Modified Monosaccharides Content of Xanthan Gum Impairs Citrus Canker Disease by Affecting the Epiphytic Lifestyle of Xanthomonas citri subsp. citri

Xanthomonas citri subsp. citri (X. citri) is a plant pathogenic bacterium causing citrus canker disease. The xanA gene encodes a phosphoglucomutase/phosphomannomutase protein that is a key enzyme required for the synthesis of lipopolysaccharides and exopolysaccharides in Xanthomonads. In this work, firstly we isolated a xanA transposon mutant (xanA::Tn5) and analyzed its phenotypes as biofilm formation, xanthan gum production, and pathogenesis on the sweet orange host. Moreover, to confirm the xanA role in the impaired phenotypes we further produced a non-polar deletion mutant (ΔxanA) and performed the complementation of both xanA mutants. In addition, we analyzed the percentages of the xanthan gum monosaccharides produced by X. citri wild-type and xanA mutant. The mutant strain had higher ratios of mannose, galactose, and xylose and lower ratios of rhamnose, glucuronic acid, and glucose than the wild-type strain. Such changes in the saccharide composition led to the reduction of xanthan yield in the xanA deficient strain, affecting also other important features in X. citri, such as biofilm formation and sliding motility. Moreover, we showed that xanA::Tn5 caused no symptoms on host leaves after spraying, a method that mimetics the natural infection condition. These results suggest that xanA plays an important role in the epiphytical stage on the leaves that is essential for the successful interaction with the host, including adaptive advantage for bacterial X. citri survival and host invasion, which culminates in pathogenicity.


Introduction
Xanthomonas citri subsp. citri (X. citri) is a Gram-negative plant pathogenic bacterium causing citrus canker, one of the most economically damaging diseases that affects almost all commercial citrus varieties worldwide [1,2]. X. citri invades the host through natural openings, such as stomata and wounds [2], and many studies have shown that biofilms formed on the leaf surface and bacterial motility are important features in the early stages of infection [3,4]. Notably, mutants of X. citri impaired in biofilm formation, and/ or motility results in the decrease of citrus canker symptoms [3][4][5][6][7][8][9]. Additionally, either biofilm or sliding motility is affected by X. citri ability to produce exopolysaccharides (EPS) [4]. Therefore, EPS production in X. citri interferes directly with citrus canker disease development.
The main EPS produced by Xanthomonads is the xanthan, which is a polymer of repeating pentasaccharide units with the mannose-(β-1,4)-glucuronic acid-(β-1,2)-mannose-(α-1,3)-cellobiose structure [10,11]. Production of xanthan involves many genes, including the gum gene cluster, composed of twelve genes (gumBCDEFGHIJKLM), and the xanA gene (XAC3579), that encodes a phosphoglucomutase/phosphomannomutase (PGM/PMM) homologous [11,12]. XanA is a conserved enzyme in the Xanthomonadaceae family that provides essential 1-phosphosugars required for the biosynthesis of lipopolysaccharide and exopolysaccharide [11,13]. Genetic and biochemical analysis showed that X. campestris XanA is involved in the biosynthesis of both glucose 1-phosphate and mannose 1-phosphate and that a single mutation in xanA led to a drastic decrease of enzyme activity, though low levels of the activity remained detectable [11]. Furthermore, colonies of the X. citri xanA mutant were less viscous/sticky than the wild type and did not show PGM activity [12], besides demonstrated late canker symptoms on infiltrated and detached Citrus aurantifolia leaves [12].
Although a previous study in X. citri indicated that the PGM activity of XanA may be involved with xanthan synthesis, xanthan gum content and yield was not analyzed. Furthermore, the method used to verify bacterial pathogenicity did not evaluate the leaf epiphytic colonization, since X. citri survive on leaf surface forming biofilm before invading the parenchyma tissue. Indeed, the epiphytical stage has an important role in the X. citri pathogenesis [3] and is positively affected by xanthan gum production, which is crucial to pathogen survival and interaction with the host [4].
In this work, we expanded the investigation of xanA function, studying two different xanA mutants, to verify the xanthan gum composition and yield, besides to analyze important lifestyle traits of X. citri, as biofilm formation and motility. Further, we used a method that mimics the natural process of infection by X. citri to test the bacterial pathogenicity on Citrus sinensis leaves. Further, we showed changes in the xanthan gum monosaccharide composition that may be involved with the impairment of pathogenicity, which gives insight into the development of a targeted strategy to reduce citrus canker disease.

Screening Of Mutants and Identification of Transposon Insertion Sites
The screening for the biofilm formation-affected mutants in a mutant library of X. citri strain 306 (National Center for Biotechnology Information, NCBI, accession No: AE008923), constructed previously [14] using the EZ::TN < KAN-2 > transposome complex (Epicentre Technologies, Madison, WI) was performed using a polystyrene 96-well plate (Nunclon surface, Nuncbrand, Roskilde, Denmark) assay system [8]. After that, was selected a clone, identified as A7 that showed a decrease in biofilm formation.
For mapping the location of transposon insertion, thermal asymmetric interlaced PCR (TAIL-PCR) was performed to amplify unknown DNA sequences contiguous to known kanamycin gene sequences, according to [15]. Three successive high-and lowstringency PCR amplifications were performed with nested sequence-specific primers with a melting temperature (Tm) > 65 • C in consecutive reactions together with a short (15-16 nucleotides) arbitrary degenerate (AD1) primer with a Tm of about 45 • C, and genomic templates from the mutant strain generated by random insertion. TAIL-PCR reactions were analyzed on a 1.0% agarose TBE gel and the brightest band was purified with a PCR Purification Kit (Wizard ® SV Gel and PCR Clean-Up System, Promega). The insertion point of transposon mutant was confirmed by DNA sequencing with the forward and reverse primers of EZ::TN (Epicentre Technologies, Madison, WI, USA) ( Table 1). Sequences were compared and aligned with sequences from the GenBank database, by using the BLAST program of the National Center for Biotechnology Information (NCBI) website ( http://www.ncbi.nlm.nih.gov/, access date: 27 January 2015). To verify the accuracy of transposon insertions in the sequence, PCR amplification was performed using primers (XAC3579F and XAC3579R) cited in Table 2, designed from the sequences flanking the gene XAC3579 (xanA). The PCR amplification was carried out in 1 cycle at 94 • C for 2 min, 35 cycles at 94 • C for 30 s, 56 • C for 30 s and 72 • C for 4 min, and 1 cycle at 72 • C for 10 min. Table 1. Primers used to transposon insertion point confirmation.
Bacterial growth in 96-well microtiter plates was measured in the Varioskan Flash Multimode Reader (Thermo Fisher Scientific) at 600 nm (OD600).

Construction of the XanA Deletion Mutant and Complementation of EZ-Tn5 Insertion and Deletion Mutants of the X. Citri
Bacterial genomic DNA and plasmid DNA were extracted using a Wizard genomic DNA purification kit and a Wizard miniprep DNA purification system according to the manufacturer's instructions (Promega, Madison, WI, USA). The concentration and purity of the DNA were determined using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). DNA was stored in Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0) at −20 • C. PCR was performed using standard procedures [20] with Pfu DNA polymerase (Promega Corporation, Madison, WI, USA). The restriction digestions and DNA ligations were performed according to the manufacturer's instructions.
To construct the xanA deletion mutant, approximately 1 kbp of the upstream and downstream regions of the xanA gene (XAC3579) was amplified using PCR from genomic DNA obtained from X. citri strain 306 using the following primer pairs: xanAF1 (5 -GGCTGGCGCCAAGCTTCCGACTGCAGCCACACATCGA-3 ) and xanAR1 (5 -CAATCA GGCGGGTAGCGTCATGGGCAAATCCTG-3 ); and xanAF2 (5 -TACCCGCCTGATTGACC CCTCTCCCACCCATAGAC-3 ) and xanAR2 (5 -TCCTGCAGAGAAGCTTGGTGTTCTGG CAATCGAGCTGGATCAC-3 ), respectively. Fragments were amplified using a highfidelity polymerase (Phusion, Thermo Scientific) and PCR reaction conditions were: 98 • C for 4 min, 35 amplification cycles were performed at 95 • C for 1 min, 60 • C for 30 s, 72 • C for 1 min, and final incubation at 72 • C for 5 min. The PCR products were digested with BamHI, and both fragments were ligated to produce a deletion construct fragment containing 2 kbp. Thus, the resulting fragment was cloned into the pNPTS138 suicide vector to generate pNPTS_xanA ( Table 2) using the HindIII restriction site, as previously described in Andrade et al. (2014) [21]. The pNPTS_xanA construction was introduced into E. coli by heat-shock at 42 • C [20] according to standard procedures and into X. citri by electroporation [22]. X. citri transformants were grown in LB medium plus kanamycin for 3 days at 28 • C and after the second recombination event X. citri cells were plated on LB medium containing ampicillin and 5% sucrose [23]. X. citri recombinant cells were grown for 2 days at 28 • C and after confirmation of the loss of both the kanamycin resistance cassette and sacB gene, a PCR was performed using the primers xanAF1 and xanAR2 mentioned above. X. citri strains carrying the xanA mutant allele were selected for further studies.
To complement the xanA EZ-Tn5 insertion and deletion mutants, a 1300 bp DNA fragment containing the entire xanA gene was amplified by PCR using total DNA obtained from the X. citri wild-type strain 306 as the template and the specific primer pair xanA_p53_F (5 ATTATTGGTACCATGACGCTACCCGCCTTCAAG3 ) and xanA_p53_R (5 ATTATTAAGCTTTCAGCCGCGCAGCAGGTTAGA3 ). The amplified DNA fragment was cloned into pUFR053 [19] at the BamHI and HindIII restriction sites to obtain the recombinant plasmid pUFR053_xanA (Table 2), which was used for genetic complementation. The construction was confirmed using sequencing. The recombinant plasmid pUFR053_xanA was transferred into xanA::Tn5 and ∆xanA mutant strains using electroporation, and cells were selected on NBY solid media using gentamicin, resulting in the strain xanA::Tn5_c and ∆xanA_c (xanA+).

Biofilm Formation on Abiotic Surfaces
Biofilms that formed on glasses and polystyrene plates were examined as previously described [3] with some modifications. Wild-type, mutants (xanA::Tn5 and ∆xanA), and the complemented (xanA::Tn5_c and ∆xanA_c) strains were grown into NBY overnight and the bacteria were centrifuged for six minutes at 4456 g. The optical density (OD) at 600 nm was adjusted to 0.1 (10 8 CFU/mL) using fresh NBY plus glucose (1% w/v). The bacterial suspension was transferred into each glass tube or polystyrene plate and incubated at 28 • C without shaking for 48 h. Culture media were removed and bacterial cells attached to the abiotic surfaces were gently washed three times with sterilized distilled water and stained with 2 mL 0.1% Crystal Violet (CV) for 30 min. The unbound CV was poured off, and the surfaces were washed with water. The CV-stained cells were solubilized in 2 mL of ethanol. The samples were measured at 590 nm using a spectrometer (UV/Vis Spectrometer Lambda Bio; Perkin Elmer). Biofilm values were normalized to bacterial growth (OD at 600 nm). Assays were repeated two times independently with six replicates each.

Xanthan Gum Quantification
Bacteria were grown overnight at 28 • C with shaking at 180 rpm in NBY medium. Bacteria were adjusted to 0.1 (10 8 CFU/mL) and 5 mL was transferred to 45 mL of NBY plus glucose (2% w/v). After 48 h of growth at 28 • C and shaking at 180 rpm, the cells were pelleted by centrifugation (4456 g for 6 min), and 3 volumes of 100% ethanol were added to the supernatant. The crude xanthan was collected using a glass rod and placed on a Petri dish to dry at 60 • C for 24 h. The assay was repeated three times independently with three replicates for each one.

Bacterial Motility Assay
Assays were performed as previously described [3,5]. Briefly, bacteria were grown overnight in NBY medium, and 3 µL of bacterial culture (10 6 CFU/mL) was then plated onto SB medium plus 0.5% (w/v) agar for sliding analysis [24]. Plates were incubated at 28 • C for 48 h. Motility analysis was assessed qualitatively by examining the circular halo formed by the bacterial growth. The assay was performed in triplicate and repeated two times independently. The diameters of the circular halos that were occupied by each strain were measured, and the resulting values were taken to indicate the motility of X. citri strains. The assay was repeated three times independently with three replicates for each one.

Pathogenicity Assay
Pathogenicity assays were performed by three different methods. X. citri strains (X. citri wild-type and xanA EZ-Tn5 insertion mutant) that were grown in selective antibiotic NBY medium overnight at 28 • C, centrifuged at 4456× g, and then suspended in a 10 mM potassium phosphate buffer (pH 7.0).
Leaves from sweet orange (Citrus sinensis cv. 'Bahia') plants were inoculated by spraying and infiltration. In the first method, the abaxial surfaces of fully expanded immature leaves of each plant were sprayed with 10 8 CFU/mL from each X. citri strain. In the second method, leaves were inoculated by pressure infiltration of 100 uL of bacterial suspension (10 4 CFU/mL) from each X. citri strain. Phosphate buffer was used as the control in noninfected plants. All plant inoculations involved a minimum of three immature leaves from each plant, and three plants were inoculated for each bacterial strain. The plants were kept in the greenhouse at Instituto Agronômico de Campinas/IAC (Campinas, SP), at a temperature of 28 ± 4 • C in high humidity for 30 days. Disease symptoms were evaluated until 30 days post-inoculation, and assays were independently repeated twice.
In the third method, sweet orange leaves were pierced with a microneedle device (0.5 mm). Care was taken to apply gentle pressure onto the leaf abaxial surface with one move horizontally over the area. Bacteria strains were inoculated using a cotton pad soaked with the different X. citri strains (10 8 CFU/mL). Three plants were inoculated for each X. citri strain and five leaves of each plant were used. The plants were kept at a temperature of 28 ± 4 • C and disease symptoms were evaluated after 15 days post-inoculation (dpi). The assay was repeated two times independently.

Xanthan Gum Composition Assessment in XanA Mutant and Wild-Type
Xanthan gum produced by wt and xanA::Tn5 mutant were evaluated by high-performance liquid chromatography (HPLC) according to the pre-established method [25] at "Laboratório de Bioquímica de Plantas" of UNESP in Jaboticabal (SP/Brazil). First of all, bacteria were grown for 72 h in NBY medium plus glucose (1% w/v) at 28 • C and 200 rpm. The cells were pelleted by centrifugation (4456 g for 6 min) and 3 volumes of 100% ethanol were added to the supernatant. The crude xanthan was collected using a glass rod. A blend of three samples, totalizing 1 mg xanthan gum, was hydrolyzed with trifluoracetic acid (4 mol/L) and the monosaccharides were marked with the addition of 1-fenil-3-metil-5-pirazolone (PMP) solution (0.5 mol/L and methanol) and sodium hydroxide solution (0.3 mol/L). The microtubes were vortexed and incubated at 70 • C for 2 h. After cooling, the mixture was neutralized by a hydrochloric acid (0.3 mol/L) addition. The monosaccharides extraction was performed by the addition of butyl ether. The organic phase (supernatant) was removed by centrifugation at 5000 g for 5 min and one mL of milli-Q water was added in the remaining phase. The monosaccharides detection was performed at 245 nm by HPLC equipped with UV/VIS detector (Shimadzu, model SPD-M10A) using column GHRC ODS-C-18 (4.6 mm i.d. × 15 cm), flow rate of 0.5 mL/min, buffers A (ammonium acetate 100 mmol/L, pH 5.5 + 10% (w/v) acetonitrile) and B (ammonium acetate 100 mmol/L, pH 5.5 + 25% (w/v) acetonitrile) and separation gradient of 0% (30 min) and 0-100% up 100 min. Pattern monosaccharides were used for standard curve construction in the following concentrations: 0.0125, 0.0250, 0.0500, 0.1000 mg/mL.

Statistical Analysis
Data from biofilm formation, sliding motility, gum production, and pathogenicity assays were statistically analyzed using a t-test (p < 0.05). The values were expressed as the means ± standard deviations of independent replicates.

XanA Encodes a Phosphoglucomutase Protein, Important to the Biofilm Formation and Xanthan Gum Production
The A7 mutant was isolated from an EZ-Tn5 transposon mutagenesis library of X. citri strain 306 [5,14] since it exhibited a reduced ability to form biofilm. Sequencing indicated that EZ-Tn5 was inserted in the position 651 downstream of the translation start site of the locus XAC_RS18095 (Figure 1a). PCR using the primers XAC3579F and XAC3579R demonstrated the increase in the length of the obtained PCR amplicon by insertion of EZ-Tn5 transposon (1.22 kb) generating a PCR product with 2.22 kb, which confirmed the mutation of the xanA gene (locus XAC_RS18095) (Figure 1b). Thus, we will refer to the A7 mutant as xanA::Tn5. The xanA gene encodes a predicted phosphoglucomutase protein, required to synthesize the xanthan gum, a pathogenesis-related exopolysaccharide in Xanthomonads [13].

Statistical Analysis
Data from biofilm formation, sliding motility, gum production, and pathogenicity assays were statistically analyzed using a t-test (p < 0.05). The values were expressed as the means ± standard deviations of independent replicates.

XanA Encodes a Phosphoglucomutase Protein, Important to the Biofilm Formation and Xanthan Gum Production
The A7 mutant was isolated from an EZ-Tn5 transposon mutagenesis library of X. citri strain 306 [5,14] since it exhibited a reduced ability to form biofilm. Sequencing indicated that EZ-Tn5 was inserted in the position 651 downstream of the translation start site of the locus XAC_RS18095 ( Figure 1A). PCR using the primers XAC3579F and XAC3579R demonstrated the increase in the length of the obtained PCR amplicon by insertion of EZ-Tn5 transposon (1.22 kb) generating a PCR product with 2.22 kb, which confirmed the mutation of the xanA gene (locus XAC_RS18095) ( Figure 1B). Thus, we will refer to the A7 mutant as xanA::Tn5. The xanA gene encodes a predicted phosphoglucomutase protein, required to synthesize the xanthan gum, a pathogenesis-related exopolysaccharide in Xanthomonads [13].
The xanA::Tn5 produced approximately 80% less biofilm compared with the X. citri wild-type (wt) strain ( Figure 1C). We further verified the importance of the mutated gene in xanthan gum production. As summarized in Figure 1D, the xanthan gum yield of xanA::Tn5 was significantly different from that of the wild-type strain, showing 83% less xanthan gum production.  The xanA::Tn5 produced approximately 80% less biofilm compared with the X. citri wild-type (wt) strain (Figure 1c). We further verified the importance of the mutated gene in xanthan gum production. As summarized in Figure 1d, the xanthan gum yield of xanA::Tn5 was significantly different from that of the wild-type strain, showing 83% less xanthan gum production.

Mutant Strain XanA::Tn5 Developed No Symptoms on Sweet Orange Host
Previous studies have verified that xanthan plays a role in X. citri pathogenesis in detached leaves [12], however, this is not a natural condition to understand the hostpathogen interaction. We then inoculated the wild-type strain and xanA::Tn5 on host leaves by two different methods: spray and infiltration. The symptoms evaluation was monitored for 30 days after inoculation. No symptoms were observed in xanA::Tn5 when inoculated by spray, in opposition to severe citrus canker symptoms caused by wt cells (Figure 2a). However, when the strains were inoculated by infiltration, xanA::Tn5 mutant was able to cause canker symptoms, even though less severe than those observed by wt strain (Figure 2b). These results indicate that xanA plays an important role in the epiphytic stage of the X. citri lifestyle, since xanA mutated allele affected this ability, thus the bacterium was not able to cause disease when inoculated by spray, a method that mimics a natural condition of X. citri infection.

xanA Plays a Critical Role in Controlling Important Features in X. citri
To verified that the Tn5 mutation did not have a polar effect, we generated a deletion mutant of the xanA gene in X. citri that was also complemented with the construct

xanA Plays a Critical Role in Controlling Important Features in X. citri
To verified that the Tn5 mutation did not have a polar effect, we generated a deletion mutant of the xanA gene in X. citri that was also complemented with the construct pUFR053-xanA. Both xanA mutants (xanA::Tn5 and ∆xanA) and wt strains were tested to determine their ability to form biofilm on the abiotic surface, sliding motility, gum production, and pathogenicity. The growth curve of the mutant strains was not different from that in the wild-type ( Figure S1).
Two independent mutant strains of xanA showed a decreased in biofilm formation, as shown in the first test (Figure 3a), and in sliding on medium plates (Figure 3b). On the plate surface, the diameters of the bacteria sliding resulted from migration away from the inoculation points in the agar surface were about 3-fold higher for wt than both xanA mutant strains after 48 h at 28 • C (Figure 3b). The complemented strain showed results like those of the wild-type strain, indicating that the sliding motility phenotype of the mutant was restored. Swimming motility analyses were also performed, but no difference was observed between the mutant and the wt strains (data not shown). roorganisms 2021, 9, x FOR PEER REVIEW 9 of 13 gum extracted from supernatants of these strains can also be visually observed on the plates (Figure 3c). Impairment of pathogenesis after bacterial inoculation on leaf surfaces that has been verified for xanA::Tn5 mutant strain was also confirmed by ΔxanA and restored in xanA complementation strains (Figure 3d). A large number of pustules was observed on the leaves surfaces at 15 days after inoculation of wt and both complemented strains (xanA::Tn5_c and ΔxanA_c). However, very few pustules were observed on the leaves surfaces inoculated with each of the mutants (xanA::Tn5 and ΔxanA) strains.

Xanthan Gum from xanA Mutant Have Altered Monosaccharides Content
Xanthan gum produced by wt and xanA::Tn5 showed different percentages of some specific monosaccharides by HPLC analysis (Table 3). Xanthan gum produced by xanA::Tn5 exhibited a higher proportion of mannose, galactose, and xylose and reduced rates of rhamnose, glucuronic acid, and glucose. Thus, monosaccharide composition analysis indicated that xanA deletion altered the xanthan composition. Man (mannose), Rha (ramnose), GlcA (glucuronic acid), GalA (galacturonic acyd), Glc (glucose), Xanthan gum amount also decreased about 4.5-fold in xanA::Tn5 and 3.4-fold in ∆xanA strain compared to the wt strain. This phenotype was restored to the wt level by complementation of the mutants, with no significant difference (p < 0.05) between xanA::tn5_c, ∆xanA_c, and wt, respectively. These differences in the amount of xanthan gum extracted from supernatants of these strains can also be visually observed on the plates (Figure 3c). Impairment of pathogenesis after bacterial inoculation on leaf surfaces that has been verified for xanA::Tn5 mutant strain was also confirmed by ∆xanA and restored in xanA complementation strains (Figure 3d). A large number of pustules was observed on the leaves surfaces at 15 days after inoculation of wt and both complemented strains (xanA::Tn5_c and ∆xanA_c). However, very few pustules were observed on the leaves surfaces inoculated with each of the mutants (xanA::Tn5 and ∆xanA) strains.

XanA Is Highly Conserved in the Xanthomonads Group
BLASTP search revealed that XAC3579 is highly conserved in other plant pathogenic species, including, X. oryzae pv. oryzae (99% identity), X. campestris pv. campestris (96% identity), and Xylella fastidiosa Temecula (84% identity) and Xylella fastidiosa 9a5c (84% identity). The phosphoglucomutase and phosphomannomutase, as well as XanA, share a conserved domain I that contains the catalytic phosphoserine (Figure 4), suggesting that this enzyme may be critical for cellular functions and give adaptative advantage for bacterial cells.

XanA Is Highly Conserved in the Xanthomonads Group
BLASTP search revealed that XAC3579 is highly conserved in other plant pathogenic species, including, X. oryzae pv. oryzae (99% identity), X. campestris pv. campestris (96% identity), and Xylella fastidiosa Temecula (84% identity) and Xylella fastidiosa 9a5c (84% identity). The phosphoglucomutase and phosphomannomutase, as well as XanA, share a conserved domain I that contains the catalytic phosphoserine (Figure 4), suggesting that this enzyme may be critical for cellular functions and give adaptative advantage for bacterial cells.

Discussion
In this work, we showed changes in the xanthan gum monosaccharide content that were associated with the xanA function in Xanthomonas. Disruption of xanA in X. citri genome, which codes for enzyme XanA that is necessary for isomerization of glucose-6-phosphate to glucose-1-phosphate and conversion of mannose-6-phosphate to mannose-1-phosphate [12,26,27], led to impairment of xanthan gum production.

Discussion
In this work, we showed changes in the xanthan gum monosaccharide content that were associated with the xanA function in Xanthomonas. Disruption of xanA in X. citri genome, which codes for enzyme XanA that is necessary for isomerization of glucose-6phosphate to glucose-1-phosphate and conversion of mannose-6-phosphate to mannose-1phosphate [12,26,27], led to impairment of xanthan gum production. PGM/PMM possibly works as a valve, rerouting the metabolic flux originating from hexose phosphates either toward the biosynthesis of lipopolysaccharides (LPS) or xanthan, or the generation of energy or building blocks, such as amino acids, for cellular growth [27,28]. Thus, with the lack of XanA synthesis, the PGM/PMM activity levels decreased and consequently, the conversion of the substrates to UDP-D-glucose and GDP-mannose was compromised, leading to a reduction of glucose and mannose activated monosaccharides. This explains the lower percentages of glucose and mannose in the xanthan composition in xanA::Tn5 mutant compared with that observed in the wild-type strain. Indeed, the xanA deletion resulted in a lack of both activated monosaccharides UDP-glucose and GDP-mannose, which led to the formation of a xanthan gum structurally different [29].
For completion of xanthan gum synthesis, different steps are involved, and they require specific substrates and specific enzymes during this biosynthetic process. If either the substrate or the enzyme is absent, the step is compromised [30]. Some authors have shown that when mutation of specific genes involved in the xanthan gum synthesis are made to simplify the repeating unit structure, the xanthan gum yield was much lower as than that produced from the wild-type strain [29]. Betlach et al. [31] constructed a mutant lacking the glucuronic acid residues and the pyruvate and as a result, the xanthan gum solution produced by this strain was highly viscous. As we showed here, mutation of xanA reflected in changes of the repeating unit in the xanthan structure, and consequently, the xanthan gum yield was much lower than that produced from the wild-type and complemented strains, although low levels of xanthan gum remained detectable. The remaining xanthan may be explained by the presence of the xanB gene in the X. citri genome which codes for the enzyme phosphomannose-isomerase-GDP-mannose pyrophosphorylase that has involvement in GDP-mannose synthesis, one of those xanthan precursors [11].
The drastic reduction of xanthan gum in xanA mutants (xanA::Tn5 and ∆xanA) was responsible for the decrease of biofilm formation, sliding motility, and pathogenicity observed in this work. As xanthan gum contributes to the bacterial attachment to surfaces [8] and biofilm development with an organized structure on both abiotic and biotic surfaces [4], we suggest that such features are associates with the xanA function in determining EPS content. These findings, together with the demonstration that biofilm formation in xanA mutants can be complemented by the pUFR53-xanA construct, strongly suggest that a structurally intact xanthan gum harboring a particular composition of monosaccharides is critical for the X. citri lifestyle. Therefore, xanthan gum can affect bacterial cell adherence on host tissue and survival under stress conditions, as had already been demonstrated by other authors [32].
Further, sliding motility was previously shown to be promoted by xanthan gum and inhibited by type IV pili in X. citri [3,23]. These previous results corroborate our data which shows that xanA mutants also had a significant reduction in sliding motility compared with the wild-type strain. Consistently, it has been proposed that xanthan acts as a surfactant or surface-wetting agent to facilitate this type of movement [33,34]. Indeed, xanA::Tn5 and ∆xanA displayed less spread on the medium surface that may be due to low levels of the xanthan produced by these mutant strains, since anterior works have shown that X. citri mutants impaired in xanthan gum production also have sliding motility altered [3][4][5]35,36].
Moreover, reduced xanthan gum has also been related to the impairment of X. citri pathogenesis [3,12,13]. In our study, likewise, we also observed fewer symptoms when the xanA::Tn5 mutant cells are infiltrated into the leaves. Since inoculation by infiltration breaks the first physical defense layer, X. citri can use subsequent pathogenicity mechanisms, such as the type III secretion system to inject specific effectors into host cells, culminating in the development of citrus canker symptoms. Accordingly, previous results showed that the xanA mutant retained its infectivity when infiltrated into leaves and the authors speculated that PGM itself is not critical to canker progression, but it is related to the inoculation method used, which does not simulate the natural process of infection by X. citri [12]. Even though infiltration does not reflect a natural infection condition, xanA has a role when the bacteria are in plant mesophyll. On the other hand, we show that xanA::Tn5 caused no symptoms in sweet orange leaves after spraying, a method that simulates the natural infection condition. These results suggest that xanA has an important role in the epiphytical stage on the leaves that is essential for the interaction with the host, including adaptive advantage for bacteria cell survival under stress conditions and invasion of parenchyma tissue, which is required for the pathogenesis of X. citri.
Our data expanded the investigation on the xanA function, showing its role in affecting the monosaccharide composition of xanthan gum. The modified xanthan produced by xanA mutant compromised important features in X. citri and strongly impaired its ability to cause disease when in the epiphytic stage. Thus, chemical compounds that target the metabolic pathways involved in the activation of monosaccharides such as UDP-D-glucose and GDP-mannose, which was shown in this work that interferes in pathogenicity, could be a strategy to impair citrus canker disease. Further, as xanA is highly conserved in many bacteria, this strategy could be applied to other plant pathogenic species.