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Jiang, Bo, Rebecca Argyros, John Bukowski, Stephanie Nelson, Nathan Sharkey, Sehoon Kim, Victoria Copeland, et al. "Inactivation of aGAL4-Like Transcription Factor Improves Cell Fitness and Product Yield in Glycoengineered Pichia pastoris Strains." Applied and Environmental Microbiology 81, no. 1 (October 24, 2014): 260–71. http://dx.doi.org/10.1128/aem.02619-14.
Повний текст джерелаАнотація:
ABSTRACTWith a completely reengineered and humanized glycosylation pathway, glycoengineeredPichia pastorishas emerged as a promising production host for the manufacture of therapeutic glycoproteins. However, the extensive genetic modifications have also negatively affected the overall fitness levels of the glycoengineered host cells. To make glycoengineeredPichiastrains more compatible with a scalable industrial fermentation process, we sought to identify genetic solutions to broadly improve cell robustness during fermentation. In this study, we report that mutations within thePichia pastorisATT1(PpATT1) gene (a homolog of theSaccharomyces cerevisiaeGAL4[ScGAL4] transcriptional activator) dramatically increased the cellular fitness levels of glycoengineeredPichiastrains. We demonstrate that deletion of thePpATT1gene enabled glycoengineeredPichiastrains to improve their thermal tolerance levels, reduce their cell lysis defects, and greatly improve fermentation robustness. The extension of the duration of fermentation enabled thePpATT1-modified glycoengineeredPichiastrains to increase their product yields significantly without any sacrifice in product quality. Because theATT1gene could be deleted from anyPichiastrains, including empty hosts and protein-expressing production strains alike, we suggest that the findings described in this study are broadly applicable to anyPichiastrains used for the production of therapeutic proteins, including monoclonal antibodies, Fc fusions, peptides, hormones, and growth factors.
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Finn, Thomas J., Sonal Shewaramani, Sinead C. Leahy, Peter H. Janssen, and Christina D. Moon. "Dynamics and genetic diversification ofEscherichia coliduring experimental adaptation to an anaerobic environment." PeerJ 5 (May 3, 2017): e3244. http://dx.doi.org/10.7717/peerj.3244.
Повний текст джерелаАнотація:
BackgroundMany bacteria are facultative anaerobes, and can proliferate in both anoxic and oxic environments. Under anaerobic conditions, fermentation is the primary means of energy generation in contrast to respiration. Furthermore, the rates and spectra of spontaneous mutations that arise during anaerobic growth differ to those under aerobic growth. A long-term selection experiment was undertaken to investigate the genetic changes that underpin how the facultative anaerobe,Escherichia coli, adapts to anaerobic environments.MethodsTwenty-one populations ofE. coliREL4536, an aerobically evolved 10,000th generation descendent of theE. coliB strain, REL606, were established from a clonal ancestral culture. These were serially sub-cultured for 2,000 generations in a defined minimal glucose medium in strict aerobic and strict anaerobic environments, as well as in a treatment that fluctuated between the two environments. The competitive fitness of the evolving lineages was assessed at approximately 0, 1,000 and 2,000 generations, in both the environment of selection and the alternative environment. Whole genome re-sequencing was performed on random colonies from all lineages after 2,000-generations. Mutations were identified relative to the ancestral genome, and based on the extent of parallelism, traits that were likely to have contributed towards adaptation were inferred.ResultsThere were increases in fitness relative to the ancestor among anaerobically evolved lineages when tested in the anaerobic environment, but no increases were found in the aerobic environment. For lineages that had evolved under the fluctuating regime, relative fitness increased significantly in the anaerobic environment, but did not increase in the aerobic environment. The aerobically-evolved lineages did not increase in fitness when tested in either the aerobic or anaerobic environments. The strictly anaerobic lineages adapted more rapidly to the anaerobic environment than did the fluctuating lineages. Two main strategies appeared to predominate during adaptation to the anaerobic environment: modification of energy generation pathways, and inactivation of non-essential functions. Fermentation pathways appeared to alter through selection for mutations in genes such asnadR, adhE, dcuS/R, andpflB. Mutations were frequently identified in genes for presumably dispensable functions such as toxin-antitoxin systems, prophages, virulence and amino acid transport. Adaptation of the fluctuating lineages to the anaerobic environments involved mutations affecting traits similar to those observed in the anaerobically evolved lineages.DiscussionThere appeared to be strong selective pressure for activities that conferred cell yield advantages during anaerobic growth, which include restoring activities that had previously been inactivated under long-term continuous aerobic evolution of the ancestor.
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Staib, Lena, and Thilo M. Fuchs. "From food to cell: nutrient exploitation strategies of enteropathogens." Microbiology 160, no. 6 (June 1, 2014): 1020–39. http://dx.doi.org/10.1099/mic.0.078105-0.
Повний текст джерелаАнотація:
Upon entering the human gastrointestinal tract, foodborne bacterial enteropathogens encounter, among numerous other stress conditions, nutrient competition with the host organism and the commensal microbiota. The main carbon, nitrogen and energy sources exploited by pathogens during proliferation in, and colonization of, the gut have, however, not been identified completely. In recent years, a huge body of literature has provided evidence that most enteropathogens are equipped with a large set of specific metabolic pathways to overcome nutritional limitations in vivo, thus increasing bacterial fitness during infection. These adaptations include the degradation of myo-inositol, ethanolamine cleaved from phospholipids, fucose derived from mucosal glycoconjugates, 1,2-propanediol as the fermentation product of fucose or rhamnose and several other metabolites not accessible for commensal bacteria or present in competition-free microenvironments. Interestingly, the data reviewed here point to common metabolic strategies of enteric pathogens allowing the exploitation of nutrient sources that not only are present in the gut lumen, the mucosa or epithelial cells, but also are abundant in food. An increased knowledge of the metabolic strategies developed by enteropathogens is therefore a key factor to better control foodborne diseases.
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Ticinesi, Andrea, Antonio Nouvenne, Nicoletta Cerundolo, Pamela Catania, Beatrice Prati, Claudio Tana, and Tiziana Meschi. "Gut Microbiota, Muscle Mass and Function in Aging: A Focus on Physical Frailty and Sarcopenia." Nutrients 11, no. 7 (July 17, 2019): 1633. http://dx.doi.org/10.3390/nu11071633.
Повний текст джерелаАнотація:
Human gut microbiota is able to influence the host physiology by regulating multiple processes, including nutrient absorption, inflammation, oxidative stress, immune function, and anabolic balance. Aging is associated with reduced microbiota biodiversity, increased inter-individual variability, and over-representation of pathobionts, and these phenomena may have great relevance for skeletal muscle mass and function. For this reason, the presence of a gut-muscle axis regulating the onset and progression of age-related physical frailty and sarcopenia has been recently hypothesized. In this narrative review, we summarize the studies supporting a possible association between gut microbiota-related parameters with measures of muscle mass, muscle function, and physical performance in animal models and humans. Reduced muscle mass has been associated with distinct microbiota composition and reduced fermentative capacity in mice, and the administration of probiotics or butyrate to mouse models of muscle wasting has been associated with improved muscle mass. However, no studies have targeted the human microbiome associated with sarcopenia. Limited evidence from human studies shows an association between microbiota composition, involving key taxa such as Faecalibacterium and Bifidobacterium, and grip strength. Similarly, few studies conducted on patients with parkinsonism showed a trend towards a different microbiota composition in those with reduced gait speed. No studies have assessed the association of fecal microbiota with other measures of physical performance. However, several studies, mainly with a cross-sectional design, suggest an association between microbiota composition and frailty, mostly assessed according to the deficit accumulation model. Namely, frailty was associated with reduced microbiota biodiversity, and lower representation of butyrate-producing bacteria. Therefore, we conclude that the causal link between microbiota and physical fitness is still uncertain due to the lack of targeted studies and the influence of a large number of covariates, including diet, exercise, multimorbidity, and polypharmacy, on both microbiota composition and physical function in older age. However, the relationship between gut microbiota and physical function remains a very promising area of research for the future.
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Wang, Zhiying, Pan Li, Lixin Luo, David J. Simpson, and Michael G. Gänzle. "Daqu Fermentation Selects for Heat-ResistantEnterobacteriaceaeand Bacilli." Applied and Environmental Microbiology 84, no. 21 (August 17, 2018). http://dx.doi.org/10.1128/aem.01483-18.
Повний текст джерелаАнотація:
ABSTRACTDaqu is a spontaneous solid-state cereal fermentation used as saccharification and starter culture in Chinese vinegar and liquor production. The evolution of microbiota in this spontaneous fermentation is controlled by the temperature profile, which reaches temperatures from 50 to 65°C for several days. Despite these high temperatures, mesophilicEnterobacteriaceae(includingCronobacter) and bacilli are present throughout Daqu fermentation. This study aimed to determine whether Daqu spontaneous solid-state fermentation selects for heat-resistant variants of these organisms. Heat resistance inEnterobacteriaceaeis mediated by the locus of heat resistance (LHR). One LHR-positive strain ofKosakonia cowaniiwas identified in Daqu, and it exhibited higher heat resistance than the LHR-negativeK. cowaniiisolated from malted oats. Heat resistance inBacillusendospores is mediated by thespoVA2moboperon. Out of 10 Daqu isolates of the speciesBacillus licheniformis,Brevibacillus parabrevis,Bacillus subtilis,Bacillus amyloliquefaciens, andBacillus velezensis, 5 did not containspoVA2mob, 3 contained one copy, and 2 contained two copies. The presence and copy number of thespoVA2moboperon increased the resistance of spores to treatment with 110°C. To confirm the selection of LHR- andspoVA2mob-positive strains during Daqu fermentation, the copy numbers of these genetic elements in Daqu samples were quantified by quantitative PCR (qPCR). The abundance of LHR and thespoVA2moboperon in community DNA relative to that of total bacterial 16S rRNA genes increased 3-fold and 5-fold, respectively, during processing. In conclusion, culture-dependent and culture-independent analyses suggest that Daqu fermentation selects for heat-resistantEnterobacteriaceaeand bacilli.IMPORTANCEDaqu fermentations select for mobile genetic elements conferring heat resistance inEnterobacteriaceaeand bacilli. The locus of heat resistance (LHR), a genomic island conferring heat resistance inEnterobacteriaceae, and thespoVA2moboperon, conferring heat resistance on bacterial endospores, were enriched 3- to 5-fold during Daqu fermentation and maturation. It is therefore remarkable that the LHR and thespoVA2moboperon are accumulated in the same food fermentation. The presence of heat-resistantKosakoniaspp. andBacillusspp. in Daqu is not of concern for food safety; however, both genomic islands are mobile and transferable to pathogenic bacteria or toxin-producing bacteria by horizontal gene transfer. The identification of the LHR and thespoVA2moboperon as indicators of fitness ofEnterobacteriaceaeand bacilli in Daqu fermentation provides insights into environmental sources of heat-resistant organisms that may contaminate the food supply.
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Lopez, Christopher A., William N. Beavers, Andy Weiss, Reece J. Knippel, Joseph P. Zackular, Walter Chazin, and Eric P. Skaar. "The Immune Protein Calprotectin Impacts Clostridioides difficile Metabolism through Zinc Limitation." mBio 10, no. 6 (November 19, 2019). http://dx.doi.org/10.1128/mbio.02289-19.
Повний текст джерелаАнотація:
ABSTRACT The intestines house a diverse microbiota that must compete for nutrients to survive, but the specific limiting nutrients that control pathogen colonization are not clearly defined. Clostridioides difficile colonization typically requires prior disruption of the microbiota, suggesting that outcompeting commensals for resources is critical to establishing C. difficile infection (CDI). The immune protein calprotectin (CP) is released into the gut lumen during CDI to chelate zinc (Zn) and other essential nutrient metals. Yet, the impact of Zn limitation on C. difficile colonization is unknown. To define C. difficile responses to Zn limitation, we performed RNA sequencing on C. difficile exposed to CP. In medium containing CP, C. difficile upregulated genes involved in metal homeostasis and amino acid metabolism. To identify CP-responsive genes important during infection, we measured the abundance of select C. difficile transcripts in a mouse CDI model relative to expression in vitro. Gene transcripts involved in selenium (Se)-dependent proline fermentation increased during infection and in response to CP. Increased proline fermentation gene transcription was dependent on CP Zn binding and proline availability, yet proline fermentation was only enhanced when Se was supplemented. CP-deficient mice could not restrain C. difficile proline fermentation-dependent growth, suggesting that CP-mediated Zn sequestration along with limited Se restricts C. difficile proline fermentation. Overall, these results highlight how C. difficile colonization depends on the availability of multiple nutrients whose abundances are dynamically influenced by the host response. IMPORTANCE Clostridioides difficile infection (CDI) is the leading cause of postantibiotic nosocomial infection. Antibiotic therapy can be successful, yet up to one-third of individuals suffer from recurrent infections. Understanding the mechanisms controlling C. difficile colonization is paramount in designing novel treatments for primary and recurrent CDI. Here, we found that limiting nutrients control C. difficile metabolism during CDI and influence overall pathogen fitness. Specifically, the immune protein CP limits Zn availability and increases transcription of C. difficile genes necessary for proline fermentation. Paradoxically, this leads to reduced C. difficile proline fermentation. This reduced fermentation is due to limited availability of another nutrient required for proline fermentation, Se. Therefore, CP-mediated Zn limitation combined with low Se levels overall reduce C. difficile fitness in the intestines. These results emphasize the complexities of how nutrient availability influences C. difficile colonization and provide insight into critical metabolic processes that drive the pathogen’s growth.
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Vuillemin, Aurèle, Sergio Vargas, Ömer K. Coskun, Robert Pockalny, Richard W. Murray, David C. Smith, Steven D’Hondt, and William D. Orsi. "Atribacteria Reproducing over Millions of Years in the Atlantic Abyssal Subseafloor." mBio 11, no. 5 (October 6, 2020). http://dx.doi.org/10.1128/mbio.01937-20.
Повний текст джерелаАнотація:
ABSTRACT How microbial metabolism is translated into cellular reproduction under energy-limited settings below the seafloor over long timescales is poorly understood. Here, we show that microbial abundance increases an order of magnitude over a 5 million-year-long sequence in anoxic subseafloor clay of the abyssal North Atlantic Ocean. This increase in biomass correlated with an increased number of transcribed protein-encoding genes that included those involved in cytokinesis, demonstrating that active microbial reproduction outpaces cell death in these ancient sediments. Metagenomes, metatranscriptomes, and 16S rRNA gene sequencing all show that the actively reproducing community was dominated by the candidate phylum “Candidatus Atribacteria,” which exhibited patterns of gene expression consistent with fermentative, and potentially acetogenic, metabolism. “Ca. Atribacteria” dominated throughout the 8 million-year-old cored sequence, despite the detection limit for gene expression being reached in 5 million-year-old sediments. The subseafloor reproducing “Ca. Atribacteria” also expressed genes encoding a bacterial microcompartment that has potential to assist in secondary fermentation by recycling aldehydes and, thereby, harness additional power to reduce ferredoxin and NAD+. Expression of genes encoding the Rnf complex for generation of chemiosmotic ATP synthesis were also detected from the subseafloor “Ca. Atribacteria,” as well as the Wood-Ljungdahl pathway that could potentially have an anabolic or catabolic function. The correlation of this metabolism with cytokinesis gene expression and a net increase in biomass over the million-year-old sampled interval indicates that the “Ca. Atribacteria” can perform the necessary catabolic and anabolic functions necessary for cellular reproduction, even under energy limitation in millions-of-years-old anoxic sediments. IMPORTANCE The deep subseafloor sedimentary biosphere is one of the largest ecosystems on Earth, where microbes subsist under energy-limited conditions over long timescales. It remains poorly understood how mechanisms of microbial metabolism promote increased fitness in these settings. We discovered that the candidate bacterial phylum “Candidatus Atribacteria” dominated a deep-sea subseafloor ecosystem, where it exhibited increased transcription of genes associated with acetogenic fermentation and reproduction in million-year-old sediment. We attribute its improved fitness after burial in the seabed to its capabilities to derive energy from increasingly oxidized metabolites via a bacterial microcompartment and utilize a potentially reversible Wood-Ljungdahl pathway to help meet anabolic and catabolic requirements for growth. Our findings show that “Ca. Atribacteria” can perform all the necessary catabolic and anabolic functions necessary for cellular reproduction, even under energy limitation in anoxic sediments that are millions of years old.
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Zeilinger, K., J. Hellmich, J. Zentek, and W. Vahjen. "Novel ex vivo screening assay to preselect farm specific pre- and probiotics in pigs." Beneficial Microbes, August 23, 2021, 1–16. http://dx.doi.org/10.3920/bm2020.0226.
Повний текст джерелаАнотація:
A novel rapid ex vivo assay was developed as part of a concept to determine potential tailor-made combinations of pre- and probiotics for individual farms. Sow faecal slurries from 20 German pig farms were anaerobically incubated with pre- and probiotics or their combinations together with pathogenic strains that are of interest in pig production. Aliquots of these slurries were then incubated with media containing antibiotic mixtures allowing only growth of the specific pathogen. Growth was monitored and lag time was used to determine the residual fitness of the pathogenic strains. The background growth could be inhibited for an Escherichia coli- and a Clostridium difficile- but not for a Clostridium perfringens strain. The prebiotic fructo-oligosaccharides (FOS) and its combination with probiotics reduced the residual fitness of the E. coli strain in some farms. However, notable exceptions occurred in other farms where FOS increased the fitness of the E. coli strain. Generally, combinations of pre- and probiotics did not show additive effects on fitness for E. coli but displayed farm dependent differences. The effects of pre- and probiotics on the residual fitness of the C. difficile strain were less pronounced, but distinct differences between single application of prebiotics and their combination with probiotics were observed. It was concluded that the initial composition of the microbiota in the samples was more determinative for incubations with the C. difficile strain than for incubations with the E. coli strain, as the presumed fermentation of prebiotic products showed less influence on the fitness of the C. difficile strain. Farm dependent differences were pronounced for both pathogenic strains and therefore, this novel screening method offers a promising approach for pre-selecting pre- and probiotics for individual farms. However, evaluation of farm metadata (husbandry, feed, management) will be crucial in future studies to determine a tailor-made solution for combinations of pre- and probiotics for individual farms. Also, refinement of the ex vivo assay in terms of on-farm processing of samples and validation of unambiguous growth for pathogenic strains from individual farms should be addressed.
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Andrews, Fraser, Matthew Faulkner, Helen S. Toogood, and Nigel S. Scrutton. "Combinatorial use of environmental stresses and genetic engineering to increase ethanol titres in cyanobacteria." Biotechnology for Biofuels 14, no. 1 (December 2021). http://dx.doi.org/10.1186/s13068-021-02091-w.
Повний текст джерелаАнотація:
AbstractCurrent industrial bioethanol production by yeast through fermentation generates carbon dioxide. Carbon neutral bioethanol production by cyanobacteria uses biological fixation (photosynthesis) of carbon dioxide or other waste inorganic carbon sources, whilst being sustainable and renewable. The first ethanologenic cyanobacterial process was developed over two decades ago using Synechococcus elongatus PCC 7942, by incorporating the recombinant pdc and adh genes from Zymomonas mobilis. Further engineering has increased bioethanol titres 24-fold, yet current levels are far below what is required for industrial application. At the heart of the problem is that the rate of carbon fixation cannot be drastically accelerated and carbon partitioning towards bioethanol production impacts on cell fitness. Key progress has been achieved by increasing the precursor pyruvate levels intracellularly, upregulating synthetic genes and knocking out pathways competing for pyruvate. Studies have shown that cyanobacteria accumulate high proportions of carbon reserves that are mobilised under specific environmental stresses or through pathway engineering to increase ethanol production. When used in conjunction with specific genetic knockouts, they supply significantly more carbon for ethanol production. This review will discuss the progress in generating ethanologenic cyanobacteria through chassis engineering, and exploring the impact of environmental stresses on increasing carbon flux towards ethanol production.
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Link, Tobias, and Matthias A. Ehrmann. "Transcriptomic profiling reveals differences in the adaptation of two Tetragenococcus halophilus strains to a lupine moromi model medium." BMC Microbiology 23, no. 1 (January 14, 2023). http://dx.doi.org/10.1186/s12866-023-02760-w.
Повний текст джерелаАнотація:
Abstract Background Tetragenococcus (T.) halophilus is a common member of the microbial consortia of food fermented under high salt conditions. These comprises salty condiments based on soy or lupine beans, fish sauce, shrimp paste and brined anchovies. Within these fermentations this lactic acid bacterium (LAB) is responsible for the formation of lactic and other short chain acids that contribute to the flavor and lower the pH of the product. In this study, we investigated the transcriptomic profile of the two T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine moromi model medium supplied with galactose. To get further insights into which genomic trait is important, we used a setup with two strains. That way we can determine if strain dependent pathways contribute to the overall fitness. These strains differ in the ability to utilize L-arginine, L-aspartate, L-arabinose, D-sorbitol, glycerol, D-lactose or D-melibiose. The lupine moromi model medium is an adapted version of the regular MRS medium supplied with lupine peptone instead of casein peptone and meat extract, to simulate the amino acid availabilities in lupine moromi. Results The transcriptomic profiles of the T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine peptone-based model media supplied with galactose, used as simulation media for a lupine seasoning sauce fermentation, were compared to the determine potentially important traits. Both strains, have a great overlap in their response to the culture conditions but some strain specific features such as the utilization of glycerol, sorbitol and arginine contribute to the overall fitness of the strain TMW 2.2256. Interestingly, although both strains have two non-identical copies of the tagatose-6P pathway and the Leloir pathway increased under the same conditions, TMW 2.2256 prefers the degradation via the tagatose-6P pathway while TMW 2.2254 does not. Furthermore, TMW 2.2256 shows an increase in pathways required for balancing out the intracellular NADH/NADH+ ratios. Conclusions Our study reveals for the first time, that both versions of tagatose-6P pathways encoded in both strains are simultaneously active together with the Leloir pathway and contribute to the degradation of galactose. These findings will help to understand the strain dependent features that might be required for a starter strain in lupine moromi.
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Raghavendran, Vijayendran, Christian Marx, Lisbeth Olsson, and Maurizio Bettiga. "The protective role of intracellular glutathione in Saccharomyces cerevisiae during lignocellulosic ethanol production." AMB Express 10, no. 1 (December 2020). http://dx.doi.org/10.1186/s13568-020-01148-7.
Повний текст джерелаАнотація:
AbstractTo enhance the competitiveness of industrial lignocellulose ethanol production, robust enzymes and cell factories are vital. Lignocellulose derived streams contain a cocktail of inhibitors that drain the cell of its redox power and ATP, leading to a decrease in overall ethanol productivity. Many studies have attempted to address this issue, and we have shown that increasing the glutathione (GSH) content in yeasts confers tolerance towards lignocellulose inhibitors, subsequently increasing the ethanol titres. However, GSH levels in yeast are limited by feedback inhibition of GSH biosynthesis. Multidomain and dual functional enzymes exist in several bacterial genera and they catalyse the GSH biosynthesis in a single step without the feedback inhibition. To test if even higher intracellular glutathione levels could be achieved and if this might lead to increased tolerance, we overexpressed the genes from two bacterial genera and assessed the recombinants in simultaneous saccharification and fermentation (SSF) with steam pretreated spruce hydrolysate containing 10% solids. Although overexpressing the heterologous genes led to a sixfold increase in maximum glutathione content (18 µmol gdrycellmass−1) compared to the control strain, this only led to a threefold increase in final ethanol titres (8.5 g L− 1). As our work does not conclusively indicate the cause-effect of increased GSH levels towards ethanol titres, we cautiously conclude that there is a limit to cellular fitness that could be accomplished via increased levels of glutathione.
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LaSarre, Breah, Adam M. Deutschbauer, Crystal E. Love, and James B. McKinlay. "Covert Cross-Feeding Revealed by Genome-Wide Analysis of Fitness Determinants in a Synthetic Bacterial Mutualism." Applied and Environmental Microbiology 86, no. 13 (April 24, 2020). http://dx.doi.org/10.1128/aem.00543-20.
Повний текст джерелаАнотація:
ABSTRACT Microbial interactions abound in natural ecosystems and shape community structure and function. Substantial attention has been given to cataloging mechanisms by which microbes interact, but there is a limited understanding of the genetic landscapes that promote or hinder microbial interactions. We previously developed a mutualistic coculture pairing Escherichia coli and Rhodopseudomonas palustris, wherein E. coli provides carbon to R. palustris in the form of glucose fermentation products and R. palustris fixes N2 gas and provides nitrogen to E. coli in the form of NH4+. The stable coexistence and reproducible trends exhibited by this coculture make it ideal for interrogating the genetic underpinnings of a cross-feeding mutualism. Here, we used random barcode transposon sequencing (RB-TnSeq) to conduct a genome-wide search for E. coli genes that influence fitness during cooperative growth with R. palustris. RB-TnSeq revealed hundreds of genes that increased or decreased E. coli fitness in a mutualism-dependent manner. Some identified genes were involved in nitrogen sensing and assimilation, as expected given the coculture design. The other identified genes were involved in diverse cellular processes, including energy production and cell wall and membrane biogenesis. In addition, we discovered unexpected purine cross-feeding from R. palustris to E. coli, with coculture rescuing growth of an E. coli purine auxotroph. Our data provide insight into the genes and gene networks that can influence a cross-feeding mutualism and underscore that microbial interactions are not necessarily predictable a priori. IMPORTANCE Microbial communities impact life on Earth in profound ways, including driving global nutrient cycles and influencing human health and disease. These community functions depend on the interactions that resident microbes have with the environment and each other. Thus, identifying genes that influence these interactions will aid the management of natural communities and the use of microbial consortia as biotechnology. Here, we identified genes that influenced Escherichia coli fitness during cooperative growth with a mutualistic partner, Rhodopseudomonas palustris. Although this mutualism centers on the bidirectional exchange of essential carbon and nitrogen, E. coli fitness was positively and negatively affected by genes involved in diverse cellular processes. Furthermore, we discovered an unexpected purine cross-feeding interaction. These results contribute knowledge on the genetic foundation of a microbial cross-feeding interaction and highlight that unanticipated interactions can occur even within engineered microbial communities.
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Hamdallah, Issam, Nadia Torok, Katarina M. Bischof, Nadim Majdalani, Sriya Chadalavada, Nonto Mdluli, Kaitlin E. Creamer, et al. "Experimental Evolution ofEscherichia coliK-12 at High pH and with RpoS Induction." Applied and Environmental Microbiology 84, no. 15 (May 25, 2018). http://dx.doi.org/10.1128/aem.00520-18.
Повний текст джерелаАнотація:
ABSTRACTExperimental evolution ofEscherichia coliK-12 W3110 by serial dilutions for 2,200 generations at high pH extended the range of sustained growth from pH 9.0 to pH 9.3. pH 9.3-adapted isolates showed mutations in DNA-binding regulators and envelope proteins. One population showed an IS1knockout ofphoB(encoding the positive regulator of the phosphate regulon). AphoB::kanRknockout increased growth at high pH.phoBmutants are known to increase production of fermentation acids, which could enhance fitness at high pH. Mutations inpcnB[poly(A) polymerase] also increased growth at high pH. Three out of four populations showed deletions oftorI, an inhibitor of TorR, which activates expression oftorCAD(trimethylamineN-oxide respiration) at high pH. All populations showed point mutations affecting the stationary-phase sigma factor RpoS, either in the coding gene or in genes for regulators of RpoS expression. RpoS is required for survival at extremely high pH. In our microplate assay,rpoSdeletion slightly decreased growth at pH 9.1. RpoS protein accumulated faster at pH 9 than at pH 7. The RpoS accumulation at high pH required the presence of one or more antiadaptors that block degradation (IraM, IraD, and IraP). Other genes with mutations after high-pH evolution encode regulators, such as those encoded byyobG(mgrB) (PhoPQ regulator),rpoN(nitrogen starvation sigma factor),malI, andpurR, as well as envelope proteins, such as those encoded byompTandyahO. Overall,E. colievolution at high pH selects for mutations in key transcriptional regulators, includingphoBand the stationary-phase sigma factor RpoS.IMPORTANCEEscherichia coliin its native habitat encounters high-pH stress such as that of pancreatic secretions. Experimental evolution over 2,000 generations showed selection for mutations in regulatory factors, such as deletion of the phosphate regulator PhoB and mutations that alter the function of the global stress regulator RpoS. RpoS is induced at high pH via multiple mechanisms.
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Pagliaccia, Deborah, Sohrab Bodaghi, Xingyu Chen, Danielle Stevenson, Elizabeth Deyett, Agustina De Francesco, James Borneman, et al. "Two Food Waste By-Products Selectively Stimulate Beneficial Resident Citrus Host-Associated Microbes in a Zero-Runoff Indoor Plant Production System." Frontiers in Sustainable Food Systems 4 (December 14, 2020). http://dx.doi.org/10.3389/fsufs.2020.593568.
Повний текст джерелаАнотація:
The global production of food waste is a far-reaching problem with sizable financial, ethical, social, and environmental costs. Over 66 million tons of food waste is produced annually in the United States alone. This waste can be converted into valuable digestate by-products that promote a circular economy within agri-food systems. The present work investigated the use of two liquid digestates of microaerobic fermentation from mixed food waste and beer mash, respectively, as biostimulants for non-bearing citrus plants (nursery stock) grown in a zero-runoff greenhouse system with recirculating irrigation. The digestates' impact on the structure and diversity of the microbiota was determined on the irrigation water, soil, leaves, roots, and rhizosphere of citrus plants. A combination of culture-dependent (selective media) and culture-independent approaches (Next-Generation Sequencing) was used to assess the composition of the microbial communities and to single out the presence of foodborne pathogens. Our results suggest that the use of digestates is safe (i.e., no human or plant pathogens were present in the digestates or enriched in the plant production system following amendments). Digestates application to the irrigation water reduced the bacterial diversity within 24–48 h and selectively and significantly stimulated beneficial resident host-associated microorganisms (Pseudomonas putida) by two to three orders of magnitude. Carbon dynamics were analyzed in the nutrient solutions by measuring dissolved organic carbon and characterizing carbon species through gas chromatography-electron ionization-mass spectrometry. Our results indicate that dissolved organic carbon in the recirculating irrigation water spikes after each digestate amendment and it is quickly metabolized by bacteria, plateauing 24 h after application. Soil carbon, nitrogen, and nutrient dynamics were also analyzed, and results suggest that digestates increased the concentration of some plant nutrients in soils without causing a surge of potentially toxic elements. This study represents a proof-of-concept for the safe re-use of organic wastes, from farming and consumers, in agriculture. Implementing this type of integrated plant production system could reduce the environmental impact of food waste and benefit the public by improving soil health, reducing agricultural footprint, and increasing crop fitness by deploying a method based on a circular economy and sustainable food production approaches.
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Russell, Shonagh, Liping Xu, Yoonseok Kam, Dominique Abrahams, Bryce Ordway, Alex S. Lopez, Marilyn M. Bui, et al. "Proton export upregulates aerobic glycolysis." BMC Biology 20, no. 1 (July 15, 2022). http://dx.doi.org/10.1186/s12915-022-01340-0.
Повний текст джерелаАнотація:
Abstract Introduction Aggressive cancers commonly ferment glucose to lactic acid at high rates, even in the presence of oxygen. This is known as aerobic glycolysis, or the “Warburg Effect.” It is widely assumed that this is a consequence of the upregulation of glycolytic enzymes. Oncogenic drivers can increase the expression of most proteins in the glycolytic pathway, including the terminal step of exporting H+ equivalents from the cytoplasm. Proton exporters maintain an alkaline cytoplasmic pH, which can enhance all glycolytic enzyme activities, even in the absence of oncogene-related expression changes. Based on this observation, we hypothesized that increased uptake and fermentative metabolism of glucose could be driven by the expulsion of H+ equivalents from the cell. Results To test this hypothesis, we stably transfected lowly glycolytic MCF-7, U2-OS, and glycolytic HEK293 cells to express proton-exporting systems: either PMA1 (plasma membrane ATPase 1, a yeast H+-ATPase) or CA-IX (carbonic anhydrase 9). The expression of either exporter in vitro enhanced aerobic glycolysis as measured by glucose consumption, lactate production, and extracellular acidification rate. This resulted in an increased intracellular pH, and metabolomic analyses indicated that this was associated with an increased flux of all glycolytic enzymes upstream of pyruvate kinase. These cells also demonstrated increased migratory and invasive phenotypes in vitro, and these were recapitulated in vivo by more aggressive behavior, whereby the acid-producing cells formed higher-grade tumors with higher rates of metastases. Neutralizing tumor acidity with oral buffers reduced the metastatic burden. Conclusions Therefore, cancer cells which increase export of H+ equivalents subsequently increase intracellular alkalization, even without oncogenic driver mutations, and this is sufficient to alter cancer metabolism towards an upregulation of aerobic glycolysis, a Warburg phenotype. Overall, we have shown that the traditional understanding of cancer cells favoring glycolysis and the subsequent extracellular acidification is not always linear. Cells which can, independent of metabolism, acidify through proton exporter activity can sufficiently drive their metabolism towards glycolysis providing an important fitness advantage for survival.