Isolation and characterization of novel acetogenic Moorella strains for employment as potential thermophilic biocatalysts.

Thermophilic acetogenic bacteria have attracted attention as promising candidates for biotechnological applications such as syngas fermentation, microbial electrosynthesis, and methanol conversion. Here, we aimed to isolate and characterize novel thermophilic acetogens from diverse environments. Enrichment of heterotrophic and autotrophic acetogens was monitored by 16S rRNA gene-based bacterial community analysis. Seven novel Moorella strains were isolated and characterized by genomic and physiological analyses. Two Moorella humiferrea isolates showed considerable differences during autotrophic growth. The M. humiferrea LNE isolate (DSM 117358) fermented carbon monoxide (CO) to acetate, while the M. humiferrea OCP isolate (DSM 117359) transformed CO to hydrogen and carbon dioxide (H2 + CO2), employing the water-gas shift reaction. Another carboxydotrophic hydrogenogenic Moorella strain was isolated from the covering soil of an active charcoal burning pile and proposed as the type strain (ACPsT) of the novel species Moorella carbonis (DSM 116161T and CCOS 2103T). The remaining four novel strains were affiliated with Moorella thermoacetica and showed, together with the type strain DSM 2955T, the production of small amounts of ethanol from H2 + CO2 in addition to acetate. The physiological analyses of the novel Moorella strains revealed isolate-specific differences that considerably increase the knowledge base on thermophilic acetogens for future applications.

Genome reduction in novel, obligately methyl-reducing Methanosarcinales isolated from arthropod guts (Methanolapillus gen. nov. and Methanimicrococcus).

Recent metagenomic studies have identified numerous lineages of hydrogen-dependent, obligately methyl-reducing methanogens. Yet only a few representatives have been isolated in pure culture. Here, we describe six new species with this capability in the family Methanosarcinaceae (order Methanosarcinales), which makes up a substantial fraction of the methanogenic community in arthropod guts. Phylogenomic analysis placed the isolates from cockroach hindguts into the genus Methanimicrococcus (M. hacksteinii, M. hongohii, M. stummii) and the isolates from millipede hindguts into a new genus, Methanolapillus (M. africanus, M. millepedarum, M. ohkumae). Members of this intestinal clade, which includes also uncultured representatives from termites and vertebrates, have substantially smaller genomes (1.6-2.2 Mbp) than other Methanosarcinales. Genome reduction was accompanied by the loss of the upper part of the Wood-Ljungdahl pathway, several energy-converting membrane complexes (Fpo, Ech, Rnf), and various biosynthetic pathways. However, genes involved in the protection against reactive oxygen species (catalase and superoxide reductase) were conserved in all genomes, including cytochrome bd (CydAB), a high-affinity terminal oxidase that may confer the capacity for microaerobic respiration. Since host-associated Methanosarcinales are nested within omnivorous lineages, we conclude that the specialization on methyl groups is an adaptation to the intestinal environment.

Isolation and characterization of novel acetogenic strains of the genera Terrisporobacter and Acetoanaerobium.

Due to their metabolic versatility in substrate utilization, acetogenic bacteria represent industrially significant production platforms for biotechnological applications such as syngas fermentation, microbial electrosynthesis or transformation of one-carbon substrates. However, acetogenic strains from the genera Terrisporobacter and Acetoanaerobium remained poorly investigated for biotechnological applications. We report the isolation and characterization of four acetogenic Terrisporobacter strains and one Acetoanaerobium strain. All Terrisporobacter isolates showed a characteristic growth pattern under a H2 + CO2 atmosphere. An initial heterotrophic growth phase was followed by a stationary growth phase, where continuous acetate production was indicative of H2-dependent acetogenesis. One of the novel Terrisporobacter isolates obtained from compost (strain COMT) additionally produced ethanol besides acetate in the stationary growth phase in H2-supplemented cultures. Genomic and physiological characterizations showed that strain COMT represented a novel Terrisporobacter species and the name Terrisporobacter vanillatitrophus is proposed (=DSM 116160T = CCOS 2104T). Phylogenomic analysis of the novel isolates and reference strains implied the reclassification of the T. petrolearius/T. hibernicus phylogenomic cluster to the species T. petrolearius and of the A. noterae/A. sticklandii phylogenomic cluster to the species A. sticklandii. Furthermore, we provide first insights into active prophages of acetogens from the genera Terrisporobacter and Acetoanaerobium.

Corrigendum: Genome reduction in Paenibacillus polymyxa DSM 365 for chassis development.

[This corrects the article DOI: 10.3389/fbioe.2024.1378873.].

Complete genome sequence of Sporomusa sphaeroides DSM 2875T isolated from mud of the Leine river and Sporomusa ovata DSM 2662T isolated from sugar beet leaf silage.

We report the closed genome sequences of the acetogen Sporomusa sphaeroides ET (DSM 2875T) and of Sporomusa ovata H1T (DSM 2662T). The S. sphaeroides ET genome harbors a chromosome (4,956,256 bp) and a plasmid (59,087 bp). The genome of S. ovata H1T harbors one chromosome (5,433,971 bp).

Comparative genome analyses of clinical and non-clinical Clostridioides difficile strains.

The pathogenic bacterium Clostridioides difficile is a worldwide health burden with increasing morbidity, mortality and antibiotic resistances. Therefore, extensive research efforts are made to unravel its virulence and dissemination. One crucial aspect for C. difficile is its mobilome, which for instance allows the spread of antibiotic resistance genes (ARG) or influence strain virulence. As a nosocomial pathogen, the majority of strains analyzed originated from clinical environments and infected individuals. Nevertheless, C. difficile can also be present in human intestines without disease development or occur in diverse environmental habitats such as puddle water and soil, from which several strains could already be isolated. We therefore performed comprehensive genome comparisons of closely related clinical and non-clinical strains to identify the effects of the clinical background. Analyses included the prediction of virulence factors, ARGs, mobile genetic elements (MGEs), and detailed examinations of the pan genome. Clinical-related trends were thereby observed. While no significant differences were identified in fundamental C. difficile virulence factors, the clinical strains carried more ARGs and MGEs, and possessed a larger accessory genome. Detailed inspection of accessory genes revealed higher abundance of genes with unknown function, transcription-associated, or recombination-related activity. Accessory genes of these functions were already highlighted in other studies in association with higher strain virulence. This specific trend might allow the strains to react more efficiently on changing environmental conditions in the human host such as emerging stress factors, and potentially increase strain survival, colonization, and strain virulence. These findings indicated an adaptation of the strains to the clinical environment. Further, implementation of the analysis results in pairwise genome comparisons revealed that the majority of these accessory genes were encoded on predicted MGEs, shedding further light on the mobile genome of C. difficile. We therefore encourage the inclusion of non-clinical strains in comparative analyses.

Redirecting electron flow in Acetobacterium woodii enables growth on CO and improves growth on formate.

Anaerobic, acetogenic bacteria are well known for their ability to convert various one-carbon compounds, promising feedstocks for a future, sustainable biotechnology, to products such as acetate and biofuels. The model acetogen Acetobacterium woodii can grow on CO2, formate or methanol, but not on carbon monoxide, an important industrial waste product. Since hydrogenases are targets of CO inhibition, here, we genetically delete the two [FeFe] hydrogenases HydA2 and HydBA in A. woodii. We show that the ∆hydBA/hydA2 mutant indeed grows on CO and produces acetate, but only after a long adaptation period. SNP analyzes of CO-adapted cells reveal a mutation in the HycB2 subunit of the HydA2/HydB2/HydB3/Fdh-containing hydrogen-dependent CO2 reductase (HDCR). We observe an increase in ferredoxin-dependent CO2 reduction and vice versa by the HDCR in the absence of the HydA2 module and speculate that this is caused by the mutation in HycB2. In addition, the CO-adapted ∆hydBA/hydA2 mutant growing on formate has a final biomass twice of that of the wild type.

Roseobacter fucihabitans sp. nov., isolated from the brown alga Fucus spiralis.

A Gram-negative, aerobic, pink-pigmented, and bacteriochlorophyll a-containing bacterial strain, designated B14T, was isolated from the macroalga Fucus spiralis sampled from the southern North Sea, Germany. Based on 16S rRNA gene sequences, species of the genera Roseobacter and Sulfitobacter were most closely related to strain B14T with sequence identities ranging from 98.15 % (Roseobacter denitrificans Och 114T) to 99.11 % (Roseobacter litoralis Och 149T), whereas Sulfitobacter mediterraneus CH-B427T exhibited 98.52 % sequence identity. Digital DNA-DNA hybridization and average nucleotide identity values between the genome of the novel strain and that of closely related Roseobacter and Sulfitobacter type strains were <20 % and <77 %, respectively. The novel strain contained ubiquinone-10 as the only respiratory quinone and C18 : 1 ω7c, C16 : 0, C18 : 0, C12 : 1 ω7c, C18 : 2 ω7,13c, and C10 : 0 3-OH as the major cellular fatty acids. The predominant polar lipids of strain B14T were phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol. The genome of strain B14T comprises a chromosome with a size of 4.5 Mbp, one chromid, and four plasmids. The genome contains the complete gene cluster for aerobic anoxygenic photosynthesis required for a photoheterotrophic lifestyle. The results of this study indicate that strain B14T (=DSM 116946T=LMG 33352T) represents a novel species of the genus Roseobacter for which the name Roseobacter fucihabitans sp. nov. is proposed.

Novel insights into phage biology of the pathogen Clostridioides difficile based on the active virome.

The global pathogen Clostridioides difficile is a well-studied organism, and researchers work on unraveling its fundamental virulence mechanisms and biology. Prophages have been demonstrated to influence C. difficile toxin expression and contribute to the distribution of advantageous genes. All these underline the importance of prophages in C. difficile virulence. Although several C. difficile prophages were sequenced and characterized, investigations on the entire active virome of a strain are still missing. Phages were mainly isolated after mitomycin C-induction, which does not resemble a natural stressor for C. difficile. We examined active prophages from different C. difficile strains after cultivation in the absence of mitomycin C by sequencing and characterization of particle-protected DNA. Phage particles were collected after standard cultivation, or after cultivation in the presence of the secondary bile salt deoxycholate (DCA). DCA is a natural stressor for C. difficile and a potential prophage-inducing agent. We also investigated differences in prophage activity between clinical and non-clinical C. difficile strains. Our experiments demonstrated that spontaneous prophage release is common in C. difficile and that DCA presence induces prophages. Fourteen different, active phages were identified by this experimental procedure. We could not identify a definitive connection between clinical background and phage activity. However, one phage exhibited distinctively higher activity upon DCA induction in the clinical strain than in the corresponding non-clinical strain, although the phage is identical in both strains. We recorded that enveloped DNA mapped to genome regions with characteristics of mobile genetic elements other than prophages. This pointed to mechanisms of DNA mobility that are not well-studied in C. difficile so far. We also detected phage-mediated lateral transduction of bacterial DNA, which is the first described case in C. difficile. This study significantly contributes to our knowledge of prophage activity in C. difficile and reveals novel aspects of C. difficile (phage) biology.

Balancing economic and ecological functions in smallholder and industrial oil palm plantations.

The expansion of the oil palm industry in Indonesia has improved livelihoods in rural communities, but comes at the cost of biodiversity and ecosystem degradation. Here, we investigated ways to balance ecological and economic outcomes of oil palm cultivation. We compared a wide range of production systems, including smallholder plantations, industrialized company estates, estates with improved agronomic management, and estates with native tree enrichment. Across all management types, we assessed multiple indicators of biodiversity, ecosystem functions, management, and landscape structure to identify factors that facilitate economic-ecological win-wins, using palm yields as measure of economic performance. Although, we found that yields in industrialized estates were, on average, twice as high as those in smallholder plantations, ecological indicators displayed substantial variability across systems, regardless of yield variations, highlighting potential for economic-ecological win-wins. Reducing management intensity (e.g., mechanical weeding instead of herbicide application) did not lower yields but improved ecological outcomes at moderate costs, making it a potential measure for balancing economic and ecological demands. Additionally, maintaining forest cover in the landscape generally enhanced local biodiversity and ecosystem functioning within plantations. Enriching plantations with native trees is also a promising strategy to increase ecological value without reducing productivity. Overall, we recommend closing yield gaps in smallholder cultivation through careful intensification, whereas conventional plantations could reduce management intensity without sacrificing yield. Our study highlights various pathways to reconcile the economics and ecology of palm oil production and identifies management practices for a more sustainable future of oil palm cultivation.

Genome reduction in Paenibacillus polymyxa DSM 365 for chassis development.

The demand for highly robust and metabolically versatile microbes is of utmost importance for replacing fossil-based processes with biotechnological ones. Such an example is the implementation of Paenibacillus polymyxa DSM 365 as a novel platform organism for the production of value-added products such as 2,3-butanediol or exopolysaccharides. For this, a complete genome sequence is the first requirement towards further developing this host towards a microbial chassis. A genome sequencing project has just been reported for P. polymyxa DSM 365 showing a size of 5,788,318 bp with a total of 47 contigs. Herein, we report the first complete genome sequence of P. polymyxa DSM 365, which consists of 5,889,536 bp with 45 RNAs, 106 tRNAs, 5,370 coding sequences and an average GC content of 45.6%, resulting in a closed genome of P. polymyxa 365. The additional nucleotide data revealed a novel NRPS synthetase that may contribute to the production of tridecaptin. Building on these findings, we initiated the top-down construction of a chassis variant of P. polymyxa. In the first stage, single knock-out mutants of non-essential genomic regions were created and evaluated for their biological fitness. As a result, two out of 18 variants showed impaired growth. The remaining deletion mutants were combined in two genome-reduced P. polymyxa variants which either lack the production of endogenous biosynthetic gene clusters (GR1) or non-essential genomic regions including the insertion sequence ISPap1 (GR2), with a decrease of the native genome of 3.0% and 0.6%, respectively. Both variants, GR1 and GR2, showed identical growth characteristics to the wild-type. Endpoint titers of 2,3-butanediol and EPS production were also unaffected, validating these genome-reduced strains as suitable for further genetic engineering.

Proteomic and transcriptomic analysis of selenium utilization in Methanococcus maripaludis.

Methanococcus maripaludis utilizes selenocysteine- (Sec-) containing proteins (selenoproteins), mostly active in the organism's primary energy metabolism, methanogenesis. During selenium depletion, M. maripaludis employs a set of enzymes containing cysteine (Cys) instead of Sec. The genes coding for these Sec-/Cys-containing isoforms were the only genes known of which expression is influenced by the selenium status of the cell. Using proteomics and transcriptomics, approx. 7% and 12%, respectively, of all genes/proteins were found differentially expressed/synthesized in response to the selenium supply. Some of the genes identified involve methanogenesis, nitrogenase functions, and putative transporters. An increase of transcript abundance for putative transporters under selenium depletion indicated the organism's effort to tap into alternative sources of selenium. M. maripaludis is known to utilize selenite and dimethylselenide as selenium sources. To expand this list, a selenium-responsive reporter strain was assessed with nine other, environmentally relevant selenium species. While the effect of some was very similar to that of selenite, others were effectively utilized at lower concentrations. Conversely, selenate and seleno-amino acids were only utilized at unphysiologically high concentrations and two compounds were not utilized at all. To address the role of the selenium-regulated putative transporters, M. maripaludis mutant strains lacking one or two of the putative transporters were tested for the capability to utilize the different selenium species. Of the five putative transporters analyzed by loss-of-function mutagenesis, none appeared to be absolutely required for utilizing any of the selenium species tested, indicating they have redundant and/or overlapping specificities or are not dedicated selenium transporters. IMPORTANCE: While selenium metabolism in microorganisms has been studied intensively in the past, global gene expression approaches have not been employed so far. Furthermore, the use of different selenium sources, widely environmentally interconvertible via biotic and abiotic processes, was also not extensively studied before. Methanococcus maripaludis JJ is ideally suited for such analyses, thanks to its known selenium usage and available genetic tools. Thus, an overall view on the selenium regulon of M. maripaludis was obtained via transcriptomic and proteomic analyses, which inspired further experimentation. This led to demonstrating the use of selenium sources M. maripaludis was previously not known to employ. Also, an attempt-although so far unsuccessful-was made to pinpoint potential selenium transporter genes, in order to deepen our understanding of trace element utilization in this important model organism.

Complete genome sequences of Blautia hydrogenotrophica DSM 10507T isolated from human feces and Blautia coccoides DSM 935T isolated from mouse feces.

We report on the closed genome sequences of the acetogen Blautia hydrogenotrophica S5a33T (DSM 10507T) and of Blautia coccoides CLC-1T (DSM 935T). The B. hydrogenotrophica S5a33T genome harbors a chromosome (3,590,609 bp) and a plasmid (7,176 bp). The B. coccoides CLC-1T genome consists of a single chromosome (6,097,890 bp).

The energy-converting hydrogenase Ech2 is important for the growth of the thermophilic acetogen Thermoanaerobacter kivui on ferredoxin-dependent substrates.

Thermoanaerobacter kivui is the thermophilic acetogenic bacterium with the highest temperature optimum (66°C) and with high growth rates on hydrogen (H2) plus carbon dioxide (CO2). The bioenergetic model suggests that its redox and energy metabolism depends on energy-converting hydrogenases (Ech). Its genome encodes two Echs, Ech1 and Ech2, as sole coupling sites for energy conservation during growth on H2 + CO2. During growth on other substrates, its redox activity, the (proton-gradient-coupled) oxidation of H2 may be essential to provide reduced ferredoxin (Fd) to the cell. While Ech activity has been demonstrated biochemically, the physiological function of both Ech's is unclear. Toward that, we deleted the complete gene cluster encoding Ech2. Surprisingly, the ech2 mutant grew as fast as the wild type on sugar substrates and H2 + CO2. Hence, Ech1 may be the essential enzyme for energy conservation, and either Ech1 or another enzyme may substitute for H2-dependent Fd reduction during growth on sugar substrates, putatively the H2-dependent CO2 reductase (HDCR). Growth on pyruvate and CO, substrates that are oxidized by Fd-dependent enzymes, was significantly impaired, but to a different extent. While ∆ech2 grew well on pyruvate after four transfers, ∆ech2 did not adapt to CO. Cell suspensions of ∆ech2 converted pyruvate to acetate, but no acetate was produced from CO. We analyzed the genome of five T. kivui strains adapted to CO. Strikingly, all strains carried mutations in the hycB3 subunit of HDCR. These mutations are obviously essential for the growth on CO but may inhibit its ability to utilize Fd as substrate. IMPORTANCE: Acetogens thrive by converting H2+CO2 to acetate. Under environmental conditions, this allows for only very little energy to be conserved (∆G'<-20 kJ mol-1). CO2 serves as a terminal electron acceptor in the ancient Wood-Ljungdahl pathway (WLP). Since the WLP is ATP neutral, energy conservation during growth on H2 + CO2 is dependent on the redox metabolism. Two types of acetogens can be distinguished, Rnf- and Ech-type. The function of both membrane-bound enzyme complexes is twofold-energy conversion and redox balancing. Ech couples the Fd-dependent reduction of protons to H2 to the formation of a proton gradient in the thermophilic bacterium Thermoanaerobacter kivui. This bacterium may be utilized in gas fermentation at high temperatures, due to very high conversion rates and the availability of genetic tools. The physiological function of an Ech hydrogenase in T. kivui was studied to contribute an understanding of its energy and redox metabolism, a prerequisite for future industrial applications.

Culture-independent detection of low-abundant Clostridioides difficile in environmental DNA via PCR.

Clostridioides difficile represents a major burden to public health. As a well-known nosocomial pathogen whose occurrence is highly associated with antibiotic treatment, most examined C. difficile strains originated from clinical specimen and were isolated under selective conditions employing antibiotics. This suggests a significant bias among analyzed C. difficile strains, which impedes a holistic view on this pathogen. In order to support extensive isolation of C. difficile strains from environmental samples, we designed a detection PCR that targets the hpdBCA-operon and thereby identifies low abundances of C. difficile in environmental samples. This operon encodes the 4-hydroxyphenylacetate decarboxylase, which catalyzes the production of the antimicrobial compound para-cresol. Amplicon-based analyses of diverse environmental samples demonstrated that the designed PCR is highly specific for C. difficile and successfully detected C. difficile despite its absence in general 16S rRNA gene-based detection strategies. Further analyses revealed the potential of the hpdBCA detection PCR sequence for initial phylogenetic classification, which allows assessment of C. difficile diversity in environmental samples via amplicon sequencing. Our findings furthermore showed that C. difficile strains isolated under antibiotic treatment from environmental samples were originally dominated by other strains according to PCR amplicon results. This provided evidence for selective cultivation of under-represented but antibiotic-resistant isolates. Thereby, we revealed a substantial bias in C. difficile isolation and research.IMPORTANCEClostridioides difficile is a main cause of diarrheic infections after antibiotic treatment with serious morbidity and mortality worldwide. Research on this pathogen and its virulence has focused on bacterial isolation from clinical specimens under antibiotic treatment, which implies a substantial bias in isolated strains. Comprehensive studies, however, require an unbiased strain collection, which is accomplished by isolation of C. difficile from diverse environmental samples and avoidance of antibiotic-based enrichment strategies. Thus, isolation can significantly benefit from our C. difficile-specific detection PCR, which rapidly verifies C. difficile presence in environmental samples and further allows estimation of the C. difficile diversity by using next-generation sequencing.

Bacteria use a catabolic patchwork pathway of apparently recent origin for degradation of the synthetic buffer compound TRIS.

The synthetic buffer compound TRIS (2-amino-2-(hydroxymethyl)propane-1,3-diol) is used in countless applications, and no detailed information on its degradation has been published so far. Herein, we describe the discovery of a complete bacterial degradation pathway for TRIS. By serendipity, a Pseudomonas strain was isolated from sewage sludge that was able to grow with TRIS as only carbon and nitrogen source. Genome and transcriptome analyses revealed two adjacent gene clusters embedded in a mobile genetic element on a conjugative plasmid to be involved in TRIS degradation. Heterologous gene expression revealed cluster I to encode a TRIS uptake protein, a TRIS alcohol dehydrogenase, and a TRIS aldehyde dehydrogenase, catalyzing the oxidation of TRIS into 2-hydroxymethylserine. Gene cluster II encodes a methylserine hydroxymethyltransferase (mSHMT) and a d-serine dehydratase that plausibly catalyze the conversion of 2-hydroxymethylserine into pyruvate. Conjugational plasmid transfer into Pseudomonas putida KT2440 enabled this strain to grow with TRIS and with 2-hydromethylserine, demonstrating that the complete TRIS degradation pathway can be transmitted by horizontal gene transfer. Subsequent enrichments from wastewater purification systems led to the isolation of further TRIS-degrading bacteria from the Pseudomonas and Shinella genera carrying highly similar TRIS degradation gene clusters. Our data indicate that TRIS degradation evolved recently via gene recruitment and enzyme adaptation from multiple independent metabolic pathways, and database searches suggest that the TRIS degradation pathway is now globally distributed. Overall, our study illustrates how engineered environments can enhance the emergence of new microbial metabolic pathways in short evolutionary time scales.

Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineering.

BACKGROUND: The genus Eubacterium is quite diverse and includes several acetogenic strains capable of fermenting C1-substrates into valuable products. Especially, Eubacterium limosum and closely related strains attract attention not only for their capability to ferment C1 gases and liquids, but also due to their ability to produce butyrate. Apart from its well-elucidated metabolism, E. limosum is also genetically accessible, which makes it an interesting candidate to be an industrial biocatalyst. RESULTS: In this study, we examined genomic, phylogenetic, and physiologic features of E. limosum and the closest related species E. callanderi as well as E. maltosivorans. We sequenced the genomes of the six Eubacterium strains 'FD' (DSM 3662T), 'Marburg' (DSM 3468), '2A' (DSM 2593), '11A' (DSM 2594), 'G14' (DSM 107592), and '32' (DSM 20517) and subsequently compared these with previously available genomes of the E. limosum type strain (DSM 20543T) as well as the strains 'B2', 'KIST612', 'YI' (DSM 105863T), and 'SA11'. This comparison revealed a close relationship between all eleven Eubacterium strains, forming three distinct clades: E. limosum, E. callanderi, and E. maltosivorans. Moreover, we identified the gene clusters responsible for methanol utilization as well as genes mediating chain elongation in all analyzed strains. Subsequent growth experiments revealed that strains of all three clades can convert methanol and produce acetate, butyrate, and hexanoate via reverse ß-oxidation. Additionally, we used a harmonized electroporation protocol and successfully transformed eight of these Eubacterium strains to enable recombinant plasmid-based expression of the gene encoding the fluorescence-activating and absorption shifting tag (FAST). Engineered Eubacterium strains were verified regarding their FAST-mediated fluorescence at a single-cell level using a flow cytometry approach. Eventually, strains 'FD' (DSM 3662T), '2A' (DSM 2593), '11A' (DSM 2594), and '32' (DSM 20517) were genetically engineered for the first time. CONCLUSION: Strains of E. limosum, E. callanderi, and E. maltosivorans are outstanding candidates as biocatalysts for anaerobic C1-substrate conversion into valuable biocommodities. A large variety of strains is genetically accessible using a harmonized electroporation protocol, and FAST can serve as a reliable fluorescent reporter protein to characterize genetically engineered cells. In total eleven strains have been assigned to distinct clades, providing a clear and updated classification. Thus, the description of respective Eubacterium species has been emended, improved, aligned, and is requested to be implemented in respective databases.

Pan-genome analysis of six Paracoccus type strain genomes reveal lifestyle traits.

The genus Paracoccus capable of inhabiting a variety of different ecological niches both, marine and terrestrial, is globally distributed. In addition, Paracoccus is taxonomically, metabolically and regarding lifestyle highly diverse. Until now, little is known on how Paracoccus can adapt to such a range of different ecological niches and lifestyles. In the present study, the genus Paracoccus was phylogenomically analyzed (n = 160) and revisited, allowing species level classification of 16 so far unclassified Paracoccus sp. strains and detection of five misclassifications. Moreover, we performed pan-genome analysis of Paracoccus-type strains, isolated from a variety of ecological niches, including different soils, tidal flat sediment, host association such as the bluespotted cornetfish, Bugula plumosa, and the reef-building coral Stylophora pistillata to elucidate either i) the importance of lifestyle and adaptation potential, and ii) the role of the genomic equipment and niche adaptation potential. Six complete genomes were de novo hybrid assembled using a combination of short and long-read technologies. These Paracoccus genomes increase the number of completely closed high-quality genomes of type strains from 15 to 21. Pan-genome analysis revealed an open pan-genome composed of 13,819 genes with a minimal chromosomal core (8.84%) highlighting the genomic adaptation potential and the huge impact of extra-chromosomal elements. All genomes are shaped by the acquisition of various mobile genetic elements including genomic islands, prophages, transposases, and insertion sequences emphasizing their genomic plasticity. In terms of lifestyle, each mobile genetic elements should be evaluated separately with respect to the ecological context. Free-living genomes, in contrast to host-associated, tend to comprise (1) larger genomes, or the highest number of extra-chromosomal elements, (2) higher number of genomic islands and insertion sequence elements, and (3) a lower number of intact prophage regions. Regarding lifestyle adaptations, free-living genomes share genes linked to genetic exchange via T4SS, especially relevant for Paracoccus, known for their numerous extrachromosomal elements, enabling adaptation to dynamic environments. Conversely, host-associated genomes feature diverse genes involved in molecule transport, cell wall modification, attachment, stress protection, DNA repair, carbon, and nitrogen metabolism. Due to the vast number of adaptive genes, Paracoccus can quickly adapt to changing environmental conditions.

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification / Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas

In order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.

Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST), se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un gen codificante de una hipotetica lipasa/esterasa. La secuencia deducida de amino acidos (RBest1) contiene los motivos conservados de enzimas lipoliticas y esta relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la unica enzima procariota estudiada perteneciente al subgrupo 4.4 de a/ß hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-esteres, respectivamente, revelaron que la enzima es altamente especifica para compuestos butiricos (C4), comportandose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquimicamente. La actividad optima de esterasa fue observada a pH 6,5 y las temperaturas optimas fueron entre 38 y 45 °C. Se establecio que la actividad enzimatica, determinada por hidrolisis de p-nitrofenil esteres, es afectada en presencia de diferentes solventes organicos miscibles y no miscibles, y tambien sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas ionicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptacion molecular en presencia de compuestos organicos, asi como la resistencia de las proteinas halofilas.

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification / Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas

In order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.(AU)

Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST), se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un gen codificante de una hipotetica lipasa/esterasa. La secuencia deducida de amino acidos (RBest1) contiene los motivos conservados de enzimas lipoliticas y esta relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la unica enzima procariota estudiada perteneciente al subgrupo 4.4 de a/ß hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-esteres, respectivamente, revelaron que la enzima es altamente especifica para compuestos butiricos (C4), comportandose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquimicamente. La actividad optima de esterasa fue observada a pH 6,5 y las temperaturas optimas fueron entre 38 y 45 °C. Se establecio que la actividad enzimatica, determinada por hidrolisis de p-nitrofenil esteres, es afectada en presencia de diferentes solventes organicos miscibles y no miscibles, y tambien sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas ionicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptacion molecular en presencia de compuestos organicos, asi como la resistencia de las proteinas halofilas.(AU)