Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+,K(+)-ATPase.

Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.     

Key NAD+-binding residues in human 15-hydroxyprostaglandin dehydrogenase

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins, and is believed to be the key enzyme responsible for the biological inactivation of these biologically potent eicosanoids. The enzyme utilizes NAD(+) specifically as a coenzyme. Potential amino acid residues involved in binding NAD(+) and facilitating enzyme catalysis have been partially identified. In this report, we propose that three more residues in 15-PGDH, Ile-17, Asn-91, and Val-186, are also involved in the interaction with NAD(+). Site-directed mutagenesis was used to examine their roles in binding NAD(+). Several mutants (I17A, I17V, I17L, I17E, I17K, N91A, N91D, N91K, V186A, V186I, V186D, and V186K) were prepared, expressed as glutathione S-transferase (GST) fusion enzymes in Escherichia coli, and purified by GSH-agarose affinity chromatography. Mutants I17E, I17K, N91L, N91K, and V186D were found to be inactive. Mutants N91A, N91D, V186A, and V186K exhibited comparable activities to the wild type enzyme. However, mutants I17A, I17V, I17L, and V186I had higher activity than the wild type. Especially, the activities of I17L and V186I were increased nearly 4- and 5-fold, respectively. The k(cat)/K(m) ratios of all active mutants for PGE(2) were similar to that of the wild type enzyme. However, the k(cat)/K(m) ratios of mutants I17A and N91A for NAD(+) were decreased 5- and 10-fold, respectively, whereas the k(cat)/K(m) ratios of mutants I17V, N91D, V186I, and V186K for NAD(+) were comparable to that of the wild type enzyme. The k(cat)/K(m) ratios of mutants I17L and V186A for NAD(+) were increased over nearly 2-fold. These results suggest that Ile-17, Asn-91, and Val-186 are involved in the interaction with NAD(+) and contribute to the full catalytic activity of 15-PGDH.     

Thymidylate synthesis and aminopterin resistance in Bacillus subtilis

Wilson, Melba Carr (Brown University, Providence, R.I.), James L. Farmer, and Frank Rothman. Thymidylate synthesis and aminopterin resistance in Bacillus subtilis. J. Bacteriol. 92:186-196. 1966.-The thymine-requirement of Bacillus subtilis 168 thy results from mutation in two unlinked genes (i.e., genetic loci) designated thyA and thyB. The thyB gene is located between the met and ile markers. Both thyA(+)thyB and thyA thyB(+) strains are phenotypically thy(+). ThyA(+)thyB strains resemble the wild type in their sensitivity to aminopterin, poor incorporation of exogenous thymine into deoxyribonucleic acid (DNA), and high level of thymidylate synthetase activity in crude extracts. ThyA thyB(+) strains are resistant to aminopterin in the presence of thymine, incorporate exogenous thymine into DNA, and have no detectable thymidylate synthetase activity. Experiments designed to elucidate the role of the thyB gene indicate that it specifies an alternate pathway of thymidylate synthesis, similar to thymidylate synthetase but requiring a cofactor other than tetrahydrofolate. The mechanism of selection of thymine-requiring mutants by aminopterin is revealed by these results.     

Chinese hamster ovary cell mutants resistant to DNA polymerase inhibitors. II. Segregational analysis and DNA transfection of the aphr gene

Genetic characterizations of the Chinese hamster ovary cell mutants resistant to the DNA polymerase inhibitors (aphidicolin, ara-A and ara-C) have been described. Resistance to all three inhibitors showed dominance among the progeny of somatic cell crosses between the wild type and mutant parents. Analysis of the segregation of the drug-resistant character among 566 hybrid progeny from somatic crosses between the wild type (aphs, ara-As, and ara-Cs) and the triple mutants (aphr, ara-Ar, ara-Cr) showed the involvement of at least three unlinked genes in controlling the expression of the resistance to different DNA polymerase inhibitors. The mutant (aphr) DNA was used to transfect aphidicolin resistance to recipient mouse Ltk- cells either directly or in combination with the plasmid pTK2 DNA. The aphidicolin resistance of the transfected cells was found to be a stable phenotype and could be used in multiple rounds of transfection, indicating the chromosomal integration of the transfecting gene.     

Regulation of the Pool Size of Valine in Escherichia coli K-12

Three mutations (ilvH611, ilvH612, and ilvH613) are described which make Escherichia coli K-12 resistant to valine inhibition and are located near leu. The expression of the ilv genes appears to be normal in these mutants since the isoleucine-valine biosynthetic enzymes are not derepressed relative to the wild type. The intracellular concentration of valine is, however, higher in the mutants than in the isogenic ilvH(+) strain. These mutants also excrete valine, probably because of the high intracellular concentration of this amino acid. The pool size of valine is regulated independently from that of isoleucine and leucine. The increased intracellular concentration of valine is due to a decreased feedback inhibition that valine exerts on its own biosynthetic pathway. In fact, acetolactate synthase activity assayed in extracts of ilvH612 and ilvH613 mutants is more resistant to valine inhibition than the activity assayed in the ilvH(+) isogenic strain. Two forms of acetolactate synthase activity can be separated from these extracts by adsorption and elution on hydroxylapatite. One of them is as sensitive to valine inhibition as that of the wild type, the other is more resistant to valine inhibition.     

K plus-dependent deplasmolysis of a marine pseudomonad plasmolyzed in a hypotonic solution

When cells of a marine pseudomonad were washed with a solution consisting of 0.3 m NaCl, 0.05 m MgSO(4), and 0.01 m KCl (complete salts), they maintained their normal morphology. When washed with a solution of 0.05 m MgSO(4), they became plasmolyzed as indicated by both phase and electron microscopy. Suspensions of cells washed with 0.05 m MgSO(4) showed an increase in optical density (OD) when 0.3 m NaCl was added, and this was followed by a decrease in OD upon the further addition of 0.01 m KCl. Salts of other monovalent cations were not effective in replacing K(+) in producing the OD decrease. Phase-contrast microscopy revealed that the increase in OD was accompanied by a decrease in cell size, and the decrease in OD, by an increase in the cell size. Both phase and electron microscopy showed that the K(+)-dependent decrease in OD was accompanied by deplasmolysis of the cells. Na(+) was required in the suspending medium in addition to K(+) to obtain deplasmolysis. The intracellular K(+) concentration in cells which had been washed with complete salts and which had retained their normal morphology was found to be 0.290 m. In cells plasmolyzed by washing with 0.05 m MgSO(4), the intracellular K(+) concentration was 0.004 m. Deplasmolyzed cells contained 0.330 m K(+). The membrane profile of plasmolyzed cells was retained when protoplasts were formed. The protoplasts became spherical if incubated in a solution permitting the deplasmolysis of the parent cells. The evidence obtained indicates that plasmolysis and deplasmolysis under the conditions described was due to the loss and gain, respectively, of K(+) by the cells. The effect of Na(+) could be ascribed to its capacity to control the porosity of the cytoplasmic membrane of this organism.     

Analysis of sorbose resistance in Neurospora crassa in heterokaryons of sorbose resistant mutants; contribution to the genetics of active transport

Summary Suitable auxotrophic markers were introduced into sorbose-resistant mutants and the sorbose-sensitive wildtype strain. Pairwise combinations of one resistant and one sensitive strain each as well as of two sensitive strains were then grown on minimal-agar to obtain forced heterocaryons. The growth behaviour of these on minimal-agar with and without added sorbose was compared.Of seven resistant mutants, representing six separate genes, among which were genes A and B, six mutants were recessive to the wildtype. The seventh, representing gene C, was recessive only with regard to colony-size, but intermediate with regard to germination counts. Heterocaryons forced between pairs of 2 closely linked mutants (intragenic case of the type A^– ₁+A^– ₂) were resistant, as were the separate mutants. However two heterocaryons forced between pairs of unlinked mutants (intergenic case of the type A^–+B^–) were sorbose sensitive. Heterocaryons forced between A or B-mutants and the C-mutant mentioned, unlinked to either A or B (intergenic cases of the type A^–+C^– and B^–+C^–) were more sensitive than the separate mutants but more resistant than the wildtype.It follows that sorbose-resistant mutants in heterocaryons of the intergenic types can complement each others defects (no growth complementation), but can not do so in heterocaryons of the intragenic type. Their complementation is considered to be the result of the activity of the intact wildtype genes homologous to the defective ones that are contained together in the multinucleate cells of the heterocaryons. This complementation may be taken as evidence for the recessiveness resp. intermediate expression of the different resistant mutants.Since none of the mutants checked so far were dominant compared to the wildtype, none of them can be a regulator-mutant. The possibility of explaining them as suppressor mutants is restricted by their recessiveness to mechanisms of suppression giving a recessive phenotype. An alternative explanation suggests that the respective wildtype genes may contain structural information for the synthesis of permeases involved in sorbose transport. The mutants would then be resistant due to defective permeases. Their recessiveness is in full accord with this suggestion.

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II. Teil einer Habilitationsschrift bei der Naturwissenschaftlichen Fakultät der Universität München.     

Physiological and Genetic Aspects of Abortive Infection of a Shigella sonnei Strain by Coliphage T7

Phage T7 adsorbed to and lysed cells of Shigella sonnei D(2) 371-48, although the average burst size was only 0.1 phage per cell (abortive infection). No mechanism of host-controlled modification was involved. Upon infection, T7 rapidly degraded host deoxyribonucleic acid (DNA) to acid-soluble material. Phage-directed DNA synthesis was initiated normally, but after a few minutes the pool of phage DNA, including the parental DNA, was degraded. Addition of chloramphenicol, at the time of phage infection, prevented both the initiation of phage-directed DNA synthesis and the degradation of parental phage DNA. Addition of chloramphenicol 4.5 min after phage was added permitted the onset of phage-directed DNA synthesis but prevented breakdown of phage DNA. Mutants of T7 (ss(-) mutants) have been isolated which show normal growth in strain D(2) 371-48. Upon mixed infection of this strain with T7 wild type and an ss(-) mutant, infection was abortive; no complementation occurred. The DNA of the ss(-) mutants was degraded in mixed infection like that of the wild type. Revertant mutants which have lost their ability to grow on D(2) 371-48 were isolated from ss(-) mutants; they are, in essence, phenotypically like T7 wild type. Independently isolated revertants of ss(-) mutants did not produce ss(-) recombinants when they were crossed among themselves. When independently isolated ss(-) mutants were crossed with each other, wild-type recombinants were found; ss(-) mutants could then be mapped in a cluster compatible with the length of one cistron. We concluded that T7 codes for an active, chloramphenicol-sensitive function [ss(+) function (for suicide in Shigella)] which leads to the breakdown of phage DNA in the Shigella host.     

Role of K(+) binding residues in stabilization of heme spin state of Leishmania major peroxidase

The endogenous cation in peroxidases may contribute to the type of heme coordination. Here a series of ferric and ferrous derivatives of wild-type Leishmania major peroxidase (LmP) and of engineered K(+) site mutants of LmP, lacking potassium cation binding site, has been examined by electronic absorption spectroscopy at 25°C. Using UV-visible spectrophotometry, we show that the removal of K(+) binding site causes substantial changes in spin states of both the ferric and ferrous forms. The spectral changes are interpreted to be, most likely, due to the formation of a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH 7.0. Stopped flow spectrophotometric techniques revealed that characteristics of Compound I were not observed in the K(+) site double mutants in the presence of H(2)O(2). Similarly electron donor oxidation rate was two orders less for the K(+) site double mutants compared to the wild type. These data show that K(+) functions in preserving the protein structure in the heme surroundings as well as the spin state of the heme iron, in favor of the enzymatically active form of LmP.     

Membrane potential defect in hygromycin B-resistant pma1 mutants of Saccharomyces cerevisiae

The proton transport properties of hygromycin B-resistant pma1 mutants which show kinetic defects in the plasma membrane H+-ATPase were examined. It was found that net proton efflux, as measured by whole cell medium acidification in the presence of 25 mM KCl, was similar for normal and pma1 mutant cells. However, in the absence of added KCl, the extent of net proton efflux was considerably less in wild type than in pma1 mutant cells. The cellular membrane potential was implicated as an important factor in regulating net proton transport and was determined from [14C]tetraphenylphosphonium uptake studies to be considerably depolarized in the pma1 mutants. The growth of wild type cells, which is normally inhibited by hygromycin B at 200 micrograms/ml, was found to be resistant to the antibiotic by the addition of 50 mM KCl to the growth medium. These results suggest that the electrogenic behavior of proton transport by the H+-ATPase may be altered in pma1 mutants and that resistance to hygromycin B may be mediated via depolarization of the cellular membrane potential.     

Genetic requirements for potassium ion-dependent colony spreading in Bacillus subtilis

Undomesticated strains of Bacillus subtilis exhibit extensive colony spreading on certain soft agarose media: first the formation of dendritic clusters of cells, followed by spreading (pellicle-like) growth to cover the entire surface. These phases of colonization are dependent on the level of potassium ion (K(+)) but independent of flagella, as verified with a mutant with a hag gene replacement; this latter finding highlights the importance of sliding motility in colony spreading. Exploring the K(+) requirement, directed mutagenesis of the higher-affinity K(+) transporter KtrAB, but not the lower-affinity transporter KtrCD, was found to inhibit surface colonization unless sufficient KCl was added. To identify other genes involved in K(+)-dependent colony spreading, transposon insertion mutants in wild-type strain 3610 were screened. Disruption of genes for pyrimidine (pyrB) or purine (purD, purF, purH, purL, purM) biosynthetic pathways abolished the K(+)-dependent spreading phase. Consistent with a requirement for functional nucleic acid biosynthesis, disruption of purine synthesis with the folic acid antagonist sulfamethoxazole also inhibited spreading. Other transposon insertions disrupted acetoin biosynthesis (the alsS gene), acidifying the growth medium, glutamine synthetase (the glnA gene), and two surfactin biosynthetic genes (srfAA, srfAB). This work identified four classes of surface colonization mutants with defective (i) potassium transport, (ii) surfactin formation, (iii) growth rate or yield, or (iv) pH control. Overall, the ability of B. subtilis to colonize surfaces by spreading is highly dependent on balanced nucleotide biosynthesis and nutrient assimilation, which require sufficient K(+) ions, as well as growth conditions that promote sliding motility.     

Ion exchangers NHX1 and NHX2 mediate active potassium uptake into vacuoles to regulate cell turgor and stomatal function in Arabidopsis

Intracellular NHX proteins are Na(+),K(+)/H(+) antiporters involved in K(+) homeostasis, endosomal pH regulation, and salt tolerance. Proteins NHX1 and NHX2 are the two major tonoplast-localized NHX isoforms. Here, we show that NHX1 and NHX2 have similar expression patterns and identical biochemical activity, and together they account for a significant amount of the Na(+),K(+)/H(+) antiport activity in tonoplast vesicles. Reverse genetics showed functional redundancy of NHX1 and NHX2 genes. Growth of the double mutant nhx1 nhx2 was severely impaired, and plants were extremely sensitive to external K(+). By contrast, nhx1 nhx2 mutants showed similar sensitivity to salinity stress and even greater rates of Na(+) sequestration than the wild type. Double mutants had reduced ability to create the vacuolar K(+) pool, which in turn provoked greater K(+) retention in the cytosol, impaired osmoregulation, and compromised turgor generation for cell expansion. Genes NHX1 and NHX2 were highly expressed in guard cells, and stomatal function was defective in mutant plants, further compromising their ability to regulate water relations. Together, these results show that tonoplast-localized NHX proteins are essential for active K(+) uptake at the tonoplast, for turgor regulation, and for stomatal function.     

Electrostatic contributions to the binding of Ca2+ in calbindin D9k

A set of accurate experimental data is provided for Ca2+ ion binding to calbindin D9k, a protein in the calmodulin superfamily of intracellular regulatory proteins. The study comprises both the role of protein surface charges and the effects of added electrolyte. The two macroscopic Ca2(+)-binding constants K1 and K2 are determined for the wild-type and eight mutant calbindins in 0, 0.05, 0.10, and 0.15 M KCl from titrations in the presence of Quin 2 or 5,5'-Br2BAPTA. The mutations involve replacement of surface carboxylates (of Glu17, Asp19, Glu26, and Glu60) with the corresponding amides. It is found that K1K2 may decrease by a factor of up to 2.5 x 10(5) (triple mutant in 0.15 M KCl as compared to the wild-type protein in 0 M KCl). Ca2(+)-binding constants of the individual Ca2+ sites (microscopic binding constants) have also been determined. The positive cooperativity of Ca2+ binding, previously observed at low salt concentration [Linse et al. (1987) Biochemistry 26, 6723-6735], is also present at physiological ionic strength and amounts to 5 kJ.mol-1 at 0.15 M KCl. The electrolyte concentration and some of the mutations are found to affect the cooperativity. 39K NMR studies show that K+ binds weakly to calbindin. Two-dimensional 1H NMR studies show, however, that potassium binding does not change the protein conformation, and the large effect of KCl on the Ca2+ affinity is thus of unspecific nature. Two-dimensional 1H NMR has also been used to assess the structural consequences of the mutations through assignments of the backbone NH and C alpha H resonances of six mutants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Constitutive penicillinase formation in Staphylococcus aureus owing to a mutation unlinked to the penicillinase plasmid

Previously described penicillinase-constitutive mutations in Staphylococcus aureus are caused by genetic lesions in a regulator gene (or genes) on the penicillinase plasmid in close linkage to the structural gene. This report describes a new class (R2(-)) of penicillinase-constitutive mutants of S. aureus unlinked to the plasmid. By transductional analysis, the penicillinase plasmids in these mutants were wild type. Wild-type plasmids transduced into penicillinase-negative (plasmid loss) derivatives of R2(-) mutants produced penicillinase constitutively in amounts comparable to a fully induced culture of the wild-type strain. Penicillinase production in R2(-) mutants was maximal at 30 to 32 C and was much reduced at 40 C.     

Residues in Na(+) channel D3-S6 segment modulate both batrachotoxin and local anesthetic affinities

Batrachotoxin (BTX) alters the gating of voltage-gated Na(+) channels and causes these channels to open persistently, whereas local anesthetics (LAs) block Na(+) conductance. The BTX and LA receptors have been mapped to several common residues in D1-S6 and D4-S6 segments of the Na(+) channel alpha-subunit. We substituted individual residues with lysine in homologous segment D3-S6 of the rat muscle mu1 Na(+) channel from F1274 to N1281 to determine whether additional residues are involved in BTX and LA binding. Two mutant channels, mu1-S1276K and mu1-L1280K, when expressed in mammalian cells, become completely resistant to 5 microM BTX during repetitive pulses. The activation and/or fast inactivation gating of these mutants is substantially different from that of wild type. These mutants also display approximately 10-20-fold reduction in bupivacaine affinity toward their inactivated state but show only approximately twofold affinity changes toward their resting state. These results demonstrate that residues mu1-S1276 and mu1-L1280 in D3-S6 are critical for both BTX and LA binding interactions. We propose that LAs interact readily with these residues from D3-S6 along with those from D1-S6 and D4-S6 in close proximity when the Na(+) channel is in its inactivated state. Implications of this state-dependent binding model for the S6 alignment are discussed.     

Isolation of a suppressor host bacterium in Staphylococcus aureus

A bacterial mutant of Staphylococcus aureus NCTC 8325 has been isolated which has the properties of a suppressor host mutant. The mutant was isolated as a one-step phenotypic revertant to wild type of a strain containing mutations in two unlinked markers concerned with metabolism of lactose via the phosphoenol pyruvate-dependent phosphotransferase system. The revertant (called sup1(+)) has been used to isolate seventy conditional lethal mutants of the phage O11. The phage mutants, which plate on sup1(+) but not on the original 8325 strain, have been assigned by complementation studies into 10 groups. It is probable that this technique for the isolation of suppressor hosts would be applicable to other Staphylococcus strains.     

Genes from Debaryomyces hansenii increase salt tolerance in Saccharomyces cerevisiae W303

The yeast Debaryomyces hansenii has been chosen as a model for molecular studies of tolerance to NaCl. A gene library was built and transformants of Saccharomyces cerevisiae W303 containing genes from D. hansenii were selected for their ability to grow in the presence of high concentrations of NaCl and/or low concentrations of KCl. In three of these transformants 500 mM NaCl improved growth at pH 7.6 like in D. hansenii but not in S. cerevisiae. One of the plasmids restored growth at 50 microM KCl and K(+) uptake in a mutant of S. cerevisiae lacking genes that encode K(+) transporters.     

Mutants of Arabidopsis thaliana Capable of Germination under Saline Conditions

Three mutant strains of Arabidopsis thaliana var Columbia were selected for their ability to germinate in elevated concentrations of NaCl. They were not more tolerant than wild type at subsequent development stages. Wild-type strains could not germinate at concentrations > 125 mM NaCl. Two of mutant strains, RS17 and RS20, could withstand up to 225 mM, whereas RS19 was resistant to 175 mM. The RS mutants could also germinate under even lower osmotic potentials imposed by high concentrations of exogenous mannitol (550 mM), whereas the effects of elevated levels of KCl, K2SO4, and LiCl were similar among the mutants and wild type. Therefore, the mutants are primarily osmotolerant, but they also possess a degree of ionic tolerance for sodium. Sodium and potassium contents of seeds exposed to high salinities indicated that the NaCl-tolerant mutants absorbed more of these respective cations during imbibition. These higher internal concentrations of potassium and sodium could contribute to the osmotic adjustment of the germinating seeds to the low osmotic potential of the external medium. Genetic analysis of F1 and F2 progeny of outcrosses suggest that the salt-tolerant mutations are recessive and that they define three complementation groups.