, 2005). The B. pseudomallei K96243 bpss1516
gene sequence was compared with homologues in other available B. pseudomallei genomes, that is, Pasteur 52237, 576, DM98, 1710b, 305 and 1106a. This revealed that bpss1516 in K96243 was probably misannotated as the start codon for this ORF in K96243 was assigned 120 nucleotides downstream of the 5′ end annotated in other strains (data not shown). Therefore, we concluded that the gene is likely to be 40 codons longer than originally annotated. With this correction, B. pseudomallei bpss1516 encodes a 509 amino acid-long protein, with predicted molecular weight of 55.7 kDa. BPSS1516 has no high sequence homology to any protein in the available databases.
It CHIR-99021 price is conserved in B. pseudomallei and Burkholderia mallei, but absent in Burkholderia thailandensis (data not shown). As most T3SS effectors can be detected within bacterial culture supernatant in vitro, we incubated wild-type B. pseudomallei 10276 and the secretion deficient bsaZ mutant strain in LB medium under Bsa-inducing conditions. The secreted proteins and the whole-cell lysates were then separated by SDS-PAGE and analysed by Western http://www.selleckchem.com/products/ABT-737.html blotting using anti-BPSS1516 antibodies. A protein band migrating with an apparent molecular weight of approximately 56 kDa (the expected size for BPSS1516) was detected with anti-BPSS1516 antibodies in the total cell lysates of both B. pseudomallei strains, but only in the supernatant from the wild-type strain (Fig. 2). These data show that BPSS1516 is secreted by the Bsa T3SS. The level of the intracellular expression of BPSS1516 in the bsaZ mutant strain was slightly lower than that in the wild-type strain (Fig. 2). This phenomenon has been observed for the expression of many T3SS substrates in mutant
strains lacking T3SS structural components in other bacterial species, possibly through a negative feed-back mechanism (Francis et al., 2001; Parsot et al., 2005). It has been reported that many T3SS effectors interact with T3SS chaperones and this interaction has Non-specific serine/threonine protein kinase been proposed to stabilize effectors in the bacterial cells and to maintain their export-competent state for targeting to the T3SS apparatus (Cornelis, 2006). As the putative BPSS1516 effector seems to form an operon with BPSS1517, a protein with sequence similarity to the CesT family of T3SS chaperones (Pallen et al., 2005), we designed a series of experiments to investigate if the two proteins could interact in vitro. GST-BPSS1516 fusion protein (GST1516; Fig. 3a) was expressed in E. coli and immobilized on glutathione sepharose-4B beads. The beads were incubated with a clarified cell lysate from E. coli expressing a His6-tagged BPSS1517 (His1517; Fig. 3a) and a GST pull-down assay followed by immunoblotting with anti-His-tag and anti-BPSS1516 antibodies was performed.