Untersuchungen zu Mechanismen der Stressantwort und des Kaliumtransportes in Corynebacterium glutamicum

Die Stressantwort von Corynebacterium glutamicum auf verschiedene abiotische Faktoren wurde physiologisch und biochemisch untersucht. Bei tiefen externen pH-Werten und unter hyperosmotischen Bedingungen ist Kalium für das optimale Wachstum von C. glutamicum essentiell. Bei niedrigen pH-Werten kommt es durch die endogene Produktion von H2O2 zu oxidativem Stress, und die Limitation der Aktivität der Cystathionin-β-Lyase führt zu einer Störung der Thiolhomöostase, was das Wachstum der Zellen einschränkt. Kalium ist unter sauren Bedingungen für die Stimulation der Atmungskettenaktivität essentiell. Unter hyperosmotischen Bedingungen wird Kalium in großen Mengen intern akkumuliert, wo es essentiell zum Pool der Osmolyte beiträgt. Kalium ist dabei für die induzierte osmoregulierte Expression der Gene betP und proP notwendig. Der kaliumspezifische Kanal CglK fungiert in C. glutamicum sowohl bei niedrigen pH-Werten, als auch bei Osmostress als Hauptaufnahmesystem für Kalium, während der putative sekundär aktive Kup-Transporter keine Funktion hat. Die mRNA von cglK wird in ein membranintegriertes CglK-Protein und zusätzlich in ein cytoplasmatisches KTN-Protein translatiert. Die Analyse der Funktion von CglK wurde in C. glutamicum- und E. coli-Zellen durchgeführt, wobei in Proteoliposomen und in E. coli-Sphäroplasten elektrophysiologische Untersuchungen möglich waren. Die C terminalen KTN-Domänen von CglK sind für dessen Schließung essentiell, während die separaten KTN-Proteine an der vollständigen Öffnung und möglicherweise an einer pH-Wert abhängigen Regulation des Kanals beteiligt sind. Die Kaliumaufnahme über den CglK-Kanal ist für C. glutamicum unter habitatähnlichen Bedingungen für das Wachstum und die Aminosäureproduktion ausreichend. Unter extremen Stressbedingungen in Verbindung mit einer Kaliumlimitation hat die heterologe Expression eines aktiven Kaliumtransporters einen positiven Effekt auf die Stressresistenz der C. glutamicum-Zellen

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, Kraemer R, et al. Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis. BMC Genomics. 2009;10(1):621.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

Osmotic stress response in C. glutamicum: impact of channel- and transporter-mediated potassium accumulation

Potassium accumulation is an essential aspect of bacterial response to diverse stress situations; consequently its uptake plays a pivotal role. Here, we show that the Gram-positive soil bacterium Corynebacterium glutamicum which is employed for the large-scale industrial production of amino acids requires potassium under conditions of ionic and non-ionic osmotic stress. Besides the accumulation of high concentrations of potassium contributing significantly to the osmotic potential of the cytoplasm, we demonstrate that glutamate is not the counter ion for potassium under these conditions. Interestingly, potassium is required for the activation of osmotic stress-dependent expression of the genes betP and proP. The Kup-type potassium transport system which is present in C. glutamicum in addition to the potassium channel CglK does not contribute to potassium uptake at conditions of hyperosmotic stress. Furthermore, we established a secondary carrier of the KtrAB type from C. jeikeium in C. glutamicum thus providing an experimental comparison of channel-and carrier-mediated potassium uptake under osmotic stress. While at low potassium availability, the presence of the KtrAB transporter improves both potassium accumulation and growth of C. glutamicum upon osmotic stress, at proper potassium supply, the channel CglK is sufficient

Potassium Transport in Corynebacterium glutamicum Is Facilitated by the Putative Channel Protein CglK, Which Is Essential for pH Homeostasis and Growth at Acidic pH▿

We studied the requirement for potassium and for potassium transport activity for the biotechnologically important bacterium Corynebacterium glutamicum, which is used for large-scale production of amino acids. Different from many other bacteria, at alkaline or neutral pH, C. glutamicum is able to grow without the addition of potassium, resulting in very low cytoplasmic potassium concentrations. In contrast, at acidic pH, the ability for growth was found to depend on the presence of K+. For the first time, we provide experimental evidence that a potential potassium channel (CglK) acts as the major potassium uptake system in a bacterium and proved CglK's function directly in its natural membrane environment. A full-length CglK protein and a separate soluble protein harboring the RCK domain can be translated from the cglK gene, and both are essential for full CglK functionality. As a reason for potassium-dependent growth limitation at acidic pH, we identified the impaired capacity for internal pH homeostasis, which depends on the availability and internal accumulation of potassium. Potassium uptake via CglK was found to be relevant for major physiological processes, like the activity of the respiratory chain, and to be crucial for maintenance of the internal pH, as well as for the adjustment of the membrane potential in C. glutamicum