The Lysine 299 Residue Endows the Multisubunit Mrp1 Antiporter with Dominant Roles in Na ⁺ Resistance and pH Homeostasis in Corynebacterium glutamicum

Abstract

is generally regarded as a moderately salt- and 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 Δ Δ 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 were differentially expressed in response to specific salt or alkaline stress, and an alkaline stimulus particularly induced transcript levels of the Mrp-type antiporters. When the major Mrp1 antiporter was overwhelmed, 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 K, Mrp1C I, Mrp1A H, and Mrp1D E 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 stress acclimatization. The capacity to adapt to harsh environments is crucial for bacterial survival and product yields, including industrially useful Although exhibits a marked resistance to salt-alkaline stress, the possible mechanism for these adaptations is still unclear. Here, we present the physiological functions and expression patterns of putative Na/H antiporters and conserved residues of Mrp1 subunits, which respond to different salt and alkaline stresses. We found that the Mrp-type antiporters, particularly the Mrp1 antiporter, played a predominant role in maintaining intracellular nontoxic Na levels and alkaline pH homeostasis. Loss of the major Mrp1 antiporter had a profound effect on gene expression of other antiporters under salt or alkaline conditions. The lysine 299 residue may play its essential roles in conferring salt and alkaline tolerance by affecting the ion translocation channel of the Mrp1A subunit. These findings will contribute to a better understanding of Na/H antiporters in sodium antiport and pH regulation.

An Uncharacterized Major Facilitator Superfamily Transporter From Planococcus maritimus Exhibits Dual Functions as a Na+(Li+, K+)/H+ Antiporter and a Multidrug Efflux Pump

“…Bacterial Na + /H + antiporters are a category of secondary transmembrane transporters that extrude Na + and Li + and sometimes also K + in exchange for external H + , which play crucial roles in the maintenance of toxic monovalent cations at an acceptable cytoplasmic level and Na + /K + -dependent intracellular pH homeostasis under alkaline conditions ( Krulwich et al, 2011 ; Quinn et al, 2012 ; Meng et al, 2014 ; Padan, 2014 ). Known Na + /H + antiporters can be classified on the basis of the number of encoding genes into three major groups: (i) single-gene Na + /H + antiporters including NhaA and NhaB ( Herz et al, 2003 ), NhaC ( Ito et al, 1997 ), NhaD ( Zhang et al, 2014 ; Wang et al, 2017 ; Yang et al, 2018 ), NheE ( Sousa et al, 2013 ), NhaG ( Gouda et al, 2001 ), NhaH ( Jiang et al, 2013b ), NhaP ( Utsugi et al, 1998 ), NapA ( Waser et al, 1992 ), and GerN ( Southworth et al, 2001 ); (ii) double-gene Na + /H + antiporters including PsmrAB ( Jiang et al, 2013a ) and UmpAB ( Meng et al, 2017 ); and (iii) multiple-gene Na + /H + antiporters such as Mrp ( Cheng et al, 2016 ; Xu et al, 2018 ), Mnh ( Hiramatsu et al, 1998 ), Pha ( Jiang et al, 2004 ; Yang et al, 2006a ) or Sha ( Kosono et al, 1999 ). In addition, three MFS multi-drug efflux pumps such as MdfA ( Edgar and Bibi, 1997 ), MdtM ( Holdsworth and Law, 2013 ) and Tet(L) ( Cheng et al, 1994 ), and a HCT (2-hydroxy-carboxylate transporter) family transporter MleN ( Wei et al, 2000 ), and a primary Na + pump Nap of NDH (NADH dehydrogenase) family ( Yang et al, 2006b ); and an UPF0118 family protein ( Dong et al, 2017 ), and a RDD family protein ( Shao et al, 2018 ) have also been increasingly reported to be able to function as Na + /H + antiporters.…”

Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter

“…This raises the question as to why a so much more complicated protein assembly is needed to catalyse a similar reaction. Since under the extreme environmental conditions Mrp is essential for cell survival and cannot be replaced by single subunit antiporters ( Cheng et al, 2016 ; Xu et al, 2018 ), one of the proposals is that the Mrp complex can support cell growth at very high pH due to its large surface area, where only a few available external protons can still be gathered for translocation into the cell ( Ito et al, 2017 ).…”

Transposon Mutagenesis of Pseudomonas syringae Pathovars syringae and morsprunorum to Identify Genes Involved in Bacterial Canker Disease of Cherry

“…Herz et al [ 22 ] created a triple mutant of three Na+/H+ mutants in Vibrio cholerae and found no significant reduction in exponential growth in high pH environment. One theory to explain this phenomenon is that the bacterium may employ alternative regulatory strategies, for example functional redundancy or compensatory upregulation mechanisms, to rescue negative effects caused by the single-antiporter gene disruptions [ 23 ]. Three mutants involved in arginine metabolism were observed—two showing hypervirulence and one loss of virulence.…”