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2021
Bueno, Danilo; Pedrolli, Danielle B.; Martins, Paula M. M.; Bocchini, Daniela A.; Moraes, Karen C. M.; Facincani, Agda P.; Ferro, Jesus A.; Varani, Alessandro M.; Pena, Michelle M.; Ferreira, Henrique
Riboswitch theo/metE as a Transcription Regulation Tool for Xanthomonas citri subsp. citri Journal Article
In: Microorganisms 2021, Vol. 9, Page 329, vol. 9, iss. 2, pp. 329, 2021, ISSN: 2076-2607.
Abstract | Links | BibTeX | Tags: <i>parB</i>, Chromosome segregation, Citrus canker, pNPTS138 sequence, regulation of gene expression
@article{Bueno2021,
title = {Riboswitch theo/metE as a Transcription Regulation Tool for Xanthomonas citri subsp. citri},
author = {Danilo Bueno and Danielle B. Pedrolli and Paula M. M. Martins and Daniela A. Bocchini and Karen C. M. Moraes and Agda P. Facincani and Jesus A. Ferro and Alessandro M. Varani and Michelle M. Pena and Henrique Ferreira},
url = {https://www.mdpi.com/2076-2607/9/2/329/htm https://www.mdpi.com/2076-2607/9/2/329},
doi = {10.3390/MICROORGANISMS9020329},
issn = {2076-2607},
year = {2021},
date = {2021-01-01},
journal = {Microorganisms 2021, Vol. 9, Page 329},
volume = {9},
issue = {2},
pages = {329},
publisher = {Multidisciplinary Digital Publishing Institute},
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.},
keywords = {<i>parB</i>, Chromosome segregation, Citrus canker, pNPTS138 sequence, regulation of gene expression},
pubstate = {published},
tppubtype = {article}
}
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.
2017
Lacerda, Lilian A.; Cavalca, Lucia B.; Martins, Paula M. M.; Govone, José S.; Bacci, Maurício; Ferreira, Henrique
Protein depletion using the arabinose promoter in Xanthomonas citri subsp. citri Journal Article
In: Plasmid, vol. 90, pp. 44-52, 2017, ISSN: 0147-619X.
Abstract | Links | BibTeX | Tags: Bacterial gene knockout, Chromosome segregation, Citrus canker
@article{Lacerda2017,
title = {Protein depletion using the arabinose promoter in Xanthomonas citri subsp. citri},
author = {Lilian A. Lacerda and Lucia B. Cavalca and Paula M. M. Martins and José S. Govone and Maurício Bacci and Henrique Ferreira},
doi = {10.1016/J.PLASMID.2017.03.005},
issn = {0147-619X},
year = {2017},
date = {2017-01-01},
journal = {Plasmid},
volume = {90},
pages = {44-52},
publisher = {Academic Press},
abstract = {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.},
keywords = {Bacterial gene knockout, Chromosome segregation, Citrus canker},
pubstate = {published},
tppubtype = {article}
}
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.