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Microbial activity is a major contributor to the biogeochemical cycles that make up the life support system of planet Earth. A 613 m deep geomicrobiological perforation and a systematic multi-analytical characterization revealed an unexpected diversity associated with the rock matrix microbiome that operates in the subsurface of the Iberian Pyrite Belt (IPB). Members of 1 class and 16 genera were deemed the most representative microorganisms of the IPB deep subsurface and selected for a deeper analysis. The use of fluorescence in situ hybridization allowed not only the identification of microorganisms but also the detection of novel activities in the subsurface such as anaerobic ammonium oxidation (ANAMMOX) and anaerobic methane oxidation, the co-occurrence of microorganisms able to maintain complementary metabolic activities and the existence of biofilms. The use of enrichment cultures sensed the presence of five different complementary metabolic activities along the length of the borehole and isolated 29 bacterial species. Genomic analysis of nine isolates identified the genes involved in the complete operation of the light-independent coupled C, H, N, S and Fe biogeochemical cycles. This study revealed the importance of nitrate reduction microorganisms in the oxidation of iron in the anoxic conditions existing in the subsurface of the IPB., (© 2022 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

COVID-19 is an acute, highly transmissible respiratory infection that is potentially lethal, but often mild, sometimes asymptomatic, especially in the young. However, it has become clear that, in some patients, there may be sequelae involving tissues other than the lung, resulting in other types of morbidity, and sometimes longer term consequences that are often termed 'long covid'. In this Lilliput, we summarize recent findings about COVID-19 sequelae, with a particular focus on long covid. We also discuss some of the long scars that COVID-19 and long covid will collectively leave on society that we term Societal Long Covid., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The origin of the SARS-CoV-2 virus remains enigmatic. It is likely to be a continuum resulting from inevitable mutations and recombination events. These genetic changes keep developing in the present epidemic. Mutations tending to deplete the genome in its cytosine content will progressively lead to attenuation as a consequence of Muller's ratchet, but this is counteracted by recombination when different mutants co-infect the same host, in particular, in clusters of infection. Monitoring as a function of time the genome sequences in closely related cases is critical to anticipate the future of SARS-CoV-2 and hence of COVID-19., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Countries of Southern Europe are currently suffering from severe socio-economic pain resulting from high debt levels and austerity measures which constrain investment in innovation-based recovery strategies that are essential for entry into a long-term sustainable period of increasing employment and wealth creation. Young university-educated people are particularly innovative, and hence vital to the development of such strategies, but employment opportunities are poor and many are forced to seek employment that neither profits from their training nor satisfies their justified career expectations, or to emigrate. They are the 'lost generation'. A strategy is proposed here for the creation of Pipelines for New Chemicals, national centre-network partnerships for the discovery-synthesis of new chemicals obtained though harvesting new biological diversity, and their exploitation to develop new medicines, agrochemicals, materials, and other products and applications. The goal is to create new regional motors of economic growth and development, by harnessing the knowledge, motivation and innovation potential of the excellently educated young people of Europe to catalyse the development of new small, medium and large enterprises centred around novel chemicals, and the value chains that will evolve with them, and thereby develop a powerful sector of sustainable growth in employment and social and economic prosperity in Southern Europe., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Here, we propose an advanced method for recently developed fingerprinting strategies to analyse microbial populations by direct detection of 16S rRNA sequences occurring in natural habitats. The differential display (DD) technique, which is widely used to analyse for eukaryotic gene expression, was optimized to assess bacterial rRNA diversity in environmental samples. Double-stranded cDNAs of rRNAs were synthesized without a forward primer digested with endonuclease and ligated with a double-stranded adapter. The fragments obtained were then amplified using an adapter-specific extended primer and a 16S rDNA universal reverse primer pair displayed by electrophoresis on a polyacrylamide gel. We validated this approach by characterization of a microbial community colonizing a geothermal (48 degrees C) vent system located close to the eruption zone of the south-east crater of the Mount Etna volcano, Sicily. Analysis of the patterns of abundant 16S rRNA revealed a considerable diversity of metabolically active bacteria phylogenetically clustering within the Crenarchaeota, Cyanobacteria, Firmicutes, Planctomycetales and Thermus divisions. Two sequence phylotypes were affiliated with uncultivated representatives of the recently described candidate division OP10 from a Yellowstone hot spring.

Engineering barriers to the spread of specific genes are of great interest both to increase the predictability of recombinant microorganisms used for environmental applications and to study the role of gene transfer in the adaptation of microbial communities to changing environments. We report here a new gene containment circuit based on a toxin-antidote pair that targets the cell DNA, i.e. the type II EcoRI restriction-modification system. The set-up involved linkage of the ecoRIR lethal gene encoding the EcoRI endonuclease (toxin) to the contained character in a plasmid and chromosomal insertion of the ecoRIM gene encoding the cognate EcoRI methylase (antidote) that protects the target DNA from restriction. Transfer of the contained character to a recipient cell lacking the antidote caused EcoRI-mediated chromosomal breaks, leading to cell death, thereby preventing gene spread. Using transformation and conjugation as mechanisms of DNA transfer and different environmentally relevant bacteria as recipients, we have shown that the potentially universal EcoRI-based containment system decreases gene transfer frequencies by more than four orders of magnitude. Analyses of the survivors escaping killing revealed a number of possible inactivation mechanisms.

Biofilm formation on a low-energy substratum floating on the surface of a water column overlying a polychlorinated biphenyl (PCB)-contaminated sandy clay soil was followed by light and electron microscopy. The biofilms that developed consisted of a dense lawn of clay aggregates, each one of which contained one or more bacteria, phyllosilicates and grains of iron oxide material, all held together by bacterial extracellular polysaccharides (EPS). The clay leaflets were arranged in the form of 'houses of cards' and gave the aggregates the appearance of 'hutches' housing the bacteria. Interestingly, although the soil is poor in carbon, and the weakly bioavailable PCBs constitute the principal source of carbon in this system, the bacteria contained electron-transparent structures presumed to be carbon storage granules. These, and the EPS material present in the hutches, indicate that carbon is not limiting in this system and, as PCBs have been found associated with the clay mineral fraction of the floating substratum, the clay particles may serve as carbon shuttles. The interesting possibilities that the 'clay hutches' may represent a 'soil microhabitat', a 'minimal nutritional sphere' and an 'effective survival unit' for autochthonous bacteria are noted. The formation of clay hutches by bacteria would seem to merit further investigation, particularly regarding their roles in bacterial processes in soil and in geological processes.

A systematic evaluation of the value and potential of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure has been undertaken. The reproducibility and robustness of the method has been assessed using environmental DNA samples isolated directly from PCB-polluted or pristine soil, and subsequent polymerase chain reaction (PCR) amplification of total community 16S rDNA. An initial investigation to assess the variability both within and between different polyacrylamide gel electrophoresis (PAGE) runs showed that almost identical community profiles were consistently produced from the same sample. Similarly, very little variability was observed as a result of variation between replicate restriction digestions, PCR amplifications or between replicate DNA isolations. Decreasing concentrations of template DNA produced a decline in both the complexity and the intensity of fragments present in the community profile, with no additional fragments detected in the higher dilutions that were not already present when more original template DNA was used. Reducing the number of cycles of PCR produced similar results. The greatest variation between profiles generated from the same DNA sample was produced using different Taq DNA polymerases, while lower levels of variability were found between PCR products that had been produced using different annealing temperatures. Incomplete digestion by the restriction enzyme may, as a result of the generation of partially digested fragments, lead to an overestimation of the overall diversity within a community. The results obtained indicate that, once standardized, T-RFLP analysis is a highly reproducible and robust technique that yields high-quality fingerprints consisting of fragments of precise sizes, which, in principle, could be phylogenetically assigned, once an appropriate database is constructed.

The presumptive metabolically active members of a bacterial community in a moorland soil in Germany, highly polluted with polychlorinated biphenyls (PCBs), were identified by sequencing of cloned reverse transcription-polymerase chain reaction (RT-PCR) amplification products of 16S rRNA generated from total RNA extracts. Analysis of the 16S rRNA clone library revealed a considerable diversity of metabolically active bacteria in the soil, despite the acidic pH and high concentrations of PCBs. Cloned sequence types clustered within the Proteobacteria (34% alpha-, 33% beta- and 7% gamma-subclasses), the Holophaga-Acidobacterium phylum (14%), the Actinobacteria (6.5%) and the Planctomycetales (2%). Three cloned sequence types were not affiliated to any described phylogenetic group. An unusual feature of this soil was the abundance of sequence types within the beta-subclass of the Proteobacteria, most of which were similar to the 16S rRNA gene sequences of species from only two genera, Burkholderia and Variovorax. Three other numerous 16S rRNA sequence types were similar to the sequences of Sphingomonas species, members of the Rhodopila globiformis group and Acidobacterium capsulatum. Some of the sequence types retrieved were similar to the 16S rRNA sequences of bacterial isolates able to degrade a variety of organic pollutants, including PCBs. As the PCB contamination is the major source of measurable carbon in this soil, some of the 16S rRNA sequence types detected and presumed to represent the metabolically active members of the community indicate the organisms likely to be involved, directly or indirectly, in the utilization of the PCBs as carbon and energy sources.

Although much information on metabolic pathways within individual organisms is available, little is known about the pathways operating in natural communities in which extensive sharing of nutritional resources is the rule. In order to analyse such a consortium pathway, we have investigated the flow of 4-chlorosalicylate as carbon substrate within a simple chemostat microbial community using 13C-labelled metabolites and isotopic ratio mass spectrometric analysis of label enrichment in immunocaptured member populations of the community. A complex pathway network of carbon sharing was thereby revealed, involving two different metabolic routes, one of which is completely novel and involves the toxic metabolite protoanemonin. The high stability of the community results, at least in part, from interdependencies based on carbon sharing and the rapid removal of toxic metabolites.