Čestmír Vlček

Active and total microbial communities in forest soil are largely different and highly stratified during decomposition

Soils of coniferous forest ecosystems are important for the global carbon cycle, and the identification of active microbial decomposers is essential for understanding organic matter transformation in these ecosystems. By the independent analysis of DNA and RNA, whole communities of bacteria and fungi and its active members were compared in topsoil of a Picea abies forest during a period of organic matter decomposition. Fungi quantitatively dominate the microbial community in the litter horizon, while the organic horizon shows comparable amount of fungal and bacterial biomasses. Active microbial populations obtained by RNA analysis exhibit similar diversity as DNA-derived populations, but significantly differ in the composition of microbial taxa. Several highly active taxa, especially fungal ones, show low abundance or even absence in the DNA pool. Bacteria and especially fungi are often distinctly associated with a particular soil horizon. Fungal communities are less even than bacterial ones and show higher relative abundances of dominant species. While dominant bacterial species are distributed across the studied ecosystem, distribution of dominant fungi is often spatially restricted as they are only recovered at some locations. The sequences of cbhI gene encoding for cellobiohydrolase (exocellulase), an essential enzyme for cellulose decomposition, were compared in soil metagenome and metatranscriptome and assigned to their producers. Litter horizon exhibits higher diversity and higher proportion of expressed sequences than organic horizon. Cellulose decomposition is mediated by highly diverse fungal populations largely distinct between soil horizons. The results indicate that low-abundance species make an important contribution to decomposition processes in soils.

A Mouse Speciation Gene Encodes a Meiotic Histone H3 Methyltransferase

Speciation genes restrict gene flow between the incipient species and related taxa. Three decades ago, we mapped a mammalian speciation gene, hybrid sterility 1 (Hst1), in the intersubspecific hybrids of house mouse. Here, we identify this gene as Prdm9, encoding a histone H3 lysine 4 trimethyltransferase. We rescued infertility in male hybrids with bacterial artificial chromosomes carrying Prdm9 from a strain with the “fertility” Hst1f allele. Sterile hybrids display down-regulated microrchidia 2B (Morc2b) and fail to compartmentalize γH2AX into the pachynema sex (XY) body. These defects, seen also in Prdm9-null mutants, are rescued by the Prdm9 transgene. Identification of a vertebrate hybrid sterility gene reveals a role for epigenetics in speciation and opens a window to a hybrid sterility gene network.

Novel structurally distinct family of leucocyte surface glycoproteins including CD9, CD37, CD53 and CD63.

Several of the recently described leucocyte surface (glyco)‐proteins with significant amino acid sequence similarity (human CD9, CD37, CD53, CD63, TAPA‐1, CO‐029 and R2 and several homologues of other species) are distinguished by the polypeptide chain apparently four times crossing the membrane. Although the biological role of none of these molecules is known, their structure, associations with other membrane components and the effects of specific monoclonal antibodies suggest that they may constitute a family of ion channels or other transport molecules.

Melanoma cells influence the differentiation pattern of human epidermal keratinocytes

BackgroundNodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC).MethodsComparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK.ResultsEpidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes.ConclusionWe conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

Assembly of the cnidarian camera-type eye from vertebrate-like components

Animal eyes are morphologically diverse. Their assembly, however, always relies on the same basic principle, i.e., photoreceptors located in the vicinity of dark shielding pigment. Cnidaria as the likely sister group to the Bilateria are the earliest branching phylum with a well developed visual system. Here, we show that camera-type eyes of the cubozoan jellyfish, Tripedalia cystophora, use genetic building blocks typical of vertebrate eyes, namely, a ciliary phototransduction cascade and melanogenic pathway. Our findings indicative of parallelism provide an insight into eye evolution. Combined, the available data favor the possibility that vertebrate and cubozoan eyes arose by independent recruitment of orthologous genes during evolution.

A Eukaryote without a Mitochondrial Organelle

The presence of mitochondria and related organelles in every studied eukaryote supports the view that mitochondria are essential cellular components. Here, we report the genome sequence of a microbial eukaryote, the oxymonad Monocercomonoides sp., which revealed that this organism lacks all hallmark mitochondrial proteins. Crucially, the mitochondrial iron-sulfur cluster assembly pathway, thought to be conserved in virtually all eukaryotic cells, has been replaced by a cytosolic sulfur mobilization system (SUF) acquired by lateral gene transfer from bacteria. In the context of eukaryotic phylogeny, our data suggest that Monocercomonoides is not primitively amitochondrial but has lost the mitochondrion secondarily. This is the first example of a eukaryote lacking any form of a mitochondrion, demonstrating that this organelle is not absolutely essential for the viability of a eukaryotic cell.

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

ABSTRACT 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.

Bacterial proteins pinpoint a single eukaryotic root

Significance The root of eukaryote phylogeny formally represents the last eukaryotic common ancestor (LECA), but its position has remained controversial. Using new genome sequences, we revised and expanded two datasets of eukaryotic proteins of bacterial origin, which previously yielded conflicting views on the eukaryotic root. Analyses using state-of-the-art phylogenomic methodology revealed that both expanded datasets now support the same root position. Our results justify a new nomenclature for the two main eukaryotic groups and provide a robust phylogenetic framework to investigate the early evolution of the eukaryotic cell. The large phylogenetic distance separating eukaryotic genes and their archaeal orthologs has prevented identification of the position of the eukaryotic root in phylogenomic studies. Recently, an innovative approach has been proposed to circumvent this issue: the use as phylogenetic markers of proteins that have been transferred from bacterial donor sources to eukaryotes, after their emergence from Archaea. Using this approach, two recent independent studies have built phylogenomic datasets based on bacterial sequences, leading to different predictions of the eukaryotic root. Taking advantage of additional genome sequences from the jakobid Andalucia godoyi and the two known malawimonad species (Malawimonas jakobiformis and Malawimonas californiana), we reanalyzed these two phylogenomic datasets. We show that both datasets pinpoint the same phylogenetic position of the eukaryotic root that is between “Unikonta” and “Bikonta,” with malawimonad and collodictyonid lineages on the Unikonta side of the root. Our results firmly indicate that (i) the supergroup Excavata is not monophyletic and (ii) the last common ancestor of eukaryotes was a biflagellate organism. Based on our results, we propose to rename the two major eukaryotic groups Unikonta and Bikonta as Opimoda and Diphoda, respectively.

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 13C-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. 13C-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 13C 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.

Nucleotide sequence of the late region of Bacillus phage φ29 completes the 19285-bp sequence of φ29 genome. Comparison with the homologous sequence of phage PZA

The 12,177-bp nucleotide (nt) sequence of the late region of Bacillus phage luminal diameter 29 genome was determined. This sequence completes the entire 19,285-bp sequence of phage luminal diameter 29 DNA. Eleven open reading frames were found in this region, and these were assigned to eleven late genes. Ribosome-binding sites and a potential transcriptional promoter and terminator are considered. The nt sequence was compared to the homologous region of the closely related phage PZA and tolerated variations at the nt and amino acid (aa) level were evaluated. The most frequent changes are silent nt substitutions in the third position of codons, but aa substitutions are also found.