Abstract
Xanthomonas citri subsp. citri (X. citri) is the causal agent of Asiatic Citrus Canker (ACC), a disease that affects citrus. ACC has no cure, and growers must rely on special agricultural practices to prevent bacterial spreading. Understanding X. citri basic biology is essential to foresee potential genetic targets to control ACC. Traditionally, microbial genetics use gene deletion/disruption to investigate gene function. However, essential genes are difficult to study this way. Techniques based on small-RNAs and antisense-RNAs are powerful for gene characterization, but not yet fully explored in prokaryotes. One alternative is riboswitches, which derive from bacteria, and can control transcription/translation. Riboswitches are non-coding RNAs able to modulate gene expression in the presence of specific ligands. Here we demonstrate that the riboswitch theo/metE decreases parB expression in X. citri in a platform responsive to theophylline. By monitoring cell respiration, we showed that higher concentrations of the ligand interfered with bacterial viability. Therefore, we determined the safe dose of theophylline to be used with X. citri. Finally, in downstream investigations of parB transcription modulation, we show evidence for the fact that ParB is stable, remains functional throughout the cell cycle, and is inherited by the daughter cells upon cell division.
Highlights
The Gram-negative bacterium Xanthomonas citri subsp. citri [1] is the causal agent of Asiatic Citrus Canker, one of the major causes of yield losses in the production of sweet oranges in citrus growing areas around the world [2,3]
There is no treatment for citrus canker, and nowadays, control of this disease is done by the application of a set of agricultural measures intended to avoid the spread of X. citri amongst orchards and to areas free of its occurrence [2]
PNPTS138 is replicative in E. coli and a suicide vector in many other Gram-negative bacteria, including X. citri [24,26,27,37]
Summary
The Gram-negative bacterium Xanthomonas citri subsp. citri [1] is the causal agent of Asiatic Citrus Canker, one of the major causes of yield losses in the production of sweet oranges in citrus growing areas around the world [2,3]. The investigation of gene expression and functionality are, in general, conducted based on either transposon disruption or gene deletion/complementation seeking to understand their roles in several aspects of the cell life cycle. These are the basis for genetic analyses in any living organism, many drawbacks hinder effectiveness. Upon obtaining cells having this extra copy of the essential gene that will be characterized, the original one can be deleted, and modulation of gene expression from the inducible promoter allows the functional study of the essential gene This strategy was already used in X. citri to characterize the protein ParB [6].
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