Effects of chromosomal deletion of the operon encoding the multiple resistance and pH-related antiporter in Vibrio cholerae

Abstract

To examine the possible physiological significance of Mrp, a multi-subunit cation/proton antiporter from Vibrio cholerae, a chromosomal deletion Δmrp of V. cholerae was constructed and characterized. The resulting mutant showed a consistent early growth defect in LB broth that became more evident at elevated pH of the growth medium and increasing Na+ or K+ loads. After 24 h incubation, these differences disappeared likely due to the concerted effort of other cation pumps in the mrp mutant. Phenotype MicroArray analyses revealed an unexpected systematic defect in nitrogen utilization in the Δmrp mutant that was complemented by using the mrpA'-F operon on an arabinose-inducible expression vector. Deletion of the mrp operon also led to hypermotility, observable on LB and M9 semi-solid agar. Surprisingly, Δmrp mutation resulted in wild-type biofilm formation in M9 despite a growth defect but the reverse was true in LB. Furthermore, the Δmrp strain exhibited higher susceptibility to amphiphilic anions. These pleiotropic phenotypes of the Δmrp mutant demonstrate how the chemiosmotic activity of Mrp contributes to the survival potential of V. cholerae despite the presence of an extended battery of cation/proton antiporters of varying ion selectivity and pH profile operating in the same membrane.

Sodium antiporters of Pseudomonas aeruginosa in challenging conditions: effects on growth, biofilm formation, and swarming motility

“…Our previously published work on Mrp in V. cholerae [1] identified susceptibility of the Mrp mutant to three weak acids: docusate, also known as dioctyl sulfosuccinate, n-lauroylsarcosine, and probenecid. Therefore, those weak acids were tested in this assay.…”

“…Then, 200 μl of 95% ethanol was added to the wells to solubilize the stained biofilm and consequently incubated for 15 min at room temperature. Optical density at 570 nm was measured in the iMark plate reader to assess the biofilm formation, and the biofilm ratio (OD 570 /OD 595 ) was calculated to account for growth deficiencies [1]. These experiments were independently repeated five times for each pH.…”

“…Interestingly, Pa-Mrp contributed to pathogenicity when assayed in mice, but had no influence in antibiotic resistance [26]. Overall, Mrp has also been shown to confer bile acid resistance when expressed in Escherichia coli [13] and Vibrio cholerae [13,22], and played a role in nitrogen metabolism, cell motility and biofilm formation in V. cholerae [1]. In addition, putative mrp operons have been found in a large variety of pathogens and environmental strains that often survive in very extreme conditions [45].…”

Mrp Antiporters Have Important Roles in Diverse Bacteria and Archaea

“…In a study of the group 2 Mrp antiporter of Vibrio cholerae , expressed in a major Na + /H + antiporter-deficient E. coli strain, EP432, this antiporter had Na + (Li + , K + )/H + antiport activity with optimal pH at pH 9–9.5 and also showed bile acid resistance in E. coli ( Dzioba-Winogrodzki et al, 2009 ). A deletion mutant of the group 2 mrp gene cluster from V. cholerae revealed mutant physiological defects in nitrogen metabolism, cell motility, and biofilm formation ( Aagesen et al, 2016 ).…”

Metabolic Reprogramming of Vibrio cholerae Impaired in Respiratory NADH Oxidation Is Accompanied by Increased Copper Sensitivity

“…The NQR is a membrane-bound flavo-iron sulfur protein complex composed of six subunits (9). The enzyme is of central importance for V. cholerae, since the electrochemical Na ϩ gradient generated by the NQR drives many other cellular processes, such as flagellar rotation, exchange of cations, and expulsion of antibiotics (10)(11)(12).…”