Pyridoxal 5-phosphate (PLP) is an essential cofactor for numerous enzymes involved in a diversity of cellular processes in living organisms. DXP-independent pathway [5, 6]. and other members of the -subdivision of proteobacteria adopt the DXP-dependent pathway, which involves two enzymes PdxA and PdxJ. In contrast, the DXP-independent pathway is found in most eubacteria, fungi, protozoa, archaea, plants and metazoan, and requires PdxS and PdxT enzymes [5, 7]. As shown in Fig 1A, by converting FLN1 glutamine into glutamate, PdxT generates ammonia, which is used by PdxS to synthesize PLP from a 5 and a 3 carbon sugar, such as ribose 5-phosphate and glyceraldehyde 3-phosphate [8, 9]. Although the PLP biosynthesis pathways have been found in nonpathogenic bacteria, such as and [5, 10], the relation between PLP biosynthesis pathways and bacterial pathogenicity remains poorly comprehended. However, a previous study on suggested that PLP biosynthesis was also essential for bacterial survival and virulence [8]. Fig 1 Enzymatic activity of PdxS and PdxT. is a Gram-negative bacterial pathogen responsible for porcine pleuropneumonia, which is a severely contagious respiratory disease that causes major economic losses for the swine industry worldwide [11]. Effective survival and persistence in pigs is usually a critical hindrance for eradication [11, 12]. Recent analysis of the S-8 genome sequence revealed the presence of the and genes [13]. Additionally, a previous study revealed that both and genes were downregulated after inactivation of the gene which is required for stress tolerance in [14]. To date, the vitamin B6 biosynthesis pathway has not been characterized in adopts the DXP-independent pathway or whether the PLP synthases PdxS/PdxT are required for viability, stress tolerance, and virulence of remain unclear. LY341495 In the present study, we identified and characterized the function of enzymes PdxS and PdxT LY341495 in the vitamin B6 biosynthesis pathway in and genes, respectively and investigated the role of PdxS and PdxT in viability, stress tolerance, and virulence of strains were cultured in a brain heart infusion (BHI) medium supplemented with 10 g/mL nicotinamide dinucleotide (NAD) (Sigma-Aldrich, USA). For the culture of transconjugants (single crossovers), BHI medium LY341495 was supplemented with 10 g/mL of NAD and 7 g/mL of chloramphenicol. 2155 was grown in Luria-Bertani (LB) medium supplemented LY341495 with 1 mM diaminopimelic acid (DAP) (Sigma-Aldrich, USA). The chemically defined medium (CDM) was prepared as previously described [15], without the addition of NH4Cl. All strains were routinely produced at 37C. Protein expression and purification The coding sequences of and genes were PCR-amplified from S-8 genomic DNA using specific primers SF/SR and TF/TR (S1 Table). The digested PCR products were ligated with NdeI/XhoI-digested pET22b(+) (Novagen). The recombinant plasmids were confirmed by sequencing and used to transform into BL21 (DE3). The expression of the each target protein was induced for 18 h at 16C with 0.5 mM isopropyl 1-thio–D-galactopyranoside (IPTG) in LB broth made up of 50 g/ml ampicillin. The His6-tag fusion proteins were loaded onto a Ni Sepharose 6 Fast Flow column (GE Healthcare, United States) and purified as previously described [16]. The recombinant protein concentrations were decided using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). Glutaminase activity assay Glutaminase activity was measured as described previously [17]. Samples of 8 M PdxS, 8 M PdxT, or 8 M mixture of both proteins.