Defective cation-coupling mutants of Escherichia coli Na+/proline symport carrier. Characterization and localization of mutations

A major proline carrier in Escherichia coli encoded by the putP gene mediates proline/Na+ or Li+ symport. Proline carrier mutants with altered cation specificity were obtained by mutagenesis with nitrous acid in vitro of a plasmid carrying the wild-type putP gene. Two mutant strains harboring plasmid pMOP4135 and pMOP4141 could transport proline efficiently only in the presence of an increased concentration of sodium ion. Mutations of these plasmids, putP4135 and putP4141, caused reduction of affinity for Na+ of proline transport and binding, without remarkable change in the affinity for proline or in production of the carriers. Consistent with the lower affinity of the putP4141 carrier for Na+, the mutant carrier was supersensitive to N-ethylmaleimide inhibition. The pH dependence of proline binding was also changed in these mutant carriers. The lesions of putP4135 and putP4141 were located in the N-terminal part of the putP gene (ClaI-PvuII fragment) by in vitro recombination and subsequent examination of the phenotype of the transformants. DNA sequencing of these fragments revealed one base alteration of G to A at nucleotides 299 and 656 in pMOP4141 and pMOP4135, respectively, which corresponded to amino acid changes from Gly22 to glutamic acid and Cys141 to tyrosine, respectively.     

Purification and reconstitution of Escherichia coli proline carrier using a site specifically cleavable fusion protein

We previously constructed a bifunctionally active membrane-bound fusion protein, in which Escherichia coli proline carrier (the product of the putP gene) was linked with beta-galactosidase (the product of the lacZ gene) through a collagen linker (Hanada, K., Yamato, I., and Anraku, Y. (1987) J. Biol. Chem. 262, 14100-14104). The proline carrier was purified from this site specifically cleavable fusion protein. Cytoplasmic membranes overproducing the fusion protein were solubilized with dodecylmaltoside, and the solubilized fraction was subjected to anti-beta-galactosidase IgG-Sepharose chromatography. The fusion protein was specifically adsorbed to the immunoaffinity resin and then treated with collagenase for splitting the proline carrier moiety of the fusion protein from the beta-galactosidase moiety. The collagenase used for the collagenolysis was then removed by anti-collagenase IgG-Sepharose chromatography. In this way, the proline carrier was purified to more than 95% homogeneity of the protein. Proline transport in proteoliposomes reconstituted with the purified carrier was dependent on the membrane potential and the chemical gradient of Na+ across the membrane with apparent Michaelis constants for proline and for Na+ stimulation of 3.6 microM and 31 microM, respectively. These results indicated that the proline carrier mediates electrogenic Na+/proline symport.     

Mechanism of proline transport in Escherichia coli K12. II. Effect of alkaline cations on binding of proline to a H+/proline symport carrier in cytoplasmic membrane vesicles

The substrate binding reaction of the proline carrier was investigated in nonenergized conditions using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The binding activity specifically required both alkaline cations (X+), Na+ and Li+, and protons. The Na+-dependent binding activity was dependent on the proline carrier, which is the product of the putP gene, and was not affected by ionophores and energy transduction inhibitors. The parameters of proline binding were determined by double reciprocal plots in reaction media with various combinations of Na+ and H+ concentrations. The apparent dissociation constant was greatly affected by the Na+ and H+ concentrations of the medium and could be expressed as a combination of the reciprocals of the Na+ and H+ concentrations, while the maximum number of binding sites remained constant. The characteristics of proline binding to the carrier can be explained by a mechanism in which the unloaded carrier forms a carrier/H+/X+ (CH+X+) complex by a random equilibrium and only the CH+X+ complex binds substrate in nonenergized conditions, as proposed for the Na+/H+/glutamate symport carrier of E. coli B ( Fujimura , T., Yamato , I., and Anraku , Y. (1983) Biochemistry 22, 1954-1959).     

Yeast CAL1 is a structural and functional homologue to the DPR1 (RAM) gene involved in ras processing

A 2.3-kilobase pair DNA fragment of the yeast CAL1 gene was cloned by complementation of the cal1-1 mutation, which causes a defect in nuclear division and bud formation (Ohya, Y., Ohsumi, Y., and Anraku, Y. (1984) Mol. & Gen. Genet. 193, 389-394). Nucleotide sequencing of this fragment revealed a single open reading frame (ORF) encoding a polypeptide of 376 amino acids. Comparative analysis of the predicted amino acid sequence has shown that the CAL1 product has similarity to two yeast proteins: the DPR1 (RAM) gene product that is involved in processing of ras protein at the farnesylation step, and the essential ORF2 protein whose structural gene has a head-to-head arrangement with PRP4, which is involved in mRNA processing. Functional homology between CAL1 and DPR1 has also been suggested from genetic evidence that multiple copies of the CAL1 gene suppress the growth defects of a dpr1 null mutant at high temperature. This suppression is Ca(2+)-dependent, since it was not observed in complete medium containing 200 microM CaCl2 but was apparent in medium containing 100 mM CaCl2. From sequence analysis of the cal1-1 mutation, together with the alignment of the three gene products, we have concluded that the conserved Gly328 in the C terminus is important for activity. We suggest that the CAL1 protein participates in a ras-like C-terminal modification of proteins involved in nuclear division and bud growth.     

Characterization of a gamma-glutamyl kinase from Escherichia coli that confers proline overproduction and osmotic tolerance.

Mutation(s) in the proBA operon of Escherichia coli confers proline overproduction and enhanced osmotic tolerance in enteric bacteria (L. N. Csonka, Mol. Gen. Genet. 182:82-86, 1981; M. J. Mahan and L. N. Csonka, J. Bacteriol. 156:1249-1262, 1983). A glutamate-dependent ATPase assay was developed and used to determine proB-encoded gamma-glutamyl kinase activity in the absence of glutamate-gamma-semialdehyde dehydrogenase. This assay indicated that the feedback insensitivity of mutant gamma-glutamyl kinase was independent of glutamate-gamma-semialdehyde dehydrogenase. However, the capacity of glutamate-gamma-semialdehyde dehydrogenase from the osmotolerant mutant to interact with the kinase was altered in thermal stability, suggesting that mutations in both proB and proA may be required for osmotolerance.     

Detection and characterization of Tn2501, a transposon included within the lactose transposon Tn951

The DNA sequence spanning coordinates 9.9 to 16.4 kilobases of the lactose transposon Tn951 ( Cornelis et al., Mol. Gen. Genet. 160:215-224, 1978) constitutes a transposable element by itself. Unlike Tn951 ( Cornelis et al., Mol. Gen. Genet. 184:241-248, 1981), this element, called Tn2501 , transposes in the absence of any other transposon. Transposition of Tn2501 proceeds through transient cointegration and duplicates 5 base pairs of host DNA. Tn2501 is flanked by nearly perfect inverted repeats (44 of 48), related to the inverted repeats of Tn21 ( Zheng et al., Nucleic Acids Res. 9:6265-6278, 1982). Unlike Tn21 , Tn2501 does not confer mercury resistance.     

Mutations of putP that alter the lithium sensitivity of Salmonella typhimurium

The putP gene encodes the major proline permease in Salmonella typhimurium that couples transport of proline to the sodium electrochemical gradient. To identify residues involved in the cation binding site, we have isolated putP mutants that confer resistance to lithium during growth on proline. Wild-type S. typhimurium can grow well on proline as the sole carbon source in media supplemented with NaCl, but grows poorly when LiCl is substituted for NaCl. In contrast to the growth phenotype, proline permease is capable of transporting proline via Na+/proline or Li+/proline symport. Therefore, we selected mutants that grow well on media containing proline as the sole carbon source in the presence of lithium ions. All of the mutants assayed exhibit decreased rates of Li+/proline and Na+/proline cotransport relative to wild type. The location of each mutation was determined by deletion mapping: the mutations cluster in two small deletion intervals at the 5' and 3' termini of the putP gene. The map positions of these lithium resistance mutations are different from the locations of the previously isolated substrate specificity mutations. These results suggest that Lir mutations may define domains of the protein that fold to form the cation binding site of proline permease.     

Cloning and characterization of the histidine biosynthetic gene cluster of Streptomyces coelicolor A3(2)

Biochemical and genetic data indicate that in Streptomyces coelicolor A3(2) the majority of the genes involved in the biosynthesis of histidine are clustered in a small region of the chromosome [Carere et al., Mol. Gen. Genet. 123 (1973) 219-224; Russi et al., Mol. Gen. Genet. 123 (1973) 225-232]. To investigate the structural organization and the regulation of these genes, we have constructed genomic libraries from S. coelicolor A3(2) in pUC vectors. Recombinant clones were isolated by complementation of an Escherichia coli hisBd auxotroph. A recombinant plasmid containing a 3.4-kb fragment of genomic DNA was further characterized. When cloned in the plasmid vector, pIJ699, this fragment was able to complement S. coelicolor A3(2) hisB mutants. Overlapping clones spanning a 15-kb genomic region were isolated by screening other libraries with labeled DNA fragments obtained from the first clone. Derivative clones were able to complement mutations in four different cistrons of the his cluster of S. coelicolor A3(2). Nucleotide sequence analysis of a 4-kb region allowed the identification of five ORFs which showed significant homology with the his gene products of E. coli. The order of the genes in S. coelicolor A3(2) (5'--hisD-hisC-hisBd-hisH-hisA-3') is the same as in the his operon of E. coli.     

Proline transport in Salmonella typhimurium: putP permease mutants with altered substrate specificity.

The putP gene encodes a proline permease required for Salmonella typhimurium LT2 to grow on proline as the sole source of nitrogen. The wild-type strain is sensitive to two toxic proline analogs (azetidine-2-carboxylic acid and 3,4-dehydroproline) also transported by the putP permease. Most mutations in putP prevent transport of all three substrates. Such mutants are unable to grow on proline and are resistant to both of the analogs. To define domains of the putP gene that specify the substrate binding site, we used localized mutagenesis to isolate rare mutants with altered substrate specificity. The position of the mutations in the putP gene was determined by deletion mapping. Most of the mutations are located in three small (approximately 100-base-pair) deletion intervals of the putP gene. The sensitivity of the mutants to the proline analogs was quantitated by radial streaking to determine the affinity of the mutant permeases for the substrates. Some of the mutants showed apparent changes in the kinetics of the substrates transported. These results indicate that the substrate specificity mutations are probably due to amino acid substitutions at or near the active site of proline permease.     

Two proline porters in Escherichia coli K-12

Escherichia coli mutants defective at putP and putA lack proline transport via proline porter I and proline dehydrogenase activity, respectively. They retain a proline uptake system (proline porter II) that is induced during tryptophan-limited growth and are sensitive to the toxic L-proline analog, 3,4-dehydroproline. 3,4-Dehydroproline-resistant mutants derived from a putP putA mutant lack proline porter II. Auxotrophic derivatives derived from putP+ or putP bacteria can grow if provided with proline at low concentration (25 microM); those derived from the 3,4-dehydroproline-resistant mutants require high proline for growth (2.5 mM). We conclude that E. coli, like Salmonella typhimurium, possesses a second proline porter that is inactivated by mutations at the proP locus.     

Cloning and characterization of dnaA(Cs), a mutation which leads to overinitiation of DNA replication in Escherichia coli K-12.

The product of the dnaA gene is essential for the initiation of chromosomal DNA replication in Escherichia coli K-12. A cold-sensitive mutation, dnaA(Cs), was originally isolated as a putative intragenic suppressor of the temperature sensitivity of a dnaA46 mutant (G. Kellenberger-Gujer, A. J. Podhajska, and L. Caro, Mol. Gen. Genet. 162:9-16, 1978). The cold sensitivity of the dnaA(Cs) mutant was attributed to a loss of replication control resulting in overinitiation of DNA replication. We cloned and sequenced the dnaA gene from the dnaA(Cs) mutant and showed that it contains three point mutations in addition to the original dnaA46(Ts) mutation. The dnaA(Cs) mutation was dominant to the wild-type allele. Overproduction of the DnaA(Cs) protein blocked cell growth. In contrast, overproduction of wild-type DnaA protein reduced the growth rate of cells but did not stop cell growth. Thus, the effect of elevated levels of the DnaA(Cs) protein was quite different from that of the wild-type protein under the same conditions.     

High-efficiency transformation of Streptococcus lactis protoplasts by plasmid DNA

Streptococcus lactis IL1403 protoplasts were transformed by plasmid pIL204 (5.5 kilobases), which conferred erythromycin resistance with an average efficiency of 5 X 10(6) transformants per microgram of supercoiled DNA. The procedure used and transformation efficiencies obtained were close to those described for Bacillus subtilis (G. Chang and S. N. Cohen, Mol. Gen. Genet. 168:111-115, 1979).     

DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae.

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.     

Altered induction of the adaptive response to alkylation damage in Escherichia coli recF mutants.

Escherichia coli recF mutants are hypermutable when treated with methyl methanesulfonate (G. C. Walker, Mol. Gen. Genet. 152:93-103, 1977). In this study, methylation hypermutability of recF mutant strains was examined, and it was found that recF+ is required for normal induction of the adaptive response to alkylation damage. Although this regulatory effect of recF mutations results in reduced levels of enzymes that specifically repair methyl lesions in DNA, it only partially explains the hypermutability. Further examination showed that methylation hypermutability of recF mutant strains required a functional umuDC operon, a component of the SOS response. These results lead to the hypothesis that methylation hypermutability results from the effects of recF mutations on the induction of both the SOS response and the adaptive response.     

Cell-cycle mutations among the collection of Saccharomyces cerevisiae dna mutants

Abstract The temperature-sensitive dna mutants of the budding yeast Saccharomyces cerevisiae (Dumas et al. (1982) Mol. Gen. Genet. 187, 42–46) are more inhibited in DNA synthesis than in protein synthesis. These properties are also characteristics of many yeast mutations that inhibit progress through the cell cycle. Therefore we surveyed the collection of dna mutants for cell-cycle mutations. By genetic complementation we found that dna 1 = cdc 22, dna 6 = cdc 34, dna 19 = cdc 36, and dna 39 = dbf 3. Furthermore, by direct gene cloning we found that the dna26 mutation is allelic to prt1 mutations, which are known to exert primary inhibition on protein synthesis. This protein-synthesis mutation exerts a dna phenotype due to cell-cycle inhibition: prt1 mutations can block the regulatory step of the cell cycle while allowing significant amounts of protein synthesis to continue. Our non-exhausive screening suggests that the dna mutants may house other mutations that affect the yeast cell cycle.     

Lactose carrier protein of Escherichia coli. Structure and expression of plasmids carrying the Y gene of the lac operon

The previously described hybrid plasmid pC7 which carries lacI+O+delta(Z)Y+A+ on a 12.3 X 10(6)-Mr DNA fragment [Teather et al. (1978) Mol. Gen. Genet. 159, 239-248] was partially digested with the restriction endonuclease EcoRI under conditions reducing the recognition sequence to d(A-A-T-T) and ligated to the vector pB322. lac Y-carrying inserts of various sized (Mr 1.5-4.7 X 10(6)) were obtained. Hybrid plasmid pTE18 (2300-base-pair insert) carries part of the I (repressor) gene, the promotor-operator region, part of the Z (beta-galactosidase) gene, the Y (lactose carrier) gene and part of the A (transacetylase) gene. Upon induction of pTE18-harbouring strains the Y-gene product is expressed at a nearly constant rate for several generations and accumulates to a level of 12-16% of the total cytoplasmic membrane protein. Integration into the membrane leads to active carrier as judged by binding and transport measurements.     

New genes involved in carbon catabolite repression and derepression in the yeast Saccharomyces cerevisiae.

A mutation causing resistance to carbon catabolite repression in gene HEX2, mutant allele hex2-3, causes an extreme sensitivity to maltose when in combination with the genes necessary for maltose metabolism. This provided a convenient system for the selective isolation of mutations in genes specifically required for maltose metabolism and other genes involved in general carbon catabolite repression. In addition to reversion of the hex2-3 allele, mutations in three other genes were detected. These genes were called CAT1, CAT3, and MUR1 and in a mutated form abolished maltose inhibition caused by mutant allele hex2-3. Mutant alleles cat1 and cat3 also restored normal repression in the presence of the hex2-3 allele. Segregants having only mutant alleles cat1 or cat3 were obtained by tetrad analysis. These segregants could not grow on nonfermentable carbon sources. Mutant alleles of gene CAT1 were allelic to a mutant allele cat1-1 previously isolated (Zimmermann et al., Mol. Gen. Genet. 151:95-103). Such mutants prevented derepression not only of the maltose catabolizing system, the selected property, but also of glyoxylate shunt and gluconeogenic enzymes. However, respiratory activities and invertase formation were not affected under derepressing conditions. cat3 mutants had the same phenotypic properties as cat1 mutants. This showed that carbon metabolism in yeast cells is under a very complex and ramified control of repressing and derepressing genes, which are interdependent.