Visualization of Glutamine Transporter Activities in Living Cells Using Genetically Encoded Glutamine Sensors

Glutamine plays a central role in the metabolism of critical biological molecules such as amino acids, proteins, neurotransmitters, and glutathione. Since glutamine metabolism is regulated through multiple enzymes and transporters, the cellular glutamine concentration is expected to be temporally dynamic. Moreover, differentiation in glutamine metabolism between cell types in the same tissue (e.g. neuronal and glial cells) is often crucial for the proper function of the tissue as a whole, yet assessing cell-type specific activities of transporters and enzymes in such heterogenic tissue by physical fractionation is extremely challenging. Therefore, a method of reporting glutamine dynamics at the cellular level is highly desirable. Genetically encoded sensors can be targeted to a specific cell type, hence addressing this knowledge gap. Here we report the development of Föster Resonance Energy Transfer (FRET) glutamine sensors based on improved cyan and yellow fluorescent proteins, monomeric Teal Fluorescent Protein (mTFP)1 and venus. These sensors were found to be specific to glutamine, and stable to pH-changes within a physiological range. Using cos7 cells expressing the human glutamine transporter ASCT2 as a model, we demonstrate that the properties of the glutamine transporter can easily be analyzed with these sensors. The range of glutamine concentration change in a given cell can also be estimated using sensors with different affinities. Moreover, the mTFP1-venus FRET pair can be duplexed with another FRET pair, mAmetrine and tdTomato, opening up the possibility for real-time imaging of another molecule. These novel glutamine sensors will be useful tools to analyze specificities of glutamine metabolism at the single-cell level

Emended classification of xanthomonad pathogens on citrus

In the paper by Schaad et al. [24] on reclassification of several xanthomonads, nomenclatural errors were made. The name Xanthomonas smithii subsp. citri proposed for the former taxon X. campestris pv. citri ( = X. axonopodis pv. citri) is illegitimate. Following the reinstatement of X. citri (ex Hasse 1915) Gabriel et al. [9] as a validly published name, Young et al. [34] wrote that the reinstatement of this epithet was based on a description that was inadequate in terms of modern practice for the purpose of formal classification. This report was subsequently summarized by the International Committee on the Systematics of Bacteria (ICSB) Subcommittee on the Taxonomy of the Genus Pseudomonas and Related Organisms [32] as implying rejection of the epithet, which the Subcommittee itself appeared to endorse. As we now understand, in accord with the International Code of Nomenclature of Prokaryotes (‘the Code’—hitherto the International Code of Nomenclature of Bacteria [14]) the Judicial Commission of the ICSP only may reject a name for precisely specified reasons (Rule 56a). We also misinterpreted the subsequent establishment of the pathovar ‘‘citri’’ within Xanthomonas axonopodis [29] as further evidence for rejection of reinstatement of X. citri [9]. Finally, believing that the epithet ‘‘citri’’ had been rejected, we followed rule 23a of the Code [14] and proposed an illegitimate specific epithet ‘‘smithii’’ (which also required establishing the subspecies epithet ‘‘smithii’’ replacing ‘‘malvacearum’’; see rule 13a [14]). In fact, X. citri Gabriel et al. 1989 was a legitimate, validly published name that was allowed to fall into abeyance because of the inadequacies perceived in its description. Schaad et al. [24] indicated their support for the conclusions of Gabriel et al. [9] but included DNA–DNA reassociation data indicated as necessary by for modern classification [26,31]. One purpose of this note is to recognize by effective publication the species related to pathogenic xanthomonads of citrus. The second purpose is to avoid confusion in plant pathological literature by replacing the illegitimate subspecies name X. smithii subsp. ‘‘smithii’’ with X. citri subsp. ‘‘malvacearum’’. For that purpose, corrected protologues for those species and subspecies are reported here: X. citri subsp. citri and X. citri subsp. malvacearum; X. fuscans subsp. fuscans and X. fuscans subsp. aurantifolii; and X. alfalfae subsp. alfalfae and X. alfalfae subsp. citrumelonis. We also present (Table 1) GenBank accession numbers for the intergeneric spacer (ITS) sequences for the type strains proposed in this note [24]

Reclassification of \u3ci\u3eXanthomonas campestris\u3c/i\u3e pv. \u3ci\u3ecitri \u3c/i\u3e (ex Hasse 1915) Dye 1978 forms A, B/C/D, and E as \u3ci\u3eX. smithii \u3c/i\u3esubsp. \u3ci\u3ecitri \u3c/i\u3e (ex Hasse) sp. nov. nom. rev. comb. nov., \u3ci\u3eX. fuscans\u3c/i\u3e subsp. \u3ci\u3eaurantifolii\u3c/i\u3e (ex Gabriel 1989) sp. nov. nom. rev. comb. nov., and \u3ci\u3eX. alfalfae\u3c/i\u3e subsp. \u3ci\u3ecitrumelo\u3c/i\u3e (ex Riker and Jones) Gabriel et al., 1989 sp. nov. nom. rev. comb. nov.; \u3ci\u3eX. campestris\u3c/i\u3e pv \u3ci\u3emalvacearum\u3c/i\u3e (ex Smith 1901) Dye 1978 as \u3ci\u3eX. smithii\u3c/i\u3e subsp. \u3ci\u3esmithii\u3c/i\u3e nov. comb. nov. nom. nov.; \u3ci\u3eX. campestris\u3c/i\u3e pv. \u3ci\u3ealfalfae\u3c/i\u3e (ex Riker and Jones, 1935) Dye 1978 as \u3ci\u3eX. alfalfae\u3c/i\u3e subsp. \u3ci\u3ealfalfae\u3c/i\u3e (ex Riker et al., 1935) sp. nov. nom. rev.; and ‘‘var. fuscans’’ of \u3ci\u3eX. campestris\u3c/i\u3e pv. \u3ci\u3ephaseoli\u3c/i\u3e (ex Smith, 1987) Dye 1978 as \u3ci\u3eX. fuscans\u3c/i\u3e subsp. \u3ci\u3efuscans\u3c/i\u3e sp. nov.

Bacterial canker of citrus is a serious disease of citrus worldwide. Five forms of the disease have been described, cankers “A”, “B”, “C”, “D”, and “E”. Although considerable genetic diversity has been described among the causal agents of the five forms of citrus canker and supports multiple taxons, the causal agents currently are classified as pathovars citri (“A”), aurantifolii (“B/C/D”) and citrumelo (“E”) of a single species, Xanthomonas campestris pv. citri (or X. axonopodis pv. citri).To determine the taxonomic relatedness among strains of X. campestris pv. citri, we conducted DNA–DNA relatedness assays, sequenced the 16S-23S intergenic spacer (ITS) regions, and performed amplified fragment length polymorphism (AFLP) analysis, using 44 strains representative of the five recognized forms of citrus canker.Under stringent DNA reassociation conditions (Tm -15 °C), three distinct genotypes of citrus pathogens were revealed: taxon I included all “A” strains; taxon II contained all “B”, “C”, and “D” strains; and taxon III contained all “E” strains. The three citrus taxa showed less than 50% (mean) DNA–DNA relatedness to each other and less than 30% (mean) to X. campestris pv. campestris and X. axonopodis pv. axonopodis. Taxa I and II strains share over 70% DNA relatedness to X. campestris pv. malvacearum and X. campestris pv. phaseoli var. fuscans, respectively (at Tm -15 °C).Tax on III strains share 70% relatedness to X. campestris pv. alfalfae. Previous and present phenotypic data support these DNA reassociation data. Taxon II strains grow more slowly on agar media than taxa I and III strains. Taxa I and III strains utilize maltose, and liquefy gelatin whereas taxon II strains do not. Tax on I strains hydrolyze pectate (pH 7.0) whereas Taxon II strains do not. Taxon III strains utilize raffinose whereas Taxon I strains do not. Each taxon can be differentiated by serology and pathogenicity. We propose taxa I, II, and III citrus strains be named, respectively, Xanthomonas smithii subsp. citri (ex Hasse, 1915) sp. nov. nom. rev. comb. nov ., Xanthomonas fuscans subsp. aurantifolii (ex Gabriel et al., 1989) sp. nov. nom. rev. comb. nov. , and Xanthomonas alfalfae subsp. citrumelo (ex Riker and Jones) Gabriel et al., 1989 nov. rev. comb. nov. Furthermore, based on the analysis of 40 strains of 19 other xanthomonads, we propose to reclassify X. campestris pv. malvacearum (ex Smith, 1901) Dye 1978 asX. smithii subsp. smithii sp. nov. comb. nov. nom. nov.; X. campestris pv. alfalfae (ex Riker and Jones) Dye 1978 as X. alfalfae subsp. alfalfae (ex Riker et al., 1935) sp. nov. nov. rev. ; and “var. fuscans” (ex Burkholder 1930) of X. campestris pv. phaseoli (ex Smith, 1897) as X. fuscans subsp. fuscans sp. nov

Affinities and substrate specificities of FLIPQ-TV3.0 sensors.

<p>(A) Saturation curves of FLIPQ-TV3.0 sensors with altered affinities. (B) Substrate specificities of FLIPQ-TV3.0_1.5 μ (black), 50 μ (hatched), 100 μ (white), 2 m (horizontal stripes), and 8 m (gray) sensors to Gln, Glu, Asn and Asp.</p

Relatedness of Chromosomal and Plasmid DNAs of Erwinia pyrifoliae and Erwinia amylovora

The plant pathogen Erwinia pyrifoliae has been classified as a separate species from Erwinia amylovora based in part on differences in molecular properties. In this study, these and other molecular properties were examined for E. pyrifoliae and for additional strains of E. amylovora, including strains from brambles (Rubus spp.). The nucleotide composition of the internal transcribed spacer (ITS) region was determined for six of the seven 16S-23S rRNA operons detected in these species with a 16S rRNA gene probe. Each species contained four operons with a tRNA(Glu) gene and two with tRNA(Ile) and tRNA(Ala) genes, and analysis of the operons from five strains of E. amylovora indicated a high degree of ITS variability among them. One tRNA(Glu)-containing operon from E. pyrifoliae Ep1/96 was identical to one in E. amylovora Ea110, but three tRNA(Glu) operons and two tRNA(Ile) and tRNA(Ala) operons from E. pyrifoliae contained unique nucleotide changes. When groEL sequences were used for species-specific identification, E. pyrifoliae and E. amylovora were the closest phylogenetic relatives among a set of 12 bacterial species. The placement of E. pyrifoliae distinct from E. amylovora corroborated molecular hybridization data indicating low DNA-DNA similarity between them. Determination of the nucleotide sequence of plasmid pEP36 from E. pyrifoliae Ep1/96 revealed a number of presumptive genes that matched genes previously found in pEA29 from E. amylovora and similar organization for the genes and origins of replication. Also, pEP36 and pEA29 were incompatible with clones containing the reciprocal origin regions. Finally, the ColE1-like plasmid pEP2.6 from strain Ep1/96 contained sequences found in small plasmids in E. amylovora strains IL-5 and IH3-1

Responses of cos7 cells co-expressing the FLIPQ-TV3.0_8 m sensor and ASCT2-mCherry to external glutamine in the absence (A) or in the presence of 13.4 mM (∼10% of normal Hank’s buffer) of extracellular sodium.

<p>Timepoints when 5 mM extracellular glutamine (red box) or 5 mM Ala (blue box) are indicated as boxes above the graph. Solid and dashed lines represent two individual cells measured in the same experiment.</p

Configuration of a FRET glutamine sensor.

<p>(A) Open (cyan) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038591#pone.0038591-Hsiao1" target="_blank">[36]</a> and closed (yellow, glutamine in the binding pocket is indicated in red) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038591#pone.0038591-Sun1" target="_blank">[37]</a> conformation of glnH, glutamine binding protein from <i>E.coli</i>. The position of the internal hairpin permissive to an insertion of FP is marked in magenta. (B) Schematic representations of chimeric fusions between mTFP1, glnH and venus sequences.</p

Affinities of FLIPQ-TV3.0 point mutants.

<p>Affinities of FLIPQ-TV3.0 point mutants.</p