Glucose 1-phosphate is then converted to UDP-glucose by GalU and mannose 1-phosphate to GDP-mannose by mannose 1-phosphate guanylyltransferase. These nucleotide sugars are directly implicated in EPS synthesis
[30, 31]. Production of EPS was measured in X. citri, the hrp mutants and the hrpB −c strains and results showed that EPS production in these mutants was over 1.7 times that in X. citri and hrpB −c SB-715992 in vitro strain (p < 0.05) (Figure 6A). Additionally, the expression of gumD, a see more gene encoding a protein of the EPS biosynthetic pathway, was analyzed by RT-qPCR in all the strains. The results showed that the transcript levels of gumD were over 17 times higher in hrp mutant strains as compared to X.
citri and the hrpB −c strain (p < 0.05) (Figure 6B). Moreover, the proteomic analysis also showed a down-regulation of the outer membrane protein XAC0019 in the hrpB − mutant (Table 1) and recently, it has been shown that this protein is necessary for X. citri swimming . Furthermore, CcmA that is required for bacterial motility [33, 34] was also down-regulated in the hrpB − mutant (Table 1). Therefore, bacterial motility was assayed for the hrp mutants and results showed that X. citri and the hrpB −c strain moved about 2.5 and 1.25 further in swimming and swarming plates respectively, than the hrp mutants DNA Damage inhibitor (p < 0.05) (Figure 6C) (Additional file 2: Figure S2). Figure 6 EPS production and bacterial motility assays in X. citri , the hrp mutants and the hrpB − c strains. (A) Quantification of EPS present in the supernatant fraction of cultures of the different strains. Quadruplicate measurements were made for each strain and an average of all measurements was obtained. Error bars indicate standard deviations. (B) RT-qPCR assay to determine gumD expression of the different stains relative to X. citri. Values are the means
of four biological replicates with three technical replicates each. (C) Quantification of bacterial swimming and swarming motility. Results are the average of the motility zones of 16 Petri dishes per strain. Error bars indicate the standard deviation. Avelestat (AZD9668) Discussion The role of T3SS in bacterial pathogenesis as a machine involved in effector protein delivery is well established, however, little is known about other functions in bacterial behavior that this system may have. Given that biofilm formation is required for X. citri to achieve full virulence, we used X. citri as a model to gain further insights into the functional role of T3SS in biofilm formation. By comparing the capacity of biofilm formation of three T3SS mutants and X. citri and also performing a proteomic assay with the hrpB − mutant, which revealed differentially expressed proteins between both strains, we demonstrated that T3SS is involved in biofilm formation in X. citri. To date the involvement of X.