TY - JOUR
T1 - The lysine 299 residue endows the multisubunit Mrp1 antiporter with dominant roles in Na+ resistance and pH homeostasis in Corynebacterium glutamicum
AU - Xu, Ning
AU - Zheng, Yingying
AU - Wang, Xiaochen
AU - Krulwich, Terry A.
AU - Ma, Yanhe
AU - Liu, Jun
N1 - Publisher Copyright:
© 2018 American Society for Microbiology.
PY - 2018/5/1
Y1 - 2018/5/1
N2 - Corynebacterium glutamicum is generally regarded as a moderately saltand alkali-tolerant industrial organism. However, relatively little is known about the molecular mechanisms underlying these specific adaptations. Here, we found that the Mrp1 antiporter played crucial roles in conferring both environmental Na+ resistance and alkali tolerance whereas the Mrp2 antiporter was necessary in coping with high-KCl stress at alkaline pH. Furthermore, the δmrp1 δmrp2 double mutant showed the most-severe growth retardation and failed to grow under high-salt or alkaline conditions. Consistent with growth properties, the Na+/H+ antiporters of C. glutamicum were differentially expressed in response to specific salt or alkaline stress, and an alkaline stimulus particularly induced transcript levels of the Mrptype antiporters. When the major Mrp1 antiporter was overwhelmed, C. glutamicum might employ alternative coordinate strategies to regulate antiport activities. Site-directed mutagenesis demonstrated that several conserved residues were required for optimal Na+ resistance, such as Mrp1A K299, Mrp1C I76, Mrp1A H230, and Mrp1D E136. Moreover, the chromosomal replacement of lysine 299 in the Mrp1A subunit resulted in a higher intracellular Na+ level and a more alkaline intracellular pH value, thereby causing a remarkable growth attenuation. Homology modeling of the Mrp1 subcomplex suggested two possible ion translocation pathways, and lysine 299 might exert its effect by affecting the stability and flexibility of the cytoplasm-facing channel in the Mrp1A subunit. Overall, these findings will provide new clues to the understanding of salt-alkali adaptation during C. glutamicum stress acclimatization.
AB - Corynebacterium glutamicum is generally regarded as a moderately saltand alkali-tolerant industrial organism. However, relatively little is known about the molecular mechanisms underlying these specific adaptations. Here, we found that the Mrp1 antiporter played crucial roles in conferring both environmental Na+ resistance and alkali tolerance whereas the Mrp2 antiporter was necessary in coping with high-KCl stress at alkaline pH. Furthermore, the δmrp1 δmrp2 double mutant showed the most-severe growth retardation and failed to grow under high-salt or alkaline conditions. Consistent with growth properties, the Na+/H+ antiporters of C. glutamicum were differentially expressed in response to specific salt or alkaline stress, and an alkaline stimulus particularly induced transcript levels of the Mrptype antiporters. When the major Mrp1 antiporter was overwhelmed, C. glutamicum might employ alternative coordinate strategies to regulate antiport activities. Site-directed mutagenesis demonstrated that several conserved residues were required for optimal Na+ resistance, such as Mrp1A K299, Mrp1C I76, Mrp1A H230, and Mrp1D E136. Moreover, the chromosomal replacement of lysine 299 in the Mrp1A subunit resulted in a higher intracellular Na+ level and a more alkaline intracellular pH value, thereby causing a remarkable growth attenuation. Homology modeling of the Mrp1 subcomplex suggested two possible ion translocation pathways, and lysine 299 might exert its effect by affecting the stability and flexibility of the cytoplasm-facing channel in the Mrp1A subunit. Overall, these findings will provide new clues to the understanding of salt-alkali adaptation during C. glutamicum stress acclimatization.
KW - Corynebacterium glutamicum
KW - Expression pattern
KW - Mrp-type system
KW - Na/H antiporter
KW - Site mutations
UR - http://www.scopus.com/inward/record.url?scp=85046751318&partnerID=8YFLogxK
U2 - 10.1128/AEM.00110-18
DO - 10.1128/AEM.00110-18
M3 - Article
C2 - 29523552
AN - SCOPUS:85046751318
SN - 0099-2240
VL - 84
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
IS - 10
M1 - e00110-18
ER -