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Microbiology education has a serious handicap - the lack of visibility of the players of the subject and their interactions - which engenders a disproportionate reliance upon multimedia teaching aids (MTAs). The International Microbiology Literacy Initiative (IMiLI) is creating educational resources in societally-relevant microbiology complemented by appropriate MTAs. However, proper guidance supporting microbiology educators in locating and selecting, or commissioning the creation of, adequate MTAs for different target audiences and learning objectives is lacking. The aims of this study were to (i) identify important considerations regarding educational/didactical standards and the design of educational multimedia and (ii) create an evidence-based guideline for selecting and appraising existing, and informing the creation of new, microbiology MTAs. This investigation is based on an exploratory, mixed-methods approach. The results of two literature reviews (covering educational and good practice multimedia design) informed the collation of a preliminary appraisal guideline for videos, animations, comics, and video games. A web-scraping approach was utilised to locate and retrieve existing exemplars of the four multimedia types and create four pertinent multimedia databases (including metadata). The preliminary guideline was piloted (and revised accordingly) by appraising quasi-random (or purposive) samples of each multimedia type. Educational multimedia experts were interviewed to discuss the findings. Finally, the guideline was updated to reflect the expert comments together with the results of the pilot appraisals. The final guideline has four components: (i) central considerations for selecting and appraising multimedia for specific audiences and educational purposes, (ii) multimedia selection tool, (iii) multimedia appraisal tools, and (iv) extensive background information as appendices linking all sections for further comprehension. Broad utilisation of the guideline has significant potential for simplifying and systematising multimedia selection/creation, leading to superior multimedia-based learning outcomes, establishing a rapid selection database (pre-appraised multimedia), reducing disparities in microbiology education and incentivising educational content creators., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

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., (© 2022 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic., (© 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

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.

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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

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 N 2 O to molecular nitrogen (N 2 ) 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 N 2 O reductase NosZ through NosR. A nitrate transporter (NarK2), the corresponding regulatory system NarXL, various nitrite (NirEJMNQ) and N 2 O 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., (© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

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.

Unlabelled: 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 → N2O → N2 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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: 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 d1 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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Whereas osmotic stress response induced by solutes has been well-characterized in fungi, less is known about the other activities of environmentally ubiquitous substances. The latest methodologies to define, identify and quantify chaotropicity, i.e. substance-induced destabilization of macromolecular systems, now enable new insights into microbial stress biology (Cray et al. in Curr Opin Biotechnol 33:228-259, 2015a, doi: 10.1016/j.copbio.2015.02.010 ; Ball and Hallsworth in Phys Chem Chem Phys 17:8297-8305, 2015, doi: 10.1039/C4CP04564E ; Cray et al. in Environ Microbiol 15:287-296, 2013a, doi: 10.1111/1462-2920.12018 ). We used Aspergillus wentii, a paradigm for extreme solute-tolerant fungal xerophiles, alongside yeast cell and enzyme models (Saccharomyces cerevisiae and glucose-6-phosphate dehydrogenase) and an agar-gelation assay, to determine growth-rate inhibition, intracellular compatible solutes, cell turgor, inhibition of enzyme activity, substrate water activity, and stressor chaotropicity for 12 chemically diverse solutes. These stressors were found to be: (i) osmotically active (and typically macromolecule-stabilizing kosmotropes), including NaCl and sorbitol; (ii) weakly to moderately chaotropic and non-osmotic, these were ethanol, urea, ethylene glycol; (iii) highly chaotropic and osmotically active, i.e. NH4NO3, MgCl2, guanidine hydrochloride, and CaCl2; or (iv) inhibitory due primarily to low water activity, i.e. glycerol. At ≤0.974 water activity, Aspergillus cultured on osmotically active stressors accumulated low-M r polyols to ≥100 mg g dry weight(-1). Lower-M r polyols (i.e. glycerol, erythritol and arabitol) were shown to be more effective for osmotic adjustment; for higher-M r polyols such as mannitol, and the disaccharide trehalose, water-activity values for saturated solutions are too high to be effective; i.e. 0.978 and 0.970 (25 ºC). The highly chaotropic, osmotically active substances exhibited a stressful level of chaotropicity at physiologically relevant concentrations (20.0-85.7 kJ kg(-1)). We hypothesized that the kosmotropicity of compatible solutes can neutralize chaotropicity, and tested this via in-vitro agar-gelation assays for the model chaotropes urea, NH4NO3, phenol and MgCl2. Of the kosmotropic compatible solutes, the most-effective protectants were trimethylamine oxide and betaine; but proline, dimethyl sulfoxide, sorbitol, and trehalose were also effective, depending on the chaotrope. Glycerol, by contrast (a chaotropic compatible solute used as a negative control) was relatively ineffective. The kosmotropic activity of compatible solutes is discussed as one mechanism by which these substances can mitigate the activities of chaotropic stressors in vivo. Collectively, these data demonstrate that some substances concomitantly induce chaotropicity-mediated and osmotic stresses, and that compatible solutes ultimately define the biotic window for fungal growth and metabolism. The findings have implications for the validity of ecophysiological classifications such as 'halophile' and 'polyextremophile'; potential contamination of life-support systems used for space exploration; and control of mycotoxigenic fungi in the food-supply chain.

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 (a(w)) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650-0.605 a(w). Here, we present evidence that halophilic prokayotes can grow down to water activities of <0.755 for Halanaerobium lacusrosei (0.748), Halobacterium strain 004.1 (0.728), Halobacterium sp. NRC-1 and Halococcus morrhuae (0.717), Haloquadratum walsbyi (0.709), Halococcus salifodinae (0.693), Halobacterium noricense (0.687), Natrinema pallidum (0.681) and haloarchaeal strains GN-2 and GN-5 (0.635 a(w)). Furthermore, extrapolation of growth curves (prone to giving conservative estimates) indicated theoretical minima down to 0.611 aw for extreme, obligately halophilic Archaea and Bacteria. These were compared with minima for the most solute-tolerant Bacteria in high-sugar (or other non-saline) media (Mycobacterium spp., Tetragenococcus halophilus, Saccharibacter floricola, Staphylococcus aureus and so on) and eukaryotic microbes in saline (Wallemia spp., Basipetospora halophila, Dunaliella spp. and so on) and high-sugar substrates (for example, Xeromyces bisporus, Zygosaccharomyces rouxii, Aspergillus and Eurotium spp.). We also manipulated the balance of chaotropic and kosmotropic stressors for the extreme, xerophilic fungi Aspergillus penicilloides and X. bisporus and, via this approach, their established water-activity limits for mycelial growth (∼0.65) were reduced to 0.640. Furthermore, extrapolations indicated theoretical limits of 0.632 and 0.636 a(w) for A. penicilloides and X. bisporus, respectively. Collectively, these findings suggest that there is a common water-activity limit that is determined by physicochemical constraints for the three domains of life.

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., (© 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

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., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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., (© 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

The marine hydrocarbonoclastic bacterium Alcanivorax borkumensis is able to degrade mixtures of n-alkanes as they occur in marine oil spills. However, investigations of growth behavior and physiology of these bacteria when cultivated with n-alkanes of different chain lengths (C6 to C30) as the substrates are still lacking. Growth rates increased with increasing alkane chain length up to a maximum between C12 and C19, with no evident difference between even- and odd-numbered chain lengths, before decreasing with chain lengths greater than C19. Surface hydrophobicity of alkane-grown cells, assessed by determination of the water contact angles, showed a similar pattern, with maximum values associated with growth rates on alkanes with chain lengths between C11 and C19 and significantly lower values for cells grown on pyruvate. A. borkumensis was found to incorporate and modify the fatty acid intermediates generated by the corresponding n-alkane degradation pathway. Cells grown on distinct n-alkanes proved that A. borkumensis is able to not only incorporate but also modify fatty acid intermediates derived from the alkane degradation pathway. Comparing cells grown on pyruvate with those cultivated on hexadecane in terms of their tolerance toward two groups of toxic organic compounds, chlorophenols and alkanols, representing intensely studied organic compounds, revealed similar tolerances toward chlorophenols, whereas the toxicities of different n-alkanols were significantly reduced when hexadecane was used as a carbon source. As one adaptive mechanism of A. borkumensis to these toxic organic solvents, the activity of cis-trans isomerization of unsaturated fatty acids was proven. These findings could be verified by a detailed transcriptomic comparison between cultures grown on hexadecane and pyruvate and including solvent stress caused by the addition of 1-octanol as the most toxic intermediate of n-alkane degradation.

Background: In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains., Results: Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs., Conclusion: The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co-localization studies. In addition, the new tools facilitate metabolic engineering and yield assessment for cytoplasmic or periplasmic protein production in a number of different expression hosts when yields in one initially selected are insufficient.

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 1.5 cm 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.)

Mudflats and salt marshes are habitats at the interface of aquatic and terrestrial systems that provide valuable services to ecosystems. Therefore, it is important to determine how catastrophic incidents, such as oil spills, influence the microbial communities in sediment that are pivotal to the function of the ecosystem and to identify the oil-degrading microbes that mitigate damage to the ecosystem. In this study, an oil spill was simulated by use of a tidal chamber containing intact diatom-dominated sediment cores from a temperate mudflat. Changes in the composition of bacteria and diatoms from both the sediment and tidal biofilms that had detached from the sediment surface were monitored as a function of hydrocarbon removal. The hydrocarbon concentration in the upper 1.5 cm of sediments decreased by 78% over 21 days, with at least 60% being attributed to biodegradation. Most phylotypes were minimally perturbed by the addition of oil, but at day 21, there was a 10-fold increase in the amount of cyanobacteria in the oiled sediment. Throughout the experiment, phylotypes associated with the aerobic degradation of hydrocarbons, including polycyclic aromatic hydrocarbons (PAHs) (Cycloclasticus) and alkanes (Alcanivorax, Oleibacter, and Oceanospirillales strain ME113), substantively increased in oiled mesocosms, collectively representing 2% of the pyrosequences in the oiled sediments at day 21. Tidal biofilms from oiled cores at day 22, however, consisted mostly of phylotypes related to Alcanivorax borkumensis (49% of clones), Oceanospirillales strain ME113 (11% of clones), and diatoms (14% of clones). Thus, aerobic hydrocarbon biodegradation is most likely to be the main mechanism of attenuation of crude oil in the early weeks of an oil spill, with tidal biofilms representing zones of high hydrocarbon-degrading activity.

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., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

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., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Background: Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a cell factory for production of scFvs., Results: We have generated new broad host range scFv expression constructs and assessed their production in the Pseudomonas putida KT2440 host. Two scFvs bind either to human C-reactive protein or to mucin1, proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme scFv. The KT2440 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for P. putida, and medium on scFv yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active scFv fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the Ptac expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an E. coli K-12 expression host., Conclusions: Pseudomonas putida KT2440 is a good cell factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High cell density cultivation and further optimization and refinement of the KT2440 cell factory will achieve additional increases in the yields of scFvs.

The human gastrointestinal tract microbiota, despite its key roles in health and disease, remains a diverse, variable and poorly understood entity. Current surveys reveal a multitude of undefined bacterial taxa and a low diversity of methanogenic archaea. In an analysis of the microbiota in colonic mucosal biopsies from patients with inflammatory bowel disease we found 16S rDNA sequences representing a phylogenetically rich diversity of halophilic archaea from the Halobacteriaceae (haloarchaea), including novel phylotypes. As the human colon is not considered a salty environment and haloarchaea are described as extreme halophiles, we evaluated and further discarded the possibility that these sequences originated from pre-colonoscopy saline lavage solutions. Furthermore, aerobic enrichment cultures prepared from a patient biopsy at low salinity (2.5% NaCl) yielded haloarchaeal sequence types. Microscopic observation after fluorescence in situ hybridization provided evidence of the presence of viable archaeal cells in these cultures. These results prove the survival of haloarchaea in the digestive system and suggest that they may be members of the mucosal microbiota, even if present in low numbers in comparison with methanogenic archaea. Investigation of a potential physiological basis of this association may lead to new insights into gastrointestinal health and disease., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Isoprene is a volatile and climate-altering hydrocarbon with an atmospheric concentration similar to that of methane. It is well established that marine algae produce isoprene; however, until now there was no specific information about marine isoprene sinks. Here we demonstrate isoprene consumption in samples from temperate and tropical marine and coastal environments, and furthermore show that the most rapid degradation of isoprene coincides with the highest rates of isoprene production in estuarine sediments. Isoprene-degrading enrichment cultures, analysed by denaturing gradient gel electrophoresis and 454 pyrosequencing of the 16S rRNA gene and by culturing, were generally dominated by Actinobacteria, but included other groups such as Alphaproteobacteria and Bacteroidetes, previously not known to degrade isoprene. In contrast to specialist methane-oxidizing bacteria, cultivated isoprene degraders were nutritionally versatile, and nearly all of them were able to use n-alkanes as a source of carbon and energy. We therefore tested and showed that the ubiquitous marine hydrocarbon-degrader, Alcanivorax borkumensis, could also degrade isoprene. A mixture of the isolates consumed isoprene emitted from algal cultures, confirming that isoprene can be metabolized at low, environmentally relevant concentrations, and suggesting that, in the absence of spilled petroleum hydrocarbons, algal production of isoprene could maintain viable populations of hydrocarbon-degrading microbes. This discovery of a missing marine sink for isoprene is the first step in obtaining more robust predictions of its flux, and suggests that algal-derived isoprene provides an additional source of carbon for diverse microbes in the oceans.

We describe a sensitive metabolite array for genome sequence-independent functional analysis of metabolic phenotypes and networks, the reactomes, of cell populations and communities. The array includes 1676 dye-linked substrate compounds collectively representing central metabolic pathways of all forms of life. Application of cell extracts to the array leads to specific binding of enzymes to cognate substrates, transformation to products, and concomitant activation of the dye signals. Proof of principle was shown by reconstruction of the metabolic maps of model bacteria. Utility of the array for unsequenced organisms was demonstrated by reconstruction of the global metabolisms of three microbial communities derived from acidic volcanic pool, deep-sea brine lake, and hydrocarbon-polluted seawater. Enzymes of interest are captured on nanoparticles coated with cognate metabolites, sequenced, and their functions unequivocally established.

Aims: To investigate the feasibility of applying sorbent material X-Oil in marine oil spill mitigation and to survey the interactions of oil, bacteria and sorbent., Methods and Results: In a series of microcosms, 25 different treatments including nutrient amendment, bioaugmentation with Alcanivorax borkumensis and application of sorbent were tested. Microbial community dynamics were analysed by DNA fingerprinting methods, RISA and DGGE. Results of this study showed that the microbial communities in microcosms with highly active biodegradation were strongly selected in favour of A. borkumensis. Oxygen consumption measurements in microcosms and gas chromatography of oil samples indicated the fast and intense depletion of linear alkanes as well as high oxygen consumption within 1 week followed by consequent slower degradation of branched and polyaromatic hydrocarbons., Conclusion: Under given conditions, A. borkumensis was an essential organism for biodegradation, dominating the biofilm microbial community formation and was the reason of emulsification., Significance and Impact of the Study: This study strongly emphasizes the pivotal importance of A. borkumensis as an essential organism in the initial steps of marine hydrocarbon degradation. Interaction with the sorbent material X-Oil proved to be neutral to beneficial for biodegradation and also promoted the growth of yet unknown micro-organisms.

An experimental prototype oil boom including oil sorbents, slow-release fertilizers and biomass of the marine oil-degrading bacterium, Alcanivorax borkumensis, was applied for sorption and degradation of heavy fuel oil in a 500-L mesocosm experiment. Fingerprinting of DNA and small subunit rRNA samples for microbial activity conducted to study the changes in microbial communities of both the water body and on the oil sorbent surface showed the prevalence of A. borkumensis on the surface of the oil sorbent. Growth of this obligate oil-degrading bacterium on immobilized oil coincided with a 30-fold increase in total respiration. A number of DNA and RNA signatures of aromatic hydrocarbon-degrading bacteria were detected both in samples of water body and on oil sorbent. Ultimately, the heavy fuel oil in this mesocosm study was effectively removed from the water body. This is the first study to successfully investigate the fate of oil-degrading microbial consortia in an experimental prototype for a bioremediation strategy in offshore, coastal or ship-bound oil spill mitigation using a combination of mechanical and biotechnological techniques.

A strictly anaerobic, Gram-positive, short-rod/coccobacillus-shaped bacterial strain, designated 7-10-1-b(T), was isolated from the colon of a patient suffering from acute Crohn's disease. The isolate formed small, pale-white, semi-translucent colonies on solid cultivation media. The strain was catalase-positive and metabolized only a small number of carbon sources. Whole-cell fatty acids consisted predominantly of saturated fatty acids (89 %), of which 15 : 0 anteiso was the major component. The polar lipids phosphatidylglycerol and diphosphatidylglycerol as well as four glycolipids were identified. 16S rRNA gene sequence analysis revealed that the isolate represents a distinct lineage within the family Coriobacteriaceae and has 94.6 % identity to the type strain of [Eggerthella] hongkongensis, the phylogenetically closest bacterial species. On the basis of the analyses performed, the new genus and species Gordonibacter pamelaeae gen. nov., sp. nov. is described, with strain 7-10-1-b(T) (=DSM 19378(T) =CCUG 55131(T)) as the type and only strain of Gordonibacter pamelaeae. Also, based on the chemotaxonomic data obtained for all type strains of the neighbouring genus Eggerthella, we propose that Eggerthella hongkongensis Lau et al. 2006 be transferred to a new genus as Paraeggerthella hongkongensis gen. nov., comb. nov.; the type strain of Paraeggerthella hongkongensis is HKU10(T) (=DSM 16106(T) =CCUG 49250(T)).

Molecular signatures of new, as yet uncultured mollicute-like organisms (MLOs) have been detected in total rRNA and DNA extracted from tissues, gut contents and casts of four species of the earthworm family Lumbricidae. The MLO 16S rRNA sequences exhibited low identity to those of known Mollicutes species and formed a monophyletic cluster distantly affiliated to the 'Candidatus Bacilloplasma' (84.9%) and almost equidistant to the other main phylogenetic group of Mollicutes (< 79.8%) and the classes Bacilli (< 79.5%) and Clostridia (< 76.1%). SSCP profiling and sequence analysis of bands and bacterial clones derived from the earthworms and substrata revealed high phylogenetic relatedness of MLOs in earthworms from different geographic locations (Russia and Germany), with no obvious host species specificity being observed. Fluorescence in situ hybridization (FISH) analysis with a nucleotide probe specific for the new MLO group localized them to the coelomic fluids of earthworms. A new taxonomic group within the Mollicutes, designated 'Candidatus Lumbricincola', is proposed to include these as yet uncultured organisms.