Diversities and similarities in pH dependency among bacterial NhaB-like Na+/H+ antiporters.

NhaB-like antiporters were the second described class of Na(+)/H(+) antiporters, identified in bacteria more than 20 years ago. While nhaB-like gene sequences have been found in a number of bacterial genomes, only a few of the NhaB-like antiporters have been functionally characterized to date. Although earlier studies have identified a few pH-sensitive and -insensitive NhaB-like antiporters, the mechanisms that determine their pH responses still remain elusive.

Critical involvement of the E373-D434 region in the acid sensitivity of a NhaB-type Na(+)/H(+) antiporter from Vibrio alginolyticus.

It has been well established that VaNhaB, a NhaB-type Na(+)/H(+) antiporter found in Vibrio alginolyticus, exhibits a striking acid sensitivity. However, the molecular basis of the pH-dependent regulatory mechanism of the antiport activity is yet to be investigated. In this study, we generated various chimeric proteins composed of VaNhaB and a pH insensitive ortholog found in Escherichia coli (EcNhaB) and analyzed the pH responses of their Na(+)/H(+) antiport activities to search for the key residues or domains that are involved in the pH sensitivity of VaNhaB.

Identification and characterization of the Streptomyces globisporus 1912 regulatory gene lndYR that affects sporulation and antibiotic production.

Here, we report the identification and functional characterization of the Streptomyces globisporus 1912 gene lndYR, which encodes a GntR-like regulator of the YtrA subfamily. Disruption of lndYR arrested sporulation and antibiotic production in S. globisporus.

Glutamate 85 is involved in the sodium/proton exchange activity of the Escherichia coli ChaA.

Hitherto, the roles of specific amino acid residues of ChaA, one of three Na(+)/H(+) antiporters in Escherichia coli, in exchange activity have not been reported. Here we examined the role of acidic amino acid residues, Glu-85 and Glu-325, on the hydrophobic transmembrane domains. It was found that ChaA is involved in salt tolerance at alkaline pH.

The implication of YggT of Escherichia coli in osmotic regulation.

An Escherichia coli mutant lacking three major K(+) uptake systems, Trk, Kup, and Kdp, did not grow under low K(+)and high Na(+) concentrations. The introduction of fkuA and of fkuB of a marine bacterium, Vibrio alginolyticus, has been reported to compensate for the growth defect by accelerating the rate of K(+) uptake (Nakamura, Katoh, Shimizu, Matsuba, and Unemoto, Biochim. Biophys.

An Mrp-like cluster in the halotolerant cyanobacterium Aphanothece halophytica functions as a Na+/H+ antiporter.

The mrp homolog gene cluster mrpCD1D2EFGAB (Ap-mrp) was found in a halotolerant cyanobacterium, Aphanothece halophytica, amplified, and expressed in Escherichia coli mutant TO114. Ap-mrp complemented the salt-sensitive phenotype of TO114 and exhibited Na(+)/H(+) and Li(+)/H(+) exchange activities, indicating that Ap-Mrp functions as a Na(+)/H(+) antiporter.

Characterization and analysis of the regulatory network involved in control of lipomycin biosynthesis in Streptomyces aureofaciens Tü117.

Analysis of the alpha-lipomycin biosynthesis gene cluster of Streptomyces aureofaciens Tü117 led to the identification of five putative regulatory genes, which are congregated into a subcluster. Analysis of the lipReg1-4 and lipX1 showed that they encode components of two-component signal transduction systems (LipReg1 and LipReg2), multiple antibiotics resistance-type regulator (LipReg3), large ATP-binding regulators of the LuxR family-type regulator (LipReg4), and small ribonuclease (LipRegX1), respectively. A combination of targeted gene disruptions, complementation experiments, lipomycin production studies, and gene expression analysis via RT-PCR suggests that all regulatory lip genes are involved in alpha-lipomycin production.

pH-dependent regulation of the multi-subunit cation/proton antiporter Pha1 system from Sinorhizobium meliloti.

The pha1 gene cluster (pha1A'-G) of Sinorhizobium meliloti has previously been characterized as a necessary component for proper invasion into plant root tissue. It has been suggested to encode a multi-subunit K(+)/H(+) antiporter, since mutations in the pha1 region rendered S. meliloti cells sensitive to K(+) and alkali, and because there is high amino acid sequence similarity to previously characterized multi-subunit cation/H(+) antiporters (Mrp antiporters).

Identification and characterization of the Na+/H+ antiporter Nhas3 from the thylakoid membrane of Synechocystis sp. PCC 6803.

Na+/H+ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na+/H+ antiporters, nhaS1-5 and sll0556.

An ABC transporter encoding gene lndW confers resistance to landomycin E.

Streptomyces globisporus 1912 produces a polyketide antibiotic landomycin E (LaE), which possesses anticancer activity. A 1.8 kb DNA fragment at the end of landomycin E biosynthetic gene cluster was sequenced.

Function of lanI in regulation of landomycin A biosynthesis in Streptomyces cyanogenus S136 and cross-complementation studies with Streptomyces antibiotic regulatory proteins encoding genes.

The transcriptional regulator of landomycin A biosynthesis encoded by lanI gene has been inactivated within the chromosome of Streptomyces cyanogenus S136. The obtained mutant strain did not produce landomycin A and its known intermediates. Loss of landomycin A production caused significant changes in morphology of the lanI deficient strain.

Potassium/proton antiport system of Escherichia coli.

The intracellular level of potassium (K(+)) in Escherichia coli is regulated through multiple K(+) transport systems. Recent data indicate that not all K(+) extrusion system(s) have been identified (15). Here we report that the E.

Carboxyl-terminal hydrophilic tail of a NhaP type Na+/H+ antiporter from cyanobacteria is involved in the apparent affinity for Na+ and pH sensitivity.

Little information is available on the C-terminal hydrophilic tails of prokaryotic Na(+)/H(+) antiporters. To address functional properties of the C-terminal tail, truncation mutants in this domain were constructed. Truncation of C-terminal amino acid residues of NhaP1 type antiporter from Synechocystis PCC6803 (SynNhaP1) did not change the V(max) values, but increased the K(m) values for Na(+) and Li(+) about 3 to 15-fold.

Expression of the regulatory protein LndI for landomycin E production in Streptomyces globisporus 1912 is controlled by the availability of tRNA for the rare UUA codon.

The gene lndI encodes the activator of landomycin biosynthesis. The utilization of LndI-EGFP fusions led us to investigate the temporal pattern of this gene expression and demonstrated the delay between lndI transcription and translation. The TTA codon in lndI is thought to be the reason for this delay.

A putative proteinase gene is involved in regulation of landomycin E biosynthesis in Streptomyces globisporus 1912.

The prx gene, which is highly homologous to putative proteinases, has been identified by sequencing in the vicinity of the biosynthetic gene cluster for landomycin E (LaE) biosynthesis (lnd) in Streptomyces globisporus 1912. The S. globisporus Pro6 gene, deficient in prx, produced fivefold less LaE than the parental strain.

Cloning, functional expression and primary characterization of Vibrio parahaemolyticus K+/H+ antiporter genes in Escherichia coli.

The regulation of internal Na(+) and K(+) concentrations is important for bacterial cells, which, in the absence of Na(+) extrusion systems, cannot grow in the presence of high external Na(+). Likewise, bacteria require K(+) uptake systems when the external K(+) concentration becomes too low to support growth. At present, we have little knowledge of K(+) toxicity and bacterial outward-directed K(+) transport systems.

All four putative selectivity filter glycine residues in KtrB are essential for high affinity and selective K+ uptake by the KtrAB system from Vibrio alginolyticus.

The subunit KtrB of bacterial Na+-dependent K+-translocating KtrAB systems belongs to a superfamily of K+ transporters. These proteins contain four repeated domains, each composed of two transmembrane helices connected by a putative pore loop (p-loop). The four p-loops harbor a conserved glycine residue at a position equivalent to a glycine selectivity filter residue in K+ channels.

Survival of two Orientia tsutsugamushi bacterial strains that infect mouse macrophages with varying degrees of virulence.

Orientia tsutsugamushi, an intracellular parasitic bacterium, comprises numerous strains of differing virulence. When BALB/c mice were infected intraperitoneally with this pathogen, a virulent strain known as Karp was found to multiply in the intraperitoneal macrophages and kill the mouse. In contrast, an avirulent strain, Kuroki, was shown to invade macrophages but be eliminated from the cells, allowing mouse survival.

Halotolerant cyanobacterium Aphanothece halophytica contains NapA-type Na+/H+ antiporters with novel ion specificity that are involved in salt tolerance at alkaline pH.

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium Synechocystis sp.

Na+-dependent K+ uptake Ktr system from the cyanobacterium Synechocystis sp. PCC 6803 and its role in the early phases of cell adaptation to hyperosmotic shock.

Transmembrane ion transport processes play a key role in the adaptation of cells to hyperosmotic conditions. Previous work has shown that the disruption of a ktrB/ntpJ-like putative Na(+)/K(+) transporter gene in the cyanobacterium Synechocystis sp. PCC 6803 confers increased Na(+) sensitivity, and inhibits HCO(3)(-) uptake.

Generation of Streptomyces globisporus SMY622 strain with increased landomycin E production and it's initial characterization.

Landomycin E (LaE) overproducing strain Streptomyces globisporus SMY6222 has been developed using UV induced mutagenesis and selection for streptomycin resistance. SMY622 has been shown by HPLC to produce 200-fold higher amounts of LaE when comparing with parental strain. The levels of transcription of regulatory gene lndI and oxygenase gene lndE are two times higher in the mutant than in the wild type.

Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria.

Genome sequences of cyanobacteria, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Thermosynechococcus elongatus BP-1 revealed the presence of a single Ca2+/H+ antiporter in these organisms.

Molecular dissection of the contribution of negatively and positively charged residues in S2, S3, and S4 to the final membrane topology of the voltage sensor in the K+ channel, KAT1.

Voltage-dependent ion channels control changes in ion permeability in response to membrane potential changes. The voltage sensor in channel proteins consists of the highly positively charged segment, S4, and the negatively charged segments, S2 and S3. The process involved in the integration of the protein into the membrane remains to be elucidated.

Introduction of a Na+/H+ antiporter gene from Atriplex gmelini confers salt tolerance to rice.

We engineered a salt-sensitive rice cultivar (Oryza sativa cv. Kinuhikari) to express a vacuolar-type Na+/H+ antiporter gene from a halophytic plant, Atriplex gmelini (AgNHX1). The activity of the vacuolar-type Na+/H+ antiporter in the transgenic rice plants was eight-fold higher than that in wild-type rice plants.

Functional characterization of betaine/proline transporters in betaine-accumulating mangrove.

Betaine is an important osmoprotectant in many plants, but its transport activity has only been demonstrated using a proline transporter from tomato, a betaine-nonaccumulating plant. In this study, two full-length and one partial transporter genes were isolated from betaine-accumulating mangrove Avicennia marina. Their homologies to betaine transporters from bacteria and betaine/4-aminobutyrate transporters from mammalian cells were low but were high to proline transporters from Arabidopsis and tomato.