Rhodobacter capsulatus functions independently from BioM and BioN in a recombinant Escherichia coli transport model, BioMN increases biotin transport affinity. Thus it is unclear if the orphaned BioY transporters function as high or low affinity transporters or perhaps interact with highly divergent AT modules not recognized through similarity searches. Analysis of chlamydial genomes identified differences across species in how biotin is acquired. Some species possess the biotin synthesis genes, the biotin transporter bioY, or both biotin synthesis and transport genes. We used recombinant E. coli expressing BioY from C. trachomatis 434/Bu to determine that BioY functions as a high affinity and capacity transporter in the absence of the BioM and BioN AT module. This is the first study characterizing an orphaned type II ECF BioY and our results indicate that some S components might not require an AT module for high affinity substrate transport. Mammalian acquisition of biotin via protein transporters indicates that cell membranes are poorly permeable or impermeable to biotin. Hence, the host derived Chlamydia inclusion membrane also may require a biotin 
transporter. Immunofluorescence microscopy studies on infected cells demonstrated that SMVT colocalizes with the chlamydial inclusion membrane. These findings indicate that Chlamydia redirects a portion of the SMVT pool away from the cell membrane to the inclusion membrane to facilitate transport of biotin into the inclusion space where it can then be transported by BioY into the bacterium. SMVT also transports the essential compounds lipoic acid and pantothenic acid into the host cell. While previous work has shown that Chlamydia redirect cellular proteins to the inclusion membrane for its benefit, this is the first example to our knowledge of Chlamydia recruiting a cellular transporter and may serve as a paradigm for how small molecules gain access to the intra-inclusion space. Results Utilization of biotin by Chlamydia Chlamydia encode at least two well characterized biotin utilizing proteins, the biotin carboxyl carrier protein AccB and the biotin protein ligase BirA. AccB acts in concert with AccACD to form acetyl CoA carboxylase, which converts acetyl-CoA to malonylCoA in the first committed step in fatty acid synthesis. The biotin ligase, BirA, ligates biotin to the e-amino group in a conserved lysine residue buy AZD 0530 within AccB. The chlamydial AccB proteins share 2532% amino acid sequence identity with the E. coli homolog and possess an altered biotin-ligation motif sequence . The chlamydial BirA is a Class I biotin ligase, which lacks the conserved C-terminal region found in many Class I and II BirA proteins . In addition to AccB and BirA, Chlamydia spp. encode accACD along with genes comprising downstream fatty acid biosynthesis pathways indicating that Chlamydia utilize biotin for fatty acid synthesis. Western blot analysis of EB samples and cells infected for 20 hours identified at least one Chlamydia specific biotinylated protein. E. coli was used as a positive control. Samples were run under denaturing conditions resulting in the detection of only covalently modified biotinylated proteins. These blots identified a unique biotinylated protein at,20 kDa present in chlamydial samples that was absent in control cell lysates. The predicted mass of the chlamydial AccB is 18.2 kDa. Under our conditions, the E. coli AccB ran larger than its predicted mass of 17.7 kDa. Banding patterns at th
