coli K-12 was impaired in surface binding, intercellular
adhesion, and biofilm formation [19]. Mutation of orfN in Pseudomonas aeruginosa PAK affected the flagellin glycosylation [20]. In X. campestris pv. campestris strain 8004, mutation of xagB (XC_3555) led to decreased EPS production, abolished biofilm formation and attenuated bacterial resistance to oxidative stress [21], and the XC_3814 mutant was significantly reduced both in EPS production and virulence on host plants [22]; while the rfbC mutation in Xac strain 306 resulted in altered O-antigen of LPS, reduced biofilm formation and attenuated bacterial resistance to environmental stresses MI-503 in vivo [23]. In our previous work, an EZ-Tn5 transposon mutant of Xac strain 306 with an insertion in the XAC3110 locus was isolated in a screening that aimed at identifying genes involved in biofilm formation. The XAC3110 locus was named as bdp24 for mTOR inhibitor biofilm-defective phenotype and the mutant was observed to be affected in EPS and LPS biosynthesis, cell motility and biofilm formation on abiotic
surfaces [24]. Due to the nature of our previous study in genome-wide identification of biofilm related genes, we focused on big picture rather than Crenigacestat individual genes. It is necessary to further characterize the novel genes identified in our previous study and provide conclusive genetic evidence in complementation. In this study, we further characterized the bdp24 (XAC3110) gene (renamed as gpsX) that encodes a putative glycosyltransferase using genetic complementation assays. The data obtained confirmed that the novel gene gpsX plays a role in EPS and LPS biosynthesis, cell motility, biofilm formation on abiotic surfaces and host leaves, stress tolerance, growth in planta, and host virulence of the citrus canker bacterium. These findings suggest that the gpsX gene contributes to the adaptation of Xac to the host microenvironments at early stage of infection and thus is required for full virulence on host plants. Results The gpsX gene encodes a Doxacurium chloride glycosyltransferase involved in polysaccharide biosynthesis in X. citri subsp. citri
The XAC3110 locus was identified as a biofilm formation-related gene of bdp24 that may be involved in EPS and LPS biosynthesis, following screening a transposon insertion mutant library of Xac strain 306 in our earlier work [24]. The XAC3110 open reading frame (ORF) is 2028 bp in length and located in the genome sequence at position 3655217-3657244 (Figure 1). XAC3110 consists of a single transcriptional unit, whereas the adjacent upstream and downstream genes were transcribed separately from this ORF in reverse orientation [25]. XAC3110 was annotated as a 675 aa glycosyltransferase [7]. The predicted pI and molecular weight (MW) of the putative enzyme are 6.67 and 73.9 kD (http://web.expasy.org/compute_pi/), respectively. The predicted protein contained a glycosyltransferase family 2 domain (PF00535, 2.