A combination of in vivo mutational analysis and primer extension allowed us to locate the PatzT promoter and map the transcriptional start site. Similarly, we used deletion and point mutation analyses, along with in vivo expression studies and in vitro binding assays, to locate the NtrC, IHF, and AtzR binding sites and address their functionality. Our results suggest a regulatory model in which NtrC activates PatzT transcription via DNA looping, while AtzR acts as an antiactivator that diminishes expression by interfering with the activation process. P seudomonas sp. strain ADP (31) is the model organism for bacterial degradation of the s-triazine herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine). Pseudomonas sp. strain ADP uses atrazine as the sole nitrogen source via a six-step hydrolytic pathway, carried by the 108-kbp catabolic plasmid pADP-1 (35). The six genes involved in atrazine mineralization are localized in two distinct regions of the plasmid: atzA, atzB, and atzC, responsible for atrazine conversion to cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine), are harbored at three distant positions within a large (Ͼ40-kbp) unstable region featuring multiple direct repeats and transposable elements. The genes involved in cyanuric acid cleavage and ammonium release are clustered in the stable portion of pADP-1 to form the atzDEF operon (reviewed in reference 51).Our previous work has shown that atrazine utilization by Pseudomonas sp. strain ADP is regulated by nitrogen availability in a manner resembling general nitrogen control (15), and the cyanuric acid utilization operon atzDEF is one of the targets for such regulation (16). Expression of atzDEF is induced by the substrate of the pathway, cyanuric acid, and repressed in the presence of a preferential nitrogen source. The product of atzR, the gene transcribed divergently from atzDEF, is a LysR-type transcriptional regulator (LTTR) required for both nitrogen-and cyanuric aciddependent control. Transcription of atzR is initiated from the Ndependent PatzR promoter, activated by the general nitrogen control protein NtrC, and repressed by AtzR (16). NtrC-dependent activation of atzR is unusual in that it does not require interaction with any upstream or downstream sequence elements (41). AtzR binds a single site overlapping...

Mechanism of Antiactivation at the Pseudomonas sp. Strain ADP σN-Dependent PatzTPromoter

“…3, lane 2). This was previously observed (22) and suggests that the NtrC D55E,S161F -PatzT complex is unstable during gel electrophoresis. In contrast, AtzR-His 6 retarded the PatzT probe to form a single stable complex (Fig.…”

“…The location of the AtzR binding motif, centered at position Ϫ112, far upstream from the E-N binding motif and in the vicinity of the NtrC binding site NtrC2 (centered at Ϫ130), and the fact that AtzR cannot repress PatzT transcription when it is activated by NtrC in an UAS-independent fashion (22) suggest that rather than tampering with RNA polymerase function, AtzR may antagonize NtrC interaction with the PatzT promoter region (22). To test this possibility, gel mobility shift assays were performed in which PatzT-bound NtrC D55E,S161F was challenged with increasing concentrations of AtzR-His 6 and vice versa (Fig.…”

“…In addition, AtzR represses its own synthesis (17)(18)(19)(20)(21). Recently, we showed that atzR is cotranscribed with five additional genes: atzS, encoding a putative outer membrane protein, and the atzTUVW cluster, encoding an ABCtype transport system that is involved in high-affinity uptake of cyanuric acid (22,23). However, an additional N -dependent promoter, PatzT, located within the atzS coding region, is responsible for most of the transcription of the atzTUVW cluster (22).…”

“…1). Nevertheless, PatzT also displays significant levels of UAS-independent activation, suggesting a strong interaction between E-N and its highly conserved binding site (22). AtzR binds the PatzT promoter region at a single site, containing a strong recognition motif centered at position Ϫ112 (Fig.…”

“…Recently, we showed that atzR is cotranscribed with five additional genes: atzS, encoding a putative outer membrane protein, and the atzTUVW cluster, encoding an ABCtype transport system that is involved in high-affinity uptake of cyanuric acid (22,23). However, an additional N -dependent promoter, PatzT, located within the atzS coding region, is responsible for most of the transcription of the atzTUVW cluster (22). Similarly to PatzR, PatzT is activated by NtrC in response to nitrogen limitation and is repressed by AtzR in a cyanuric acidindependent fashion.…”