Citrus canker is a disease caused by the gram-negative bacterium Xanthomonas citri subp. citri (X. citri), which affects all commercially important varieties of citrus and can lead to significant losses. Fruit sanitization with products such as chlorine-based ones can reduce the spread of the disease. While effective, their use raises concerns about safety of the workers. This work proposes essential oils (EOs) as viable alternatives for fruit sanitization. EOs from Cymbopogon species were evaluated as to their antibacterial activity, their effect on the bacterial membrane, and their ability to sanitize citrus fruit. The in vitro assays revealed that the EOs from C. schoenanthus and C. citratus had a lower bactericidal concentration at 312 mg L⁻¹, followed by 625 mg L⁻1 for C. martini and C. winterianus. Microscopy assay revealed that the bacterial cell membranes were disrupted after 15 min of contact with all EOs tested. Regarding the sanitizing potential, the EOs with higher proportions of geraniol were more effective in sanitizing acid limes. Fruit treated with C. shoenanthus and C. martini showed a reduction of ∼68% in the recovery of viable bacterial cells. Therefore, these EOs can be used as viable natural alternatives in citrus fruit disinfection.

Peptidoglycan hydrolases are enzymes responsible for breaking the peptidoglycan present in the bacterial cell wall, facilitating cell growth, cell division and peptidoglycan turnover. Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker, encodes an Escherichia coli M23 peptidase EnvC homolog. EnvC is a LytM factor essential for cleaving the septal peptidoglycan, thereby facilitating the separation of daughter cells. In this study, the investigation focused on EnvC contribution to the virulence and cell separation of X. citri. It was observed that disruption of the X. citri envC gene (ΔenvC) led to a reduction in virulence. Upon inoculation into leaves of Rangpur lime (Citrus limonia Osbeck), the X. citri ΔenvC exhibited a delayed onset of citrus canker symptoms compared with the wild-type X. citri. Mutant complementation restored the wild-type phenotype. Sub-cellular localization confirmed that X. citri EnvC is a periplasmic protein. Moreover, the X. citri ΔenvC mutant exhibited elongated cells, indicating a defect in cell division. These findings support the role of EnvC in the regulation of cell wall organization, cell division, and they clarify the role of this peptidase in X. citri virulence.

Microorganisms have a limited and highly adaptable repertoire of genes capable of encoding proteins containing single or variable multidomains. The phytopathogenic bacteria Xanthomonas citri subsp. citri (X. citri) (Xanthomonadaceae family), the etiological agent of Citrus Canker (CC), presents a collection of multidomain and multifunctional enzymes (MFEs) that remains to be explored. Recent studies have shown that multidomain enzymes that act on the metabolism of the peptidoglycan and bacterial cell wall, belonging to the Lytic Transglycosylases (LTs) superfamily, play an essential role in X. citri biology. One of these LTs, named XAC4296, apart from the Transglycosylase SLT_2 and Peptidoglycan binding-like domains, contains an unexpected aldose 1-epimerase domain linked to the central metabolism; therefore, resembling a canonical MFE. In this work, we experimentally characterized XAC4296 revealing its role as an MFE and demonstrating its probable gene fusion origin and evolutionary history. The XAC4296 is expressed during plant-pathogen interaction, and the Δ4296 mutant impacts CC progression. Moreover, Δ4296 exhibited chromosome segregation and cell division errors, and sensitivity to ampicillin, suggesting not only LT activity but also that the XAC4296 may also contribute to resistance to β-lactams. However, both Δ4296 phenotypes can be restored when the mutant is supplemented with sucrose or glutamic acid as a carbon and nitrogen source, respectively; therefore, supporting the epimerase domain’s functional relationship with the central carbon and cell wall metabolism. Taken together, these results elucidate the role of XAC4296 as an MFE in X. citri, also bringing new insights into the evolution of multidomain proteins and antimicrobial resistance in the Xanthomonadaceae family.

Peptidoglycan hydrolases are enzymes that cleave the peptidoglycan of the bacterial cell wall, facilitating cell growth, cell division, and peptidoglycan turnover. Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker, encodes an Escherichia coli M23 peptidase EnvC homolog. EnvC is a LytM factor required for septal peptidoglycan splitting and daughter cell separation. In this study we investigate how EnvC contributes to the virulence and cell separation of X. citri. We observed that disruption of the X. citri envC gene (ΔenvC) led to a reduction in virulence. Upon inoculation into leaves of Rangpur lime (Citrus limonia Osbeck), the X. citri ΔenvC mutant showed a delay in causing citrus canker symptoms compared with the wild-type X. citri strain. Mutant complementation restored the X. citri wild type phenotype. Subcellular localization confirmed that X. citri EnvC is a periplasmic protein. Moreover, the X. citri ΔenvC mutant exhibited elongated cells, indicative of a cell division defect. These findings support the role of EnvC in regulating virulence, cell wall organization, and cell division in X. citri.