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Slow decomposition rates of cereal crop residues can lead to agronomic challenges, such as nutrient immobilization, delayed soil warming, and increased pest pressures. In this regard, microbial inoculation with efficient strains offers a viable and eco-friendly solution to accelerating the decomposition process of crop residues. However, this solution often focuses mostly on selecting microorganisms based on the appropriate enzymic capabilities and neglects the metabolic versatility required to utilize both structural and non-structural components of residues. Therefore, this study aimed to address these limitations by assessing the metabolic profiles of five previously identified cellulolytic bacterial strains, including Bacillus pumilus 1G17, Micromonospora chalcea 1G49, Bacillus mobilis 5G17, Streptomyces canus 1TG5, and Streptomyces achromogenes 3TG21 using Biolog Phenotype Microarray analysis. Moreover, this study evaluated the impact of wheat straw inoculation with single strains and a bacterial consortium on soil organic carbon and nitrogen content in a pot experiment. Results revealed that, beyond the core subset of 12 carbon sources, the strains exhibited diverse metabolic capacities in utilizing 106 carbon sources. All strains demonstrated effective straw biomass degradation compared to the negative control, with significant differences detected only in oil seed rape straw biodegradation estimations. Furthermore, wheat straw inoculated with a bacterial consortium showed a significant increase in soil organic carbon content after 180 days in the pot experiment. Overall, these findings underscore the critical role of metabolic profiling in gaining a deeper understanding of microbial capabilities and addressing the complexities of residue composition and environmental variability. [ABSTRACT FROM AUTHOR]

Aims Carbon source is a necessary nutrient for bacterial strain growth. In industrial production, the cost of using different carbon sources varies greatly. Moreover, the complex environment in space may cause metabolic a series of changes in the strain, and this method has been successfully applied in some basic research. To date, space mutagenesis is still limited number of studies, particularly in carbon metabolism of probiotics. Methods and results HG-R7970-41 was isolated from bacterium suspension (Probio-M9) after space flight, which can produce capsular polysaccharide after space mutagenesis. Phenotype Microarray (PM) was used to evaluated the metabolism of HG-R7970-41 in 190 single carbon sources. RNA sequencing and total protein identification of two strains revealed their different carbon metabolism mechanisms. PM results demonstrated the metabolism of 10 carbon sources were different between Probio-M9 and HG-R7970-41. Transcriptomic and proteomic analyses revealed that this change in carbon metabolism of HG-R7970-41 mainly related to changes in phosphorylation and the glycolysis pathway. Based on the metabolic mechanism of different carbon sources and related gene cluster analysis, we found that the final metabolic activities of HG-R7970-41 and Probio-M9 were mainly regulated by PTS-specific membrane embedded permease, carbohydrate kinase and two rate-limiting enzymes (phosphofructokinase and pyruvate kinase) in the glycolysis pathway. The expanded culture test also confirmed that HG-R7970-41 had different metabolic characteristics from original strain. Conclusions These results suggested that space environment could change carbon metabolism of Probio-M9. The new isolate (HG-R7970-41) showed a different carbon metabolism pattern from the original strain mainly by the regulation of two rate-limiting enzymes. [ABSTRACT FROM AUTHOR]

Slow decomposition rates of cereal crop residues can lead to agronomic challenges, such as nutrient immobilization, delayed soil warming, and increased pest pressures. In this regard, microbial inoculation with efficient strains offers a viable and eco-friendly solution to accelerating the decomposition process of crop residues. However, this solution often focuses mostly on selecting microorganisms based on the appropriate enzymic capabilities and neglects the metabolic versatility required to utilize both structural and non-structural components of residues. Therefore, this study aimed to address these limitations by assessing the metabolic profiles of five previously identified cellulolytic bacterial strains, including Bacillus pumilus 1G17, Micromonospora chalcea 1G49, Bacillus mobilis 5G17, Streptomyces canus 1TG5, and Streptomyces achromogenes 3TG21 using Biolog Phenotype Microarray analysis. Moreover, this study evaluated the impact of wheat straw inoculation with single strains and a bacterial consortium on soil organic carbon and nitrogen content in a pot experiment. Results revealed that, beyond the core subset of 12 carbon sources, the strains exhibited diverse metabolic capacities in utilizing 106 carbon sources. All strains demonstrated effective straw biomass degradation compared to the negative control, with significant differences detected only in oil seed rape straw biodegradation estimations. Furthermore, wheat straw inoculated with a bacterial consortium showed a significant increase in soil organic carbon content after 180 days in the pot experiment. Overall, these findings underscore the critical role of metabolic profiling in gaining a deeper understanding of microbial capabilities and addressing the complexities of residue composition and environmental variability.

A microbial fungicide developed from Bacillus subtilis NCD-2 has been registered for suppressing verticillium wilt in crops in China. Spores are the main ingredient of this fungicide and play a crucial role in suppressing plant disease. Therefore, increasing the number of spores of strain NCD-2 during fermentation is important for reducing the cost of the fungicide. In this study, five kinds of carbon sources were found to promote the metabolism of strain NCD-2 revealed via Biolog Phenotype MicroArray (PM) technology. L-arabinose showed the strongest ability to promote the growth and sporulation of strain NCD-2. L-arabinose increased the bacterial concentration and the sporulation efficiency of strain NCD-2 by 2.04 times and 1.99 times compared with D-glucose, respectively. Moreover, L-arabinose significantly decreased the autolysis of strain NCD-2. Genes associated with arabinose metabolism, sporulation, spore resistance to heat, and spore coat formation were significantly up-regulated, and genes associated with sporulation-delaying protein were significantly down-regulated under L-arabinose treatment. The deletion of msmX, which is involved in arabinose transport in the Bacillus genus, decreased growth and sporulation by 53.71% and 86.46% compared with wild-type strain NCD-2, respectively. Complementing the mutant strain by importing an intact msmX gene restored the strain's growth and sporulation. [ABSTRACT FROM AUTHOR]

Abstract Background Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium used to treat various gastrointestinal diseases. EcN is increasingly being used as a chassis for the engineering of advanced microbiome therapeutics. To aid in future engineering efforts, our aim was to construct an updated metabolic model of EcN with extended secondary metabolite representation. Results An updated high-quality genome-scale metabolic model of EcN, iHM1533, was developed based on comparison with 55 E. coli/Shigella reference GEMs and manual curation, including expanded secondary metabolite pathways (enterobactin, salmochelins, aerobactin, yersiniabactin, and colibactin). The model was validated and improved using phenotype microarray data, resulting in an 82.3% accuracy in predicting growth phenotypes on various nutrition sources. Flux variability analysis with previously published 13C fluxomics data validated prediction of the internal central carbon fluxes. A standardised test suite called Memote assessed the quality of iHM1533 to have an overall score of 89%. The model was applied by using constraint-based flux analysis to predict targets for optimisation of secondary metabolite production. Modelling predicted design targets from across amino acid metabolism, carbon metabolism, and other subsystems that are common or unique for influencing the production of various secondary metabolites. Conclusion iHM1533 represents a well-annotated metabolic model of EcN with extended secondary metabolite representation. Phenotype characterisation and the iHM1533 model provide a better understanding of the metabolic capabilities of EcN and will help future metabolic engineering efforts.

The surveillance of foods for Salmonella is hindered by bias in common enrichment media where serovars implicated in human illness are outgrown by less virulent serovars. We examined four Salmonella serovars, two common in human illness (Enteritidis and Typhimurium) and two that often dominate enrichments (Give and Kentucky), for factors that might influence culture bias. The four serovars had similar growth kinetics in Tryptic Soy Broth and Buffered Peptone Water. Phenotype microarray analysis with 950 chemical substrates to assess nutrient utilization and stress resistance revealed phenotype differences between serovars. Strains of S. Enteritidis had better utilization of plant-derived sugars such as xylose, mannitol, rhamnose, and fructose, while S. Typhimurium strains were able to metabolize tagatose. Strains of S. Kentucky used more compounds as phosphorus sources and grew better with inorganic phosphate as the sole phosphorus source. The sequences of nine genes involved in phosphate metabolism were compared, and there were differences between serovars in the catalytic ATP-binding domain of the histidine kinase phoR. Analysis of the predicted PhoR amino acid sequences from additional Salmonella genomes indicated a conservation of sequences each within the Typhimurium, Give, and Enteritidis serovars. However, three different PhoR versions were observed in S. Kentucky. [ABSTRACT FROM AUTHOR]

Background: Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium used to treat various gastrointestinal diseases. EcN is increasingly being used as a chassis for the engineering of advanced microbiome therapeutics. To aid in future engineering efforts, our aim was to construct an updated metabolic model of EcN with extended secondary metabolite representation. Results: An updated high-quality genome-scale metabolic model of EcN, iHM1533, was developed based on comparison with 55 E. coli/Shigella reference GEMs and manual curation, including expanded secondary metabolite pathways (enterobactin, salmochelins, aerobactin, yersiniabactin, and colibactin). The model was validated and improved using phenotype microarray data, resulting in an 82.3% accuracy in predicting growth phenotypes on various nutrition sources. Flux variability analysis with previously published 13C fluxomics data validated prediction of the internal central carbon fluxes. A standardised test suite called Memote assessed the quality of iHM1533 to have an overall score of 89%. The model was applied by using constraint-based flux analysis to predict targets for optimisation of secondary metabolite production. Modelling predicted design targets from across amino acid metabolism, carbon metabolism, and other subsystems that are common or unique for influencing the production of various secondary metabolites. Conclusion: iHM1533 represents a well-annotated metabolic model of EcN with extended secondary metabolite representation. Phenotype characterisation and the iHM1533 model provide a better understanding of the metabolic capabilities of EcN and will help future metabolic engineering efforts. [ABSTRACT FROM AUTHOR]

Herein, Bacillus subtilis PBE-8′s biocontrol efficacy was evaluated through physiological and metabolic approaches against Fusarium oxysporum f.sp. lycopersici (FOL). The study elaborates on PBE-8′s cell-free filtrate (CFF) antifungal activity through mycelial growth inhibition, metabolite profiling, and substrates utilization patterns. Additionally, under different CFF concentrations, reduction in spore count (94%–55%), biomass (50%), and cytoplasmic bulbous protrusions in mycelia were also observed. Furthermore, the effect of bacterial CFF on FOL metabolism was confirmed through GC-MS. CFF suppresses the concentration of aliphatic amino acids like L-valine, L-leucine, L-Isoleucine, glycine, and fatty acids such as linoleic acid and α- linolenic acid during the co-culturing conditions, which are essential for pathogenicity and resistance against host's systemic acquired resistance. The phenotype microarray assay revealed that CFF-treated FOL shows phenotype loss in 507 (56.58%) out of 896 substrates. Among 507, twenty-seven substrates showed significant phenotype loss, among which four substrates such as L-glutamic acid, L-glutamine, ammonia, and L-arginine are common in different crucial metabolic pathways of FOL, like alanine, aspartate, and glutamate metabolism, arginine and proline, carbon metabolism, arginine biosynthesis, nitrogen metabolism, amino-acyl tRNA synthesis, and biosynthesis of amino acids. The results suggest that PBE-8 CFF has certain antifungal metabolites that hinder the fungal metabolic pathways. [ABSTRACT FROM AUTHOR]

The surveillance of foods for Salmonella is hindered by bias in common enrichment media where serovars implicated in human illness are outgrown by less virulent serovars. We examined four Salmonella serovars, two common in human illness (Enteritidis and Typhimurium) and two that often dominate enrichments (Give and Kentucky), for factors that might influence culture bias. The four serovars had similar growth kinetics in Tryptic Soy Broth and Buffered Peptone Water. Phenotype microarray analysis with 950 chemical substrates to assess nutrient utilization and stress resistance revealed phenotype differences between serovars. Strains of S. Enteritidis had better utilization of plant-derived sugars such as xylose, mannitol, rhamnose, and fructose, while S. Typhimurium strains were able to metabolize tagatose. Strains of S. Kentucky used more compounds as phosphorus sources and grew better with inorganic phosphate as the sole phosphorus source. The sequences of nine genes involved in phosphate metabolism were compared, and there were differences between serovars in the catalytic ATP-binding domain of the histidine kinase phoR. Analysis of the predicted PhoR amino acid sequences from additional Salmonella genomes indicated a conservation of sequences each within the Typhimurium, Give, and Enteritidis serovars. However, three different PhoR versions were observed in S. Kentucky.

Food preservatives are crucial in fruit production, but fungal resistance is a challenge. The main objective was to compare the sensitivity of Neosartorya spp. isolates to preservatives used in food security applications and to assess the role of metabolic properties in shaping Neosartorya spp. resistance. Sodium metabisulfite, potassium sorbate, sodium bisulfite and sorbic acid showed inhibitory effects, with sodium metabisulfite the most effective. Tested metabolic profiles included fungal growth intensity and utilization of amines and amides, amino acids, polymers, carbohydrates and carboxylic acids. Significant decreases in the utilization of all tested organic compound guilds were observed after fungal exposure to food preservatives compared to the control. Although the current investigation was limited in the number of predominately carbohydrate substrates and the breadth of metabolic responses, extensive sensitivity panels are logical step in establishing a course of action against spoilage agents in food production being important approach for innovative food chemistry. [Display omitted] • Tested preservatives impeded Neosartorya spp. growth in disc-diffusion inhibition test. • Sodium metabisulfite inhibits Neosartorya spp. growth and decreases fungal biomass production on tested carbon sources. • Amino acids were the least used for Neosartorya spp. growth in optimal cultivation conditions. • Sodium cyanate, copper (II) chloride dihydrate, cisplatin and hydroxyurea caused the most prominent growth inhibition. • Sensitivity panels are effective against spoilage agents in food production and an innovative approach for food chemistry. [ABSTRACT FROM AUTHOR]

The increased resistance of bacteria to antimicrobials, as well as the growing interest in innovative and sustainable alternatives to traditional food additives, are driving research towards the use of natural food preservatives. Among these, hydrolates (HYs) have gained attention as "mild" alternatives to conventional antimicrobial compounds. In this study, the response of L. monocytogenes ATCC 7644 exposed to increasing concentrations of Coridothymus capitatus HY (CHY) for 1 h at 37 °C was evaluated by means of Phenotype Microarray, modelling the kinetic data obtained by inoculating control and treated cells into GEN III microplates, after CHY removal. The results revealed differences concerning the growth dynamics in environmental conditions commonly encountered in food processing environments (different carbon sources, pH 6.0, pH 5.0, 1–8% NaCl). More specifically, for treated cells, the lag phase was extended, the growth rate was slowed down and, in most cases, the maximum concentration was diminished, suggesting the persistence of stress even after CHY removal. Confocal Laser Scanner Microscopy evidenced a diffuse aggregation and suffering of the treated cells, as a response to the stress encountered. In conclusion, the treatment with HY caused a stressing effect that persisted after its removal. The results suggest the potential of CHY application to control L. monocytogenes in food environments. [ABSTRACT FROM AUTHOR]

The phenotype-genotype landscape is a projection coming from detailed phenotypic and genotypic data under environmental pressure. Although phenome of microbes or microbial consortia mirrors the functional expression of a genome or set of genomes, metabolic traits rely on the phenotype. Phenomics has the potential to revolution functional genomics. In this review, we discuss why and how phenomics was developed. We described how phenomics may extend our understanding of the assembly of microbial consortia and their functionality, and then we outlined the novel applications within the study of phenomes using Omnilog platform together with a revision of its current application to study lactic acid bacteria (LAB) metabolic traits during food processing. LAB were proposed as a suitable model system to analyze and discuss the implementation and exploitation of this emerging omics approach. We introduced the ‘phenotype switching’, as a new phenotype microarray approach to get insights in bacterial physiology. An overview of methodologies and tools to manage and analyze the generated data was provided. Finally, pro and cons of pipelines developed so far, including the most innovative ones were critically analyzed. We propose an R pipeline, recently deposited, which allows to automatically analyze Omnilog data integrating the latest approaches and implementing the new concepts described here.

A central unresolved issue in evolutionary biology is how metabolic innovations emerge. Low-level enzymatic side activities are frequent and can potentially be recruited for new biochemical functions. However, the role of such underground reactions in adaptation toward novel environments has remained largely unknown and out of reach of computational predictions, not least because these issues demand analyses at the level of the entire metabolic network. Here, we provide a comprehensive computational model of the underground metabolism in Escherichia coli. Most underground reactions are not isolated and 45% of them can be fully wired into the existing network and form novel pathways that produce key precursors for cell growth. This observation allowed us to conduct an integrated genome-wide in silico and experimental survey to characterize the evolutionary potential of E. coli to adapt to hundreds of nutrient conditions. We revealed that underground reactions allow growth in new environments when their activity is increased. We estimate that at least ∼20% of the underground reactions that can be connected to the existing network confer a fitness advantage under specific environments. Moreover, our results demonstrate that the genetic basis of evolutionary adaptations via underground metabolism is computationally predictable. The approach used here has potential for various application areas from bioengineering to medical genetics.

Background: Leptospirosis, an acute febrile disease caused by the pathogenic species of genus Leptospira, is one of the neglected emerging zoonoses that is of global public health concern. The recent genus-wide sequencing of Leptospira isolates led to the need for better understanding of the complex metabolic mechanisms of this organism. However, majority of the published studies on Leptospira metabolism were still the pioneering works of Baseman and Cox in the 60’s and their contemporaries. Knowledge on the carbon sources that supports the growth of a Leptospira species will not only contribute to the limited metabolic studies but will further support the reported genes and metabolic pathways of this organism. Objective: Thus, this study aimed to describe the carbon utilization phenome of Leptospira interrogans serovar Manilae strain K64, one of the dominantly circulating pathogenic Leptospira in the Philippines. Methods: A previously optimized BiologTM Gen III sole carbon utilization phenotype microarray assay protocol for leptospires was adapted. Results: L. interrogans serovar Manilae strain K64 showed utilization of 29 carbon sources belonging to sugars and sugar derivatives, amino acids, methyl ester, carboxylic acid and fatty acids. These were N-acetyl-β-D-mannosamine, N-acetyl-D-galactosamine, N-acetyl neuraminic acid, D-fructose, D-galactose, 3-methyl glucose, D-fucose, L-fucose, L-rhamnose, inosine, D-fructose-6-phosphate, D-gluconic, D-glucuronic acid, glucuronamide, D-saccharic acid, D-aspartic acid, D-serine, L-alanine, L-arginine, L-histidine, L-pyroglutamic acid, L-serine, D-lactic acid methyl ester, citric acid, D-malic acid, L-malic acid, alpha ketoglutaric acid, alpha ketobutyric acid, and acetoacetic acid. Discussion and Conclusion: The carbon sources utilized by L. interrogans serovar Manilae strain K64 agreed well with the identified genes and metabolic pathways among Leptospira species. Moreover, these 29 carbon sources have been previously reported to be associated in the biosynthesis of peptidoglycan, lipopolysaccharide, histidine, sulfur, amino acids, and isoleucine and in other metabolic pathways such as glycolysis, pentose-phosphate, pyruvate and fatty acid in Leptospira spp.

Pea (Pisum sativum) is one of the most popular legume crops used in agriculture. Because of the high demand and relatively reasonable price, Lithuania has increased the cultivation of this crop and invested in the research of new effective breeding lines in the last years. Rhizobial inoculants contribute to increasing yield in legumes through N2 fixation. Therefore, the objective of this work was to identify rhizobial strains able to increase the activity of two pea breeding lines ('DS 3637–2' and 'DS 3795–3') known for high productivity, resistance to biotic and abiotic stresses, and competitiveness in respect to weeds. Six rhizobial strains isolated from pea plants were identified as members of the Rhizobium leguminosarum group and phenotypically characterized in depth by Phenotype Microarray (PM). Phenotypic differences observed were linked to their phylogeny. Then, strains were tested for their ability to stimulate the growth of the breeding lines 'DS 3637–2' and 'DS 3795–3'. Reference strain Rhizobium anhuiense Z1 and Rhizobium leguminosarum sv. viciae 14ZE showed the best symbiotic performances with breeding lines 'DS 3637–2' and 'DS 3795–3', respectively. Based on the obtained results, R. leguminosarum sv. viciae strain 14ZE appears to be a new effective inoculant of peas. [ABSTRACT FROM AUTHOR]

The increased resistance of bacteria to antimicrobials, as well as the growing interest in innovative and sustainable alternatives to traditional food additives, are driving research towards the use of natural food preservatives. Among these, hydrolates (HYs) have gained attention as “mild” alternatives to conventional antimicrobial compounds. In this study, the response of L. monocytogenes ATCC 7644 exposed to increasing concentrations of Coridothymus capitatus HY (CHY) for 1 h at 37 °C was evaluated by means of Phenotype Microarray, modelling the kinetic data obtained by inoculating control and treated cells into GEN III microplates, after CHY removal. The results revealed differences concerning the growth dynamics in environmental conditions commonly encountered in food processing environments (different carbon sources, pH 6.0, pH 5.0, 1–8% NaCl). More specifically, for treated cells, the lag phase was extended, the growth rate was slowed down and, in most cases, the maximum concentration was diminished, suggesting the persistence of stress even after CHY removal. Confocal Laser Scanner Microscopy evidenced a diffuse aggregation and suffering of the treated cells, as a response to the stress encountered. In conclusion, the treatment with HY caused a stressing effect that persisted after its removal. The results suggest the potential of CHY application to control L. monocytogenes in food environments.

• Biolog analysis demonstrated that Halomonas TD01 exhibit a wide-ranging substrate spectrum and can utilize 140 of the 190 test compounds. • Acetate, glycerol, ethanol and lactate could be efficiently used as alternative low-cost carbon sources by Halomonas TD01. • Halomonas TD01 possess high tolerance to organic acid salts, eg. sodium acetate, succinate and itaconate. Halomonas TD01, which can grow under non-sterile and continuous processes at high pH and high salt concentrations, is a robust platform for PHA production from glucose. For extending other low-cost sustainable substrates and increasing the potential application in other value-added products, a better understanding of substrates utilization and chemicals tolerance is necessary. In this study, the substrate profiling of TD01 was analyzed via Biolog. Phenotype microarray results demonstrated that TD01 has a wide-ranging substrate spectrum and can utilize 140 of the 190 test compounds. Some cheap, abundant carbon sources, such as sodium acetate, glycerol, ethanol and lactate can well support the growth of TD01 in shake-flask, and are therefore suggested to be its alternative low-cost substrates for chemicals production in future. Furthermore, the tolerance of TD01 to various chemicals was tested. The results showed that the tolerability of TD01 to high concentrations of organic acid salts is prominent. When adding 75 g/L sodium acetate, 100 g/L succinic acid and 100 g/L itaconic acid in the medium, the growth rate reduced 56.14%, 52.63% and 47.37%, respectively. All these results highlight TD01 as a promising, next generation industrial workhorse in chemicals biomanufacturing from cheap organic acid salts. [ABSTRACT FROM AUTHOR]

Nitrogen metabolism in the rhizosphere microbiome plays an important role in mediating plant nutrition, particularly under low inputs of mineral fertilizers. However, there is relatively little mechanistic information about which genes and metabolic pathways are induced by rhizosphere bacterial strains to utilize diverse nitrogen substrates. Here we investigate nitrogen substrate utilization in three taxonomically diverse bacterial strains previously isolated from Arabidopsis roots. The three strains represent taxa that are consistently detected as core members of the plant microbiome: Pseudomonas, Streptomyces, and Rhizobium. We use phenotype microarrays to determine the nitrogen substrate preferences of these strains, and compare the experimental results vs. computational simulations of genome-scale metabolic network models obtained with EnsembleFBA. Results show that all three strains exhibit generalistic nitrogen substrate preferences, with substrate utilization being well predicted by EnsembleFBA. Using label-free quantitative proteomics, we document hundreds of proteins in each strain that exhibit differential abundance values following cultivation on five different nitrogen sources: ammonium, glutamate, lysine, serine, and urea. The proteomic response to these nitrogen sources was strongly strain-dependent, with lysine nutrition eliciting widespread protein-level changes in Pseudomonas sp. Root9 , whereas Rhizobium sp. Root491 showed relatively stable proteome composition across different nitrogen sources. Our results give new protein-level information about the specific transporters and enzymes induced by diverse rhizosphere bacterial strains to utilize organic nitrogen substrates. [ABSTRACT FROM AUTHOR]

Limited information is available on the phenotypic characteristics of Listeria monocytogenes that may affect its virulence of L. monocytogenes. The study focused on comparing the differences between two strains of L. monocytogenes , one hemolytic (LLO+) and one non-hemolytic (tLLO). Using Phenotype Microarray (PM) technology, carbon, nitrogen, and phosphorus-sulfur source utilization by the strains was performed and the KEGG pathway involvement in substrate utilization was analyzed. Of the 285 substrates screened, the results suggested that d -gluconic acid and l -serine may play a role in L. monocytogenes hemolytic activity towards LLO production by generating pyruvate as an intermediate and amino acid biosynthesis, which may play an important role in virulence protein translation. d -gluconic acid and l -serine showed no effect on the tLLO strain in PM analysis and validation tests. Notably, the scope of this study was limited to these two strains of L. monocytogenes. While these findings provide valuable insights into the metabolic differences between hemolytic and non-hemolytic strains, further research is necessary to expand this understanding to a broader range of strains and explore additional factors that may influence LLO functionality. • There are metabolism differences between L. monocytogenes producing active LLO and truncated LLO. • l -serine and d -gluconic acid were identified as key metabolites that may affect hemolytic activity. • l -serine and d -gluconic acid metabolism generates PRPP and pyruvate as intermediates, respectively. • The metabolism of the intermediates synthesized amino acids may be involved in LLO formation. [ABSTRACT FROM AUTHOR]

Adaptive laboratory evolution (ALE) is used to find causal mutations that underlie improved strain performance under the applied selection pressure. ALE studies have revealed that mutator populations tend to outcompete their non-mutator counterparts following the evolutionary trajectory. Among them, mutS-inactivated mutator cells, characterize d by a dysfunctional methyl-mismatch repair system, are frequently found in ALE experiments. Here, we examined mutS inactivation as an approach to facilitate ALE of Escherichia coli. The wild-type E. coli MG1655 and mutS knock-out derivative (ΔmutS) were evolved in parallel for 800 generations on lactate or glycerol minimal media in a serial-transfer experiment. Whole-genome re-sequencing of each lineage at 100-generation intervals revealed that (1) mutations emerge rapidly in the ΔmutS compared to in the wild-type strain; (2) mutations were more than fourfold higher in the ΔmutS strain at the end-point populations compared to the wild-type strain; and (3) a significant number of random mutations accumulated in the ΔmutS strains. We then measured the fitness of the end-point populations on an array of non-adaptive carbon sources. Interestingly, collateral fitness increases on non-adaptive carbon sources were more pronounced in the ΔmutS strains than the parental strain. Fitness measurement of single mutants revealed that the collateral fitness increase seen in the mutator lineages can be attributed to a pool of random mutations. Together, this study demonstrates that short-term mutator ALE extensively expands possible genotype space, resulting in versatile bacteria with elevated fitness levels across various carbon sources.

A combination of phenotype microarrays, targeted stress resistance and virulence assays and comparative genome analysis was used to compare a set of Listeria monocytogenes strains including those involved in previous Swiss foodborne listeriosis outbreaks. Despite being highly syntenic in gene content these strains showed significant phenotypic variation in utilization of different carbon (C)-sources as well as in resistance of osmotic and pH stress conditions that are relevant to host and food associated environments. An outbreak strain from the 2005 Swiss Tomme cheese listeriosis outbreak (Lm3163) showed the highest versatility in C-sources utilized whereas the strain responsible for the 1983 to 1987 Vacherin Montd’or cheese listeriosis outbreak (LL195) showed the highest tolerance to both osmotic and pH stress conditions among the examined strains. Inclusion of L-norvaline led to enhanced resistance of acidic stress in all the examined strains and there were strain–strain-specific differences observed in the ability of other amino acids and urea to enhance acid stress resistance in L. monocytogenes. A strain dependent inhibition pattern was also observed upon inclusion of β-phenylethylamine under alkaline stress conditions. In targeted phenotypic analysis the strain-specific differences in salt stress tolerance uncovered in phenotypic microarrays were corroborated and variations in host cell invasion and virulence among the examined strains were also revealed. Outbreak associated strains representing lineage I serotype 4b showed superior pathogenicity in a zebrafish infection model whilst Lm3163 a lineage II serotype 1/2a outbreak strain demonstrated the highest cellular invasion capacity amongst the tested strains. A genome wide sequence comparison of the strains only revealed few genetic differences between the strains suggesting that variations in gene regulation and expression are largely responsible for the phenotypic differences revealed among the examined strains. Our results have generated data that provides a potential basis for the future design of improved Listeria specific media to enhance routine detection and isolation of this pathogen as well as provide knowledge for developing novel methods for its control in food.

Bacterial rhizosphere colonization is critical for phytobeneficial rhizobacteria such as phenazine-producing Pseudomonas spp. To better understand this colonization process, potential metabolic and genomic determinants required for rhizosphere colonization were identified using a collection of 60 phenazine-producing Pseudomonas strains isolated from multiple plant species and representative of the worldwide diversity. Arabidopsis thaliana and Solanum tuberosum (potato) were used as host plants. Bacterial rhizosphere colonization was measured by quantitative PCR using a newly designed primer pair and TaqMan probe targeting a conserved region of the phenazine biosynthetic operon. The metabolic abilities of the strains were assessed on 758 substrates using Biolog phenotype microarray technology. These data, along with available genomic sequences for all strains, were analyzed in light of rhizosphere colonization. Strains belonging to the P. chlororaphis subgroup colonized the rhizospheres of both plants more efficiently than strains belonging to the P. fluorescens subgroup. Metabolic results indicated that the ability to use amines and amino acids was associated with an increase in rhizosphere colonization capability in A. thaliana and/or in S. tuberosum. The presence of multiple genetic determinants in the genomes of the different strains involved in catabolic pathways and plant-microbe and microbe-microbe interactions correlated with increased or decreased rhizosphere colonization capabilities in both plants. These results suggest that the metabolic and genomic traits found in different phenazine-producing Pseudomonas strains reflect their rhizosphere competence in A. thaliana and S. tuberosum. Interestingly, most of these traits are associated with similar rhizosphere colonizing capabilities in both plant species. IMPORTANCE Rhizosphere colonization is crucial for plant growth promotion and biocontrol by antibiotic-producing Pseudomonas spp. This colonization process relies on different bacterial determinants which partly remain to be uncovered. In this study, we combined a metabolic and a genomic approach to decipher new rhizosphere colonization determinants which could improve our understanding of this process in Pseudomonas spp. Using 60 distinct strains of phenazine-producing Pseudomonas spp., we show that rhizosphere colonization abilities correlated with both metabolic and genomic traits when these bacteria were inoculated on two distant plants, Arabidopsis thaliana and Solanum tuberosum. Key metabolic and genomic determinants presumably required for efficient colonization of both plant species were identified. Upon further validation, these targets could lead to the development of simple screening tests to rapidly identify efficient rhizosphere colonizers. [ABSTRACT FROM AUTHOR]

Microbial biofilms are undesired in food manufacturing, drinking water distribution systems, and clinical realms. Yeast biofilms are particularly problematic because of the strong capacity of yeast cells to adhere to abiotic surfaces, cells, and tissues. Novel approaches have been developed over recent years to prevent the establishment of microbial biofilms, such as through the use of small molecules with inhibiting and dispersing properties. Here, we studied the inhibitory activity of 11 different amino acids on the biofilm formation ability of three wild-type Saccharomyces cerevisiae strains and the reference strain ∑1278b. Subsequent evaluation of different concentrations of the two most effective amino acids, namely, arginine and cysteine, revealed that they acted in different ways. Arginine prevented biofilm formation by reducing FLO11 gene expression; its addition did not affect cell viability and was even found to enhance cell metabolism (vitality marker) as determined by phenotype microarray (PM) analysis. On the contrary, the addition of cysteine reduced both cell viability and vitality as well as FLO11 expression. Thus, the use of cysteine and arginine as agents against biofilm formation can be diversified depending on the most desired action towards yeast growth. [ABSTRACT FROM AUTHOR]

Adaptive laboratory evolution (ALE) is used to find causal mutations that underlie improved strain performance under the applied selection pressure. ALE studies have revealed that mutator populations tend to outcompete their non-mutator counterparts following the evolutionary trajectory. Among them, mutS- inactivated mutator cells, characterize d by a dysfunctional methyl-mismatch repair system, are frequently found in ALE experiments. Here, we examined mutS inactivation as an approach to facilitate ALE of Escherichia coli. The wild-type E. coli MG1655 and mutS knock-out derivative (Δ mutS) were evolved in parallel for 800 generations on lactate or glycerol minimal media in a serial-transfer experiment. Whole-genome re-sequencing of each lineage at 100-generation intervals revealed that (1) mutations emerge rapidly in the Δ mutS compared to in the wild-type strain; (2) mutations were more than fourfold higher in the Δ mutS strain at the end-point populations compared to the wild-type strain; and (3) a significant number of random mutations accumulated in the Δ mutS strains. We then measured the fitness of the end-point populations on an array of non-adaptive carbon sources. Interestingly, collateral fitness increases on non-adaptive carbon sources were more pronounced in the Δ mutS strains than the parental strain. Fitness measurement of single mutants revealed that the collateral fitness increase seen in the mutator lineages can be attributed to a pool of random mutations. Together, this study demonstrates that short-term mutator ALE extensively expands possible genotype space, resulting in versatile bacteria with elevated fitness levels across various carbon sources. [ABSTRACT FROM AUTHOR]

The cold-tolerant yeast Saccharomyces cerevisiae is industrially useful for lager fermentation, high-quality wine, and frozen dough production. S. cerevisiae Cheongdo is a recent isolate from frozen peach samples which has a good fermentation performance at low temperatures and desirable flavor profiles. Here, phenotype microarray was used to investigate industrial potentials of S. cerevisiae Cheongdo using 192 carbon sources. Compared to commercial wine yeast S. cerevisiae EC1118, Cheongdo showed significantly different growth rates on 34 substrates. The principal component analysis of the results highlighted that the better growth of Cheongdo on galactose than on EC1118 was the most significant difference between the two strains. The intact GAL4 gene and the galactose fermentation performance at a low temperatures suggested that S. cerevisiae Cheongdo is a promising host for industrial fermentation rich in galactose, such as lactose and agarose.

Background: Pseudogymnoascus destructans, a psychrophile, causes bat white-nose syndrome (WNS). Pseudogymnoascus pannorum, a closely related fungus, causes human and canine diseases rarely. Both pathogens were reported from the same mines and caves in the United States, but only P. destructans caused WNS. Earlier genome comparisons revealed that P. pannorum contained more deduced proteins with ascribed enzymatic functions than P. destructans. Methods: We performed metabolic profiling with Biolog PM microarray plates to confirm in silico gene predictions. Results: P. pannorum utilized 78 of 190 carbon sources (41%), and 41 of 91 nitrogen compounds (43%) tested. P. destructans used 23 carbon compounds (12%) and 23 nitrogen compounds (24%). P. destructans exhibited more robust growth on the phosphorous compounds and nutrient supplements (83% and 15%, respectively) compared to P. pannorum (27% and 1%, respectively.). P. pannorum exhibited higher tolerance to osmolytes, pH extremes, and a variety of chemical compounds than P. destructans. Conclusions: An abundance of carbohydrate degradation pathways combined with robust stress tolerance provided clues for the soil distribution of P. pannorum. The limited metabolic profile of P. destructans was compatible with in silico predictions of far fewer proteins and enzymes. P. destructans ability to catabolize diverse phosphorous and nutrient supplements might be critical in the colonization and invasion of bat tissues. The present study of 1,047 different metabolic activities provides a framework for future gene-function investigations of the unique biology of the psychrophilic fungi.

Background: Pseudogymnoascus destructans, a psychrophile, causes bat white-nose syndrome (WNS). Pseudogymnoascus pannorum, a closely related fungus, causes human and canine diseases rarely. Both pathogens were reported from the same mines and caves in the United States, but only P. destructans caused WNS. Earlier genome comparisons revealed that P. pannorum contained more deduced proteins with ascribed enzymatic functions than P. destructans. Methods: We performed metabolic profiling with Biolog PM microarray plates to confirm in silico gene predictions. Results: P. pannorum utilized 78 of 190 carbon sources (41%), and 41 of 91 nitrogen sources (43%) tested. P. destructans used 23 carbon compounds (12%) and 23 nitrogen compounds (24%). P. destructans exhibited more robust growth on the phosphorous sources and nutrient supplements (83% and 15%, respectively) compared to P. pannorum (27% and 1%, respectively.). P. pannorum exhibited higher tolerance to osmolytes, pH extremes, and a variety of chemical compounds than P. destructans. Conclusions: An abundance of carbohydrate degradation pathways combined with robust stress tolerance provided clues for the soil distribution of P. pannorum. The limited metabolic profile of P. destructans validated in silico predictions of far fewer proteins and enzymes. P. destructans ability to catabolize diverse phosphorous and nutrient supplements might be critical in the colonization and invasion of bat tissues. The present study of 1,047 different metabolic activities provides a framework for future gene-function investigations of the unique biology of the psychrophilic fungi.

Hypoxia plays a significant role in solid tumors by the increased expression of hypoxia-inducible factor-1α (HIF-1α), which is known to promote cancer invasion and metastasis. Cancer-cell invasion dynamically begins with the degradation of the extracellular matrix (ECM) via invadopodia formation. The chemical substrates that are utilized by hypoxic cells as fuel to drive invadopodia formation are still not fully understood. Therefore, the aim of the study was to maintain MDA-MB-231 cells under hypoxia conditions to allow cells to form a large number of invadopodia as a model, followed by identifying their nutrient utilization. The results of the study revealed an increase in the number of cells forming invadopodia under hypoxia conditions. Moreover, Western blot analysis confirmed that essential proteins for hypoxia and invadopodia, including HIF-1α, vascular endothelial growth factor (VEGF), metallopeptidase-2 (MMP-2), and Rho guanine nucleotide exchange factor 7 (β-PIX), significantly increased under hypoxia. Interestingly, phenotype microarray showed that only 11 chemical substrates from 367 types of substrates were significantly metabolized in hypoxia compared to in normoxia. This is thought to be fuel for hypoxia to drive the invasion process. In conclusion, we found 11 chemical substrates that could have potential energy sources for hypoxia-induced invadopodia formation of these cells. This may in part be a target in the hypoxic tumor and invadopodia formation. Additionally, these findings can be used as potential carrier targets in cancer-drug discovery, such as the usage of dextrin.

Feline infectious peritonitis (FIP) is a fatal feline immune-mediated disease caused by feline infectious peritonitis virus (FIPV). Little is known about the biological pathways associated in FIP pathogenesis. This is the first study aiming to determine the phenotypic characteristics on the cellular level in relation to specific metabolic pathways of importance to FIP pathogenesis. Methods: The internalization of type II FIPV WSU 79-1146 in Crandell-Rees Feline Kidney (CrFK) cells was visualized using a fluorescence microscope, and optimization prior to phenotype microarray (PM) study was performed. Then, four types of Biolog Phenotype MicroArray™ plates (PM-M1 to PM-M4) precoated with different carbon and nitrogen sources were used to determine the metabolic profiles in FIPV-infected cells. Results: The utilization of palatinose was significantly low in FIPV-infected cells; however, there were significant increases in utilizing melibionic acid, L-glutamine, L-glutamic acid and alanyl-glutamine (Ala-Gln) compared to non-infected cells. Conclusion: This study has provided the first insights into the metabolic profiling of a feline coronavirus infection in vitro using PMs and deduced that glutamine metabolism is one of the essential metabolic pathways for FIPV infection and replication. Further studies are necessary to develop strategies to target the glutamine metabolic pathway in FIPV infection.

Since the recognition of the name Frankia in the Approved Lists of bacterial names (1980), few amendments have been given to the genus description. Successive editions of Bergey's Manual of Systematics of Archaea and Bacteria have broadly conflicting suprageneric treatments of the genus without any advances for subgeneric classification. This review focuses on recent results from taxongenomics and phenoarray approaches to the positioning and the structuring of the genus Frankia. Based on phylogenomic analyses, Frankia should be considered the single member of the family Frankiaceae within the monophyletic order, Frankiales. A polyphasic strategy incorporating genome to genome data and omniLog® phenoarrays, together with classical approaches, has allowed the designation and an amended description of a type strain of the type species Frankia alni, and the recognition of at least 10 novel species covering symbiotic and non symbiotic taxa within the genus. Genome to phenome data will be shortly incorporated in the scheme for proposing novel species including those recalcitrant to isolation in axenic culture. [ABSTRACT FROM AUTHOR]

Oomycetes in the genus Phytophthora are among the most damaging plant pathogens worldwide. Two important species are Phytophthora cinnamomi, which causes root rot in thousands of native and agricultural plants, and Phytophthora agathidicida, which causes kauri dieback disease in New Zealand. As is the case for other Phytophthora species, management options for these two pathogens are limited. Here, we have screened over 100 compounds for their anti-oomycete activity, as a potential first step toward identifying new control strategies. Our screening identified eight compounds that showed activity against both Phytophthora species. These included five antibiotics, two copper compounds and a quaternary ammonium cation. These compounds were tested for their inhibitory action against three stages of the Phytophthora life cycle: mycelial growth, zoospore germination, and zoospore motility. The inhibitory effects of the compounds were broadly similar between the two Phytophthora species, but their effectiveness varied widely among life cycle stages. Mycelial growth was most successfully inhibited by the antibiotics chlortetracycline and paromomycin, and the quaternary ammonium salt benzethonium chloride. Copper chloride and copper sulfate were most effective at inhibiting zoospore germination and motility, whereas the five antibiotics showed relatively poor zoospore inhibition. Benzethonium chloride was identified as a promising antimicrobial, as it is effective across all three life cycle stages. While further testing is required to determine their efficacy and potential phytotoxicity in planta, we have provided new data on those agents that are, and those that are not, effective against P. agathidicida and P. cinnamomi. Additionally, we present here the first published protocol for producing zoospores from P. agathidicida, which will aid in the further study of this emerging pathogen.

Understanding the driving forces behind the shifts in the ecological balance of the oral microbiota will become essential for the future management and treatment of periodontitis. As the use of competitive approaches for modulating bacterial outgrowth is unexplored in the oral ecosystem, our study aimed to investigate both the associations among groups of functional compounds and the impact of individual substrates on selected members of the oral microbiome. We employed the Phenotype Microarray high-throughput technology to analyse the microbial cellular phenotypes of 15 oral bacteria. Multivariate statistical analysis was used to detect respiratory activity triggers and to assess similar metabolic activities. Carbon and nitrogen were relevant for the respiration of health-associated bacteria, explaining competitive interactions when grown in biofilms. Carbon, nitrogen, and peptides tended to decrease the respiratory activity of all pathobionts, but not significantly. None of the evaluated compounds significantly increased activity of pathobionts at both 24 and 48 h. Additionally, metabolite requirements of pathobionts were dissimilar, suggesting that collective modulation of their respiratory activity may be challenging. Flow cytometry indicated that the metabolic activity detected in the Biolog plates may not be a direct result of the number of bacterial cells. In addition, damage to the cell membrane may not influence overall respiratory activity. Our methodology confirmed previously reported competitive and collaborative interactions among bacterial groups, which could be used either as marker of health status or as targets for modulation of the oral environment.

Objectives: Clostridium difficile infection (CDI) is the leading cause of infectious diarrhea in North America and Europe. The risk of CDI increases significantly in the case where antimicrobial treatment reduces the number of competing bacteria in the gut, thus leading to the increased availability of nutrients and loss of colonization resistance. The objective of this study was to determine comprehensive nutritional utilization and the chemical sensitivity profile of historic and newer C. difficile isolates and to examine the possible role of the phenotype diversity in C. difficile virulence. Methods: Phenotype microarrays (PMs) were used to elucidate the complete nutritional and chemical sensitivity profile of six C. difficile isolates. Results: Of the 760 nutrient sources tested, 285 compounds were utilized by at least one strain. Among the C. difficile isolates compared, R20291, a recent hypervirulent outbreak-associated strain, appears to have an expanded nutrient utilization profile when compared to all other strains. Conclusions: The expanded nutritional utilization profile of some newer C. difficile strains could be one of the reasons for infections in patients who are not exposed to the hospital environment or not undergoing antibiotic treatment. This nutritional profile could be used to design tube feeding formulas that reduce the risk of CDI.

Oomycetes in the genus Phytophthora are among the most damaging plant pathogens worldwide. Two important species are Phytophthora cinnamomi, which causes root rot in thousands of native and agricultural plants, and Phytophthora agathidicida, which causes kauri dieback disease in New Zealand. As is the case for other Phytophthora species, management options for these two pathogens are limited. Here, we have screened over 100 compounds for their anti-oomycete activity, as a potential first step toward identifying new control strategies. Our screening identified eight compounds that showed activity against both Phytophthora species. These included five antibiotics, two copper compounds and a quaternary ammonium cation. These compounds were tested for their inhibitory action against three stages of the Phytophthora life cycle: mycelial growth, zoospore germination, and zoospore motility. The inhibitory effects of the compounds were broadly similar between the two Phytophthora species, but their effectiveness varied widely among life cycle stages. Mycelial growth was most successfully inhibited by the antibiotics chlortetracycline and paromomycin, and the quaternary ammonium salt benzethonium chloride. Copper chloride and copper sulfate were most effective at inhibiting zoospore germination and motility, whereas the five antibiotics showed relatively poor zoospore inhibition. Benzethonium chloride was identified as a promising antimicrobial, as it is effective across all three life cycle stages. While further testing is required to determine their efficacy and potential phytotoxicity in planta, we have provided new data on those agents that are, and those that are not, effective against P. agathidicida and P. cinnamomi. Additionally, we present here the first published protocol for producing zoospores from P. agathidicida, which will aid in the further study of this emerging pathogen. [ABSTRACT FROM AUTHOR]

Background Mycobacterium avium subsp. hominissuis (MAH) is an emerging opportunistic human pathogen. It can cause pulmonary infections, lymphadenitis and disseminated infections in immuno-compromised patients. In addition, MAH is widespread in the environment, since it has been isolated from water, soil or dust. In recent years, knowledge on MAH at the molecular level has increased substantially. In contrast, knowledge of the MAH metabolic phenotypes remains limited. Methods In this study, for the first time we analyzed the metabolic substrate utilization of ten MAH isolates, five from a clinical source and five from an environmental source. We used BIOLOG Phenotype MicroarrayTM technology for the analysis. This technology permits the rapid and global analysis of metabolic phenotypes. Results The ten MAH isolates tested showed different metabolic patterns pointing to high intra-species diversity. Our MAH isolates preferred to use fatty acids such as Tween, caproic, butyric and propionic acid as a carbon source, and L-cysteine as a nitrogen source. Environmental MAH isolates resulted in being more metabolically active than clinical isolates, since the former metabolized more strongly butyric acid (p = 0.0209) and propionic acid (p = 0.00307). Discussion Our study provides new insight into the metabolism of MAH. Understanding how bacteria utilize substrates during infection might help the developing of strategies to fight such infections.

Background Helicobacter pylori colonizes almost half of the human population worldwide. H. pylori strains are genetically diverse, and the specific genotypes are associated with various clinical manifestations including gastric adenocarcinoma, peptic ulcer disease (PUD), and nonulcer dyspepsia (NUD). However, our current knowledge of the H. pylori metabolism is limited. To understand the metabolic differences among H. pylori strains, we investigated four Malaysian H. pylori clinical strains, which had been previously sequenced, and a standard strain, H. pylori J99, at the phenotypic level. Materials and Methods The phenotypes of the H. pylori strains were profiled using the Biolog Phenotype Microarray system to corroborate genomic data. We initiated the analyses by predicting carbon and nitrogen metabolic pathways from the H. pylori genomic data from the KEGG database. Biolog PM aided the validation of the prediction and provided a more intensive analysis of the H. pylori phenomes. Results We have identified a core set of metabolic nutrient sources that was utilized by all strains tested and another set that was differentially utilized by only the local strains. Pentose sugars are the preferred carbon nutrients utilized by H. pylori. The amino acids l-aspartic acid, d-alanine, and l-asparagine serve as both carbon and nitrogen sources in the metabolism of the bacterium. Conclusion The phenotypic profile based on this study provides a better understanding on the survival of H. pylori in its natural host. Our data serve as a foundation for future challenges in correlating interstrain metabolic differences in H. pylori. [ABSTRACT FROM AUTHOR]

In the present study, the response of Rhodococcus aetherivorans BCP1 and Rhodococcus opacus R7 to various stress conditions and several antimicrobials was examined by PM in relation with genetic determinants, as revealed by annotation analysis of the two genomes. Comparison between metabolic activities and genetic features of BCP1 and R7 provided new insight into the environmental persistence of these two members of the genus Rhodococcus . [ABSTRACT FROM AUTHOR]

We constructed the genome-scale metabolic network of Bacillus licheniformis ( B. licheniformis ) WX-02 by combining genomic annotation, high-throughput phenotype microarray (PM) experiments and literature-based metabolic information. The accuracy of the metabolic network was assessed by an OmniLog PM experiment. The final metabolic model iWX1009 contains 1009 genes, 1141 metabolites and 1762 reactions, and the predicted metabolic phenotypes showed an agreement rate of 76.8% with experimental PM data. In addition, key metabolic features such as growth yield, utilization of different substrates and essential genes were identified by flux balance analysis. A total of 195 essential genes were predicted from LB medium, among which 149 were verified with the experimental essential gene set of B. subtilis 168. With the removal of 5 reactions from the network, pathways for poly-γ-glutamic acid (γ-PGA) synthesis were optimized and the γ-PGA yield reached 83.8 mmol/h. Furthermore, the important metabolites and pathways related to γ-PGA synthesis and bacterium growth were comprehensively analyzed. The present study provides valuable clues for exploring the metabolisms and metabolic regulation of γ-PGA synthesis in B. licheniformis WX-02. [ABSTRACT FROM AUTHOR]

Bioconversion processes of organic acid or acid hydrolysis of raw material for microbial metabolism often suffer limitations as a result of microbial sensitivity in low-pH conditions. We adopted a three-step method called RAndom Insertional-deletional Strand Exchange mutagenesis (RAISE) to engineer the components of global regulator Sigma D factor (RpoD) of Escherichia coli to improve its acid tolerance. The best strain Mutant VII was identified from random mutagenesis libraries based on the growth performance, which exhibited much higher growth rate than the control (0.22 h −1 vs. 0.15 h −1 ) at pH as low as 3.17. Combined transcriptome and phenome analysis of E. coli was carried out to better understand the global effects of RpoD on the regulatory networks. Our analysis showed that 95 (2.1%) of all E. coli genes were induced and 178 (4.0%) genes were repressed, including those for trehalose biosynthesis, nucleotides biosynthesis, carbon metabolism, amino acid utilization, except for acid resistance. Also regulated were the master regulators (ArcA, EvgA, H-NS and RpoS) and gene/operon-specific transcription factors (GadX, GadW, AppY, YdeO, KdgR). These results demonstrated that RpoD acts as global regulator in the growth phase of E. coli and consequently improves acid tolerances. [ABSTRACT FROM AUTHOR]

Metabolic modeling provides the means to define metabolic processes at a systems level; however, genome-scale metabolic models often remain incomplete in their description of metabolic networks and may include reactions that are experimentally unverified. This shortcoming is exacerbated in reconstructed models of newly isolated algal species, as there may be little to no biochemical evidence available for the metabolism of such isolates. The Phenotype Microarray (PM) technology (Biolog, Hayward, CA, USA) provides an efficient, high throughput method to functionally define cellular metabolic activities in response to a large array of entry metabolites. The platform can experimentally verify many of the unverified reactions in a network model as well as identify missing or new reactions in the reconstructed metabolic model. The PM technology has been used for metabolic phenotyping of non-photosynthetic bacteria and fungi but it has not been reported for the phenotyping of microalgae. Here we introduce the use of PM assays in a systematic way to the study of microalgae, applying it specifically to the green microalgal model species Chlamydomonas reinhardtii. The results obtained in this study validate a number of existing annotated metabolic reactions and identify a number of novel and unexpected metabolites. The obtained information was used to expand and refine the existing COBRA-based C. reinhardtii metabolic network model iRC1080. Over 254 reactions were added to the network, and the effects of these additions on flux distribution within the network are described. The novel reactions include the support of metabolism by a number of D-amino acids, L-dipeptides, and L-tripeptides as nitrogen sources, as well as support of cellular respiration by cysteamine-S-phosphate as a phosphorus source. The protocol developed here can be used as a foundation to functionally profile other microalgae such as known microalgae mutants and novel isolates.