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 GC3 was extracted from the permanent and unpublished draft available at 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

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

ORFs identified and involved in autoinducer biosynthesis in HH01 and closely related microorganisms.

<p>−, autoinducer synthase not detected, +, autoinducer synthase identified; (+), weak similarity observed to the known autoinducer I synthases from <i>C. violaceum</i> CviI and related species.</p

TEM image of HH01.

<p>A single flagellum attached to its cell pole is visible. Active cells were stained by uranyl acetate.</p

Predicted structures resulting from cluster 2–6.

<p>The predicted configuration is indicated by R- or S-nomenclature. All compounds are shown in the linear form but might be cyclic (for details see text). The HH01 genome was analyzed for secondary metabolite biosynthesis gene clusters using the AntiSMASH program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055045#pone.0055045-Medema1" target="_blank">[61]</a>. Additionally, the genome was manually searched for genes encoding adenylation (A) and ketosynthase (KS) domains using a local BLAST server. All identified genes and/or gene clusters encoding the respective enzymes were then manually inspected and the predicted natural products resulting from the identified enzyme activities were drawn.</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

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