RESUMEN
Gordonia polyisoprenivorans VH2 harbours two latex clearing proteins, which are responsible for the cleavage of poly(cis-1,4-isoprene) into oligoisoprenes, thereby allowing growth in presence of, e.g. natural rubber. A gene coding for a putative regulator of the TetR-family (lcpRVH2) is located 131 bp upstream of lcp1VH2. We heterologously expressed lcpRVH2 in Escherichia coli, and purified and characterized the protein with respect to its ability to bind to the operator region of lcp1VH2. LcpRVH2 forms a dimer in its native state. The size of the dimer was determined to be 52.7 kDa by size exclusion chromatography, whereas the calculated size of a monomer was 24.1 kDa. Electrophoretic mobility shift assays (EMSAs) with the purified protein revealed a shift upon binding to the intergenic region between lcpRVH2 and lcp1VH2. Within this region, an inverted repeat was identified in silico, probably being the binding site of LcpRVH2. This binding sequence was confirmed by a DNase I footprinting assay. A shift also occurred in EMSAs with this 44 bp sequence only. Interestingly, no regulator was detected upstream of the second lcp (lcp2VH2). Therefore, we performed EMSA studies with LcpRVH2 and the putative operator region upstream of lcp2VH2, and discovered by DNase I footprinting another binding sequence upstream of lcp2VH2. Hence, we concluded that LcpRVH2 binds the operator region of both lcps and, most likely, regulates their expression in G. polyisoprenivorans VH2.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacteria Gordonia/genética , Látex/metabolismo , Transactivadores/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión , Biodegradación Ambiental , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Bacteria Gordonia/metabolismo , Hemiterpenos/metabolismo , Peso Molecular , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transactivadores/química , Transactivadores/genética , Transactivadores/aislamiento & purificaciónRESUMEN
The environmental fate of plastic particles in water bodies is influenced by microbial biofilm formation. Invertebrate grazers may be affected when foraging biofilms on plastics compared to biofilms on natural substrata but the mechanistic basis for these effects is unknown. For analyzing these effects in ecotoxicological assays stable and reproducible biofilm communities are required that are related to the environmental site of interest. Here, a defined biofilm community was established and used to perform grazing experiments with a freshwater snail. For this, snippets of different plastic materials were incubated in the photic zone of three different freshwater sites. Amplicon sequencing of biofilms formed on these snippets showed that the site of incubation and not the plastic material dominated the microbial community composition. From these biofilms, individual microbial strains as well as photoautotrophic consortia were isolated; these consortia consisted of heterotrophic bacteria that were apparently nourished by microalga. While biofilms formed by defined dual cultures of a microalga and an Alphaproteobacterium were not accepted by the snail P. fontinalis, a photoautotrophic consortium (Co_3) sustained growth and metabolism of this grazer. Amplicon sequencing revealed that consortium Co_3, which could be stably maintained on solid medium under photoautotrophic conditions, reproducibly formed biofilms of a defined composition on three different plastic materials and on glass surfaces. In conclusion, our study shows that the generation of domesticated photoautotrophic microbial communities is a valid novel approach for establishing laboratory ecotoxicological assays with higher environmental relevance than those based on defined microbiota.
RESUMEN
Low-density microplastics are frequently found in sediments of many lakes and reservoirs. The processes leading to sedimentation of initially buoyant polymers are poorly understood for inland waters. This study investigated the impact of biofilm formation and aggregation on the density of buoyant polyethylene microplastics. Biofilm formation on polyethylene films (4 × 4 × 0.15 mm) was studied in a eutrophic reservoir (Bautzen, Saxony, Germany). Additionally, aggregation dynamics of small PE microplastics (~85 µm) with cyanobacteria were investigated in laboratory experiments. During summer phototrophic sessile cyanobacteria (Chamaesiphon spp. and Leptolyngbya spp.) precipitated calcite while forming biofilms on microplastics incubated in Bautzen reservoir. Subsequently the density of the biofilms led to sinking of roughly 10% of the polyethylene particles within 29 days of incubation. In the laboratory experiments planktonic cyanobacteria (Microcystis spp.) formed large and dense cell aggregates under the influence of elevated Ca2+ concentrations. These aggregates enclosed microplastic particles and led to sinking of a small portion (~0.4 %) of polyethylene microplastics. This study showed that both sessile and planktonic phototrophic microorganisms mediate processes influenced by calcium which facilitates densification and sinking of microplastics in freshwater reservoirs. Loss of buoyancy leads to particle sedimentation and could be a prerequisite for the permanent burial of microplastics within reservoir sediments.