Structural basis for inhibition of homologous recombination by the RecX protein

The RecA/RAD51 nucleoprotein filament is central to the reaction of homologous recombination (HR). Filament activity must be tightly regulated in vivo as unrestrained HR can cause genomic instability. Our mechanistic understanding of HR is restricted by lack of structural information about the regulatory proteins that control filament activity. Here, we describe a structural and functional analysis of the HR inhibitor protein RecX and its mode of interaction with the RecA filament. RecX is a modular protein assembled of repeated three-helix motifs. The relative arrangement of the repeats generates an elongated and curved shape that is well suited for binding within the helical groove of the RecA filament. Structure-based mutagenesis confirms that conserved basic residues on the concave side of RecX are important for repression of RecA activity. Analysis of RecA filament dynamics in the presence of RecX shows that RecX actively promotes filament disassembly. Collectively, our data support a model in which RecX binding to the helical groove of the filament causes local dissociation of RecA protomers, leading to filament destabilisation and HR inhibition

Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity

Expression systems based on the Escherichia coli arabinose operon P(BAD) promoter exhibit the all-or-nothing (autocatalytic) induction of expression that was first documented in the lac operon. Under conditions of subsaturating levels of inducer, some of the cells of the population are fully induced, whereas other cells remain uninduced. Recently, a new AraE transporter system was reported to have circumvented the problem of autocatalytic expression in the pBAD expression vectors and to provide graded and homogeneous cell-to-cell expression in the presence of variable inducer concentrations [Khlebnikov, A., Risa, O., Skaug, T., Carrier, T. A. & Keasling, J. D. (2000) J. Bacteriol. 182, 7029–7034]. However, we report that nonuniform gene expression in the AraE system was readily detectable by the use of mutant green fluorescent proteins that are rapidly degraded in E. coli. We report an approach to avoid all-or-nothing induction of the pBAD promoter; the use of a mutant LacY transporter in a strain deficient in both arabinose transport (araE araFGH) and degradation (araBAD). This mutant LacY protein performs facilitated diffusion of arabinose resulting in homogeneous expression of an unstable GFP that is maintained over extended incubation times at subsaturating levels of inducer. This approach is readily adapted to other sugar-regulated expression systems

Agrobacterium tumefaciens fur Has Important Physiological Roles in Iron and Manganese Homeostasis, the Oxidative Stress Response, and Full Virulence▿

In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the fur mutant was more sensitive than the wild type to an iron chelator, 2,2′-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations. A. tumefaciens sitA, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (sitABCD), was strongly repressed by Mn2+ and, to a lesser extent, by Fe2+, and this regulation was Fur dependent. Moreover, the fur mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the fur mutant showed constitutive up-expression of the manganese uptake sit operon. FurAt showed a regulatory role under iron-limiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The fur mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the fur mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4

Roles of Agrobacterium tumefaciens RirA in Iron Regulation, Oxidative Stress Response, and Virulence▿

The analysis of genetics and physiological functions of Agrobacterium tumefaciens RirA (rhizobial iron regulator) has shown that it is a transcription regulator and a repressor of iron uptake systems. The rirA mutant strain (NTLrirA) overproduced siderophores and exhibited a highly constitutive expression of genes involved in iron uptake (fhuA, irp6A, and fbpA) compared to that of the wild-type strain (NTL4). The deregulation in the iron control of iron uptake in NTLrirA led to iron overload in the cell, which was supported by the observation that the NTLrirA mutant was more sensitive than wild-type NTL4 to an iron-activated antibiotic, streptonigrin. The NTLrirA mutant was more sensitive than the parental strain to oxidants, including hydrogen peroxide, organic hydroperoxide, and a superoxide generator, menadione. However, the addition of an iron chelator, 2,2′-dipyridyl, reversed the mutant hypersensitivity to H2O2 and organic hydroperoxide, indicating the role of iron in peroxide toxicity. Meanwhile, the reduced level of superoxide dismutase (SodBIII) was partly responsible for the menadione-sensitive phenotype of the NTLrirA mutant. The NTLrirA mutant showed a defect in tumorigenesis on tobacco leaves, which likely resulted from the increased sensitivity of NTLrirA to oxidants and the decreased ability of NTLrirA to induce virulence genes (virB and virE). These data demonstrated that RirA is important for A. tumefaciens during plant-pathogen interactions