Protein depletion using the arabinose promoter in Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (X. citri) is a plant pathogen and the etiological agent of citrus canker, a severe disease that affects all the commercially important citrus varieties, and has worldwide distribution. Citrus canker cannot be healed, and the best method known to control the spread of X. citri in the orchards is the eradication of symptomatic and asymptomatic plants in the field. However, in the state of São Paulo, Brazil, the main orange producing area in the world, control is evolving to an integrated management system (IMS) in which growers have to use less susceptible plants, windshields to prevent bacterial spread out and sprays of cupric bactericidal formulations. Our group has recently proposed alternative methods to control citrus canker, which are based on the use of chemical compounds able to disrupt vital cellular processes of X. citri. An important step in this approach is the genetic and biochemical characterization of genes/proteins that are the possible targets to be perturbed, a task not always simple when the gene/protein under investigation is essential for the organism. Here, we describe vectors carrying the arabinose promoter that enable controllable protein expression in X. citri. These vectors were used as complementation tools for the clean deletion of parB in X. citri, a widespread and conserved gene involved in the essential process of bacterial chromosome segregation. Overexpression or depletion of ParB led to increased cell size, which is probably a resultant of delayed chromosome segregation with subsequent retard of cell division. However, ParB is not essential in X. citri, and in its absence the bacterium was fully competent to colonize the host citrus and cause disease. The arabinose expression vectors described here are valuable tools for protein expression, and especially, to assist in the deletion of essential genes in X. citri.

[DNA supercoiling and topoisomerases in Escherichia coli]. / Superenrollamiento del DNA y topoisomerasas de Escherichia coli.

The chromosomal DNA of all cells is under helical tension or supercoiling. There are two classes of DNA supercoiling: plectonemic and toroidal. Plectonemic supercoiling is generated by the action of DNA topoisomerases, while toroidal supercoiling is generated by DNA-protein interactions and by topoisomerase activitities. DNA supercoiling plays an important role in replication, repair, recombination, transposition and transcription. DNA topoisomerases type I are ATP-independent enzymes that cut one DNA strand and relax supercoiled molecules. DNA topoisomerases type II requiere ATP, cut both DNA strands and supercoil relaxed molecules. All organisms have more than one topoisomerase of each, type I and type II. Escherichia coli has two topoisomerases type I: topoisomerase I and topoisomerase III and two topoisomerases type II: topoisomerase II or gyrase and topoisomerase IV. In this review we discuss the concept of DNA supercoiling and present current knowledge on E. coli DNA topoisomerases.

[The bacterial nucleoid]. / El nucleoide bacteriano.

The bacterial genome is present in the cell within a complex structure, the nucleoid. The nucleoid contains the genomic DNA, and molecules of RNA and proteins. The main proteins of the nucleoid are: RNA polymerase, topoisomerases and the histone-like proteins: HU, H-NS (H1), H, HLP1, IHF and FIS. The DNA molecule in the nucleoid is under helical tension or supercoiling and is organized into 43 +/- 10 topodomains. DNA supercoiling is generated by the activity of the topoisomerases and by DNA-protein interactions. In this review, we analize current knowledge in Escherichia coli about genome organization and proteins of the nucleoid.