A novel NhaD-type Na+/H+ antiporter from the moderate halophile and alkaliphile Halomonas alkaliphila.

In this study, a NhaD-type Na+/H+ antiporter gene designated Ha-nhaD was obtained by selection of genomic DNA from the moderate halophile and alkaliphile Halomonas alkaliphila in Escherichia coli KNabc lacking 3 major Na+/H+ antiporters. The presence of Ha-NhaD conferred tolerance of E. coli KNabc to NaCl up to 0.6 mol·L-1 and to LiCl up to 0.2 mol·L-1 and to an alkaline pH. pH-dependent Na+(Li+)/H+ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc/pUC-nhaD but not those of KNabc/pUC18. Ha-NhaD exhibited Na+(Li+)/H+ antiport activity over a wide pH range from 7.0 to 9.5, with the highest activity at pH 9.0. Protein sequence alignment and phylogenetic analysis revealed that Ha-NhaD is significantly different from the 7 known NhaD-type Na+/H+ antiporters, including Dw-NhaD, Dl-NhaD, Vp-NhaD, Vc-NhaD, Aa-NhaD, He-NhaD, and Ha-NhaD1. Although Ha-NhaD showed a closer phylogenetic relationship with Ha-NhaD2, a significant difference in pH-dependent activity profile exists between Ha-NhaD and Ha-NhaD2. Taken together, Ha-nhaD encodes a novel pH-dependent NhaD-type Na+/H+ antiporter.

Effects of dietary yeast culture on health status in digestive tract of juvenile Pacific white shrimp Litopenaeus Vannamei.

A feeding trial was conducted to investigate the effects of dietary yeast culture (YC) on health status in digestive tract of juvenile Pacific white shrimp Litopenaeus Vannamei. Shrimps (initial weight: 3.33 ± 0.06 g) were fed with graded levels of dietary YC (control, 0.3%, 0.5% and 1.0%). Results of the present study showed that villus height and the ratio between villus height and crypt depth in the digestive tract of juvenile shrimp was significantly increased by dietary 0.5% and 1.0%YC (P < 0.05). Besides, dietary 0.5% and 1.0%YC significantly activities of phenoloxidase (PO), lysozyme (LZ), acid phosphatase (ACP) and alkaline phosphatase (AKP) (P < 0.05), significantly up-regulated mRNA levels of prophenoloxidase (propo), lysozyme (lz), anti-lipopolysaccharide factor (alf), crustin and penaienadin (P < 0.05) and down-regulated mRNA levels of caspase-1, nuclear factor κB p65 (nf-κbp65) myeloid differentiation primary response protein (myd88) and toll like receptor (tlr) in the digestive tract of juvenile shrimp (P < 0.05). Compared with the control, dietary 0.5%YC increased Chao1 index in the digestive tract of juvenile shrimp. In addition, compared with the control, dietary 0.5% and 1.0%YC significantly increased relative abundance of Lactobacillus (P < 0.05). It can be concluded that dietary YC made positive contribution to health status in digestive tract of juvenile shrimp through improving morphology and microbiota, enhancing immune function, and inhibiting inflammation of digestive tract.

Characterization of a Functionally Unknown Arginine-Aspartate-Aspartate Family Protein From Halobacillus andaensis and Functional Analysis of Its Conserved Arginine/Aspartate Residues.

Arginine-aspartate-aspartate (RDD) family, representing a category of transmembrane proteins containing one highly conserved arginine and two highly conserved aspartates, has been functionally uncharacterized as yet. Here we present the characterization of a member of this family designated RDD from the moderate halophile Halobacillus andaensis NEAU-ST10-40T and report for the first time that RDD should function as a novel Na+(Li+, K+)/H+ antiporter. It's more interesting whether the highly conserved arginine/aspartate residues among the whole family or between RDD and its selected homologs are related to the protein function. Therefore, we analyzed their roles in the cation-transporting activity through site-directed mutagenesis and found that D154, R124, R129, and D158 are indispensable for Na+(Li+, K+)/H+ antiport activity whereas neither R35 nor D42 is involved in Na+(Li+, K+)/H+ antiport activity. As a dual representative of Na+(Li+, K+)/H+ antiporters and RDD family proteins, the characterization of RDD and the analysis of its important residues will positively contribute to the knowledge of the cation-transporting mechanisms of this novel antiporter and the roles of highly conserved arginine/aspartate residues in the functions of RDD family proteins.

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

Within major facilitator superfamily (MFS), up to 27 unknown major facilitator families and many members of 60 well-characterized families have been functionally unknown as yet, due to their sharing no or significantly low sequence identity with characterized MFS members. Here we present the first report on the characterization of one functionally unknown MFS transporter designated MdrP with the accession version No. ANU18183.1 from the slight halophile Planococcus maritimus DS 17275T. During the screening of Na+/H+ antiporter genes, we found at first that MdrP exhibits Na+(Li+, K+)/H+ antiport activity, and propose that it should represent a novel class of Na+(Li+, K+)/H+ antiporters. However, we speculate that MdrP may possess an additional protein function. The existence of the signature Motif A of drug/H+antiporter (DHA) family members and phylogenetic analysis suggest that MdrP may also function as a drug efflux pump, which was established by minimum inhibitory concentration tests and drug efflux activity assays. Taken together, this novel MFS transporter exhibits dual functions as a Na+(Li+, K+)/H+ antiporter and a multidrug efflux pump, which will be very helpful to not only positively contribute to the function prediction of uncharacterized MFS members especially DHA1 family ones, but also broaden the knowledge of Na+/H+ antiporters.

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

In this study, genomic DNA was screened for novel Na+/H+ antiporter genes from Halomonas zhaodongensis by selection in Escherichia coli KNabc lacking three major Na+/H+ antiporters. Co-expression of two genes designated umpAB, encoding paired homologous unknown membrane proteins belonging to DUF1538 (domain of unknown function with No. 1538) family, were found to confer E. coli KNabc the tolerance to 0.4 M NaCl and 30 mM LiCl, and an alkaline pH resistance at 8.0. Western blot and co-immunoprecipitation establish that UmpAB localize as a hetero-dimer in the cytoplasmic membranes. Functional analysis reveals that UmpAB exhibit pH-dependent Na+(Li+, K+)/H+ antiport activity at a wide pH range of 6.5 to 9.5 with an optimal pH at 9.0. Neither UmpA nor UmpB showed homology with known single-gene or multi-gene Na+/H+ antiporters, or such proteins as ChaA, MdfA, TetA(L), Nap and PsmrAB with Na+/H+ antiport activity. Phylogenetic analysis confirms that UmpAB should belong to DUF1538 family, which are significantly distant with the above-mentioned proteins with Na+/H+ antiport activity. Taken together, we propose that UmpAB represent a novel two-component Na+(Li+, K+)/H+ antiporter. To the best of our knowledge, this is the first report on the functional analysis of unknown membrane proteins belonging to DUF1538 family.