RESUMO
The genome of the Honeybee bacterial pathogen, Paenibacillus larvae, encodes for protein a with substantial amino acid sequence similarity to the canonical Escherichia coli uracil transporter UraA. P. larvae expresses the uracil permease (PlUP) locus, and is sensitive to the presence of the toxic uracil analog 5-fluorouracil under vegetative growth conditions. The solute transport and binding profile of PlUP was determined by radiolabeled uptake experiments via heterologous expression in nucleobase transporter-deficient Saccharomyces cerevisiae strains. PlUP is specific for the transport of uracil and competitively binds xanthine and uric acid. Further biochemical characterization reveals that PlUP has a strong affinity for uracil with a Km 19.5⯱â¯1.6⯵M. Uracil transport is diminished in the presence of the proton disruptor carbonyl cyanide m-chlorophenylhydrazone, but not by the sodium gradient disruptor Ouabain.
Assuntos
Proteínas de Bactérias/metabolismo , Abelhas/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Paenibacillus larvae/metabolismo , Uracila/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico , Cinética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Paenibacillus larvae/química , Paenibacillus larvae/genética , Especificidade por Substrato , Uracila/química , Ácido Úrico/química , Ácido Úrico/metabolismo , Xantina/química , Xantina/metabolismoRESUMO
Two nucleobase transporters encoded in the genome of the Honey bee bacterial pathogen Paenibacillus larvae belong to the azaguanine-like transporters and are referred to as PlAzg1 and PlAzg2. PlAzg1 and 2 display significant amino acid sequence similarity, and share predicted secondary structures and functional sequence motifs with two Escherichia coli nucleobase cation symporter 2 (NCS2) members: adenine permease (EcAdeP) and guanine-hypoxanthine permease EcGhxP. However, similarity does not define function. Heterologous complementation and functional analysis using nucleobase transporter-deficient Saccharomyces cerevisiae strains revealed that PlAzg1 transports adenine, hypoxanthine, xanthine and uracil, while PlAzg2 transports adenine, guanine, hypoxanthine, xanthine, cytosine and uracil. Both PlAzg1 and 2 display high affinity for adenine with Km of 2.95 ± 0.22 and 1.92 ± 0.22 µM, respectively. These broad nucleobase transport profiles are in stark contrast to the narrow transport range observed for EcAdeP (adenine) and EcGhxP (guanine and hypoxanthine). PlAzg1 and 2 are similar to eukaryotic Azg-like transporters in that they share a broad solute transport profile, particularly the fungal Aspergillus nidulans AzgA (that transports adenine, guanine and hypoxanthine) and plant AzgA transporters from Arabidopsis thaliana and Zea mays (that collectively move adenine, guanine, hypoxanthine, xanthine, cytosine and uracil).
