bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning, mapping, and expression of genes involved in the fatty acid-degradative multienzyme complex of Escherichia coli
Authors: Spratt S K , Black P N , Ragozzino M M , Nunn W D
Published: January 2020

Two protein subunits (42,000 and 78,000 daltons) encoded by the fadAB genes form a multifunctional enzyme complex containing thiolase, 3-hydroxyacyl-coenzyme A dehydrogenase, crotonase , epimerase, and isomerase activities (S. Pawar and H. Schulz, J. Biol. Chem. 256:3894-3899, 1981). In an attempt to characterize the structural organization and regulatory properties of these genes, a 5.2-kilobase pair fragment containing the fadAB genes has been isolated. Plasmids containing this fragment (i) complement mutations in the fadAB genes; (ii) overproduce by 10- to 50-fold thiolase, 3-hydroxyacyl-coenzyme A dehydrogenase and crotonase ; and (iii) specify a 42,000- and a 78,000-dalton protein. The fadA gene, which encodes the 42,000-dalton protein, has been localized within the original clone to a 3.3-kilobase pair fragment. Thiolase activity, which is encoded by the 42,000-dalton protein, was not observed in the absence of the 78,000-dalton protein, suggesting that an intact complex is required for function. Transposon Tn5 insertional mutagenesis of the cloned fadAB genes has demonstrated that both fadA and fadB are transcribed as a single transcriptional unit with the direction of transcription from fadA to fadB . The molecular cloning and characterization of the fadAB region confirm the original genetic contention that the genes encoding the proteins for the multifunctional complex form an operon.
Method for determining whether a gene of Escherichia coli is essential: application to the polA gene
Authors: Joyce C M , Grindley N D
Published: January 2020

We have developed a general method for determining whether a gene of Escherichia coli is essential for viability. The method requires cloned DNA spanning the gene in question and a reasonably detailed genetic and physical map of the cloned segment. Using this information, one constructs a deletion of the target gene in vitro. For convenience, the deletion can be marked by an antibiotic resistance gene. A DNA segment containing the deletion is then cloned onto an att delta phage lambda vector. Integration of this phage, by homologous recombination at the target locus, and subsequent excision provide an efficient route for crossing the marked deletion onto the bacterial chromosome. Failure to delete the target gene indicates either that the resulting deletion was not viable or that the desired recombinational event did not take place. The use of prophage excision to generate the deletion allows one to estimate the fraction of deletion-producing events by analysis of the other product of the excision, the phage produced on induction of the prophage. In this way one can determine whether failure to recover a particular chromosomal deletion was due to its never having been formed, or, once formed, to its failure to survive. Applying this method to the polA gene, we found that polA is required for growth on rich medium but not on minimal medium. We repeated the experiment in the presence of plasmids carrying functional fragments of the polA gene, corresponding to the 5'-3' exonuclease and the polymerase-3'-5' exonuclease portions of DNA polymerase I. Surprisingly, either of these fragments, in the absence of the other, was sufficient to allow growth on rich medium.
Isolation of a Saccharomyces cerevisiae mutant strain deficient in deoxycytidylate deaminase activity and partial characterization of the enzyme
Authors: McIntosh E M , Haynes R H
Published: January 2020

Deoxycytidylate deaminase activity in Saccharomyces cerevisiae has been partially characterized. The yeast enzyme was found to exhibit properties similar to those of dCMP deaminases isolated from higher eucaryotes. A mutant strain completely deficient in dCMP deaminase activity was isolated by selection for resistance to 5-fluoro-2'-deoxycytidylate followed by screening for cross sensitivity to 5-fluoro-2'-deoxyuridylate, a potent inhibitor of the yeast thymidylate synthetase. We have designated this new allele dcd1 . A strain exhibiting an auxotrophic requirement for dUMP was isolated after mutagenesis of a dcd1 tup7 haploid. Genetic analysis revealed that this auxotrophic phenotype resulted from a combination of the dcd1 allele and a second, unlinked, nuclear mutation that we designated dmp1 . This allele, which by itself conveys no readily discernible phenotype, presumably impairs efficient synthesis of dUMP from UDP. The auxotrophic requirement of dcd1 dmp1 tup7 strains also can be satisfied by exogenous dTMP but not deoxyuridine.
Expression of a Thiobacillus ferrooxidans origin of replication in Escherichia coli
Authors: Rawlings D E , Pretorius I , Woods D R
Published: January 2020

A cryptic plasmid from an autotrophically grown arsenic-resistant strain of Thiobacillus ferrooxidans was isolated and cloned into pBR325. The origin of replication of pBR325 was deleted, and the recombinant plasmid was shown to replicate in Escherichia coli, using an origin of replication located on the Thiobacillus plasmid.
trans-Acting virulence functions of the octopine Ti plasmid from Agrobacterium tumefaciens
Authors: Hille J , van Kan J , Schilperoort R
Published: January 2020

All Ti plasmid-encoded virulence functions that were studied act in trans. An octopine Ti plasmid-specific vir operon, called vir-O, located on an EcoRI restriction fragment has been characterized. Sequences with promoter activity in Escherichia coli were identified for a second vir operon, called vir-C, which was located close to the position of vir-O.
Construction of a single-copy promoter vector and its use in analysis of regulation of the transposon Tn10 tetracycline resistance determinant
Authors: Bertrand K P , Postle K , Wray L V , Reznikoff W S
Published: January 2020

The construction and characterization of a promoter expression vector, lambda RS205 , is described. lambda RS205 can be used for the in vitro construction of transcriptional (operon) fusions to the lacZ gene of Escherichia coli K-12. The level of beta-galactosidase activity in lysogens of lambda RS205 fusion phages provides a quantitative measure of promoter function under single-copy conditions. The regulation of the Tn10 tetracycline resistance gene ( tetA ) and the Tn10 tet repressor gene (tetR) was examined by inserting DNA fragments that span the tetR- tetA promoter-operator region into lambda RS205 . Levels of beta-galactosidase in tetA -lacZ and tetR-lacZ fusion strains indicate that the tetA and tetR promoters are strong promoters; the tetA promoter is fourfold more active than the tetR promoter. Introduction of tetR+ plasmids into tetA -lacZ and tetR-lacZ fusion strains represses beta-galactosidase synthesis 15- to 60-fold and 6- to 15-fold, respectively. The concentration of tetracycline required to induce half-maximal beta-galactosidase synthesis in these tetR+ tet-lac strains depends on both the tetracycline resistance phenotype and the level of tetR repressor in the fusion strain. However, the induction of beta-galactosidase in isogenic tetA -lacZ and tetR-lacZ strains is coordinate. The data presented here support the current model of Tn10 tet gene organization and regulation and provide quantitative information about the regulation of tetA and tetR in vivo.
S-phase, G2, and nuclear division mutants of Aspergillus nidulans
Authors: Bergen L G , Upshall A , Morris N R
Published: January 2020

Twenty-two temperature-sensitive cell cycle mutants of the fungus Aspergillus nidulans, which block in interphase at restrictive temperature, were analyzed by the reciprocal shift method of Jarvik and Botstein (Proc. Nath Acad. Sci. U.S.A. 70:2046-2050, 1973) and Hereford and Hartwell (J. Mol. Biol. 84:445-461, 1974) to determine whether these mutations were blocked at the G1, S, or G2 phase of the cell cycle. We found five mutants to be blocked in S and nine to be blocked in G2. Two of the G2 mutants were atypical in that they were not able to accomplish the G2 to M transition at restrictive temperature but nevertheless could initiate subsequent cycles of DNA replication. None was blocked in G1. There were nine strains that could not be classified. The block imposed by restrictive temperature was irreversible in three of these strains, and the six other strains were unclassifiable due to their aberrant terminal nuclear phenotypes.
Regulation by molecular oxygen and organic substrates of hydrogenase synthesis in Alcaligenes eutrophus
Authors: Cangelosi G A , Wheelis M L
Published: January 2020

Chemoautotrophic growth of Alcaligenes eutrophus 17707 is inhibited by 20% oxygen in the gas phase. Lowering the oxygen concentration to 4% results in chloramphenicol-sensitive derepression of soluble and membrane-bound hydrogenase activity (and of soluble hydrogenase antigen), showing that oxygen inhibition is due at least in part to repression of hydrogenase synthesis. Mutations resulting in derepression of hydrogenase activity (and antigen) under 25% oxygen (Ose-) mobilized with a self-transmissable plasmid which is already known to carry genes necessary for hydrogenase expression. Plasmid-borne mutations resulting in loss of soluble hydrogenase activity have no effect on the Ose phenotype, but chromosomal mutations resulting in reduction or loss of both hydrogenase activities cannot be made Ose-. The Ose- mutation does not alter the thermostability of either hydrogenase, and soluble hydrogenase in the mutant reacts with complete identity with that of the wild type, indicating that the Ose- phenotype does not result from structural alterations in either enzyme. Ose- mutants are also relieved of normal hydrogenase repression by organic substrates, which aggravates hydrogenase-mediated inhibition of heterotrophic growth by hydrogen. Regulation of hydrogenase in Ose- strains of A. eutrophus 17707 is nearly identical to that of wild-type A. eutrophus strains H1 and H16.
Control of bacteriophage lambda CII activity by bacteriophage and host functions
Authors: Rattray A , Altuvia S , Mahajna G , Oppenheim A B , Gottesman M
Published: January 2020

We have studied the regulation of the lambda cII gene in vivo using cloned lambda fragments. Lambda N protein stimulated cII expression. Surprisingly, although very high cII protein levels were detected by gel electrophoresis, little cII protein activity, measured as stimulation of the lambda pI and pE promoters, was observed. The half-life of cII protein depended critically on its initial level. At low concentrations its half-life was as short as 1.5 min, whereas at high cII protein levels, it could be as long as 22 min. The Escherichia coli mutant ER437 directs lambda towards lysogeny; cII protein was more stable in this strain than in the wild type. On the other hand, although cyclic AMP is required for efficient lysogeny, it did not appear to influence the synthesis, stability, or activity of cII protein.
Evidence for antitermination in Escherichia coli RRNA transcription
Authors: Aksoy S , Squires C L , Squires C
Published: January 2020

The stable RNA operons of Escherichia coli do not exhibit polarity, even though they make an RNA product that is not translated. By contrast, most E. coli operons that specify proteins exhibit polarity if their translation is interrupted. The transcriptional component of this polarity depends on the action of Rho protein on the exposed mRNA, which results in premature transcription termination. Here we examine how a stable RNA operon (rrnG) transcript is protected from the Rho protein-mediated polarity response. We compared transcription from the ara and the rrnG promoters through a 16S DNA segment. In each case, the promoter-16S sequences were joined to a trp-lac fusion, and lacZ mRNA was examined in rho+ and rho-115 strains. We found significant Rho protein-dependent termination of transcripts from the ara promoter but little or no Rho protein effect on transcription from the rrnG promoter. We concluded that the transcript of the 16S ribosomal DNA segment does contain Rho protein-dependent transcription terminators, but there is an antitermination system in the rrnG control region that allows it to transcribe through those terminators.
Exogenous induction of the iron dicitrate transport system of Escherichia coli K-12
Authors: Zimmermann L , Hantke K , Braun V
Published: January 2020

Streptonigrin was used to select mutants impaired in the citrate-dependent iron transport system of Escherichia coli K-12. Mutants in fecA and fecB could not transport iron via citrate. fecA-lac and fecB-lac operon fusions were constructed with the aid of phage Mu dl(Ap lac). Strains deficient in ferric dicitrate transport which were mutated in fecB were as inducible as transport-active strains. They expressed the FecA outer membrane protein and beta-galactosidase of the fecB-lac operon fusions. In contrast, all fecA::lac mutants and fecA mutants induced with N-methyl-N'-nitro-N-nitrosoguanidine did not respond to ferric dicitrate supplied in the growth medium. tonB fecB mutants which were lacking all tonB-related functions were not inducible. We conclude that binding of iron in the presence of citrate to the outer membrane receptor protein is required for induction of the transport system. In addition, the tonB gene has to be active. However, iron and citrate must not be transported into the cytoplasm for the induction process. These data support our previous conclusion of an exogenous induction mechanism. Mutants in fur expressed the transport system nearly constitutively. In wild-type cells limiting the iron concentration in the medium enhanced the expression of the transport system. Thus, the citrate-dependent iron transport system shares regulatory devices with the other iron transport systems in E. coli and, in addition, requires ferric dicitrate for induction.
Properties of salicylate hydroxylase and hydroxyquinol 1,2-dioxygenase purified from Trichosporon cutaneum
Authors: Sze I S , Dagley S
Published: January 2020

Salicylate hydroxylase (salicylate 1-monooxygenase, EC 1.14.13.1) was purified from the soil yeast Trichosporon cutaneum. The enzyme contained flavin adenine dinucleotide and was monomeric, with a molecular weight of 45,300. In addition to salicylate, the four isomeric dihydroxybenzoates having one hydroxyl adjacent to carboxyl in the benzene nucleus were oxidatively decarboxylated without formation of hydrogen peroxide. One of these isomers, gentisate, was rapidly oxidized to hydroxyquinol by the enzyme but did not serve as an effective single carbon source for T. cutaneum; however, when growing with salicylate, cells also readily utilized gentisate for growth. Hydroxyquinol 1,2-dioxygenase (EC 1.13.11....) is a newly investigated enzyme which was purified from T. cutaneum grown with 4-hydroxybenzoate. The enzyme was red, contained ferric iron, and was specific for hydroxyquinol; catechol and pyrogallol were oxidized at less than 1% of the rate for hydroxyquinol, and no activity could be detected against seven other catechols. The enzyme was composed of two nonidentical subunits having molecular weights of 39,600 and 38,200 and was apparently dimeric.
Cloning of nitrate reductase genes from the cyanobacterium Anacystis nidulans
Authors: Kuhlemeier C J , Logtenberg T , Stoorvogel W , van Heugten H A , Borrias W E , van Arkel G A
Published: January 2020

Anacystis nidulans, a non-nitrogen-fixing cyanobacterium, can fulfill its nitrogen requirement by the assimilation of nitrate. The first step in the pathway, the reduction of nitrate to nitrite, is catalyzed by the molybdo-protein nitrate reductase. In this study, newly developed techniques for gene cloning in A. nidulans R2 were used for the isolation of two genes involved in nitrate reduction. One gene was cloned by complementation of the corresponding mutant; the other gene was picked up from a cosmid gene library by using a restriction fragment containing the transposon-inactivated gene as a probe. Both genes were unlinked single-copy chromosomal genes. Transformation studies provided evidence for the existence of a third locus involved in nitrate reduction.
Transition of deletion mutants of the composite resistance plasmid NR1 in Escherichia coli and Salmonella typhimurium
Authors: Huffman G A , Rownd R H
Published: January 2020

Derivatives of the composite R plasmid NR1 from which a portion of the resistance determinants (r-determinants) component had been deleted were found to undergo amplification of the remaining r-determinants region in Escherichia coli and Salmonella typhimurium. The wild-type NR1 plasmid does not amplify in these genera, although all of these plasmids undergo amplification in Proteus mirabilis. The deletion mutants retained the mercuric ion resistance operon (mer) but conferred a much lower level of sulfonamide resistance than NR1. The remaining r-determinants region, which is bounded by direct repeats of the insertion element IS1, formed multiple tandem duplications in E. coli, S. typhimurium, and P. mirabilis after subculturing the host cells in medium containing high concentrations of sulfonamide. Gene amplification was characterized by restriction endonuclease analysis, analytical buoyant density centrifugation, DNA-DNA hybridization, and sedimentation in sucrose gradients. The tandem repeats remained attached to the resistance transfer factor component of the plasmid in at least part of the plasmid population; autonomous tandem repeats of r-determinants were probably also present. Amplification did not occur in host recA mutants. Amplified strains subcultured in drug-free medium lost the amplified r-determinants. By using a strain temperature sensitive for the recA gene, it was possible to obtain gene amplification at the permissive temperature. Loss of r-determinants took place at the permissive temperature, but not at the nonpermissive temperature. The termini of the deletions of several independent mutants which conferred low sulfonamide resistance were found to be located within the adjacent streptomycin-spectinomycin resistance gene.
Conduction of nonconjugative plasmids by F' lac is not necessarily associated with transposition of the gamma delta sequence
Authors: Goto N , Shoji A , Horiuchi S , Nakaya R
Published: January 2020

A nonconjugative kanamycin-resistant (Kmr) recombinant plasmid, pNR5311, transferred at a low frequency from an Hfr or F' lac Escherichia coli donor to an F- lac- recipient. Among the transconjugants, two types of Kmr plasmids were found: one was indistinguishable from pNR5311 (type A), and the other was a recombinant between pNR5311 and the gamma delta sequence (type B). When the F' lac strain was used as a donor, 5% of lactose-fermenting (Lac+) and 75% of lactose-nonfermenting (Lac-) transconjugants had type A plasmids. A kinetic study revealed that type A plasmids were transferred more readily in short mating periods than were type B plasmids. Involvement of Tn903, which is present in pNR5311, in transfer of type A plasmids was unlikely since there was no discernible change in the F' lac molecule coexisting with the type A plasmid in the transconjugant cells. The non-gamma delta-associated conduction of pNR5311 by F' lac did not require the recA+ function of the donor. Conduction of pBR322 by F' lac was also carried out, and two types of plasmids with and without gamma delta were found, as with pNR5311. These findings suggest that the transfer of nonconjugative plasmids is conducted by a novel pathway which is not associated with translocation of transposable elements into either plasmid.
Cytochrome o as a terminal oxidase and receptor for aerotaxis in Salmonella typhimurium
Authors: Laszlo D J , Fandrich B L , Sivaram A , Chance B , Taylor B L
Published: January 2020

Cytochrome o was the only oxidase of the electron transport system that was present in exponentially growing Salmonella typhimurium ST1. Identification of cytochrome o was made by the (CO-reduced)-minus-(reduced) difference spectra and by the photochemical action spectrum of the relief, by light, of CO-inhibited respiration. Cytochrome o also functioned as the receptor for chemotaxis to oxygen (aerotaxis). The concentration of oxygen that elicits the maximum response for aerotaxis (0.7 microM) was similar to the Km for respiration (0.74 microM), and both aerotaxis and respiration were blocked 5 mM KCN.
Flocculation of Saccharomyces cerevisiae tup1 mutants
Authors: Lipke P N , Hull-Pillsbury C
Published: January 2020

Strains of Saccharomyces cerevisiae carrying a mutation in the TUP1 locus exhibited calcium-dependent flocculation. The flocculation had none of the characteristics of sexual agglutination. The flocculation differed from that exhibited by a FLO1 strain in the effect of pH on cation dependence and sensitivity to chemical inactivation.
Multiple copies of hemolysin genes and associated sequences in the chromosomes of uropathogenic Escherichia coli strains
Authors: Knapp S , Hacker J , Then I , Müller D , Goebel W
Published: January 2020

The O6 serogroup Escherichia coli strain 536 carries two hemolysin (hly) determinants integrated into the chromosome. The two hly determinants are not completely identical, either functionally or structurally, as demonstrated by spontaneous deletion mutants carrying only one of them and by cloning each of the two determinants separately into cosmid vectors. Each hly determinant is independently deleted at a frequency of 10(-4), leading to variants which exhibit similar levels of internal hemolysin but different amounts of secreted hemolysin. The two hly determinants were also identified in the O4 E. coli strain 519. The three E. coli strains 251, 764, and 768, which belong to the serogroup O18, and the O4 strain 367 harbor a single chromosomal hly determinant, as demonstrated by hybridization with hly-gene-specific probes. However, a hybridization probe derived from a sequence adjacent to the hlyC-proximal end of the plasmid pHly 152-encoded hly determinant hybridizes with several additional chromosomal bands in hemolytic O18 and O6 E. coli strains and even in E. coli K-12. The size of the probe causing the multiple hybridization suggests a 1,500- to 1,800-base pair sequence directly flanking hlyC. Spontaneous hemolysin-negative mutants were isolated from strains 764 and 768, which had lost the entire hly determinant but retained all copies of the hlyC-associated sequence.2+.
Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein
Authors: Vasantha N , Thompson L D , Rhodes C , Banner C , Nagle J , Filpula D
Published: January 2020

The genes for alkaline protease (apr[BamP]) and neutral protease (npr[BamP]) from Bacillus amyloliquefaciens have been isolated and expressed in Bacillus subtilis. The DNA sequences of apr[BamP] and npr[BamP] revealed, in each case, the presence of a large open reading frame. The inferred amino acid sequence of either gene contained a signal sequence and an additional polypeptide sequence ('pro' sequence) preceding the mature protein. Based on DNA sequence, the start point of translation has been identified as amino acid residue - 107 for apr[BamP] and -221 for npr[BamP]. To demonstrate that the start point of translation of apr[BamP] in vivo is probably at codon -107, codon -103 (AAA) was changed to an ochre (TAA) by site-directed mutagenesis. Alkaline protease was produced from this ochre mutant derivative of apr[BamP] only when the host strain was Su+. The presence of a pro sequence may be common to all of the secreted proteases from bacilli.
Biosynthesis of iron-molybdenum cofactor in the absence of nitrogenase
Authors: Ugalde R A , Imperial J , Shah V K , Brill W J
Published: January 2020

Klebsiella pneumoniae accumulates molybdenum during nitrogenase derepression. The molybdenum is primarily in nitrogenase component I in the form of iron-molybdenum cofactor (FeMo-co). Mutations in any of three genes (nifB, nifN, and nifE) involved in the biosynthesis of FeMo-co resulted in very low molybdenum accumulation and in a molybdenum-free nitrogenase component I. A mutant lacking both subunits of nitrogenase component I accumulated 60% of the amount of molybdenum present in the wild type. The molybdenum was in protein-bound form and behaved differently than that in the wild type with respect to electrophoretic mobility, size, and extractability by organic solvents. Two forms of molybdenum could be extracted from the protein fraction of the mutant; one of them was not detected in the wild type, and the other behaved like FeMo-co in nonaqueous gel filtration chromatography. Crude extracts of this mutant were able to complement in vitro K. pneumoniae or Azotobacter vinelandii mutants unable to produce FeMo-co. These data show that biosynthesis of FeMo-co does not require the presence of nitrogenase component I. In its absence, FeMo-co is accumulated on a different protein, presumably an intermediate in the normal FeMo-co biosynthetic pathway.
Periplasmic gel: new concept resulting from the reinvestigation of bacterial cell envelope ultrastructure by new methods
Authors: Hobot J A , Carlemalm E , Villiger W , Kellenberger E
Published: January 2020

Bacterial cell envelope ultrastructure was investigated both by the progressive lowering of temperature embedding technique and freeze-substitution, using conventional and scanning transmission electron microscopy. Comparison with standard embedding procedures revealed a new aspect of cell envelope structure in specimens at low temperatures. The envelope was delimited by an electron-dark layer, beneath which was a uniform matter-containing layer lying between the outer and inner membranes. There was no empty periplasmic space. Buoyant densities of isolated peptidoglycan obtained in Percoll (1.02 to 1.07 g ml-1) and CsCl2 (1.44 g ml-1) led to a calculated hydration of the peptidoglycan which was more than was previously assumed. Peptidoglycan therefore possibly fills the entire space between the inner and outer membranes in the form of a periplasmic gel. The new model of cell envelope organization is discussed with respect to the current knowledge on bacterial cell wall structure and function.
Cloning and characterization of recA genes froM Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r
Authors: Keener S L , McNamee K P , McEntee K
Published: January 2020

The recA genes of Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r have been isolated and introduced into Escherichia coli K-12. All the heterologous genes restore resistance to killing by UV irradiation and the mutagen 4-nitroquinoline-1-oxide in RecA- E. coli K-12 hosts. Recombination proficiency is also restored as measured by formation of Lac+ recombinants from duplicated mutant lacZ genes and the ability to propagate phage lambda derivatives requiring host recombination functions for growth (Fec-). The cloned heterologous genes increase the spontaneous induction of lambda prophage in lysogens of a recA strain. Addition of mitomycin C stimulates phage production in cells carrying the E. coli B/r and S. flexneri recA genes, but little or no stimulation is seen in cells carrying the E. carotovora and P. vulgaris recA genes. After treatment with nalidixic acid, the heterologous RecA proteins are synthesized at elevated levels, a result consistent with their regulation by the E. coli K-12 LexA repressor. Southern hybridization and preliminary restriction analysis indicate divergence among the coding sequences, but antibodies prepared against the E. coli K-12 RecA protein cross-react with the heterologous enzymes, indicating structural conservation among these proteins.
Saccharomyces cerevisiae aldolase mutants
Authors: Lobo Z
Published: January 2020

Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase. The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type. The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis. This suggests that the aldolase activity is absent in vivo.
Purification and biochemical characterization of pyruvate oxidase from Lactobacillus plantarum
Authors: Sedewitz B , Schleifer K H , Götz F
Published: January 2020

Pyruvate oxidase (EC 1.2.3.3) was isolated and characterized from Lactobacillus plantarum. The enzyme catalyzes the oxidative decarboxylation of pyruvate in the presence of phosphate and oxygen, yielding acetyl phosphate, carbon dioxide, and hydrogen peroxide. This pyruvate oxidase is a flavoprotein, with the relatively tightly bound cofactors flavin adenine dinucleotide, thiamine pyrophosphate, and a divalent metal ion, with Mn2+ being the most effective. The enzyme is only slightly inhibited by EDTA, implying that the enzyme-bound metal ion is poorly accessible to EDTA. Only under relatively drastic conditions, such as acid ammonium sulfate precipitation, could a colorless and entirely inactive apoenzyme be obtained. A partial reactivation of the enzyme was only possible by the combined addition of flavin adenine dinucleotide, thiamine pyrophosphate, and MnSO4. The enzyme has a molecular weight of ca. 260,000 and consists of four subunits with apparently identical molecular weights of 68,000. For catalytic activity the optimum pH is 5.7, and the optimum temperature is 30 degrees C. The Km values for pyruvate, phosphate, and arsenate are 0.4, 2.3, and 1.2 mM, respectively. The substrate specificity revealed that the enzyme reacts also with certain aldehydes and that phosphate can be replaced by arsenate. In addition to oxygen, several artificial compounds can function as electron acceptors.
Iron transport in Streptomyces pilosus mediated by ferrichrome siderophores, rhodotorulic acid, and enantio-rhodotorulic acid
Authors: Müller G , Matzanke B F , Raymond K N
Published: January 2020

Streptomyces pilosus is one of several microbes which produce ferrioxamine siderophores. In the accompanying paper (G. Müller and K. Raymond, J. Bacteriol. 160:304-312), the mechanism of iron uptake mediated by the endogenous ferrioxamines B, D1, D2, and E was examined. Here we report iron transport behavior in S. pilosus as mediated by the exogenous siderophores ferrichrome, ferrichrysin, rhodotorulic acid (RA), and synthetic enantio-RA. In each case iron acquisition depended on metabolic energy and had uptake rates comparable to that of [55Fe]ferrioxamine B. However, the synthetic ferric enantio-RA (which has the same preferred chirality at the metal center as ferrichrome) was twice as effective in supplying iron as was the natural ferric RA complex, suggesting that stereospecific recognition at the metal center is involved in the transport process. Iron uptake mediated by ferrichrome and ferric enantio-RA was strongly inhibited by kinetically inert chromic complexes of desferrioxamine B. These inhibition experiments indicate that iron from these exogenous siderophores is transported by the same uptake system as ferrioxamine B. Since the ligands have no structural similarity to ferrioxamine B except for the presence of three hydoxamate groups, we conclude that only the hydroxamate iron center and its direct surroundings are important for recognition and uptake. This hypothesis is supported by the fact that ferrichrome A and ferrirubin, which are both substituted at the hydroxamate carbonyl groups, were not (or were poorly) effective in supplying iron to S. pilosus.
Properties of ATP-dependent protein kinase from Streptococcus pyogenes that phosphorylates a seryl residue in HPr, a phosphocarrier protein of the phosphotransferase system
Authors: Reizer J , Novotny M J , Hengstenberg W , Saier M H
Published: January 2020

Transport of sugars across the cytoplasmic membranes of gram-positive bacteria appears to be regulated by the action of a metabolite-activated, ATP-dependent protein kinase that phosphorylates a seryl residue in the phosphocarrier protein of the phosphotransferase system, HPr. We have developed a quantitative assay for measuring the activity of this enzyme from Streptococcus pyogenes. The product of the in vitro protein kinase-catalyzed reaction was shown to be phosphoseryl-HPr by several independent criteria (rates of hydrolysis in the presence of various agents, detection of serine-phosphate in acid hydrolysates, immunological assay, and electrophoretic migration rates). HPrs isolated from four different gram-positive bacteria (S. pyogenes, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis) were shown to be phosphorylated by the kinase from S. pyogenes. In contrast, Escherichia coli HPr was not a substrate of this enzyme. The soluble kinase released from the particulate fraction of the cells with high salt in the presence of a protease inhibitor was shown to have an approximate molecular weight of 60,000 as estimated by gel filtration. Its activity was dependent on divalent cations, with Mg2+ and Mn2+ being most active. EDTA, Pi, and high concentrations of salt were strongly inhibitory. The enzyme was optimally active at pH 7.0, exhibited high affinity for its substrates, and was dependent on the presence of one of several metabolites. Of these compounds, fructose 1-6-diphosphate was most active, with gluconate 6-phosphate, 2-phosphoglycerate, 2,3-diphosphoglycerate, phosphoenolpyruvate, and pyruvate exhibiting moderate to low stimulatory activities. Other compounds tested, including a variety of sugar phosphates, pyridine nucleotides, and other metabolites were without effect. The ATP-dependent phosphorylation of HPr on the seryl residue was strongly inhibited by phosphoenolpyruvate-dependent phosphorylation of the active histidyl residue of this protein. Treatment of the kinase with diethyl pyrocarbonate strongly inhibited the ATP-dependent phosphorylation activity, although the sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, and iodoacetate were without effect. These results serve to characterize the HPr (serine) kinase, which apparently regulates the rates of carbohydrate transport in streptococcal cells via the phosphotransferase system. A primary role of this kinase in the control of cellular inducer levels and carbohydrate metabolic rates is proposed.
Genetic system for analyzing Escherichia coli thymidylate synthase
Authors: Belfort M , Pedersen-Lane J
Published: January 2020

Random in vitro mutagenesis of the thyA gene is being used to delineate its regulatory elements as well as the functional domains of its product, thymidylate synthase (EC 2.1.1.45). Streamlined procedures have been developed for the isolation and characterization of the mutants. Positive selection for synthase-deficient thyA Escherichia coli permitted the isolation of 400 mutants, which are being categorized by phenotypic and genetic criteria. An in situ 5-fluorodeoxyuridylate binding assay was devised to rapidly probe the substrate binding domain, whereas facile mapping procedures, based on pBR322- or M13-borne thyA deletion derivatives, were developed to localize mutations. The sequence changes of one amber mutation and another mutation that abolishes catalysis while maintaining substrate binding activity are presented. The orientation of the thyA gene on the E. coli chromosome was established.
Enzymatic and nucleotide sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from thermophilic bacilli in comparison with that encoded by plasmid pUB110
Authors: Matsumura M , Katakura Y , Imanaka T , Aiba S
Published: January 2020

The product of a kanamycin resistance gene encoded by plasmid pTB913 isolated from a thermophilic bacillus was identified as a kanamycin nucleotidyltransferase which is similar to that encoded by plasmid pUB110 from a mesophile, Staphylococcus aureus. The enzyme encoded by pTB913 was more thermostable than that encoded by pUB110. In view of a close resemblance of restriction endonuclease cleavage maps around the BglII site in the structural genes of both enzymes, ca. 1,200 base pairs were sequenced, followed by amino-terminal amino acid sequencing of the enzyme. The two nucleotide sequences were found to be identical to each other except for only one base in the midst of the structural gene. Each structural gene, initiating from a GUG codon as methionine, was composed of 759 base pairs and 253 amino acid residues (molecular weight, ca. 29,000). The sole difference was transversion from a cytosine (pUB110) to an adenine (pTB913) at a position + 389, counting the first base of the initiation codon as + 1. That is, a threonine at position 130 for the pUB110-coded kanamycin nucleotidyltransferase was replaced by a lysine for the pTB913-coded enzyme. The difference in thermostability between the two enzymes caused by a single amino acid replacement is discussed in light of electrostatic effects.
Physiological role of pyruvate oxidase in the aerobic metabolism of Lactobacillus plantarum
Authors: Sedewitz B , Schleifer K H , Götz F
Published: January 2020

Under aerobic growth conditions Lactobacillus plantarum produced acetic acid in addition to lactic acid. It was found that lactic acid was predominantly produced at first, and then when the carbohydrate was nearly exhausted, lactic acid was metabolized further to acetic acid. The most likely enzyme involved in the aerobic metabolism of L. plantarum is pyruvate oxidase. Its activity is enhanced in the presence of oxygen and is reduced in the presence of glucose. The specific activity of pyruvate oxidase is highest at the beginning of the stationary-growth phase, where a strong increase in acetic acid production was also observed.
Betaine is the main compatible solute of halophilic eubacteria
Authors: Imhoff J F , Rodriguez-Valera F
Published: January 2020

A number of moderately halophilic bacteria of diverse taxonomic groups have been studied to determine the intracellular concentrations of organic compounds at various salt concentrations. Betaine was accumulated in all of these organisms in proportion to the salinity of the medium, suggesting that this compound plays a major role in osmoregulation.
Isolation of pigmented and nonpigmented mutants of Serratia marcescens with reduced cell surface hydrophobicity
Authors: Rosenberg M
Published: January 2020

Enrichment for nonhydrophobic mutants of Serratia marcescens yielded two types: (i) a nonpigmented mutant which exhibited partial hydrophobic characteristics compared with the wild type, as determined by adherence to hexadecane and polystyrene; and (ii) a pigmented, nonhydrophobic mutant whose colonies were translucent with respect to those of the wild type. The data suggest that the pronounced cell surface hydrophobicity of the wild type is mediated by a combination of several surface factors.
Mobilization of a Sym plasmid from a fast-growing cowpea Rhizobium strain
Authors: Morrison N A , Cen Y H , Chen H C , Plazinski J , Ridge R , Rolfe B G
Published: January 2020

A large Sym plasmid from a fast-growing cowpea Rhizobium species was made mobilizable by cointegration with plasmid pSUP1011, which carries the oriT region of RP4. This mobilizable Sym plasmid was transferred to a number of Rhizobium strains, in which nodulation and nitrogen fixation functions for symbiosis with plants of the cowpea group were expressed.
Inverted repeat sequences flank a Bacillus thuringiensis crystal protein gene
Authors: Kronstad J W , Whiteley H R
Published: January 2020

Two sets of inverted repeat DNA sequences, IR2150 and IR1750, were discovered flanking the crystal protein gene on the 75-kilobase plasmid of Bacillus thuringiensis subsp. kurstaki HD73. A restriction map of ca. 40 kilobases around the crystal protein gene was constructed, and the positions of the copies of IR2150 and IR1750 were determined. Three copies of IR2150 were found flanking the crystal protein gene in an inverted orientation, and one partial and three intact copies of IR1750 were found in both inverted and direct orientations around the gene. Hybridization experiments with fragments from within IR2150 and IR1750 demonstrated the presence of multiple copies of these sequences on the chromosome of B. thuringiensis subsp. kurstaki HD73 and also revealed a strong correlation between the presence of these sequences and the presence of the crystal protein gene on plasmids from 14 strains of B. thuringiensis.
Genetic analysis of mutants of Aspergillus nidulans blocked at an early stage of sporulation
Authors: Butnick N Z , Yager L N , Kurtz M B , Champe S P
Published: January 2020

Three mutants of Aspergillus nidulans, selected to have a block at an early stage of conidiation (asexual sporulation), exhibit similar pleiotropic phenotypes. Each of these mutants, termed preinduction mutants, also are blocked in sexual sporulation and secrete a set of phenolic metabolites at level much higher than wild type or mutants blocked at later stages of conidiation. Backcrosses of these mutants to wild type showed that the three phenotypes always cosegregated. Diploids containing the mutant alleles in all pairwise combinations were normal for all phenotypes, showing that the three mutations are nonallelic. This conclusion was confirmed by the finding that the mutations map at three unlinked or distantly linked loci. Ten revertants of the two least leaky preinduction mutants, selected for ability to conidiate, were found in each case to arise by a second-site suppressor mutation. All of the revertants still showed accumulation of some of the phenolic metabolites but differed from each other in certain components. Three of the revertants retained the block in sexual sporulation. In these cases the suppressor has thus uncoupled the block in asexual sporulation from the block in sexual sporulation. These results are understandable in terms of a model in which preinduction mutations and their suppressors affect steps in a single metabolic pathway whose intermediates include an effector specific for asexual sporulation and a second effector specific for sexual sporulation.
Purification and properties of glutamate synthase from Bacillus licheniformis
Authors: Schreier H J , Bernlohr R W
Published: January 2020

Glutamate synthase [L-glutamate:NADP+ oxidoreductase (transaminating); EC 1.4.1.13](GltS) was purified to homogeneity from Bacillus licheniformis A5. The native enzyme had a molecular weight of approximately 220,000 and was composed of two nonidentical subunits (molecular weights, approximately 158,000 and approximately 54,000). The enzyme was found to contain 8.1 +/- 1 iron atoms and 8.1 +/- 1 acid-labile sulfur atoms per 220,000-dalton dimer. Two flavin moieties were found per 220,000-dalton dimer, with a ratio of flavin adenine dinucleotide to flavin mononucleotide of 1.2. The UV-visible spectrum of the enzyme exhibited maxima at 263,380 and 450 nm. The GltS from B. licheniformis had a requirement for NADPH, alpha-ketoglutarate, and glutamine. Classical hyperbolic kinetics were seen for NADPH affinity, which resulted in an apparent Km value of 13 microM. Nonhyperbolic kinetics were obtained for alpha-ketoglutarate and glutamine affinities, and the reciprocal plots obtained for these substrates were biphasic. The apparent Km values obtained for glutamine were 8 and 100 microM, and the apparent Km values obtained for alpha-ketoglutarate were 6 and 50 microM. GltS activity was found to be relatively insensitive to inhibition by amino acids, keto acids, or various nucleotides. L-Methionine-DL-sulfoximine, L-methionine sulfone, and DL-methionine sulfoxide were found to be potent inhibitors of GltS activity, yielding I0.5 values of 150, 11, and 250 microM, respectively. GltSs were purified from cells grown in the presence of ammonia and nitrate as sole nitrogen sources and were compared. Both yielded identical final specific activities and identical physical (UV-visible spectra, flavin, and iron-sulfur composition) and kinetic characteristics.
Anthranilate hydroxylase from Aspergillus niger: new type of NADPH-linked nonheme iron monooxygenase
Authors: Subramanian V , Vaidyanathan C S
Published: January 2020

Anthranilate hydroxylase from Aspergillus niger catalyzes the oxidative deamination and dihydroxylation of anthranilic acid to 2,3-dihydroxybenzoic acid. This enzyme has been purified to homogeneity and has a molecular weight of 89,000. The enzyme is composed of two subunits of 42,000 with 2 gram-atoms of nonheme iron per mol. Fe2+-chelators like alpha,alpha'-dipyridyl and o-phenanthroline are potent inhibitors of the enzyme activity. Absorption and fluorescence spectra of the enzyme offer no evidence for the presence of other cofactors like flavin. Flavins and flavin-specific inhibitors like atebrin have no effect on the activity of the enzyme. The enzyme incorporates one atom of oxygen each from 18O2 and H218O into the product 2,3-dihydroxybenzoic acid. Based on these studies, it is concluded that anthranilate hydroxylase from A. niger is a new type of NADPH-linked nonheme iron monooxygenase.
Regulation of amino acid utilization in Neurospora crassa: effect of nmr-1 and ms-5 mutations
Authors: DeBusk R M , Ogilvie S
Published: January 2020

The effect of the nmr-1 and ms-5 mutations, which lead to insensitivity to glutamine-mediated nitrogen metabolite repression, was examined with respect to extracellular deaminase production by Neurospora crassa. Deaminase production normally requires nitrogen limitation, but these mutations eliminated this requirement and allowed production of deaminase activity under nitrogen metabolite repressing conditions. Demonstration of normal glutamine transport by both strains eliminated the possibility that these mutations exerted their effects through repressor exclusion. We have proposed a new working model for nitrogen regulation in Neurospora based on the findings that these mutations affected a nitrogen-regulated activity in addition to those activities originally reported and that the mutations are genetically very closely linked and likely allelic.
Anaerobic iron uptake by Escherichia coli
Authors: Lodge J S , Emery T
Published: January 2020

Assimilation and uptake of iron in anaerobic cultures of Escherichia coli were supported by iron supplied as ferrienterobactin, ferrichrome, and ferrous ascorbate; however, as in the aerobic cultures, ferrichrome A was a poor iron source. Albomycin inhibited both aerobically and anaerobically grown cells. The siderophore outer membrane receptor proteins FepA and FhuA were produced under anaerobic iron-deficient conditions. Anaerobic transport of ferrienterobactin and ferrichrome was inhibited by KCN and dinitrophenol. The Km for ferrienterobactin uptake in anaerobically grown cells was 0.8 microM, and the Vmax was 38 pmol/min per mg, compared with 0.1 microM and 80 pmol/min per mg, respectively, in aerobically grown cells.
Simultaneous release of penicilloic acid and phenylacetyl glycine by penicillin-binding proteins 5 and 6 of Escherichia coli
Authors: Amanuma H , Strominger J L
Published: January 2020

Penicillin-binding proteins (PBPs) 5 and 6 of Escherichia coli released the bound penicilloyl moiety at an intermediate rate relative to, e.g., Staphylococcus aureus PBPs 4 (rapid) and 1 or 2 (slow). Each of these E. coli PBPs released the bound penicilloyl moiety as both penicilloic acid (hydrolysis) and phenylacetyl glycine (scission of the C-5--C-6 bond followed by hydrolysis).