Bacillus cereus Group-Type Strain-Specific Diagnostic Peptides.

The Bacillus cereus group consists of eight very closely related species and comprises both harmless and human pathogenic species such as Bacillus anthracis, Bacillus cereus, and Bacillus cytotoxicus. Numerous efforts have been undertaken to allow presumptive differentiation of B. cereus group species from one another. However, methods to rapidly and accurately distinguish these species are currently lacking. We confirmed that classical matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) biotyping cannot achieve reliable identification of each type strain. We therefore assigned type strain-specific diagnostic peptides to the B. cereus group based on comparisons of their proteomic profiles. The number of diagnostic peptides varied remarkably in a type strain-dependent manner. The accuracy of the reference database was crucial to validate candidate diagnostic peptides and led to a noteworthy reduction of verified diagnostic peptides. Diagnostic peptides ranged from one for B. weihenstephanensis to 62 for B. pseudomycoides and were associated with proteins involved in diverse biological processes, e.g. amino acid biosynthesis, cell envelope, cellular processes, energy metabolism, and transport processes. However, 45.6% of all diagnostic peptides comprised currently unclassified proteins or proteins of unknown function. In addition, a phylogenetic tree based on clustering of theoretical precursor masses deduced from in silico-generated tryptic peptides was reconstructed.

SWEET17, a facilitative transporter, mediates fructose transport across the tonoplast of Arabidopsis roots and leaves.

Fructose (Fru) is a major storage form of sugars found in vacuoles, yet the molecular regulation of vacuolar Fru transport is poorly studied. Although SWEET17 (for SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS17) has been characterized as a vacuolar Fru exporter in leaves, its expression in leaves is low. Here, RNA analysis and SWEET17-ß-glucuronidase/-GREEN FLUORESCENT PROTEIN fusions expressed in Arabidopsis (Arabidopsis thaliana) reveal that SWEET17 is highly expressed in the cortex of roots and localizes to the tonoplast of root cells. Expression of SWEET17 in roots was inducible by Fru and darkness, treatments that activate accumulation and release of vacuolar Fru, respectively. Mutation and ectopic expression of SWEET17 led to increased and decreased root growth in the presence of Fru, respectively. Overexpression of SWEET17 specifically reduced the Fru content in leaves by 80% during cold stress. These results intimate that SWEET17 functions as a Fru-specific uniporter on the root tonoplast. Vacuoles overexpressing SWEET17 showed increased [14C]Fru uptake compared with the wild type. SWEET17-mediated Fru uptake was insensitive to ATP or treatment with NH4Cl or carbonyl cyanide m-chlorophenyl hydrazone, indicating that SWEET17 functions as an energy-independent facilitative carrier. The Arabidopsis genome contains a close paralog of SWEET17 in clade IV, SWEET16. The predominant expression of SWEET16 in root vacuoles and reduced root growth of mutants under Fru excess indicate that SWEET16 also functions as a vacuolar transporter in roots. We propose that in addition to a role in leaves, SWEET17 plays a key role in facilitating bidirectional Fru transport across the tonoplast of roots in response to metabolic demand to maintain cytosolic Fru homeostasis.

Vacuolar transport of abscisic acid glucosyl ester is mediated by ATP-binding cassette and proton-antiport mechanisms in Arabidopsis.

Abscisic acid (ABA) is a key plant hormone involved in diverse physiological and developmental processes, including abiotic stress responses and the regulation of stomatal aperture and seed germination. Abscisic acid glucosyl ester (ABA-GE) is a hydrolyzable ABA conjugate that accumulates in the vacuole and presumably also in the endoplasmic reticulum. Deconjugation of ABA-GE by the endoplasmic reticulum and vacuolar ß-glucosidases allows the rapid formation of free ABA in response to abiotic stress conditions such as dehydration and salt stress. ABA-GE further contributes to the maintenance of ABA homeostasis, as it is the major ABA catabolite exported from the cytosol. In this work, we identified that the import of ABA-GE into vacuoles isolated from Arabidopsis (Arabidopsis thaliana) mesophyll cells is mediated by two distinct membrane transport mechanisms: proton gradient-driven and ATP-binding cassette (ABC) transporters. Both systems have similar Km values of approximately 1 mm. According to our estimations, this low affinity appears nevertheless to be sufficient for the continuous vacuolar sequestration of ABA-GE produced in the cytosol. We further demonstrate that two tested multispecific vacuolar ABCC-type ABC transporters from Arabidopsis exhibit ABA-GE transport activity when expressed in yeast (Saccharomyces cerevisiae), which also supports the involvement of ABC transporters in ABA-GE uptake. Our findings suggest that the vacuolar ABA-GE uptake is not mediated by specific, but rather by several, possibly multispecific, transporters that are involved in the general vacuolar sequestration of conjugated metabolites.