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Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis.

Follmann M, Ochrombel I, Krämer R, Trötschel C, Poetsch A, Rückert C, Hüser A, Persicke M, Seiferling D, Kalinowski J, Marin K - BMC Genomics (2009)

Bottom Line: Regulators involved were identified and thereby participation of numerous signaling modules in pH response was found.Methionine and cysteine synthesis were found to be activated via the McbR regulator, cysteine accumulation was observed and addition of cysteine was shown to be toxic under acidic conditions.The results offer new insights into bacterial stress physiology and new starting points for bacterial strain design or pathogen defense.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Cologne, Institute of Biochemistry, Zuelpicher Str, 47, 50674 Cologne, Germany. martin.follmann@uni-koeln.de

ABSTRACT

Background: The maintenance of internal pH in bacterial cells is challenged by natural stress conditions, during host infection or in biotechnological production processes. Comprehensive transcriptomic and proteomic analyses has been conducted in several bacterial model systems, yet questions remain as to the mechanisms of pH homeostasis.

Results: Here we present the comprehensive analysis of pH homeostasis in C. glutamicum, a bacterium of industrial importance. At pH values between 6 and 9 effective maintenance of the internal pH at 7.5 +/- 0.5 pH units was found. By DNA microarray analyses differential mRNA patterns were identified. The expression profiles were validated and extended by 1D-LC-ESI-MS/MS based quantification of soluble and membrane proteins. Regulators involved were identified and thereby participation of numerous signaling modules in pH response was found. The functional analysis revealed for the first time the occurrence of oxidative stress in C. glutamicum cells at neutral and low pH conditions accompanied by activation of the iron starvation response. Intracellular metabolite pool analysis unraveled inhibition of the TCA and other pathways at low pH. Methionine and cysteine synthesis were found to be activated via the McbR regulator, cysteine accumulation was observed and addition of cysteine was shown to be toxic under acidic conditions.

Conclusions: Novel limitations for C. glutamicum at non-optimal pH values were identified by a comprehensive analysis on the level of the transcriptome, proteome, and metabolome indicating a functional link between pH acclimatization, oxidative stress, iron homeostasis, and metabolic alterations. The results offer new insights into bacterial stress physiology and new starting points for bacterial strain design or pathogen defense.

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The pH dependent impact of cysteine on growth of C. glutamicum. Wild type cells were exposed to different pH values in MTP and growth rates were determined in absence (black bars) or presence 10 mM cysteine (white bars).
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Figure 5: The pH dependent impact of cysteine on growth of C. glutamicum. Wild type cells were exposed to different pH values in MTP and growth rates were determined in absence (black bars) or presence 10 mM cysteine (white bars).

Mentions: From the observed metabolic imbalance we inferred that accumulation of intermediates of the methionine pathway upstream of AecD or the lower pool size of the downstream intermediates could contribute to the growth defect of C. glutamicum cells at acidic pH. In order to test this hypothesis we performed growth experiments at pH 7.5 and 6 in absence or presence of 10 mM cystathionine, cysteine, homocysteine, or methionine. Based on these assumptions, the addition of cystathionine or cysteine should increase pH dependent growth inhibition whereas homocysteine and methionine should supplement a putative demand for these compounds at pH 6. The addition of cystathionine, homocysteine and methionine had no significant effect on C. glutamicum growth at pH 6 (data not shown). However, addition of cysteine significantly decreased growth rates of cells exposed to acidic pH values. Further experiments revealed that the extent of growth inhibition by cysteine was indeed pH dependent. Whereas at pH 9 and 7.5 cysteine addition had no effect on the growth rate, at pH 7 growth was retarded and at pH 6.5 and 6 cells were hardly able to grow (Fig. 5).


Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis.

Follmann M, Ochrombel I, Krämer R, Trötschel C, Poetsch A, Rückert C, Hüser A, Persicke M, Seiferling D, Kalinowski J, Marin K - BMC Genomics (2009)

The pH dependent impact of cysteine on growth of C. glutamicum. Wild type cells were exposed to different pH values in MTP and growth rates were determined in absence (black bars) or presence 10 mM cysteine (white bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807442&req=5

Figure 5: The pH dependent impact of cysteine on growth of C. glutamicum. Wild type cells were exposed to different pH values in MTP and growth rates were determined in absence (black bars) or presence 10 mM cysteine (white bars).
Mentions: From the observed metabolic imbalance we inferred that accumulation of intermediates of the methionine pathway upstream of AecD or the lower pool size of the downstream intermediates could contribute to the growth defect of C. glutamicum cells at acidic pH. In order to test this hypothesis we performed growth experiments at pH 7.5 and 6 in absence or presence of 10 mM cystathionine, cysteine, homocysteine, or methionine. Based on these assumptions, the addition of cystathionine or cysteine should increase pH dependent growth inhibition whereas homocysteine and methionine should supplement a putative demand for these compounds at pH 6. The addition of cystathionine, homocysteine and methionine had no significant effect on C. glutamicum growth at pH 6 (data not shown). However, addition of cysteine significantly decreased growth rates of cells exposed to acidic pH values. Further experiments revealed that the extent of growth inhibition by cysteine was indeed pH dependent. Whereas at pH 9 and 7.5 cysteine addition had no effect on the growth rate, at pH 7 growth was retarded and at pH 6.5 and 6 cells were hardly able to grow (Fig. 5).

Bottom Line: Regulators involved were identified and thereby participation of numerous signaling modules in pH response was found.Methionine and cysteine synthesis were found to be activated via the McbR regulator, cysteine accumulation was observed and addition of cysteine was shown to be toxic under acidic conditions.The results offer new insights into bacterial stress physiology and new starting points for bacterial strain design or pathogen defense.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Cologne, Institute of Biochemistry, Zuelpicher Str, 47, 50674 Cologne, Germany. martin.follmann@uni-koeln.de

ABSTRACT

Background: The maintenance of internal pH in bacterial cells is challenged by natural stress conditions, during host infection or in biotechnological production processes. Comprehensive transcriptomic and proteomic analyses has been conducted in several bacterial model systems, yet questions remain as to the mechanisms of pH homeostasis.

Results: Here we present the comprehensive analysis of pH homeostasis in C. glutamicum, a bacterium of industrial importance. At pH values between 6 and 9 effective maintenance of the internal pH at 7.5 +/- 0.5 pH units was found. By DNA microarray analyses differential mRNA patterns were identified. The expression profiles were validated and extended by 1D-LC-ESI-MS/MS based quantification of soluble and membrane proteins. Regulators involved were identified and thereby participation of numerous signaling modules in pH response was found. The functional analysis revealed for the first time the occurrence of oxidative stress in C. glutamicum cells at neutral and low pH conditions accompanied by activation of the iron starvation response. Intracellular metabolite pool analysis unraveled inhibition of the TCA and other pathways at low pH. Methionine and cysteine synthesis were found to be activated via the McbR regulator, cysteine accumulation was observed and addition of cysteine was shown to be toxic under acidic conditions.

Conclusions: Novel limitations for C. glutamicum at non-optimal pH values were identified by a comprehensive analysis on the level of the transcriptome, proteome, and metabolome indicating a functional link between pH acclimatization, oxidative stress, iron homeostasis, and metabolic alterations. The results offer new insights into bacterial stress physiology and new starting points for bacterial strain design or pathogen defense.

Show MeSH
Related in: MedlinePlus