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The updated Wobble Hypothesis reasonably explains why some 40 tRNA species are sufficient to decode the 61 amino acid codons of the Universal Genetic Code. However, we still have no clue why eubacteria lack tRNA isoacceptors with ANN anticodons, whereas eukaryotes universally lack eight GNN anticodons, only one of which is also absent in bacteria. Direct tRNA sequencing could resolve the patterns of nucleoside modification that had been driving the divergent evolution in prokaryotes and eukaryotes, but this task will require the development of AI-supported base-callers that can recognize modified nucleosides without any subsequent analytical verification. Our knowledge of the bacterial tRNA landscape is moreover broadened by the recent discovery of antisense tRNAs and tRNA-derived fragments that should be examined in their roles for gene expression, translation, bacterial physiology or metabolism.

Summary Microbial technology is exceptional among human activities and endeavours in its range of applications that benefit humanity, even exceeding those of chemistry. What is more, microbial technologists are among the most creative scientists, and the scope of the field continuously expands as new ideas and applications emerge. Notwithstanding this diversity of applications, given the dire predictions for the fate of the surface biosphere as a result of current trajectories of global warming, the future of microbial biotechnology research must have a single purpose, namely to help secure the future of life on Earth. Everything else will, by comparison, be irrelevant. Crucially, microbes themselves play pivotal roles in climate (Cavicchioli et al., Nature Revs Microbiol 17: 569–586, 2019). To enable realization of their full potential in humanity’s effort to survive, development of new and transformative global warming‐relevant technologies must become the lynchpin of microbial biotechnology research and development. As a consequence, microbial biotechnologists must consider constraining their usual degree of freedom, and re‐orienting their focus towards planetary‐biosphere exigences. And they must actively seek alliances and synergies with others to get the job done as fast as humanly possible; they need to enthusiastically embrace and join the global effort, subordinating where necessary individual aspirations to the common good (the amazing speed with which new COVID‐19 diagnostics and vaccines were developed and implemented demonstrates what is possible given creativity, singleness of purpose and funding). In terms of priorities, some will be obvious, others less so, with some only becoming revealed after dedicated effort yields new insights/opens new vistas. We therefore refrain from developing a priority list here. Rather, we consider what is likely to happen to the Earth’s biosphere if we (and the rest of humanity) fail to rescue it. We do so with the aim of galvanizing the formulation and implementation of strategic and financial science policy decisions that will maximally stimulate the development of relevant new microbial technologies, and maximally exploit available technologies, to repair existing environmental damage and mitigate against future deterioration., Clearly, to avoid, or at least delay, a scenario like that depicted above, humanity must immediately institute radical changes that drastically reduce greenhouse gas emissions and other planet‐destroying activities, and single‐mindedly seek to develop and exploit new technologies to repair existing damage. Moreover, we must enter into more cooperative relationships with microbes and other life‐forms and, with them, devise new and transformative technologies to solve or mitigate current and future global, regional and local environmental problems. This will necessitate changes in mindsets (e.g. see Timmis and Ramos, Microb Biotechnol 14: 769–797, 2021). The next 15 years of microbial biotechnology must largely be devoted to the discovery and development of new microbial technologies that contribute significantly towards sustainability. Hopefully, during this time, humankind, with microbial assistance, will reorient its developmental trajectory to one that is consistent with the survival of Earth’s surface biosphere.

Summary The global propagation of SARS‐CoV‐2 and the detection of a large number of variants, some of which have replaced the original clade to become dominant, underscores the fact that the virus is actively exploring its evolutionary space. The longer high levels of viral multiplication occur – permitted by high levels of transmission –, the more the virus can adapt to the human host and find ways to success. The third wave of the COVID‐19 pandemic is starting in different parts of the world, emphasizing that transmission containment measures that are being imposed are not adequate. Part of the consideration in determining containment measures is the rationale that vaccination will soon stop transmission and allow a return to normality. However, vaccines themselves represent a selection pressure for evolution of vaccine‐resistant variants, so the coupling of a policy of permitting high levels of transmission/virus multiplication during vaccine roll‐out with the expectation that vaccines will deal with the pandemic, is unrealistic. In the absence of effective antivirals, it is not improbable that SARS‐CoV‐2 infection prophylaxis will involve an annual vaccination campaign against ‘dominant’ viral variants, similar to influenza prophylaxis. Living with COVID‐19 will be an issue of SARS‐CoV‐2 variants and evolution. It is therefore crucial to understand how SARS‐CoV‐2 evolves and what constrains its evolution, in order to anticipate the variants that will emerge. Thus far, the focus has been on the receptor‐binding spike protein, but the virus is complex, encoding 26 proteins which interact with a large number of host factors, so the possibilities for evolution are manifold and not predictable a priori. However, if we are to mount the best defence against COVID‐19, we must mount it against the variants, and to do this, we must have knowledge about the evolutionary possibilities of the virus. In addition to the generic cellular interactions of the virus, there are extensive polymorphisms in humans (e.g. Lewis, HLA, etc.), some distributed within most or all populations, some restricted to specific ethnic populations and these variations pose additional opportunities for/constraints on viral evolution. We now have the wherewithal – viral genome sequencing, protein structure determination/modelling, protein interaction analysis – to functionally characterize viral variants, but access to comprehensive genome data is extremely uneven. Yet, to develop an understanding of the impacts of such evolution on transmission and disease, we must link it to transmission (viral epidemiology) and disease data (patient clinical data), and the population granularities of these. In this editorial, we explore key facets of viral biology and the influence of relevant aspects of human polymorphisms, human behaviour, geography and climate and, based on this, derive a series of recommendations to monitor viral evolution and predict the types of variants that are likely to arise.

Summary Soil provides a multitude of services that are essential to a healthily functioning biosphere and continuity of the human race, such as feeding the growing human population and the sequestration of carbon needed to counteract global warming. Healthy soil availability is the limiting parameter in the provision of a number of these services. As a result of anthropogenic abuses, and natural and global warming‐promoted extreme weather events, Planet Earth is currently experiencing an unprecedented crisis of soil deterioration, desertification and erosive loss that increasingly prejudices the services it provides. Such services are pivotal to the Sustainability Development Goals formulated by the United Nations. Immediate and coordinated action on a global scale is urgently required to slow and ultimately reverse the loss of healthy soils. Despite the ‘dirt‐dust’, non‐vital appearance of soil, it is a highly dynamic living entity, whose life is overwhelmingly microbial. The soil microbiota, which constitutes the greatest reservoir and donor of microbial diversity on Earth, acts as a vast bioreactor, mediating a myriad of chemical reactions that turn the biogeochemical cycles, recycle wastes, purify water, and underpin the multitude of other services soil provides. Fuelling the belowground microbial bioreactor is the aboveground plant and photosynthetic surface microbial life which captures solar energy, fixes inorganic CO2 to organic carbon, and channels fixed carbon and energy into soil. In order to muster an effective response to the crisis, to avoid further deterioration, and to restore unhealthy soils, we need a new and coherent approach, namely to deal with soils worldwide as patients in need of health care and create (i) a public health system for development of effective policies for land use, conservation, restoration, recommendations of prophylactic measures, monitoring and identification of problems (epidemiology), organizing crisis responses, etc., and (ii) a healthcare system charged with soil care: the promotion of good practices, implementation of prophylaxis measures, and institution of therapies for treatment of unhealthy soils and restoration of drylands. These systems need to be national but there is also a desperate need for international coordination. To enable development of effective, evidence‐based strategies that will underpin the efforts of soil healthcare systems, a substantial investment in wide‐ranging interdisciplinary research on soil health and disease is mandatory. This must lead to a level of understanding of the soil:biota functionalities underlying key ecosystem services that enables formulation of effective diagnosis‐prophylaxis‐therapy pathways for sustainable use, protection and restoration of different types of soil resources in different climatic zones. These conservation‐regenerative‐restorative measures need to be complemented by an educative‐political‐economic‐legislative framework that provides incentives encouraging soil care: knowledge, policy, economic and others, and laws which promote international adherence to the principles of restorative soil management. And: we must all be engaged in improving soil health; everyone has a duty of care (https://www.bbc.co.uk/ideas/videos/why‐soil‐is‐one‐of‐the‐most‐amazing‐things‐on‐eart/p090cf64). Creative application of microbes, microbiomes and microbial biotechnology will be central to the successful operation of the healthcare systems.

Summary 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.

This year, 2022, Microbial Biotechnology (MBT) celebrates its 15th birthday. In journal terms, 15 is adulthood. It has been a privilege to develop the idea, launch the journal, nurture it in its infancy and stimulate and support it during its adolescence and early adulthood. The success of MBT - its growth over the last 4 years averaging > 30%, and the highest impact factor in the field, making it the leading research journal in applied microbiology/microbial biotechnology and the most attractive to publish in - gives us enormous pleasure and satisfaction, and not a little pride.

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. ispartof: ENVIRONMENTAL MICROBIOLOGY vol:23 issue:8 pages:4077-4091 ispartof: location:England status: published

This Crystal Ball speculates on the potential of molecular decoys for prevention and therapy in infectious diseases. It is dedicated to the memory of Singh Chhatwal, who pioneered research on disguises and decoys produced by Streptococcus, and so much more.

We have recently argued that, because microbes have pervasive – often vital – influences on our lives, and that therefore their roles must be taken into account in many of the decisions we face, society must become microbiology‐literate, through the introduction of relevant microbiology topics in school curricula (Timmis et al. 2019. Environ Microbiol 21: 1513‐1528). The current coronavirus pandemic is a stark example of why microbiology literacy is such a crucial enabler of informed policy decisions, particularly those involving preparedness of public‐health systems for disease outbreaks and pandemics. However, a significant barrier to attaining widespread appreciation of microbial contributions to our well‐being and that of the planet is the fact that microbes are seldom visible: most people are only peripherally aware of them, except when they fall ill with an infection. And it is disease, rather than all of the positive activities mediated by microbes, that colours public perception of ‘germs’ and endows them with their poor image. It is imperative to render microbes visible, to give them life and form for children (and adults), and to counter prevalent misconceptions, through exposure to imagination‐capturing images of microbes and examples of their beneficial outputs, accompanied by a balanced narrative. This will engender automatic mental associations between everyday information inputs, as well as visual, olfactory and tactile experiences, on the one hand, and the responsible microbes/microbial communities, on the other hand. Such associations, in turn, will promote awareness of microbes and of the many positive and vital consequences of their actions, and facilitate and encourage incorporation of such consequences into relevant decision‐making processes. While teaching microbiology topics in primary and secondary school is key to this objective, a strategic programme to expose children directly and personally to natural and managed microbial processes, and the results of their actions, through carefully planned class excursions to local venues, can be instrumental in bringing microbes to life for children and, collaterally, their families. In order to encourage the embedding of microbiology‐centric class excursions in current curricula, we suggest and illustrate here some possibilities relating to the topics of food (a favourite pre‐occupation of most children), agriculture (together with horticulture and aquaculture), health and medicine, the environment and biotechnology. And, although not all of the microbially relevant infrastructure will be within reach of schools, there is usually access to a market, local food store, wastewater treatment plant, farm, surface water body, etc., all of which can provide opportunities to explore microbiology in action. If children sometimes consider the present to be mundane, even boring, they are usually excited with both the past and the future so, where possible, visits to local museums (the past) and research institutions advancing knowledge frontiers (the future) are strongly recommended, as is a tapping into the natural enthusiasm of local researchers to leverage the educational value of excursions and virtual excursions. Children are also fascinated by the unknown, so, paradoxically, the invisibility of microbes makes them especially fascinating objects for visualization and exploration. In outlining some of the options for microbiology excursions, providing suggestions for discussion topics and considering their educational value, we strive to extend the vistas of current class excursions and to: (i) inspire teachers and school managers to incorporate more microbiology excursions into curricula; (ii) encourage microbiologists to support school excursions and generally get involved in bringing microbes to life for children; (iii) urge leaders of organizations (biopharma, food industries, universities, etc.) to give school outreach activities a more prominent place in their mission portfolios, and (iv) convey to policymakers the benefits of providing schools with funds, materials and flexibility for educational endeavours beyond the classroom UK Research and Innovation/[ST/S001425/1]/UKRI/Reino Unido Functionality of Urban Soils/[NE/S005560/1]/FUSED/Reino Unido UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)

ispartof: ENVIRONMENTAL MICROBIOLOGY vol:22 issue:6 pages:1986-1996 ispartof: location:England status: published

Standards are the basis of technology: they allow rigorous description and exact measurement of properties, reliable reproducibility and a common “language” that enables different communities to work together. Molecular biology was in part created by physicists; yet, the field did not inherit the focus on the quantitation, the definition of system boundaries and the robust, unequivocal language that is characteristic of the other natural sciences. However, synthetic biology (SynBio) increasingly requires scientific, technical, operational and semantic standards for the field to become a full‐fledged engineering discipline with a high level of accuracy in the design, manufacturing and performance of biological artefacts. Although the benefits of adopting standards are clear, the community is still largely reluctant to accept them, owing to concerns about adoption costs and losses in flexibility., This work was funded by the European Union through the BioRoboost Project, H2020‐NMBP‐TR‐IND‐2018‐2020/BIOTEC‐01‐2018 (CSA), Project ID 820699. Jake Beal was also supported in part by NSF Expeditions in Computing Program Award #1522074.

Cyanobacteria are ecologically versatile microorganisms inhabiting most environments, ranging from marine systems to arid deserts. Although they possess several pathways for light-independent energy generation, until now their ecological range appeared to be restricted to environments with at least occasional exposure to sunlight. Here we present molecular, microscopic, and metagenomic evidence that cyanobacteria predominate in deep subsurface rock samples from the Iberian Pyrite Belt Mars analog (southwestern Spain). Metagenomics showed the potential for a hydrogen-based lithoautotrophic cyanobacterial metabolism. Collectively, our results suggest that they may play an important role as primary producers within the deep-Earth biosphere. Our description of this previously unknown ecological niche for cyanobacteria paves the way for models on their origin and evolution, as well as on their potential presence in current or primitive biospheres in other planetary bodies, and on the extant, primitive, and putative extraterrestrial biospheres., This work was supported by the European Research Council Advanced Grant ERC250350-IPBSL, and the Spanish Ministerio de Economía y Competitividad/Fondo Europeo de Desarrollo Regional Grants AYA2011-24803 and ESP2015-69540-R. F.P.-S. is currently funded by the Spanish Ministerio de Economía y Competitividad/Fondo Europeo de Desarrollo Regional Grant CTM2016-80095-C2-1-R.

Summary The most efficient means of generating cellular energy is through aerobic respiration. Under anaerobic conditions, several prokaryotes can replace oxygen by nitrate as final electron acceptor. During denitrification, nitrate is reduced via nitrite, NO and N2O to molecular nitrogen (N2) by four membrane‐localized reductases with the simultaneous formation of an ion gradient for ATP synthesis. These four multisubunit enzyme complexes are coupled in four electron transport chains to electron donating primary dehydrogenases and intermediate electron transfer proteins. Many components require membrane transport and insertion, complex assembly and cofactor incorporation. All these processes are mediated by fine‐tuned stable and transient protein–protein interactions. Recently, an interactomic approach was used to determine the exact protein–protein interactions involved in the assembly of the denitrification apparatus of Pseudomonas aeruginosa. Both subunits of the NO reductase NorBC, combined with the flavoprotein NosR, serve as a membrane‐localized assembly platform for the attachment of the nitrate reductase NarGHI, the periplasmic nitrite reductase NirS via its maturation factor NirF and the N2O reductase NosZ through NosR. A nitrate transporter (NarK2), the corresponding regulatory system NarXL, various nitrite (NirEJMNQ) and N2O reductase (NosFL) maturation proteins are also part of the complex. Primary dehydrogenases, ATP synthase, most enzymes of the TCA cycle, and the SEC protein export system, as well as a number of other proteins, were found to interact with the denitrification complex. Finally, a protein complex composed of the flagella protein FliC, nitrite reductase NirS and the chaperone DnaK required for flagella formation was found in the periplasm of P. aeruginosa. This work demonstrated that the interactomic approach allows for the identification and characterization of stable and transient protein–protein complexes and interactions involved in the assembly and function of multi‐enzyme complexes.

Summary Healthcare systems worldwide are confronted with major economic, organizational and logistical challenges. Historic evolution of health care has led to significant healthcare sector fragmentation, resulting in systemic inefficiencies and suboptimal resource exploitation. To attain a sustainable healthcare model, fundamental, system‐wide improvements that effectively network, and ensure fulfilment of potential synergies between sectors, and include and facilitate coherent strategic planning and organisation of healthcare infrastructure are needed. Critically, they must be specifically designed to sustainably achieve peak performance within the current policy environment for cost‐control, and efficiency and quality improvement for service delivery. We propose creation of a new healthcare cluster, to be embedded in existing healthcare systems. It consists of (i) local 24/7 walk‐in virtually autonomous do‐it‐yourself Digital Medical Centres performing routine diagnosis, monitoring, prevention, treatment and standardized documentation and health outcome assessment/reporting, which are online interfaced with (ii) regional 24/7 eClinician Centres providing on‐demand clinical supervision/assistance to Digital Medical Centre patients. Both of these are, in turn, online interfaced with (iii) the National Clinical Informatics Centre, which houses the national patient data centre (cloud) and data analysis units that conduct patient‐ and population‐level, personalized and predictive(‐medicine) intervention optimization analyses. The National Clinical Informatics Centre also interfaces with biomedical research and prioritizes and accelerates the translation of new discoveries into clinical practice. The associated Health Policy Innovation and Evaluation Centre rapidly integrates new findings with health policy/regulatory discussions. This new cluster would synergistically link all health system components in a circular format, enable not only access by all arms of the health service to latest patient data, but also automatic algorithm analysis and prediction of clinical development of individual patients, reduce bureaucratic burden on medical professionals by enabling a greater level of focus of their expertise on non‐routine medical tasks, lead to automatic translation of aggregate patient data/new knowledge into medical practice, and orient future evolution of health systems towards greater cohesion/integration and hence efficiency. A central plank of the proposed concept is increased emphasis on reduction of disease incidence and severity, to diminish both patient suffering and treatment costs. This will be achieved at the individual and population levels, through (i) significantly improved access to medical services, (ii) stronger focus on primary and secondary prevention and early treatment measures, and disease susceptibility prediction via personalized medicine, involving inter alia genome analysis at birth and periodic analysis of microbiomes and biomarkers, and integration with other patient health and epidemiology parameters, (iii) improved surveillance and (iv) intervention outcome benchmarking. The dMCs will become drivers of innovation and integrative evolution in health systems, of disease reduction and efficiency gains, and thus major contributors to development of sustainability of health care.

Non

In the Anthropocene, in which we now live, climate change is impacting most life on Earth. Microorganisms support the existence of all higher trophic life forms. To understand how humans and other life forms on Earth (including those we are yet to discover) can withstand anthropogenic climate change, it is vital to incorporate knowledge of the microbial ‘unseen majority’. We must learn not just how microorganisms affect climate change (including production and consumption of greenhouse gases) but also how they will be affected by climate change and other human activities. This Consensus Statement documents the central role and global importance of microorganisms in climate change biology. It also puts humanity on notice that the impact of climate change will depend heavily on responses of microorganisms, which are essential for achieving an environmentally sustainable future., Nature Reviews Microbiology, 17 (9), ISSN:1740-1526, ISSN:1740-1534

Oxidative phosphorylation using multiple-component, membrane-associated protein complexes is the most effective way for a cell to generate energy. Here, we systematically investigated the multiple protein-protein interactions of the denitrification apparatus of the pathogenic bacterium Pseudomonas aeruginosa . During denitrification, nitrate (Nar), nitrite (Nir), nitric oxide (Nor), and nitrous oxide (Nos) reductases catalyze the reaction cascade of NO 3− → NO 2− → NO → N 2 O → N 2 . Genetic experiments suggested that the nitric oxide reductase NorBC and the regulatory protein NosR are the nucleus of the denitrification protein network. We utilized membrane interactomics in combination with electron microscopy colocalization studies to elucidate the corresponding protein-protein interactions. The integral membrane proteins NorC, NorB, and NosR form the core assembly platform that binds the nitrate reductase NarGHI and the periplasmic nitrite reductase NirS via its maturation factor NirF. The periplasmic nitrous oxide reductase NosZ is linked via NosR. The nitrate transporter NarK2, the nitrate regulatory system NarXL, various nitrite reductase maturation proteins, NirEJMNQ, and the Nos assembly lipoproteins NosFL were also found to be attached. A number of proteins associated with energy generation, including electron-donating dehydrogenases, the complete ATP synthase, almost all enzymes of the tricarboxylic acid (TCA) cycle, and the Sec system of protein transport, among many other proteins, were found to interact with the denitrification proteins. This deduced nitrate respirasome is presumably only one part of an extensive cytoplasmic membrane-anchored protein network connecting cytoplasmic, inner membrane, and periplasmic proteins to mediate key activities occurring at the barrier/interface between the cytoplasm and the external environment. IMPORTANCE The processes of cellular energy generation are catalyzed by large multiprotein enzyme complexes. The molecular basis for the interaction of these complexes is poorly understood. We employed membrane interactomics and electron microscopy to determine the protein-protein interactions involved. The well-investigated enzyme complexes of denitrification of the pathogenic bacterium Pseudomonas aeruginosa served as a model. Denitrification is one essential step of the universal N cycle and provides the bacterium with an effective alternative to oxygen respiration. This process allows the bacterium to form biofilms, which create low-oxygen habitats and which are a key in the infection mechanism. Our results provide new insights into the molecular basis of respiration, as well as opening a new window into the infection strategies of this pathogen.

Pseudomonas aeruginosa is a ubiquitously occurring environmental bacterium and opportunistic pathogen responsible for various acute and chronic infections. Obviously, anaerobic energy generation via denitrification contributes to its ecological success. To investigate the structural basis for the interconnection of the denitrification machinery to other essential cellular processes, we have sought to identify the protein interaction partners of the denitrification enzyme nitrite reductase NirS in the periplasm. We employed NirS as an affinity-purifiable bait to identify interacting proteins in vivo . Results obtained revealed that both the flagellar structural protein FliC and the protein chaperone DnaK form a complex with NirS in the periplasm. The interacting domains of NirS and FliC were tentatively identified. The NirS-interacting stretch of amino acids lies within its cytochrome c domain. Motility assays and ultrastructure analyses revealed that a nirS mutant was defective in the formation of flagella and correspondingly in swimming motility. In contrast, the fliC mutant revealed an intact denitrification pathway. However, deletion of the nirF gene, coding for a heme d 1 biosynthetic enzyme, which leads to catalytically inactive NirS, did not abolish swimming ability. This pointed to a structural function for the NirS protein. FliC and NirS were found colocalized with DnaK at the cell surface of P. aeruginosa . A function of the detected periplasmic NirS-DnaK-FliC complex in flagellum formation and motility was concluded and discussed. IMPORTANCE Physiological functions in Gram-negative bacteria are connected with the cellular compartment of the periplasm and its membranes. Central enzymatic steps of anaerobic energy generation and the motility mediated by flagellar activity use these cellular structures in addition to multiple other processes. Almost nothing is known about the protein network functionally connecting these processes in the periplasm. Here, we demonstrate the existence of a ternary complex consisting of the denitrifying enzyme NirS, the chaperone DnaK, and the flagellar protein FliC in the periplasm of the pathogenic bacterium P. aeruginosa . The dependence of flagellum formation and motility on the presence of an intact NirS was shown, structurally connecting both cellular processes, which are important for biofilm formation and pathogenicity of the bacterium.

Archaea and Bacteria constitute a majority of life systems on Earth but have long been considered inferior to Eukarya in terms of solute tolerance. Whereas the most halophilic prokaryotes are known for an ability to multiply at saturated NaCl (water activity (aw) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650–0.605 aw. Here, we present evidence that halophilic prokayotes can grow down to water activities of

Whether the extreme conditions of acidity and heavy metal pollution of streams and rivers originating in pyritic formations are caused primarily by mining activities or by natural activities of metal-oxidizing microbes living within the geological formations is a subject of considerable controversy. Most microbiological studies of such waters have so far focused on acid mine drainage sites, which are heavily human-impacted environments, so it has been problematic to eliminate the human factor in the question of the origin of the key metal compounds. We have studied the physico-chemistry and microbiology of the Río Sucio in the Braulio Carrillo National Park of Costa Rica, 22 km from its volcanic rock origin. Neither the remote origin, nor the length of the river to the sampling site, have experienced human activity and are thus pristine. The river water had a characteristic brownish-yellow color due to high iron-dominated minerals, was slightly acidic, and rich in chemolithoautotrophic iron- and sulfur-oxidizing bacteria, dominated by Gallionella spp. Río Sucio is thus a natural acid-rock drainage system whose metal-containing components are derived primarily from microbial activities. Universidad de Costa Rica/[809-B4-282]/UCR/Costa Rica European Commission-Science and Technology Development for in situ detection and characterization of subsurface life on the Iberian Pyritic Belt/[ERC250350-IPBSL]/ERC IPBSL/Unión Europea USA National Science Foundation/[0959894]//Estados Unidos Consejo Superior de Investigaciones Científicas//CSIC/España European Union FP7 programme /[607346]/EU/ UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA) UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Química UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)

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Since a key requirement of known life forms is available water (water activity; aw), recent searches for signatures of past life in terrestrial and extraterrestrial environments have targeted places known to have contained significant quantities of biologically available water. However, early life on Earth inhabited high-salt environments, suggesting an ability to withstand low water-activity. The lower limit of water activity that enables cell division appears to be ∼ 0.605 which, until now, was only known to be exhibited by a single eukaryote, the sugar-tolerant, fungal xerophile Xeromyces bisporus. The first forms of life on Earth were, though, prokaryotic. Recent evidence now indicates that some halophilic Archaea and Bacteria have water-activity limits more or less equal to those of X. bisporus. We discuss water activity in relation to the limits of Earth's present-day biosphere; the possibility of microbial multiplication by utilizing water from thin, aqueous films or non-liquid sources; whether prokaryotes were the first organisms able to multiply close to the 0.605-aw limit; and whether extraterrestrial aqueous milieux of ≥ 0.605 aw can resemble fertile microbial habitats found on Earth.

The aim of this work was to determine the effect of light crude oil on bacterial communities during an experimental oil spill in the North Sea and in mesocosms (simulating a heavy, enclosed oil spill), and to isolate and characterize hydrocarbon-degrading bacteria from the water column. No oil-induced changes in bacterial community (3 m below the sea surface) were observed 32 h after the experimental spill at sea. In contrast, there was a decrease in the dominant SAR11 phylotype and an increase in Pseudoalteromonas spp. in the oiled mesocosms (investigated by 16S rRNA gene analysis using denaturing gradient gel electrophoresis), as a consequence of the longer incubation, closer proximity of the samples to oil, and the lack of replenishment with seawater. A total of 216 strains were isolated from hydrocarbon enrichment cultures, predominantly belonging to the genus Pseudoaltero monas; most strains grew on PAHs, branched and straight-chain alkanes, as well as many other carbon sources. No obligate hydrocarbonoclastic bacteria were isolated or detected, highlighting the potential importance of cosmopolitan marine generalists like Pseudoalteromonas spp. in degrading hydrocarbons in the water column beneath an oil slick, and revealing the susceptibility to oil pollution of SAR11, the most abundant bacterial clade in the surface ocean.

Environmental microbes oscillate between feast and famine and need to carefully manage utilization, storage and conversion of reserve products to exploitable sources of carbon and energy. Polyhydroxyalkanoates (PHAs) are storage polymers that serve bacteria as sources of food materials under physiological conditions of carbon demand. In order to obtain insights into the role of PHA depolymerase (PhaZ) and its relationship to a PHA polymerase (PhaC2) in the carbon management activity of Pseudomonas putida strain U, we created a polymerase hyperexpression strain and a depolymerase knockout mutant of this strain, and examined their synthesis of PHA and expression of their PHA genes. This study revealed that hyperexpression of PhaC2 led to the accumulation of higher amounts of PHA (44%wt) than in the wild-type strain (24%wt) after 24 h of cultivation, which then returned to wild-type levels by 48 h, as a result of elevated depolymerization. The phaZ mutant, however, accumulated higher levels of PHA than the parental strain (62%wt), which were maintained for at least 96 h. Transcriptional analysis of the pha cluster by RT-PCR revealed that PHA operon proteins, including depolymerase, are expressed from the beginning of the growth phase. Hyperexpression of the PhaC2 polymerase was accompanied by an increase in the expression of the PhaZ depolymerase and a decrease in expression of another PHA polymerase, PhaC1. This suggests tight regulatory coupling of PHA polymerase and depolymerase activities that act in synergy, and in concert with other PHA proteins, to provide dynamic PHA granule synthesis and remodelling that rapidly and sensitively respond to changes in availability of carbon and the physiological-metabolic needs of the cell, to ensure optimal carbon resource management.

Coastal and estuarine ecosystems are highly susceptible to crude oil pollution. Therefore, in order to examine the resilience of benthic phototrophs that are pivotal to coastal ecosystem functioning, we simulated an oil spill in tidal mesocosms consisting of intact sediment cores from a mudflat at the mouth of the Colne Estuary, UK. At day 21, fluorescence imaging revealed a bloom of cyanobacteria on the surface of oiled sediment cores, and the upper 1.5cm thick sediment had 7.2 times more cyanobacterial and 1.7 times more diatom rRNA sequences when treated with oil. Photosystem II operating efficiency (Fq′/Fm′) was significantly reduced in oiled sediments at day 7, implying that the initial diatom-dominated community was negatively affected by oil, but this was no longer apparent by day 21. Oil addition significantly reduced numbers of the key deposit feeders, and the decreased grazing pressure is likely to be a major factor in the increased abundance of both diatoms and cyanobacteria. By day 5 concentrations of dissolved inorganic nitrogen were significantly lower in oiled mesocosms, likely resulting in the observed increase in nifH-containing, and therefore potentially dinitrogen-fixing, cyanobacteria. Thus, indirect effects of oil, rather than direct inhibition, are primarily responsible for altering the microphytobenthos. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Aims: The purpose of this study was to isolate new and potentially better polyhydroxyalkanoate (PHA)-producing bacteria, with a view to obtaining high yields from inexpensive substrates like glycerol, a major by-product of the biodiesel process. Methods and Results: Eleven new plant original isolates of the genus Massilia, a poorly studied lineage within the Betaproteobacteria, were isolated and characterized. Two isolates, 2C4 and 4D3c, could not be assigned to a validated Massilia species and probably represent new species. Six isolates were found to produce poly-3-hydroxybutyrate (P3HB) when cultured with glucose or glycerol as carbon source. Isolate 4D6 accumulated up to 50 wt% of cell mass as polyhydroxybutyrate (PHB) when grown on glycerol. Conclusions: The phyllosphere may be a good source of bacteria unrelated or weakly related to human/animal pathogens for screening for new PHA producers for industrial application. Isolate 4D6 was capable of accumulating particularly high levels of PHB from glycerol. Significance and Impact of the Study: With the increase in biodiesel production, which generates increasing amounts of glycerol as a by-product, there is a major interest in exploiting this compound as feedstock for the synthesis of interesting products, like biopolymers, such as PHA. The new Massilia sp. 4D6 isolate described in this study may be a useful candidate as a cell factory for the industrial production of PHA from glycerol.

The marine oil-degrading bacterium Alcanivorax borkumensis SK2 has attracted significant interest due to its hydrocarbonoclastic lifestyle, its alkane-centered metabolism, and for playing an important ecological role in cleaning up marine oil spills. In this study, we used microarray technology to characterize the transcriptional responses of A. borkumensis to n-hexadecane exposure as opposed to pyruvate, which led to the identification of a total of 220 differentially expressed genes, with 109 genes being upregulated and 111 genes being downregulated. Among the genes upregulated on alkanes are systems predicted to be involved in the terminal oxidation of alkanes, biofilm formation, signal transduction, and regulation.