Utjecaj diklofenaka na bakterijsku populaciju aktivnog mulja u reaktoru s pritokom supstrata

Research background. The occurrence and environmental toxicity of pharmaceuticals have recently attracted increasing attention. Diclofenac is a highly consumed non-steroidal anti-inflammatory drug, which is often detected in wastewaters, but investigations of its influence on bacteria are scarce. Experimental approach. We investigated the influence of this pharmaceutical on bacterial community in activated sludge exposed to increasing concentrations of diclofenac in fed-batch reactors over 41 days. Nitrification activity of the activated sludge was measured and changes in bacterial community structure were followed using culture-independent molecular method (terminal restriction fragment length polymorphism, T-RFLP) and by the cultivation approach. Results and conclusions. Nitrification activity was not detectably influenced by addition of diclofenac, while the main change of the bacterial community structure was detected only at the end of incubation (after 41 days) when diclofenac was added to artificial wastewater as the only carbon source. Changes in community composition due to enrichment were also observed using cultivation approach. However, taxonomic affiliation of isolates did not match taxons identified by T-RFLP community profiling. Isolates obtained from inoculum activated sludge belonged to five genera: Comamonas, Arthrobacter, Acinetobacter, Citrobacter and Aeromonas, known for their potential to degrade aromatic compounds. However, only Pseudomonas species were isolated after the last enrichment step on minimal agar plates with diclofenac added as the sole carbon source. Novelty and scientific contribution. Our results suggested that the selected recalcitrant and commonly detected pharmaceutical does not strongly influence the sensitive and important nitrification process of wastewater treatment neither. Moreover, the isolated strains obtained after enrichment procedure that were able to grow on minimal agar plates with diclofenac added as the only carbon source could serve as potential model bacteria to study bacterial diclofenac degradation.Pozadina istraživanja. U zadnje vrijeme prisutnost i toksičnost farmaceutskih proizvoda u okolišu pobuđuju veliki interes javnosti. Diklofenak je nesteroidni protuupalni lijek koji se uvelike koristi, pa često dospijeva u otpadne vode, no usprkos tome postoji vrlo malo istraživanja o njegovom utjecaju na bakterije aktivnog mulja. Eksperimentalni pristup. Ispitali smo utjecaj ovog lijeka na bakterijsku populaciju aktivnog mulja u reaktoru s pritokom supstrata kojem smo dodavali diklofenak tijekom 41 dana. Izmjerena je nitrifikacijska aktivnost bakterija aktivnog mulja, a promjena sastava bakterijske populacije praćena je molekularnom metodom za izravnu identifikaciju (T-RFLP) te uzgojem bakterija. Rezultati i zaključci. Dodatak diklofenaka nije vidljivo utjecao na nitrifikacijsku aktivnost bakterija, a veća je promjena sastava bakterijske populacije opažena tek na kraju inkubacije (nakon 41 dana), kad je diklofenak bio dodan kao jedini izvor ugljika u otopinu koja simulira sastav otpadnih voda. Promjene sastava bakterijske populacije nakon obogaćivanja podloge diklofenakom primijećene su nakon izolacije i praćenja rasta bakterija na minimalnoj podlozi. Međutim, taksonomska pripadnost izolata nije odgovarala taksonima identificiranim metodom T-RFLP. Izolati dobiveni iz aktivnog mulja te korišteni kao inokulum spadali su u pet rodova bakterija: Comamonas, Arthrobacter, Acinetobacter, Citrobacter i Aeromonas, koje su poznate po sposobnosti razgradnje aromatskih spojeva. Međutim, nakon posljednjeg dodatka diklofenaka kao jedinog izvora ugljika minimalnoj podlozi izolirane su jedino bakterije vrste Pseudomonas. Novina i znanstveni doprinos. Rezultati pokazuju da ispitani lijek, koji ima ograničenu biorazgradivost a često ga nalazimo u otpadnim vodama, ne utječe u velikoj mjeri na osjetljiv i bitan postupak nitrifikacije otpadnih voda. Osim toga, zaključeno je da izolirani sojevi bakterija koji su rasli na minimalnoj podlozi s diklofenakom kao jedinim izvorom ugljika mogu poslužiti kao model za proučavanje bakterijske razgradnje diklofenaka

Social Interactions and Biofilm Formation in Bacillus subtilis

Međustanična je komunikacija (engl. quorum sensing, QS) oblik kooperativnog socijalnog ponašanja bakterija što se oslanja na prepoznavanje izvanstaničnih signalnih molekula. Signalna se molekula veže na receptor i inducira promjenu transkripcije gena, odgovornih za stvaranje biofilma, proizvodnju izvanstaničnih enzima i druge kooperativne značajke populacije. Svrha je ovoga rada bila dati pregled objavljenih znanstvenih radova koji se bave kooperativnim socijalnim ponašanjem bakterije Bacillus subtilis, a naročito doprinosom komunikacijskog sustava ComQXPA. Sustav QS obuhvaća četiri komponente koje su u međusobnoj interakciji: izoprenil transferazu ComQ što procesira i modificira signal, peptid ComX koji ima ulogu signala, receptor ComP i transkripcijski regulator ComA. Fosforilirani ComA kontrolira transkripciju brojnih gena, uključujući i one odgovorne za proizvodnju surfaktina te izvanstaničnog matriksa, važnog za nastajanje biofilma. Sustav ComQXPA QS ima visok stupanj genetičkog polimorfizma, što je vidljivo iz činjenice da se sojevi Bacillus subtilis mogu podijeliti u četiri skupine. Sojevi jedne skupine (ferotipa) mogu razmjenjivati signale i informacije, dok to nije moguće između različitih ferotipova. Nedavno smo pokazali da je ovaj fenomen djelomično posljedica ekološke raznolikosti sojeva, ali su moguće i alternativne hipoteze, koje daju prednost socijalnoj evoluciji. Između ostalog, sustav ComQXPA kontrolira i proizvodnju izvanstaničnog matriksa, koji se sastoji od polisaharida, proteina i nukleinskih kiselina. U ovom je radu dan pregled trenutnih spoznaja o regulaciji, strukturi, kemijskom sastavu i funkciji izvanstaničnog matriksa. Usprkos mnogim važnim nedavnim otkrićima u području regulacije formiranja biofilma B. subtilis, molekularne interakcije među komponentama matriksa i njihov utjecaj na QS i stabilnost biofilma nisu još dobro poznati, pa se u ovom radu razmatraju i moguća rješenja ovih zanimljivih pitanja.Quorum sensing (QS) is a form of cooperative social behaviour which relies on extracellular signalling molecules that elicit the QS response across many cells and controls the development of many cooperative traits including biofilm formation. The main aim of this work is to review the published work on cooperative social behaviour of Bacillus subtilis and especially its QS system ComQXPA. This QS system involves four interacting components: the signal-processing enzyme ComQ, the ComX signal, the ComP receptor and the ComA transcriptional regulator. Phosphorylated ComA controls the transcription of many genes including those responsible for the production of surfactin and extracellular matrix, essential for biofilm formation. The ComQXPA QS shows a high degree of genetic polymorphism, which manifests itself in the separation of Bacillus subtilis strains into four different communication groups (pherotypes). The information exchange is possible between members of the same pherotype but not across pherotypes. We have recently suggested that this phenomenon is at least in part driven by the ecological divergence of strains, but may also be induced by frequency-dependent selection. The ComQXPA QS system controls the production of extracellular matrix (ECM) components: polysaccharides, proteins and nucleic acids. We will address the present understanding of the ECM structure-function relationships in B. subtilis biofilms and review published results on regulation, composition and distribution of ECM components. Despite many important recent discoveries on regulation of B. subtilis biofilm development, we know little about the molecular interactions in the ECM and the role they play in the QS and stability of the biofilm. Future research needs to address these questions better

The ComX Quorum Sensing Peptide of Bacillus subtilis Affects Biofilm Formation Negatively and Sporulation Positively

Quorum sensing (QS) is often required for the formation of bacterial biofilms and is a popular target of biofilm control strategies. Previous studies implicate the ComQXPA quorum sensing system of Bacillus subtilis as a promoter of biofilm formation. Here, we report that ComX signaling peptide deficient mutants form thicker and more robust pellicle biofilms that contain chains of cells. We confirm that ComX positively affects the transcriptional activity of the P$_{epsA}$ promoter, which controls the synthesis of the major matrix polysaccharide. In contrast, ComX negatively controls the P$_{tapA}$ promoter, which drives the production of TasA, a fibrous matrix protein. Overall, the biomass of the mutant biofilm lacking ComX accumulates more monosaccharide and protein content than the wild type. We conclude that this QS phenotype might be due to extended investment into growth rather than spore development. Consistent with this, the ComX deficient mutant shows a delayed activation of the pre-spore specific promoter, P$_{spoIIQ}$, and a delayed, more synchronous commitment to sporulation. We conclude that ComX mediated early commitment to sporulation of the wild type slows down biofilm formation and modulates the coexistence of multiple biological states during the early stages of biofilm development

Microbial Community Structure and Function in Peat Soil

Mnoga su tresetišta u Europi izlagana melioraciji i odvodnji, što je dovelo do promjena u procesima kruženja hranjivih tvari u tlu. Ovaj je rad sažetak objavljenih studija o mikrobnim procesima vezanim uz transformaciju ugljika i dušika u tlu Ljubljanskog barja. To je drenirano tresetište, smješteno nedaleko od Ljubljane, glavnoga grada Slovenije, poznato po bogatstvu biljnog i životinjskog svijeta. U radu je po prvi put dan širi pregled raznolikosti zajednice bakterija i arheja u tom organskoj tvari bogatom tlu, koje je izvor stakleničkih plinova, dušikovog oksida i ugljikovog dioksida, te ponor metana. U Ljubljanskom je barju metanogeneza ograničena velikim udjelom željeza, koje konkurira ostalim akceptorima elektrona. Osim toga, tlo je bogato vrlo aktivnim metanotrofima, naročito u slojevima tla s promjenjivom razinom podzemne vode. Denitrifikacija je ograničena akceptorima elektrona, a u dubljim slojevima tla i manjkom ugljičnih supstrata dostupnih za mikrobiološku razgradnju. Nitrifikacija je tla posljedica aktivnosti bakterija i arheja koje oksidiraju amonijak, pa je stupanj oksidacije amonijaka u Ljubljanskom barju među najvišim u svijetu. Zanimljivo, arheje iz odjeljka Thaumarchaeota u kiselim tresetištima uspijevaju samo na amonijaku što potječe iz organskog izvora i ne mogu oksidirati amonijak iz mineralnog izvora. U tlima je Ljubljanskoga barja pronađeno veliko bogatstvo gena što kodiraju bakterijske oksidoreduktaze slične lakazama. Uloga je tih enzima uglavnom nepoznata, a prema spoznajama o lakazama iz gljiva može se zaključiti da bakterijski enzimi sudjeluju u degradaciji lignina, oksidaciji različitih aromatskih i fenolnih spojeva, te oksidaciji metala. Budući su izazovi u ovom području istražiti specifične fiziološke uloge fenolnih oksidaza i ostalih enzima što sudjeluju u transformaciji tresetišta. Naše je poznavanje različitosti mikroorganizama u tresetištima, njihove funkcije i utjecaja na ekosustav još uvijek ograničeno, iako neophodno za učinkovito održavanje tresetišta, tih izvanrednih, ekološki značajnih, no osjetljivih staništa.Many peatlands in Europe have been subjected to land reclamation and systematic drainage, which have substantially affected nutrient cycles in the soil. This work reviews published studies on microbial processes linked to carbon and nitrogen transformations in the soils of the Ljubljana marsh, a drained peatland positioned close to Ljubljana, the capital of Slovenia. This region is known for its dramatic diversity of animal and plant life, but below ground it hides diverse bacterial and archaeal communities that are highly responsive to environmental changes and make the Ljubljana marsh soils a good source of N2O and CO2, and a sink for CH4. Methanogenesis is highly restricted in these soils due to competition for electron donors with iron reducers. In addition, methane is efficiently removed by methanotrophs, which are highly active, especially in the soil layers exposed to the changing water table. Denitrification is limited by electron acceptors and in deeper soil layers also by carbon, which becomes more recalcitrant with depth. Nitrification involves bacterial and archaeal ammonia oxidisers with ammonia oxidation rates being among the highest in the world. Interestingly, ammonia-oxidising Thaumarchaeota in acidic bog soils thrive only on ammonia released through mineralisation of organic matter and are incapable of oxidising added mineral ammonia. The soils of the Ljubljana marsh are rich in bacterial laccase-like genes, which may encode enzymes involved in lignin degradation and are therefore interesting for bioexploitations. Future challenges involve designing studies that will reveal specific physiological functions of phenol oxidases and other enzymes involved in peat transformations and address relations between microbial diversity, function and ecosystem responses to anthropogenic disturbances

Identification and Characterization of a Novel Plasmid-Encoded Laccase-Like Multicopper Oxidase from Ochrobactrum sp. BF15 Isolated from an On-Farm Bio-Purification System

Research background. In recent decades, laccases (p-diphenol-dioxygen oxidoreductases; EC 1.10.3.2) have attracted the attention of researchers due to their wide range of biotechnological and industrial applications. Laccases can oxidize a variety of organic and inorganic compounds, making them suitable as biocatalysts in biotechnological processes. Even though the most traditionally used laccases in the industry are of fungal origin, bacterial laccases have shown an enormous potential given their ability to act on several substrates and in multiple conditions. The present study aims to characterize a plasmid-encoded laccase-like multicopper oxidase (LMCO) from Ochrobactrum sp. BF15, a bacterial strain previously isolated from polluted soil. Experimental approach. We used in silico profile hidden Markov models to identify novel laccase-like genes in Ochrobactrum sp. BF15. For laccase characterization, we performed heterologous expression in Escherichia coli, purification and activity measurement on typical laccase substrates. Results and conclusions. Profile hidden Markov models allowed us to identify a novel LMCO, named Lac80. In silico analysis of Lac80 revealed the presence of three conserved copper oxidase domains characteristic of three-domain laccases. We successfully expressed Lac80 heterologously in E. coli, allowing us to purify the protein for further activity evaluation. Of thirteen typical laccase substrates tested, Lac80 showed lower activity on 2,2\u2019-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), pyrocatechol, pyrogallol and vanillic acid, and higher activity on 2,6-dimethoxyphenol. Novelty and scientific contribution. Our results show Lac80 as a promising laccase for use in industrial applications. The present work shows the relevance of bacterial laccases and highlights the importance of environmental plasmids as valuable sources of new genes encoding enzymes with potential use in biotechnological processes

Bioinformatic Analysis Reveals High Diversity of Bacterial Genes for Laccase-Like Enzymes

Fungal laccases have been used in various fields ranging from processes in wood and paper industries to environmental applications. Although a few bacterial laccases have been characterized in recent years, prokaryotes have largely been neglected as a source of novel enzymes, in part due to the lack of knowledge about the diversity and distribution of laccases within Bacteria. In this work genes for laccase-like enzymes were searched for in over 2,200 complete and draft bacterial genomes and four metagenomic datasets, using the custom profile Hidden Markov Models for two- and three- domain laccases. More than 1,200 putative genes for laccase-like enzymes were retrieved from chromosomes and plasmids of diverse bacteria. In 76% of the genes, signal peptides were predicted, indicating that these bacterial laccases may be exported from the cytoplasm, which contrasts with the current belief. Moreover, several examples of putatively horizontally transferred bacterial laccase genes were described. Many metagenomic sequences encoding fragments of laccase-like enzymes could not be phylogenetically assigned, indicating considerable novelty. Laccase-like genes were also found in anaerobic bacteria, autotrophs and alkaliphiles, thus opening new hypotheses regarding their ecological functions. Bacteria identified as carrying laccase genes represent potential sources for future biotechnological applications

Multiscale spatial segregation analysis in digital images of biofilms

Quantifying the degree of spatial segregation of two bacterial strains in mixed biofilms is an important topic in microbiology. Spatial segregation is dependent on spatial scale as two strains may appear to be well mixed if observed from a distance, but a closer look can reveal strong separation. Typically, this information is encoded in a digital image that represents the binary system, e.g., a microscopy image of a two species biofilm. To decode spatial segregation information, we have developed quantitative measures for evaluating the degree of the spatial scale-dependent segregation of two bacterial strains in a digital image. The constructed algorithm is based on the new segregation measures and overcomes drawbacks of existing approaches for biofilm segregation analysis. The new approach is implemented in a freely available software and was successfully applied to biofilms of two strains and bacterial suspensions for detection of the different spatial scale-dependent segregation levels

A MecA Paralog, YpbH, Binds ClpC, Affecting both Competence and Sporulation

ComK, the master regulator of competence, is degraded by the general stress-related protease ClpCP but must be targeted to this protease by binding to the adapter protein MecA. The genome of Bacillus subtilis contains a paralog of mecA, ypbH. We show in the present study that YpbH, like MecA, binds ClpC and that its elimination or overproduction affects competence and sporulation