Relevance and Diversity of <i>Nitrospira</i> Populations in Biofilters of Brackish RAS

<div><p>Lithoautotrophic nitrite-oxidizing bacterial populations from moving-bed biofilters of brackish recirculation aquaculture systems (RAS; shrimp and barramundi) were tested for their metabolic activity and phylogenetic diversity. Samples from the biofilters were labeled with ¹³C-bicarbonate and supplemented with nitrite at concentrations of 0.3, 3 and 10 mM, and incubated at 17 and 28°C, respectively. The biofilm material was analyzed by fatty acid methyl ester - stable isotope probing (FAME-SIP). High portions of up to 45% of <i>Nitrospira</i>-related labeled lipid markers were found confirming that <i>Nitrospira</i> is the major autotrophic nitrite oxidizer in these brackish systems with high nitrogen loads. Other nitrite-oxidizing bacteria such as <i>Nitrobacter</i> or <i>Nitrotoga</i> were functionally not relevant in the investigated biofilters. <i>Nitrospira</i>-related 16S rRNA gene sequences were obtained from the samples with 10 mM nitrite and analyzed by a cloning approach. Sequence studies revealed four different phylogenetic clusters within the marine sublineage IV of <i>Nitrospira</i>, though most sequences clustered with the type strain of <i>Nitrospira marina</i> and with a strain isolated from a marine RAS. Three lipids dominated the whole fatty acid profiles of nitrite-oxidizing marine and brackish enrichments of <i>Nitrospira</i> sublineage IV organisms. The membranes included two marker lipids (16∶1 <i>cis</i>7 and 16∶1 <i>cis</i>11) combined with the non-specific acid 16∶0 as major compounds and confirmed these marker lipids as characteristic for sublineage IV species. The predominant labeling of these characteristic fatty acids and the phylogenetic sequence analyses of the marine <i>Nitrospira</i> sublineage IV identified organisms of this sublineage as main autotrophic nitrite-oxidizers in the investigated brackish biofilter systems.</p></div

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

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

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

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