bGenomic sequences are available through the NCBI genomic BLAST service. cThe putative signal sequence selleck chemicals cleavage sites were determined using the SignalP 4.1 server. *The B. pseudomallei strain DD503 is a derivative of isolate 1026b in which the AmrAB-OprA antibiotic efflux pump has been deleted to facilitate mutant construction . The BMA1027 orthologs of strains DD503 and 1026b are identical (confirmed by nucleotide sequence analysis, data not shown). The published genomic sequence of the B. pseudomallei strain K96243 was
found to specify a BMA1027 ortholog (locus tag # BPSL1631, Figure 1B) that is 89% identical to that of B. mallei ATCC 23344. The BMA1027 ortholog was sequenced from the B. pseudomallei strain used HM781-36B in our laboratory, DD503, and was predicted to encode a protein that is 97% and 87% identical to that of B. pseudomallei K96243 and B. mallei ATCC 23344, respectively (Figure 1C). Database searches with the NCBI genomic BLAST service also identified orthologs in several B. pseudomallei and B. mallei isolates. Seven B. mallei and twenty-nine B. pseudomallei strains for which sequences are available through the service were found to have the gene. Characteristics of these ORFs are listed in the Additional files 1 and 2. Overall, the proteins are 87-100% identical and differ primarily in the number and/or arrangement
of SLST repeats, YadA stalk domains, and/or NSTA elements in their passenger domains. Based on these results, we conclude that BMA1027 orthologs are well-conserved gene products shared by B. mallei and B. pseudomallei. While preparing this article, Campos and colleagues published a report in which they functionally characterized autotransporter genes specified by the B. pseudomallei strain 1026b . One of these molecules corresponds to the BMA1027 ortholog (locus tag # BP1026B_1575), which the authors designated bpaC. Henceforth, Nintedanib (BIBF 1120) BMA1027 and orthologs will be referred to as BpaC. Expression and functional properties of the BpaC protein in E. coli Because of sequence and structural similarities to known bacterial
adhesins, we speculated that BpaC mediates adherence to Protein Tyrosine Kinase inhibitor epithelial cells. To test this hypothesis, the bpaC gene of B. pseudomallei DD503 was cloned and expressed in the E. coli strain EPI300. This organism does not adhere well to epithelial cells [8, 53, 55, 62] and therefore provides a suitable heterologous genetic background to study the adherence properties of BpaC. To verify protein expression, whole cell lysates were prepared from E. coli EPI300 harboring the plasmid pCC1.3 (control) or pCCbpaC (specifies B. pseudomallei DD503 bpaC) and analyzed by western blot. Figure 2A shows that α-BpaC Abs (directed against aa 392–1098, part of surface-exposed passenger domain) react specifically with a band of 100-kDa in E.