bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of Pseudomonas putida PpF1 mutants defective in the toluene dioxygenase enzyme system
Authors: Finette B A , Subramanian V , Gibson D T
Published: January 2020

Pseudomonas putida PpF1 degraded toluene via a dihydrodiol pathway to tricarboxylic acid cycle intermediates. The initial reaction was catalyzed by a multicomponent enzyme, toluene dioxygenase, which oxidized toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol). The enzyme consisted of three protein components: NADH-ferredoxintol oxidoreductase (reductasetol), ferredoxintol, and a terminal oxygenase which is an iron-sulfur protein (ISPtol). Mutants blocked in each of these components were isolated after mutagenesis with nitrosoguanidine. Mutants occurred as colony morphology variants when grown in the presence of toluene on indicator plates containing agar, mineral salts, a growth-supporting nutrient (arginine), 2,3,5-triphenyltetrazolium chloride (TTC), and Nitro Blue Tetrazolium (NBT). Under these conditions, wild-type colonies appeared large and red as a result of TTC reduction. Colonies of reductasetol mutants were white or white with a light blue center, ferredoxintol strains were light blue with a dark blue center, and strains that lacked ISPtol gave dark blue colonies. Blue color differences in the mutant colonies were due to variations in the extent of NBT reduction. Strains lacking all three components appeared white. Toluene dioxygenase mutants were characterized by assaying toluene dioxygenase activity in crude cell extracts which were complemented with purified preparations of each protein component. Between 40 and 60% of the putative mutants selected from the NBT-TTC indicator plates were unable to grow with toluene as the sole source of carbon and energy. This method should prove extremely useful in isolating mutants in other multicomponent oxygenase enzyme systems.
Cyclic AMP levels during induction and repression of cellulase biosynthesis in Thermomonospora curvata
Authors: Wood W E , Neubauer D G , Stutzenberger F J
Published: January 2020

Specific cellulase production rates (SCPR) were compared with intracellular cyclic AMP (cAMP) levels in the thermophilic actinomycete, Thermomonospora curvata, during growth on several carbon sources in a chemically defined medium. SCPR and cAMP levels were 0.03 U (endoglucanase [EG] units) and 2 pmol per mg of dry cells, respectively, during exponential growth on glucose. These values increased to about 6 and 25, respectively, during growth on cellulose. Detectable EG production ceased when cAMP levels dropped below 10. Cellobiose (usually considered to be a cellulase inducer) caused a sharp decrease in cAMP levels and repressed EG production when added to cellulose-grown cultures. 2-deoxy-D-glucose, although nonmetabolizable in T. curvata, depressed cAMP to levels observed with glucose, but unlike glucose, the 2DG effect persisted until cells were washed and transferred to fresh medium. SCPR values and cAMP levels in cells grown in continuous culture under conditions of cellobiose limitation were markedly influenced by dilution rate (D). The maxima for both occurred at D = 0.085 (culture generation time of 11.8 h). When D was held constant and cellobiose concentration was increased over a 14-fold range to support higher steady state population levels, SCPR values decreased about fivefold, indicating that extracellular catabolite accumulation may be a factor in EG repression. The role of cAMP in the mechanism of this repression appears to be neither simple nor direct, since large changes (up to 200-fold) in SCPR accompany relatively small changes (10-fold) in cellular cAMP levels.
In vivo evidence for the role of the epsilon subunit as an inhibitor of the proton-translocating ATPase of Escherichia coli
Authors: Klionsky D J , Brusilow W S , Simoni R D
Published: January 2020

The function of the epsilon subunit of the Escherichia coli proton-translocating ATPase has been examined by using a mutant defective in the uncC gene. Strains with a defective uncC gene show a reduction in both growth yield and growth rate that is more severe than for other unc mutants; this deleterious effect is shown to be a result of the ATPase activity of the F1 complex which is missing the epsilon subunit. In addition, the epsilon-deficient F1 is bound less tightly to the membrane. These data suggest that, in vivo, the epsilon subunit is capable of inhibiting the ATPase activity of F1 and also functions in the binding of F1 to F0.
Return of Streptococcus faecalis DNA cloned in Escherichia coli to its original host via transformation of Streptococcus sanguis followed by conjugative mobilization
Authors: Smith M D , Clewell D B
Published: January 2020

Cloning vectors were introduced into Streptococcus faecalis by conjugation. A conjugative plasmid (pVA797) and cloning vector pVA838 recombined in Streptococcus sanguis at homologous sequences, forming a cointegrate. The pVA797::pVA838 cointegrate transferred to S. faecalis by conjugation. Recombination between homologous sequences resolved the cointegrate in the S. faecalis transconjugants, and pVA797 and pVA838 segregated because of incompatibility. S. faecalis strains that received pVA838 by this mechanism contained plasmids indistinguishable from authentic pVA838 from Escherichia coli. Other plasmids, including pVA736, were introduced into S. faecalis by this method. This approach should facilitate the introduction of cloned DNA into S. faecalis.
Regulation of expression of the ilvB operon in Salmonella typhimurium
Authors: Weinberg R A , Burns R O
Published: January 2020

The ilvB gene of Salmonella typhimurium encodes the valine-sensitive form of acetohydroxy acid synthase, acetohydroxy acid synthase I, which catalyzes the first step in the parallel biosynthesis of isoleucine and valine. Although nearly all of the other genes involved in this pathway are clustered at minute 83, ilvB was found to lie at minute 80.5. Expression of ilvB was shown to be nearly completely repressed by the end products leucine and valine. Studies in which we used strains with mutations in cya (adenylate cyclase) and crp (cAMP receptor protein) demonstrated that synthesis of acetohydroxy acid synthase I is enhanced by the cAMP-cAMP receptor protein complex. Although no stimulation was achieved by growth on poor carbon sources, introduction of crp on a multicopy plasmid led to markedly increased expression. Strains of S. typhimurium lacking valine-resistant acetohydroxy acid synthase II (ilvG) are like Escherichia coli K-12 in that they are not able to grow in the presence of L-valine owing to a conditional isoleucine auxotrophy. The valine toxicity of these ilvG mutants of S. typhimurium was overcome by increasing the level of acetohydroxy acid synthase I. Enzyme activity could be elevated either by maximally derepressing expression with severe leucine limitation, by introduction of either ilvB or crp on a multicopy plasmid, or by the presence of the ilv-513 mutation. This mutation, which is closely linked to genes encoding the phosphoenol pyruvate:sugar phosphotransferase system (pts), causes highly elevated expression of ilvB that is refractory to repression by leucine and valine, as is the major ilv operon. The response of ilvB to the cAMP-cAMP receptor protein complex was not affected by this lesion. Data obtained by using this mutant led us to propose that the two modes of regulation act independently. We also present some evidence which suggests that ilvB expression may be affected by the phosphoenol pyruvate:sugar phosphotransferase system.
Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway
Authors: Vander Wauven C , Piérard A , Kley-Raymann M , Haas D
Published: January 2020

Pseudomonas aeruginosa PAO was able to grow in the absence of exogenous terminal electron acceptors, provided that the medium contained 30 to 40 mM L-arginine and 0.4% yeast extract. Under strictly anaerobic conditions (O2 at less than 1 ppm), growth could be measured as an increase in protein and proceeded in a non-exponential way; arginine was largely converted to ornithine but not entirely consumed at the end of growth. In the GasPak anaerobic jar (Becton Dickinson and Co.), the wild-type strain PAO1 grew on arginine-yeast extract medium in 3 to 5 days; mutants could be isolated that were unable to grow under these conditions. All mutants (except one) were defective in at least one of the three enzymes of the arginine deiminase pathway (arcA, arcB, and arcC mutants) or in a novel function that might be involved in anaerobic arginine uptake (arcD mutants). The mutations arcA (arginine deiminase), arcB (catabolic ornithine carbamoyltransferase), arcC (carbamate kinase), and arcD were highly cotransducible and mapped in the 17-min chromosome region. Some mutations in the arc cluster led to low, noninducible levels of all three arginine deiminase pathway enzymes and thus may affect control elements required for induction of the postulated arc operon. Two fluorescent pseudomonads (P. putida and P. fluorescens) and P. mendocina, as well as one PAO mutant, possessed an inducible arginine deiminase pathway and yet were unable to grow fermentatively on arginine. The ability to use arginine-derived ATP for growth may provide P. aeruginosa with a selective advantage when oxygen and nitrate are scarce.
Generation of a large, protonophore-sensitive proton motive force and pH difference in the acidophilic bacteria Thermoplasma acidophilum and Bacillus acidocaldarius
Authors: Michels M , Bakker E P
Published: January 2020

The mechanism by which acidophilic bacteria generate and maintain their cytoplasmic pH close to neutrality was investigated. For this purpose we determined the components of proton motive force in the eubacterium Bacillus acidocaldarius and the archaebacterium Thermoplasma acidophilum. After correction for probe binding, the proton motive force of untreated cells was 190 to 240 mV between external pH 2 and 4. Anoxia diminished total proton motive force and the transmembrane pH difference by 60 to 80 mV. The protonophore 2,4-dinitrophenol abolished the total proton motive force almost completely and diminished the transmembrane pH difference by at least two units. However, even after correction for probe binding, protonophore-treated cells maintained a pH difference of approximately one unit.
Anaerobic degradation of 2-fluorobenzoate by benzoate-degrading, denitrifying bacteria
Authors: Schennen U , Braun K , Knackmuss H J
Published: January 2020

Three strains of anaerobically benzoate-degrading, denitrifying bacteria of the genus Pseudomonas were able to grow on 2-fluorobenzoate as the sole carbon and energy source. Fluoride ion release was stoichiometric, and the reduction of dissolved organic carbon indicated total degradation. Cells grown anaerobically with benzoate were adapted for immediate growth with 2-fluorobenzoate, and both compounds were substrates for an inducible benzoyl-coenzyme A synthetase, the initial enzyme of anaerobic degradation. It is proposed that fluoride is eliminated gratuitously by a regioselective reaction in a sequence common to both carbon sources. Benzoate, but not 2-fluorobenzoate, was oxidized by aerobically grown cells.
Locus affecting regulation of the colicin I receptor by iron
Authors: Worsham P L , Konisky J
Published: January 2020

Using a strain containing a cir-lac operon fusion and a selective medium, we isolated a regulatory mutant of the colicin I receptor, which we have designated cirR. Cells carrying the cirR mutation were defective in the transcriptional regulation of cir by iron, but synthesis of other iron-regulated proteins was unaffected. cirR was found to be cis dominant. This is in contrast to previously described mutations in iron regulation which are pleiomorphic and trans dominant. Temperature regulation of colicin I receptor production was unaffected by cirR.
Germination-initiated spores of Bacillus brevis Nagano retain their resistance properties
Authors: Daher E , Rosenberg E , Demain A L
Published: January 2020

Initiated spores and vegetative cells of the gramicidin S-producing Bacillus brevis Nagano were compared with respect to their resistance to various forms of stress (osmotic shock-starvation, exposure to ethanol, sonic oscillation, and heat). The resistance of initiated spores to all of these stress situations was considerably greater than that of vegetative cells and approached that of dormant spores. The period during which the initiated spores remained resistant to heat was extended by addition of gramicidin S. The antibiotic may therefore be of survival value to the species in nature by slowing down the development of initiated spores in the outgrowth phase of germination, thereby extending the period during which the cells are resistant to environmental stress.
Generation of transducing particles in Staphylococcus aureus
Authors: Dyer D W , Rock M I , Lee C Y , Iandolo J J
Published: January 2020

Transduction of plasmid pC194 and bacteriophage phi 11de varied inversely with the multiplicity of infection. As the multiplicity of infection decreased from 10(-1) to 10(-5) PFU/CFU, the transduction frequency of pC194 increased 10(4)-fold; the transduction frequency of phi 11de increased 300-fold with a 100-fold decrease in multiplicity of infection. Physical and genetic analysis of the transduced DNA showed that pC194 resided in the phage particle as a random, circularly permuted linear concatemer. In DNA prepared from phage that cotransduced pC194 and phi 11de, pC194 resided in the transducing phage primarily as a linear multimer of 15.8 kilobases, or about 5.4 pC194 monomers. The pC194 multimer was randomly inserted into the phi 11 genome.
Location and characteristics of enzymes involved in the breakdown of polygalacturonic acid by Bacteroides thetaiotaomicron
Authors: McCarthy R E , Kotarski S F , Salyers A A
Published: January 2020

When Bacteroides thetaiotaomicron is grown in medium which contains polygalacturonic acid (PGA) as the sole carbon source, two different polygalacturonases are produced: a PGA lyase (EC 4.2.2.2) and a PGA hydrolase (EC 3.2.1.15). Both enzymes are cell associated. The PGA hydrolase appears to be an inner membrane protein. The PGA lyase is a soluble protein that associates with membranes under certain conditions. The PGA lyase was purified to apparent homogeneity. It has a molecular weight (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 74,000, a pH optimum of 8.7, a pI of 7.5, and a Km for PGA of 40 to 70 micrograms/ml. It requires calcium for maximal activity. The main product of this enzyme appears to be a disaccharide that contains a delta 4,5-unsaturated galacturonic acid residue. The PGA hydrolase can be solubilized from membranes with 2% Triton X-100 and has been partially purified. It has a pH optimum of 5.4 to 5.5, a pI of 4.7 to 4.9, and a Km for PGA of 350 to 400 micrograms/ml. The main product of this enzyme appears to be galacturonic acid. The specific activities of both PGA hydrolase and PGA lyase increase at the same rate when bacteria are exposed to PGA. The two enzymes therefore appear to be similarly regulated.
Isolation of Bacillus subtilis mutants altered in expression of a gene transcribed in vitro by a minor form of RNA polymerase (E-sigma 37)
Authors: Truitt C L , Ray G L , Trempy J E , Da-Jian Z , Haldenwang W G
Published: January 2020

To develop a technique for identifying Bacillus subtilis genes whose products affect transcription from promoters recognized by sigma 37-containing RNA polymerase (E-sigma 37), we cloned the promoter region of a gene (ctc) that is actively transcribed in vitro by E-sigma 37 into a plasmid (pPL603B) so that a transcriptional fusion was created between ctc and a plasmid-borne chloramphenicol acetyltransferase (CAT) gene. CAT levels in B. subtilis carrying the ctc/CAT fusion plasmid varied in a manner that was consistent with the known pattern of ctc RNA synthesis. Mutagenesis of cells harboring the ctc/CAT plasmid led to the isolation of bacterial clones which displayed altered chloramphenicol resistance. Analysis of the mutants demonstrated that CAT activity was substantially changed in the mutant cells. Several of the B. subtilis mutants, both CAT overproducers and underproducers, also had acquired a sporulation-deficient phenotype. The mutations responsible for altered CAT expression were not carried on the plasmid. Analysis of RNA synthesized by mutant cells indicates that at least a portion of the mutants may be altered in the level of transcription from the ctc promoter and, hence, are likely to define B. subtilis genes which influence this process.
Regulation of transcription of the repA1 gene in the replication control region of IncFII plasmid NR1 by gene dosage of the repA2 transcription repressor protein
Authors: Dong X , Womble D D , Luckow V A , Rownd R H
Published: January 2020

Transcription of the repA1 gene of the IncFII plasmid NR1 is initiated at two promoters in the replication control region. Transcription from the upstream promoter is constitutive at a low level, whereas transcription from the downstream promoter is regulated. The 5' end of the constitutively synthesized transcript also encodes the transcription repressor protein for the regulated downstream promoter. Therefore, the level of the repressor protein in the cell is gene dosage dependent. Using both lac gene fusions and quantitative hybridization methods, we have determined the in vivo relationship between the rate of transcription from the regulated promoter and the repressor protein concentration as a function of gene dosage. At the wild-type copy number of NR1, transcription from the regulated promoter is 96% repressed, but substantial derepression occurs when the copy number falls below the normal value. At or above the normal plasmid copy number, the basal level of repA1 mRNA is provided by transcription from the constitutive upstream promoter.
Cloning and characterization of a gene (fadR) involved in regulation of fatty acid metabolism in Escherichia coli
Authors: DiRusso C C , Nunn W D
Published: January 2020

The regulatory gene fadR has been previously characterized by classical genetic means as a diffusible protein which exerts negative control over fatty acid degradation and acetate metabolism. fadR has also been implicated in the regulation of unsaturated fatty acid biosynthesis. To facilitate the identification of the product of the fadR gene and to study the mechanism by which this multifunctional regulatory gene exerts its control, we cloned a segment of DNA containing the fadR gene in the phage vector lambda L47. Subsequent subcloning of a segment of the chromosomal DNA from the lambda fadR+ phage into various plasmid vectors resulted in the isolation of the fadR gene on a 1.3-kilobase-pair HindIII-EcoRV fragment. fadR strains harboring the cloned fadR+ gene showed inducible levels of fatty acid oxidation and crotonase (enoyl-coenzyme A-hydratase, fadB) activity. The cloned gene exerted transcriptional control over beta-galactosidase synthesis in an fadR strain that had a lambda phi (fad-lacZ+) operon fusion. An fadR mutation in fabA(Ts) strains prevents growth at permissive temperatures without unsaturated fatty acid supplementation (Nunn et al., J. Bacteriol. 154:554-560, 1983). Plasmids carrying the fadR+ gene suppress this unsaturated fatty acid auxotrophy in fadR fabA(Ts) strains at the permissive condition. Maxicell analysis identified a 29,000-dalton protein encoded by the 1.3-kilobase fragment which appeared to be associated with functional fadR gene activity.
Identity of the photoproduct that causes lacI mutations in UV-irradiated Escherichia coli
Authors: Lawrence C W , Christensen R B , Christensen J R
Published: January 2020

Estimates of the capacity of photoreactivation to act specifically on premutational lesions were obtained by conjugational transfer of an F' lac plasmid from a UV-irradiated, photoreactivated donor to a delta (pro-lac) recipient that had been UV irradiated and allowed to induce SOS functions for 30 min. This treatment reduced the frequency of induced lacI mutations by 70 to 80%, indicating that cyclobutane dimers cause most mutations in this system.
Expression of a Saccharomyces cerevisiae photolyase gene in Escherichia coli
Authors: Sancar G B
Published: January 2020

A 3.3-kilobase PvuII fragment carrying the PHR1 gene of Saccharomyces cerevisiae has been cloned into an Escherichia coli expression vector and introduced into E. coli strains deficient in DNA photolyase. Complementation of the E. coli phr-1 mutation was observed, strongly suggesting that the yeast PHR1 gene encodes a DNA photolyase.
Occurrence of 2-keto-deoxyoctonic acid 5-phosphate in lipopolysaccharides of Vibrio cholerae Ogawa and Inaba
Authors: Brade H
Published: January 2020

A phosphorylated 2-keto-3-deoxyoctonic acid (KDO) was released from the lipopolysaccharides of Vibrio cholerae Ogawa and Inaba after strong acid hydrolysis. The phosphorylated KDO was identified by gas-liquid chromatography and mass spectrometry after reduction and permethylation as KDO-5-phosphate and an isomer of it being phosphorylated at position 7 or 8. After treatment with alkaline phosphatase, KDO was detected by gas-liquid chromatography and mass spectrometry. It was indistinguishable from authentic 2-keto-3-deoxy-D-manno-octonic acid.
Overproduction and nucleotide sequence of the respiratory D-lactate dehydrogenase of Escherichia coli
Authors: Rule G S , Pratt E A , Chin C C , Wold F , Ho C
Published: January 2020

Recombinant DNA plasmids containing the gene for the membrane-bound D-lactate dehydrogenase (D-LDH) of Escherichia coli linked to the promoter PL from lambda were constructed. After induction, the levels of D-LDH were elevated 300-fold over that of the wild type and amounted to 35% of the total cellular protein. The nucleotide sequence of the D-LDH gene was determined and shown to agree with the amino acid composition and the amino-terminal sequence of the purified enzyme. Removal of the amino-terminal formyl-Met from D-LDH was not inhibited in cells which contained these high levels of D-LDH.
Instability of Candida albicans hybrids
Authors: Whelan W L , Markie D M , Simpkin K G , Poulter R M
Published: January 2020

Total cellular DNA content, determined by a colorimetric method, was used as an index of ploidy in Candida albicans. Mononucleate hybrids were formed by fusion of spheroplasts derived from diploid parent strains. Five hybrids, of six studied, were taken to be tetraploid on the basis of estimated DNA content. One hybrid was taken to be hexaploid or near-hexaploid. Selection for increased resistance to 5-fluorocytosine in the hybrids, which were heterozygous for resistance, resulted in isolation of variants which were of lower ploidy than the hybrids from which they originated. Variants were obtained which corresponded (in measured DNA content) to aneuploid, triploid, and diploid states. These results may form the basis of a cyclic parasexual system (2n X 2n----4n----2n) for genetic analysis of this asexual species.
Differential expression of protein S genes during Myxococcus xanthus development
Authors: Downard J S , Zusman D R
Published: January 2020

Protein S, the most abundant protein synthesized during development of the fruiting bacterium Myxococcus xanthus, is coded by two highly homologous genes called protein S gene 1 (ops) and protein S gene 2 (tps). The expression of these genes was studied with fusions of the protein S genes to the lacZ gene of Escherichia coli. The gene fusions were constructed so that expression of beta-galactosidase activity was dependent on protein S gene regulatory sequences. Both the gene 1-lacZ fusion and the gene 2-lacZ fusion were expressed exclusively during fruiting body formation (development) in M. xanthus. However, distinct patterns of induction of fusion protein activity were observed for the two genes. Gene 2 fusion activity was detected early during development on an agar surface and could also be observed during nutritional downshift in dispersed liquid culture. Gene 1 fusion activity was not detected until much later in development and was not observed after downshift in liquid culture. The time of induction of gene 1 fusion activity was correlated with the onset of sporulation, and most of the activity was spore associated. This gene fusion was expressed during glycerol-induced sporulation when gene 2 fusion activity could not be detected. The protein S genes appear to be members of distinct regulatory classes of developmental genes in M. xanthus.
Effects of beta-pinene on yeast membrane functions
Authors: Uribe S , Ramirez J , Peña A
Published: January 2020

The effects of beta-pinene on yeast cells were studied. This terpene inhibited respiration with glucose or ethanol as the substrate. The inhibition depended on the ratio of the terpene to the amount of yeast cells; for a fixed concentration of pinene, inhibition decreased as the amount of yeast cells increased. Pinene also inhibited the pumping of protons and K+ transport, but this inhibition was more marked with with ethanol than with glucose as the substrate, indicating the mitochondrial localization of the inhibition. The studies on isolated mitochondria showed a series of effects, starting with the disappearance of the respiratory control and deenergization of the organelles and followed by an inhibition of respiration at higher concentrations of the terpene. The effect on respiration could be localized to the cytochrome b region of the electron transport chain. No effect could be detected on the activity of ATPase. The effects can be ascribed to a localization of pinene on membranes which was also accompanied by a decrease in the fluorescence polarization of diphenyl hexatriene, probably meaning an increase in the fluidity of the membrane, localized preferentially to the mitochondria.
Mutational analysis of the Klebsiella pneumoniae nitrogenase promoter: sequences essential for positive control by nifA and ntrC (glnG) products
Authors: Ow D W , Xiong Y , Gu Q , Shen S C
Published: January 2020

ntr (nitrogen regulated) and nif (nitrogen fixation) promoters are structurally similar to each other but bear no resemblance to canonic Escherichia coli promoters. ntr promoters are normally activated by the ntrC (glnG) product, but they can also be activated by the ntrC-related Klebsiella pneumoniae nifA product. In contrast, nif promoters of K. pneumoniae such as the nitrogenase (nifH) promoter can only be nifA activated. In this paper, we report the isolation and characterization of 28 mutants of the K. pneumoniae nifH promoter. Class A mutants no longer respond to nifA-mediated transcription, and class B mutants can now respond to ntrC-mediated activation. These two classes of mutants define sequences important to nifA- and ntrC-mediated transcription. Most surprising is that a single base change is sufficient to convert a nifA-activated promoter into an ntrC-activated one.
Isolation of genes (nif/hup cosmids) involved in hydrogenase and nitrogenase activities in Rhizobium japonicum
Authors: Hom S S , Graham L A , Maier R J
Published: January 2020

Recombinant cosmids containing a Rhizobium japonicum gene involved in both hydrogenase (Hup) and nitrogenase (Nif) activities were isolated. An R. japonicum gene bank utilizing broad-host-range cosmid pLAFR1 was conjugated into Hup- Nif- R. japonicum strain SR139. Transconjugants containing the nif/hup cosmid were identified by their resistance to tetracycline (Tcr) and ability to grow chemoautotrophically (Aut+) with hydrogen. All Tcr Aut+ transconjugants possessed high levels of H2 uptake activity, as determined amperometrically. Moreover, all Hup+ transconjugants tested possessed the ability to reduce acetylene (Nif+) in soybean nodules. Cosmid DNAs from 19 Hup+ transconjugants were transferred to Escherichia coli by transformation. When the cosmids were restricted with EcoRI, 15 of the 19 cosmids had a restriction pattern with 13.2-, 4.0-, 3.0-, and 2.5-kilobase DNA fragments. Six E. coli transformants containing the nif/hup cosmids were conjugated with strain SR139. All strain SR139 transconjugants were Hup+ Nif+. Moreover, one nif/hup cosmid was transferred to 15 other R. japonicum Hup- mutants. Hup+ transconjugants of six of the Hup- mutants appeared at a frequency of 1.0, whereas the transconjugants of the other nine mutants remained Hup-. These results indicate that the nif/hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in H2 uptake activity.
Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol
Authors: Allen L N , Hanson R S
Published: January 2020

Four new cloning vectors have been constructed from the broad-host-range cloning vector pRK290. These vectors, pLA2901, pLA2905, pLA2910, and pLA2917, confer resistance to kanamycin and tetracycline. The latter two are cosmid derivatives of pLA2901. The new vectors can be mobilized into, and are stably maintained in, a variety of gram-negative bacteria. A Sau3A genomic bank of Methylobacterium organophilum strain xx DNA has been constructed in pLA2917, and complementation analysis, with a variety of mutants unable to grow on methanol, revealed at least five separate regions necessary for growth on methanol. Complementation analysis and Tn5 mutagenesis data suggest that at least three genes are responsible for expression of active methanol dehydrogenase.
Identification of multiple cell surface antigens associated with the sex pheromone response of Streptococcus faecalis
Authors: Tortorello M L , Dunny G M
Published: January 2020

High-molecular-weight surface antigens, obtained by ammonium sulfate precipitation of culture supernatants and identified in Western blots of sodium dodecyl sulfate-polyacrylamide gels, have been correlated with the sex pheromone response of Streptococcus faecalis donor cells. Pheromone-induced cells carrying the conjugative plasmid pCF10 produced both an antigenic component (C130) composed of at least four bands in the range of 130 kilodaltons and a 73-kilodalton antigen (SA 73). The concentration of the C130 antigen in culture supernatants increased with time after exposure of donor cells to pheromone preparations. Gel filtration studies indicated that this antigen exists in the native state as a very large complex that is more than 180,000 daltons in size. The C130 antigen was susceptible to digestion by proteinase K and was not reactive with either concanavalin A or wheat germ agglutinin. The antigenicity of C130 was not destroyed by treatment of blots with trypsin, chymotrypsin, or papain before development with antibody, whereas the antigenicity of SA73 was susceptible to these treatments.
Induction of superoxide dismutases in Escherichia coli B by metal chelators
Authors: Pugh S Y , Fridovich I
Published: January 2020

The effects of metal salts, chelating agents, and paraquat on the superoxide dismutases (SODs) of Escherichia coli B were explored. Mn(II) increased manganese-containing SOD (MnSOD), whereas Fe(II) increased iron-containing SOD (FeSOD). Chelating agents induced MnSOD but decreased FeSOD and markedly increased the degree of induction seen with Mn(II). Paraquat also exerted a synergistic effect with Mn(II). High levels of MnSOD were achieved in the combined presence of Mn(II), chelating agent, and paraquat. All of these effects were dependent on the presence of oxygen. MnSOD, not ordinarily present in anaerobically grown E. coli cells, was present when the cells were grown anaerobically in the presence of chelating agents. These results are accommodated by a scheme which incorporates autogenous repression by the apoSODs and competition between Fe(II) and Mn(II) for the metal-binding sites of the apoSODs. It is further supposed that oxygenation and intracellular O2- production favor MnSOD production because O2- oxidizes Mn(II) to Mn(III), which competes favorably with Fe(II) for the apoSODs.
Cloning of genes involved in myo-inositol transport in a Pseudomonas sp
Authors: Gauchat-Feiss D , Frey J , Belet M , Deshusses J
Published: January 2020

A soil isolate of a Pseudomonas sp. can utilize myo-inositol (MI) as the sole carbon source. In this strain, MI is transported through the membrane by a high-affinity transport system in which a periplasmic binding protein is involved. Mutants impaired in the transport system were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently identified by their slow growth rate at low MI concentrations. Strains with a low linear initial rate of MI uptake were analyzed. Using a broad-host-range cosmid cloning system, we have constructed a gene bank of the wild-type Pseudomonas sp. in an Escherichia coli recA-host. A rapid mating technique enabled us to screen the gene library for clones which are able to restore the active transport of MI in the mutant. An 11.5-kilobase segment containing genes involved in the MI transport has been isolated, and its restriction enzyme cleavage map has been determined.
Isolation and characterization of mutant strains of Escherichia coli altered in H2 metabolism
Authors: Lee J H , Patel P , Sankar P , Shanmugam K T
Published: January 2020

A positive selection procedure is described for the isolation of hydrogenase-defective mutant strains of Escherichia coli. Mutant strains isolated by this procedure can be divided into two major classes. Class I mutants produced hydrogenase activity (determined by using a tritium-exchange assay) and formate hydrogenlyase activity but lacked the ability to reduce benzyl viologen or fumarate with H2 as the electron donor. Class II mutants failed to produce active hydrogenase and hydrogenase-dependent activities. All the mutant strains produced detectable levels of formate dehydrogenase-1 and -2 and fumarate reductase. The mutation in class I mutants mapped near 65 min of the E. coli chromosome, whereas the mutation in class II mutants mapped between srl and cys operons (58 and 59 min, respectively) in the genome. The class II Hyd mutants can be further subdivided into two groups (hydA and hydB) based on the cotransduction characteristics with cys and srl. These results indicate that there are two hyd operons and one hup operon in the E. coli chromosome. The two hyd operons are needed for the production of active hydrogenase, and all three are essential for hydrogen-dependent growth of the cell.
Construction and application of a promoter-probe plasmid that allows chromogenic identification in Streptomyces lividans
Authors: Horinouchi S , Beppu T
Published: January 2020

We cloned a Streptomyces coelicolor A3(2) DNA fragment which directed synthesis of a brown pigment, presumably a shunt product in the actinorhodin biosynthetic pathway, on the plasmid vector pIJ41 in Streptomyces lividans. The pigment production was observed only when the DNA fragment was inserted downstream from a functional promoter sequence. By subcloning the fragment together with in vitro manipulation, a promoter-probe plasmid vector (pARC1) with a unique BamHI cloning site was constructed that allows chromogenic identification of transcriptional control signals in Streptomyces lividans based on the expression of the cloned pigment gene(s). The Escherichia coli tac (trp-lac hybrid) promoter, consisting of 92 base pairs and a promoter region including the leader sequence of erythromycin resistance gene (ermC) on staphylococcal plasmid pE194, when ligated in the correct orientation in the BamHI site of pARC1, promoted expression of the cloned pigment gene(s) in Streptomyces lividans, whereas the Saccharomyces cerevisiae GAL7 promoter did not. In the case of the ermC, induction of the pigment production by the addition of either erythromycin or lincomycin, but not virginiamycin, was observed. The system was also shown to be useful and convenient in isolating transcriptional control signals of Streptomyces chromosomal DNA and estimating their activities.
Localization of acyl carrier protein in Escherichia coli
Authors: Jackowski S , Edwards H H , Davis D , Rock C O
Published: January 2020

Acyl carrier protein was localized by immunoelectron microscopy in the cytoplasm of Escherichia coli. These data are inconsistent with the previous report of an association between acyl carrier protein and the inner membrane (H. Van den Bosch, J.R. Williamson, and P.R. Vagelos, Nature [London] 228:338-341, 1970). Moreover, bacterial membranes did not bind a significant amount of acyl carrier protein or its thioesters in vitro. A thioesterase activity specific for long-chain acyl-acyl carrier protein was associated with the inner membrane.
Physical analysis of deletion mutations in the ilvGEDA operon of Escherichia coli K-12
Authors: Driver R P , Lawther R P
Published: January 2020

DNA-DNA hybridization of cloned segments of the Escherichia coli K-12 ilvGEDA operon to genomic blots was used to determine the physical dimensions of a series of deletion mutations of the ilvGEDA operon. The smallest mutation resulted from the deletion of approximately 200 base pairs from within ilvD, whereas the largest mutation resulted from the deletion of 17 kilobases including the rep gene. The structure of three of these mutants indicates that formation of the deletions was mediated by Tn5 (or Tn5-131) that is retained in the chromosome. This is the first observation of this type of Tn5-mediated event. Our analysis of the total acetohydroxy acid synthase activity of strains containing deletions of ilvG indicates that the truncated ilvG polypeptide of wild-type E. coli K-12 lacks enzyme activity. The small 200-base-pair deletion of ilvD confirms the presence of a strong polar site 5' to ilvA. The detailed structure of these deletions should prove useful for the investigation of other genes in this region. This genomic analysis demonstrates that the ilv restriction site map that was established previously by the analysis of recombinant bacteriophage and plasmids is identical to that on the genome.
Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum
Authors: Carlson T A , Guerinot M L , Chelm B K
Published: January 2020

We have isolated the Bradyrhizobium japonicum gene encoding glutamine synthetase I (glnA) from a phage lambda library by using a fragment of the Escherichia coli glnA gene as a hybridization probe. The rhizobial glnA gene has homology to the E. coli glnA gene throughout the entire length of the gene and can complement an E. coli glnA mutant when borne on an expression plasmid in the proper orientation to be transcribed from the E. coli lac promoter. High levels of glutamine synthetase activity can be detected in cell-free extracts of the complemented E. coli. The enzyme encoded by the rhizobial gene was identified as glutamine synthetase I on the basis of its sedimentation properties and resistance to heat inactivation. DNA sequence analysis predicts a high level of amino acid sequence homology among the amino termini of B. japonicum, E. coli, and Anabaena sp. strain 7120 glutamine synthetases. S1 nuclease protection mapping indicates that the rhizobial gene is transcribed from a single promoter 131 +/- 2 base pairs upstream from the initiation codon. This glnA promoter is active when B. japonicum is grown both symbiotically and in culture with a variety of nitrogen and carbon sources. There is no detectable sequence homology between the constitutively expressed glnA promoter and the differentially regulated nif promoters of the same B. japonicum strain.
Mevinolinic acid biosynthesis by Aspergillus terreus and its relationship to fatty acid biosynthesis
Authors: Greenspan M D , Yudkovitz J B
Published: January 2020

Mevinolinic acid, the open acid form of mevinolin, which is a metabolite of Aspergillus terreus, has been shown to be a competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (Alberts et al., Proc. Natl. Acad. Sci. U.S.A. 77:3957-3961, 1980). The biosynthesis of mevinolinic acid was studied by examining the incorporation of [1-14C]acetate and [methyl-14C]methionine into the molecule. These isotopes were rapidly incorporated into mevinolinic acid, with [1-14C]acetate and [methyl-14C]methionine incorporation being linear for at least 10 and 30 min, respectively. A comparison of acetate incorporation into mevinolinic acid and fatty acids indicated that mevinolinic acid biosynthesis increased with a maximum between days 3 and 5 of growth; at this time cell growth had ceased and fatty acid biosynthesis was negligible. Hydrolysis of the mevinolinic acid and isolation of the products showed that [1-14C]acetate and [methyl-14C]methionine were incorporated into the 2-methylbutyric acid side chain as well as into the main (alcohol) portion of the molecule.
Evidence for a common siderophore transport system but different siderophore receptors in Neurospora crassa
Authors: Huschka H , Naegeli H U , Leuenberger-Ryf H , Keller-Schierlein W , Winkelmann G
Published: January 2020

Uptake and competition experiments were performed with Neurospora crassa and Penicillium parvum by using 14C-labeled coprogen and 55Fe-labeled ferrichrome-type siderophores. Several siderophores of the ferrichrome family, such as ferrichrome, ferricrocin, ferrichrysin, and tetraglycyl-ferrichrome as well as the semisynthetic ferricrocin derivatives O-(phenyl-carbamoyl)-ferricrocin and O-(sulfanilyl-carbamoyl)-ferricrocin were taken up by N. crassa. The ferrichrome-type siderophores used vary in the structure of the peptide backbone but possess a common lambda-cis configuration about the iron center and three identical ornithyl-delta-N-acetyl groups as surrounding residues. This suggests that these ferrichrome-type siderophores are recognized by a common ferrichrome receptor. We also concluded that the ferrichrome receptor is lambda-cis specific from the inability to take up the synthetic enantiomers, enantio-ferrichrome and enantio-ferricrocin, possessing a delta-cis configuration about the iron center. On the other hand, we found that coprogen, possessing a delta-absolute configuration and two trans-anhydromevalonic acid residues around the metal center, was also taken up by N. crassa and was competitively inhibited by the ferrichrome-type siderophores. We therefore propose the existence of a common siderophore transport system but the presence of different siderophore receptors in N. crassa. In addition, ferrirubin, which is very slowly transported by N. crassa, inhibited both coprogen and ferrichrome-type siderophore transport. Contrary to the findings with N. crassa, transport experiments with P. parvum revealed the presence of a ferrichrome receptor but the absence of a coprogen receptor; coprogen was neither transported nor did it inhibit the ferrichrome transport.
Some mercurial resistance plasmids from different incompatibility groups specify merR regulatory functions that both repress and induce the mer operon of plasmid R100
Authors: Foster T J , Ginnity F
Published: January 2020

Transcription of the mer genes of plasmid R100 is regulated by the product of the merR gene. The merR gene negatively regulates its own expression and also controls the transcription of the merTCA operon both negatively (in the absence of inducer) and positively (in the presence of inducer). We used transcriptional mer-lac fusions of R100-1 in complementation tests to measure the ability of the merR products of different mercury-resistant transposons and plasmids to functionally interact with R100-1. Plasmids from incompatibility groups C, B, S, L, and P, as well as the Pseudomonas transposons Tn501 and Tn3401, regulated the expression of the R100 mer genes in a similar fashion to the R100-1 merR product itself, suggesting that these elements are closely related. Only plasmid R391 (IncJ) did not complement.
UDP-N-acetylmuramylpentapeptide as acceptor in murein biosynthesis in Escherichia coli membranes and ether-permeabilized cells
Authors: Kraus W , Glauner B , Höltje J V
Published: January 2020

Two widely used in vitro systems of Escherichia coli capable of synthesizing murein were evaluated by using high-pressure liquid chromatography for murein analysis. Comparison of the composition of murein synthesized by either a membrane preparation or ether-treated cells with native murein revealed that both in vitro systems failed to synthesize murein that was identical to murein formed in vivo. Furthermore, neither system attached the lipoprotein to the murein. Ether-treated cells, however, were superior to the membrane preparation in catalyzing the formation of the remarkable A2pm-A2pm cross-linkage. In both systems an atypical transpeptidation reaction was found to take place in which exogenously supplied UDP-N-acetylmuramylpentapeptide was directly linked to the murein without participation of the bactoprenol lipid carrier. The direct transpeptidation yields preferentially trimeric peptide bridges with the UDP-linked muramylpentapeptide serving as the acceptor.
Selection for mutants altered in the expression or export of outer membrane porin OmpF
Authors: Sodergren E J , Davidson J , Taylor R K , Silhavy T J
Published: January 2020

Strains in which the lacZ gene (which specifies beta-galactosidase) is fused to a gene encoding an envelope protein often exhibit a phenotype termed overproduction lethality. In such strains, high-level synthesis of the cognate hybrid protein interferes with the process of protein export, and this leads ultimately to cell death. A variation of this phenomenon has been discovered with lacZ fusions to the gene specifying the major outer membrane porin protein OmpF. In this case, we find that lambda transducing phage carrying an ompF-lacZ fusion will not grow on a host strain that constitutively overexpresses ompF. We have exploited this observation to develop a selection for ompF mutants. Using this protocol, we have isolated mutants altered in ompF expression and have identified mutations that block OmpF export. Our results suggest that it should be possible to adapt this selection for use with other genes specifying exported proteins.
Heat shock response of Neurospora crassa: protein synthesis and induced thermotolerance
Authors: Plesofsky-Vig N , Brambl R
Published: January 2020

At elevated temperatures, germinating conidiospores of Neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 Mr and one minor heat shock protein of 30,000 Mr. Postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 Mr and a minor heat shock protein of 34,000 Mr. The three heat shock proteins of lower molecular weight are associated with mitochondria. This exclusive synthesis of heat shock proteins is transient, and after 60 min of exposure to high temperatures, restoration of the normal pattern of protein synthesis is initiated. Despite the transiency of the heat shock response, spores incubated continuously at 45 degrees C germinate very slowly and do not grow beyond the formation of a germ tube. The temperature optimum for heat shock protein synthesis is 45 degrees C, but spores incubated at other temperatures from 40 through 47 degrees C synthesize heat shock proteins at lower rates. Survival was high for germinating spores exposed to temperatures up to 47 degrees C, but viability declined markedly at higher temperatures. Germinating spores survived exposure to the lethal temperature of 50 degrees C when they had been preexposed to 45 degrees C; this thermal protection depends on the synthesis of heat shock proteins, since protection was abolished by cycloheximide. During the heat shock response mitochondria also discontinue normal protein synthesis; synthesis of the mitochondria-encoded subunits of cytochrome c oxidase was as depressed as that of the nucleus-encoded subunits.