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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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Functional distribution and abundance fold change (blue diamond) of differentially represented protein spots resulting from the proteomic comparison between anaerobic growth on FEC and NAC. Fold change and spot number refer to Table 1. A. a. stands for Amino acid. Met. stands for metabolism. Cof. biosynth. stands for Cofactor biosynthesis.
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Figure 3: Functional distribution and abundance fold change (blue diamond) of differentially represented protein spots resulting from the proteomic comparison between anaerobic growth on FEC and NAC. Fold change and spot number refer to Table 1. A. a. stands for Amino acid. Met. stands for metabolism. Cof. biosynth. stands for Cofactor biosynthesis.

Mentions: The differential proteomic analysis revealed up-regulation of citrate lyase α- and β-subunit (CitE and CitF), oxaloacetate decarboxylase α-subunit (OadA), pyruvate formate lyase (PFL), phosphotrans-acetylase (PAT) and acetate/propionate kinase (ACK) in anaerobic FEC with respect to both aerobic FEC and anaerobic NAC (Table 1; Figures 2 and 3). These proteins are known to be involved in anaerobic citrate fermentation, eventually converting citrate to acetate with production of ATP. In particular, CitF and CitE are part of the citrate lyase complex, the key enzyme in initiating the anaerobic utilization of citrate, which is responsible for catalyzing the conversion of citrate into acetate and oxaloacetate. The oxaloacetate decarboxylase complex, constituted by alpha, beta and gamma subunits, catalyzes the second step of citrate fermentation by converting oxaloacetate into pyruvate, the latter being the substrate of PFL, a typical enzyme of enterobacteria growing under anaerobic conditions [25], that generates formate and acetyl-CoA. Phosphotrans-acetylase (PAT) catalyzes conversion of acetyl-CoA into acetyl-P, which finally transfers the phosphate group to ADP to yield acetic acid and ATP in the ACK-catalyzed last step of citrate fermentation. Different forms of CitE, CitF, OadA and PFL were identified in K. oxytoca BAS-10 2D-protein maps (Table 1, Figures 2 and 3; Additional file 1 Table S1 and Figure S2). The occurrence of several protein forms having Mw and/or pI different from the predicted one would imply that protein activity could be controlled by post-translational modifications (PTMs), like covalently-bound charged small molecules (as in the case horizontal spot trains) or proteolytic digestion (as in the case of protein isoform with reduced Mw). Indeed, PTMs, like acetylation, succinylation and radical formation have already been shown for PFL activity regulation [26-28], thus giving count for the generation of different protein spots in 2D-maps. In the case of OadA, biotinylation in lysine, reported to be crucial for oxaloacetate decarboxylase activity [29], could explain the slight increase of the measured Mw while the generation of reduced Mw species is likely to be due to proteolytic fragmentation. Concerning CitE and CitF, this is the first study suggesting PTM regulatory events. The differential analysis showed that anaerobic conditions promote citrate fermentation enzymes accumulation, similarly to what already reported in other enterobacteria [10,30,31]. In addition and more interestingly, during BAS-10 anaerobic growth, Fe(III) positively controls the accumulation of citrate fermentative enzymes better than Na(I). In fact, in K. oxytoca BAS-10 citrate fermentation appears much more up-regulated in presence of Fe(III) than Na(I), making this process unique for this microorganism among enterobacteria which usually ferment citrate in a NA(I)-dependent manner [1,8-10].


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)

Functional distribution and abundance fold change (blue diamond) of differentially represented protein spots resulting from the proteomic comparison between anaerobic growth on FEC and NAC. Fold change and spot number refer to Table 1. A. a. stands for Amino acid. Met. stands for metabolism. Cof. biosynth. stands for Cofactor biosynthesis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Functional distribution and abundance fold change (blue diamond) of differentially represented protein spots resulting from the proteomic comparison between anaerobic growth on FEC and NAC. Fold change and spot number refer to Table 1. A. a. stands for Amino acid. Met. stands for metabolism. Cof. biosynth. stands for Cofactor biosynthesis.
Mentions: The differential proteomic analysis revealed up-regulation of citrate lyase α- and β-subunit (CitE and CitF), oxaloacetate decarboxylase α-subunit (OadA), pyruvate formate lyase (PFL), phosphotrans-acetylase (PAT) and acetate/propionate kinase (ACK) in anaerobic FEC with respect to both aerobic FEC and anaerobic NAC (Table 1; Figures 2 and 3). These proteins are known to be involved in anaerobic citrate fermentation, eventually converting citrate to acetate with production of ATP. In particular, CitF and CitE are part of the citrate lyase complex, the key enzyme in initiating the anaerobic utilization of citrate, which is responsible for catalyzing the conversion of citrate into acetate and oxaloacetate. The oxaloacetate decarboxylase complex, constituted by alpha, beta and gamma subunits, catalyzes the second step of citrate fermentation by converting oxaloacetate into pyruvate, the latter being the substrate of PFL, a typical enzyme of enterobacteria growing under anaerobic conditions [25], that generates formate and acetyl-CoA. Phosphotrans-acetylase (PAT) catalyzes conversion of acetyl-CoA into acetyl-P, which finally transfers the phosphate group to ADP to yield acetic acid and ATP in the ACK-catalyzed last step of citrate fermentation. Different forms of CitE, CitF, OadA and PFL were identified in K. oxytoca BAS-10 2D-protein maps (Table 1, Figures 2 and 3; Additional file 1 Table S1 and Figure S2). The occurrence of several protein forms having Mw and/or pI different from the predicted one would imply that protein activity could be controlled by post-translational modifications (PTMs), like covalently-bound charged small molecules (as in the case horizontal spot trains) or proteolytic digestion (as in the case of protein isoform with reduced Mw). Indeed, PTMs, like acetylation, succinylation and radical formation have already been shown for PFL activity regulation [26-28], thus giving count for the generation of different protein spots in 2D-maps. In the case of OadA, biotinylation in lysine, reported to be crucial for oxaloacetate decarboxylase activity [29], could explain the slight increase of the measured Mw while the generation of reduced Mw species is likely to be due to proteolytic fragmentation. Concerning CitE and CitF, this is the first study suggesting PTM regulatory events. The differential analysis showed that anaerobic conditions promote citrate fermentation enzymes accumulation, similarly to what already reported in other enterobacteria [10,30,31]. In addition and more interestingly, during BAS-10 anaerobic growth, Fe(III) positively controls the accumulation of citrate fermentative enzymes better than Na(I). In fact, in K. oxytoca BAS-10 citrate fermentation appears much more up-regulated in presence of Fe(III) than Na(I), making this process unique for this microorganism among enterobacteria which usually ferment citrate in a NA(I)-dependent manner [1,8-10].

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