Diversity, Phylogeny and Expression Patterns of Pou and Six Homeodomain Transcription Factors in Hydrozoan Jellyfish Craspedacusta sowerbyi

Formation of all metazoan bodies is controlled by a group of selector genes including homeobox genes, highly conserved across the entire animal kingdom. The homeobox genes from Pou and Six classes are key members of the regulation cascades determining development of sensory organs, nervous system, gonads and muscles. Besides using common bilaterian models, more attention has recently been targeted at the identification and characterization of these genes within the basal metazoan phyla. Cnidaria as a diploblastic sister group to bilateria with simple and yet specialized organs are suitable models for studies on the sensory organ origin and the associated role of homeobox genes. In this work, Pou and Six homeobox genes, together with a broad range of other sensory-specific transcription factors, were identified in the transcriptome of hydrozoan jellyfish Craspedacusta sowerbyi. Phylogenetic analyses of Pou and Six proteins revealed cnidarian-specific sequence motifs and contributed to the classification of individual factors. The majority of the Craspedacusta sowerbyi Pou and Six homeobox genes are predominantly expressed in statocysts, manubrium and nerve ring, the tissues with sensory and nervous activities. The described diversity and expression patterns of Pou and Six factors in hydrozoan jellyfish highlight their evolutionarily conserved functions. This study extends the knowledge of the cnidarian genome complexity and shows that the transcriptome of hydrozoan jellyfish is generally rich in homeodomain transcription factors employed in the regulation of sensory and nervous functions

Biphenyl-Metabolizing Bacteria in the Rhizosphere of Horseradish and Bulk Soil Contaminated by Polychlorinated Biphenyls as Revealed by Stable Isotope Probing▿ †

DNA-based stable isotope probing in combination with terminal restriction fragment length polymorphism was used in order to identify members of the microbial community that metabolize biphenyl in the rhizosphere of horseradish (Armoracia rusticana) cultivated in soil contaminated with polychlorinated biphenyls (PCBs) compared to members of the microbial community in initial, uncultivated bulk soil. On the basis of early and recurrent detection of their 16S rRNA genes in clone libraries constructed from [13C]DNA, Hydrogenophaga spp. appeared to dominate biphenyl catabolism in the horseradish rhizosphere soil, whereas Paenibacillus spp. were the predominant biphenyl-utilizing bacteria in the initial bulk soil. Other bacteria found to derive carbon from biphenyl in this nutrient-amended microcosm-based study belonged mostly to the class Betaproteobacteria and were identified as Achromobacter spp., Variovorax spp., Methylovorus spp., or Methylophilus spp. Some bacteria that were unclassified at the genus level were also detected, and these bacteria may be members of undescribed genera. The deduced amino acid sequences of the biphenyl dioxygenase α subunits (BphA) from bacteria that incorporated [13C]into DNA in 3-day incubations of the soils with [13C]biphenyl are almost identical to that of Pseudomonas alcaligenes B-357. This suggests that the spectrum of the PCB congeners that can be degraded by these enzymes may be similar to that of strain B-357. These results demonstrate that altering the soil environment can result in the participation of different bacteria in the metabolism of biphenyl

Identification of bacteria utilizing biphenyl, benzoate, and naphthalene in long-term contaminated soil.

Bacteria were identified associated with biodegradation of aromatic pollutants biphenyl, benzoate, and naphthalene in a long-term polychlorinated biphenyl- and polyaromatic hydrocarbon-contaminated soil. In order to avoid biases of culture-based approaches, stable isotope probing was applied in combination with sequence analysis of 16 S rRNA gene pyrotags amplified from (13)C-enriched DNA fractions. Special attention was paid to pyrosequencing data analysis in order to eliminate the errors caused by either generation of amplicons (random errors caused by DNA polymerase, formation of chimeric sequences) or sequencing itself. Therefore, sample DNA was amplified, sequenced, and analyzed along with the DNA of a mock community constructed out of 8 bacterial strains. This warranted that appropriate tools and parameters were chosen for sequence data processing. (13)C-labeled metagenomes isolated after the incubation of soil samples with all three studied aromatics were largely dominated by Proteobacteria, namely sequences clustering with the genera Rhodanobacter Burkholderia, Pandoraea, Dyella as well as some Rudaea- and Skermanella-related ones. Pseudomonads were mostly labeled by (13)C from naphthalene and benzoate. The results of this study show that many biphenyl/benzoate-assimilating bacteria derive carbon also from naphthalene, pointing out broader biodegradation abilities of some soil microbiota. The results also demonstrate that, in addition to traditionally isolated genera of degradative bacteria, yet-to-be cultured bacteria are important players in bioremediation. Overall, the study contributes to our understanding of biodegradation processes in contaminated soil. At the same time our results show the importance of sequencing and analyzing a mock community in order to more correctly process and analyze sequence data

<i>Craspedacusta sowerbyi</i> body plan scheme.

<p>Radial symmetric body plan of <i>Craspedacusta sowerbyi</i> is divided in subumbrella and exumbrella regions from the side projection. The most distinct features are four gonads and the presence of up to two hundreds of tentacles. Particular organs are described in the scheme. Body size is up to 2.5 cm in diameter.</p

Comparison of phylogenetic trees of HD (A) and SD (B) regions of Six class transcription factors.

<p>The phylogenetic trees were calculated by the maximum likelihood method (WAG protein substitution model, bootstrap 1000) and processed by NJ Plot software. Phylogenetically representative sequences from the following metazoan animals were selected for comparison: sponges (<i>Chalinula loosanoffi, Ephydatia fluviatilis, Sycon calcaravis</i>), cnidarians (<i>Nematostella vectensis</i>, <i>Anthopleura japonica</i>, <i>Podocoryne carnea</i>, <i>Hydra magnipapillata</i>, <i>Cladonema radiatum</i>, <i>Aurelia aurita</i>), invertebrates (<i>Saccoglossus kowalevskii</i>, <i>Drosophila melanogaster</i>, <i>Branchistoma floridae</i>) and vertebrates (<i>Danio rerio</i> and <i>Homo sapiens</i>). For the list of ID numbers of reference sequences see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036420#pone.0036420.s011" target="_blank">Text S1</a>.</p

Number of sequences obtained after sequence processing, after subtracting sequences detected also in control DNA (valid sequences), and after normalizing.

<p>Number of sequences obtained after sequence processing, after subtracting sequences detected also in control DNA (valid sequences), and after normalizing.</p

Bacterial strains used for the preparation of the mock community.

<p>Bacterial strains used for the preparation of the mock community.</p

Top OTUs detected in ¹³C-DNA after incubation of soil with ¹³C-biphenyl, ¹³C-benzoate, and ¹³C-naphthalene.

<p>Identification was performed by mothur-implemented RDP reference files <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040653#pone.0040653-Wang1" target="_blank">[78]</a> and the closest type strain was determined by RDP Seqmatch with the representative sequence of each OTU <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040653#pone.0040653-Cole1" target="_blank">[76]</a>. The entire dataset is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040653#pone.0040653.s002" target="_blank">Table S1</a>.</p>a<p>Relative abundance of sequences.</p>b<p>Identification of OTU based on identification of the representative at the level of genus as determined by RDP classifier (using 50% threshold).</p>c<p>Determined by RDP Seqmatch.</p>d<p>Score represents S_ab score – the number of (unique) 7-base oligomers shared between the sequence data and a given RDP sequence divided by the lowest number of unique oligos in either of the two sequences.</p>e<p>Refers to samples where the same OTU was detected.</p

Analysis of inorganic nutrients and contaminants available in the soil.

<p>Results shown are averages from 5 independently measured samples (performed commercially).</p>a<p>Based on method CZ_SOP_D06_07_121.</p>b<p>Based on method US EPA 8082.</p>c<p>Based on methods EPA 8270, EPA 8131, EPA 8091.</p>d<p>Based on method US EPA 200.7.</p

Matrix-Assisted Laser Desorption Ionization (MALDI)-Time of Flight Mass Spectrometry- and MALDI Biotyper-Based Identification of Cultured Biphenyl-Metabolizing Bacteria from Contaminated Horseradish Rhizosphere Soil▿

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with Arthrobacter, Serratia, Rhodococcus, and Rhizobium being predominant. Arthrobacter isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases