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Three different fungi were tested for their ability to degrade 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid and for the role of laccases and cytochromes P450-type in this process. We studied a white-rot fungus Rigidoporus sp. FMD21, which has a high laccase activity, for its efficiency to degrade these herbicides. A positive correlation was found between its laccase activity and the corresponding herbicide degradation rate. Even more, the doubling of the enzyme activity in this phase corresponded with a doubling of the herbicide degradation rate. It is, therefore, tempting to speculate that laccase is the most dominant enzyme in the degradation of 2,4-D and 2,4,5-T under these conditions. In addition, it was shown that Rigidoporus sp. FMD21 partly relies on cytochromes P450-type for the breakdown of the herbicides as well. Two filamentous fungi were isolated from soil contaminated with herbicides and dioxins located at Bien Hoa airbase. They belong to genera Fusarium and Verticillium of the phylum Ascomycota as judged by their 18S rRNA gene sequences. Both isolated fungi were able to degrade the herbicides but with different rates. Their laccase activity, however, was very low and did not correlate with the rate of breakdown of the herbicides. These data indicate that the white-rot fungus most likely synthesizes laccase and cytochromes P450-type for the breakdown of the herbicides, while the types of enzyme used for the breakdown of the herbicides by the two Ascomycota remain unclear.

Background Genomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology. To investigate a small to medium number of genes or to confirm large scale microarray studies, Quantitative Reverse Transcriptase PCR (QRT-PCR) can achieve high accuracy of quantification when key standards, such as normalization, are carefully set. In this study, we validated potential reference genes for their use as endogenous controls under different chemical and physical stresses in two species of soil-living Collembola, Folsomia candida and Orchesella cincta . Treatments for F. candida were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for O. cincta cadmium, desiccation, heat shock and starvation. Results Eight potential reference genes for F. candida and seven for O. cincta were ranked by their stability per stress factor using the programs geNorm and Normfinder. For F. candida the succinate dehydrogenase ( SDHA ) and eukaryotic transcription initiation factor 1A ( ETIF ) genes were found the most stable over the different treatments, while for O. cincta , the beta actin ( ACTb ) and tyrosine 3-monooxygenase ( YWHAZ ) genes were the most stable. Conclusion We present a panel of reference genes for two emerging ecological genomic model species tested under a variety of treatments. Within each species, different treatments resulted in differences in the top stable reference genes. Moreover, the two species differed in suitable reference genes even when exposed to similar stresses. This might be attributed to dissimilarity of physiology. It is vital to rigorously test a panel of reference genes for each species and treatment, in advance of relative quantification of QRT-PCR gene expression measurements.

Horizontal transfer of genes is widespread among prokaryotes, but is less common between microorganisms and animals. Here, we present evidence for the presence of a gene encoding functional isopenicillin N synthase, an enzyme in the β-lactam antibiotics biosynthesis pathway, in the genome of the soil-living collembolan species, Folsomia candida (FcIPNS). At present, this gene is only known from bacteria and fungi, as is the capacity to produce β-lactam antibiotics. The FcIPNS gene was located on two genomic contigs, was physically linked to a predicted insect ATP-binding cassette transporter gene, and contained three introns each flanked by eukaryotic splicing recognition sites (GT/AG). Homology searches revealed no similarity between these introns and the FcIPNS regions of bacteria or fungi. All amino acids conserved across bacteria and fungi were also conserved in F. candida. Recombinant FcIPNS was able to convert its substrate amino δ-(l-α-aminoadipyl)–l-cysteinyl–d-valine into isopenicillin N, providing strong evidence that FcIPNS is functional. Phylogenetic analysis clustered FcIPNS outside the bacterial IPNS clade, and also outside the fungal IPNS clade, suggesting an ancient gene transfer followed by divergence in the F. candida genome. In conclusion, the data suggest that the soil-living collembolan F. candida has assimilated the capacity for antibacterial activity by horizontal gene transfer, which may be an important adaptive trait in the microbe-dominated soil ecosystem.

The soil worm Enchytraeus crypticus (Oligochaeta) is an ecotoxicology model species that, until now, was without genome or transcriptome sequence information. The present research aims at studying the transcriptome of Enchytraeus crypticus, sampled from multiple test conditions, and the construction of a high-density microarray for functional genomic studies. Over 1.5 million cDNA sequence reads were obtained representing 645 million nucleotides. After assembly, 27,296 contigs and 87,686 singletons were obtained, from which 44% and 25% are annotated as protein-coding genes, respectively, sharing homology with other animal proteomes. Concerning assembly quality, 84% of the contig sequences contain an open reading frame with a start codon while E. crypticus homologs were identified for 92% of the core eukaryotic genes. Moreover, 65% and 77% of the singletons and contigs without known homologs, respectively, were shown to be transcribed in an independent microarray experiment. An Agilent 180 K microarray platform was designed and validated by hybridizing cDNA from 4 day zinc- exposed E. crypticus to the concentration corresponding to 50% reduction in reproduction after three weeks (EC50). Overall, 70% of all probes signaled expression above background levels (mean signal + 1x standard deviation). More specifically, the probes derived from contigs showed a wider range of average intensities when compared to probes derived from singletons. In total, 522 significantly differentially regulated transcripts were identified upon zinc exposure. Several significantly regulated genes exerted predicted functions (e.g. zinc efflux, zinc transport) associated with zinc stress. Unexpectedly, the microarray data suggest that zinc exposure alters retro transposon activity in the E. crypticus genome. An initial investigation of the E. crypticus transcriptome including an associated microarray platform for future studies proves to be a valuable resource to investigate functional genomics mechanisms of toxicity in soil environments and to annotate a potentially large number of lineage specific genes that are responsive to environmental stress conditions.

ABSTRACT We present here the draft genome of Bacillus toyonensis VU-DES13, which was isolated from the midgut of the soil-living springtail Folsomia candida. Previous research revealed the presence of gene clusters for the biosynthesis of various secondary metabolites, including β-lactam antibiotics, in the host's genome. The genome data are discussed in the light of the antimicrobial properties against fungi and oomycetes and a high level of β-lactam resistance of the isolate.

Several plant parasitic nematode genera were identified in the Robusta coffee ( Coffea canephora Pierre ex A. Froehn) tree roots and surrounding soil samples from three different coffee groups: coffee planted at 5 years (CYG), 18 years (CBG) and 40 years (COG) in Central Highland, Vietnam. They included Meloidogyne spp., Pratylenchus spp., Apratylenchus spp., Criconemella spp., Xiphinema spp. and Rotylenchulus spp. Meloidogyne spp. was the most abundant genus, at 77% across all three groups. Endophytic bacteria were isolated from healthy tissues of roots of the same Robusta coffee trees. In total, 77 bacterial strains were isolated and determined to be Bacillus spp., Serratia spp., Paenibacillus spp., Enterobacter spp. and Streptomyces spp. based on colony morphological and 16S rRNA gene sequence analysis. Overall, Streptomyces was the dominant genus and accounted for 49.35% of total isolated strains. Using statistical methods, we found a tendency in the abundance of endophytic bacterial isolates with the elevation or decrease of several nematode populations, indicating a relation between endophytic bacteria occurrence and nematode distribution. In in vitro anti nematode screening test, a Streptomyces sp. strain named CBG9 showed significant nematicidal activities against Meloidogyne incognita , inhibiting egg hatching (85.8%) and causing mortality of secondary stage juveniles (85%). This study explored the anti-nematode potency of endophytic bacteria isolated from coffee trees, which could provide a future application in suppression and management of pathogenic nematodes without the use of chemical pesticides.

The characteristic ground colour and banding patterns on shells of the land snail Cepaea nemoralis form a classic study system for genetics and adaptation as it varies widely between individuals. We use RNAseq analysis to identify candidate genes underlying this polymorphism. We sequenced cDNA from the foot and the mantle (the shell-producing tissue) of four individuals of two phenotypes and produced a de novo transcriptome of 147,397 contigs. Differential expression analysis identified a set of 1,961 transcripts that were upregulated in mantle tissue. Sequence variant analysis resulted in a set of 2,592 transcripts with single nucleotide polymorphisms (SNPs) that differed consistently between the phenotypes. Inspection of the overlap between the differential expression analysis and SNP analysis yielded a set of 197 candidate transcripts, of which 38 were annotated. Four of these transcripts are thought to be involved in production of the shell’s nacreous layer. Comparison with morph-associated Restriction-site Associated DNA (RAD)-tags from a published study yielded eight transcripts that were annotated as metallothionein, a protein that is thought to inhibit the production of melanin in melanocytes. These results thus provide an excellent starting point for the elucidation of the genetic regulation of the Cepaea nemoralis shell colour polymorphism.

Enchytraeus albidus is a terrestrial earthworm widespread along the coasts of northern Europe and the Arctic. This species tolerates freezing of body fluids and survives winters in a frozen state. Their acclimatory physiological mechanisms behind freeze tolerance involve increased fluidity of membrane lipids during cold exposure and accumulation of cryoprotectants (glucose) during the freezing process. Gene regulatory processes of these physiological responses have not been studied, partly because no gene expression tools were developed. The main aim of this study was to understand whether the freeze tolerance mechanisms have a transcriptomic basis in E. albidus. For that purpose, first the transcriptome of E. albidus was assembled with RNAseq data. Second, two strains from contrasting thermal environments (Germany and Greenland) were compared by mapping barcoded RNAseq data onto the assembled transcriptome. Both of these strains are freeze tolerant, but Greenland is extremely freeze tolerant. Results showed more plastic responses in the Greenland strain as well as higher constitutive expression of particular stress response genes. These altered transcriptional networks are associated with an adapted homeostasis coping with prolonged freezing conditions in Greenland animals. Previously identified physiological alterations in freeze‐tolerant strains of E. albidus are underpinned at the transcriptome level. These processes involve anion transport in the hemolymph, fatty acid metabolism, metabolism, and transport of cryoprotective sugars as well as protection against oxidative stress. Pathway analysis supported most of these processes, and identified additional differentially expressed pathways such as peroxisome and Toll‐like receptor signaling. We propose that the freeze‐tolerant phenotype is the consequence of genetic adaptation to cold stress and may have driven evolutionary divergence of the two strains. We studied transcriptional regulatory networks underlying cold tolerance in a natural Enchytraeus albidus population from Greenland as compared to a freeze‐sensitive reference population. Constitutive overexpression of stress response genes as well as increased transcriptional plasticity of genes involved in cold tolerance‐associated physiological processes seem to have shaped genetic adaptation to prolonged freezing conditions.

Plastic waste is a global environmental burden and long-lasting plastic polymers, including ubiquitous and toxic polyurethanes (PUs), rapidly accumulate in the water environments. In this study, samples were collected from the three alkaline groundwater occurrences in the geotectonic regions of the Pannonian basin of northern Serbia (Torda and Slankamen Banja) and Inner Dinarides of western Serbia (Mokra Gora) with aim to isolate and identify bacteria with plastic- and lignocellulose-degrading potential, that could be applied to reduce the burden of environmental plastic pollution. The investigated occurrences belong to cold, mildly alkaline (pH: 7.6–7.9) brackish and hyperalkaline (pH: 11.5) fresh groundwaters of the SO 4 – Na + K, Cl – Na + K and OH, Cl – Ca, Na + K genetic type. Full-length 16S rDNA sequencing, using Oxford Nanopore sequencing device, was performed with DNA extracted from colonies obtained by cultivation of all groundwater samples, as well as with DNA extracted directly from one groundwater sample. The most abundant genera belong to Pseudomonas , Acidovorax , Kocuria and Methylotenera . All screened isolates (100%) had the ability to grow on at least 3 of the tested plastic and lignocellulosic substrates, with 53.9% isolates degrading plastic substrate Impranil® DLN-SD (SD), a model compound for PUs degradation. Isolates degrading SD that were identified by partial 16S rDNA sequencing belong to the Stenotrophomonas , Pseudomonas , Paraburkholderia , Aeromonas , Vibrio and Acidovorax genera. Taking into account that plastics, including commonly produced PUs, are widespread in groundwater, identification of PUs-degrading bacteria may have potential applications in bioremediation of groundwater polluted with this polymer. Summary Points The most abundant bacteria identified by next-generation full-length 16S rDNA sequencing of cultivated and direct samples of alkaline groundwater occurrences in the geotectonic regions of the Pannonian basin of northern Serbia and Inner Dinarides of western Serbia belong to Pseudomonas , Acidovorax , Kocuria and Methylotenera . Of all screened groundwater isolates, 53.9% isolates demonstrated the ability to degrade plastic substrate Impranil® DLN-SD, a model compound for polyurethane degradation. Investigated groundwaters contain polyurethane-degrading bacteria, including Stenotrophomonas , Pseudomonas , Paraburkholderia , Aeromonas , Vibrio and Acidovorax . Identified isolates should be further explored as a potential resource for bioremediation treatments of polyurethane-polluted groundwater.

Global plastic waste accumulation has become omnipresent in public discourse and the focus of scientific research. Ranking as the sixth most produced polymer globally, polyurethanes (PU) significantly contribute to plastic waste and environmental pollution due to the toxicity of their building blocks, such as diisocyanates. In this study, the effects of PU on soil microbial communities over 18 months were monitored revealing that it had marginal effects on microbial diversity. However, Streptomyces sp. PU10, isolated from this PU‐contaminated soil, proved exceptional in the degradation of a soluble polyester‐PU (Impranil) across a range of temperatures with over 96% degradation of 10 g/L in 48 h. Proteins involved in PU degradation and metabolic changes occurring in this strain with Impranil as the sole carbon source were further investigated employing quantitative proteomics. The proposed degradation mechanism implicated the action of three enzymes: a polyester‐degrading esterase, a urethane bond‐degrading amidase and an oxidoreductase. Furthermore, proteome data revealed that PU degradation intermediates were incorporated into Streptomyces sp. PU10 metabolism via the fatty acid degradation pathway and subsequently channelled to polyketide biosynthesis. Most notably, the production of the tri‐pyrrole undecylprodigiosin was confirmed paving the way for establishing PU upcycling strategies to bioactive metabolites using Streptomyces strains. Streptomyces sp. PU10 degrades Impranil and funnels degradation intermediates into polyketide biosynthesis via the fatty acid degradation pathway.