Constitutional complaint and civil proceedings

Raw data for the analysis of N2, CB4856, and the npr-1 mutants. Sheet 1, Lawn leaving behavior of N2 and CB4856 from Bacillus thuringiensis - Raw data for Fig. 1; sheet 2, Lawn leaving behavior of mutants from Bacillus thuringiensis - Raw data for Fig. 4; sheet 3, Surival of N2 and CB4856 on Bacillus thuringiensis - Raw data for Additional file 9; sheet 4, Surival of mutants on Bacillus thuringiensis - Raw data for Fig. 5; sheet 5, Lawn leaving behavior of N2 and CB4856 from Pseudomonas aeruginosa PA14 - Raw data for Additional file 10; sheet 6, Lawn leaving behavior of mutants from Pseudomonas aeruginosa PA14 - Raw data for Fig. 6; sheet 7, Surival of mutants on Pseudomonas PA14 - Raw data for Fig. 5. (XLS 482 kb

Additional file 15: of Contrasting invertebrate immune defense behaviors caused by a single gene, the Caenorhabditis elegans neuropeptide receptor gene npr-1

Table with the list of differentially expressed genes after exposure of the C. elegans N2 wildtype or npr-1 mutant to pathogenic B. thuringiensis B-18247, pathogenic P. aeruginosa PA14, or non-pathogenic E. coli. (XLS 2096 kb

Additional file 13: of Contrasting invertebrate immune defense behaviors caused by a single gene, the Caenorhabditis elegans neuropeptide receptor gene npr-1

Table on the statistical results for the comparison of C. elegans survival rate on P. aeruginosa PA14 versus the control E. coli OP50. (PDF 74 kb

General features of the HH01 chromosome and closely related microorganisms.

<p>The genome of <i>C. violaceum</i> ATCC12472 was derived from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055045#pone.0055045-BrazilianConsortium1" target="_blank">[13]</a>; the genome information on <i>Janthinobacterium</i> sp. PAMC 25724 was obtained from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055045#pone.0055045-Kim1" target="_blank">[12]</a> the genome information on <i>Janthinobacterium</i> sp. Marseille was derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055045#pone.0055045-Audic1" target="_blank">[11]</a>; additional information and the genome information for <i>Janthinobacterium</i> sp. GC3 was extracted from the permanent and unpublished draft available at <a href="http://www.jgi.doe.gov/and" target="_blank">http://www.jgi.doe.gov/and</a> using the IMG software at <a href="https://img.jgi.doe.gov/cgi-bin/er/main.cgi" target="_blank">https://img.jgi.doe.gov/cgi-bin/er/main.cgi</a>.</p

BlastP comparison of the <i>Janthinobacterium</i> sp. HH01 genome compared against genomes of closely related species.

<p>The innermost rings indicate the GC content (black) and GC skew (purple/green). The outer rings represent the genomes of the following microbes in different colorings: <i>Janthinobacterium</i> sp. Marseille, blue; <i>Janthinobacterium</i> sp. PAMC 25724, red; <i>Janthinobacterium</i> sp. GC3, green; and <i>C. violaceum</i> ATCC 12472, black. The genome map was created using BRIG (Blast Ring Image Generator; <a href="http://sourceforge.net/projects/brig" target="_blank">http://sourceforge.net/projects/brig</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055045#pone.0055045-Alikhan1" target="_blank">[46]</a>.</p

Phylogenetic analysis of <i>cqsA-, jqsA</i>- and <i>lqsA</i>-like autoinducer synthases in Gram-negative bacteria.

<p>The neighbor-joining phylogenetic analysis was performed using the MEGA5 software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055045#pone.0055045-Tamura1" target="_blank">[45]</a> version 5.1 and comparing amino acid sequences of the corresponding synthases. Homology searches for orthologous proteins were done in the IMG genome database in September 2012 with 3,938 completed or draft bacterial genomes present. The autoinducer synthase sequences of the following strains are included, numbers in parenthesis indicate the corresponding accession number: <i>R. eutropha</i> H16 (YP_728640), <i>C. necator</i> N-1 (YP_004680649), <i>C. taiwanensis</i> LMG19424 (YP_001796752), <i>C. fungivorans</i> Ter331 (YP_004750816), <i>P. naphthalenivorans</i> CJ2 (YP_983733<i>), R. tataouinensis</i> TTB310 (YP_004617950), <i>R. vannielii</i> ATCC 17100 (YP_004010985), <i>S. shabanensis</i> E1L3A (ZP_08550556), <i>N. mobilis</i> Nb-231 (ZP_01127067), <i>B. xenovorans</i> LB400 (YP_555293), <i>C. phaeobacteroides</i> DSM 266 (YP_912394), <i>C. ferrooxidans</i> DSM 13031 (ZP_01385258), <i>C. limicola</i> DSM 245 (YP_001942557), <i>P. aestuarii</i> DSM 271 (YP_002015366), <i>Photobacterium</i> sp. SKA34 (ZP_01162832), <i>V. cholerae</i> CIRS 101 (ZP_05420646), <i>P. profundum</i> SS9 (YP_133409), <i>V. parahaemolyticus</i> RIMD 2210633 (NP_800221), <i>V. alginolyticus</i> 12G01 (ZP_01260612), <i>V. harveyi</i> ATCC BAA-1116 (YP_001448208), <i>V. splendidus</i> 12B01 (ZP_00990208). a) <i>Marinomonas</i> sp. is summarized for: <i>M. mediterranea</i> MMB-1 (ATCC 700492); <i>M. posidonica</i> IVIA-Po-181. b) <i>L. pneumophila</i> is summarized for: Philadelphia-1 (YP_096734), Paris (YP_125092) and Lens (YP_127984). Bacterial genera that have previously not been reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055045#pone.0055045-Tiaden1" target="_blank">[17]</a> to contain a <i>cqsA/lqsA</i> homologue are marked with an asterisk.</p

HH01 violacein biosynthesis affects <i>C. elegans</i> survival and nematode development.

<p>A) Decreased survival of <i>C. elegans</i> exposed to violacein-producing HH01. <i>C. elegans</i> grown on the violacein-producing parent strain HH01 died faster than worms on the <i>E. coli</i> control (p<0.001), while there was no significant difference in survival between worms grown on the violacein biosynthesis mutant and the <i>E. coli</i> control (p = 0.0375). B-D) Developmental arrest of <i>C. elegans</i> on violacein-producing janthinobacteria. B) DIC image (10x magnification) of a 4-day-old worm grown on <i>E. coli</i>. C) DIC image (10x magnification) of a 4-day-old worm grown on the violacein-negative mutant HH5-1. D) DIC image (40x magnification) of a 4-day-old worm grown on HH01. <i>C. elegans</i> grown on the violacein biosynthesis mutant and <i>E. coli</i> developed normally to the adult stage, whereas worms grown on the violacein-producing parent strain HH01 showed larval arrest.</p

Effect of the known or assumed autoinducer molecules of different strains on the HH01 or HH02 violacein production.

<p>A) Effects of extracted possible JAI-1 and CAI-1 autoinducer molecules on HH01 parent strain violacein production. The autoinducers were extracted from <i>E. coli</i> DH5α, carrying either the <i>jqsA</i> or the <i>cqsA</i> gene in pBBR1MCS-2. Autoinducers were purified as described in the material and methods section. 10 µl of these extracts were applied to HH01 growing cultures during early exponential growth phase. The control strain carried the empty vector. B) Effects of extra copies of the HH01 <i>jqsA, the V. cholerae cqsA</i> and the <i>L. pneumophila lqsA</i> genes in the parent strain HH01. The corresponding genes were inserted into the broad host range vector pBBR1MCS-2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055045#pone.0055045.s003" target="_blank">Table S1</a>). C) Violacein produced by the Δ<i>jqsA</i> mutant HH02, HH01 and HH02 carrying either the native <i>jqsA</i>, the <i>V. cholerae cqsA</i> or the <i>L. pneumophila lqsA</i> in pBBR1MCS-2. HH02 carrying an empty pBBR1MCS-2 produced similarly low amounts of violacein compared to HH02 without the empty control vector. Error bars indicate the simple standard deviations. Violacein values were calculated per ml of culture supernatant and normalized with respect to culture density at OD600 nm.</p

Transmission and scanning electron microscopic images of HH01 as well as a 16S rRNA-based tree.

<p>A) Transmission and B) scanning electron microscopic images of HH01. Arrows indicate observed vesicles on the HH01 outer cell surface. Scale bars of 200 nm are indicated in the images. C) 16S rRNA-based tree showing the phylogenetic affiliation of HH01. The tree was constructed using the neighbor-joining algorithm in MEGA5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055045#pone.0055045-Tamura1" target="_blank">[45]</a>. Topology was evaluated by bootstrap analysis (1000 repeats, with <i>N. europae</i>a as an outgroup). Only sequences longer than 1450 nucleotides of representatives of the next relative (≥97% similarity) species validly described were included. Numbers in parenthesis indicate the corresponding GenBank entries. Bootstrap values are shown as percentages at the branch points. The scale bar represents the expected number of changes per nucleotide position.</p

Violacein operon of HH01 and other violacein-producing bacteria.

<p>Conserved organization of the violacein biosynthesis operon of HH01 in comparison to violacein operons from <i>Pseudoalteromonas tunicata</i> strain D2 and <i>C. violaceum</i> ATCC 12472. Flanking genes not associated with the violacein biosynthesis are in grey; genes directly associated with violacein biosynthesis are in black. Arrows indicate direction of transcription. Jab_2c08770, two component regulator; Jab_2c08780, histidine sensor kinase, Jab_2c08790, FOG-domain containing hypothetical protein, Jab_2c08800, histidine sensor kinase; <i>cmlR</i>, potential chloramphenicol resistance protein; <i>luxQ2, luxQ</i> homologue; PTD2_19522, MATE efflux pump related protein; PTD2_19492 tryptophanyl t-RNA synthetase; CV3275, SpH family like protein; CV3276, hypothetical protein; CV3277, hypothetical protein; CV3278, cytochrome b561 protein; CV3266–C3269 hypothetical proteins. <i>P. tunicata</i> genes and ORFs were extracted from GenBank entry AAOH00000000 and the corresponding <i>C. violaceum</i> genes from GenBank entry NC_005085.1.</p