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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 and aerobic growth on FEC. Fold change and spot number refer to Table 1. A. a. stands for Amino acid. Met. stands for metabolism.
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Figure 2: Functional distribution and abundance fold change (blue diamond) of differentially represented protein spots resulting from the proteomic comparison between anaerobic and aerobic growth on FEC. Fold change and spot number refer to Table 1. A. a. stands for Amino acid. Met. stands for metabolism.

Mentions: To investigate iron-dependent cell processes and regulatory networks thereof associated to citrate fermentation and EPS production, BAS-10 was cultivated: i) under aerobic conditions on Fe(III)-citrate (FEC) as sole carbon source; ii) under anaerobic conditions using FEC or iii) Na(I)-citrate (NAC) as sole carbon source. Then, bacterial proteomes were analyzed using anaerobic FEC growth as pivotal condition. In particular, the “anaerobic FEC Vs aerobic FEC” differential analysis was carried out to reveal proteins that are required for anaerobic growth on FEC while “anaerobic FEC Vs anaerobic NAC” differential analysis was carried out to reveal proteins whose abundance is iron-dependent during anaerobic growth. In all cases, BAS-10 biomass samples were all harvested at late exponential growth phases, when anaerobic growth on FEC is coupled with iron-binding EPS production [12,16] at an average yield of 6.7 g/L (dry weight EPS per culture volume). Proteomic 2D-DIGE maps showed the occurrence of 869 reproducible protein spots (i. e. detected in all 2D-Gels) which were used to create the match set for comparative analysis. According to the criteria reported in the experimental section for identification of deregulated proteins, comparative analysis between FEC medium cultivations under anaerobic or aerobic conditions revealed 27 and 32 protein spots up- and down-regulated under anaerobic conditions, respectively (Table 1; Additional file 1 Figure S2). On the other hand, comparative analysis between cultivations in FEC and NAC media under anaerobic conditions revealed 28 and 42 protein spots as up- and down-regulated in the presence of Fe(III) (Table 1; Additional file 1 Figure S2). Among these protein spots, 46 resulted differentially abundant with a concordant profile in both analyses (anaerobic FEC vs aerobic FEC and anaerobic FEC vs anaerobic NAC) (Table 1), thus indicating that these proteins are specifically associated with anaerobic growth on FEC. The selected differentially abundant protein spots (83 in number) were alkylated, trypsinolyzed and identified by MALDI-TOF-peptide mass fingerprinting (PMF) and nanoLC-ESI-LIT-MS/MS procedures (Table 1 and Additional file 1 Table S1). Corresponding protein entries were clustered into functional groups, according to KEGG (http://www.genome.ad.jp/kegg/kegg2.html) [22], EcoCyc (http://ecocyc.org/) [23] and ExPASy (http://www.expasy.org) [24] databases (Additional file 1 Figure S3). Among the identified proteins, the most represented ones were related to central carbon metabolism -including glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, citrate fermentation, pentose phosphate (PP) pathway -and membrane transport (Table 1; Additional file 1 Figure S3).


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 and aerobic growth on FEC. Fold change and spot number refer to Table 1. A. a. stands for Amino acid. Met. stands for metabolism.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Functional distribution and abundance fold change (blue diamond) of differentially represented protein spots resulting from the proteomic comparison between anaerobic and aerobic growth on FEC. Fold change and spot number refer to Table 1. A. a. stands for Amino acid. Met. stands for metabolism.
Mentions: To investigate iron-dependent cell processes and regulatory networks thereof associated to citrate fermentation and EPS production, BAS-10 was cultivated: i) under aerobic conditions on Fe(III)-citrate (FEC) as sole carbon source; ii) under anaerobic conditions using FEC or iii) Na(I)-citrate (NAC) as sole carbon source. Then, bacterial proteomes were analyzed using anaerobic FEC growth as pivotal condition. In particular, the “anaerobic FEC Vs aerobic FEC” differential analysis was carried out to reveal proteins that are required for anaerobic growth on FEC while “anaerobic FEC Vs anaerobic NAC” differential analysis was carried out to reveal proteins whose abundance is iron-dependent during anaerobic growth. In all cases, BAS-10 biomass samples were all harvested at late exponential growth phases, when anaerobic growth on FEC is coupled with iron-binding EPS production [12,16] at an average yield of 6.7 g/L (dry weight EPS per culture volume). Proteomic 2D-DIGE maps showed the occurrence of 869 reproducible protein spots (i. e. detected in all 2D-Gels) which were used to create the match set for comparative analysis. According to the criteria reported in the experimental section for identification of deregulated proteins, comparative analysis between FEC medium cultivations under anaerobic or aerobic conditions revealed 27 and 32 protein spots up- and down-regulated under anaerobic conditions, respectively (Table 1; Additional file 1 Figure S2). On the other hand, comparative analysis between cultivations in FEC and NAC media under anaerobic conditions revealed 28 and 42 protein spots as up- and down-regulated in the presence of Fe(III) (Table 1; Additional file 1 Figure S2). Among these protein spots, 46 resulted differentially abundant with a concordant profile in both analyses (anaerobic FEC vs aerobic FEC and anaerobic FEC vs anaerobic NAC) (Table 1), thus indicating that these proteins are specifically associated with anaerobic growth on FEC. The selected differentially abundant protein spots (83 in number) were alkylated, trypsinolyzed and identified by MALDI-TOF-peptide mass fingerprinting (PMF) and nanoLC-ESI-LIT-MS/MS procedures (Table 1 and Additional file 1 Table S1). Corresponding protein entries were clustered into functional groups, according to KEGG (http://www.genome.ad.jp/kegg/kegg2.html) [22], EcoCyc (http://ecocyc.org/) [23] and ExPASy (http://www.expasy.org) [24] databases (Additional file 1 Figure S3). Among the identified proteins, the most represented ones were related to central carbon metabolism -including glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, citrate fermentation, pentose phosphate (PP) pathway -and membrane transport (Table 1; Additional file 1 Figure S3).

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