YggB of Corynebacterium glutamicum - Dual function in osmotic stress response and glutamate production

The membrane protein YggB of Corynebacterium glutamicum was previously described to belong to the MscS-type family of mechanosensitive (MS) channels functioning as emergency valves upon osmotic downshift. Bacterial cells respond to rapid water influx by immediate activation of MS channels that allow efflux of compatible solutes to prevent cell lysis. Recently, YggB was also connected to the glutamate export in C. glutamicum under glutamate productive conditions. The mechanism of glutamate export is not fully understood so far although C. glutamicum has been used for the industrial production of amino acids for decades. Deletion of yggB led to a drastic decrease in glutamate excretion while truncation of 110 AA resulted in continuous export of glutamate. In this work YggB was characterized with respect to its dual function as MS channel on the one hand and in the excretion of glutamate on the other. Using the patch clamp technique and additional physiological approaches it was shown that YggB harbors the functions of a pressuresensitive MS channel similar to the E. coli homolog MscS. However, for the first time also an involvement of a MS channel in bacterial response to hyperosmotic conditions was shown. A so called "pump and leak" model including active betaine uptake (via BetP) and passive betaine efflux (via YggB) to accurately adjust the internal solute concentration, which are accumulated under hyperosmotic conditions to balance the osmotic gradient, is proposed. Concerning the second function of YggB in the glutamate production of C. glutamicum the integrity of the C-terminal domain was shown to have a strong effect on the inducibility of glutamate production. However, the exact function of the C-terminal domain cannot be unequivocally clarified. Additionally, this work provides strong evidence that glutamate excretion is triggered directly by the C. glutamicum MS channel YggB

Glutamate efflux mediated by Corynebacterium glutamicum MscCG, Escherichia coli MscS, and their derivatives

AbstractCorynebacterium glutamicum is used in microbial biotechnology for the production of amino acids, in particular glutamate. The mechanism of glutamate excretion, however, is not yet fully understood. Recently, evidence was provided that the NCgl1221 gene product from C. glutamicum ATCC 13869, a MscS-type mechanosensitive efflux channel, is responsible for glutamate efflux [1]. The major difference of NCgl1221 and the homologous protein MscCG of C. glutamicum ATCC 13032 from Escherichia coli MscS and most other MscS-type proteins is the presence of an additional, 247 amino acid long C-terminal domain. By topology analysis, we show that this domain in MscCG carries a transmembrane segment. We have generated selected C-terminal truncations of MscCG, gain-of-function and loss-of-function constructs of both E. coli MscS and C. glutamicum MscCG, as well as fusion constructs of the two proteins. These mutant proteins were investigated for mechanosensitive efflux, MS channel activity, glutamate excretion and their impact on membrane potential. We provide evidence that the channel domain of MscCG mediates glutamate efflux in response to penicillin treatment, and that the E. coli MscS channel is to some extent able to function in a similar manner. We further show that the C-terminal domain of MscCG has a significant impact for function and/or regulation of MscCG. Significantly, a positive effect on glutamate efflux of the C-terminal extension of MscCG from C. glutamicum was also observed when fused to the E. coli MscS channel

Significance of the corynebacterium glutamicum MscCG mechanosensitive channel in fine-tuning of compatible solute accumulation

Many types of micro-organisms including bacteria are frequently exposed to changes in environmental osmotic conditions. This in particular applies to soil bacteria such as Corynebacterium glutamicum. A change in external osmolarity has direct impact on the bacterial cell volume and hydration. To counteract deleterious effects of sudden hyper- and hypo- osmotic conditions to which they are frequently exposed, these bacteria have developed a range of cellular responses including mechanosensitive channels, which are activated upon sudden hypo- osmotic shifts resulting in the immediate release of small solutes. Based on structural similarity, MscCG (the YggB gene product) of C. glutamicum belongs to the family of MscS-type mechanosensitive channels..

The properties and contribution of the Corynebacterium glutamicum MscS variant to fine-tuning of osmotic adaptation

Based on sequence similarity, the mscCG gene product of Corynebacterium glutamicum belongs to the family of MscS-type mechanosensitive channels. In order to investigate the physiological significance of MscCG in response to osmotic shifts in detail, we studied its properties using both patch-clamp techniques and betaine efflux kinetics. After heterologous expression in an Escherichia coli strain devoid of mechanosensitive channels, in patch-clamp analysis of giant E. coli spheroplasts MscCG showed the typical pressure dependent gating behavior of a stretch-activated channel with a current/voltage dependence indicating a strongly rectifying behavior. Apart from that, MscCG is characterized by significant functional differences with respect to conductance, ion selectivity and desensitation behavior as compared to MscS from E. coli. Deletion and complementation studies in C. glutamicum showed a significant contribution of MscCG to betaine efflux in response to hypoosmotic conditions. A detailed analysis of concomitant betaine uptake (by the betaine transporter BetP) and efflux (by MscCG) under hyperosmotic conditions indicates that MscCG may act in osmoregulation in C. glutamicum by fine-tuning the steady state concentration of compatible solutes in the cytoplasm which are accumulated in response to hyperosmotic stress