Metabolic Network Analysis of Streptomyces tenebrarius, a Streptomyces Species with an Active Entner-Doudoroff Pathway

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. ¹³C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.     

Catabolic pathways of glucose in Bacillus circulans var. alkalophilus

Enzymes and the metabolic pathways of glucose catabolism of Bacillus circulans var. alkalophilus were studied. The metabolism of the microbe was mixed acid fermentative yielding mainly acetic and formic acids as end products from glucose. It was estimated that B. circulans var. alkalophilus partitions 90%–93% of the carbon from glucose into the Embden-Meyerhof-Parnas (EMP) pathway and 7%–10% into the hexose monophosphate (HMP) and Entner-Doudoroff (ED) pathways. Rather low activities of glucose dehydrogenase and gluconokinase appeared in the early logarithmic and late stationary phases, whereas NADH oxidase was markedly high. This result can be explained by a demand to reduce NADH to NAD⁺ for the EMP pathway; when acetic and formic acids are produced, no NADH is regenerated to NAD⁺, which is required in the early steps of EMP and HMP pathways. A small percentage (1.6%–2.4%) of the total CO₂ was formed from (6-C) of glucose, which means that the tricarboxylic acid cycle was functional but its contribution was insignificant. Large differences do not seem to exist between alkaliphilic and neutrophilic bacilli in the use of glucose pathways. Received: January 29, 1999 / Accepted: July 30, 1999     

Carbohydrate metabolism of the phase variants of purple photosynthetic bacteria

The R and M phase variants of Rhodobacter sphaeroides and Rhodobacter capsulatus were isolated. The growth rates in the dark and in the light in glucose-containing media were much higher for the Rba. sphaeroides R variant than for the M variant. For the Rba. capsulatus R and M variants, growth rates in the dark and in the light in fructose- or glucose-containing media differed insignificantly. The cells of Rba. sphaeroides and Rba. capsulatus phase variants growing in media with glucose and fructose exhibited differences in activity of the key enzymes of the Embden–Meyerhof–Parnas (EMP) and Entner–Doudoroff (ED) pathways. The oxidative pentose phosphate pathway (PPP) does not participate in glucose and fructose metabolism in the studied bacteria. Specific activity of the ED pathway enzymes was higher in dark-grown R and M variants of both Rba. sphaeroides and Rba. capsulatus than in the cells grown under light. Specific activity of the EMP enzymes was higher for the R and M variants of both cultures grown in the light than for those grown in the dark. Activities of the 2-keto-3-deoxy-6-phosphogluconate and fructose bisphosphate aldolases, the key enzymes of the ED and EMP pathways in Rba. sphaeroides M variant grown in the medium with glucose in the light or in the dark, were approximately twice those of the R variant. In the medium with fructose activities of these enzymes in both R and M variants did not change significantly depending on growth conditions. Activities of the enzymes of the EMP and ED pathways in the extracts of the Rba. capsulatus R and M cells grown with glucose or fructose did not change significantly. Cultivation of Rba. sphaeroides and Rba. capsulatus phase variants in the medium with fructose resulted in a considerably increased synthesis of 1-phosphofructokinase. Induction of 1-phosphofructokinase synthesis in Rba. sphaeroides occurred only in the light, while in Rba. capsulatus induction of this enzyme in the medium with fructose was observed both in the dark and in the light. Thus, under aerobic conditions in the dark the phase variants of both bacteria probably assimilated glucose and fructose via the ED pathway, while in the light the EMP pathway was active.     

Phosphoketolase pathway dominates in Lactobacillus reuteri ATCC 55730 containing dual pathways for glycolysis

Metabolic flux analysis indicated that the heterofermentative Lactobacillus reuteri strain ATCC 55730 uses both the Embden-Meyerhof pathway (EMP) and phosphoketolase pathway (PKP) when glucose or sucrose is converted into the three-carbon intermediate stage of glycolysis. In all cases studied, the main flux is through the PKP, while the EMP is used as a shunt. In the exponential growth phase, 70%, 73%, and 84% of the flux goes through the PKP in cells metabolizing (i) glucose plus fructose, (ii) glucose alone, and (iii) sucrose alone, respectively. Analysis of the genome of L. reuteri ATCC 55730 confirmed the presence of the genes for both pathways. Further evidence for the simultaneous operation of two central carbon metabolic pathways was found through the detection of fructose-1,6-bisphosphate aldolase, phosphofructokinase, and phosphoglucoisomerase activities and the presence of phosphorylated EMP and PKP intermediates using in vitro ³¹P NMR. The maximum specific growth rate and biomass yield obtained on glucose were twice as low as on sucrose. This was the result of low ATP levels being present in glucose-metabolizing cells, although the ATP production flux was as high as in sucrose-metabolizing cells due to a twofold increase of enzyme activities in both glycolytic pathways. Growth performance on glucose could be improved by adding fructose as an external electron acceptor, suggesting that the observed behavior is due to a redox imbalance causing energy starvation.     

Carbohydrate Metabolism and Carbon Fixation in Roseobacter denitrificans OCh114

The Roseobacter clade of aerobic marine proteobacteria, which compose 10–25% of the total marine bacterial community, has been reported to fix CO₂, although it has not been determined what pathway is involved. In this study, we report the first metabolic studies on carbohydrate utilization, CO₂ assimilation, and amino acid biosynthesis in the phototrophic Roseobacter clade bacterium Roseobacter denitrificans OCh114. We develop a new minimal medium containing defined carbon source(s), in which the requirements of yeast extract reported previously for the growth of R. denitrificans can be replaced by vitamin B₁₂ (cyanocobalamin). Tracer experiments were carried out in R. denitrificans grown in a newly developed minimal medium containing isotopically labeled pyruvate, glucose or bicarbonate as a single carbon source or in combination. Through measurements of ¹³C-isotopomer labeling patterns in protein-derived amino acids, gene expression profiles, and enzymatic activity assays, we report that: (1) R. denitrificans uses the anaplerotic pathways mainly via the malic enzyme to fix 10–15% of protein carbon from CO₂; (2) R. denitrificans employs the Entner-Doudoroff (ED) pathway for carbohydrate metabolism and the non-oxidative pentose phosphate pathway for the biosynthesis of histidine, ATP, and coenzymes; (3) the Embden-Meyerhof-Parnas (EMP, glycolysis) pathway is not active and the enzymatic activity of 6-phosphofructokinase (PFK) cannot be detected in R. denitrificans; and (4) isoleucine can be synthesized from both threonine-dependent (20% total flux) and citramalate-dependent (80% total flux) pathways using pyruvate as the sole carbon source.     

Comparative genome analysis of the <Emphasis Type="Italic">Flavobacteriales</Emphasis> bacterium strain UJ101, isolated from the gut of <Emphasis Type="Italic">Atergatis reticulatus</Emphasis>

Here we report the comparative genomic analysis of strain UJ101 with 15 strains from the family Flavobacteriaceae, using the CGExplorer program. Flavobacteriales bacterium strain UJ101 was isolated from a xanthid crab, Atergatis reticulatus, from the East Sea near Korea. The complete genome of strain UJ101 is a 3,074,209 bp, single, circular chromosome with 30.74% GC content. While the UJ101 genome contains a number of annotated genes for many metabolic pathways, such as the Embden–Meyerhof pathway, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle, genes for the Entner-Douddoroff pathway are not found in the UJ101 genome. Overall, carbon fixation processes were absent but nitrate reduction and denitrification pathways were conserved. The UJ101 genome was compared to genomes from other marine animals (three invertebrate strains and 5 fish strains) and other marine animal- derived genera. Notable results by genome comparisons showed that UJ101 is capable of denitrification and nitrate reduction, and that biotin-thiamine pathway participation varies among marine bacteria; fish-dwelling bacteria, freeliving bacteria, invertebrate-dwelling bacteria, and strain UJ101. Pan-genome analysis of the 16 strains in this study included 7,220 non-redundant genes that covered 62% of the pan-genome. A core-genome of 994 genes was present and consisted of 8% of the genes from the pan-genome. Strain UJ101 is a symbiotic hetero-organotroph isolated from xanthid crab, and is a metabolic generalist with nitrate-reducing abilities but without the ability to synthesize biotin. There is a general tendency of UJ101 and some fish pathogens to prefer thiamine-dependent glycolysis to gluconeogenesis. Biotin and thiamine auxotrophy or prototrophy may be used as important markers in microbial community studies.     

Carbohydrate metabolism in Thermoproteus tenax: in vivo utilization of the non-phosphorylative Entner-Doudoroff pathway and characterization of its first enzyme, glucose dehydrogenase

Thermoproteus tenax is a hyperthermophilic, facultative heterotrophic archaeum. In this organism the utilization of the two catabolic pathways, a variant of the Embden-Meyerhof-Parnas (EMP) pathway and the modified (nonphosphorylative) Entner-Doudoroff (ED) pathway, was investigated and the first enzyme of the ED pathway, glucose dehydrogenase, was characterized. The distribution of the ¹³C label in alanine synthesized by cells grown with [1-¹³C]glucose indicated that in vivo the EMP pathway and the modified ED pathway operate parallel, with glucose metabolization via the EMP pathway being prominent. To initiate studies on the regulatory mechanisms governing carbon flux via these pathways, the first enzyme of the ED pathway, glucose dehydrogenase, was purified to homogeneity and its phenotypic properties were characterized. The pyridine-nucleotide-dependent enzyme used both NAD⁺ and NADP⁺ as cosubstrates, showing a 100-fold higher affinity for NADP⁺. Besides glucose, xylose was used as substrate, but with significantly lower affinity. These data suggest that the physiological function of the enzyme is the oxidation of glucose by NADP⁺. A striking feature was the influence of NADP⁺ and NAD⁺ on the quaternary structure and activity state of the enzyme. Without cosubstrate, the enzyme was highly aggregated (mol. mass > 600 kDa) but inactive, whereas in the presence of the cosubstrate the aggregates dissociated into enzymatically active, homomeric dimers with a mol. mass of 84 kDa (mol. mass of subunits: 41 kDa). The N-terminal amino acid sequence showed striking similarity to the respective partial sequences of alcohol dehydrogenases and sorbitol dehydrogenases, but no resemblance to the known pyridine-nucleotide-dependent archaeal and bacterial glucose dehydrogenases. Received: 25 October 1996 / Accepted: 15 April 1997     

13C and 1H Nuclear Magnetic Resonance Study of Glycogen Futile Cycling in Strains of the Genus Fibrobacter

We investigated the carbon metabolism of three strains of Fibrobacter succinogenes and one strain of Fibrobacter intestinalis. The four strains produced the same amounts of the metabolites succinate, acetate, and formate in approximately the same ratio (3.7/1/0.3). The four strains similarly stored glycogen during all growth phases, and the glycogen-to-protein ratio was close to 0.6 during the exponential growth phase. ¹³C nuclear magnetic resonance (NMR) analysis of [1-¹³C]glucose utilization by resting cells of the four strains revealed a reversal of glycolysis at the triose phosphate level and the same metabolic pathways. Glycogen futile cycling was demonstrated by ¹³C NMR by following the simultaneous metabolism of labeled [¹³C]glycogen and exogenous unlabeled glucose. The isotopic dilutions of the CH₂ of succinate and the CH₃ of acetate when the resting cells were metabolizing [1-¹³C]glucose and unlabeled glycogen were precisely quantified by using ¹³C-filtered spin-echo difference ¹H NMR spectroscopy. The measured isotopic dilutions were not the same for succinate and acetate; in the case of succinate, the dilutions reflected only the contribution of glycogen futile cycling, while in the case of acetate, another mechanism was also involved. Results obtained in complementary experiments are consistent with reversal of the succinate synthesis pathway. Our results indicated that for all of the strains, from 12 to 16% of the glucose entering the metabolic pathway originated from prestored glycogen. Although genetically diverse, the four Fibrobacter strains studied had very similar carbon metabolism characteristics.     

Metabolic network analysis of Streptomyces tenebrarius, a Streptomyces species with an active entner-doudoroff pathway

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. (13)C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.     

Chemostat culture of Bacillus caldotenax at 65°C: Activity and regulation of the main metabolic pathways

Bacillus caldotenax was cultivated in chemostat experiments at 65°C with a chemically defined minimal medium. Glycolysis, tricarboxylic acid cycle, pentose phosphate pathway and the respiratory chain were active as demonstrated by measuring the corresponding enzymes. No enzyme activity of the Entner-Doudoroff pathway could be detected. The specific activities of the citrate cycle enzymes were up to 10 times higher as compared to the enzymes of glycolysis. At dilution rates between 0.3 and 2.2 h^-1 none of the main metabolic pathways was regulated. In contrast the isocitrate lyase was regulated (drop of activity with increasing growth rates). As a result of a batch culture with glucose and acetate as carbon sources a regulation model was proposed: glucose, or a metabolite of glucose, represses the isocitrate lyase; in the absence of glucose acetate acts as an inducer.Abbreviations DCIP dichlorphenol indophenol - ED Entner-Doudoroff pathway - EMP Emden-Meyerhof-Parnas pathway - ICL isocitrate lyase - PP pentose phosphate pathway - TCC tricarbonic acid cycle     

Growth phase‐dependent global protein and metabolite profiles of Phaeobacter gallaeciensis strain DSM 17395, a member of the marine Roseobacter‐clade

The marine heterotrophic roseobacter Phaeobacter gallaeciensis DSM 17395 was grown with glucose in defined mineral medium. Relative abundance changes of global protein (2‐D DIGE) and metabolite (GC‐MS) profiles were determined across five different time points of growth. In total, 215 proteins were identified and 147 metabolites detected (101 structurally identified), among which 60 proteins and 87 metabolites displayed changed abundances upon entry into stationary growth phase. Glucose breakdown to pyruvate apparently proceeds via the Entner–Doudoroff (ED) pathway, since phosphofructokinase of the Embden–Meyerhof–Parnas pathway is missing and the key metabolite of the ED‐pathway, 2‐keto‐3‐desoxygluconate, was detected. The absence of pfk in other genome‐sequenced roseobacters suggests that the use of the ED pathway is an important physiological property among these heterotrophic marine bacteria. Upon entry into stationary growth phase (due to glucose starvation), sulfur assimilation (including cysteine biosynthesis) and parts of cell envelope synthesis (e.g. the lipid precursor 1‐monooleoylglycerol) were down‐regulated and cadaverine formation up‐regulated. In contrast, central carbon catabolism remained essentially unchanged, pointing to a metabolic “stand‐by” modus as an ecophysiological adaptation strategy. Stationary phase response of P. gallaeciensis differs markedly from that of standard organisms such as Escherichia coli, as evident e.g. by the absence of an rpoS gene.     

Glucose metabolic flux distribution of Lactobacillus amylophilus during lactic acid production using kitchen waste saccharified solution

The ¹³C isotope tracer method was used to investigate the glucose metabolic flux distribution and regulation in Lactobacillus amylophilus to improve lactic acid production using kitchen waste saccharified solution (KWSS). The results demonstrate that L. amylophilus is a homofermentative bacterium. In synthetic medium, 60.6% of the glucose entered the Embden–Meyerhof–Parnas (EMP) to produce lactic acid, whereas 36.4% of the glucose entered the pentose phosphate metabolic pathway (HMP). After solid–liquid separation of the KWSS, the addition of Fe³⁺ during fermentation enhanced the NADPH production efficiency and increased the NADH content. The flux to the EMP was also effectively increased. Compared with the control (60.6% flux to EMP without Fe³⁺ addition), the flux to the EMP with the addition of Fe³⁺ (74.3%) increased by 23.8%. In the subsequent pyruvate metabolism, Fe³⁺ also increased lactate dehydrogenase activity, and inhibited alcohol dehydrogenase, pyruvate dehydrogenase and pyruvate carboxylase, thereby increasing the lactic acid production to 9.03 g l⁻¹, an increase of 8% compared with the control. All other organic acid by-products were lower than in the control. However, the addition of Zn²⁺ showed an opposite effect, decreasing the lactic acid production. In conclusion it is feasible and effective means using GC-MS, isotope experiment and MATLAB software to integrate research the metabolic flux distribution of lactic acid bacteria, and the results provide the theoretical foundation for similar metabolic flux distribution.     

A transferable plasticity region in Campylobacter coli allows isolates of an otherwise non‐glycolytic food‐borne pathogen to catabolize glucose

Thermophilic Campylobacter species colonize the intestine of agricultural and domestic animals commensally but cause severe gastroenteritis in humans. In contrast to other enteropathogenic bacteria, Campylobacter has been considered to be non‐glycolytic, a metabolic property originally used for their taxonomic classification. Contrary to this dogma, we demonstrate that several Campylobacter coli strains are able to utilize glucose as a growth substrate. Isotopologue profiling experiments with ¹³C‐labeled glucose suggested that these strains catabolize glucose via the pentose phosphate and Entner‐Doudoroff (ED) pathways and use glucose efficiently for de novo synthesis of amino acids and cell surface carbohydrates. Whole genome sequencing of glycolytic C. coli isolates identified a genomic island located within a ribosomal RNA gene cluster that encodes for all ED pathway enzymes and a glucose permease. We could show in vitro that a non‐glycolytic C. coli strain could acquire glycolytic activity through natural transformation with chromosomal DNA of C. coli and C. jejuni subsp. doylei strains possessing the ED pathway encoding plasticity region. These results reveal for the first time the ability of a Campylobacter species to catabolize glucose and provide new insights into how genetic macrodiversity through intra‐ and interspecies gene transfer expand the metabolic capacity of this food‐borne pathogen.     

Temperature and nutrient induced responses of Lake Fryxell sulfate-reducing prokaryotes and description of Desulfovibrio lacusfryxellense, sp. nov., a pervasive, cold-active, sulfate-reducing bacterium from Lake Fryxell, Antarctica

The effects of temperature and carbon substrate availability on the stimulation of sulfate reduction by indigenous populations of sulfate-reducing prokaryotes (SRP) in permanently ice-covered Lake Fryxell, Antarctica were investigated. Psychrophilic and halotolerant, lactate-degrading SRP showed significant metabolic activity throughout all sampled depths of the water column, suggesting that such organisms, possibly of marine origin, may be key contributors to carbon and sulfur cycling in Lake Fryxell. Planktonic and benthic strains of lactate-oxidizing sulfate-reducing bacteria (SRB) were isolated from samples of various depths of the anoxic water column and from surficial sediments. Phylogenetic analyses of 16S rRNA gene sequences placed the Fryxell sulfate-reducer (FSR) strains within the Deltaproteobacteria and showed them to be most closely related to the Arctic marine species of SRB Desulfovibrio frigidus and Desulfovibrio ferrireducens. Based on phylogenetic and phenotypic differences between the Antarctic FSR strains and related species of the genus Desulfovibrio, strain FSRs^T (=DSM 23315^T =ATCC BAA-2083^T) is proposed as the type strain of a novel species of cold-active SRB, Desulfovibrio lacusfryxellense, sp. nov.     

The characteristics and comparative analysis of methanotrophs reveal genomic insights into Methylomicrobium sp. enriched from marine sediments

Methanotrophic bacteria are widespread and use methane as a sole carbon and energy source. They also play a crucial role in marine ecosystems by preventing the escape of methane into the atmosphere from diverse methane sources, such as methane seeps and hydrothermal vents. Despite their importance for methane carbon cycling, relatively few marine methanotrophic bacteria have been isolated and studied at the genomic level. Herein, we report the genome of a marine methanotrophic member of the genus Methylomicrobium, metagenome-assembled genome (MAG) wino1, which was obtained through enrichment using methane as the sole carbon source. Phylogenetic analysis based on 16S rRNA sequences and comparison of pmoA genes supported the close relationship of MAG-wino1 to the genus Methylomicrobium and it possessed a genome of 5.06 Mb encoding many specialized methanotrophic genes. A comparison of MAG-wino1 with the genomes of other strains (Methylomicrobium alcaliphilum 20Z^T and Methylomicrobium buryatense 5G) showed that genes (e.g. ectABC, ask, and mscLS) involved in the accumulation of compatible solutes required for survival in marine environments might be conserved. Methane utilization genes, including methanol dehydrogenase, and key enzymes related to ribulose monophosphate (RuMP) metabolism were identified. The wino1 genome harbored nitrogen fixation, urease, urea and nitrate transporter genes involved in the exploitation of nitrogen sources. Poly-β-hydroxybutyrate degradation and glycogen synthesis-related genes may facilitate survival under nutrient-limiting conditions. Additionally, genome analysis revealed three dominant taxa in the enrichment culture, methanotroph Methylomicrobium sp., methylotroph Methyloceanibacter sp., and non-methylotroph Labrenzia sp., which provided insights into microbial associations in marine sediments.