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The Janthinobacterium sp. HH01 genome encodes a homologue of the V. cholerae CqsA and L. pneumophila LqsA autoinducer synthases

Janthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes

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

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

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

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