Ana Bielen

The SGNH-hydrolase of Streptomyces coelicolor has (aryl)esterase and a true lipase activity

The Streptomyces coelicolor A3(2) gene SCI11.14c was overexpressed and purified as a His-tagged protein from heterologous host, Streptomyces lividans. The purification procedure resulted in 34.1-fold increase in specific activity with an overall yield of 21.4%. Biochemical and physical properties of the purified enzyme were investigated and it was shown that it possesses (aryl)esterase and a true lipase activity. The enzyme was able to hydrolyze p-nitrophenyl-, alpha- and beta-naphthyl esters and poly(oxyethylene) sorbitan monoesters (Tween 20-80). It showed pronounced activity towards p-nitrophenyl and alpha- and beta-naphthyl esters of C(12)-C(16). Higher activity was observed with alpha-naphthyl esters. The enzyme hydrolyzed triolein (specific activity: 91.9 U/mg) and a wide range of oils with a preference for those having higher content of linoleic or oleic acid (C18:2; C18:1, cis). The active-site serine specific inhibitor 3,4-dichloroisocoumarin (DCI) strongly inhibited the enzyme, while tetrahydrofurane and 1,4-dioxane significantly increased (2- and 4- fold, respectively) hydrolytic activity of lipase towards p-nitrophenyl caprylate. The enzyme exhibited relatively high temperature optimum (55 degrees C) and thermal stability. CD analysis revealed predominance of alpha-helical structure (54% alpha-helix, 21% beta-sheet) and a T(m) value at 66 degrees C. Systematic bioinformatic analysis of deduced amino acid sequence of S. coelicolor enzyme placed it to the SGNH-hydrolase family. Phylogenetic analysis of the predicted protein homologous to the S. coelicolor SGNH-hydrolase generated three distinct groups consisting of proteins from Actinomycetales, Ascomycota and Nematoda. At present it seems that these enzymes are most conserved among soil inhabiting organisms.

Differences in tolerance to anthropogenic stress between invasive and native bivalves.

Tolerance towards environmental stress has been frequently considered as one of the key determinants of invasion success. However, empirical evidence supporting the assumption that invasive species can better endure unfavorable conditions compared with native species is limited and has yielded opposing results. In this study, we examined the tolerance to different stress conditions (thermal stress and trace metal zinc pollution stress) in two phylogenetically related and functionally similar freshwater bivalve species, the native Anodonta anatina and the invasive Sinanodonta woodiana. We assessed potential differences in response to stress conditions using several cellular response assays: efficiency of the multixenobiotic resistance mechanism, respiration estimate (INT reduction capacity), and enzymatic biomarkers. Our results demonstrated that the invasive species overall coped much better with unfavorable conditions. The higher tolerance of S. woodiana was evident from (i) significantly decreased Rhodamine B accumulation indicating more efficient multixenobiotic resistance mechanism; (ii) significantly higher INT reduction capacity and (iii) less pronounced alterations in the activity of stress-related enzymes (glutathione-S-transferase, catalase) and of a neurotoxicity biomarker (cholinesterase) in the majority of treatment conditions in both stress trials. Higher tolerance to thermal extremes may provide physiological benefit for further invasion success of S. woodiana in European freshwaters, especially in the context of climate change.

Exploring Actinobacteria assemblages in coastal marine sediments under contrasted Human influences in the West Istria Sea, Croatia

The exploration of marine Actinobacteria has as major challenge to answer basic questions of microbial ecology that, in turn, will provide useful information to exploit Actinobacteria metabolisms in biotechnological processes. The ecological functions performed by Actinobacteria in marine sediments are still unclear and belongs to the most burning basic questions. The comparison of Actinobacteria communities inhabiting marine sediments that are under the influence of different contamination types will provide valuable information in the adaptation capacities of Actinobacteria to colonize specific ecological niche. In the present study, the characterization of different Actinobacteria assemblages according to contamination type revealed the ecological importance of Actinobacteria for maintaining both general biogeochemical functions through a “core” Actinobacteria community and specific roles associated with the presence of contaminants. Indeed, the results allowed to distinguish Actinobacteria genera and species operational taxonomic units (OTUs) able to cope with the presence of either (i) As, (ii) metals Ni, Fe, V, Cr, and Mn, or (iii) polycyclic aromatic hydrocarbons (PAHs) and toxic metals (Hg, Cd, Cu, Pb, and Zn). Such observations highlighted the metabolic capacities of Actinobacteria and their potential that should be taken into consideration and advantage during the implementation of bioremediation processes in marine ecosystems.

First evidence of the P-glycoprotein gene expression and multixenobiotic resistance modulation in earthworm

Summary Multixenobiotic resistance (MXR) is an important mechanism of cellular efflux mediated by ATP binding cassette (ABC) transporters that bind and actively remove toxic substrates from the cell. This study was the first to identify ABC transporter P-glycoprotein (P-gp/ABCB1) as a representative of the MXR phenotype in earthworm (Eisenia fetida). The identified partial cDNA sequence of ABCB1 overlapped with ABCB1 homologues of other organisms from 58.5 % to 72.5 %. We also studied the effect of five modulators (verapamil, cyclosporine A, MK571, probenecid, and orthovanadate) on the earthworm’s MXR activity by measuring the accumulation of model substrates rhodamine B and rhodamine 123 in whole body tissue of the adult earthworm. MK571, orthovanadate, and verapamil significantly inhibited MXR activity, and rhodamine 123 turned out to better reflect MXR activity in that species than rhodamine B. Our results show that E. fetida can serve well as a test organism for environmental pollutants that inhibit MXR activity. Sažetak Mehanizam multiksenobiotičke otpornosti (MXR) prisutan je u mnogim organizmima kao važan stanični detoksikacijski mehanizam. Posredovan je aktivnošću ABC prijenosnika koji vežu i aktivno izbacuju različite toksične tvari iz stanice. U ovom radu dani su podaci o molekularnoj identifikaciji ABC prijenosnika (eksportera) - P-glikoproteina (P-gp/Abcb1), kao jednog od predstavnika MXR fenotipa, u gujavici Eisenia fetida. Određen je djelomični slijed identificiranoga gena Abcb1, njegov predviđeni aminokiselinski slijed uspoređen je s ABCB1 homolozima iz drugih organizama i utvrđena je identičnost od 58,5 do72,5 %. Uz to, istraživali smo učinak pet modulatora (verapamil, ciklosporin, MK571, probenecid, ortovanadat) na aktivnost MXR mehanizma tih gujavica. Kako bismo potvrdili modulirajuće djelovanje istraživanih modelnih inhibitora na MXR mehanizam u E. fetida, mjerili smo akumulaciju modelnih supstrata rodamina B (RB) i rodamina 123 (R123) u tijelu spolno zrelih jedinki gujavica testom izlaganja na filtar papiru. Rezultati su pokazali da svi istraživani modulatori značajno inhibiraju MXR transportnu aktivnost. Naši podaci prvi upućuju na prisutnost P-gp/Abcb1 srodnih gena u gujavici E. fetida. Osim toga, ukazali smo na veliku važnost MXR-a kao specifičnog detoksikacijskog mehanizma koji omogućuje preživljavanje gujavica u onečišćenom okolišu.

Multilevel ecotoxicity assessment of environmentally relevant bisphenol A concentrations using the soil invertebrate Eisenia fetida.

Bisphenol A (BPA) presents a serious threat to soil ecosystems, yet its effects on soil-inhabiting organisms are mostly unexplored. Therefore, the impact of environmentally relevant BPA concentrations on a terrestrial model organism, the earthworm Eisenia fetida, was assessed. Animals were cutaneously exposed to 100nM and 10μM BPA up to 10days (10-d). Next, a battery of biomarkers was used for ecotoxicological evaluation on a cellular, tissue and behavioural level. HPLC analysis showed that after a 10-d exposure, BPA accumulation reached a maximum of 2.50μg BPA per g of wet tissue weight. On the cellular level, up to 3-d BPA exposure caused increased lipid oxidation indicating oxidative stress. Histopathological assessment of cell wall and ovaries after 7- and 10-d BPA exposure showed multiple abnormalities, i.e. hyperplasia of epidermis, increased body wall thickness and ovarian atrophy. Detection of these changes was facilitated by a newly proposed semi-quantitative scoring system. Finally, behavioural changes were detected after only 3days of exposure to 100nM BPA. Altogether, the presented multilevel toxicity evaluation indicates high sensitivity of earthworms to low BPA doses.

Integron diversity in marine environments

Integrons are bacterial genetic elements known to be active vectors of antibiotic resistance among clinical bacteria. They are also found in bacterial communities from natural environments. Although integrons have become especially efficient for bacterial adaptation in the particular context of antibiotic usage, their role in natural environments in other contexts is still unknown. Indeed, most studies have focused on integrons and the spread of antibiotic resistance in freshwater or soil impacted by anthropogenic activities, with only few on marine environments. Notably, integrons show a wider diversity of both gene cassettes and integrase gene in natural environments than in clinical environments, suggesting a general role of integrons in bacterial adaptation. This article reviews the current knowledge on integrons in marine environments. We also present conclusions of our studies on polluted and nonpolluted backgrounds.

Comparative analysis of three different methods for monitoring the use of green bridges by wildlife.

Green bridges are used to decrease highly negative impact of roads/highways on wildlife populations and their effectiveness is evaluated by various monitoring methods. Based on the 3-year monitoring of four Croatian green bridges, we compared the effectiveness of three indirect monitoring methods: track-pads, camera traps and active infrared (IR) trail monitoring system. The ability of the methods to detect different species and to give good estimation of number of animal crossings was analyzed. The accuracy of species detection by track-pad method was influenced by granulometric composition of track-pad material, with the best results obtained with higher percentage of silt and clay. We compared the species composition determined by track-pad and camera trap methods and found that monitoring by tracks underestimated the ratio of small canids, while camera traps underestimated the ratio of roe deer. Regarding total number of recorder events, active IR detectors recorded from 11 to 19 times more events then camera traps and app. 80% of them were not caused by animal crossings. Camera trap method underestimated the real number of total events. Therefore, an algorithm for filtration of the IR dataset was developed for approximation of the real number of crossings. Presented results are valuable for future monitoring of wildlife crossings in Croatia and elsewhere, since advantages and disadvantages of used monitoring methods are shown. In conclusion, different methods should be chosen/combined depending on the aims of the particular monitoring study.

Camera Traps on Wildlife Crossing Structures as a Tool in Gray Wolf (Canis lupus) Management - Five-Years Monitoring of Wolf Abundance Trends in Croatia

The conservation of gray wolf (Canis lupus) and its coexistence with humans presents a challenge and requires continuous monitoring and management efforts. One of the non-invasive methods that produces high-quality wolf monitoring datasets is camera trapping. We present a novel monitoring approach where camera traps are positioned on wildlife crossing structures that channel the animals, thereby increasing trapping success and increasing the cost-efficiency of the method. In this way we have followed abundance trends of five wolf packs whose home ranges are intersected by a motorway which spans throughout the wolf distribution range in Croatia. During the five-year monitoring of six green bridges we have recorded 28 250 camera-events, 132 with wolves. Four viaducts were monitored for two years, recording 4914 camera-events, 185 with wolves. We have detected a negative abundance trend of the monitored Croatian wolf packs since 2011, especially severe in the northern part of the study area. Further, we have pinpointed the legal cull as probable major negative influence on the wolf pack abundance trends (linear regression, r2 > 0.75, P < 0.05). Using the same approach we did not find evidence for a negative impact of wolves on the prey populations, both wild ungulates and livestock. We encourage strict protection of wolf in Croatia until there is more data proving population stability. In conclusion, quantitative methods, such as the one presented here, should be used as much as possible when assessing wolf abundance trends.

An effective approach for annotation of protein families with low sequence similarity and conserved motifs: identifying GDSL hydrolases across the plant kingdom

BackgroundThe massive accumulation of protein sequences arising from the rapid development of high-throughput sequencing, coupled with automatic annotation, results in high levels of incorrect annotations. In this study, we describe an approach to decrease annotation errors of protein families characterized by low overall sequence similarity. The GDSL lipolytic family comprises proteins with multifunctional properties and high potential for pharmaceutical and industrial applications. The number of proteins assigned to this family has increased rapidly over the last few years. In particular, the natural abundance of GDSL enzymes reported recently in plants indicates that they could be a good source of novel GDSL enzymes. We noticed that a significant proportion of annotated sequences lack specific GDSL motif(s) or catalytic residue(s). Here, we applied motif-based sequence analyses to identify enzymes possessing conserved GDSL motifs in selected proteomes across the plant kingdom.ResultsMotif-based HMM scanning (Viterbi decoding-VD and posterior decoding-PD) and the here described PD/VD protocol were successfully applied on 12 selected plant proteomes to identify sequences with GDSL motifs. A significant number of identified GDSL sequences were novel. Moreover, our scanning approach successfully detected protein sequences lacking at least one of the essential motifs (171/820) annotated by Pfam profile search (PfamA) as GDSL. Based on these analyses we provide a curated list of GDSL enzymes from the selected plants. CLANS clustering and phylogenetic analysis helped us to gain a better insight into the evolutionary relationship of all identified GDSL sequences. Three novel GDSL subfamilies as well as unreported variations in GDSL motifs were discovered in this study. In addition, analyses of selected proteomes showed a remarkable expansion of GDSL enzymes in the lycophyte, Selaginella moellendorffii. Finally, we provide a general motif-HMM scanner which is easily accessible through the graphical user interface (http://compbio.math.hr/).ConclusionsOur results show that scanning with a carefully parameterized motif-HMM is an effective approach for annotation of protein families with low sequence similarity and conserved motifs. The results of this study expand current knowledge and provide new insights into the evolution of the large GDSL-lipase family in land plants.

Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments

Environments polluted by direct discharges of effluents from antibiotic manufacturing are important reservoirs for antibiotic resistance genes (ARGs), which could potentially be transferred to human pathogens. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. We applied functional metagenomics to explore the resistome of two Croatian antibiotic manufacturing effluents and sediments collected upstream of and at the effluent discharge sites. Metagenomic libraries built from an azithromycin-production site were screened for resistance to macrolide antibiotics, whereas the libraries from a site producing veterinary antibiotics were screened for resistance to sulfonamides, tetracyclines, trimethoprim, and beta-lactams. Functional analysis of eight libraries identified a total of 82 unique, often clinically relevant ARGs, which were frequently found in clusters and flanked by mobile genetic elements. The majority of macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolide phosphotransferases, and transporters. Potentially novel macrolide resistance genes included one most similar to a 23S rRNA methyltransferase from Clostridium and another, derived from upstream unpolluted sediment, to a GTPase HflX from Emergencia. In libraries deriving from sediments exposed to lower levels of veterinary antibiotics, we found 8 potentially novel ARGs, including dihydrofolate reductases and beta-lactamases from classes A, B, and D. In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta-lactamase. Taken together, in addition to finding known gene types, we report the discovery of novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments, providing a qualitative basis for monitoring the dispersal of ARGs from environmental hotspots such as discharge sites of pharmaceutical effluents.