An Erwinia amylovora uracil transporter mutant retains virulence on immature apple and pear fruit.

Erwinia amylovora is the causal agent of fire blight, a devastating disease of apples and pears. A previous study revealed that an E. amylovora uracil auxotroph was still virulent and can cause disease, suggesting that uracil can be obtained from the host environment. The E. amylovora genome contains a locus encoding for a uracil transporter belonging to the nucleobase cation symporter 2 family, displaying a high level of amino acid sequence similarity to the Escherichia coli UraA. Expression of E. amylovora UraA in nucleobase transporter-deficient E. coli strains, coupled with radiolabeled uptake studies reveal that E. amylovora UraA is a high affinity uracil transporter with a Km of 0.57 µM. Both E. coli and E. amylovora carrying extra copies of E. amylovora UraA are sensitive to growth on the toxic analog 5-fluorouracil. An E. amylovora ΔuraA::Camr mutant is still able to grow and cause disease symptoms on immature pears and apples.

The solute transport and binding profile of a novel nucleobase cation symporter 2 from the honeybee pathogen Paenibacillus larvae.

Here, we report that a novel nucleobase cation symporter 2 encoded in the genome of the honeybee bacterial pathogen Paenibacillus larvae reveals high levels of amino acid sequence similarity to the Escherichia coli and Bacillus subtilis uric acid and xanthine transporters. This transporter is named P. larvae uric acid permease-like protein (PlUacP). Even though PlUacP displays overall amino acid sequence similarities, has common secondary structures, and shares functional motifs and functionally important amino acids with E. coli xanthine and uric acid transporters, these commonalities are insufficient to assign transport function to PlUacP. The solute transport and binding profile of PlUacP was determined by radiolabeled uptake experiments via heterologous expression in nucleobase transporter-deficient Saccharomyces cerevisiae strains. PlUacP transports the purines adenine and guanine and the pyrimidine uracil. Hypoxanthine, xanthine, and cytosine are not transported by PlUacP, but, along with uric acid, bind in a competitive manner. PlUacP has strong affinity for adenine Km 7.04 ± 0.18 µm, and as with other bacterial and plant NCS2 proteins, PlUacP function is inhibited by the proton disruptor carbonyl cyanide m-chlorophenylhydrazone. The solute transport and binding profile identifies PlUacP as a novel nucleobase transporter.

Functional characterization of the uracil transporter from honeybee pathogen Paenibacillus larvae.

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 µM. Uracil transport is diminished in the presence of the proton disruptor carbonyl cyanide m-chlorophenylhydrazone, but not by the sodium gradient disruptor Ouabain.