Anaerobic Clostridium Research Articles

Diverse YqeK Diadenosine Tetraphosphate Hydrolases Control Biofilm Formation in an Iron-Dependent Manner

YqeK is a bacterial HD-domain metalloprotein that hydrolyzes the putative second messenger diadenosine tetraphosphate (Ap4A). Elevated Ap4A levels are primarily observed upon exposure of bacteria to factors such as heat or oxidative stress and cause pleiotropic effects, including antibiotic sensitivity and disrupted biofilm formation. Ap4A thus plays a central role in bacterial physiology and metabolism, and its hydrolysis by YqeK is intimately linked to the ability of these microbes to cope with stress. Although YqeK is reported to hydrolyze Ap4A under aerobic conditions, all four existing crystal structures reveal an active site that consists of a diiron center, portraying a cryptic chemical nature for the active metallocofactor. This study examines two YqeK proteins from two ecologically diverse parent organisms: the obligate anaerobe Clostridium acetobutylicum and the facultative aerobe Bacillus halodurans. Both enzymes utilize Fe-based cofactors for catalysis, while under ambient or oxidative conditions, Bh YqeK hydrolyzes Ap4A more efficiently compared to Ca YqeK. This redox-dependent activity difference stems from the following two molecular mechanisms: the incorporation of mixed-metal, Fe-based bimetallic cofactors, in which the second metal is redox inert (i.e., Fe–Zn) and the upshift of the Fe–Fe cofactor reduction potentials. In addition, three strictly conserved, positively charged residues vicinal to the active site are critical for tuning Ap4A hydrolysis. In conclusion, YqeK is an Fe-dependent phosphohydrolase that appears to have evolved to permit Ap4A hydrolysis under different environmental niches (aerobic vs. anaerobic) by expanding its cofactor configuration and O2 tolerance.

Isolation and characterization of a strain Clostridium butyricum B3 from the intestine of Pelteobagrus fulvidraco and its potential probiotic roles

The use of host-associated probiotics in aquaculture have been limited due to difficulties in their isolation from the host gastrointestinal tract and validation of their biological function. Here, anaerobic Clostridium butyricum strain B3 was isolated for the first time from the intestines of yellow catfish (Pelteobagrus fulvidraco). Alignment of 16 S rDNA sequences revealed 99 % identify between strain B3 and wild-type C. butyricum. C. butyricum B3 was found to tolerate a wide range of temperature (up to 80 °C) and pH (1 to 13) levels. In vitro microbial growth experiments revealed that C. butyricum B3 produced acetic acid (890.00 ± 23.00 mg/L) and butyric acid (672.67 ± 51.67 mg/L) highly productively within 24 h. Further in vitro characterization findings also suggested that C. butyricum B3 is sensitive to several antibiotics, including ampicillin and chloramphenicol, and potently inhibits the growth of pathogenic bacteria such as Aeromonas hydrophila. In a feeding experiments, 30 days of dietary supplementation with 3.0 × 107 to 109 CFU/g B3 significantly increased the specific growth rates of yellow catfish juveniles by more than 61.5 %. Activities of trypsin, α-amylase and lipase were significantly enhanced in group fed with 3.0 × 107 B3 compared to the control group (P < 0.05). Furthermore, ACP activity was significantly increased by more than 25.0 %, whereas malondialdehyde content significantly decreased by at least 38.5 % in all B3 feeding groups (P < 0.05). In addition, hepatocyte density increased, and goblet cells in the intestinal epithelial cells expanded. Meanwhile, C. butyricum B3 feeding significantly activated genes (such as grhpr and enpp2) involved in lipid metabolism and proliferator-activated receptor signaling pathway, increased bacterial diversity, and reduced the bacterial richness of the gut microbiota (P < 0.05). In conclusion, dietary inclusion of 3.0 × 107 CFU/g of C. butyricum B3 enhanced the growth performance, immune response, and lipid metabolism of P. fulvidraco.

Gallic acid as biofilm inhibitor can improve transformation efficiency of Ruminiclostridium papyrosolvens.

Ruminiclostridium papyrosolvens is an anaerobic, mesophilic, and cellulolytic clostridia, promising consolidated bioprocessing (CBP) candidate for producing renewable green chemicals from cellulose, but its genetic transformation has been severely impeded by extracellular biofilm. Here, we analyzed the effects of five different inhibitors with gradient concentrations on R. papyrosolvens growth and biofilm formation. Gallic acid was proved to be a potent inhibitor of biofilm synthesis of R. papyrosolvens. Furthermore, the transformation efficiency of R. papyrosolvens was significantly increased when the cells were treated by the gallic acid, and the mutant strain was successfully obtained by the improved transformation method. Thus, inhibition of biofilm formation of R. papyrosolvens by using gallic acid will contribute to its genetic transformation and efficient metabolic engineering.

An efficient cre-based workflow for genomic integration and expression of large biosynthetic pathways in Eubacterium limosum.

Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single-carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis-independent recombinase-assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum. We then integrated a non-ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum, which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.

Botulinum toxin: a new differential diagnosis for a lytic bone lesion

BackgroundBotulinum toxin, produced by the Gram-positive anaerobe Clostridium botulinum, is composed of seven antigenic subtypes (A, B, C, D, E, F, and G). Currently, only Botulinum toxin type A, commonly referred to as “Botox,” is approved for clinical use, given its relatively safe clinical profile. Botulinum toxin type A has a wide range of therapeutic indications, including treatment for dystonia, migraine headache, neurogenic bladder, and large muscle spastic disorders. However, the toxin is most widely known for its cosmetic effects in treating wrinkles and facial lines.Case presentationThis article describes a 62-year-old Caucasian female who presented for investigation and workup of an isolated lytic lesion of her frontal bone a few weeks after administration of botulinum toxin injection into the corresponding site in the frontalis muscle. This presented as a large, palpable, painless forehead lump causing significant psychological distress. After no neoplastic cause for the lesion was found and histopathology was performed, our researchers concluded that the most likely explanation was that the bony lytic lesion resulted from inadvertent injection of the “Botox” neurotoxin through the intended target muscle and into the cortex of the underlying bone.ConclusionsOur search of the literature failed to identify any previous cases of this occurring. However, as the popularity of this cosmetic procedure only increases, we believe that this represents an important possible differential for isolated lytic lesion after administration of “Botox” injection.

Genetic disruption of the bacterial raiA motif noncoding RNA causes defects in sporulation and aggregation

Several structured noncoding RNAs in bacteria are essential contributors to fundamental cellular processes. Thus, discoveries of additional ncRNA classes provide opportunities to uncover and explore biochemical mechanisms relevant to other major and potentially ancient processes. A candidate structured ncRNA named the "raiA motif" has been found via bioinformatic analyses in over 2,500 bacterial species. The gene coding for the RNA typically resides between the raiA and comFC genes of many species of Bacillota and Actinomycetota. Structural probing of the raiA motif RNA from the Gram-positive anaerobe Clostridium acetobutylicum confirms key features of its sophisticated secondary structure model. Expression analysis of raiA motif RNA reveals that the RNA is constitutively produced but reaches peak abundance during the transition from exponential growth to stationary phase. The raiA motif RNA becomes the fourth most abundant RNA in C. acetobutylicum, excluding ribosomal RNAs and transfer RNAs. Genetic disruption of the raiA motif RNA causes cells to exhibit substantially decreased spore formation and diminished ability to aggregate. Restoration of normal cellular function in this knock-out strain is achieved by expression of a raiA motif gene from a plasmid. These results demonstrate that raiA motif RNAs normally participate in major cell differentiation processes by operating as a trans-acting factor.

A comparative assessment of microbiocenosis of saliva and oropharynx in patients with migraine

To identify changes in the microbiome of saliva and to compare it with the microbiome of the oropharynx of patients with migraine. Sixty patients with migraine (21-56 years old), were examined using a headache diary, MIDAS and VAS. A microbiological examination of saliva and smear from the mucosa of the posterior wall of the oropharynx with evaluation by the method of mass spectrometry of microbial markers (MSMM) with the determination of 57 microorganisms was performed. All patients had comorbid chronic diseases of the gastrointestinal tract and upper respiratory tract (URT), according to anamnestic data and examination by specialists. A significant increase in the content of markers of resident (conditionally pathogenic) microorganisms characteristic of chronic diseases of URT (strepto- and staphylococci); markers of transient microorganisms characteristic of intestinal microflora (clostridia, gram-negative rods, anaerobes) that are normally absent; viral markers of cytomegaloviruses and herpes groups; a decrease in the content of fungi were identified in saliva. A comparative analysis of the microbiome of saliva and oropharynx showed: 1) a significant decrease in the concentration of coccal flora Enterococcus spp., Streptococcus mutans, Staphylococcus aureus, anaerobic bacteria Clostridium difficile and Clostridium perfringens in saliva; enterobacteria Helicobacter pylori; gram-negative rods Kingella spp., fungi and Epstein-Barr virus; 2) an increase in salivary concentrations of Staphylococcus epidermidis, anaerobic Clostridium ramosum and Fusobacterium spp./Haemophilus spp. and gram-negative bacilli Porphyromonas spp. A comparative assessment of the microbiota of a smear from the posterior wall of the oropharynx and saliva using MMSM showed the presence of dysbiosis both in the oropharynx and in the saliva of patients with migraine. However, there were fewer deviations from the norm in saliva, therefore, for diagnostic purposes, a smear from the posterior wall of the oropharynx is more significant as a biomarker for patients with migraine.

Caproate production from Enset fiber in one-pot two-step fermentation using anaerobic fungi (Neocallimastix cameroonii strain G341) and Clostridium kluyveri DSM 555

BackgroundLignocellulosic biomass plays a crucial role in creating a circular bioeconomy and minimizing environmental impact. Enset biomass is a byproduct of traditional Ethiopian Enset food processing that is thrown away in huge quantities. This study aimed to produce caproate from Enset fiber using Neocallimastix cameroonii strain G341 and Clostridium kluyveri DSM 555 in one-pot two-step fermentation.ResultsThe process started by growing N. cameroonii on Enset fiber as a carbon source for 7 days. Subsequently, the fungal culture was inoculated with active C. kluyveri preculture and further incubated. The results showed that N. cameroonii grew on 0.25 g untreated Enset fiber as the sole carbon source and produced 1.16 mmol acetate, 0.51 mmol hydrogen, and 1.34 mmol formate. In addition, lactate, succinate, and ethanol were detected in small amounts, 0.17 mmol, 0.08 mmol, and 0.7 mmol, respectively. After inoculating with C. kluyveri, 0.3 mmol of caproate and 0.48 mmol of butyrate were produced, and hydrogen production also increased to 0.95 mmol compared to sole N. cameroonii fermentation. Moreover, after the culture was supplemented with 2.18 mmol of ethanol during C. kluyveri inoculation, caproate, and hydrogen production was further increased to 1.2 and 1.36 mmol, respectively, and the consumption of acetate also increased.ConclusionA novel microbial cell factory was developed to convert untreated lignocellulosic Enset fiber into the medium chain carboxylic acid caproate and H2 by a co-culture of the anaerobic fungi N. cameroonii and C. kluyveri. This opens a new value chain for Enset farmers, as the process requires only locally available raw materials and low-price fermenters. As the caproate production was mainly limited by the available ethanol, the addition of locally produced ethanol-containing fermentation broth (“beer”) would further increase the titer.Graphical

Identification, activity and delivery of new LysFA67 endolysin to target cheese spoilage Clostridium tyrobutyricum

Bacteriophages and their endolysins are potential biocontrol agents for the anaerobic spoilage organism Clostridium tyrobutyricum, which causes cheese late blowing defect. This study sequenced and compared the genomes of eight bacteriophages from Spanish dairy farms that were active against C. tyrobutyricum, to identify novel species and phage proteins. Phages vB_CtyS-FA67 and vB_CtyS-FA70 shared >94% intergenomic similarity to each other but neither phage had significant similarity to ΦCTP1, the unique C. tyrobutyricum phage sequenced to date. Taxonomic analysis indicated that both phages belong to the class Caudoviricetes and are related to dsDNA viruses with long non-contractile tails. vB_CtyS-FA67 had no other close relatives and encoded a novel endolysin, LysFA67, predicted to belong to the glycoside hydrolase GH24 family. LysFA67 lysed 93% of C. tyrobutyricum cells after 4 min in turbidity reduction assays, retaining lytic activity at pHs 4.2–8.1 and at 30–45 °C. The endolysin remained stable after 30 d storage at 4, 12 and 25 °C, while its activity decreased at −20 °C. LysFA67 lysed several clostridia species, while common dairy bacteria were not affected. Lactococcus lactis INIA 437, used as a cheese starter, was engineered to deliver LysFA67 and red fluorescent LysFA67-mCherry to dairy products. We demonstrated that these engineered strains were able to maintain lytic activity and fluorescence without affecting their technological properties in milk.

Cesin, a short natural variant of nisin, displays potent antimicrobial activity against major pathogens despite lacking two C-terminal macrocycles.

Nisin is a widely used lantibiotic owing to its potent antimicrobial activity and its food-grade status. Its mode of action includes cell wall synthesis inhibition and pore formation, which are attributed to the lipid II binding and pore-forming domains, respectively. We discovered cesin, a short natural variant of nisin, produced by the psychrophilic anaerobe Clostridium estertheticum. Unlike other natural nisin variants, cesin lacks the two terminal macrocycles constituting the pore-forming domain. The current study aimed at heterologous expression and characterization of the antimicrobial activity and physicochemical properties of cesin. Following the successful heterologous expression of cesin in Lactococcus lactis, the lantibiotic demonstrated a broad and potent antimicrobial profile comparable to that of nisin. Determination of its mode of action using lipid II and lipoteichoic acid binding assays linked the potent antimicrobial activity to lipid II binding and electrostatic interactions with teichoic acids. Fluorescence microscopy showed that cesin lacks pore-forming ability in its natural form. Stability tests have shown the lantibiotic is highly stable at different pH values and temperature conditions, but that it can be degraded by trypsin. However, a bioengineered analog, cesin R15G, overcame the trypsin degradation, while keeping full antimicrobial activity. This study shows that cesin is a novel (small) nisin variant that efficiently kills target bacteria by inhibiting cell wall synthesis without pore formation. IMPORTANCE The current increase in antibiotic-resistant pathogens necessitates the discovery and application of novel antimicrobials. In this regard, we recently discovered cesin, which is a short natural variant of nisin produced by the psychrophilic Clostridium estertheticum. However, its suitability as an antimicrobial compound was in doubt due to its structural resemblance to nisin(1-22), a bioengineered short variant of nisin with low antimicrobial activity. Here, we show by heterologous expression, purification, and characterization that the potency of cesin is not only much higher than that of nisin(1-22), but that it is even comparable to the full-length nisin, despite lacking two C-terminal rings that are essential for nisin's activity. We show that cesin is a suitable scaffold for bioengineering to improve its applicability, such as resistance to trypsin. This study demonstrates the suitability of cesin for future application in food and/or for health as a potent and stable antimicrobial compound.

Development of an Efficient Recombinant Protein Expression System in Clostridium saccharoperbutylacetonicum Based on the Bacteriophage T7 System.

Recombinant proteins have broad applications. However, there is a lack of a recombinant protein expression system specifically for large-scale production in anaerobic hosts. Here, we developed a powerful and stringently inducible protein expression system based on the bacteriophage T7 system in the strictly anaerobic solvent-producing Clostridium saccharoperbutylacetonicum. With the integration of a codon optimized T7 RNA polymerase into the chromosome, a single plasmid carrying a T7 promoter could efficiently drive high-level expression of the target gene in an orthogonal manner, which was tightly regulated by a lactose-inducible system. Furthermore, by deleting beta-galactosidase genes involved in lactose metabolism, the transcriptional strength was further improved. In the ultimately optimized strain TM-07, the transcriptional strength of the T7 promoter showed 9.5-fold increase compared to the endogenous strong promoter Pthl. The heterologous NADP+-dependent 3-hydroxybutyryl-CoA dehydrogenase (Hbd1) from C. kluyveri was expressed in TM-07, and the yield of the recombinant protein reached 30.4-42.4% of the total cellular protein, surpassing the strong protein expression systems in other Gram-positive bacteria. The relative activity of Hbd1 in the crude enzyme was 198.0 U/mg, which was 8.3-fold higher than the natural activity in C. kluyveri. The relative activity of the purified enzyme reached 467.4 U/mg. To the best of our knowledge, this study represents the first application of the T7 expression system in Clostridium species, and this optimized expression system holds great potential for large-scale endotoxin-free recombinant protein production under strictly anaerobic conditions. This development paves the way for significant advancements in biotechnology and opens up new avenues for industrial applications.

Severity of Clinical Mastitis and Bacterial Shedding.

The aim of this cross-sectional study was to investigate associated factors of the severity of clinical mastitis (CM). Milk samples of 249 cases of CM were microbiologically examined, of which 27.2% were mild, 38.5% moderate, and 34.3% severe mastitis. The samples were incubated aerobically and anaerobically to investigate the role of aerobic and anaerobic microorganisms. In addition, the pathogen shedding was quantitatively examined, and animal individual data, outside temperature and relative humidity, were collected to determine associated factors for the severity of CM. The pathogen isolated the most was Escherichia coli (35.2%), followed by Streptococcus spp. (16.4%). Non-aureus staphylococci (NaS) (15.4%) and other pathogens (e.g., Staphylococcus aureus, coryneforms) (15.4%) were the pathogens that were isolated the most for mild mastitis. Moderate mastitis was mostly caused by E. coli (38%). E. coli was also the most common pathogen in severe mastitis (50.6%), followed by Streptococcus spp. (16.4%), and Klebsiella spp. (10.3%). Obligate anaerobes (Clostridium spp.) were isolated in one case (0.4%) of moderate mastitis. The mortality rate (deceased or culled due to the mastitis in the following two weeks) was 34.5% for severe mastitis, 21.7% for moderate mastitis, and 4.4% for mild mastitis. The overall mortality rate of CM was 21.1%. The pathogen shedding (back logarithmized) was highest for severe mastitis (55,000 cfu/mL) and E. coli (91,200 cfu/mL). High pathogen shedding, low previous somatic cell count (SCC) before mastitis, high outside temperature, and high humidity were associated with severe courses of mastitis.

Composition and fate of heat-resistant anaerobic spore-formers in the milk powder production line

Anaerobic spore-forming bacteria are a continuous threat to the dairy industry due to their ability to withstand processing conditions, such as those during heat treatment. These ubiquitous microorganisms have ample opportunity for multiple entry points into the milk chain, creating food quality and safety issues. Certain spore-formers, namely bacilli and clostridia, are more problematic due to their ability to spoil dairy products and pathogenicity. In this study, we investigated how milk treatment and milk powder production influenced the composition and survival of anaerobic spore-formers. Samples were obtained on three different days (replicate blocks) during the production of dairy powders and examined in a culture-dependent manner using the most probable number method coupled with 16S rRNA amplicon sequencing and metagenomic analysis of the enriched samples. Results revealed that the milk separation greatly affected the spore-former presence and composition which were detected along the entire production line from raw material to milk powders. Throughout the various points of the production line, the occurrence of species belonging to the Bacillus cereus sensu lato was higher than that of clostridia. Sequence variants (SVs) belonging to the anaerobic spore-forming genus Clostridium were taxonomically assigned to two SVs groups and were detected in all three replicate blocks. A total of 19 metagenome-assembled genomes were recovered from nine enrichments. Four near-complete and two medium-quality genomes were found in raw milk/milk powder samples and further assigned as Clostridium tyrobutyricum and Clostridium diolis, which may constitute a problem in the finished dairy product. In conclusion, our findings highlight spore-formers' importance on dairy quality and may aid in their intervention and control in the dairy production line.

Assessment of correlation between clinical, radiographic, microbiological, and histopathological examinations in identification of pulpal diseases - a single-centre study

AimThis study aimed to analyse the presence of pulpitis using different techniques and compare the findings of the various examination methods. MethodsA total of 108 patients were enrolled and randomly divided into two groups: 56 patients whose pulp samples were sent for histopathological analysis and 52 patients whose samples were sent for microbiological analysis. All participants underwent endodontic procedures, with clinical evaluation and assessment using periapical radiography. Bacteria were isolated and identified using agar culture and VITEK 2 identification cards. ResultsHistopathology confirmed chronic pulpitis in 33 samples (58.9%) and acute pulpitis in 23 samples (41.1 %). For chronic pulpitis, the histopathological diagnosis agreed with the clinical evaluation diagnosis in 65.2% of cases, and a similar percentage of agreement was observed for acute pulpitis. Chronic pulpitis was observed in 34.8% of patients on clinical examination; however, according to histopathology, these cases were acute. Dilated blood vessels were detected in 56.5% of patients with acute pulpitis and 15.2% of patients with chronic pulpitis. Neutrophilic leucocytes were observed in 43.5% of patients with acute pulpitis and 69.7% of patients with chronic pulpitis. Lymphocytes were observed in 17.4% of acute pulpitis samples but zero chronic pulpitis samples. Microbiological analysis identified gram-positive bacilli in 22 samples, gram-positive cocci in 51 samples, and fungi in 2 samples. Acute pulpitis was typically found to be associated with anaerobic Clostridium bifermentans, aerobic Streptococcus mitis, and Granulicatella elegans, whereas chronic pulpitis was more often associated with two facultative anaerobes, Streptococcus oralis and Streptococcus mitis. ConclusionComparison of clinical, radiographic, and histological examination techniquesrevealed several notable discrepancies. Radiographic imaging only suggested the presence of pulpal pathologies; therefore, histopathological analysis of the pulp material was still ultimately required to verify the clinical diagnosis and exclude other pathologies. Although histopathology remains the gold standard for assessing pulpal disease, performing additional examinations may provide the most comprehensive, and perhaps the most effective, approach.

Mineral-Bound Trace Metals as Cofactors for Anaerobic Biological Nitrogen Fixation.

Nitrogenase is the only known biological enzyme capable of reducing N2 to bioavailable NH3. Most nitrogenases use Mo as a metallocofactor, while alternative cofactors V and Fe are also viable. Both geological and bioinformatic evidence suggest an ancient origin of Mo-based nitrogenase in the Archean, despite the low concentration of dissolved Mo in the Archean oceans. This apparent paradox would be resolvable if mineral-bound Mo were bioavailable for nitrogen fixation by ancient diazotrophs. In this study, the bioavailability of mineral-bound Mo, V, and Fe was determined by incubating an obligately anaerobic diazotroph Clostridium kluyveri with Mo-, V-, and Fe-bearing minerals (molybdenite, cavansite, and ferrihydrite, respectively) and basalt under diazotrophic conditions. The results showed that C. kluyveri utilized mineral-associated metals to express nitrogenase genes and fix nitrogen, as measured by the reverse transcription quantitative polymerase chain reaction and acetylene reduction assay, respectively. C. kluyveri secreted chelating molecules to extract metals from the minerals. As a result of microbial weathering, mineral surface chemistry significantly changed, likely due to surface coating by microbial exudates for metal extraction. These results provide important support for the ancient origin of Mo-based nitrogenase, with profound implications for coevolution of the biosphere and geosphere.

A detailed genome-scale metabolic model of Clostridium thermocellum investigates sources of pyrophosphate for driving glycolysis

Lignocellulosic biomass is an abundant and renewable source of carbon for chemical manufacturing, yet it is cumbersome in conventional processes. A promising, and increasingly studied, candidate for lignocellulose bioprocessing is the thermophilic anaerobe Clostridium thermocellum given its potential to produce ethanol, organic acids, and hydrogen gas from lignocellulosic biomass under high substrate loading. Possessing an atypical glycolytic pathway which substitutes GTP or pyrophosphate (PPi) for ATP in some steps, including in the energy-investment phase, identification, and manipulation of PPi sources are key to engineering its metabolism. Previous efforts to identify the primary pyrophosphate have been unsuccessful. Here, we explore pyrophosphate metabolism through reconstructing, updating, and analyzing a new genome-scale stoichiometric model for C. thermocellum, iCTH669. Hundreds of changes to the former GEM, iCBI655, including correcting cofactor usages, addressing charge and elemental balance, standardizing biomass composition, and incorporating the latest experimental evidence led to a MEMOTE score improvement to 94%. We found agreement of iCTH669 model predictions across all available fermentation and biomass yield datasets. The feasibility of hundreds of PPi synthesis routes, newly identified and previously proposed, were assessed through the lens of the iCTH669 model including biomass synthesis, tRNA synthesis, newly identified sources, and previously proposed PPi-generating cycles. In all cases, the metabolic cost of PPi synthesis is at best equivalent to investment of one ATP suggesting no direct energetic advantage for the cofactor substitution in C. thermocellum. Even though no unique source of PPi could be gleaned by the model, by combining with gene expression data two most likely scenarios emerge. First, previously investigated PPi sources likely account for most PPi production in wild-type strains. Second, alternate metabolic routes as encoded by iCTH669 can collectively maintain PPi levels even when previously investigated synthesis cycles are disrupted. Model iCTH669 is available at github.com/maranasgroup/iCTH669.

Biotechnological Approaches to Generate Biogenic Solvents and Energy Carriers from Renewable Resources

Abstract Background: Current threats connected to the ongoing depletion of fossil resources and elevated levels of greenhouse gases accelerating climate change and global warming provoke a renaissance of biotechnological production of various organic bulk chemicals, which, particularly during the second half of the 20th century, were almost exclusively produced from fossil resources via chemosynthetic processes. Scope: Besides the manufacture of bioethanol, a product obtained by microbial fermentation, biogenic production of solvents and energy carriers like acetone, isopropanol, 2,3-butanediol, or 1-butanol, hence, processes known since the beginning of the last century, experiences now a substantial revival. Summary of new synthesis and conclusions reached in the review: The review illustrates how to produce these products by resorting to fossil raw materials instead of petrochemical production processes, and how this can be accomplished by the cultivation of anaerobic organisms, namely facultatively anaerobic yeasts and bacteria (production of ethanol or 2,3-butanediol), and strictly anaerobic Clostridia (1-butanol, acetone, or isopropanol) on renewable resources. Moreover, novel methods for producing biodiesel-like methyl-esters of aerobically produced bacterial polyhydroxyalkanoate biopolyester building blocks combine the synthesis of microbial biopolyesters from wastewater with the progress of innovative renewable energy carriers. The biochemical background, the current state of research and development, and the status of industrialization of these processes are reviewed. Conclusion: Challenges to make these bioprocesses, based on inexpensive renewable resources, competitive with or even superior to petrochemical production routes in terms of sustainability, scalability, and economic feasibility still exist: however, they can be overcome by the concerted action of various scientific disciplines.

Current scenario of recently rising up cases of invasive group A streptococcal (iGAS) infections in younger children in many European nations: clinical management and prospective counteracting measures – an update

Current scenario of recently rising up cases of invasive group A streptococcal (iGAS) infections in younger children in many European nations: clinical management and prospective counteracting measures – an update

Development of an efficient ClosTron system for gene disruption in Ruminiclostridium papyrosolvens.

Ruminiclostridium papyrosolvens is an anaerobic, mesophilic, and cellulolytic clostridia, promising consolidated bioprocessing (CBP) candidate for producing renewable green chemicals from cellulose, but its metabolic engineering is limited by lack of genetic tools. Here, we firstly employed the endogenous xylan-inducible promoter to control ClosTron system for gene disruption of R. papyrosolvens. The modified ClosTron can be easily transformed into R. papyrosolvens and specifically disrupt targeting genes. Furthermore, a counter selectable system based on uracil phosphoribosyl-transferase (Upp) was successfully established and introduced into the ClosTron system, which resulted in plasmid curing rapidly. Thus, the combination of xylan-inducible ClosTron and upp-based counter selectable system makes the gene disruption more efficient and convenient for successive gene disruption in R. papyrosolvens. KEY POINTS: • Limiting expression of LtrA enhanced the transformation of ClosTron plasmids in R. papyrosolvens. • DNA targeting specificity can be improved by precise management of the expression of LtrA. • Curing of ClosTron plasmids was achieved by introducing the upp-based counter selectable system.

Evaluation of Laminaria Digitata Hydrolysate for the Production of Bioethanol and Butanol by Fermentation

Seaweeds (macroalgae) are gaining attention as potential sustainable feedstock for the production of fuels and chemicals. This comparative study focuses on the characterization of the microbial production of alcohols from fermentable carbohydrates in the hydrolysate of the macroalgae Laminaria digitata as raw material. The potential of a hydrolysate as a carbon source for the production of selected alcohols was tested, using three physiologically different fermentative microbes, in two main types of processes. For the production of ethanol, Saccharomyces cerevisiae was used as a benchmark microorganism and compared with the strictly anaerobic thermophile Thermoanaerobacterium strain AK17. For mixed production of acetone/isopropanol, butanol, and ethanol (A/IBE), three strictly anaerobic Clostridium strains were compared. All strains grew well on the hydrolysate, and toxicity constraints were not observed, but fermentation performance and product profiles were shown to be both condition- and strain-specific. S. cerevisiae utilized only glucose for ethanol formation, while strain AK17 utilized glucose, mannitol, and parts of the glucan oligosaccharides. The clostridia strains tested showed different nutrient requirements, and were able to utilize glucan, mannitol, and organic acids in the hydrolysate. The novelty of this study embodies the application of different inoculates for fermenting a common brown seaweed found in the northern Atlantic Ocean. It provides important information on the fermentation properties of different microorganisms and pinpoints the value of carbon source utilization when selecting microbes for efficient bioconversion into biofuel and chemical products of interest.