Cloning and identification of Group 1 mrp operon encoding a novel monovalent cation/proton antiporter system from the moderate halophile Halomonas zhaodongensis

Abstract

The novel species Halomonas zhaodongensis NEAU-ST10-25(T) recently identified by our group is a moderate halophile which can grow at the range of 0-2.5 M NaCl (optimum 0.5 M) and pH 6-12 (optimum pH 9). To explore its halo-alkaline tolerant mechanism, genomic DNA was screened from NEAU-ST10-25(T) in this study for Na(+)(Li(+))/H(+) antiporter genes by selection in Escherichia coli KNabc lacking three major Na(+)(Li(+))/H(+) antiporters. One mrp operon could confer tolerance of E. coli KNabc to 0.8 M NaCl and 100 mM LiCl, and an alkaline pH. This operon was previously mainly designated mrp (also mnh, pha or sha) due to its multiple resistance and pH-related activity. Here, we will also use mrp to designate the homolog from H. zhaodongensis (Hz_mrp). Sequence analysis and protein alignment showed that Hz_mrp should belong to Group 1 mrp operons. Further phylogenetic analysis reveals that Hz_Mrp system should represent a novel sub-class of Group 1 Mrp systems. This was confirmed by a significant difference in pH-dependent activity profile or the specificity and affinity for the transported monovalent cations between Hz_Mrp system and all the known Mrp systems. Therefore, we propose that Hz_Mrp should be categorized as a novel Group 1 Mrp system.

Alkaline Response of a Halotolerant Alkaliphilic Halomonas Strain and Functional Diversity of Its Na+(K+)/H+ Antiporters

“…However, Ha-Mrp exhibited a different cation export profile; other Mrp systems from alkaliphiles function as an electrogenic Na ϩ (Li ϩ )/H ϩ antiporter with no detectable K ϩ /H ϩ antiport ability. Differently, by catalyzing Na ϩ , Li ϩ , and K ϩ efflux for exchanging protons, Ha-Mrp exhibited a similar substrate spectrum to the Mrp system of B. subtilis and the recently reported Hz-Mrp from H. zhaodongensis (8,22). We suspected that these combined properties mediate the ability to survive in environments with high salinity and alkalinity in Halomonas sp.…”

“…Another putative Na ϩ /H ϩ antiporter was annotated as unknown but showed ϳ43% sequence identity to the K ϩ (Na ϩ )/H ϩ NhaP-type antiporters from V. parahemeolyticus and Vibrio cholerae (18,19) and ϳ31% identity to those Na ϩ /H ϩ antiporters of moderately halophilic bacteria, Haloba-cillus dabanensis D-8T and Halobacillus aidingensis AD-6T (20,21). The predicted seven subunits of the mrp operon were phylogenetically related to the recently reported Mrp protein from Halomonas zhaodongensis, with ϳ85% identity (22). In addition, Ha-Mrp was closely related to Mrp from B. pseudofirmus OF4, with 37% identity when comparing MrpA sequences and 48% identity with MrpD sequences (23).…”

Characterization of a novel two-component Na+(Li+, K+)/H+ antiporter from Halomonas zhaodongensis

“…Combined on the number of encoding genes with the specificity for the substrates, Na + /H + antiporters are divided into two major categories: one category of Na + /H + antiporters mainly with the substrates for Na + and Li + are encoded by a single gene such as nhaA 5, 8 , nhaB 9, 10 , nhaC 11 , nhaD 12–16 , nheE 17 , napA 18 , nhaP 19 , nhaG 20 or nhaH 21 , all of which are grouped into the monovalent Cation/Proton Antiporter 1 (CPA-1) family with the exception of NapA sharing the high identity with K + /H + antiporters that are grouped into the CPA-2 family 22 . The other category of monovalent cation/proton antiporters with the substrates for Na + and Li + , and sometimes for K + , are encoded by a multi-cistronic operon containing six or seven genes with different designations such as mrp 23–25 , mnh 26 , pha 27–30 or sha 31 , which are grouped into the CPA-3 family due to its distinctive multi-gene structural properties 22 . In addition to two above-mentioned major categories, some non-specific Na + /H + antiporters were also continually shown to exhibit Na + /H + antiport activity.…”

“…coli KNabc lacking three major Na + (Li + )/H + antiporters. Of several resultant genes, one Group 1 mrp operon has been identified to encode a novel monovalent cation/proton antiporter 25 . In contrast, other genes were predicted to encode proteins that have not been reported to possess Na + (Li + )/H + antiport activity as yet.…”

A UPF0118 family protein with uncharacterized function from the moderate halophile Halobacillus andaensis represents a novel class of Na+(Li+)/H+ antiporter

“…In present, Na + (Li + )/H + antiporters are divided into two main categories based on the number of encoding genes: one category of Na + (Li + )/H + antiporters are encoded by a single gene such as nhaA 68, nhaB 910, nhaC 11, nhaD 8111213141516, nhaE 17, napA 18, nhaP 19, nhaG 20 or nhaH 21, all of which are grouped into the monovalent C ation/ P roton A ntiporter 1 (CPA-1) family with the exception of NapA sharing the high identity with K + /H + antiporters that are grouped into the CPA-2 family22. The other category containing six or seven subunits are encoded by a multi-cistronic operon with the different designations such as mrp 2232425, mnh 26, pha 27282930 or sha 31, which are grouped into the CPA-3 family due to its distinctive multigene structural properties22. In addition to two above mentioned main categories, other genes with Na + (Li + )/H + antiport activity were also continually shown to exhibit Na + /H + antiport activity.…”