bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired incision of ultraviolet-irradiated deoxyribonucleic acid in uvrC mutants of Escherichia coli
Authors: Seeberg E , Rupp W D , Strike P
Published: January 2020

The production of single-strand breaks in the deoxyribonucleic acid of irradiated uvrC mutants of Escherichia coli K-12 was studied both in vivo and in vitro. In vivo, uvrC mutants displayed a slow accumulation of breaks after irradiation, and in this respect appeared different from uvrA mutants, in which very few breaks could be detected. The breakage observed in uvrC mutants differed from that observed in wild-type strains in both the slow rate of break accumulation and the very limited dose response. The behavior of the uvrC lig-7(Ts) double mutant was shown not to be consistent with the suggestion of ligase reversal as the explanation for the lower rate and limited dose response of break formation observed in ultraviolet-irradiated uvrC mutants in vivo. Rather, there appeared to be a real defect in incision. In toluene-treated cells, we studied the effect of the ligase inhibitor nicotinamide mononucleotide on strand incision. Whereas uvrC mutants displayed more strand breakage in the presence of this inhibitor, the same amount of breakage was seen in uvrA mutants, and as such the breakage could be judged as not due to the main excision repair pathway. Experiments using a cell-free system comprising the partially purified uvr+ gene products demonstrated clearly that there is a requirement for the uvrC+ gene product for strand incision. We suggest that in vivo in the absence of the uvrC+ gene product, a partial analog of this protein may allow some abnormal incision.
Developmental regulation of laccase levels in Aspergillus nidulans
Authors: Law D J , Timberlake W E
Published: January 2020

Asexual spores (conidia) of Aspergillus nidulans contain a dark green pigment which is not present in other cell types. Synthesis of this pigment is catalyzed, in part, by a developmentally controlled p-diphenol oxidase, or laccase, encoded at the gamma A genetic locus (A. J. Clutterbuck, J. Gen. Microbiol. 70:423-435, 1972). We have investigated the mechanisms regulating expression of the gamma A gene of A. nidulans. Vegetative hyphae grown in submerged culture lacked detectable laccase enzyme activity and neither contained nor synthesized immunoprecipitable laccase protein. When such cultures were induced to conidiate by harvesting the cells onto filter papers and aerating them, laccase levels began to increase after 10 to 16 h, reached a peak at 20 to 36 h, and then declined slowly. Immunological assays showed that increases in laccase enzyme activity were (i) proceded by a transient rise in the relative rate of laccase protein synthesis and (ii) closely paralleled by increases in the amount of laccase protein. Addition of cycloheximide to cultures at any time after inducing conidiation inhibited further accumulation of laccase enzyme activity. These data are most consistent with increases in laccase levels being due to regulated, de novo synthesis of laccase protein. Addition of inhibitors of ribonucleic acid synthesis to conidiating cultures also inhibited further accumulation of laccase, suggesting that laccase expression is regulated by alterations in the transcriptional activity of the gamma A locus.
Cholera toxin is synthesized in precursor form on free polysomes in Vibrio cholerae 569B
Authors: Nichols J C , Tai P C , Murphy J R
Published: January 2020

Membrane-bound and free polysomes have been isolated from Vibrio cholerae 569B. Nacent polypeptide chains were completed in a cell-free translation mixture containing Escherichia coli S-300 extracts and [3H]leucine or [35S]methionine. Cholera toxin-related polypeptides synthesized in vitro were immunologically detected after treatment with either anti-subunit A or anti-subunit B serum. Immunoreactive translation products were removed from reaction mixtures with formalinized Cowan's strain of Staphylococcus aureus, electrophoresed on sodium dodecyl sulfate-polyacrylamide gels, and visualized by fluorography. Anti-subunit A serum precipitated two major polypeptide species (molecular weights 52,000 and 45,000) from translation mixtures programed with free polysomes, whereas anti-subunit B serum precipitated only the 45,000-molecular-weight polypeptide. No cholera toxin-related polypeptides were detectable in translation mixtures programed with membrane-bound polysomes. Purified subunit A and cholera toxin competed for anti-subunit A binding sites and blocked the immunoprecipitation of the 35S-labeled 52,000- and 45,000-dalton polypeptides from in vitro translation mixtures. The data presented suggest that cholera toxin is synthesized in the cytoplasm in a precursor form on free polysomes and is secreted post-translationally.
Physiological characterization of a Neurospora crassa mutant with impaired regulation of nitrate reductase
Authors: Premakumar R , Sorger G J , Gooden D
Published: January 2020

This report describes the isolation and characterization of a Neurospora crassa mutant with an impaired regulation of nitrate reductase. Glutamine, which prevents the induction of nitrate reductase in N. crassa, did so relatively ineffectively in this mutant. The mutation did not affect the regulation of all enzymes regulated by "nitrogen metabolite regulation"; it did affect the regulation of nitrate reductase, nitrite reductase, histidase, and acetamidase, as well as that of thiourea sensitivity. The mutation was not allelic with nit-2, the gene controlling a general positive effector of nitrogen metabolite-regulated enzyme formation.
Relationship of plasmids responsible for hairy root and crown gall tumorigenicity
Authors: White F F , Nester E W
Published: January 2020

Three strains of Agrobacterium rhizogenes were examined for plasmids. Strains 15834 and A4 contained essentially identical large plasmids, pAr15834c and pArA4c, respectively (approximately 260 x 10(6) daltons). These plasmids can dissociate to two smaller plasmid species. Strain TR105 contained only a single plasmid, which was homologous with the dissociation product of pAr15834c, pAr15834b. Plasmid pAr15834c shared little overall sequence homology with other Ti plasmids. One region of conserved homology between pAr15834c and a region of the octopine type plasmid pTiB6806 which contains oncogenicity functions was detected. Lower levels of homology were detected with sequences which are distributed throughout 65% of pTiB6806. Homology with the so-called common deoxyribonucleic acid in the integrated plasmid deoxyribonucleic acid region was detected only after lowering the stringency of hybridization (Tm, -41 degrees C). Furthermore, the A. rhizogenes plasmid is compatible with other Ti plasmids. Therefore, the results suggest that the virulence plasmids of A. rhizogenes are functionally similar to other Ti plasmids, yet have diverged sufficiently from an ancestral Ti plasmid that they now represent a distinct plasmid type based on homology, compatibility, and virulence.
Chemiluminescence by Listeria monocytogenes
Authors: Roth J A , Kaeberle M L
Published: January 2020

Listeria monocytogenes cells suspended in brain heart infusion broth or in carbonated saline solution emitted light (chemiluminescence) that could be detected by a liquid scintillation spectrometer. This chemiluminescence was inhibited by superoxide dismutase and catalase but not by the hydroxyl radical scavengers mannitol and benzoate; it was also dependent upon and proportional to the carbonate ion concentration in the medium. Organisms suspended in carbonated saline solution which had ceased to chemiluminesce immediately began to chemiluminesce again when acetaldehyde was added but not when glucose, sucrose, or xanthine was added. Acetaldehyde-induced chemiluminescence was inhibited by suproxide dismutase and catalase but not by allopurinol. Our data indicate that the superoxide anion, hydrogen peroxide, and the carbonate ion are involved in chemiluminescence by L. monocytogenes. Chemiluminescence is apparently initiated by the extracellular generation of superoxide anon by this organism. The mechanism for the production of the superoxide anion is not known, but xanthine oxidase does not appear to be involved.
Transposed LEU2 gene of Saccharomyces cerevisiae is regulated normally
Authors: Kohlhaw G B , Hsu Y P , Lemmon R D , Petes T D
Published: January 2020

The repression of beta-isopropylmalate dehydrogenase, the LEU2 gene product, by leucine and leucine plus threonine was unaffected by the transposition of LEU2 from its original locus on chromosome III to a new locus within the ribosomal deoxyribonucleic acid gene cluster on chromosome XII. Since the expression of the LEU2 gene is probably controlled at a pretranslational level, we conclude that the recombinant plasmid used for transformation carries regulatory information in addition to LEU2 structural information.
Identification of an actin-like protein and of its messenger ribonucleic acid in Saccharomyces cerevisiae
Authors: Water R D , Pringle J R , Kleinsmith L J
Published: January 2020

We have identified a yeast protein that resembles actins from other eucaryotes in its tight binding to pancreatic deoxyribonuclease I, its copolymerizaton with purified muscle actin, its one-dimensional peptide map, and its apparent polymerization into 7-nm filaments. The yeast actin-like protein yielded a single spot on two-dimensional polyacrylamide gel electrophoresis, suggesting that a single protein species was present. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the actin-like protein had an apparent molecular weight of 45,000 compared with 42,000 for muscle actin. In an attempt to identify the messenger ribonucleic acid coding for the actin-like protein, yeast polyadenylic acid-rich ribonucleic acid was translated in wheat germ and reticulocyte cell-free protein-synthesizing systems. The actin-like protein was identified among the translation products of the reticulocyte system by its tight binding to deoxyribonuclease I, its comigration with the in vivo-synthesized actin-like protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an the similarity of its peptide map to that of the in vivo-synthesized protein. A yeast protein synthesized in the wheat-germ system was also found to bind to deoxyribonuclease I and to copolymerize with muscle actin. However, its apparent molecular weight was about 35,000, suggesting that it was a product either of incomplete translation or of proteolytic cleavage of the actin-like protein.
Effects of penicillin on macromolecular synthesis and surface growth of a tolerant streptococcus as studied by computer reconstruction methods
Authors: Higgins M L , McDowell T D , Sleytr U B , Mychajlonka M , Shockman G D
Published: January 2020

Strains of Streptococcus mutans are very susceptible to growth inhibition by benzylpenicillin, but are tolerant to lysis when exposed to even high concentrations of this drug. These properties enabled this study of S. mutans GS-5 surface growth and peptidoglycan, ribonucleic acid, protein, and deoxyribonucleic acid syntheses in the absence of osmotic stabilization. Inhibition of syntheses of peptidoglycan, ribonucleic acid, and protein was dose dependent. Synthesis of peptidoglycan was most susceptible. Substantial but less severe inhibitions of ribonucleic acid and protein syntheses rapidly followed decreased peptidoglycan synthesis, whereas inhibition of deoxyribonucleic acid synthesis was delayed and minimal. Computer-assisted reconstructions of surface growth zones and poles observed in electron micrographs of replicas were performed and indicated that at low concentrations of benzylpenicillin (0.03 micrograms/ml), growth sites reached abnormally large sizes and surface/volume ratios. The observed shifts in surface/volume ratio were attributed to an inhibition of the normal constrictive division mechanism. The poles of these cells also increased in size over those of the controls, but the relatively smaller change in surface/volume ratio confirmed the visual impression that the shape of the poles was much less altered than the shape of the growth sites. As the concentration of benzylpenicillin used was raised from 0.03 to 2 micrograms/ml, the ability of growth sites and poles to enlarge was restricted in a manner that most closely agreed with the extent of inhibition of peptidoglycan (rather than deoxyribonucleic acid, ribonucleic acid, or protein) synthesis. This correlation suggested that increases in cell size may be regulated by the supply of peptidoglycan precursors.
Overproduction of outer membrane protein suppresses envA-induced hyperpermeability
Authors: Grundström T , Normark S , Magnusson K E
Published: January 2020

A quantitative study on outer membrane components was performed in a number of envelope mutants of Escherichia coli K-12 exhibition different permeability properties for antimicrobial agents. The envA1 allele causing an increased influx for both hydrophobic and hydrophilic drugs was found to be associated with a deficiency in the amount of lipopolysaccharides. The sefA1 envA1 double mutant was found to have a higher outer membrane buoyant density, apparently due to an increase in protein content. This double mutant was still low in lipopolysaccharide content.
Characterization of promoter-cloning plasmids: analysis of operon structure in the rif region of Escherichia coli and isolation of an enhanced internal promoter mutant
Authors: An G , Friesen J D
Published: January 2020

Using the promotor-cloning vehicle described by An and Friesen (J. Bacteriol. 140:400-410, 1979), Escherichia coli chromosomal deoxyribonucleic acid fragments derived from the lambda drifd18 transducing phage were cloned in one of several unique restriction endonuclease sites adjacent to tetracycline(tet) genes that lack their own promotor. One of these plasmids has been used to isolate nine variants having mutations that lie in a putative internal promoter which is located between rplL and rpoB. Deoxyribonucleic acid sequence analysis revealed that, in all nine mutants, a single base change, C to T, in the ribonucleic acid polymerase recognition site led to a large increase in promoter activity. Analysis of a variety of plasmids in which tet is fused to various promoters yielded the following results: (i) rplK and rplA, genes for ribosomal protein L11 and L1, respectively, were cotranscribed from a common promoter located upstream from rplK; (ii) there was a strong promoter in the region between the rplKA operon and rplJ, the gene for ribosomal protein L10; (iii) an attenuator region was located between rplL, the gene for ribosomal protein L12, and rpoB, the gene for ribonucleic acid polymerase subunit beta; (iv) transcription terminated immediately after rpoC, the gene for ribonucleic acid polymerase subunit beta'; (v) a gene coding for unknown protein U, which is located between tufB and the rplKA operon, had its own promoter; (vi) the tufB gene was separated from all of the genes described above and had its own promoter.
Inhibition of sterol transmethylation by S-adenosylhomocysteine analogs
Authors: McCammon M T , Parks L W
Published: January 2020

Structural analogs of S-adenosylhomocysteine were tested in vitro for inhibition of the yeast S-adenosylmethionine:delta 24-sterol-C-methyltransferase enzyme. A wide inhibitory range by these compounds was observed, suggesting which structural features of the parent compound are important for binding to the enzyme. No analog tested had inhibitory activity specific only for this enzyme. The most active compound was sinefungin, a metabolite of Streptomyces griseolus, which was also able to inhibit growth of yeast cultures. Sterol extracts of cells grown in the presence of sinefungin revealed a dramatic increase in the levels of zymosterol, the sterol substrate in the transmethylation under study, and a concomitant decrease in the levels of ergosterol. Evidence is presented that sinefungin is transported inside the cell by the same permease as S-adenosylmethionine. We conclude that sinefungin is blocking the in vivo methylation of sterols in yeast. The implications of this finding are discussed.
Two interacting mutations causing temperature-sensitive phosphatidylglycerol synthesis in Escherichia coli membranes
Authors: Nishijima M , Bulawa C E , Raetz C R
Published: January 2020

A conditionally lethal mutant of Escherichia coli lacking phosphatidylglycerol in vivo at 42 degrees C has been previously isolated by two-stage mutagenesis (M. Nishijima and C. R. H. Raetz, J. Biol. Chem. 254:7837-7844, 1979). In the first step (designated pgsA444) the phosphatidylglycerophosphate synthetase is partially inactivated, but the resulting strain continues to make about two-thirds of the normal level of phosphatidylglycerol and is not temperature sensitive. The second lesion, termed pgsB1, causes temperature-sensitive growth and phosphatidylglycerol synthesis in strains harboring pgsA444. The pgsA locus appears to be the structural gene for the synthetase and maps near min 42. In the present study we mapped the pgsB1 mutation and characterized its interaction with pgsA444 by genetic and biochemical methods. Unexpectedly, pgsB1 was not a second lesion in the pgsA structural gene, but rather mapped at a distinct site near minute 4. P1 vir-mediated contransduction suggested the gene order pantonA-dapD-pgsB-dnaE (clockwise). Independent evidence for the genetic mapping was provided by the identification of two hybrid ColE1 plasmids (pLC26-43 and pLC34-20. L. Clarke and J. Carbon, Cell 9:91-99, 1976) which both carry pgsB+ and dnaE+. Introduction of either the pgsA+ or the pgsB+ gene (via episomes, hybrid plasmids or P1 vir transduction) suppressed the temperature sensitivity of the double mutant (pgsA444 pgsB1) and restored normal levels of phosphatidylglycerol at 42 degrees C. In addition, strains with the pgsA+ pgsB1 genotype produced a novel lipid (X) at all temperatures, whereas the double mutant (pgsA444 pgsB1) contained two unusual lipids (X and Y) after 3 h at 42 degrees C. Both X and Y are precursors of lipopolysaccharide, and introduction of pgsB+ into the double mutant caused the disappearance of X and Y. Although the biochemical basis of the pgsB1 lesion is unknown, its existence suggests a previously unrecognized link between lipopolysaccharide and phosphatidylglycerol syntheses in E. coli.
Role of membranes of mycelial Mucor rouxii in synthesis and secretion of cell wall matrix polymers
Authors: Dow J M , Carreon R R , Villa V D
Published: January 2020

Membrane fractions of the mycelial form of Mucor rouxii contained endogenous polyuronides and glycoproteins with sugar compositions similar to the cell wall polyuronide (mucoran) and extracellular (wall and filtrate) glycoproteins, respectively. The polyuronide pool was rapidly labeled with D-[U-14C]glucose and on pulse-chase experiments appeared to turn over, suggesting that these polymers were precursors of the wall mucoran. In contrast, the glycoproteins appeared to accumulate. Although the membrane-associated glycoproteins had molecular weights similar to those of the extracellular glycoproteins, the bulk of the membrane-associated polyuronides were of a molecular weight lower than that of the wall polyuronide.
L-cysteine oxidase activity in the membrane of Neisseria meningitidis
Authors: Yu E K , DeVoe I W
Published: January 2020

Among the L-amino acids, only L-cysteine was oxidized by isolated washed membranes of group B Neisseria meningitidis SD1C. The cysteine oxidase in the membrane obeyed Michaelis-Menten kinetics and was heat labile. The pH optimum for the maximum velocity of the reaction was 9.8. Specific activity of the enzyme increased as cell growth progressed through the exponential phase toward the stationary phase of growth. The enzyme activity was markedly sensitive to inhibition by metal chelators, but was resistant to inhibitors of terminal oxidases with the exception of cyanide. All known cytochromes in the membrane, except b563, were reduced with L-cysteine. The additive nature of L-cysteine oxidase and succinate oxidase activities suggests that an unidentified oxidase is involved in the oxidation of cysteine.
Glyoxylate bypass enzymes in Yersinia species and multiple forms of isocitrate lyase in Yersinia pestis
Authors: Hillier S , Charnetzky W T
Published: January 2020

Isocitrate lyase and malate synthase, the two unique enzymes of the glyoxylate cycle, were detected in crude extracts of Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica. Y. pestis, unlike Escherichia coli and the other yersiniae tested, yielded two forms of isocitrate lyase during growth on acetate. These forms differed in electrophoretic mobility and temperature optima. One form (A) was present during growth on acetate, but was absent during growth on alternate carbon sources such as glucose. The second form (B) was not constitutive, but was found during growth on acetate, glucose, xylose, or other complex carbon sources. Itaconate, a succinate analog which inhibited both forms of isocitrate lyase in crude extracts, did not affect the growth of Y. pestis under conditions where little isocitrate lyase activity was detected. This inhibitor, however, retarded the growth of Y. pestis under conditions where acetate was provided as the primary carbon and energy source as well as under all conditions in which either form of isocitrate lyase was evident. This suggests that the B form may play an important role in the growth of this bacterium under conditions where a requirement for the classical anaplerotic sequence involving this enzyme is not apparent.
Isolation and electron microscopic observations of intracytoplasmic inclusions containing Chlamydia psittaci
Authors: Matsumoto A
Published: January 2020

Intracytoplasmic inclusions containing Chlamydia psittaci were isolated by a newly established method. Infected L-cells at 20 h after infection were suspended in 0.25 M sucrose-tris(hydroxymethyl)aminomethane buffer containing ethylene-diaminetetraacetic acid, homogenized in a Dounce tissue grinder, and filtered through a 2,000-mesh screen. Isolated inclusions were stabilized in 5% bovine serum albumin in 10 mM tris(hydroxymethyl)aminomethane buffer. Electron microscopic observations revealed the presence of surface projections on the vegetative, reticulate bodies and a direct connection between the reticulate bodies and the inclusion membrane by means of projections.
Localization and partial characterization of soybean lectin-binding polysaccharide of Rhizobium japonicum
Authors: Tsien H C , Schmidt E L
Published: January 2020

Immunoelectron microscopy was combined with partial characterization of isolated exopolysaccharide to study binding of soybean lectin by Rhizobium japonicum strain USDA 138. Lectin-binding activity resided in two forms of exopolysaccharide produced during growth: an apparently very high-molecular-weight capsular form and a lower-molecular-weight diffusible form. At low-speed centrifugation, the capsular form cosedimented with cells to form a viscous, white, cell-gel complex which was not diffusible in 1% agar, and the diffusible form remained in the cell-free supernatant. Electron microscopic observation of the cell-gel complex after labeling with soybean lectin-ferritin conjugate revealed that capsular polysaccharides, frequently attached to one end of the cells, were receptors for lectin. The outer membrane of the cell bound no lectin. Various preparations of exopolysaccharide isolated from the culture supernatant were tested for lectin binding, interaction with homologous somatic antigen, and the presence of 2-keto-3-deoxyoctonate and were chromatographed in Sepharose 4B and 6B gel beds. Lectin binding was restricted to a polysaccharide component designated as lectin-binding polysaccharide. This polysaccharide, as present in the cell-free culture supernatant, was a diffusible acidic polysaccharide devoid of 2-keto-3-deoxyoctonate, with a molecular weight of 2 X 10(6) to 5 X 10(6). It was concluded that the soybean lectin-binding component of R. japonicum is an extracellular polysaccharide and not a lipopolysaccharide and that the diffusible lectin-binding polysaccharide probably differs from the very high-molecular-weight lectin-binding polysaccharide of the loose capsule (slime) only in the degree of polymerization.
Occurrence of pili on and adhesive properties of Haemophilus parainfluenzae
Authors: Kahn M E , Gromkova R
Published: January 2020

Of 20 clinical isolates of Haemophilus parainfluenzae, 13 produced a mannose-resistant hemagglutinin that agglutinated erythrocytes from chickens, horses, rabbits, and sheep. Examination with the electron microscope showed that only strain HR-885 was pilate. Grown in static liquid culture, the 12 hemagglutinating, nonpilate isolates formed small, tightly packed clumps, whereas strain HR-885 formed large, loosely packed clumps. However, seven isolates did not produce a hemagglutinin, did not clump, and were nonpilate.
Synthesis and metabolism of uracil-containing deoxyribonucleic acid in Escherichia coli
Authors: Warner H R , Duncan B K , Garrett C , Neuhard J
Published: January 2020

Significant amounts of uracil were found in the deoxyribonucleic acids (DNAs) of Escherichia coli mutants deficient in both uracil-DNA glycosylase (ung) and deoxyuridine 5'-triphosphate nucleotidohydrolase (dut) activities, whereas little uracil was found in the DNAs of wild-type cells and cells deficient in only one of these two activities. The amounts of uracil found in the DNAs of dut ung mutants were directly related to the growth temperature of the cultures, apparently because the deoxyuridine 5'-triphosphate nucleotidohydrolase synthesized by dut mutants was temperature sensitive. The dut mutant used failed to grow exponentially, became filamentous at temperatures above 25 degrees C, and exhibited a hyperrec phenotype; however, the ung mutation suppressed all of these effects. Although the dut ung mutants grew exponentially at all temperatures, their growth rates were always slower than the growth rate of the wild type. Since pool size measurements indicated that both deoxyuridine triphosphate and deoxythymidine triphosphate pools were markedly elevated in dut mutants, the reduced growth rate of dut ung cells apparently was due to the actual presence of uracil in the DNA, rather than to a deficiency of deoxyuridine triphosphate and deoxyribosylthymine triphosphate for DNA synthesis. The presence of uracil in E. coli donor DNA also markedly reduced the recombination frequency when the recipient cells were ung+, indicating that DNA repair commenced before the entering DNA could be replicated.
TRNA2Gln Su+2 mutants that increase amber suppression
Authors: Bradley D , Park J V , Soll L
Published: January 2020

We selected mutants of lambda pSu+2 which had an increased ability to suppress on Escherichia coli trp B9601 amber mutation on translationally stringent rpsL594 streptomycin-resistant ribosomes. tRNA2Gin Su+2 molecules produced from eight independent mutants were purified, and their ribonucleic acid sequences were determined. Two types of mutations were mapped to the tRNA2Gin Su+2(glnV) gene by this method. Both altered the pseudouridine at position 37 of the tRNA anticodon loop. Seven of the isolates were transitions (pseudouridine to cytosine), and one was a transversion (pseudouridine to adenine). These mutations resulted in Su+ transfer ribonucleic acid molecules that exhibited higher transmission coefficients than their parent Su+2 transfer ribonucleic acids. As judged by their suppressor spectra on T4 amber mutants, which were almost identical to that of Su+2, the two mutant Su+ transfer ribonucleic acids inserted glutamine at amber sites.
Regulation of derepressed synthesis of arylsulfatase by tyramine oxidase in Salmonella typhimurium
Authors: Murooka Y , Harada T
Published: January 2020

The participation of tyramine oxidase in the regulation of arylsulfatase synthesis in Salmonella typhimurium was studied. Arylsulfatase synthesis was repressed by inorganic sulfate, cysteine, methionine, or taurine. This repression was relieved by tyramine, octopamine, or dopamine, which induced tyramine oxidase synthesis, although the level of arylsulfatase activity was very low. The induction of tyramine oxidase and derepression of arylsulfatase by tyramine were strongly inhibited by glucose and ammonium chloride, and the repression of both enzymes was relieved by use of xylose as a carbon source after consumption of glucose or by use of tyramine as the sole source of nitrogen, irrespective of the carbon source used. The initial rates of tyramine uptake by cells grown with glucose and xylose were similar. Results with tyramine oxidase-constitutive mutants showed that constitutive expression of the tyramine oxidase gene resulted in derepression of arylsulfatase synthesis in the absence of tyramine. Thus, catabolite and ammonium repressions of arylsulfatase synthesis and the induction of the enzyme by tyramine seem to reflect the levels of tyramine oxidase synthesis. These results in S. typhimurium support our previous finding that the specific regulation system of arylsulfatase synthesis by tyramine oxidase is conserved in enteric bacteria.
Transformation of a Bacillus subtilis L-form with bacteriophage deoxyribonucleic acid
Authors: White T B , Doyle R J , Streips U N
Published: January 2020

A stable L-form, sal-1, of Bacillus subtilis was transformed with deoxyribonucleic acid (DNA) from bacteriophages phi 25 and phi 29 to determine whether exogenous DNA can be introduced into this organism. The viral transformation (transfection) was successful with the use of polyethylene glycol. In the presence of the fusogen, bacteriophage phi 25 DNA initiated a single cycle of infection. When compared with transfection of competent cells of Bacillus subtilis, the appearance of viral particles was delayed and their production occurred over a longer time period. L-form cells were best able to support intracellular replication of phi 25 viral particles when in balanced growth in a rich medium. The addition of polyethylene glycol also induced infection of sal-1 with whole bacteriophage phi 25 particles which could not otherwise infect the L-form and enhanced infection by intact phi 29 particles. Primary recombination was shown to be required for polyethylene glycol-mediated phi 25 transfection, but not phi 29 transfection or for whole bacteriophage phi 25 infection mediated by polyethylene glycol. Successful transfection of sal-1 suggests that the L-form may be amenable to genetic modification with exogenous DNA.
Corresponding changes in kynurenine hydroxylase activity, membrane fluidity, and sterol composition in Saccharomyces cerevisiae mitochondria
Authors: McLean-Bowen C A , Parks L W
Published: January 2020

The effect of sterol composition on the properties of the mitochondrial membrane of Saccharomyces cerevisiae was investigated. The physical state of mitochondrial membranes from wild-type strains and sterol mutants was compared, using a fluorescence polarization technique with 1,6-diphenyl-1,3-5-hexatriene. Changes in the rate of depolarization of the probe molecule as a function of temperature suggest the occurrence of a phase transition in the mitochondrial membranes isolated from the sterol mutants but not in the membranes isolated from the wild types. Arrhenius kinetics of the mitochondrial membrane-bound enzyme L-kynurenine-3-hydroxylase exhibited changes in activation energy at temperatures similar to those observed in the fluorescence polarization study. The ratio of mitochondrial sterol to phospholipid and the phospholipid fatty acid composition of the organisms were characterized.
Adenylate energy charge in Escherichia coli CR341T28 and properties of heat-sensitive adenylate kinase
Authors: Glembotski C C , Chapman A G , Atkinson D E
Published: January 2020

Escherichia coli strain CR341T28 will not grow at temperatures above 34 degrees C in liquid medium, and the adenylate kinase of this strain is heat sensitive. When a culture was shifted from a permissive (30 degrees C) to a nonpermissive (36 degrees C) temperature, the adenylate energy charge fell from 0.9 to 0.2, with a concurrent decrease in the number of viable cells and in the specific activity of adenylate kinase. When cultures of the temperature-sensitive strain were grown at temperatures above 30 degrees C, the adenylate energy charge, the specific activity of adenylate kinase, and the growth rate were lower than the corresponding parameters for the parental strain. By isotopic labeling of the adenine nucleotides in vivo, it was determined that increasing growth temperatures between 30 and 34 degrees C for the heat-sensitive strain resulted in a decrease in the adenosine triphosphate-to-adenosine monophosphate and adenosine triphosphate-to-adenosine diphosphate ratios. Between 26 and 30 degrees C the adenosine triphosphate-to-adenosine diphosphate ratio was essentially normal in the temperature-sensitive strain, but the adenosine triphosphate-to-adenosine diphosphate ratio was decreased. The adenylate ratios in the parental strain did not change between 30 and 34 degrees C. The adenylate kinase mass action ratio for each strain was essentially constant under all growth conditions. When assayed at 30 degrees C, the affinities of the enzyme from the mutant strain were somewhat lower than those of the parent adenylate kinase. The mutant enzyme also did not exhibit the substrate inhibition that was observed at high adenosine monophosphate concentrations with the parental enzyme. An increase in the assay temperature from 30 degrees to 40 degrees C had little or no effect on the Km values determined for the parental adenylate kinase, but caused the Km values determined for the mutant adenylate kinase to increase by a factor of two or more.
Versatile cloning vector for Pseudomonas aeruginosa
Authors: Wood D O , Hollinger M F , Tindol M B
Published: January 2020

A pBR322:RSF1010 composite plasmid, constructed in vitro, was used as a cloning vector in Pseudomonas aeruginosa. This nonamplifiable plasmid, pMW79, has a molecular weight of 8.4 X 10(6) and exists as a multicopy plasmid in both P. aeruginosa and Escherichia coli. In P. aeruginosa strain PAO2003, pMW79 conferred resistance to carbenicillin and tetracycline. Characterization of pMW79 with restriction enzymes revealed that four enzymes (BamHI, SalI, HindIII, and HpaI) cleaved the plasmid at unique restriction sites. Cloning P. aeruginosa chromosomal deoxyribonucleic acid fragments into the BamHI or SalI site of pMW79 inactivated the tetracycline resistance gene. Thus, cells carrying recombinant plasmids could be identified by their carbenicillin resistance, tetracycline sensitivity phenotype. Deoxyribonucleic acid fragments of approximately 0.5 to 7.0 megadaltons were inserted into pMW79, and the recombinant plasmids were stably maintained in a recombination-deficient (recA) P. aeruginosa host.
Temperature- and cyclic nucleotide-induced phase transitions of Histoplasma capsulatum
Authors: Sacco M , Maresca B , Kumar B V , Kobayashi G S , Medoff G
Published: January 2020

The transition from yeast to mycelia of Histoplasma capsulatum could be accomplished by shifting the temperature of incubation from 37 to 25 degrees C. It was accompanied by many changes in cellular metabolism, including changes in respiration, intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels, and activities of two enzymes specific for the yeast phase, cystine reductase (EC 1.6.4.1) and cysteine oxidase (EC 1.13.11.20). Even at 37 degrees C, the yeast to mycelial transition could be induced by cAMP and agents which raise the intracellular levels of cAMP (theophylline, acetylsalicylic acid, prostaglandin E1, and nerve growth factor). During this morphogenesis the same pattern of changes occurred as in the temperature-induced transition. Therefore, these changes were not simply dependent on a shift in temperature, but rather were part of the process of the phase transition.
Tn2001, a transposon encoding chloramphenicol resistance in Pseudomonas aeruginosa
Authors: Iyobe S , Sagai H , Mitsuhashi S
Published: January 2020

We isolated a new transposon, Tn2001, from the group P-2 plasmid Rms159-1 in Pseudomonas aeruginosa. Tn2001-encoded chloramphenicol resistance did not result from the formation of chloramphenicol acetyltransferase. Tn2001 was transposable between temperate phages and conjugative and nonconjugative plasmids belonging to various incompatibility groups, including P-1, P-3, P-4, P-5, P-7, and P-8 in P. aeruginosa. Transposition occurred independently of the general recombination ability of the Pseudomonas host, and its frequency varied between 10(-1) and 10(-8), depending upon the donor and recipient replicons. Tn2001 transposition also occurred in a recombination-deficient strain of Escherichia coli. Agarose gel electrophoresis and electron microscopic observations revealed that Tn2001 could transpose to different sites in the RP4 replicon and that the transposed deoxyribonucleic acid fragment was 2.1 kilobases long.
Control of membrane lipids in Mycoplasma gallisepticum: effect on lipid order
Authors: Le Grimellec C , Cardinal J , Giocondi M C , Carrière S
Published: January 2020

Adaptation of Mycoplasma gallisepticum, a sterol-requiring Mycoplasma sp., to growth in a serum-free medium supplemented with cholesterol in decreasing concentrations and with various saturated or unsaturated fatty acids enabled us to control both the cholesterol levels and the membrane fatty acid composition. An estimate of the membrane physical state from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene indicated that the membrane lipids of native M. gallisepticum were highly ordered. Elongation of the saturated fatty acid chains from 14 to 18 carbon atoms caused only a small increase in the membrane lipid ordering, whereas the introduction of a cis double bond reduced it significantly. Lipid-phase transitions were observed in low-cholesterol-adapted organisms, whose membrane lipids were still highly ordered at the growth temperature.
General method, using Mu-Mud1 dilysogens, to determine the direction of transcription of and generate deletions in the glnA region of Escherichia coli
Authors: MacNeil D
Published: January 2020

A general, genetic technique for determining the direction of transcription for bacterial genes is presented. By comparing the phenotype of Mu-Mud1 dilysogens with the phenotype of deletion-containing derivatives, the direction of transcription for the gene containing Mud1 can be unambiguously determined. This method can generate a series of strains containing deletions with predetermined endpoints, and strains with duplications of the region containing the Mud1 insertion. In Escherichia coli, the glnA and glnG genes are transcribed in the same direction.
Induction of citric acid cycle enzymes during initiation of sporulation by guanine nucleotide deprivation
Authors: Uratani-Wong B , Lopez J M , Freese E
Published: January 2020

In Bacillus subtilis, conditions causing partial deprivation of guanine nucleotides initiated sporulation and caused the synthesis of citrate synthase, aconitase, and alpha-ketoglutarate dehydrogenase. Alpha-ketoglutarate dehydrogenase could also be induced by acetate, and the specific activity of this enzyme was elevated in mutants that had high intracellular acetyl coenzyme A concentrations because they lacked citrate synthase activity. After deprivation of guanine nucleotides, the intracellular concentration of acetyl coenzyme A also increased, which explained the induction of alpha-ketoglutarate dehydrogenase. Furthermore, the decreases in alpha-ketoglutarate and L-malate concentrations observed during this deprivation accounted for the observed increases in citrate synthase activity (which was repressed by alpha-ketoglutarate and malate) and aconitase activity (which was repressed by alpha-ketoglutarate).
Mitomycin C-induced synthesis of cloacin DF13 and lethality in cloacinogenic Escherichia coli cells
Authors: van Tiel-Menkveld G J , Veltkamp E , De Graaf F K
Published: January 2020

Treatment of cloacinogenic cultures with increasing concentrations of mitomycin C induced an increasing synthesis of cloacin DF13 accompanied by a decreasing number of colony-forming cells. Cells grown in the presence of glucose required a 10-fold-higher concentration of mitomycin C for optimal induction of cloacin production than did cells grown with lactate. Release of the cloacin was hampered in glucose-grown cells. Experiments with various CloDF13 insertion and deletion mutants revealed that the transcription of CloDF13 deoxyribonucleic acid sequences adjacent to the cloacin structural gene was essential for mitomycin C-induced lethality.
Isolation of Bacillus subtilis genes from a charon 4A library
Authors: Ferrari E , Henner D J , Hoch J A
Published: January 2020

A library of Bacillus subtilis chromosomal deoxyribonucleic acid (DNA) was constructed, using lambda charon 4A as a cloning vector. Partially cleaved Bacillus subtilis DNA was prepared by partial methylation with EcoRI methylase, followed by complete EcoRI endonuclease digestion. More than 95% of the phage particles carried B. subtilis DNA inserts. When this library was screened for transforming activity, using competent cells, 70% of the genetic markers tested were found in a sample of 1,710 plaques. Cloned genetic loci were found to be about 100-fold more efficient in transforming activity than chromosomal DNA. Intact phage particles containing the pheA locus were found to be able to transform competent recipients with approximately the same efficiency as phage DNA. Transformation by intact particles was insensitive to deoxyribonuclease.
Transport of Ammonium and Methylammonium Ions by
Azotobacter vinelandii
Authors: Barnes Eugene M. , Zimniak Piotr
Published: January 2020

Ammonium and methylammonium are rapidly taken up by cultures of
Azotobacter vinelandii
respiring in the presence of succinate. The rate of methylamine uptake increased with external pH from 5.5 to 7.5 but increasing the pH further to 8.5 had little effect on activity, indicating that methylammonium cation rather than uncharged methylamine is the permeant species. The kinetics of methylammonium entry followed the Michaelis-Menten relationship, yielding a

K
m

of 25 μM and a
V
max
of 3.8 nmol/min per mg of cell protein. At saturating concentrations ammonium was taken up at rates 30-fold higher than those for methylammonium. Ammonium was a competitive inhibitor of methylammonium uptake and gave an inhibition constant of 1 μM. Ammonium derivatives were inhibitors of methylammonium entry in order of effectiveness: hydrazine > methylhydrazine > formamidine > guanidine > dimethylamine > ethylamine; amides and amino acids did not block uptake. Likewise, metal cations inhibited in the order Tl
+
> Cs
+
> Rb
+
, whereas Na
+
, K
+
, and Li
+
produced no significant effect. Methylammonium uptake was blocked in cells exposed to an uncoupler,
p
-trifluorome-thoxycarbonyl cyanide-phenyl hydrazone or gramicidin D, but not with dicyclo-hexylcarbodiimide or arsenate. Valinomycin stimulated methylammonium entry into cells in a K
+
-free medium but prevented entry in the presence of 10 mM K
+
. Monensin and nigericin had little effect on transport. These results indicate that methylammonium and ammonium ions enter
A. vinelandii
electrogenically via a specific transporter.
Gene rfaH, which affects lipopolysaccharide core structure in Salmonella typhimurium, is required also for expression of F-factor functions
Authors: Sanderson K E , Stocker B A
Published: January 2020

Mutations in gene rfaH of Salmonella typhimurium at 84 units on the linkage map make lipopolysaccharide of chemotypes Ra, Rb2, Rb3, and Rc (A. A. Lindberg and C. G. Hellerqvist, J. Gen. Microbiol, 116:25--32, 1980). F-factor expression in RfaH- strains was reduced in the following properties when compared with RfaH+ strains: transfer of Flac, number of phage f2 infective centers, lysis by and propagation of phages f2 and M13, proportion of cells with visible F-pili, and formation of mating aggregates with F- cells. Inhibition of multiplication of Br60, a female-specific phage, was not reduced in RfaH- Flac strains. Plasmid transfer from RfaH- strains was reduced for Inc groups FI, FII, and T, unaffected for Inc groups beta, I alpha, L, N, P, and W. and increased for Inc group M when compared with plasmid transfer from RfaH+ strains. Reduced F-factor function in RfaH- strains was not due to defective lipopolysaccharide since strains with mutations in other rfa genes were unaffected in plasmid transfer. Gene rfaH appears to be homologous with gene sfrB in Escherichia coli K-12, which maps at the same location, influences F-factor function, and affects synthesis of lipopolysaccharide. The gene product of sfrB has been proposed to be a transcription antiterminator.
Toxicity of leucine-containing peptides in Escherichia coli caused by circumvention of leucine transport regulation
Authors: Tavori H , Kimmel Y , Barak Z
Published: January 2020

A variety of leucine-containing peptides (LCP), Phe-Leu, Gly-Leu, Pro-Leu, Ala-Leu, Ala-Leu-Lys, Leu-Phe-Ala, Leu-Leu-Leu, and Leu-Gly-Gly, inhibited the growth of a prototrophic strain of Escherichia coli K-12 at concentrations between 0.05 and 0.28 mM. Toxicity requires normal uptake of peptides. When peptide transport was impaired by mutations, strains became resistant to the respective LCP. Inhibition of growth occurred immediately after the addition of LCP, and was relieved when 0.4 mM isoleucine was added. The presence of Gly-Leu in the medium correlated with the inhibition of growth, and the bacteria began to grow at the normal rate 70 min after Gly-Leu became undetectable. Disappearance of the peptide corresponded with the appearance of free leucine and glycine in the medium. The concentration of leucine inside the LCP-treated bacteria was higher than that in the leucine-treated and the control cultures. We suggest that entry of LCP into the cells via peptide transport systems circumvents the regulation of leucine transport, thereby causing abnormality high concentrations of leucine inside the cells. This accumulation of leucine interferes with the biosynthesis of isoleucine and inhibits the growth of the bacteria.
Intracellular mannitol, a product of glucose metabolism in staphylococci
Authors: Edwards K G , Blumenthal H J , Khan M , Slodki M E
Published: January 2020

Mannitol (Mtl), not previously reported as an intracellular component of bacteria, although it has been found as an extracellular end product of anaerobic carbohydrate metabolism, accumulated within strains of all 10 staphylococcal species tested after aerobic incubation of washed cell suspensions in phosphate-buffered 1% glucose for 2 h. Phenol extracts of the cells, before and after incubation, were analyzed for Mtl content by periodate utilization and paper chromatography and for Mtl 1-phosphate content, with Mtl 1-phosphate dehydrogenase. In Staphylococcus aureus Towler, the content of Mtl increased from a 0-h value of less than 2.4 to 16 mumol/g (dry weight) after incubation, and the level of Mtl 1-phosphate increased from a 0-h value of 1 to 8 mumol/g. The identification of Mtl was confirmed as the per-O-acetyl ester by gas-liquid chromatography and as the per-O-methyl ether by mass spectrometry. Also tested were 5 additional S. aureus strains and 32 coagulase-negative staphylococcal strains. All strains accumulated Mtl, even those strains that could not utilize exogenous Mtl during aerobic growth, usually in the range 4 to 25 mumol/g. Furthermore, three strains accumulated very high Mtl levels. Bacteria from several other genera were tested, and some were found to accumulate low to moderate levels of Mtl under similar incubation conditions. The metabolic conversion of glucose to intracellular Mtl, probably via Mtl 1-phosphate, is a common feature of staphylococci and also occurs in some other bacteria.