Additional file 1: Table S1. of Phylogenetic and comparative genomics of the family Leptotrichiaceae and introduction of a novel fingerprinting MLVA for Streptobacillus moniliformis

Analysis of clusters of orthologous groups (COGs) of the Leptotrichiaceae members used in this study. COGs were assessed as described in the Materials and Methods. (XLSX 16 kb

Phenotypic characterization of the Rps6 phosphorylation deficiency.

<p>A) WT strain BY4742 and the indicated mutants were tested for growth in YPD medium. Growth was monitored at the indicated times. Data are presented as the mean ± S.E.M. of triplicate determinations and correspond to a representative experiment among three. Statistical analysis was performed by using two-way ANOVA. WT <i>vs</i>. <i>rp6bΔ</i> (significant: p < 0.001), WT vs. <i>rps6a</i>^{<i>S232A S233A</i>}<i>rp6bΔ</i> (significant: p = 0.001), <i>rp6bΔ v</i>s. <i>rps6a</i>^{<i>S232A S233A</i>}<i>rp6bΔ</i> (not significant: p = 0.153). B) Cell size of the indicated strains was measured. Two-way ANOVA analysis was performed. **, p<0.01; ns, not significant. C) WT strain BY4742 and the indicated mutants were spotted onto YPD plates containing either 4 ng/ml of rapamycin or drug vehicle alone. Plates were grown for 3 days at 30°C. Pictures correspond to a representative experiment among three. D) Model of regulation of Rps6 phosphorylation by TORC1-Ypk3.</p

Yeast strains used in this work.

<p>Yeast strains used in this work.</p

Characterization of the phospho-specific Rps6 antibody.

<p>A) C-terminal peptide sequence of human S6 and its orthologs in <i>S</i>. <i>pombe</i> and <i>S</i>. <i>cerevisiae</i>. The residues phosphorylated by S6K are highlighted in red. B) Immunoblot analysis of <i>S</i>. <i>cerevisiae</i> Rps6 phosphorylation in several <i>rps6</i> mutants in the BY4742 background. Samples from exponentially growing cultures of the indicated strains were taken. Total lysates were resolved by SDS—PAGE on 10% polyacrylamide gels and analyzed by immunoblot. Actin was used as loading control. C) Samples of exponentially growing TB50a cells were taken. Cells were lysed in lysis buffer either in the absence (lanes 1 and 2) or presence (lane 3) of phosphatase inhibitor mixture (PPi). Total protein lysates were incubated with 2 U of alkaline phosphatase (Roche) for 30 min at 37°C and analyzed by immunoblot as in B. Ponceau S staining of the membrane indicates the presence of alkaline phosphatase. D) Immunoblot analysis of Rps6 phosphorylation in WT and rapamycin-resistant <i>TOR1–1 TOR2–1</i> cells. Cells were treated with rapamycin (200 ng/ml) for the indicated times. Total lysates were analyzed as in B. E) Immunoblot analysis of <i>S</i>. <i>cerevisiae</i> Rps6 phosphorylation in WT cells. Exponentially growing cells in SC medium were shifted to nitrogen-free medium and cultured for 1 h. Then, SC medium containing 200 ng/ml rapamycin or drug vehicle was added and growth was resumed for 1 h. Total lysates were analyzed as in B. F) Immunoblot analysis of Sch9 and Rps6 phosphorylation upon glutamine stimulation. YSBN9 cells expressing Sch9–3xHA were grown in YMM with proline as the only nitrogen source. Then, glutamine (final concentration of 0.5 g/l) was added and samples were taken at the indicated times. To assess the Sch9 C-terminal phosphorylation state, protein samples were chemically cleaved with NTCB.</p

The Change of a Medically Important Genus: Worldwide Occurrence of Genetically Diverse Novel <i>Brucella</i> Species in Exotic Frogs

<div><p>The genus <i>Brucella</i> comprises various species of both veterinary and human medical importance. All species are genetically highly related to each other, sharing intra-species average nucleotide identities (ANI) of > 99%. Infections occur among various warm-blooded animal species, marine mammals, and humans. Until recently, amphibians had not been recognized as a host for <i>Brucella</i>. In this study, however, we show that novel <i>Brucella</i> species are distributed among exotic frogs worldwide. Comparative <i>recA</i> gene analysis of 36 frog isolates from various continents and different frog species revealed an unexpected high genetic diversity, not observed among classical <i>Brucella</i> species. In phylogenetic reconstructions the isolates consequently formed various clusters and grouped together with atypical more distantly related brucellae, like <i>B</i>. <i>inopinata</i>, strain BO2, and Australian isolates from rodents, some of which were isolated as human pathogens. Of one frog isolate (10RB9215) the genome sequence was determined. Comparative genome analysis of this isolate and the classical <i>Brucella</i> species revealed additional genetic material, absent from classical <i>Brucella</i> species but present in <i>Ochrobactrum</i>, the closest genetic neighbor of <i>Brucella</i>, and in other soil associated genera of the <i>Alphaproteobacteria</i>. The presence of gene clusters encoding for additional metabolic functions, flanked by tRNAs and mobile genetic elements, as well as by bacteriophages is suggestive for a different ecology compared to classical <i>Brucella</i> species. Furthermore it suggests that amphibian isolates may represent a link between free living soil saprophytes and the pathogenic <i>Brucella</i> with a preferred intracellular habitat. We therefore assume that brucellae from frogs have a reservoir in soil and, in contrast to classical brucellae, undergo extensive horizontal gene transfer.</p></div

<i>Phylogenetic tree</i> from maximum likelihood analysis of the <i>recA</i> gene alignment of <i>Brucella</i> isolates from exotic frogs including classical and ‘atypical’ <i>Brucella</i> species.

<p>The tree was calculated with 100 bootstrap repetitions. <i>Ochrobactrum</i> served as outgroup. Bar: 0.08 substitutions per site. Accession numbers are given in brackets.</p

History and origin of ‘atypical’ <i>Brucella</i> strains isolated from amphibian host species.

<p>(wc): wild caught, (cb): captive bred.</p

Core-genome-based phylogenetic neighbor-joining tree with 200 repetitions.

<p>Bar: 0.002 substitutions per site. Isolate 10RB9215 is indicated in bold letters. Accession numbers are given in brackets.</p

Field isolates and reference strains as well as origins, clinical symptoms and host species of <i>Streptobacillus moniliformis</i>, <i>Streptobacillus</i> spp. and <i>Sebaldella termitidis</i> used in this study.

<p>^T: type strain; ATCC: American Type Culture Collection, Rockville, USA; NCTC: National Collection of Type Cultures, London, UK; CIP: Collection Institut Pasteur, Paris, France; IPDH: Institut für Geflügelkrankheiten, Hannover, Germany; RIVM: Rijksinstituut voor Volksgezondheid en Milieuhygiene, Bilthoven, The Netherlands; AHL: Animal Health Laboratory, South Perth, Australia; ZfV: Zentralinstitut für Versuchstierzucht, Hannover, Germany; DKFZ: Deutsches Krebsforschungszentrum, Heidelberg, Germany; TiHo: Tierärztliche Hochschule Hannover, Germany; RBF: rat bite fever</p><p>Field isolates and reference strains as well as origins, clinical symptoms and host species of <i>Streptobacillus moniliformis</i>, <i>Streptobacillus</i> spp. and <i>Sebaldella termitidis</i> used in this study.</p

Maximum-likelihood tree showing the phylogenetic position within the family <i>Leptotrichiaceae</i>.

<p>The tree was generated in Geneious using PhyML [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134312#pone.0134312.ref035" target="_blank">35</a>] and based on 16S rRNA gene sequences. GenBank accession numbers are KR001904-1922, KP657489, KP657490-KP657495, and HG421076. Numbers at branch nodes refer to bootstrap values (100 replicates). Bar: 0.06 nucleotide substitutions per side.</p