In this work, we analyzed the community structure and metabolic potential of sediment microbial communities in high-latitude coastal environments subjected to low to moderate levels of chronic pollution. Subtidal sediments from four low-energy inlets located in polar and subpolar regions from both Hemispheres were analyzed using large-scale 16S rRNA gene and metagenomic sequencing. Communities showed high diversity (Shannon's index 6.8 to 10.2), with distinct phylogenetic structures (<40% shared taxa at the Phylum level among regions) but similar metabolic potential in terms of sequences assigned to KOs. Environmental factors (mainly salinity, temperature, and in less extent organic pollution) were drivers of both phylogenetic and functional traits. Bacterial taxa correlating with hydrocarbon pollution included families of anaerobic or facultative anaerobic lifestyle, such as Desulfuromonadaceae, Geobacteraceae, and Rhodocyclaceae. In accordance, biomarker genes for anaerobic hydrocarbon degradation (bamA, ebdA, bcrA, and bssA) were prevalent, only outnumbered by alkB, and their sequences were taxonomically binned to the same bacterial groups. BssA-assigned metagenomic sequences showed an extremely wide diversity distributed all along the phylogeny known for this gene, including bssA sensu stricto, nmsA, assA, and other clusters from poorly or not yet described variants. This work increases our understanding of microbial community patterns in cold coastal sediments, and highlights the relevance of anaerobic hydrocarbon degradation processes in subtidal environments.

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.

Alginates are abundant polysaccharides in brown algae that constitute an important energy source for marine heterotrophic bacteria. Despite the key role of alginate degradation processes in the marine carbon cycle, little information is available on the bacterial populations involved in these processes. The aim of this work was to gain a better understanding of alginate utilization capabilities in cold coastal environments. Sediment metagenomes from four high-latitude regions of both Hemispheres were interrogated for alginate lyase gene homologue sequences and their genomic context. Sediments contained highly abundant and diverse bacterial assemblages with alginolytic potential, including members of Bacteroidetes and Proteobacteria, as well as several poorly characterized taxa. The microbial communities in Arctic and Antarctic sediments exhibited the most similar alginolytic profiles, whereas brackish sediments showed distinct structures with a higher proportion of novel genes. Examination of the gene neighbourhood of the alginate lyase homologues revealed distinct patterns depending on the potential lineage of the scaffolds, with evidence of evolutionary relationships among alginolytic gene clusters from Bacteroidetes and Proteobacteria. This information is relevant for understanding carbon fluxes in cold coastal environments and provides valuable information for the development of biotechnological applications from brown algae biomass.

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study, we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF103 of the isolate Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

Community structures of active bacterial populations were investigated along a vertical redox profile in coastal Baltic Sea sediments by terminal-restriction fragment length polymorphism (T-RFLP) and clone library analysis. According to correspondence analysis of T-RFLP results and sequencing of cloned 16S rRNA genes, the microbial community structures at three redox depths (179, -64 and -337 mV) differed significantly. The bacterial communities in the community DNA differed from those in bromodeoxyuridine (BrdU)-labelled DNA, indicating that the growing members of the community that incorporated BrdU were not necessarily the most dominant members. The structures of the actively growing bacterial communities were most strongly correlated to organic carbon followed by total nitrogen and redox potentials. Bacterial identification by sequencing of 16S rRNA genes from clones of BrdU-labelled DNA and DNA from reverse transcription polymerase chain reaction showed that bacterial taxa involved in nitrogen and sulfur cycling were metabolically active along the redox profiles. Several sequences had low similarities to previously detected sequences, indicating that novel lineages of bacteria are present in Baltic Sea sediments. Also, a high number of different 16S rRNA gene sequences representing different phyla were detected at all sampling depths.

Soil teems with microbial genetic information that can be exploited for biotechnological innovation. Because only a fraction of the soil microbiota is cultivable, our ability to unlock this genetic complement has been hampered. Recently developed molecular tools, which make it possible to utilize genomic DNA from soil, can bypass cultivation and provide information on the collective soil metagenome with the aim to explore genes that encode functions of key interest to biotechnology. The metagenome of disease-suppressive soils is of particular interest given the expected prevalence of antibiotic biosynthetic clusters. However, owing to the complexity of soil microbial communities, deciphering this key genetic information is challenging. Here, we examine crucial issues and challenges that so far have hindered the metagenomic exploration of soil by drawing on experience from a trans-European project on disease-suppressive soils denoted METACONTROL.

Antibiotic administration is known to cause short-term disturbances in the microbiota of the human gastrointestinal tract, but the potential long-term consequences have not been well studied. The aims of this study were to analyse the long-term impact of a 7-day clindamycin treatment on the faecal microbiota and to simultaneously monitor the ecological stability of the microbiota in a control group as a baseline for reference. Faecal samples from four clindamycin-exposed and four control subjects were collected at nine different time points over 2 years. Using a polyphasic approach, we observed highly significant disturbances in the bacterial community that persisted throughout the sampling period. In particular, a sharp decline in the clonal diversity of Bacteroides isolates, as assessed by repetitive sequence-based PCR (rep-PCR) and long-term persistence of highly resistant clones were found as a direct response to the antibiotic exposure. The Bacteroides community never returned to its original composition during the study period as assessed using the molecular fingerprinting technique, terminal restriction fragment length polymorphism (T-RFLP). Furthermore, using real-time PCR we found a dramatic and persistent increase in levels of specific resistance genes in DNA extracted from the faeces after clindamycin administration. The temporal variations in the microbiota of the control group were minor compared to the large and persistent shift seen in the exposed group. These results demonstrate that long after the selection pressure from a short antibiotic exposure has been removed, there are still persistent long term impacts on the human intestinal microbiota that remain for up to 2 years post-treatment.

Objectives: The aim was to study the long-term consequences of 1 week clindamycin administration regarding selection and persistence of resistance, resistance determinants and diversity of the Bacteroides spp. in the intestinal microflora. Methods: A total of 1306 Bacteroides isolates were collected from constitutively cultured faecal samples during a 2 year period from eight healthy volunteers. The strains were identified by biochemical and genotyping methods. MIC values were determined by the agar dilution method and presence of resistance genes was screened by real-time PCR. Results: Ecological changes in the intestinal microflora persisting up to 24 months were recorded after a 7 day clindamycin administration to four healthy volunteers. Compared to a control group, not exposed to clindamycin, an enrichment and stabilization of resistant Bacteroides strains and resistance determinants were discovered up to 2 years after clindamycin exposure. Conclusions: The results indicate that even a short-term antibiotic administration can cause long-term alterations in the commensal microbiota of individual subjects, detectable 2 years after dosing. The recorded selection and persistence of resistant strains and resistance genes, illustrates the importance of increasing our knowledge of the role of the abundant intestinal microbial community as a reservoir for spread of resistance.

This study evaluates the potential of Paenibacillus brasilensis strain PB177 to inhibit phytopathogenic fungi commonly causing maize diseases and to colonize maize plants. In vitro assays demonstrated antagonistic activity against the fungal pathogens, Fusarium moniliforme and Diplodia macrospora. The PB177 strain was tagged with the gfp gene, encoding the green fluorescent protein (GFP) and GFP-tagged bacteria were detected attached to maize roots by stereo- and confocal microscopy. The GFP-tagged bacteria were also used to treat maize seeds before challenging the seeds with two phytopathogenic fungi. The results demonstrated that the bacterial cells are mobilized to the maize roots in the presence of the fungal pathogens. The ability of P. brasilensis PB177 to inhibit fungal growth in vitro and its capability of colonization of maize roots in vivo suggest a potential application of this strain as a biological control agent. This is the first report on the successful introduction of the GFP marker gene into a P. brasilensis strain, enabling the direct observation of these promising plant growth promoting bacteria on maize roots in situ.

Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as Eubacterium spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic Lactobacillus strains were easily tracked by T-RFLP and were shown to be the dominant Lactobacillus community members in the intestinal microflora of subjects who received the probiotic.