Comparison of microbial communities and the profile of sulfate-reducing bacteria in patients with ulcerative colitis and their association with bowel diseases: a pilot study.

Considerable evidence has accumulated regarding the molecular relationship between gut microbiota (GM) composition and the onset (clinical presentation and prognosis of ulcerative colitis (UC)). In addition, it is well documented that short-chain fatty acid (SCFA)-producing bacteria may play a fundamental role in maintaining an anti-inflammatory intestinal homeostasis, but sulfate- and sulfite reducing bacteria may be responsible for the production of toxic metabolites, such as hydrogen sulfide and acetate. Hence, the present study aimed to assess the GM composition - focusing on sulfate-reducing bacteria (SRB) - in patients with severe, severe-active and moderate UC. Each one of the six enrolled patients provided two stool samples in the following way: one sample was cultivated in a modified SRB-medium before 16S rRNA sequencing and the other was not cultivated. Comparative phylogenetic analysis was conducted on each sample. Percentage of detected gut microbial genera showed considerable variation based on the patients' disease severity and cultivation in the SRB medium. In detail, samples without cultivation from patients with moderate UC showed a high abundance of the genera Bacteroides, Bifidobacterium and Ruminococcus, but after SRB cultivation, the dominant genera were Bacteroides, Klebsiella and Bilophila. On the other hand, before SRB cultivation, the main represented genera in patients with severe UC were Escherichia-Shigella, Proteus, Methanothermobacter and Methanobacterium. However, after incubation in the SRB medium Bacteroides, Proteus, Alistipes and Lachnoclostridium were predominant. Information regarding GM compositional changes in UC patients may aid the development of novel therapeutic strategies (e.g., probiotic preparations containing specific bacterial strains) to counteract the mechanisms of virulence of harmful bacteria and the subsequent inflammatory response that is closely related to the pathogenesis of inflammatory bowel diseases.

Bafilomycin A1 Molecular Effect on ATPase Activity of Subcellular Fraction of Human Colorectal Cancer and Rat Liver.

Bafilomycin A1 inhibits V-type H+ ATPases on the molecular level, which acidifies endo-lysosomes. The main objective of the study was to assess the effect of bafilomycin A1 on Ca2+ content, NAADP-induced Ca2+ release, and ATPase activity in rat hepatocytes and human colon cancer samples. Chlortetracycline (CTC) was used for a quantitative measure of stored calcium in permeabilized rat hepatocytes. ATPase activity was determined by orthophosphate content released after ATP hydrolysis in subcellular post-mitochondrial fraction obtained from rat liver as well as from patients' samples of colon mucosa and colorectal cancer samples. In rat hepatocytes, bafilomycin A1 decreased stored Ca2+ and prevented the effect of NAADP on stored Ca2+. This effect was dependent on EGTA-Ca2+ buffers in the medium. Bafilomycin A1 significantly increased the activity of Ca2+ ATPases of endoplasmic reticulum (EPR), but not plasma membrane (PM) Ca2+ ATPases in rat liver. Bafilomycin A1 also prevented the effect of NAADP on these pumps. In addition, bafilomycin A1 reduced Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in the subcellular fraction of rat liver. Concomitant administration of bafilomycin A1 and NAADP enhanced these effects. Bafilomycin A1 increased the activity of the Ca2+ ATPase of EPR in the subcellular fraction of normal human colon mucosa and also in colon cancer tissue samples. In contrast, it decreased Ca2+ ATPase PM activity in samples of normal human colon mucosa and caused no changes in colon cancer. Bafilomycin A1 decreased Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in normal colon mucosa samples and in human colon cancer samples. It can be concluded that bafilomycin A1 targets NAADP-sensitive acidic Ca2+ stores, effectively modulates ATPase activity, and assumes the link between acidic stores and EPR. Bafilomycin A1 may be useful for cancer therapy.

NADH and NADPH peroxidases as antioxidant defense mechanisms in intestinal sulfate-reducing bacteria.

Animal and human feces typically include intestinal sulfate-reducing bacteria (SRB). Hydrogen sulfide and acetate are the end products of their dissimilatory sulfate reduction and may create a synergistic effect. Here, we report NADH and NADPH peroxidase activities from intestinal SRB Desulfomicrobium orale and Desulfovibrio piger. We sought to compare enzymatic activities under the influence of various temperature and pH regimes, as well as to carry out kinetic analyses of enzymatic reaction rates, maximum amounts of the reaction product, reaction times, maximum rates of the enzyme reactions, and Michaelis constants in cell-free extracts of intestinal SRB, D. piger Vib-7, and D. orale Rod-9, collected from exponential and stationary growth phases. The optimal temperature (35 °C) and pH (7.0) for both enzyme's activity were determined. The difference in trends of Michaelis constants (Km) during exponential and stationary phases are noticeable between D. piger Vib-7 and D. orale Rod-9; D. orale Rod-9 showed much higher Km (the exception is NADH peroxidase of D. piger Vib-7: 1.42 ± 0.11 mM) during the both monitored phases. Studies of the NADH and NADPH peroxidases-as putative antioxidant defense systems of intestinal SRB and detailed data on the kinetic properties of this enzyme, as expressed by the decomposition of hydrogen peroxide-could be important for clarifying evolutionary mechanisms of antioxidant defense systems, their etiological role in the process of dissimilatory sulfate reduction, and their possible role in the development of bowel diseases.

Eight Up-Coming Biotech Tools to Combat Climate Crisis.

Biotechnology has a high potential to substantially contribute to a low-carbon society. Several green processes are already well established, utilizing the unique capacity of living cells or their instruments. Beyond that, the authors believe that there are new biotechnological procedures in the pipeline which have the momentum to add to this ongoing change in our economy. Eight promising biotechnology tools were selected by the authors as potentially impactful game changers: (i) the Wood-Ljungdahl pathway, (ii) carbonic anhydrase, (iii) cutinase, (iv) methanogens, (v) electro-microbiology, (vi) hydrogenase, (vii) cellulosome and, (viii) nitrogenase. Some of them are fairly new and are explored predominantly in science labs. Others have been around for decades, however, with new scientific groundwork that may rigorously expand their roles. In the current paper, the authors summarize the latest state of research on these eight selected tools and the status of their practical implementation. We bring forward our arguments on why we consider these processes real game changers.

Analysis of biomass productivity and physiology of Nitrososphaera viennensis grown in continuous culture.

Microbial ammonia oxidation is the first and usually rate limiting step in nitrification and is therefore an important step in the global nitrogen cycle. Ammonia-oxidizing archaea (AOA) play an important role in nitrification. Here, we report a comprehensive analysis of biomass productivity and the physiological response of Nitrososphaera viennensis to different ammonium and carbon dioxide (CO2) concentrations aiming to understand the interplay between ammonia oxidation and CO2 fixation of N. viennensis. The experiments were performed in closed batch in serum bottles as well as in batch, fed-batch, and continuous culture in bioreactors. A reduced specific growth rate (µ) of N. viennensis was observed in batch systems in bioreactors. By increasing CO2 gassing µ could be increased to rates comparable to that of closed batch systems. Furthermore, at a high dilution rate (D) in continuous culture (≥ 0.7 of µmax) the biomass to ammonium yield (Y(X/NH3)) increased up to 81.7% compared to batch cultures. In continuous culture, biofilm formation at higher D prevented the determination of D crit. Due to changes in Y(X/NH3) and due to biofilm, nitrite concentration becomes an unreliable proxy for the cell number in continuous cultures at D towards µmax. Furthermore, the obscure nature of the archaeal ammonia oxidation prevents an interpretation in the context of Monod kinetics and thus the determination of K S. Our findings indicate that the physiological response of N. viennensis might be regulated with different enzymatic make-ups, according to the ammonium catalysis rate. We reveal novel insights into the physiology of N. viennensis that are important for biomass production and the biomass yield of AOA. Moreover, our study has implications to the field of archaea biology and microbial ecology by showing that bioprocess technology and quantitative analysis can be applied to decipher environmental factors affecting the physiology and productivity of AOA.

Lipidomics and Comparative Metabolite Excretion Analysis of Methanogenic Archaea Reveal Organism-Specific Adaptations to Varying Temperatures and Substrate Concentrations.

Methanogenic archaea possess diverse metabolic characteristics and are an ecologically and biotechnologically important group of anaerobic microorganisms. Although the scientific and biotechnological value of methanogens is evident with regard to their methane-producing physiology, little is known about their amino acid excretion, and virtually nothing is known about the lipidome at different substrate concentrations and temperatures on a quantitative comparative basis. Here, we present the lipidome and a comprehensive quantitative analysis of proteinogenic amino acid excretion as well as methane, water, and biomass production of the three autotrophic, hydrogenotrophic methanogens Methanothermobacter marburgensis, Methanothermococcus okinawensis, and Methanocaldococcus villosus under varying temperatures and nutrient supplies. The patterns and rates of production of excreted amino acids and the lipidome are unique for each tested methanogen and can be modulated by varying the incubation temperature and substrate concentration, respectively. Furthermore, the temperature had a significant influence on the lipidomes of the different archaea. The water production rate was much higher, as anticipated from the rate of methane production for all studied methanogens. Our results demonstrate the need for quantitative comparative physiological studies connecting intracellular and extracellular constraints of organisms to holistically investigate microbial responses to environmental conditions. IMPORTANCE Biological methane production by methanogenic archaea has been well studied for biotechnological purposes. This study reveals that methanogenic archaea actively modulate their lipid inventory and proteinogenic amino acid excretion pattern in response to environmental changes and the possible utilization of methanogenic archaea as microbial cell factories for the targeted production of lipids and amino acids.

Actin cytoskeleton and complex cell architecture in an Asgard archaeon.

Asgard archaea are considered to be the closest known relatives of eukaryotes. Their genomes contain hundreds of eukaryotic signature proteins (ESPs), which inspired hypotheses on the evolution of the eukaryotic cell1-3. A role of ESPs in the formation of an elaborate cytoskeleton and complex cellular structures has been postulated4-6, but never visualized. Here we describe a highly enriched culture of 'Candidatus Lokiarchaeum ossiferum', a member of the Asgard phylum, which thrives anaerobically at 20 °C on organic carbon sources. It divides every 7-14 days, reaches cell densities of up to 5 × 107 cells per ml and has a significantly larger genome compared with the single previously cultivated Asgard strain7. ESPs represent 5% of its protein-coding genes, including four actin homologues. We imaged the enrichment culture using cryo-electron tomography, identifying 'Ca. L. ossiferum' cells on the basis of characteristic expansion segments of their ribosomes. Cells exhibited coccoid cell bodies and a network of branched protrusions with frequent constrictions. The cell envelope consists of a single membrane and complex surface structures. A long-range cytoskeleton extends throughout the cell bodies, protrusions and constrictions. The twisted double-stranded architecture of the filaments is consistent with F-actin. Immunostaining indicates that the filaments comprise Lokiactin-one of the most highly conserved ESPs in Asgard archaea. We propose that a complex actin-based cytoskeleton predated the emergence of the first eukaryotes and was a crucial feature in the evolution of the Asgard phylum by scaffolding elaborate cellular structures.

Scale-up of biomass production by Methanococcus maripaludis.

The development of a sustainable energy economy is one of the great challenges in the current times of climate crisis and growing energy demands. Industrial production of the fifth-generation biofuel methane by microorganisms has the potential to become a crucial biotechnological milestone of the post fossil fuel era. Therefore, reproducible cultivation and scale-up of methanogenic archaea (methanogens) is essential for enabling biomass generation for fundamental studies and for defining peak performance conditions for bioprocess development. This study provides a comprehensive revision of established and optimization of novel methods for the cultivation of the model organism Methanococcus maripaludis S0001. In closed batch mode, 0.05 L serum bottles cultures were gradually replaced by 0.4 L Schott bottle cultures for regular biomass generation, and the time for reaching peak optical density (OD578) values was reduced in half. In 1.5 L reactor cultures, various agitation, harvesting and transfer methods were compared resulting in a specific growth rate of 0.16 h-1 and the highest recorded OD578 of 3.4. Finally, a 300-fold scale-up from serum bottles was achieved by growing M. maripaludis for the first time in a 22 L stainless steel bioreactor with 15 L working volume. Altogether, the experimental approaches described in this study contribute to establishing methanogens as essential organisms in large-scale biotechnology applications, a crucial stage of an urgently needed industrial evolution toward sustainable biosynthesis of energy and high value products.

Isolation and Characterization of Cell Envelope Fragments Comprising Archaeal S-Layer Proteins.

The outermost component of cell envelopes of most bacteria and almost all archaea comprise a protein lattice, which is termed Surface (S-)layer. The S-layer lattice constitutes a highly porous structure with regularly arranged pores in the nm-range. Some archaea thrive in extreme milieus, thus producing highly stable S-layer protein lattices that aid in protecting the organisms. In the present study, fragments of the cell envelope from the hyperthermophilic acidophilic archaeon Saccharolobus solfataricus P2 (SSO) have been isolated by two different methods and characterized. The organization of the fragments and the molecular sieving properties have been elucidated by transmission electron microscopy and by determining the retention efficiency of proteins varying in size, respectively. The porosity of the archaeal S-layer fragments was determined to be 45%. S-layer fragments of SSO showed a retention efficiency of up to 100% for proteins having a molecular mass of ≥ 66 kDa. Moreover, the extraction costs for SSO fragments have been reduced by more than 80% compared to conventional methods, which makes the use of these archaeal S-layer material economically attractive.

Quantitative Analysis of Core Lipid Production in Methanothermobacter marburgensis at Different Scales.

Archaeal lipids have a high biotechnological potential, caused by their high resistance to oxidative stress, extreme pH values and temperatures, as well as their ability to withstand phospholipases. Further, methanogens, a specific group of archaea, are already well-established in the field of biotechnology because of their ability to use carbon dioxide and molecular hydrogen or organic substrates. In this study, we show the potential of the model organism Methanothermobacter marburgensis to act both as a carbon dioxide based biological methane producer and as a potential supplier of archaeal lipids. Different cultivation settings were tested to gain an insight into the optimal conditions to produce specific core lipids. The study shows that up-scaling at a constant particle number (n/n = const.) seems to be a promising approach. Further optimizations regarding the length and number of the incubation periods and the ratio of the interaction area to the total liquid volume are necessary for scaling these settings for industrial purposes.

No Correlation between Biofilm-Forming Capacity and Antibiotic Resistance in Environmental Staphylococcus spp.: In Vitro Results.

The production of biofilms is a critical factor in facilitating the survival of Staphylococcus spp. in vivo and in protecting against various environmental noxa. The possible relationship between the antibiotic-resistant phenotype and biofilm-forming capacity has raised considerable interest. The purpose of the study was to assess the interdependence between biofilm-forming capacity and the antibiotic-resistant phenotype in 299 Staphylococcus spp. (S. aureus n = 143, non-aureus staphylococci [NAS] n = 156) of environmental origin. Antimicrobial susceptibility testing and detection of methicillin resistance (MR) was performed. The capacity of isolates to produce biofilms was assessed using Congo red agar (CRA) plates and a crystal violet microtiter-plate-based (CV-MTP) method. MR was identified in 46.9% of S. aureus and 53.8% of NAS isolates (p > 0.05), with resistance to most commonly used drugs being significantly higher in MR isolates compared to methicillin-susceptible isolates. Resistance rates were highest for clindamycin (57.9%), erythromycin (52.2%) and trimethoprim-sulfamethoxazole (51.1%), while susceptibility was retained for most last-resort drugs. Based on the CRA plates, biofilm was produced by 30.8% of S. aureus and 44.9% of NAS (p = 0.014), while based on the CV-MTP method, 51.7% of S. aureus and 62.8% of NAS were identified as strong biofilm producers, respectively (mean OD570 values: S. aureus: 0.779±0.471 vs. NAS: 1.053±0.551; p < 0.001). No significant differences in biofilm formation were observed based on MR (susceptible: 0.824 ± 0.325 vs. resistant: 0.896 ± 0.367; p = 0.101). However, pronounced differences in biofilm formation were identified based on rifampicin susceptibility (S: 0.784 ± 0.281 vs. R: 1.239 ± 0.286; p = 0.011). The mechanistic understanding of the mechanisms Staphylococcus spp. use to withstand harsh environmental and in vivo conditions is crucial to appropriately address the therapy and eradication of these pathogens.

The Historical Development of Cultivation Techniques for Methanogens and Other Strict Anaerobes and Their Application in Modern Microbiology.

The cultivation and investigation of strictly anaerobic microorganisms belong to the fields of anaerobic microbial physiology, microbiology, and biotechnology. Anaerobic cultivation methods differ from classic microbiological techniques in several aspects. The requirement for special instruments, which are designed to prevent the contact of the specimen with air/molecular oxygen by different means of manipulation, makes this field more challenging for general research compared to working with aerobic microorganisms. Anaerobic microbiological methods are required for many purposes, such as for the isolation and characterization of new species and their physiological examination, as well as for anaerobic biotechnological applications or medical indications. This review presents the historical development of methods for the cultivation of strictly anaerobic microorganisms focusing on methanogenic archaea, anaerobic cultivation methods that are still widely used today, novel methods for anaerobic cultivation, and almost forgotten, but still relevant, techniques.

Comparison of Carbonic Anhydrases for CO2 Sequestration.

Strategies for depleting carbon dioxide (CO2) from flue gases are urgently needed and carbonic anhydrases (CAs) can contribute to solving this problem. They catalyze the hydration of CO2 in aqueous solutions and therefore capture the CO2. However, the harsh conditions due to varying process temperatures are limiting factors for the application of enzymes. The current study aims to examine four recombinantly produced CAs from different organisms, namely CAs from Acetobacterium woodii (AwCA or CynT), Persephonella marina (PmCA), Methanobacterium thermoautotrophicum (MtaCA or Cab) and Sulphurihydrogenibium yellowstonense (SspCA). The highest expression yields and activities were found for AwCA (1814 WAU mg-1 AwCA) and PmCA (1748 WAU mg-1 PmCA). AwCA was highly stable in a mesophilic temperature range, whereas PmCA proved to be exceptionally thermostable. Our results indicate the potential to utilize CAs from anaerobic microorganisms to develop CO2 sequestration applications.

Molecular Physiology of Anaerobic Phototrophic Purple and Green Sulfur Bacteria.

There are two main types of bacterial photosynthesis: oxygenic (cyanobacteria) and anoxygenic (sulfur and non-sulfur phototrophs). Molecular mechanisms of photosynthesis in the phototrophic microorganisms can differ and depend on their location and pigments in the cells. This paper describes bacteria capable of molecular oxidizing hydrogen sulfide, specifically the families Chromatiaceae and Chlorobiaceae, also known as purple and green sulfur bacteria in the process of anoxygenic photosynthesis. Further, it analyzes certain important physiological processes, especially those which are characteristic for these bacterial families. Primarily, the molecular metabolism of sulfur, which oxidizes hydrogen sulfide to elementary molecular sulfur, as well as photosynthetic processes taking place inside of cells are presented. Particular attention is paid to the description of the molecular structure of the photosynthetic apparatus in these two families of phototrophs. Moreover, some of their molecular biotechnological perspectives are discussed.

SepF is the FtsZ anchor in archaea, with features of an ancestral cell division system.

Most archaea divide by binary fission using an FtsZ-based system similar to that of bacteria, but they lack many of the divisome components described in model bacterial organisms. Notably, among the multiple factors that tether FtsZ to the membrane during bacterial cell constriction, archaea only possess SepF-like homologs. Here, we combine structural, cellular, and evolutionary analyses to demonstrate that SepF is the FtsZ anchor in the human-associated archaeon Methanobrevibacter smithii. 3D super-resolution microscopy and quantitative analysis of immunolabeled cells show that SepF transiently co-localizes with FtsZ at the septum and possibly primes the future division plane. M. smithii SepF binds to membranes and to FtsZ, inducing filament bundling. High-resolution crystal structures of archaeal SepF alone and in complex with the FtsZ C-terminal domain (FtsZCTD) reveal that SepF forms a dimer with a homodimerization interface driving a binding mode that is different from that previously reported in bacteria. Phylogenetic analyses of SepF and FtsZ from bacteria and archaea indicate that the two proteins may date back to the Last Universal Common Ancestor (LUCA), and we speculate that the archaeal mode of SepF/FtsZ interaction might reflect an ancestral feature. Our results provide insights into the mechanisms of archaeal cell division and pave the way for a better understanding of the processes underlying the divide between the two prokaryotic domains.

Anoxygenic Photosynthesis in Photolithotrophic Sulfur Bacteria and Their Role in Detoxication of Hydrogen Sulfide.

Hydrogen sulfide is a toxic compound that can affect various groups of water microorganisms. Photolithotrophic sulfur bacteria including Chromatiaceae and Chlorobiaceae are able to convert inorganic substrate (hydrogen sulfide and carbon dioxide) into organic matter deriving energy from photosynthesis. This process takes place in the absence of molecular oxygen and is referred to as anoxygenic photosynthesis, in which exogenous electron donors are needed. These donors may be reduced sulfur compounds such as hydrogen sulfide. This paper deals with the description of this metabolic process, representatives of the above-mentioned families, and discusses the possibility using anoxygenic phototrophic microorganisms for the detoxification of toxic hydrogen sulfide. Moreover, their general characteristics, morphology, metabolism, and taxonomy are described as well as the conditions for isolation and cultivation of these microorganisms will be presented.

Microscopic Methods for Identification of Sulfate-Reducing Bacteria from Various Habitats.

This paper is devoted to microscopic methods for the identification of sulfate-reducing bacteria (SRB). In this context, it describes various habitats, morphology and techniques used for the detection and identification of this very heterogeneous group of anaerobic microorganisms. SRB are present in almost every habitat on Earth, including freshwater and marine water, soils, sediments or animals. In the oil, water and gas industries, they can cause considerable economic losses due to their hydrogen sulfide production; in periodontal lesions and the colon of humans, they can cause health complications. Although the role of these bacteria in inflammatory bowel diseases is not entirely known yet, their presence is increased in patients and produced hydrogen sulfide has a cytotoxic effect. For these reasons, methods for the detection of these microorganisms were described. Apart from selected molecular techniques, including metagenomics, fluorescence microscopy was one of the applied methods. Especially fluorescence in situ hybridization (FISH) in various modifications was described. This method enables visual identification of SRB, determining their abundance and spatial distribution in environmental biofilms and gut samples.

Hyperthermophilic methanogenic archaea act as high-pressure CH4 cell factories.

Bioprocesses converting carbon dioxide with molecular hydrogen to methane (CH4) are currently being developed to enable a transition to a renewable energy production system. In this study, we present a comprehensive physiological and biotechnological examination of 80 methanogenic archaea (methanogens) quantifying growth and CH4 production kinetics at hyperbaric pressures up to 50 bar with regard to media, macro-, and micro-nutrient supply, specific genomic features, and cell envelope architecture. Our analysis aimed to systematically prioritize high-pressure and high-performance methanogens. We found that the hyperthermophilic methanococci Methanotorris igneus and Methanocaldococcoccus jannaschii are high-pressure CH4 cell factories. Furthermore, our analysis revealed that high-performance methanogens are covered with an S-layer, and that they harbour the amino acid motif Tyrα444 Glyα445 Tyrα446 in the alpha subunit of the methyl-coenzyme M reductase. Thus, high-pressure biological CH4 production in pure culture could provide a purposeful route for the transition to a carbon-neutral bioenergy sector.

Intestinal Microbiota and Perspectives of the Use of Meta-Analysis for Comparison of Ulcerative Colitis Studies.

Meta-analysis is a statistical process summarizing comparable data from a number of scientific papers. The use of meta-analysis in microbiology allows decision-making that has an impact on public health policy. It can happen that the primary researches come to different conclusions, although these are targeted with the same research question. It is, therefore, inevitable to have the means to systematically evaluate information and compare research results. Ulcerative colitis together with Crohn's disease are among the two main inflammatory bowel diseases. This chronic disease of the gastrointestinal tract, with an as yet unclear etiology, is presented by an uncontrolled inflammatory immune response in genetically predisposed individuals to as yet undefined environmental factors in interaction with the intestinal microbiota itself. In patients with ulcerative colitis (UC), changes in the composition and relative abundance of microorganisms could be observed. Sulfate-reducing bacteria (SRB), which commonly occur in the large intestine as part of the commensal microbiota of animals and humans involved in the pathogenesis of the disease, have been shown to occur. SRB are anaerobic organisms affecting short-chain fatty acid metabolism. This work outlines the perspectives of the use of meta-analysis for UC and changes in the representation of intestinal organisms in these patients.