XAC4296 Is a Multifunctional and Exclusive Xanthomonadaceae Gene Containing a Fusion of Lytic Transglycosylase and Epimerase Domains

Abstract

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.

Keywords: Xanthomonas citri; antimicrobial resistance; cell wall synthesis; citrus canker; gene fusion; multi-domains enzymes.

Figure 1

Genome context, protein domain, and structure of XAC4296. (A). Genome context of XAC4296 from X. citri genome. (B). Protein domain and structure of XAC4296. XAC4296 has 720 aa with the LT domain associated with 3B family: Transglycosylase SLT domain (SLT_2) (IPR031304.) and Peptidoglycan binding (PG_binding_1) (IPR002477) domains, and the Aldose 1-epimerase (IPR015443) domain. (C). Molecular modeling cartoon representation of the LT and epimerase XAC4296 domains.

Figure 2

(A). Maximum-likelihood phylogenetic tree of XAC4296 homologs across the Xanthomonadaceae family supports the XAC4296 potential origin before Xanthomonas, Xylella, Pseudoxanthomonas, and Stenotrophomonas differentiation. The closely-related Xylella genus lost the XAC4296 homolog. (B). Phylogenetic construction of the Xanthomonadaceae family phylogroup formed by Xanthomonas, Xylella, Pseudoxanthomonas, and Stenotrophomonas (based on [41]). The red arrow indicates the potential gene fusion event that originated the XAC4296 ancestor.

Figure 3

Morphological analysis and errors/aberrant nucleoid distribution of X. citri, Δ4296, and Δ4296c strains. Short filaments, chain phenotype, and errors/unusual nucleoid distribution were intensified without XAC4296. The figure shows microscopy phase contrast, DAPI, and overlay of the two filters for (A) X. citri WT; (B) Δ4296, and (C) Δ4296c (pMAJIIc-XAC4296) (magnification of 100×). White arrows indicate nucleoid distribution; red arrows indicate septum constriction (magnification of 100×)—scale = 5 µm.

Figure 4

XAC4296 may be related to β-lactam antibiotic resistance in X. citri (A) Ex planta bacterial growth curves performed on rich medium and ampicillin 20 µg/mL for X. citri, Δ4296, and Δ4296c. Δ4296 growth is affected in the presence of ampicillin. Error bars indicate the standard error of three independent biological and technical replicates. (B) Morphological analysis and errors/aberrant nucleoid distribution of X. citri, Δ4296, and Δ4296c strains of X. citri and Δ4296 strains on NB supplemented with ampicillin 20 µg/mL. All cultures of mutant Δ4296 exhibit short filaments and chain phenotype. The figure shows the phase contrast, DAPI, and overlay of the two images for X. citri and Δ4296 (magnification of 100×). White arrows indicate the chromosome position, and red arrows indicate septum constriction (magnification of 100×)—scale = 5 µm.

Figure 5

Short filaments and chain phenotype show partial reversion in sucrose’s presence as carbon source (A) Morphological analysis of X. citri, Δ4296, and Δ4296c strains on NB supplemented with sucrose 0.1% (w/v). (B) NB supplemented with sucrose 0.1% (w/v) and ampicillin 20 µg/mL. The figure shows the phase contrast, DAPI, and overlay of the two filters for X. citri; Δ4296 and Δ4296c. White arrows indicate the chromosome distribution, and red arrows indicate septum constriction (magnification of 100×)—scale = 5 µm.

Figure 6

Short filaments, chain phenotype, and nucleoid organization show full reversion in glutamate’s presence as carbon source (A) Morphological analysis of X. citri, Δ4296, and Δ4296c strains on NB supplemented with glutamic acid 0.1% (w/v). (B) NB supplemented with glutamic acid 0.1% (w/v) and ampicillin 20 µg/mL. The figure shows the phase contrast, DAPI, and overlay of the two filters for X. citri, Δ4296, and Δ4296c (magnification of 100×)—Scale = 5 µm.