SUMMARY Species within the genus Burkholderia exhibit remarkable phenotypic diversity. Genomic plasticity, including genome reduction and horizontal gene transfer, has been correlated with virulence traits in several species. However, the conservation of virulence genes in species otherwise considered to have limited potential for infection suggests that phenotypic diversity may not be explained solely on the basis of genetic diversity. Instead, differential organization and control of gene regulatory networks may underlie many phenotypic differences. In this review, we evaluate how regulation of gene expression by members of the multiple antibiotic resistance regulator (MarR) family of transcription factors may contribute to shaping the physiological diversity of Burkholderia species, with a focus on the clinically relevant human pathogens. All Burkholderia species encode a relatively large number of MarR proteins, a feature common to bacteria that must respond to environmental changes such as those associated with host invasion. However, evolution of gene regulatory networks has likely resulted in orthologous transcription factors controlling disparate sets of genes. Adaptation to, and survival in, diverse habitats, including a human or plant host, is key to the success of Burkholderia species as (opportunistic) pathogens, and recent reports suggest that control of virulence-associated genes by MarR proteins features prominently among the survival strategies employed by these species. We suggest that identification of MarR regulons will contribute significantly to clarification of virulence determinants and phenotypic diversity.

Fragment analysis was developed to determine the sizes of DNA fragments relative to size standards of known lengths using a capillary electrophoresis genetic analyzer. This approach has since been adapted for use in DNA footprinting. However, DNA footprinting requires accurate determination of both fragment length and intensity, imposing specific demands on the experimental design. Here we delineate essential considerations involved in optimizing the fragment analysis workflow for use in DNase I footprinting to ensure that changes in DNase I cleavage patterns may be reliably identified.

Fatty acid hydroperoxides are involved in host-pathogen interactions. In both plants and mammals, polyunsaturated fatty acids are liberated during infection and enzymatically oxidized to the corresponding toxic hydroperoxides during the defensive oxidative burst that is designed to thwart the infection. The bacterial transcription factor OhrR (organic hydroperoxide reductase regulator) is oxidized by organic hydroperoxides, as a result of which the gene encoding organic hydroperoxide reductase is induced. This enzyme converts the hydroperoxides to less toxic alcohols. We show here that OhrR from represses expression of Gene expression is induced by cumene hydroperoxide and to a lesser extent by inorganic oxidants; however, Ohr contributes to degradation only of the organic hydroperoxide. OhrR, which binds specific sites in both and promoters, as evidenced by DNase I footprinting, belongs to the 2-Cys subfamily of OhrR proteins, and its oxidation leads to reversible disulfide bond formation between conserved N- and C-terminal cysteines in separate monomers. Oxidation of the N-terminal Cys is sufficient for attenuation of DNA binding , with complete restoration of DNA binding occurring on addition of a reducing agent. Surprisingly, both overexpression of and deletion of results in enhanced survival on exposure to organic hydroperoxide While Δ cells are more virulent in a model of infection, Δ cells are less so. Taken together, our data suggest that OhrR has several unconventional features and that both OhrR and organic hydroperoxides may contribute to virulence.

The plant pathogen Pectobacterium atrosepticum encounters a stressful environment when it colonizes the plant apoplast. Chief among the stressors are the reactive oxygen species (ROS) that are produced by the host as a first line of defense. Bacterial transcription factors in turn use these signals as cues to upregulate expression of virulence-associated genes. We have previously shown that the transcription factor PecS from P. atrosepticum binds the promoters that drive expression of pecS and pecM, which encodes an efflux pump, to repress gene expression. We show here that addition of oxidant relieves repression in vivo and in vitro. While reduced PecS distorts promoter DNA on binding, oxidized PecS does not, as evidenced by DNaseI footprinting. PecS oxidation is reversible, as shown by an oxidant-dependent quenching of the intrinsic tryptophan fluorescence that is completely reversed upon addition of a reducing agent. Cysteine 45 positioned at the PecS dimer interface is the redox sensor. Reduced PecS-C45A causes less DNA distortion on binding compared to wild-type PecS; addition of an oxidant has no effect on binding, and PecS-C45A cannot repress gene expression. Our data suggest that reduced PecS distorts its cognate DNA on binding, perhaps inducing a conformation in which promoter elements are suboptimally aligned for RNA polymerase binding, resulting in transcriptional repression. In contrast, oxidized PecS binds promoter DNA such that RNA polymerase may successfully compete with PecS for binding, allowing gene expression. This mode of regulation would facilitate induction of the PecS regulon when the bacteria encounter host-derived ROS in the plant apoplast.

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