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The copper resistance determinant copARZ, which encodes a CPx-type copper ATPase efflux protein, a transcriptional regulator, and a putative intracellular copper chaperone, was functionally characterized for the phytopathogenic bacterium Agrobacterium tumefaciens. These genes are transcribed as an operon, and their expression is induced in response to increasing copper and silver ion concentrations in a copR-dependent fashion. Analysis of the copARZ promoter revealed a putative CopR binding box located within the spacer of the -35 and -10 promoter motifs. In vitro, purified CopR could specifically bind to the box. The inactivation of the copARZ operon or copZ reduces the level of resistance to copper but not to other metal ions. Also, the copARZ operon mutant shows increased sensitivity to the superoxide generators menadione and plumbagin. In addition, the loss of functional copZ does not affect the ability of copper ions to induce the copARZ promoter, indicating that CopZ is not involved in the copper-sensing mechanism of CopR. Altogether, the results demonstrate a crucial role for the copARZ operon as a component of the copper resistance machinery in A. tumefaciens.

Agrobacterium tumefaciens transferred DNA (T-DNA) transfer requires that the virulence genes (vir regulon) on the tumor-inducing (Ti) plasmid be induced by plant phenolic signals in an acidic environment. Using transcriptome analysis, we found that these acidic conditions elicit two distinct responses: (i) a general and conserved response through which Agrobacterium modulates gene expression patterns to adapt to environmental acidification and (ii) a highly specialized acid-mediated signaling response involved in Agrobacterium-plant interactions. Overall, 78 genes were induced and 74 genes were repressed significantly under acidic conditions (pH 5.5) compared to neutral conditions (pH 7.0). Microarray analysis not only confirmed previously identified acid-inducible genes but also uncovered many new acid-induced genes which may be directly involved in Agrobacterium-plant interactions. These genes include virE0, virE1, virH1, and virH2. Further, the chvG-chvI two-component system, previously shown to be critical for virulence, was also induced under acid conditions. Interestingly, acidic conditions induced a type VI secretion system and a putative nonheme catalase. We provide evidence suggesting that acid-induced gene expression was independent of the VirA-VirG two-component system. Our results, together with previous data, support the hypothesis that there is three-step sequential activation of the vir regulon. This process involves a cascade regulation and hierarchical signaling pathway featuring initial direct activation of the VirA-VirG system by the acid-activated ChvG-ChvI system. Our data strengthen the notion that Agrobacterium has evolved a mechanism to perceive and subvert the acidic conditions of the rhizosphere to an important signal that initiates and directs the early virulence program, culminating in T-DNA transfer.

Agrobacterium tumefaciens possesses three iron-containing superoxide dismutases (FeSods) encoded by distinct genes with differential expression patterns. SodBI and SodBII are cytoplasmic isozymes, while SodBIII is a periplasmic isozyme, sodBI is expressed at a high levels throughout all growth phases, sodBII expression is highly induced upon exposure to superoxide anions in a SoxR-dependent manner, sodBIII is expressed only during stationary phase. Analysis of the physiological function of sods reveals that the inactivation of sodBI markedly reduced levels of resistance to a superoxide generator, menadione. A mutant lacking all three Sod enzymes is the most sensitive to menadione treatment, indicating that all sods contribute at various levels towards the overall menadione resistance level. Sods also have important roles in A. tumefaciens virulence toward a host plant. A sodBI but not a sodBII or sodBIII mutant showed marked reduction in its ability to induce tumors on tobacco leaf discs, while the triple sod null mutant is avirulent.

In the prospect of engineering cyanobacteria for the biological photoproduction of hydrogen, we have studied the hydrogen production machine in the model unicellular strain Synechocystis PCC6803 through gene deletion, and overexpression (constitutive or controlled by the growth temperature). We demonstrate that the hydrogenase-encoding hoxEFUYH operon is dispensable to standard photoautotrophic growth in absence of stress, and it operates in cell defense against oxidative (H2O2) and sugar (glucose and glycerol) stresses. Furthermore, we showed that the simultaneous over-production of the proteins HoxEFUYH and HypABCDE (assembly of hydrogenase), combined to an increase in nickel availability, led to an approximately 20-fold increase in the level of active hydrogenase. These novel results and mutants have major implications for those interested in hydrogenase, hydrogen production and redox metabolism, and their connections with environmental conditions. [ABSTRACT FROM AUTHOR]

Sco (for the synthesis of cytochrome c oxidase) is a mitochondrial membrane protein essential for the correct assembly of cytochrome c oxidase. sco homolog genes exist in a wide variety of bacterial species. Inactivation of Agrobacterium tumefaciens sco leads to markedly decreased cytochrome c oxidase activity. This phenotype can be complemented by either supplementing the culture medium with copper or by a plasmid containing sco. The sco mutant also alters resistance to a superoxide generator menadione and H2O2. Mutational analysis of conserved cysteine residues and a histidine residue within the putative copper ions binding motif of Sco indicates that these residues are essential for the biological activity of Sco. To summarize, we find that A. tumefaciens sco is required for the delivery of copper into active cytochrome c oxidase and in maintaining optimal resistance levels to oxidative stress. [ABSTRACT FROM AUTHOR]

Singlet oxygen is a highly reactive form of molecular oxygen that is harmful to biological systems. Here, the role of three iron-containing superoxide dismutase ( sodB) genes is clearly shown in protecting Agrobacterium tumefaciens against singlet oxygen toxicity. A sodBI mutant was more sensitive to singlet oxygen than both wild-type bacteria and a double sodBII–sodBIII mutant strain. Moreover, a sodBI– sodBII double mutant had higher sensitivity to singlet oxygen than a single sodBI mutant, although the double mutant was comparable to a sodB null mutant. High-level expression of sodBI and sodBII fully complemented the singlet oxygen hypersensitivity phenotype of the sodB null mutant, while high-level expression of sodBIII encoding a periplasmic SOD only partially restored the phenotype. Taken together, our data suggest that SodBI and SodBII have novel protective roles against singlet oxygen toxicity through unknown mechanisms. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens is capable of transferring and integrating an oncogenic T-DNA (transferred DNA) from its tumor-inducing (Ti) plasmid into dicotyledonous plants. This transfer requires that the virulence genes (vir regulon) be induced by plant signals such as acetosyringone in an acidic environment Salicylic acid (SA) is a key signal molecule in regulating plant defense against pathogens. However, how SA influences Agrobacterium and its interactions with plants is poorly understood. Mere we show that SA can directly shut down the expression of the vir regulon. SA specifically inhibited the expression of the Agrobacterium virAiG two-component regulatory system that tightly controls the expression of the vir regulon including the repABC operon on the Ti plasmid. We provide evidence suggesting that SA attenuates the function of the VirA kinase domain. Independent of its effect on the vir regulon, SA up-regulated the attKLM operon, which functions in degrading the bacterial quormone N-acylhomoserine lactone. Plants defective in SA accumulation were more susceptible to Agrobacterium infection, whereas plants overproducing SA were relatively recalcitrant to tumor formation. Our results illustrate that SA, besides its well known function in regulating plant defense, can also interfere directly with several aspects of the Agrobacterium infection process. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens transferred DNA (T-DNA) transfer requires that the virulence genes (vir regulon) on the tumor-inducing (Ti) plasmid be induced by plant phenolic signals in an acidic environment. Using transcriptome analysis, we found that these acidic conditions elicit two distinct responses: (i) a general and conserved response through which Agrobacterium modulates gene expression patterns to adapt to environmental acidification and (ii) a highly specialized acid-mediated signaling response involved in Agrobacterium-plant interactions. Overall, 78 genes were induced and 74 genes were repressed significantly under acidic conditions (pH 5.5) compared to neutral conditions (pH 7.0). Microarray analysis not only confirmed previously identified acid-inducible genes but also uncovered many new acid-induced genes which may be directly involved in Agrobacterium-plant interactions. These genes include virE0, virE1, virH1, and virH2. Further, the chvG-chvI two-component system, previously shown to be critical for virulence, was also induced under acid conditions. Interestingly, acidic conditions induced a type VI secretion system and a putative nonheme catalase. We provide evidence suggesting that acid-induced gene expression was independent of the VirA-VirG two-component system. Our results, together with previous data, support the hypothesis that there is three-step sequential activation of the vir regulon. This process involves a cascade regulation and hierarchical signaling pathway featuring initial direct activation of the VirA-VirG system by the acid-activated ChvG-ChvI system. Our data strengthen the notion that Agrobacterium has evolved a mechanism to perceive and subvert the acidic conditions of the rhizosphere to an important signal that initiates and directs the early virulence program, culminating in T-DNA transfer. [ABSTRACT FROM AUTHOR]