Bacterial Domain Research Articles

Cytotoxicity, Antimicrobial, and In Silico Studies of Secondary Metabolites From Aspergillus sp. Isolated From Tecoma stans (L.) Juss. Ex Kunth Leaves.

Endophytes are prolific producers of privileged secondary metabolites with diverse therapeutic potential, although their anticancer and antimicrobial potential still have a room for further investigation. Herein, seven known secondary metabolites namely, arugosin C (1), ergosterol (2), iso-emericellin (3), sterigmatocystin (4), dihydrosterigmatocystin (5), versicolorin B (6), and diorcinol (7) were isolated from the rice culture of Aspergillus sp. retrieved from Tecoma stans (L.) Juss. ex Kunth leaves. Their anticancer and antimicrobial activities were evaluated in MTT and agar well diffusion assays, respectively. The cytotoxicity results showed that metabolite 3 displayed the best viability inhibition on the MCF-7 breast cancer cells with IC50 = 225.21 µM, while 5 on the HepG2 hepatocellular carcinoma cells with IC50 = 161.81 µM. 5 demonstrated a 60% apoptotic mode of cell death which is virtually correlated to its high docking affinity to Hsp90 ATP binding cleft (binding score −8.4 Kcal/mol). On the other side, metabolites 4 and 5 displayed promising antimicrobial activity especially on Pseudomonas aeruginosa with MIC = 125 μg/ml. The observed effect may be likely related to their excellent in silico inhibition of the bacterial DNA-gyrase kinase domain (binding score −10.28 Kcal/mol). To the best of our knowledge, this study is the first to report the promising cytotoxic and antibacterial activities of metabolites 3, 4, and 5 which needs further investigation and renovation to therapeutic leads.

Weaving of bacterial cellulose by the Bcs secretion systems

ABSTRACTCellulose is the most abundant biological compound on Earth and while it is the predominant building constituent of plants, it is also a key extracellular matrix component in many diverse bacterial species. While bacterial cellulose was first described in the 19th century, it was not until this last decade that a string of structural works provided insights into how the cellulose synthase BcsA, assisted by its inner-membrane partner BcsB, senses c-di-GMP to simultaneously polymerize its substrate and extrude the nascent polysaccharide across the inner bacterial membrane. It is now established that bacterial cellulose can be produced by several distinct types of cellulose secretion systems and that in addition to BcsAB, they can feature multiple accessory subunits, often indispensable for polysaccharide production. Importantly, the last years mark significant progress in our understanding not only of cellulose polymerization per se but also of the bigger picture of bacterial signaling, secretion system assembly, biofilm formation and host tissue colonization, as well as of structural and functional parallels of this dominant biosynthetic process between the bacterial and eukaryotic domains of life. Here, we review current mechanistic knowledge on bacterial cellulose secretion with focus on the structure, assembly and cooperativity of Bcs secretion system components.

Predicted Functional and Structural Diversity of Receiver Domains in Fungal Two-Component Regulatory Systems

ABSTRACTFungal two-component regulatory systems incorporate receiver domains into hybrid histidine kinases (HHKs) and response regulators. We constructed a nonredundant database of 670 fungal receiver domain sequences from 51 species sampled from nine fungal phyla. A much greater proportion (21%) of predicted fungal response regulators did not belong to known groups than previously appreciated. Receiver domains in Rim15 response regulators from Ascomycota and other phyla are very different from one another, as are the duplicate receiver domains in group XII HHKs. Fungal receiver domains from five known types of response regulators and 20 known types of HHKs exhibit distinct patterns of amino acids at conserved and variable positions known to be structurally and functionally important in bacterial receiver domains. We inferred structure/activity relationships from the patterns and propose multiple experimentally testable hypotheses about the mechanisms of signal transduction mediated by fungal receiver domains.

Metagenomic analysis of the diversity of microbes in the Napahai plateau wetland and their carbon and nitrogen metabolisms

Due to the special geographical location and the complex ecosystem types, plateau wetlands play important ecological roles in water supply, greenhouse gas regulation and biodiversity preservation. Napahai plateau wetland is a special wetland type with low latitude and high altitude, and its microbial diversity was rarely studied. The diversity of microbial communities in the Napahai plateau wetland was analyzed using metagenomics method. Among the microbes detected, 184 phyla, 3 262 genera and 24 260 species belong to the bacterial domain, 13 phyla and 32 genera belong to the archaeal domain, and 13 phyla and 47 genera belong to the fungal domain. Significant differences in species diversity between soil and water were observed. Acidobacteria, Proteobacteria and Actinobacteria were dominant phyla in soil, while Proteobacteria and Bacteroides were dominant phyla in water. Since the carbon and nitrogen metabolism genes were abundant, the pathways of carbon fixation and nitrogen metabolism were analyzed. Calvin cycle, reductive tricarboxylic acid cycle and 3-hydroxypropionic acid cycle were the main carbon fixation pathways, while Proteobacteria, Chloroflexi, and Crenarchaeota were the main carbon-fixing bacteria group. As for the nitrogen cycle, nitrogen fixation and dissimilatory nitrate reduction were dominant in water, while nitrification and denitrification were dominant in soil. Proteobacteria, Nitrospirae, Verrucomicrobia, Actinobacteria, Thaumarchaeota and Euryarchaeota contributed to the nitrogen cycle. The study on microbial diversity of Napahai plateau wetlands provides new knowledge for the comprehensive management and protection of wetland environment in China.

Process Performance and Microbial Community Variation in High-Rate Anaerobic Continuous Stirred Tank Reactor Treating Palm Oil Mill Effluent at Temperatures Between 55 and 70 °C

Palm oil mill effluent (POME) is wastewater that is generated from the extraction of oil from palm fruit and palm kernels and is discharged at a relatively high temperature (80–90 °C). Anaerobic digestion (AD) of POME has been mostly investigated at a mesophilic range of temperatures (30–38 °C) and at a thermophilic temperature of 55 °C. The thermophilic AD of POME at higher temperatures could be advantageous as treatment at thermophilic temperatures may increase rates of biochemical reactions. Stepwise temperature increase is a methodology for adaptation of microorganisms to higher temperatures. In this study, the effects of stepwise temperature increments of 5 °C from 55 to 70 °C on the AD of POME were investigated in a continuous stirred tank reactor (CSTR) operated under high organic loading rates (OLRs). The process performance and microbial community structure at each temperature interval were evaluated. It was observed that the methane production rates of the CSTR increased with increasing OLRs up to values of 13.7 g/L days, 25.7 g/L days, and 26.5 g/L days at operating temperatures of 55 °C, 60 °C, and 65 °C, respectively. As a result of the increasing OLRs, the maximum rate of methane production increased from 3.8 L/L days at 55 °C to 4.4 L/L days at 60 °C. The microbial community structure analysis showed that there were notable reductions in the gene copy number of the bacterial domain and the Methanosarcinales order with increasing temperatures from 55 to 60 °C and to 65 °C, whereas hydrogenotrophic methanogens, especially the genus Methanobacterium, in the order Methanobacteriales became dominant at 60 °C and 65 °C. Thus, the methanogenesis pathway was suggested to be a combination of acetoclastic and hydrogenotrophic methanogenesis at 55 °C and 60 °C with an increased contribution of hydrogenotrophic methanogenesis at 60 °C, whereas methane was mainly generated via hydrogenotrophic methanogenesis at 65 °C. The thermophilic AD of POME at 60 °C was found to be promising because the methane content in the biogas and the methane production rates were optimal, with an average methane content of approximately 73%.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy.

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700µδ6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700µδ6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700µδ6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Clec4e-Receptor Signaling in Myocardial Repair After Ischemia-Reperfusion Injury

The bacterial C-type lectin domain family 4 member E (CLEC4E) has an important role in sterile inflammation, but its role in myocardial repair is unknown. Using complementary approaches in porcine, murine, and human samples, we show that CLEC4E expression levels in the myocardium and in blood correlate with the extent of myocardial injury and left ventricular (LV) functional impairment. CLEC4E expression is markedly increased in the vasculature, cardiac myocytes, and infiltrating leukocytes in the ischemic heart. Loss of Clec4e signaling is associated with reduced acute cardiac injury, neutrophil infiltration, and infarct size. Reduced myocardial injury in Clec4e -/- translates into significantly improved LV structural and functional remodeling at 4weeks' follow-up. The early transcriptome of LV tissue from Clec4e -/- mice versus wild-type mice reveals significant upregulation of transcripts involved in myocardial metabolism, radical scavenging, angiogenesis, and extracellular matrix organization. Therefore, targeting CLEC4E in the early phase of ischemia-reperfusion injury is a promising therapeutic strategy to modulate myocardial inflammation and enhance repair after ischemia-reperfusion injury.

Nitrogen Fixation and Diazotrophs – A Review

Nitrogen fixation involves formation of ammonium from N2, which needs a high input of energy. Biological nitrogen fixation utilizes the enzyme nitrogenase and ATP to fix nitrogen. Nitrogenase contains a Fe-protein and a Mo-Fe-protein and other metal cofactors. Soil diazotrophs possess the function of fixing atmospheric N2 into biologically available ammonium in ecosystems. In Aechaea, nitrogen fixation has been reported in some methanogens such as Methanobacteriales, Methanococcales, and Methanosarcinales. Community structure and diversity of diazotrophic are correlated with soil pH. All known organisms which involve in nitrogen-fixing which are called diazatrophs are prokaryotes, and both bacterial and archaeal domains are responsible for that. Diazotrophs are categorized into two main groups namely: root-nodule bacteria and plant growth-promoting rhizobacteria. Diazotrophs include free living bacteria, such as Azospirillum, Cupriavidus, and some sulfate reducing bacteria, and symbiotic diazotrophs such Rhizobium and Frankia. Two important parameters which may affect diazotroph communities are temperature and soil moisture in different seasons. To have sustainable agriculture, replacing expensive chemical nitrogen fertilizers with environmentally friendly ways is the most accepted practice.

Identical sequences found in distant genomes reveal frequent horizontal transfer across the bacterial domain.

Horizontal gene transfer (HGT) is an essential force in microbial evolution. Despite detailed studies on a variety of systems, a global picture of HGT in the microbial world is still missing. Here, we exploit that HGT creates long identical DNA sequences in the genomes of distant species, which can be found efficiently using alignment-free methods. Our pairwise analysis of 93,481 bacterial genomes identified 138,273 HGT events. We developed a model to explain their statistical properties as well as estimate the transfer rate between pairs of taxa. This reveals that long-distance HGT is frequent: our results indicate that HGT between species from different phyla has occurred in at least 8% of the species. Finally, our results confirm that the function of sequences strongly impacts their transfer rate, which varies by more than three orders of magnitude between different functional categories. Overall, we provide a comprehensive view of HGT, illuminating a fundamental process driving bacterial evolution.

Genomic characterization of three novel Desulfobacterota classes expand the metabolic and phylogenetic diversity of the phylum.

We report on the genomic characterization of three novel classes in the phylum Desulfobacterota. One class (proposed name Candidatus 'Anaeroferrophillalia') was characterized by heterotrophic growth capacity, either fermentatively or utilizing polysulfide, tetrathionate or thiosulfate as electron acceptors. In the absence of organic carbon sources, autotrophic growth via the Wood-Ljungdahl (WL) pathway and using hydrogen or Fe(II) as an electron donor is also inferred for members of the 'Anaeroferrophillalia'. The second class (proposed name Candidatus 'Anaeropigmentia') was characterized by its capacity for growth at low oxygen concentration, and the capacity to synthesize the methyl/alkyl carrier CoM, an ability that is prevalent in the archaeal but rare in the bacterial domain. Pigmentation is inferred from the capacity for carotenoid (lycopene) production. The third class (proposed name Candidatus 'Zymogenia') was characterized by fermentative heterotrophic growth capacity, broad substrate range and the adaptation of some of its members to hypersaline habitats. Analysis of the distribution pattern of all three classes showed their occurrence as rare community members in multiple habitats, with preferences for anaerobic terrestrial, freshwater and marine environments over oxygenated (e.g. pelagic ocean and agricultural land) settings. Special preference for some members of the class Candidatus 'Zymogenia' for hypersaline environments such as hypersaline microbial mats and lagoons was observed.

Diversity and predicted inter- and intra-domain interactions in the Mediterranean Plastisphere

This study investigated the biogeography, the presence and diversity of potentially harmful taxa harbored, and potential interactions between and within bacterial and eukaryotic domains of life on plastic debris in the Mediterranean. Using a combination of high-throughput DNA sequencing (HTS), Causal Network Analysis, and Scanning Electron Microscopy (SEM), we show regional differences and gradients in the Mediterranean microbial communities associated with marine litter, positive causal effects between microbes including between and within domains of life, and how these might impact the marine ecosystems surrounding them. Adjacent seas within the Mediterranean region showed a gradient in the microbial communities on plastic with non-overlapping endpoints (Adriatic and Ligurian Seas). The largest predicted inter-domain effects included positive effects of a novel red-algal Plastisphere member on its potential microbiome community. Freshwater and marine samples housed a diversity of fungi including some related to disease-causing microbes. Algal species related to those responsible for Harmful Blooms (HABs) were also observed on plastic pieces including members of genera not previously reported on Plastic Marine Debris (PMD).

Bacterial death and TRADD-N domains help define novel apoptosis and immunity mechanisms shared by prokaryotes and metazoans.

Several homologous domains are shared by eukaryotic immunity and programmed cell-death systems and poorly understood bacterial proteins. Recent studies show these to be components of a network of highly regulated systems connecting apoptotic processes to counter-invader immunity, in prokaryotes with a multicellular habit. However, the provenance of key adaptor domains, namely those of the Death-like and TRADD-N superfamilies, a quintessential feature of metazoan apoptotic systems, remained murky. Here, we use sensitive sequence analysis and comparative genomics methods to identify unambiguous bacterial homologs of the Death-like and TRADD-N superfamilies. We show the former to have arisen as part of a radiation of effector-associated α-helical adaptor domains that likely mediate homotypic interactions bringing together diverse effector and signaling domains in predicted bacterial apoptosis- and counter-invader systems. Similarly, we show that the TRADD-N domain defines a key, widespread signaling bridge that links effector deployment to invader-sensing in multicellular bacterial and metazoan counter-invader systems. TRADD-N domains are expanded in aggregating marine invertebrates and point to distinctive diversifying immune strategies probably directed both at RNA and retroviruses and cellular pathogens that might infect such communities. These TRADD-N and Death-like domains helped identify several new bacterial and metazoan counter-invader systems featuring underappreciated, common functional principles: the use of intracellular invader-sensing lectin-like (NPCBM and FGS), transcription elongation GreA/B-C, glycosyltransferase-4 family, inactive NTPase (serving as nucleic acid receptors), and invader-sensing GTPase switch domains. Finally, these findings point to the possibility of multicellular bacteria-stem metazoan symbiosis in the emergence of the immune/apoptotic systems of the latter.

UBCG2: Up-to-date bacterial core genes and pipeline for phylogenomic analysis

Phylogenomic tree reconstruction has recently become a routine and critical task to elucidate the evolutionary relationships among bacterial species. The most widely used method utilizes the concatenated core genes, universally present in a single-copy throughout the bacterial domain. In our previous study, a bioinformatics pipeline termed Up-to-date Bacterial Core Genes (UBCG) was developed with a set of bacterial core genes selected from 1,429 species covering 28 phyla. In this study, we revised a new bacterial core gene set, named UBCG2, that was selected from the more extensive genome sequence set belonging to 3,508 species spanning 43 phyla. UBCG2 comprises 81 genes with nine Clusters of Orthologous Groups of proteins (COGs) functional categories. The new gene set and complete pipeline are available at http://leb.snu.ac.kr/ubcg2.

Inter-domain horizontal gene transfer of nickel-binding superoxide dismutase.

The ability of aerobic microorganisms to regulate internal and external concentrations of the reactive oxygen species (ROS) superoxide directly influences the health and viability of cells. Superoxide dismutases (SODs) are the primary regulatory enzymes that are used by microorganisms to degrade superoxide. SOD is not one, but three separate, non-homologous enzymes that perform the same function. Thus, the evolutionary history of genes encoding for different SOD enzymes is one of convergent evolution, which reflects environmental selection brought about by an oxygenated atmosphere, changes in metal availability, and opportunistic horizontal gene transfer (HGT). In this study, we examine the phylogenetic history of the protein sequence encoding for the nickel-binding metalloform of the SOD enzyme (SodN). The genomic potential to produce SodN is widespread among bacteria, including Actinobacteriota (Actinobacteria), Chloroflexota (Chloroflexi), Cyanobacteria, Proteobacteria, Patescibacteria, and others. The gene is also present in many archaea, with Thermoplasmatota and Nanoarchaeota representing the vast majority of archaeal sodN diversity. A comparison of organismal and SodN protein phylogenetic trees reveals several instances of HGT, including multiple inter-domain transfers of the sodN gene from the bacterial domain to the archaeal domain. Nearly half of the archaeal members with sodN live in the photic zone of the marine water column. The sodN gene is widespread and characterized by apparent vertical gene transfer in some sediment- or soil-associated lineages within the Actinobacteriota and Chloroflexota phyla, suggesting the ancestral sodN likely originated in one of these clades before expanding its taxonomic and biogeographic distribution to additional microbial groups in the surface ocean in response to decreasing iron availability. In addition to decreasing iron quotas, nickel-binding SOD has the added benefit of withstanding high reactant and product ROS concentrations without damaging the enzyme, making it particularly well suited for the modern surface ocean.

Metal-containing PAS/GAF domains in bacterial sensors

PAS and related GAF are ubiquitous and highly versatile sensing domains that associate a range of signaling effectors. Each year, new bacterial PAS and GAF domains are characterized but genomes analysis suggests this list is far from complete. This review aims to synthesize the current state of knowledge on the structure and activity of metal-binding PAS/GAF domain proteins. PAS/GAF domains can bind disparate metal cofactors with very precise coordination and high specificity concomitant with their extensive sequence diversity. Bound-metal can directly be the signal or can mediate signal detection such as gases or redox variation. Metal-containing PAS/GAF input modules are mainly associated to histidine kinases of two-component signaling systems and σ54-dependent transcription factors. In both cases, metal-binding induces conformational changes that modulate the effector activity. It appears that metal-containing PAS/GAF domains, as other PAS/GAF domains, offer great possibilities for coupling a broad range of ligand binding to various cellular responses and it is very likely that they have not yet revealed all their secrets.

Non-essential ribosomal proteins in bacteria and archaea identified using COGs.

Ribosomal proteins (RPs) are highly conserved across the bacterial and archaeal domains. Although many RPs are essential for survival, genome analysis demonstrates the absence of some RP genes in many bacterial and archaeal genomes. Furthermore, global transposon mutagenesis and/or targeted deletion showed that elimination of some RP genes had only a moderate effect on the bacterial growth rate. Here, we systematically analyze the evolutionary conservation of RPs in prokaryotes by compiling the list of the ribosomal genes that are missing from one or more genomes in the recently updated version of the Clusters of Orthologous Genes (COG) database. Some of these absences occurred because the respective genes carried frameshifts, presumably, resulting from sequencing errors, while others were overlooked and not translated during genome annotation. Apart from these annotation errors, we identified multiple genuine losses of RP genes in a variety of bacteria and archaea. Some of these losses are clade-specific, whereas others occur in symbionts and parasites with dramatically reduced genomes. The lists of computationally and experimentally defined non-essential ribosomal genes show a substantial overlap, revealing a common trend in prokaryote ribosome evolution that could be linked to the architecture and assembly of the ribosomes. Thus, RPs that are located at the surface of the ribosome and/or are incorporated at a late stage of ribosome assembly are more likely to be non-essential and to be lost during microbial evolution, particularly, in the course of genome compaction.IMPORTANCEIn many prokaryote genomes, one or more ribosomal protein (RP) genes are missing. Analysis of 1,309 prokaryote genomes included in the COG database shows that only about half of the RPs are universally conserved in bacteria and archaea. In contrast, up to 16 other RPs are missing in some genomes, primarily, tiny (<1 Mb) genomes of host-associated bacteria and archaea. Ten universal and nine archaea-specific ribosomal proteins show clear patterns of lineage-specific gene loss. Most of the RPs that are frequently lost from bacterial genomes are located on the ribosome periphery and are non-essential in Escherichia coli and Bacillus subtilis These results reveal general trends and common constraints in the architecture and evolution of ribosomes in prokaryotes.

Contribution of the NADase function of bacterial TIR domain proteins to the inhibition of innate immune responses

Abstract Pathogenic microbes have evolved various mechanisms to evade recognition by host immune responses, which can lead to the prevalence of disease and infection. Many strains of pathogenic bacteria contain genes encoding Toll/Interleukin-1 receptor (TIR) domain-containing proteins that function through the inhibition of TLR and myeloid differentiation factor 88 (MyD88) signaling. Previous research has also revealed that prokaryotic TIR domains may regulate bacterial and host metabolic pathways by acting as NAD+ glycohydrolases (NADases). We hypothesize that this NADase activity may contribute to the role of bacterial TIRs as virulence factors. In order to test this hypothesis, we observed the effects of TIR NADase activity on the binding interactions between bacterial TIRs and host MyD88, and on the ability of bacterial TIRs to block NF-kB signaling. For this, we have identified amino acids predicted to play a role in TIR NADase activity and have expressed and purified recombinant bacterial TIR proteins that have incorporated these mutations. We then tested the NADase activity of these mutants in comparison with that of wild-type TIR proteins and TIRs with mutations already known to inhibit NADase activity. We are now using mutant TIRs with deficiencies in NADase activity in pull-down assays to test their binding to MyD88 and utilizing reporter assays to analyze the effects of these mutant TIRs on NF-kB signaling. This information will allow us to gain a deeper understanding into the relationship between NADase activity in bacterial TIRs and their virulence activity, and can potentially uncover new mechanisms for pathogenic evasion of host immune defenses.

Cofactor F420: an expanded view of its distribution, biosynthesis and roles in bacteria and archaea.

ABSTRACTMany bacteria and archaea produce the redox cofactor F420. F420 is structurally similar to the cofactors FAD and FMN but is catalytically more similar to NAD and NADP. These properties allow F420 to catalyze challenging redox reactions, including key steps in methanogenesis, antibiotic biosynthesis and xenobiotic biodegradation. In the last 5 years, there has been much progress in understanding its distribution, biosynthesis, role and applications. Whereas F420 was previously thought to be confined to Actinobacteria and Euryarchaeota, new evidence indicates it is synthesized across the bacterial and archaeal domains, as a result of extensive horizontal and vertical biosynthetic gene transfer. F420 was thought to be synthesized through one biosynthetic pathway; however, recent advances have revealed variants of this pathway and have resolved their key biosynthetic steps. In parallel, new F420-dependent biosynthetic and metabolic processes have been discovered. These advances have enabled the heterologous production of F420 and identified enantioselective F420H2-dependent reductases for biocatalysis. New research has also helped resolve how microorganisms use F420 to influence human and environmental health, providing opportunities for tuberculosis treatment and methane mitigation. A total of 50 years since its discovery, multiple paradigms associated with F420 have shifted, and new F420-dependent organisms and processes continue to be discovered.

Spatiotemporal Manipulation of the Mismatch Repair System of Pseudomonas putida Accelerates Phenotype Emergence

The development of complex phenotypes in industrially relevant bacteria is a major goal of metabolic engineering, which encompasses the implementation of both rational and random approaches. In the latter case, several tools have been developed toward increasing mutation frequencies, yet the precise control of mutagenesis processes in cell factories continues to represent a significant technical challenge. Pseudomonas species are endowed with one of the most efficient DNA mismatch repair (MMR) systems found in the bacterial domain. Here, we investigated if the endogenous MMR system could be manipulated as a general strategy to artificially alter mutation rates in Pseudomonas species. To bestow a conditional mutator phenotype in the platform bacterium Pseudomonas putida, we constructed inducible mutator devices to modulate the expression of the dominant-negative mutLE36K allele. Regulatable overexpression of mutLE36K in a broad-host-range, easy-to-cure plasmid format resulted in a transitory inhibition of the MMR machinery, leading to a significant increase (up to 438-fold) in DNA mutation frequencies and a heritable fixation of mutations in the genome. Following such an accelerated mutagenesis-followed by selection approach, three phenotypes were successfully evolved: resistance to antibiotics streptomycin and rifampicin (either individually or combined) and reversion of a synthetic uracil auxotrophy. Thus, these mutator devices could be applied to accelerate the evolution of metabolic pathways in long-term evolutionary experiments, alternating cycles of (inducible) mutagenesis coupled to selection schemes toward the desired phenotype(s).

Bioprospecting Sulfuric Acid Assisted Hydrothermal Pretreatment of Sugarcane Bagasse and Microbial Community Structure for Methane Production

The complex chemical and structural arrangement of lignocellulosic biomass demand a pretreatment step to dismantle its coherent nature. The current study was designed to study H2SO4 impregnation before hydrothermal pretreatment (HP) of sugarcane bagasse for biogas production using response surface methodology based on defining factors: temperature (160, 180, 200 °C), time (5, 12, 19 min), and H2SO4 concentration (1, 2, 3% w/v). The H2SO4 impregnation had a paramount impact in combination with temperature and treatment time parameters. The lowest solid yield 12.3% was recorded in pretreatment run A-HSO (200 °C, 19 min, 3% H2SO4), up to 100% xylan removal was noted in four pretreatment runs (A-HSO; 200 °C, 19 min, 3% H2SO4, B-HSO; 200 °C, 19 min, 1% H2SO4, C-HSO; 200 °C, 5 min, 3% H2SO4, and O-HSO; 213.6 °C, 12 min, 2% H2SO4), maximum klason lignin increase in insoluble solid fraction (ISF) verified was 358.85% in pretreatment run O-HSO at 213.6 °C, while glucan content reduction (33.18–74.14%) was also observed in pretreatment runs A-HSO, C-HSO, and O-HSO. Results verified maximum methane recovery of 160.16 NmL (g TVS)−1 in pretreatment run I-SHO (180 °C, 12 min, 2% H2SO4). Microbial community analysis observed the predominance of Flavobacterium and Desulfosporosinus genera of Bacterial domain and Methanolinea of Archaea domain. H2SO4 impregnation before HP though not suitable for methane production from ISF could be employed in a biorefinery perspective to obtain valuables (chemicals and fuels) due to the production of organic acids rich liquid stream and lignin-rich solid fraction.