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. In this study, we sought to investigate the diversities and similarities among bacterial NhaB-like antiporters, with particular emphasis on their pH responsiveness. Our phylogenetic analysis of NhaB-like antiporters, combined with pH profile analyses of activities for representative members of several phylogenetic groups, demonstrated that NhaB-like antiporters could be classified into three distinct types according to the degree of their pH dependencies. Interestingly, pH-insensitive NhaB-like antiporters were only found in a limited proportion of enterobacterial species, which constitute a subcluster that appears to have diverged relatively recently among enterobacterial NhaB-like antiporters. Furthermore, kinetic property analyses of NhaB-like antiporters at different pH values revealed that the degree of pH sensitivity of antiport activities was strongly correlated with the magnitude of pH-dependent change in apparent Km values, suggesting that the dramatic pH sensitivities observed for several NhaB-like antiporters might be mainly due to the significant increases of apparent Km at lower pH. These results strongly suggested the possibility that the loss of pH sensitivity of NhaB-like antiporters had occurred relatively recently, probably via accumulation of the mutations that impair pH-dependent change of Km in the course of molecular evolution.

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. Our results revealed the significant importance of a stretch of amino acid residues within the loop 8-loop 9 regions (E373-D434) responsible for the acid sensitivity of VaNhaB, along with the possible involvement of other unidentified residues that are widely spread in the primary structure of VaNhaB. Moreover, we demonstrated that the E373-D434 region of VaNhaB was able to confer some degree of acid sensitivity on our pH insensitive chimeric antiporter that is mainly composed of EcNhaB except for seven amino acid substitutions at the N-terminal end. This result strongly suggested the possibility that the E373-D434 region is able to act, at least partially, as machinery that diminishes the activity of the NhaB-type antiporter at an acidic pH.

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. The results of in vivo and in vitro studies revealed that the ABC transporter genes lndW-lndW2 are targets of LndYR repressive action. In Streptomyces coelicolor M145, lndYR overexpression caused a significant increase in the amount of extracellular actinorhodin. We suggest that lndYR controls the transcription of transport system genes in response to an as-yet-unidentified signal. Features that distinguish lndYR-based regulation from other known regulators are discussed.

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. Mutagenesis analyses revealed the importance of Glu-85, but not Glu-325, in the exchange activity.

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. On the basis of the obtained data, we propose that LipReg2 controls the activity of LipReg1, which in its turn govern the expression of the alpha-lipomycin pathway-specific regulatory gene lipReg4. The ribonuclease gene lipX1 and the transporter regulator lipReg3 appear to work independently of genes lipReg1, lipReg2, and lipReg4.

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. Acta, 1277, 201-208 (1996)). We investigated the function of unknown genes of E. coli, yggS and yggT, homologs of fkuA and fkuB respectively. E. coli TK2420 cells, which lack the three K(+) uptake systems, did not grow under high Na(+) or mannitol concentrations. The growth defect was compensated by the introduction of the yggT gene alone: yggS was not required. Here we found that YggT endowed E. coli cells with a tolerance for osmotic shock, and discuss a possible mechanism.

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.

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). However, the detailed transport properties of the Pha1 system are yet to be determined. Interestingly, most of the Mrp antiporters are highly selective for Na(+), unlike the Pha1 system. Here, we report the functional expression of the Pha1 system in Escherichia coli and the measurement of cation/H(+) antiport activity. We showed that the Pha1 system is indeed a K(+)/H(+) antiporter with a pH optimum under mildly alkaline conditions. Moreover, we found that the Pha1 system can transport Na(+); this was unexpected based on previous phenotypic analyses of pha1 mutants. Furthermore, we demonstrated that the cation selectivity of the Pha1 system was altered when the pH was lowered from the optimum. The downregulation of Na(+)/H(+) and K(+)/H(+) antiport activities upon acidic shift appeared to occur via different processes, which might indicate the presence of distinct mechanisms for the regulation of the K(+)/H(+) and Na(+)/H(+) antiport activities of the Pha1 system.

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. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na+/H+ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na+ concentrations. NhaS3 had no K+/H+ exchange activity itself but enhanced K+ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO2 to high CO2 conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO2 consumption caused by the ceasing of O2-evolving photosynthesis. This is the first report of a Na+/H+ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H+, Na+, and K+ in supporting the conversion of photosynthetic products and in the supply of energy in the dark.

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. DNA sequence analysis of this fragment identified one complete open reading frame, designated lndW. The deduced sequence of lndW gene product revealed significant similarity to the ATP-binding domains of the ABC (ATP-binding protein cassette) superfamily of transport-related proteins. Knockout of lndW had no significant effect on resistance to LaE and its production. The expression of lndW in S. globisporus 1912 was proven via transcriptional fusion of lndW promoter to EGFP (enhanced green fluorescent protein). Overexpression of lndW in S. lividans TK24 conferred resistance to LaE. The mechanism of lndW function in LaE biosynthesis is discussed.

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. RT-PCR analysis confirmed complete cessation of transcription of certain lan genes, including lanJ (encoding putative proton dependent transporter) and lanK (presumably involved in lanJ expression regulation). Introduction of either lanI or lndI [lanI homologue controlling landomycin E biosynthesis in Streptomyces globisporus 1912, both encoding Streptomyces antibiotic regulatory proteins (SARPs)] restored landomycin A production in the mutant strain. Chimeric constructs ladI and ladR were generated by exchanging the DNA sequences corresponding to N- and C-terminal parts of LndI and LanI. None of these genes were able to activate the production of landomycins in regulatory mutants of S. cyanogenus and S. globisporus. Nevertheless, the production of novel unidentified compound was observed in the case of S. cyanogenus harboring ladI gene. Various genes encoding SARPs have been expressed in S. globisporus and S. cyanogenus regulatory mutants and the results of these complementation experiments are discussed.

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. coli Na(+) (Ca(2+))/H(+) antiporter ChaA functions as a K(+) extrusion system. Cells expressing ChaA mediated K(+) efflux against a K(+) concentration gradient. E. coli strains lacking the chaA gene were unable to extrude K(+) under conditions in which wild-type cells extruded K(+). The K(+)/H(+) antiporter activity of ChaA was detected by using inverted membrane vesicles produced using a French press. Physiological growth studies indicated that E. coli uses ChaA to discard excessive K(+), which is toxic for these cells. These results suggest that ChaA K(+)/H(+) antiporter activity enables E. coli to adapt to K(+) salinity stress and to maintain K(+) homeostasis.

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. Truncation of C-terminal tail of a halotolerant cyanobacterium Aphanothece halophytica (ApNhaP1) significantly decreased the V(max) although it did not alter the K(m) values for Na(+). The C-terminal part of SynNhaP1 was expressed in E. coli and purified as a 16kDa soluble protein. Addition of purified polypeptide to the membrane vesicles expressing the C-terminal truncated SynNhaP1 increased the exchange activities. Change of Glu519 and Glu521 to Lys in C-terminal tail altered the pH dependence of Na(+)/H(+) and Li(+)/H(+) exchange activities. These results indicate that the specific acidic amino acid residues at C-terminal domain play important roles for the K(m) and the pH dependence of the exchange activity.

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. The replacement of TTA with CTC cancelled the pause between lndI transcription and the translation. The wild-type of the lndI gene is not expressed in the Streptomyces coelicolor bldA- mutant strain, indicating the importance of the bldA tRNA in its mRNA translation.

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. The expression of prx in S. globisporus Pro6 restored LaE production to wild-type levels, whereas expression of the pathway-specific regulatory gene lndI did not. The introduction of additional copies of prx into the wild-type strain using a pSG5-based plasmid, pKC1139, led to a 2.7-fold increase in LaE production. These results indicate that prx is a novel regulatory gene for LaE biosynthesis.

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. We report here that high external concentrations of K(+) at alkaline pH are toxic and that bacteria require K(+) efflux and/or extrusion systems to avoid excessive K(+) accumulation. We have identified the first example of a bacterial K(+)(specific)/H(+) antiporter, Vp-NhaP2, from Vibrio parahaemolyticus. This protein, a member of the cation : proton antiporter-1 (CPA1) family, was able to mediate K(+) extrusion from the cell to provide tolerance to high concentrations of external KCl at alkaline pH. We also report the discovery of two V. parahaemolyticus Na(+)/H(+) antiporters, Vp-NhaA and Vp-NhaB, which also exhibit a novel ion specificity toward K(+), implying that they work as Na(+)(K(+))/H(+) exchangers. Furthermore, under specific conditions, Escherichia coli was able to mediate K(+) extrusion against a K(+) chemical gradient, indicating that E. coli also possesses an unidentified K(+) extrusion system(s).

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. We investigated whether these glycines also form a selectivity filter in KtrB. The single residues Gly70, Gly185, Gly290, and Gly402 from p-loops P(A) to P(D) of Vibrio alginolyticus KtrB were replaced with alanine, serine, or aspartate. The three alanine variants KtrB(A70), KtrB(A185), and KtrB(A290) maintained a substantial activity in KtrAB-mediated K+ uptake in Escherichia coli. This activity was associated with a decrease in the affinity for K+ by 2 orders of magnitude, with little effect on Vmax. Minor activities were also observed for three other variants: KtrB(A402), KtrB(S70), and KtrB(D185). With all of these variants, the property of Na+ dependence of K+ transport was preserved. Only the four serine variants mediated Na+ uptake, and these variants differed considerably in their K+/Na+ selectivity. Experiments on cloned ktrB in the pBAD18 vector showed that V. alginolyticus KtrB alone was still active in E. coli. It mediated Na+-independent, slow, high affinity, and mutation-specific K+ uptake as well as K+-independent Na+ uptake. These data demonstrate that KtrB contains a selectivity filter for K+ ions and that all four conserved p-loop glycine residues are part of this filter. They also indicate that the role of KtrA lies in conferring velocity and ion coupling to the Ktr complex.

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. O. tsutsugamushi invades its host cell cytoplasm through phagocytosis and disruption of phagosomal membranes but some bacteria are then killed by phago-lysosomes within 1h of infection. Microscopic observations could not differentiate the Karp and Kuroki strains during entry and subsequent cell killing by phago-lysosomes. However, the Kuroki cells failed to divide and were markedly deformed following cytoplasmic invasion at several days post-infection. These findings suggest that macrophages have a mechanism to eliminate O. tsutsugamushi in the cytoplasm, if the invading bacteria escape phagosomal clearance, and that it is this mechanism that Kuroki does not survive. Additionally, significant levels of nitric oxide (NO) are produced in macrophages by Kuroki, but not by Karp. An NO synthase inhibitor, however, does not increase the growth of Kuroki, suggesting that NO is induced in a strain-dependent manner but does not effect proliferation.

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. strain PCC 6803 contains five putative Na+/H+ antiporters, two of which are homologous to NhaP of Pseudomonas aeruginosa and three of which are homologous to NapA of Enterococcus hirae. The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in Synechocystis sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in Aphanothece halophytica. Two genes encoding polypeptides of the same size, designated Ap-napA1-1 and Ap-napA1-2, were isolated. Ap-NapA1-1 exhibited a higher level of homology to the Synechocystis ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an Escherichia coli mutant and exhibited strongly pH-dependent Na+/H+ and Li+/H+ exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K+ uptake-deficient E. coli mutant and exhibited K+ uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that A. halophytica has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.

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. Here, we report on the mechanistic basis of this effect. Heterologous expression experiments in Escherichia coli show that three Synechocystis genes are required for K(+) transport activity. They encode an NAD(+)-binding peripheral membrane protein (ktrA; sll0493), an integral membrane protein, belonging to a superfamily of K(+) transporters (ktrB; formerly ntpJ; slr1509), and a novel type of ktr gene product, not previously found in Ktr systems (ktrE; slr1508). In E. coli, Synechocystis KtrABE-mediated K(+) uptake occurred with a moderately high affinity (K(m) of about 60 microm), and depended on both Na(+) and a high membrane potential, but not on ATP. KtrABE neither mediated Na(+) uptake nor Na(+) efflux. In Synechocystis sp. PCC 6803, KtrB-mediated K(+) uptake required Na(+) and was inhibited by protonophore. A Delta ktrB strain was sensitive to long term hyperosmotic stress elicited by either NaCl or sorbitol. Hyperosmotic shock led initially to loss of net K(+) from the cells. The Delta ktrB cells shocked with sorbitol failed to reaccumulate K(+) up to its original level. These data indicate that in strain PCC 6803 K(+) uptake via KtrABE plays a crucial role in the early phase of cell turgor regulation after hyperosmotic shock.