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Formate hydrogen lyase mediates stationary-phase deacidification and increases survival during sugar fermentation in acetoin-producing enterobacteria.

Vivijs B, Haberbeck LU, Baiye Mfortaw Mbong V, Bernaerts K, Geeraerd AH, Aertsen A, Michiels CW - Front Microbiol (2015)

Bottom Line: Metabolite analysis in E. coli showed that introduction of the acetoin pathway reduced lactate and acetate production, but increased glucose consumption and formate and ethanol production.Analysis of a hycE mutant in S. plymuthica confirmed that medium deacidification in this organism is also mediated by FHL.These findings improve our understanding of the physiology and function of fermentation pathways in Enterobacteriaceae.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Food Microbiology and Leuven Food Science and Nutrition Research Centre, Department of Microbial and Molecular Systems, Faculty of Bioscience Engineering, KU Leuven Leuven, Belgium.

ABSTRACT
Two fermentation types exist in the Enterobacteriaceae family. Mixed-acid fermenters produce substantial amounts of lactate, formate, acetate, and succinate, resulting in lethal medium acidification. On the other hand, 2,3-butanediol fermenters switch to the production of the neutral compounds acetoin and 2,3-butanediol and even deacidify the environment after an initial acidification phase, thereby avoiding cell death. We equipped three mixed-acid fermenters (Salmonella Typhimurium, S. Enteritidis and Shigella flexneri) with the acetoin pathway from Serratia plymuthica to investigate the mechanisms of deacidification. Acetoin production caused attenuated acidification during exponential growth in all three bacteria, but stationary-phase deacidification was only observed in Escherichia coli and Salmonella, suggesting that it was not due to the consumption of protons accompanying acetoin production. To identify the mechanism, 34 transposon mutants of acetoin-producing E. coli that no longer deacidified the culture medium were isolated. The mutations mapped to 16 genes, all involved in formate metabolism. Formate is an end product of mixed-acid fermentation that can be converted to H2 and CO2 by the formate hydrogen lyase (FHL) complex, a reaction that consumes protons and thus can explain medium deacidification. When hycE, encoding the large subunit of hydrogenase 3 that is part of the FHL complex, was deleted in acetoin-producing E. coli, deacidification capacity was lost. Metabolite analysis in E. coli showed that introduction of the acetoin pathway reduced lactate and acetate production, but increased glucose consumption and formate and ethanol production. Analysis of a hycE mutant in S. plymuthica confirmed that medium deacidification in this organism is also mediated by FHL. These findings improve our understanding of the physiology and function of fermentation pathways in Enterobacteriaceae.

No MeSH data available.


Related in: MedlinePlus

Time profiles of glucose consumption (A) and production of the metabolites ethanol (B), acetate (C), lactate (D), succinate (E), and formate (F) during fermentative growth of E. coli MG1655 wild-type (solid lines) or ΔhycE (dashed lines) containing pTrc99A (gray) or pTrc99A-Ptrc-budAB (black) in LB medium with 5 g/l glucose, 1 mM IPTG and 100 μg/ml Ap at 37°C for 48 h. Error bars represent SD.
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Figure 4: Time profiles of glucose consumption (A) and production of the metabolites ethanol (B), acetate (C), lactate (D), succinate (E), and formate (F) during fermentative growth of E. coli MG1655 wild-type (solid lines) or ΔhycE (dashed lines) containing pTrc99A (gray) or pTrc99A-Ptrc-budAB (black) in LB medium with 5 g/l glucose, 1 mM IPTG and 100 μg/ml Ap at 37°C for 48 h. Error bars represent SD.

Mentions: To provide more direct evidence for the involvement of formate disproportionation in the deacidification capacity of budAB-containing E. coli, glucose consumption and the production of metabolites were determined by HPLC during fermentative growth in LB with glucose (Figure 4). Succinate concentrations (Figure 4E) remained low for all strains during the course of the experiment. On the other hand, the budAB genes caused a marked shift in the production of two of the major acids of the mixed-acid fermentation pathway, especially in the late exponential and stationary growth phase, with no more acetate and much less lactate being produced (Figures 4C,D, respectively). With regard to formate (Figure 4F), the highest formate accumulation was seen in the ΔhycE mutants, probably because these have lost their major route to convert formate to H2 and CO2. During the stationary growth phase (up to 48 h), the formate concentrations remained almost constant in the hycE- strains, but strongly decreased in the hycE+ strains, indicating the reuptake and conversion of formate to CO2 and H2. Interestingly, a close look at the formate accumulation curves of the ΔhycE mutants reveals a transient decline in the late exponential growth phase (onset at 4 h of growth). Also in the hycE+ background a decline (budAB-less strain) or a diminished accumulation (budAB-containing strain) of formate was observed in this phase. A possible explanation for this is the activity of the FDH-N, which also catalyzes the oxidation of formate to CO2 (Sawers, 1994). However, FDH-N transfers the electrons to nitrate (via a nitrate reductase) instead of protons and has a much higher affinity for formate than the FDH-H (Leonhartsberger et al., 2002), which could explain why it is active in an earlier growth stage. The activity of FDH-N is limited, however, because LB medium contains only a small amount of nitrate. The disproportionation of formate (Figure 4F) by the hycE+ strains lasted longer when the budAB genes were present (48 h) than when they were absent (24 h), probably because a higher amount of formate was produced. This was also reflected by an increased gas production in the presence of the budAB genes during this phase, as reported above (Figure 3). Finally, ethanol was produced in higher quantities by the budAB-containing strains (Figure 4B).


Formate hydrogen lyase mediates stationary-phase deacidification and increases survival during sugar fermentation in acetoin-producing enterobacteria.

Vivijs B, Haberbeck LU, Baiye Mfortaw Mbong V, Bernaerts K, Geeraerd AH, Aertsen A, Michiels CW - Front Microbiol (2015)

Time profiles of glucose consumption (A) and production of the metabolites ethanol (B), acetate (C), lactate (D), succinate (E), and formate (F) during fermentative growth of E. coli MG1655 wild-type (solid lines) or ΔhycE (dashed lines) containing pTrc99A (gray) or pTrc99A-Ptrc-budAB (black) in LB medium with 5 g/l glucose, 1 mM IPTG and 100 μg/ml Ap at 37°C for 48 h. Error bars represent SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4340222&req=5

Figure 4: Time profiles of glucose consumption (A) and production of the metabolites ethanol (B), acetate (C), lactate (D), succinate (E), and formate (F) during fermentative growth of E. coli MG1655 wild-type (solid lines) or ΔhycE (dashed lines) containing pTrc99A (gray) or pTrc99A-Ptrc-budAB (black) in LB medium with 5 g/l glucose, 1 mM IPTG and 100 μg/ml Ap at 37°C for 48 h. Error bars represent SD.
Mentions: To provide more direct evidence for the involvement of formate disproportionation in the deacidification capacity of budAB-containing E. coli, glucose consumption and the production of metabolites were determined by HPLC during fermentative growth in LB with glucose (Figure 4). Succinate concentrations (Figure 4E) remained low for all strains during the course of the experiment. On the other hand, the budAB genes caused a marked shift in the production of two of the major acids of the mixed-acid fermentation pathway, especially in the late exponential and stationary growth phase, with no more acetate and much less lactate being produced (Figures 4C,D, respectively). With regard to formate (Figure 4F), the highest formate accumulation was seen in the ΔhycE mutants, probably because these have lost their major route to convert formate to H2 and CO2. During the stationary growth phase (up to 48 h), the formate concentrations remained almost constant in the hycE- strains, but strongly decreased in the hycE+ strains, indicating the reuptake and conversion of formate to CO2 and H2. Interestingly, a close look at the formate accumulation curves of the ΔhycE mutants reveals a transient decline in the late exponential growth phase (onset at 4 h of growth). Also in the hycE+ background a decline (budAB-less strain) or a diminished accumulation (budAB-containing strain) of formate was observed in this phase. A possible explanation for this is the activity of the FDH-N, which also catalyzes the oxidation of formate to CO2 (Sawers, 1994). However, FDH-N transfers the electrons to nitrate (via a nitrate reductase) instead of protons and has a much higher affinity for formate than the FDH-H (Leonhartsberger et al., 2002), which could explain why it is active in an earlier growth stage. The activity of FDH-N is limited, however, because LB medium contains only a small amount of nitrate. The disproportionation of formate (Figure 4F) by the hycE+ strains lasted longer when the budAB genes were present (48 h) than when they were absent (24 h), probably because a higher amount of formate was produced. This was also reflected by an increased gas production in the presence of the budAB genes during this phase, as reported above (Figure 3). Finally, ethanol was produced in higher quantities by the budAB-containing strains (Figure 4B).

Bottom Line: Metabolite analysis in E. coli showed that introduction of the acetoin pathway reduced lactate and acetate production, but increased glucose consumption and formate and ethanol production.Analysis of a hycE mutant in S. plymuthica confirmed that medium deacidification in this organism is also mediated by FHL.These findings improve our understanding of the physiology and function of fermentation pathways in Enterobacteriaceae.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Food Microbiology and Leuven Food Science and Nutrition Research Centre, Department of Microbial and Molecular Systems, Faculty of Bioscience Engineering, KU Leuven Leuven, Belgium.

ABSTRACT
Two fermentation types exist in the Enterobacteriaceae family. Mixed-acid fermenters produce substantial amounts of lactate, formate, acetate, and succinate, resulting in lethal medium acidification. On the other hand, 2,3-butanediol fermenters switch to the production of the neutral compounds acetoin and 2,3-butanediol and even deacidify the environment after an initial acidification phase, thereby avoiding cell death. We equipped three mixed-acid fermenters (Salmonella Typhimurium, S. Enteritidis and Shigella flexneri) with the acetoin pathway from Serratia plymuthica to investigate the mechanisms of deacidification. Acetoin production caused attenuated acidification during exponential growth in all three bacteria, but stationary-phase deacidification was only observed in Escherichia coli and Salmonella, suggesting that it was not due to the consumption of protons accompanying acetoin production. To identify the mechanism, 34 transposon mutants of acetoin-producing E. coli that no longer deacidified the culture medium were isolated. The mutations mapped to 16 genes, all involved in formate metabolism. Formate is an end product of mixed-acid fermentation that can be converted to H2 and CO2 by the formate hydrogen lyase (FHL) complex, a reaction that consumes protons and thus can explain medium deacidification. When hycE, encoding the large subunit of hydrogenase 3 that is part of the FHL complex, was deleted in acetoin-producing E. coli, deacidification capacity was lost. Metabolite analysis in E. coli showed that introduction of the acetoin pathway reduced lactate and acetate production, but increased glucose consumption and formate and ethanol production. Analysis of a hycE mutant in S. plymuthica confirmed that medium deacidification in this organism is also mediated by FHL. These findings improve our understanding of the physiology and function of fermentation pathways in Enterobacteriaceae.

No MeSH data available.


Related in: MedlinePlus