bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization and complementation of pMB1 copy number mutant: effect of RNA I gene dosage on plasmid copy number and incompatibility
Authors: Moser D R , Campbell J L
Published: January 2020

A 16-base-pair insertion has been identified as the mutation responsible for the high-copy-number phenotype of the plasmid copy number mutant pFH118. The mutation is located near the plasmid origin of replication in a region of the genome that encodes two overlapping RNA transcripts. One of these transcripts, RNA I, acts as a negative regulator of plasmid replication. The second transcript is the precursor to the primer for the initiation of DNA synthesis. We demonstrate through complementation that the pFH118 DNA overproduction phenotype is a consequence of the reduced effectiveness of the mutant RNA I at inhibiting plasmid replication and not a consequence of an altered target site on the primer precursor. In addition, a series of plasmids containing multiple RNA I-coding genes was constructed for investigating the effects of RNA I gene dosage on plasmid copy number and incompatibility. The results of this study strongly support the inhibitor dilution model of plasmid copy control with RNA I as the plasmid-specified inhibitor responsible for both copy number control and incompatibility.
Regulation of glycolysis and sugar phosphotransferase activities in Streptococcus lactis: growth in the presence of 2-deoxy-D-glucose
Authors: Thompson J , Chassy B M
Published: January 2020

Streptococcus lactis K1 has the capacity to grow on many sugars, including sucrose and lactose, in the presence of high levels (greater than 500 mM) of 2-deoxy-D-glucose. Initially, growth of the organism was transiently halted by the addition of comparatively low concentrations (less than 0.5 mM) of the glucose analog to the culture. Inhibition was coincident with (i) rapid accumulation of 2-deoxy-D-glucose 6-phosphate (ca. 120 mM) and preferential utilization of phosphoenolpyruvate via the mannose:phosphotransferase system, (ii) depletion of phosphorylated glycolytic intermediates, and (iii) a 60% reduction in intracellular ATP concentration. During the 5- to 10-min period of bacteriostasis the intracellular concentration of 2-deoxy-D-glucose 6-phosphate rapidly declined, and the concentrations of glycolytic intermediates were restored to near-normal levels. When growth resumed, the cell doubling time (Td) and the steady-state levels of 2-deoxy-D-glucose 6-phosphate maintained by the cells were dependent upon the medium concentration of 2-deoxy-D-glucose. Resistance of S. lactis K1 to the potentially toxic analog was a consequence of negative regulation of the mannose:phosphotransferase system by two independent mechanisms. The first, short-term response occurred immediately after the initial "overshoot" accumulation of 2-deoxy-D-glucose 6-phosphate, and this mechanism reduced the activity (fine control) of the mannose:phosphotransferase system. The second, long-term mechanism resulted in repression of synthesis (coarse control) of enzyme IImannose. The two regulatory mechanisms reduced the rate of 2-deoxy-D-glucose translocation via the mannose:phosphotransferase system and minimized the activity of the phosphoenolpyruvate-dependent futile cycle of the glucose analog (J. Thompson and B. M. Chassy, J. Bacteriol. 151:1454-1465, 1982). Phosphoenolpyruvate was thus conserved for transport of the growth sugar and for generation of ATP required for biosynthetic and work functions of the growing cell.
Possible Correlation Between Transformability and Deficiency in Error-Prone Repair
Authors: Sicard Nicole
Published: January 2020

We have investigated the relationship between UV-induced mutability (as a measure of an error-prone repair process) and the genetic transformability of transformable and nontransformable bacterial strains. The data suggest a correlation between chromosomal transformability and a deficiency in an error-prone repair system in bacteria.
Biosynthesis of D-alanyl-lipoteichoic acid: role of diglyceride kinase in the synthesis of phosphatidylglycerol for chain elongation
Authors: Taron D J , Childs W C , Neuhaus F C
Published: January 2020

Lipophilic and hydrophilic D-alanyl-lipoteichoic acids are elongated in Lactobacillus casei by the transfer of sn-glycerol 1-phosphate units from phosphatidylglycerol to the poly(glycerophosphate) moiety of the polymer. These sn-glycerol 1-phosphate units are added to the end of the poly(glycerophosphate) which is distal to the glycolipid anchor; 1,2-diglyceride results from this addition. The presence of a diglyceride kinase was suggested by the ATP-dependent phosphorylation of 1,2-diglyceride to phosphatidic acid. Inorganic phosphate was used to initiate the synthesis of lipophilic lipoteichoic acid (LTA) and the elongation of both lipophilic and hydrophilic LTA. Three observations suggest that phosphate and other anions play a role in the in vitro synthesis of LTA and its precursors. First, the conversion of 1,2-diglyceride to phosphatidic acid by diglyceride kinase was stimulated. Second, the synthesis of phosphatidylglycerol was increased. Third, the elongation of lipophilic and hydrophilic LTA was enhanced. These observations indicated that one effect of phosphate might be to enhance the utilization of 1,2-diglyceride for the synthesis of phosphatidic acid. This phospholipid is a precursor of phosphatidylglycerol, the donor of sn-glycerol 1-phosphate for elongation of LTA.
Transposable element that causes mutations in a plant pathogenic Pseudomonas sp
Authors: Comai L , Kosuge T
Published: January 2020

A 1.3-kilobase-pair DNA element, IS51, causes a loss of virulence in the plant pathogen Pseudomonas syringae pv. savastanoi. This sequence, IS51, was first discovered in a plasmid-borne iaaM locus, which together with iaaH directs the synthesis of a virulence factor, indoleacetic acid. The spontaneous insertion of IS51 in iaaM resulted in the loss of indoleacetic acid production, attenuation of virulence, and the loss of both enzyme activities coded by iaaM and iaaH. Using a cloned IS51 element as a probe, we found that numerous homologous sequences are present in different strains of this bacterium, on both chromosomal and plasmid DNAs. By artificially positioning IS51 between a tetracycline resistance gene and a promoter region, we showed that IS51 terminated transcription in Escherichia coli.
Localization and quantitation of proteins characteristic of the complexed membrane of Bacillus subtilis
Authors: Horiuchi S , Marty-Mazars D , Tai P C , Davis B D
Published: January 2020

We prepared antibodies to four proteins (molecular weights, 68,000, 64,000, 45,000, and 31,000) that are characteristic of the complexed (ribosome-bearing) fraction of the membrane of Bacillus subtilis and found that these proteins are immunologically distinct. Quantitation by immunoprecipitation confirmed that the ribosome-free membrane fraction contains much lower concentrations of these four proteins than the complexed-membrane fraction. The 64-kilodalton protein appeared to be attached more loosely than the other proteins, since it was more readily extracted from the membrane. In addition, this protein was also present in the cytosol in an even greater amount than in the membrane. The 68-, 64-, and 31-kilodalton proteins are present in cells in stoichiometrically equivalent amounts.
Proteins of ribosome-bearing and free-membrane domains in Bacillus subtilis
Authors: Marty-Mazars D , Horiuchi S , Tai P C , Davis B D
Published: January 2020

In lysates of Bacillus subtilis a free-membrane fraction without ribosomes can be separated from the denser membrane-ribosome complexes. As determined by one-dimensional sodium dodecyl sulfate gel electrophoresis, these two fractions differ markedly in protein composition; at least six major bands (molecular weights, 130,000, 92,000, 68,000, 64,000, 45,000, and 31,000) are essentially unique to the complexed-membrane fraction (CM proteins), and two are unique to the free-membrane fraction. After growth was slowed, the proportion of the free-membrane fraction increased, but the composition of this fraction was the same, whereas after puromycin treatment, which abruptly increased the proportion of the free-membrane fraction, this fraction contained CM proteins. Thus, it appears that the two fractions recovered from growing cells represent topographically and functionally distinct domains. In addition, the effect of growth rate suggests that formation of the complexed domain is regulated at least roughly in parallel with the formation of ribosomes. The separation of these membrane fractions should facilitate the study of protein secretion, membrane topography, and morphogenesis in bacteria.
Bacteriophage in the Ixodes dammini spirochete, etiological agent of Lyme disease
Authors: Hayes S F , Burgdorfer W , Barbour A G
Published: January 2020

A bacteriophage with a B-3 morphology was detected by electron microscopy in a spirochete isolated from the tick Ixodes dammini. It has a 40- to 50-nm elongated head and a tail 50 to 70 nm in length. It appears devoid of collars or kite-tail structure. The spirochete has been identified as the causative agent of Lyme disease.
Plasmid transformation in Agmenellum quadruplicatum PR-6: construction of biphasic plasmids and characterization of their transformation properties
Authors: Buzby J S , Porter R D , Stevens S E
Published: January 2020

Biphasic, chimeric plasmids for the transformation of Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain 7002) were constructed by splicing the 3.0-megadalton cryptic plasmid from strain PR-6 into plasmids pBR322 and pBR325 from Escherichia coli. Transformants of either E. coli or strain PR-6 by these plasmids could be detected on the basis of the drug resistance marker(s) carried by the chimeric plasmids. Plasmid DNA isolated from a PR-6 transformant transformed PR-6 much more efficiently than plasmid DNA prepared from E. coli. Plasmids from which the AvaI recognition site was deleted (AvaI is an isoschizomer of the AquI restriction endonuclease of strain PR-6) also transformed strain PR-6 much more efficiently than did plasmids containing the AvaI recognition site. These and other results suggest that AquI strongly effects plasmid transformation when the donor plasmid contains an unmodified AquI recognition site. Multimeric forms of the chimeric plasmids are also much more efficient at transforming strain PR-6 than are the analogous monomeric forms.
Evolutionary analysis of the 7.1-kb beta-lactamase-specifying R-plasmid of Neisseria gonorrhoeae by restriction endonucleases
Authors: Mayer L W , Robbins K E
Published: January 2020

A physical map of restriction enzyme sites was made for the large beta-lactamase-specifying gonococcal R-plasmid (pMR0360) isolated in 1976. Single sites in the plasmid were mapped for 11 endonucleases, and multiple sites of cleavage were mapped for 17 other enzymes. Conclusions about the structure and origin of this plasmid are discussed.
Identification of the heat-inducible protein C15.4 as the groES gene product in Escherichia coli
Authors: Tilly K , VanBogelen R A , Georgopoulos C , Neidhardt F C
Published: January 2020

The product of the Escherichia coli morphogenetic gene groES (mopB) was identified as the heat-inducible protein C15.4 by two-dimensional gel analysis of the products of wild-type and mutant alleles carried on the bacterial chromosome, on a hybrid plasmid, and on a transducing phage.
Catabolism of phenylpropionic acid and its 3-hydroxy derivative by Escherichia coli
Authors: Burlingame R , Chapman P J
Published: January 2020

A number of laboratory strains and clinical isolates of Escherichia coli utilized several aromatic acids as sole sources of carbon for growth. E. coli K-12 used separate reactions to convert 3-phenylpropionic and 3-(3-hydroxyphenyl)propionic acids into 3-(2,3-dihydroxyphenyl)propionic acid which, after meta-fission of the benzene nucleus, gave succinate, pyruvate, and acetaldehyde as products. Enzyme assays and respirometry showed that all enzymes of this branched pathway were inducible and that syntheses of enzymes required to convert the two initial growth substrates into 3-(2,3-dihydroxyphenyl)propionate are under separate control. E. coli K-12 also grew with 3-hydroxycinnamic acid as sole source of carbon; the ability of cells to oxidize cinnamic and 3-phenylpropionic acids, and hydroxylated derivatives, was investigated. The lactone of 4-hydroxy-2-ketovaleric acid was isolated from enzymatic reaction mixtures and its properties, including optical activity, were recorded.
Regulation of hydrogenase activity in vegetative cells of Anabaena variabilis
Authors: Spiller H , Bookjans G , Shanmugam K T
Published: January 2020

Heterocyst-free (NH4+-grown) cultures of the cyanobacterium Anabaena variabilis produce a hydrogenase which is reversibly inhibited by light and O2. White or red light at an intensity of 5,000 lx inhibited greater than 95% of the activity. Oxygen at concentrations as low as 0.5% inhibited more than 85% of the hydrogenase in the vegetative cells of CO2-NH4+-grown cultures. The vegatative cell hydrogenase is also sensitive to strong oxidants like ferricyanide. In the presence of strong reductants like S2O4(2-), hydrogenase activity was not inhibited by light. However, hydrogenase activity in the heterocysts was insensitive to both light (greater than 5,000 lx) and O2 (10%). Heterocysts and light-insensitive hydrogenase activity appear simultaneously during differentiation of the vegetative cells into heterocysts (an NH4+-grown culture transferred to NH4+-free, N2-containing medium). This light-insensitive hydrogenase activity was detected several hours before the induction of nitrogenase activity. These results suggest a mode of regulation of hydrogenase in the vegetative cells of A. variabilis that is similar to "redox control" of hydrogenase and other "anaerobic" proteins in enteric bacteria like Escherichia coli.
Increased carbohydrate substitution of lipoteichoic acid during inhibition of protein synthesis
Authors: Kessler R E , Wicken A J , Shockman G D
Published: January 2020

Decreases in electrophoretic mobilities of intracellular lipoteichoic acid, intracellular deacylated lipoteichoic acid, and extracellular deacylated lipoteichoic acid were observed during inhibition of protein synthesis in Streptococcus faecium after exposure to chloramphenicol or valine deprivation. Increased carbohydrate content, and thus an increased mass-to-charge ratio, rather than changes in ester alanine content or novel fatty acid substitutions, appeared to account for the decreased electrophoretic mobilities. The increase in carbohydrate content, as judged from mobility measurements, was progressive over time and appeared to occur on biosynthetically new lipoteichoic acid as well as on lipoteichoic acid made before inhibition of protein synthesis.
Purine salvage pathways of Bacillus subtilis and effect of guanine on growth of GMP reductase mutants
Authors: Endo T , Uratani B , Freese E
Published: January 2020

We have isolated numerous mutants containing mutations in the salvage pathways of purine synthesis. The mutations cause deficiencies in adenine phosphoribosyltransferase (adeF), in hypoxanthine-guanine phosphoribosyltransferase (guaF), in adenine deaminase (adeC), in inosine-guanosine phosphorylase, (guaP), and in GMP reductase (guaC). The physiological properties of mutants containing one or more of these mutations and corresponding enzyme measurements have been used to derive a metabolic chart of the purine salvage pathway of Bacillus subtilis.
Alginic acid synthesis in Pseudomonas aeruginosa mutants defective in carbohydrate metabolism
Authors: Banerjee P C , Vanags R I , Chakrabarty A M , Maitra P K
Published: January 2020

Mutant cells of mucoid Pseudomonas aeruginosa isolated from cystic fibrosis patients were examined for their ability to synthesize alginic acid in resting cell suspensions. Unlike the wild-type strain which synthesizes alginic acid from glycerol, fructose, mannitol, glucose, gluconate, glutamate, or succinate, mutants lacking specific enzymes of carbohydrate metabolism are uniquely impaired. A phosphoglucose isomerase mutant did not synthesize the polysaccharide from mannitol, nor did a glucose 6-phosphate dehydrogenase mutant synthesize the polysaccharide from mannitol or glucose. Mutants lacking the Entner-Doudoroff pathway dehydrase or aldolase failed to produce alginate from mannitol, glucose, or gluconate, as a 3-phosphoglycerate kinase or glyceraldehyde 3-phosphate dehydrogenase mutant failed to produce from glutamate or succinate. These results demonstrate the primary role of the Entner-Doudoroff pathway enzymes in the synthesis of alginate from glucose, mannitol, or gluconate and the role of glyceraldehyde 3-phosphate dehydrogenase reaction for the synthesis from gluconeogenic precursors such as glutamate. The virtual absence of any activity of phosphomannose isomerase in cell extracts of several independent mucoid bacteria and the impairment of alginate synthesis from mannitol in mutants lacking phosphoglucose isomerase or glucose 6-phosphate dehydrogenase rule out free mannose 6-phosphate as an intermediate in alginate biosynthesis.
Isolation and characterization of the recA gene of Rhizobium meliloti
Authors: Better M , Helinski D R
Published: January 2020

Interspecific complementation of an Escherichia coli recA mutant with plasmids containing a gene bank of Rhizobium meliloti DNA was used to identify a clone which contains the recA gene of R. meliloti. The R. meliloti recA protein can function in recombination and in response to DNA damage when expressed in an E. coli recA host, and hybridization studies have shown that DNA sequence homology exists between the recA gene of E. coli and that of R. meliloti. The isolated R. meliloti recA DNA was used to construct a recA R. meliloti, and this bacterium was not deficient in its ability to carry out symbiotic nitrogen fixation.
Sporulation of Streptomyces griseus in submerged culture
Authors: Kendrick K E , Ensign J C
Published: January 2020

A wild-type strain of Streptomyces griseus forms spores both on solid media (aerial spores) and in liquid culture (submerged spores). Both spore types are highly resistant to sonication, but only aerial spores are resistant to lysozyme digestion. Electron micrographs suggest that lysozyme sensitivity may result from the thinner walls of the submerged spores. Studies of the life cycle indicate that neither streptomycin excretion nor extracellular protease activity is required for sporulation: the analysis of mutants, however, suggests that antibiotic production may be correlated with the ability to sporulate. A method was devised to induce the rapid sporulation of S. griseus in a submerged culture. This method, which depends on nutrient deprivation, was used to determine that either ammonia or phosphate starvation can trigger sporulation and that the enzyme glutamine synthetase may be useful as a sporulation marker after phosphate deprivation.
Involvement of a plasmid in Escherichia coli envelope alterations
Authors: Rosas S B , Calzolari A , La Torre J L , Ghittoni N E , Vásquez C
Published: January 2020

A plasmid-containing wild-type Escherichia coli strain was treated with two plasmid-curing agents, sodium dodecyl sulfate and ethidium bromide. Plasmid elimination was accompanied by drastic changes in the morphology of the colonies. Analysis of the cured strain by scanning and transmission electron microscopy showed important alterations in size and morphology of the cells. Metabolic differences were also found between the wild-type and cured cells.
Role of L-lysine-alpha-ketoglutarate aminotransferase in catabolism of lysine as a nitrogen source for Rhodotorula glutinis
Authors: Kinzel J J , Winston M K , Bhattacharjee J K
Published: January 2020

Wild-type and saccharopine dehydrogenaseless mutant strains of Rhodotorula glutinis grew in minimal medium containing lysine as the sole nitrogen source and simultaneously accumulated, in the culture supernatant, large amounts of a product identified as alpha-aminoadipic-delta-semialdehyde. The saccharopine dehydrogenase and pipecolic acid oxidase levels remained unchanged in wild-type cells grown in the presence of ammonium or lysine as the nitrogen source. Lysine-alpha-ketoglutarate aminotransferase activity was demonstrated in ammonium-grown cells. This activity was depressed in cells grown in the presence of lysine as the sole source of nitrogen.
Regeneration of protoplasts of Clostridium pasteurianum ATCC 6013
Authors: Minton N P , Morris J G
Published: January 2020

A procedure has been developed for the regeneration of Clostridium pasteurianum protoplasts with frequencies of up to 10% reversion being obtained.
Heterogeneity and variation among Neisseria meningitidis lipopolysaccharides
Authors: Tsai C M , Boykins R , Frasch C E
Published: January 2020

Eight immunotype lipopolysaccharides (LPSs) of Neisseria meningitidis were prepared by the phenol-water procedure and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and sugar analyses. By SDS-PAGE and a highly sensitive silver strain. N. meningitidis LPSs from cells grown in tryptic soy broth were shown to contain one or two predominant components and a few minor, somewhat higher-molecular-weight components. The molecular sizes of the two predominant components were approximately the same as those of two E. coli rough-type LPSs, one with a complete core and the other with an incomplete core. The molecular weight of the major LPS component varied somewhat among different immunotypes but was estimated to be in the range of 4,200 to 5,000. By sugar analyses, the eight immunotype LPSs were different in their monosaccharide compositions. All contained glucose, galactose, heptose, glucosamine, and 2-keto-3-deoxyoctonate, but in different molar ratios. The growth of N. meningitidis in tryptic soy broth under different levels of aeration resulted in a change in the two major LPS components seen on the SDS-PAGE gel. High aeration increased the amount of the smaller component, whereas low aeration increased the amount of the larger component. Sugar analyses of LPSs from high and low aeration indicated that the larger LPS component contained more galactose residues per molecule. Use of different media for cell growth may also result in small, but noticeable, variations in the LPS components and in the galactose content of the LPS. The observed heterogeneity of N. meningitidis LPS may explain why many strains of N. meningitidis appear to possess more than one immunotype.
Multicopy Tn10 tet plasmids confer sensitivity to induction of tet gene expression
Authors: Moyed H S , Nguyen T T , Bertrand K P
Published: January 2020

We inserted the Tn10 tetracycline resistance determinant (tet) into the multicopy plasmid pACYC177, and we examined the phenotype of Escherichia coli K-12 strains harboring these plasmids. In agreement with others, we find that Tn10 tet exhibits a negative gene dosage effect. Strains carrying multicopy Tn10 tet plasmids are 4- to 12-fold less resistant to tetracycline than are strains with a single copy of Tn10 in the bacterial chromosome. In addition, we find that multicopy tet strains are 30- to 100-fold less resistant to the tetracycline derivative 5a,6-anhydrotetracycline than are single-copy tet strains. Multicopy tet strains are, in fact, 10- to 25-fold more sensitive to anhydrotetracycline than are strains that lack tet altogether. The hypersensitivity of multi-copy strains to anhydrotetracycline is correlated with the effectiveness of anhydrotetracycline as an inducer of tet gene expression, rather than its effectiveness as an inhibitor of protein synthesis. Anhydrotetracycline is 50- to 100-fold more effective than tetracycline as an inducer of tetracycline resistance and as an inducer of beta-galactosidase in strains that harbor tet-lac gene fusions. In contrast, anhydrotetracycline appears to be two- to fourfold less effective than tetracycline as an inhibitor of protein synthesis. Both anhydrotetracycline and tetracycline induce synthesis of tet polypeptides in minicells harboring multicopy tet plasmids. Differences between E. coli K-12 backgrounds influence the tetracycline and anhydrotetracycline sensitivity of multicopy strains; ZnCl2 enhances the tetracycline and anhydrotetracycline sensitivity of these strains two- to threefold. We propose that the overexpression of one or more Tn10 tet gene products inhibits the growth of multicopy tet strains and accounts for their relative sensitivity to inducers of tet gene expression.
Streptococcus plasmid pAM alpha 1 is a composite of two separable replicons, one of which is closely related to Bacillus plasmid pBC16
Authors: Perkins J B , Youngman P
Published: January 2020

A tetracycline resistance plasmid of Streptococcus faecalis, pAM alpha 1, is shown to contain two independent sets of replication functions, separated from each other on either side by short (300- to 400-base-pair) sequences of homology. The homologous sequences are oriented as direct repeats and therefore permit the dissociation of pAM alpha 1 into its component replicons, referred to here as pAM alpha 1 delta 1 and pAM alpha 1 delta 2, as the reciprocal products of a simple intramolecular recombination. pAM alpha 1 delta 1 is a 4.6-kilobase plasmid which carries the tet gene, and pAM alpha 1 delta 2 is a 5.1-kilobase plasmid which carries no known selectable marker. pAM alpha 1 delta 1 is shown to replicate efficiently in Bacillus subtilis and to confer tetracycline resistance on Bacillus hosts. We demonstrate by restriction mapping analysis that pAM alpha 1 delta 1 is virtually identical to a 4.6-kilobase tetracycline resistance plasmid of Bacillus cereus, pBC16, which is known to show extensive homology to plasmid isolates from Staphylococcus species (such as pUB110), as well as from other Bacillus species. The pAM alpha 1 delta 1-pBC16-pUB110 replicon thus exists naturally in at least three different gram-positive genera, indicating that these plasmids have a high degree of interspecific functional adaptability and supporting the view that plasmid DNA is commonly exchanged among many species of gram-positive bacteria in their natural environments.
In vivo 31P and 13C nuclear magnetic resonance studies of acetate metabolism in Chromatium vinosum
Authors: Nicolay K , van Gemerden H , Hellingwerf K J , Konings W N , Kaptein R
Published: January 2020

31P and 13C nuclear magnetic resonance (NMR) experiments were performed on suspensions of the phototrophic bacterium Chromatium vinosum incubated anaerobically in the dark. 31P NMR spectra revealed that during prolonged dark incubation high ATP levels are maintained. This phenomenon was independent of the presence of the energy reserves polyglucose and polyphosphate. 13C NMR experiments revealed that the amino acids glutamate, aspartate, and alanine are the major products of acetate incorporation in the dark. Apart from these amino acids, poly-beta-hydroxybutyrate was also formed. Acetate metabolism was markedly stimulated by the presence of polyglucose. The specific 13C activity of glutamate C-2 was approximately 50% that of glutamate C-4. The idea is discussed that this difference is the consequence of the maintenance of redox balance during entry of acetate into cell metabolism.
In vivo ligation of linear DNA molecules to circular forms in the yeast Saccharomyces cerevisiae
Authors: Suzuki K , Imai Y , Yamashita I , Fukui S
Published: January 2020

The yeast Saccharomyces cerevisiae was transformed with restriction endonuclease-digested (linear) DNAs containing the replication origin of the yeast 2 microns plasmid and selectable markers with efficiencies of 10(3) to 10(4), 10(3), and 10(2) to 10(3) transformants per microgram of DNA in the cases of transformations with linear DNAs containing the same cohesive ends, flush ends, and non-complementary cohesive ends, respectively. The results of a restriction analysis of the circular plasmids recovered from transformed cells suggested that the linear DNA molecules were ligated to produce circular forms in the recipient protoplasts.
DNases of Acholeplasma spp
Authors: Roganti F S , Rosenthal A L
Published: January 2020

One strain from each of seven species of Acholeplasma was examined for the presence of DNases. Six of the strains were found to be DNase positive when assayed with DNA-methyl-green-containing agar, indicating that this method can be used to differentiate the seventh strain, Acholeplasma axanthum, from the remaining Acholeplasma spp. Electrophoretic patterns obtained from sodium dodecyl sulfate-polyacrylamide gels containing DNA revealed DNases in the cell extracts of all seven strains and in the supernatant growth medium of five of the strains. The electrophoretic patterns were highly characteristic for each strain and can be used for the rapid identification of different strains of Acholeplasma.
Transport of aromatic amino acids by Brevibacterium linens
Authors: Boyaval P , Moreira E , Desmazeaud M J
Published: January 2020

Whole metabolizing Brevibacterium linens cells were used to study the transport of aromatic amino acids. Kinetic results followed the Michaelis-Menten equation with apparent Km values for phenylalanine, tyrosine, and tryptophan of 24, 3.5, and 1.8 microM. Transport of these amino acids was optimum at pH 7.5 and 25 degrees C for phenylalanine and pH 8.0 and 35 degrees C for tyrosine and tryptophan. Crossed inhibitions were all noncompetitive. The only marked stereospecificity was for the L form of phenylalanine. Transport was almost totally inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Iodoacetate and N-ethylmaleimide were much more inhibitory for tryptophan transport than for transport of the other two aromatic amino acids.
Sequence-specific DNA modification in Neisseria gonorrhoeae
Authors: Korch C , Hagblom P , Normark S
Published: January 2020

Neisseria gonorrhoeae 82409/55(pJD1) is postulated to possess six DNA sequence-specific cytosine methyltransferases and one DNA sequence-specific N6-adenine methyltransferase. From the DNA sequencing of the plasmid pJD1 (manuscript in preparation) by a modification of the Maxam and Gilbert chemical cleavage procedure, the cytosine methylation specificities were demonstrated. Five of these methylating enzymes and their respective specificities are M . NgoI (formula; see text) does not methylate the cytosine of its recognition sequence, in agreement with a detected adenine modification. A biological implication of these different DNA methylating activities is discussed.
Absence of oligomeric murein intermediates in Escherichia coli
Authors: Goodell E W , Markiewicz Z , Schwarz U
Published: January 2020

The intermediates in the biosynthetic pathway of murein were examined in two strains of Escherichia coli to determine whether they synthesized oligomeric precursors in vivo. No oligomeric precursors could be detected; the only intermediates found were the previously described UDP-N-acetylmuramyl peptides, and the two lipid-linked compounds, N-acetylglucosamyl-N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol and N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol. It was concluded that lipid-linked monomers are directly incorporated into the murein sacculus in vivo and do not pass through an oligomeric stage.
Uracil phosphoribosyltransferase from Acholeplasma laidlawii: partial purification and kinetic properties
Authors: McIvor R S , Wohlhueter R M , Plagemann P G
Published: January 2020

Uracil phosphoribosyltransferase was purified 34-fold from sonicated extracts of Acholeplasma laidlawii by ammonium sulfate precipitation, binding to DEAE-Sephadex, Sephadex G-200 chromatography, and hydroxylapatite chromatography. The molecular weight of the enzyme by gel filtration was approximately 80,000. The pH optimum for phosphoribosylation was around 7.5, and the optimum MgCl2 concentration was 5 mM. Initial velocity studies were conducted over a wide range of both uracil and 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP) concentrations, and various equations for biomolecular reaction mechanisms were fitted to the data by nonlinear regression. When the equation for an ordered sequential mechanism was fitted to the data, the Kia thus obtained was not statistically different from zero. This is interpreted as evidence for a nonsequential ("ping-pong") reaction. Graphic analysis of the data by the Hanes-Woolf linear transform supported this conclusion. The enzyme has high affinity for uracil (KmUra = 4.2 microM; KmP-Rib-PP = 66 microM), which provides supporting evidence that this activity is responsible for the incorporation of uracil and uridine into nucleotides.
Involvement of lactose enzyme II of the phosphotransferase system in rapid expulsion of free galactosides from Streptococcus pyogenes
Authors: Reizer J , Saier M H
Published: January 2020

Streptococcus pyogenes accumulated thiomethyl-beta-galactoside as the 6-phosphate ester due to the action of the phosphoenolpyruvate:lactose phosphotransferase system. Subsequent addition of glucose resulted in rapid efflux of the free galactoside after intracellular dephosphorylation (inducer expulsion). Efflux was shown to occur in the apparent absence of the galactose permease, but was inhibited by substrate analogs of the lactose enzyme II and could not be demonstrated in a mutant of S. lactis ML3 which lacked this enzyme. The results suggest that the enzymes II of the phosphotransferase system can catalyze the rapid efflux of free sugar under appropriate physiological conditions.
Outer membrane porins are important in maintenance of the surface structure of Escherichia coli cells
Authors: Nogami T , Mizushima S
Published: January 2020

Escherichia coli cells lacking the OmpF and OmpC proteins, porin proteins of the outer membrane, are often unstable and easily revert to strains which either have regained one or both of these proteins or contain a new outer membrane protein. The structural importance of porin proteins in the cell surface was studied in the present work. Tris-hydrochloride buffer at a concentration of 120 mM caused deformation of the cell surface of a strain lacking these porins; the undulated appearance of the negatively stained cell surface changed to a smooth and expanded form. The Tris-induced deformation was seldom observed with either the wild-type strain or a pseudorevertant that possessed the OmpF protein. The role of the OmpF protein in stabilizing the cell surface against Tris treatment could be slightly taken over by the LamB protein, which shares a number of unique properties with the former proteins. The deformation of the cell surface by Tris-hydrochloride buffer was accompanied by a loss of viability, the lethal damage being especially significant when the cells lacked porins. Upon induction with maltose, cells with the undulated appearance could absorb lambda phages, whereas the deformed cells could not. These results suggest that the instability of cells lacking porins is primarily due to a structural defect of the outer membrane.
Membrane-bound fractions of R6K plasmid DNA in Escherichia coli
Authors: Archibold E R , Wilson J D , Allison D P , Sheehy R J
Published: January 2020

The intracellular location of plasmid DNA has been of interest in an effort to understand the maintenance of these molecules. We have employed a simple procedure which enables us to isolate from exponentially grown cells on sucrose gradients membrane-complexed forms of R6K plasmid DNA. Electron micrographs identified the complexing of membrane fractions to circular forms of R6K DNA. Biochemical studies of the complexed R6K molecules showed the presence of membrane-specific proteins and suggested that complexing of R6K DNA was primarily with inner membrane fractions of Escherichia coli.
Membrane-associated phosphatidylinositol kinase from Saccharomyces cerevisiae
Authors: McKenzie M A , Carman G M
Published: January 2020

Membrane-associated phosphatidylinositol kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was partially purified 93-fold from Saccharomyces cerevisiae. Activity was dependent on magnesium ions (10 mM) and the optimum pH was 8.5. The apparent Km values for ATP and phosphatidylinositol were 0.21 mM and 71 microM, respectively. Activity was stimulated by sodium cholate and inhibited by sodium, potassium, lithium, and fluoride ions.
A pair of regulatory isozymes for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase is conserved within group I pseudomonads
Authors: Byng G S , Berry A , Jensen R A
Published: January 2020

Two closely related subgroups of group I pseudomonads, which differ from one another in the overall enzymatic makeup of aromatic amino acid biosynthesis, possess in common the recently characterized major (tyrosine-sensitive) and minor (tryptophan-sensitive) isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Pseudomonas aeruginosa (17). Since these characterizations were made for strains whose phylogenetic positions have been determined by oligonucleotide cataloging, an initial perception of the evolution of aromatic pathway construction and regulation is emerging.
Transport and processing of staphylococcal alpha-toxin
Authors: Tweten R K , Christianson K K , Iandolo J J
Published: January 2020

Two larger precursors to staphylococcal alpha-toxin were identified and partially characterized. Both precursor proteins were present on the cell membrane at very low levels and appeared to be rapidly processed to the mature form. Dinitrophenol inhibited processing such that the two precursors accumulated in the membranes, whereas little extracellular (mature) alpha-toxin is formed. The peptide maps of the 35S-labeled peptides from extracellular alpha-toxin and the two precursors were almost identical. The larger precursor protein contained four additional peptides and the smaller precursor protein contained three additional peptides not found in the extracellular toxin.
A gene affecting accumulation of the RNA moiety of the processing enzyme RNase P
Authors: Dallmann G , Quinn T , Apirion D
Published: January 2020

The level of 10Sb (M1) RNA, the RNA of RNase P, is very low in growing cultures of rnpB mutants. Northern transfer experiments suggested that these strains accumulate no more than 10% of the wild-type level of 10Sb RNA. However, there is no indication that there is a limiting amount of RNase P activity in these mutants in vivo. A plasmid that directs the synthesis of 10Sb RNA does not complement the rnpB mutants, even though there is only a single gene for 10Sb RNA in the Escherichia coli genome. The 10Sb RNA synthesized from this plasmid is equivalent to wild-type 10Sb RNA since it can replace it in the reconstitution of RNase P. The 10Sb RNA, which is a rather stable molecule, is unstable in the presence of the rnpB mutation. This could explain why rnpB mutants do not accumulate 10Sb RNA. An F' plasmid that contains DNA from the rnpB region of the chromosome complements an rnpB mutant in vivo and in vitro, and it also contains the 10Sb RNA gene. A number of possible explanations for these phenomena are discussed.
Murein structure and lack of DD- and LD-carboxypeptidase activities in Caulobacter crescentus
Authors: Markiewicz Z , Glauner B , Schwarz U
Published: January 2020

High-pressure liquid chromatography of a muramidase digest of murein sacculi from Caulobacter crescentus showed that the absence of D-alanine carboxypeptidase activity in the cells was reflected by a very high content of pentapeptide in the murein. Approximately half of the pentapeptide side chains were shown to contain glycine, which replaced D-alanine as the terminal amino acid.
Molecular cloning and expression of gene fragments from corynebacteriophage beta encoding enzymatically active peptides of diphtheria toxin
Authors: Tweten R K , Collier R J
Published: January 2020

Two restriction fragments from corynebacteriophage beta vir tox+ that encode peptides similar to diphtheria toxin fragment A and the chain termination fragment, CRM45, have been cloned into Escherichia coli in plasmid pBR322. Clones containing the recombinant plasmids produced gene products that were active in catalyzing the ADP ribosylation of elongation factor 2 and were reactive with diphtheria toxin antiserum. Toxin-related peptides were found primarily in the periplasmic compartment and were degraded to nonimmunoreactive forms within 1 to 2 h of synthesis. The expression of both gene fragments appears to have originated from the diphtheria toxin promoter.
Levels of sulfhydryls and disulfides in proteins from Neurospora crassa conidia and mycelia
Authors: Brody S , Mikolajczyk S D , Fahey R C
Published: January 2020

Proteins extracted with 6 M guanidine at 90 degrees C from conidia (asexual spores) of Neurospora crassa contained ca. 25% more total protein thiol and a fivefold-higher content of disulfide bonds than proteins extracted from mycelia, as determined by labeling with iodo[14C]acetic acid. The total thiol content was 88 mumol/g of protein in conidia and 70 mumol/g of protein in mycelia. The level of protein disulfide was 18.5 mumol/g of protein in conidia and 3.5 mumol/g of protein in mycelia, by the iodo[14C]acetic acid labeling method. Confirmatory results were obtained with 5'5-dithio-bis-2-nitrobenzoic acid titration of protein thiol groups in 1% sodium dodecyl sulfate as well as by amino acid analysis of cysteic acid derivatives. Buffer-extracted proteins from conidia, but not mycelia, were found to contain enriched levels of protein thiols and disulfides per gram of protein as compared with guanidine hydrochloride extracts. It was demonstrated that the high disulfide content of crude conidial extracts was not due to measurable levels of mixed disulfides formed between protein sulfhydryl groups and cysteine. During germination of the conidia, the high disulfide levels of the conidial proteins remained constant. These data suggest that, unlike the disulfides of glutathione, the bulk of conidial protein disulfides were not reduced, excreted, or extensively degraded during germination.
Suppressors of lysine codons may be misacylated lysine tRNAs
Authors: Murgola E J , Pagel F T
Published: January 2020

We describe a novel class of missense suppressors that read the codons for lysine at two positions (211 and 234) in the trpA polypeptide of Escherichia coli. The suppressor mutations are highly linked to lysT, a gene for lysine tRNA. The results suggest that the suppressors are misacylated lysine tRNAs that carry glycine or alanine. The mutant codons are apparently suppressed better at position 211 than at position 234, indicating the existence of codon context effects in missense suppression.
Cloning of the glucokinase gene in Escherichia coli B
Authors: Fukuda Y , Yamaguchi S , Shimosaka M , Murata K , Kimura A
Published: January 2020

A gene for glucokinase (Glk) in Escherichia coli B was cloned onto vector plasmid pBR322, and the hybrid plasmid obtained was designated pGK100. The gene for Glk was located in the central MluI fragment (0.82 megadalton) of the 6.0-megadalton chromosomal DNA inserted in the HindIII site of the vector. The introduction of pGK100 into E. coli 112L having a decreased level of Glk activity resulted in the about 15-fold increase in this enzyme activity. The poor growth rate of 112L cells on glucose or mannose was also improved by the introduction of pGK100. However, removal of some portion near the glk gene prevented the growth of 112L cells, although Glk activity was high enough to support growth. Therefore, some function of Glk may be regulated by a gene(s) near the glk gene.
High-frequency induction of nodulation and nitrogen fixation mutants of Rhizobium japonicum
Authors: Skogen-Hagenson M J , Atherly A G
Published: January 2020

More than 50 symbiotic mutants of Rhizobium japonicum were isolated by purported plasmid-curing techniques. Wild-type R. japonicum strains were grown in liquid culture at 28 or 36 degrees C in different concentrations of acridine orange, ethidium bromide, or sodium dodecyl sulfate for selection of mutants. The symbiotic traits of 133 isolates from nine treatment groups were determined. Forty-two isolates were Nod- Nif+, seven were Nod+ Nif-, and two were Nod- Nif-. The nifDH genes were deleted in three mutants and consequently showed no hybridization to a nifDH probe. None of these mutants showed any detectable loss of plasmid DNA.
Action of CAP on the malT promoter in vitro
Authors: Chapon C , Kolb A
Published: January 2020

DNase I footprinting experiments demonstrated that CAP, the cyclic AMP receptor protein of Escherichia coli, binds around position -70 at the promoter of malT, the positive regulator gene of the maltose regulon. The binding of CAP in the presence of cyclic AMP favored the subsequent specific binding of RNA polymerase. Initiation of malT transcription in vitro displayed an absolute requirement for CAP at all tested RNA polymerase concentrations. However this was not the case with a mutant promoter (malTp1), which leads to CAP-independent malT expression in vivo. In that case an effect of CAP was seen only at the lower concentrations of RNA polymerase. These results, which suggest that CAP stimulates malT expression by promoting the binding of polymerase to the promoter, are compared with those obtained in other systems.
Separation and characterization of six (1 leads to 3)-beta-glucanases from Saccharomyces cerevisiae
Authors: Hien N H , Fleet G H
Published: January 2020

Using a system of chromatography through columns of DEAE-Bio-Gel, HTP-Bio-Gel, and CM-Bio-Gel, we isolated and characterized six different (1 leads to 3)-beta-glucanases from cell wall autolysates and cell extracts of Saccharomyces cerevisiae haploid strain 2180B. These enzymes were designated glucanases I, II, IIIA, IIIB, IV, and V. The haploid mating type S. cerevisiae strain 2180A and the diploid strains S. cerevisiae 2180D and S. cerevisiae 595 contained the same complex of glucanases. Glucanases II and IIIA were exoenzymes, and glucanases I, IIIB, IV, and V were endoenzymes. The enzymes exhibited different molecular weights, kinetic properties, and activities on isolated yeast cell walls. The products of substrate (laminarin) hydrolysis were quantified by using high-pressure liquid chromatography and were significantly different for the four endoglucanases.
Ultrastructure and extreme heat resistance of spores from thermophilic Clostridium species
Authors: Hyun H H , Zeikus J G , Longin R , Millet J , Ryter A
Published: January 2020

The heat resistance and ultrastructural features of spore suspensions prepared from Clostridium thermocellum LQRI, Clostridium thermosulfurogenes 4B, and Clostridium thermohydrosulfuricum 39E were compared as a function of decimal reduction time. The decimal reduction times at 121 degrees C for strains LQRI, 4B, and 39E were 0.5, 2.5, and 11 min. The higher degree of spore heat resistance was associated with a spore architecture displaying a thicker cortex layer. Heat resistance of these spores was proportional to the ratio of spore cortex volume to cytoplasmic volume. These ratios for spores of strains LQRI, 4B, and 39E were 1.4, 1.6, and 6.6, respectively. The extreme heat resistance and autoclavable nature of C. thermohydrosulfuricum spores under routine sterilization procedures is suggested as a common cause of laboratory contamination with pure cultures of thermophilic, saccharide-fermenting anaerobes.
In vitro coupled transcription-translation of linear DNA fragments in a lysate derived from a recB rna pnp strain of Escherichia coli
Authors: Bassett C L , Rawson J R
Published: January 2020

A recB21 derivative (CLB7) of an Escherichia coli rna-19 pnp-7 strain (PR7) was constructed for use in examining the in vitro coupled transcription-translation of linear DNA. The expression of linearized DNAs in CLB7 (recB21 rna-19 pnp-7) lysates was enhanced significantly when compared with expression of the same DNAs in lysates prepared from the PR7 or the original recB21 (CF300) strains. In addition, the endogenous incorporation of [35S]methionine into protein was considerably reduced in CLB7 lysates relative to lysates derived from the original recB21 strain.