Functional characterization of the adenine transporter EaAdeP from the fire blight pathogen Erwinia amylovora and its effect on disease establishment in apples and pears.

Erwinia amylovora is the causal agent of fire blight, an economically important disease of apples and pears. As part of the infection process, Er. amylovora propagates on different plant tissues each with distinct nutrient environments. Here, the biochemical properties of the Er. amylovora adenine permease (EaAdeP) are investigated. Heterologous expression of EaAdeP in nucleobase transporter-deficient Escherichia coli strains, coupled with radiolabel uptake studies, revealed that EaAdeP is a high affinity adenine transporter with a Km of 0.43 ± 0.09 µM. Both Es. coli and Er. amylovora carrying extra copies of EaAdeP are sensitive to growth on the toxic analog 8-azaadenine. EaAdeP is expressed during immature pear fruit infection. Immature pear and apple fruit virulence assays reveal that an E. amylovora ΔadeP::Camr mutant is still able to cause disease symptoms, however, with growth at a lower level, indicating that external adenine is utilized in disease establishment.

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.

The solute transport profile of two Aza-guanine transporters from the Honey bee pathogen Paenibacillus larvae.

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).