All three proteins are predicted to contain multiple trans-membrane helices, also predicted for the B. fragilis homologs, and BatD possesses a predicted signal sequence for export, suggesting that these proteins may associate with either the inner or outer membrane of L. biflexa. Figure 1 Amino acid motifs in the Bat proteins of L. biflexa . The vWF and TPR domains

are conserved among Bat homologs and have been proposed to facilitate formation of a large Bat protein complex [4]. The vWF domains identified in Bat proteins contain metal ion-dependent adhesion sites (MIDAS) shown to bind metal ions [10] and the domain overall is thought to mediate protein-protein interactions [11]. The TPR domain of BatB consists of a repeated amino acid motif previously shown to form a tertiary scaffold structure for multiprotein complex IWR1 formation (reviewed in [12]). These domains, along with the presence

of multiple transmembrane helices and a signal sequence Hydroxychloroquine price identified in BatD, suggest that the Bat proteins form a complex associated with either the inner or outer membrane of L. biflexa. Deletion of bat genes The L. biflexa bat genes are located within a contiguous stretch of 11 genes on chromosome II that are transcriptionally oriented in the same direction (Figure 2A). Two different mutations were engineered using allelic replacement with the kanamycin-resistance cassette to delete either batA alone or batABD together; flanking genes were left intact. Three mutant clones from each transformation were shown to have lost the corresponding bat loci by Southern blot analysis of genomic DNA (Figure 2B). PCR analysis also confirmed the presence of the antibiotic-resistance gene (kan) and flanking genes, but bat loci were absent, as expected (data not shown). A single transformant of each type was randomly chosen for further characterization. Figure 2 Gene organization in wild-type and mutant strains of L. biflexa . (A) Genetic organization of bat genes and

flanking genes on chromosome II of L. biflexa (not drawn to scale). The corresponding deleted regions in mutant strains Histamine H2 receptor are depicted with the respective bat genes replaced by the kanamycin-resistance cassette [13]. (B) Southern blot analysis of L. biflexa strains confirms the absence of the respective bat genes in mutant strains. Genomic DNA for the Southern blot was double-digested with restriction endonucleases NdeI and PstI. Three independently isolated transformants from each mutant were compared to wild-type and hybridized with either a labeled batA fragment or with a labeled fragment spanning batB to batD. The weak signal observed at ~3 kb in the batA mutant strains hybridized with the batA probe is likely due to cross-hybridization with batB. +, purified plasmid DNA from E. coli with a cloned region of L. biflexa DNA containing batABD.