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Adaptative biochemical pathways and regulatory networks in Klebsiella oxytoca BAS-10 producing a biotechnologically relevant exopolysaccharide during Fe(III)-citrate fermentation.

Gallo G, Baldi F, Renzone G, Gallo M, Cordaro A, Scaloni A, Puglia AM - Microb. Cell Fact. (2012)

Bottom Line: Unlikely other enterobacteria, BAS-10 is able to grow on Fe(III)-citrate as sole carbon and energy source, yielding acetic acid and CO2 coupled with Fe(III) reduction to Fe(II) and showing unusual physiological characteristics.The protein differential regulation seems to ensure efficient cell growth coupled with EPS production by adapting metabolic and biochemical processes in order to face iron toxicity and to optimize energy production.Differential proteomics provided insights on the molecular mechanisms necessary for anaeorobic utilization of Fe(III)-citrate in a biotechnologically promising enterobacteriun, also revealing genes that can be targeted for the rational design of high-yielding EPS producer strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari (STEMBIO), Università di Palermo Viale delle Scienze, ed, 16, Parco d'Orleans II, Palermo, 90128, Italy. giumir@msn.com

ABSTRACT

Background: A bacterial strain previously isolated from pyrite mine drainage and named BAS-10 was tentatively identified as Klebsiella oxytoca. Unlikely other enterobacteria, BAS-10 is able to grow on Fe(III)-citrate as sole carbon and energy source, yielding acetic acid and CO2 coupled with Fe(III) reduction to Fe(II) and showing unusual physiological characteristics. In fact, under this growth condition, BAS-10 produces an exopolysaccharide (EPS) having a high rhamnose content and metal-binding properties, whose biotechnological applications were proven as very relevant.

Results: Further phylogenetic analysis, based on 16S rDNA sequence, definitively confirmed that BAS-10 belongs to K. oxytoca species. In order to rationalize the biochemical peculiarities of this unusual enterobacteriun, combined 2D-Differential Gel Electrophoresis (2D-DIGE) analysis and mass spectrometry procedures were used to investigate its proteomic changes: i) under aerobic or anaerobic cultivation with Fe(III)-citrate as sole carbon source; ii) under anaerobic cultivations using Na(I)-citrate or Fe(III)-citrate as sole carbon source. Combining data from these differential studies peculiar levels of outer membrane proteins, key regulatory factors of carbon and nitrogen metabolism and enzymes involved in TCA cycle and sugar biosynthesis or required for citrate fermentation and stress response during anaerobic growth on Fe(III)-citrate were revealed. The protein differential regulation seems to ensure efficient cell growth coupled with EPS production by adapting metabolic and biochemical processes in order to face iron toxicity and to optimize energy production.

Conclusion: Differential proteomics provided insights on the molecular mechanisms necessary for anaeorobic utilization of Fe(III)-citrate in a biotechnologically promising enterobacteriun, also revealing genes that can be targeted for the rational design of high-yielding EPS producer strains.

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Synoptic scheme of metabolic pathways involved in Fe(III)-citrate catabolism. Anabolic routes, regulatory and membrane-associated proteins are also indicated. Reactions are reported according to KEGG [22] and EcoCy [23] databases. OM: outer membrane. PG: peptidoglycan. IM: inner membrane.
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Figure 4: Synoptic scheme of metabolic pathways involved in Fe(III)-citrate catabolism. Anabolic routes, regulatory and membrane-associated proteins are also indicated. Reactions are reported according to KEGG [22] and EcoCy [23] databases. OM: outer membrane. PG: peptidoglycan. IM: inner membrane.

Mentions: Occurrence of Fe(III) during anaerobic growth also modulated the abundance of many central carbon metabolism enzymes. In particular, pyruvate kinase (PK), glyceraldehyde phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TIM) were up-regulated whereas TCA cycle enzymes, such as dihydrolipoyllysine-residue succinyltransferase (SucB), malate dehydrogenase (MDH) and fumarate hydratase (FH) were down-regulated in anaerobic FEC with respect to both aerobic FEC and anaerobic NAC (Table 1, Figures 2 and 3). Interestingly, fumarate reductase flavoprotein subunit (FrdA) was up-regulated during the anaerobic growth on FEC with respect to both NAC and aerobic FEC (Figure 4). FrdA is part of complex II homolog menaquinol:fumarate oxidoreductase, which oxidizes menaquinol and transfers the electrons to fumarate during bacterial anaerobic respiration, with fumarate as the terminal electron acceptor [32], thus counteracting TCA cycle down-regulation. Altogether these data suggest that to efficiently divert the carbon flux towards acetate and ATP production citrate fermentation enzymes are up-regulated during anaerobic growth on FEC, whereas TCA cycle enzymes are repressed (Table 1 and Figure 4). In addition, these data indicated that the increased catabolism of citrate throughout a fermentative pathway is coupled to the synthesis of metabolic precursors necessary for anabolic processes like sugar biosynthesis. In particular, the TIM product glycerone-P is a precursor involved in rhamnose biosynthesis (Figure 4). Since rhamnose is the major sugar of EPS [17], the observed TIM up-regulation can represent an interesting link between central carbon metabolites and EPS synthesis in Klebsiella. As a consequence of the increased anaerobic citrate fermentation, the production of acetic acid may also determine an increment of H+ gradient across cell membrane, which positively affects the activity of the ATP synthase complex. In agreement with this view, ATP synthase subunit B was observed as up-regulated under anaerobic conditions in FEC medium, in the respect of both NAC and aerobic FEC (Table 1, Figures 2, 3 and 4).


Adaptative biochemical pathways and regulatory networks in Klebsiella oxytoca BAS-10 producing a biotechnologically relevant exopolysaccharide during Fe(III)-citrate fermentation.

Gallo G, Baldi F, Renzone G, Gallo M, Cordaro A, Scaloni A, Puglia AM - Microb. Cell Fact. (2012)

Synoptic scheme of metabolic pathways involved in Fe(III)-citrate catabolism. Anabolic routes, regulatory and membrane-associated proteins are also indicated. Reactions are reported according to KEGG [22] and EcoCy [23] databases. OM: outer membrane. PG: peptidoglycan. IM: inner membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539929&req=5

Figure 4: Synoptic scheme of metabolic pathways involved in Fe(III)-citrate catabolism. Anabolic routes, regulatory and membrane-associated proteins are also indicated. Reactions are reported according to KEGG [22] and EcoCy [23] databases. OM: outer membrane. PG: peptidoglycan. IM: inner membrane.
Mentions: Occurrence of Fe(III) during anaerobic growth also modulated the abundance of many central carbon metabolism enzymes. In particular, pyruvate kinase (PK), glyceraldehyde phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TIM) were up-regulated whereas TCA cycle enzymes, such as dihydrolipoyllysine-residue succinyltransferase (SucB), malate dehydrogenase (MDH) and fumarate hydratase (FH) were down-regulated in anaerobic FEC with respect to both aerobic FEC and anaerobic NAC (Table 1, Figures 2 and 3). Interestingly, fumarate reductase flavoprotein subunit (FrdA) was up-regulated during the anaerobic growth on FEC with respect to both NAC and aerobic FEC (Figure 4). FrdA is part of complex II homolog menaquinol:fumarate oxidoreductase, which oxidizes menaquinol and transfers the electrons to fumarate during bacterial anaerobic respiration, with fumarate as the terminal electron acceptor [32], thus counteracting TCA cycle down-regulation. Altogether these data suggest that to efficiently divert the carbon flux towards acetate and ATP production citrate fermentation enzymes are up-regulated during anaerobic growth on FEC, whereas TCA cycle enzymes are repressed (Table 1 and Figure 4). In addition, these data indicated that the increased catabolism of citrate throughout a fermentative pathway is coupled to the synthesis of metabolic precursors necessary for anabolic processes like sugar biosynthesis. In particular, the TIM product glycerone-P is a precursor involved in rhamnose biosynthesis (Figure 4). Since rhamnose is the major sugar of EPS [17], the observed TIM up-regulation can represent an interesting link between central carbon metabolites and EPS synthesis in Klebsiella. As a consequence of the increased anaerobic citrate fermentation, the production of acetic acid may also determine an increment of H+ gradient across cell membrane, which positively affects the activity of the ATP synthase complex. In agreement with this view, ATP synthase subunit B was observed as up-regulated under anaerobic conditions in FEC medium, in the respect of both NAC and aerobic FEC (Table 1, Figures 2, 3 and 4).

Bottom Line: Unlikely other enterobacteria, BAS-10 is able to grow on Fe(III)-citrate as sole carbon and energy source, yielding acetic acid and CO2 coupled with Fe(III) reduction to Fe(II) and showing unusual physiological characteristics.The protein differential regulation seems to ensure efficient cell growth coupled with EPS production by adapting metabolic and biochemical processes in order to face iron toxicity and to optimize energy production.Differential proteomics provided insights on the molecular mechanisms necessary for anaeorobic utilization of Fe(III)-citrate in a biotechnologically promising enterobacteriun, also revealing genes that can be targeted for the rational design of high-yielding EPS producer strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari (STEMBIO), Università di Palermo Viale delle Scienze, ed, 16, Parco d'Orleans II, Palermo, 90128, Italy. giumir@msn.com

ABSTRACT

Background: A bacterial strain previously isolated from pyrite mine drainage and named BAS-10 was tentatively identified as Klebsiella oxytoca. Unlikely other enterobacteria, BAS-10 is able to grow on Fe(III)-citrate as sole carbon and energy source, yielding acetic acid and CO2 coupled with Fe(III) reduction to Fe(II) and showing unusual physiological characteristics. In fact, under this growth condition, BAS-10 produces an exopolysaccharide (EPS) having a high rhamnose content and metal-binding properties, whose biotechnological applications were proven as very relevant.

Results: Further phylogenetic analysis, based on 16S rDNA sequence, definitively confirmed that BAS-10 belongs to K. oxytoca species. In order to rationalize the biochemical peculiarities of this unusual enterobacteriun, combined 2D-Differential Gel Electrophoresis (2D-DIGE) analysis and mass spectrometry procedures were used to investigate its proteomic changes: i) under aerobic or anaerobic cultivation with Fe(III)-citrate as sole carbon source; ii) under anaerobic cultivations using Na(I)-citrate or Fe(III)-citrate as sole carbon source. Combining data from these differential studies peculiar levels of outer membrane proteins, key regulatory factors of carbon and nitrogen metabolism and enzymes involved in TCA cycle and sugar biosynthesis or required for citrate fermentation and stress response during anaerobic growth on Fe(III)-citrate were revealed. The protein differential regulation seems to ensure efficient cell growth coupled with EPS production by adapting metabolic and biochemical processes in order to face iron toxicity and to optimize energy production.

Conclusion: Differential proteomics provided insights on the molecular mechanisms necessary for anaeorobic utilization of Fe(III)-citrate in a biotechnologically promising enterobacteriun, also revealing genes that can be targeted for the rational design of high-yielding EPS producer strains.

Show MeSH
Related in: MedlinePlus