The ability to maintain intracellular concentrations of toxic reactive oxygen species (ROS) within safe limits is essential for all aerobic life forms. In bacteria, as well as other organisms, ROS are produced during the normal course of aerobic metabolism, necessitating the constitutive expression of ROS scavenging systems. However, bacteria can also experience transient high-level exposure to ROS derived either from external sources, such as the host defense response, or as a secondary effect of other seemingly unrelated environmental stresses. Consequently, transcriptional regulators have evolved to sense the levels of ROS and coordinate the appropriate oxidative stress response. Three well-studied examples of these are the peroxide responsive regulators OxyR, PerR, and OhrR. OxyR and PerR are sensors of primarily H 2 O 2 , while OhrR senses organic peroxide (ROOH) and sodium hypochlorite (NaOCl). OxyR and OhrR sense oxidants by means of the reversible oxidation of specific cysteine residues. In contrast, PerR senses H 2 O 2 via the Fe-catalyzed oxidation of histidine residues. These transcription regulators also influence complex biological phenomena, such as biofilm formation, the evasion of host immune responses, and antibiotic resistance via the direct regulation of specific proteins.

The genes involved in organic hydroperoxide protection in Agrobacterium tumefaciens were functionally evaluated. Gene inactivation studies and functional analyses have identified ohr, encoding a thiol peroxidase, as the gene primarily responsible for organic hydroperoxide protection in A. tumefaciens. An ohr mutant was sensitive to organic hydroperoxide killing and had a reduced capacity to metabolize organic hydroperoxides. ohr is located next to, and is divergently transcribed from, ohrR, encoding a sensor and transcription regulator of organic hydroperoxide stress. Transcription of both ohr and ohrR was induced by exposure to organic hydroperoxides but not by exposure to other oxidants. This induction required functional ohrR. The results of gel mobility shift and DNase I footprinting assays with purified OhrR, combined with in vivo promoter deletion analyses, confirmed that OhrR regulated both ohrR and ohr by binding to a single OhrR binding box that overlapped the ohrR and ohr promoters. ohrR and ohr are both required for the establishment of a novel cumene hydroperoxide-induced adaptive response. Inactivation or overexpression of other Prx family genes (prx1, prx2, prx3, bcp1, and bcp2) did not affect either the resistance to, or the ability to degrade, organic hydroperoxide. Taken together, the results of biochemical, gene regulation and physiological studies support the role of ohrR and ohr as the primary system in sensing and protecting A. tumefaciens from organic hydroperoxide stress.

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The psbH gene encodes a small protein which copurifies with photosystem 2. In the cyanobacterium Synechocystis sp. PCC 6803, psbH is located upstream of the cytochrome b6-f complex genes petC and petA. In striking contrast, in the genomes of plant chloroplasts, psbH is cotranscribed with petB and petD, encoding the other two major subunits of the cytochrome b6-f complex. We report that in Synechocystis sp. PCC 6803 monocistronic psbH and dicistronic petCA transcripts are probably initiated separately, each from DNA regions bearing some similarity to Escherichia coli sigma 70 promoters. Synechocystis sp. PCC 6803 psbH null mutants were generated by cartridge mutagenesis. Studies using a rapid screening procedure involving in situ complementation showed that the PsbH protein is not absolutely required for the assembly of a functionally active photosystem 2 complex since psbH insertion and deletion strains were able to grow photoautotrophically. The rate of photoautotrophic growth was, however, slower than the wild type, and studies of oxygen evolution, chlorophyll fluorescence, and thermoluminescence indicated that this reduction in growth rate is probably due mainly to an impairment in electron flow from QA to QB. We conclude, therefore, that the PsbH protein is not an absolute requirement for photosystem 2 activity but that it functions to optimize electron flow between the two secondary plastoquinone acceptors by interacting with the QB site on the D1 protein.

The form I (cbb I ) Calvin-Benson-Bassham (CBB) reductive pentose phosphate cycle operon of Rhodobacter sphaeroides is regulated by both the transcriptional activator CbbR and the RegA/PrrA (RegB/PrrB) two-component signal transduction system. DNase I footprint analyses indicated that R. sphaeroides CbbR binds to the cbb I promoter between ؊10 and ؊70 base pairs (bp) relative to the cbb I transcription start. A cosmid carrying the R. capsulatus reg locus was capable of complementing an R. sphaeroides regA-deficient mutant to phototrophic growth with restored regulated synthesis of both photopigments and ribulose-bisphosphate carboxylase/oxygenase (Rubisco). DNase I footprint analyses, using R. capsulatus RegA*, a constitutively active mutant version of RegA, detected four RegA* binding sites within the cbb I promoter. Two sites were found within a previously identified cbb I promoter proximal regulatory region from ؊61 to ؊110 bp. One of these proximal RegA* binding sites overlapped that of CbbR. Two sites were within a previously identified promoter distal positive regulatory region between ؊301 and ؊415 bp. Expression from promoter insertion mutants showed that the function of the promoter distal regulatory region was helical phase-dependent. These results indicated that RegA exerts its regulatory affect on cbb I expression through direct interaction with the cbb I promoter.The nonsulfur purple bacterium Rhodobacter sphaeroides is capable of both dark aerobic chemoheterotrophic growth and anoxic photosynthetic growth. Under photoautotrophic growth conditions, where CO 2 functions as the sole carbon source, the Calvin-Benson-Bassham (CBB) 1 reductive pentose phosphate cycle provides nearly all cellular carbon. Photosynthetic growth in the presence of fixed carbon sources (i.e. photoheterotrophic growth) changes the primary role of the CBB cycle. Under these growth conditions, the CBB cycle facilitates the use of CO 2 as an electron sink and terminal electron acceptor for reducing equivalents generated by carbon oxidation and photosynthesis (1). In R. sphaeroides, control of CBB cycle gene (cbb) expression is achieved through the regulated expression of two major cbb operons, denoted form I (cbb I ) and form II (cbb II ) (2, 3). These operons are located on separate genetic elements in this organism (4, 5). The cbb I operon comprises structural genes that encode CBB cycle enzymes, including fructose 1,6-sedoheptulose 1,7-bisphosphatase (cbbF I ), phosphoribulokinase (cbbP I ), fructose 1,6-sedoheptulose 1,7-bisphosphate aldolase (cbbA I ), as well as the large and small subunit genes of form I (L 8 S 8 ) ribulose-bisphosphate carboxylase/oxygenase (Rubisco) (cbbL I cbbS I ) (6). The cbb II operon encodes homologs of cbbF I , P I , and A I as well as the genes for transketolase (cbbT II ), glyceraldehyde-3-phosphate dehydrogenase (cbbG II ) and the large subunit of the form II-type Rubisco (cbbM II ) (7). Studies over the years have shown that the regulation of cbb gene expression in R. sphaeroides is complex (2)...