Abstract:The alginate-producing bacterium Pseudomonas fluorescens utilizes the Entner-Doudoroff (ED) and pentose phosphate (PP) pathways to metabolize fructose, since the upper part of its Embden-Meyerhof-Parnas pathway is defective. Our previous study indicated that perturbation of the central carbon metabolism by diminishing glucose-6-phosphate dehydrogenase activity could lead to sugar phosphate stress when P. fluorescens was cultivated on fructose. In the present study, we demonstrate that PFLU2693, annotated as a … Show more
“…However, the data may also reflect a preference of the organism to internalize then further metabolize F-6-P. These parameters, including V max and K m , are also in line with what has been reported for other members of the HAD family phosphatases in bacteria (5,21,24).…”
Section: Figsupporting
confidence: 90%
“…number of mechanisms have been proposed to contribute to sugar-phosphate stress, as follows: (i) futile cycling, in which the cells expend energy to internalize and phosphorylate the sugar, but the sugar is then dephosphorylated and expelled (5); (ii) reduction of essential metabolic intermediates arising from an inability to internalize other carbohydrates (6); (iii) creation of toxic metabolic by-products, such as methylglyoxal (7,8); or (iv) deleterious effects of specific sugar-phosphates that act as allosteric modifiers of enzymes or regulatory proteins, causing dysregulation of metabolism (9). The majority of research on sugar-phosphate stress has been conducted with Gramnegative model organisms, with far fewer studies carried out with known pathogens or Gram-positive bacteria (10).…”
The dental caries pathogen Streptococcus mutans can ferment a variety of sugars to produce organic acids. Exposure of S. mutans to certain nonmetabolizable carbohydrates, such as xylitol, impairs growth and can cause cell death. Recently, the presence of a sugar-phosphate stress in S. mutans was demonstrated using a mutant lacking 1-phosphofructokinase (FruK) that accumulates fructose-1-phosphate (F-1-P). Here, we studied an operon in S. mutans, sppRA, which was highly expressed in the fruK mutant. Biochemical characterization of a recombinant SppA protein indicated that it possessed hexose-phosphate phosphohydrolase activity, with preferences for F-1-P and, to a lesser degree, fructose-6-phosphate (F-6-P). SppA activity was stimulated by Mg2+ and Mn2+ but inhibited by NaF. SppR, a DeoR family regulator, repressed the expression of the sppRA operon to minimum levels in the absence of the fructose-derived metabolite F-1-P and likely also F-6-P. The accumulation of F-1-P, as a result of growth on fructose, not only induced sppA expression, but it significantly altered biofilm maturation through increased cell lysis and enhanced extracellular DNA release. Constitutive expression of sppA, via a plasmid or by deleting sppR, greatly alleviated fructose-induced stress in a fruK mutant, enhanced resistance to xylitol, and reversed the effects of fructose on biofilm formation. Finally, by identifying three additional putative phosphatases that are capable of promoting sugar-phosphate tolerance, we show that S. mutans is capable of mounting a sugar-phosphate stress response by modulating the levels of certain glycolytic intermediates, functions that are interconnected with the ability of the organism to manifest key virulence behaviors.
IMPORTANCE Streptococcus mutans is a major etiologic agent for dental caries, primarily due to its ability to form biofilms on the tooth surface and to convert carbohydrates into organic acids. We have discovered a two-gene operon in S. mutans that regulates fructose metabolism by controlling the levels of fructose-1-phosphate, a potential signaling compound that affects bacterial behaviors. With fructose becoming increasingly common and abundant in the human diet, we reveal the ways that fructose may alter bacterial development, stress tolerance, and microbial ecology in the oral cavity to promote oral diseases.
“…However, the data may also reflect a preference of the organism to internalize then further metabolize F-6-P. These parameters, including V max and K m , are also in line with what has been reported for other members of the HAD family phosphatases in bacteria (5,21,24).…”
Section: Figsupporting
confidence: 90%
“…number of mechanisms have been proposed to contribute to sugar-phosphate stress, as follows: (i) futile cycling, in which the cells expend energy to internalize and phosphorylate the sugar, but the sugar is then dephosphorylated and expelled (5); (ii) reduction of essential metabolic intermediates arising from an inability to internalize other carbohydrates (6); (iii) creation of toxic metabolic by-products, such as methylglyoxal (7,8); or (iv) deleterious effects of specific sugar-phosphates that act as allosteric modifiers of enzymes or regulatory proteins, causing dysregulation of metabolism (9). The majority of research on sugar-phosphate stress has been conducted with Gramnegative model organisms, with far fewer studies carried out with known pathogens or Gram-positive bacteria (10).…”
The dental caries pathogen Streptococcus mutans can ferment a variety of sugars to produce organic acids. Exposure of S. mutans to certain nonmetabolizable carbohydrates, such as xylitol, impairs growth and can cause cell death. Recently, the presence of a sugar-phosphate stress in S. mutans was demonstrated using a mutant lacking 1-phosphofructokinase (FruK) that accumulates fructose-1-phosphate (F-1-P). Here, we studied an operon in S. mutans, sppRA, which was highly expressed in the fruK mutant. Biochemical characterization of a recombinant SppA protein indicated that it possessed hexose-phosphate phosphohydrolase activity, with preferences for F-1-P and, to a lesser degree, fructose-6-phosphate (F-6-P). SppA activity was stimulated by Mg2+ and Mn2+ but inhibited by NaF. SppR, a DeoR family regulator, repressed the expression of the sppRA operon to minimum levels in the absence of the fructose-derived metabolite F-1-P and likely also F-6-P. The accumulation of F-1-P, as a result of growth on fructose, not only induced sppA expression, but it significantly altered biofilm maturation through increased cell lysis and enhanced extracellular DNA release. Constitutive expression of sppA, via a plasmid or by deleting sppR, greatly alleviated fructose-induced stress in a fruK mutant, enhanced resistance to xylitol, and reversed the effects of fructose on biofilm formation. Finally, by identifying three additional putative phosphatases that are capable of promoting sugar-phosphate tolerance, we show that S. mutans is capable of mounting a sugar-phosphate stress response by modulating the levels of certain glycolytic intermediates, functions that are interconnected with the ability of the organism to manifest key virulence behaviors.
IMPORTANCE Streptococcus mutans is a major etiologic agent for dental caries, primarily due to its ability to form biofilms on the tooth surface and to convert carbohydrates into organic acids. We have discovered a two-gene operon in S. mutans that regulates fructose metabolism by controlling the levels of fructose-1-phosphate, a potential signaling compound that affects bacterial behaviors. With fructose becoming increasingly common and abundant in the human diet, we reveal the ways that fructose may alter bacterial development, stress tolerance, and microbial ecology in the oral cavity to promote oral diseases.
“…Our earlier studies in P. fluorescens indicated that even in a mucA mutant, alginate biosynthesis does not start immediately after the first alginate biosynthetic complexes have been assembled (Maleki et al, 2016). Moreover, both the P. fluorescens transposon insertion study (Ertesvåg et al, 2017) and studies focusing on the synthesis of fructose 6-phosphate (Maleki et al, 2015(Maleki et al, , 2017 clearly indicated that the cells prioritize growth and survival over alginate production. The present study indicates that the same is true for A. vinelandii.…”
Section: Alginate Biosynthesis Seems To Be Regulated By the Metabolicmentioning
confidence: 99%
“…In order to optimize bacterial production processes, it is important to understand how alginate biosynthesis is regulated as well as elucidate which other metabolic aspects have an influence on production levels. The precursor for the alginate monomers is fructose 6-phosphate, so alginate biosynthesis is closely connected to the central carbohydrate metabolism of the cells (Maleki et al, 2015(Maleki et al, , 2017Ertesvåg et al, 2017). Alginate production is furthermore an energy demanding process and is tightly controlled by a complex network of regulators which also influence other cellular processes (Urtuvia et al, 2017).…”
Azotobacter vinelandii produces the biopolymer alginate, which has a wide range of industrial and pharmaceutical applications. A random transposon insertion mutant library was constructed from A. vinelandii ATCC12518Tc in order to identify genes and pathways affecting alginate biosynthesis, and about 4,000 mutant strains were screened for altered alginate production. One mutant, containing a mucA disruption, displayed an elevated alginate production level, and several mutants with decreased or abolished alginate production were identified. The regulatory proteins AlgW and AmrZ seem to be required for alginate production in A. vinelandii, similarly to Pseudomonas aeruginosa. An algB mutation did however not affect alginate yield in A. vinelandii although its P. aeruginosa homolog is needed for full alginate production. Inactivation of the fructose phosphoenolpyruvate phosphotransferase system protein FruA resulted in a mutant that did not produce alginate when cultivated in media containing various carbon sources, indicating that this system could have a role in regulation of alginate biosynthesis. Furthermore, impaired or abolished alginate production was observed for strains with disruptions of genes involved in peptidoglycan biosynthesis/recycling and biosynthesis of purines, isoprenoids, TCA cycle intermediates, and various vitamins, suggesting that sufficient access to some of these compounds is important for alginate production. This hypothesis was verified by showing that addition of thiamine, succinate or a mixture of lysine, methionine and diaminopimelate increases alginate yield in the non-mutagenized strain. These results might be used in development of optimized alginate production media or in genetic engineering of A. vinelandii strains for alginate bioproduction.
“…During the course of model reconstruction, GSMM for P. putida Kt2440 was chosen as the basis as both P. putida Kt2440 and P. fluorescens use Entner–Doudoroff (ED) pathway for glycolysis . When catechol was selected as the substrate, both strains take β‐ketoadipate route for catechol degradation .…”
With the versatile metabolic diversity, Pseudomonas fluorescens is a potential candidate in petroleum aromatic hydrocarbon (PAH) bioremediation. Genome‐scale metabolic model (GSMM) can provide systematic information to guide the development of metabolic engineering strategy to improve microbial activity. In this study, a GSMM for P. fluorescens SBW25 was reconstructed, which is termed as lCW1057. The reconstruction was based on automatic reannotation and manual curation. The periplasmic compartment was constructed to better represent the proton gradient profile. The reconstructed proton transport chain has a P/O (ATP generated per atom oxygen consumed by the respiratory chain) ratio of 11/8. Flux balance analysis (FBA) was performed to explore the whole‐cell metabolic flow. The model suggested that instead of Embden–Meyerhof–Parnas pathway, Entner–Doudoroff pathway was used in glycolytic metabolism of P. fluorescens, indicating that the growth of P. fluorescens is more energy dependent. Furthermore, P. fluorescens can use nitrate as the terminal electron acceptor for the glucose metabolism. The β‐ketoadipate pathway was involved in catechol metabolism. The uptake of oxygen is mandatory for the aromatic ring cleavage. The in silico and in vitro maximum specific growth rate was compared, resulting in 10% difference when catechol was used as the sole carbon source.
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