bluuebunny/crossref_metadata_2025 huggingface dataset:
High-affinity glucose transport in Saccharomyces cerevisiae is under general glucose repression control
Authors: Bisson L F
Published: November 2016

Saccharomyces cerevisiae mutants defective in growth on low glucose concentration (lgn mutants) were isolated and screened for abnormal glucose transport. Nine complementation groups were identified, falling into two broad groups: those unable to significantly derepress high-affinity (low-Km) glucose uptake (lgn1, lgn4, lgn5, lgn7, and lgn8), and those with elevated repressed levels of high-affinity uptake that either derepress to normal or near normal levels of high-affinity uptake with loss of low-affinity transport (lgn2 and lgn3) or derepress only slightly, appearing to have an intermediate yet constitutive level of high-affinity transport (lgn6 and lgn9). Further analysis of the lgn mutations revealed pleiotropic phenotypes most consistent with the true defect being in regulation or expression of glucose repression and derepression. The kinetics of glucose uptake in strains carrying known mutations preventing derepression of glucose-repressible functions (snf1, snf2, snf4, and snf6) demonstrated that three of these mutations (snf1, snf4, and snf6) were similarly defective in derepression of high-affinity glucose uptake. The snf2 and snf5 mutations had no apparent effect on glucose uptake. Two mutations resulting in constitutive expression of glucose-repressible functions, cid1 and reg1, resulted in constitutive expression of high-affinity glucose uptake. These data support the conclusion that high-affinity glucose uptake in Saccharomyces cerevisiae is under general glucose repression control. The implications of other properties of these mutants are discussed.
Specificity of pyoverdine-mediated iron uptake among fluorescent Pseudomonas strains
Authors: Hohnadel D , Meyer J M
Published: November 2016

Pyoverdine-mediated iron transport was determined for seven fluorescent Pseudomonas strains belonging to different species. For all strains, cell or cell outer membrane and iron(III)-pyoverdine combinations were compared with their homologous counterparts in uptake, binding, and cross-feeding experiments. For four strains (Pseudomonas putida ATCC 12633, Pseudomonas fluorescens W, P. fluorescens ATCC 17400, and Pseudomonas tolaasii NCPPB 2192), the pyoverdine-mediated iron transport appeared to be strictly strain specific; pyoverdine-facilitated iron uptake by iron-starved cells and binding of ferripyoverdine to the purified outer membranes of such cells were efficient only in the case of the homologous systems. Cross-feeding assays, in liquid or solid cultures, resulted, however, especially for P. fluorescens ATCC 17400, in some discrepancies compared with uptake and binding assays, suggesting that growth experiments are the least likely to yield correct information on specificity of the pyoverdine-mediated iron transport. For the three other strains (P. fluorescens ATCC 13525, P. chlororaphis ATCC 9446, and P. aeruginosa ATCC 15692), cross-reactivity was demonstrated by the uptake, binding, and cross-feeding experiments. In an attempt to determine which parts of the iron transport system were responsible for the specificity, the differences in amino acid composition of the pyoverdines, together with the differences observed at the level of the iron-sensitive outer membrane protein pattern of the seven strains, are discussed.
Localization of the cis-acting regulatory DNA sequences of the Myxococcus xanthus tps and ops genes
Authors: Downard J S , Kim S H , Kil K S
Published: November 2016

The cis-acting regulatory regions of the tps and ops genes of Myxococcus xanthus were localized by analyzing the expression of fusions of these genes with the lacZ gene. A 201-base-pair (bp) fragment of tps DNa extending 95 bp upstream (-95) from the transcriptional start was sufficient to direct developmentally regulated expression of fusion gene activity. The segment of tps DNA between -95 and -81 contained information necessary for developmental regulation. A segment of ops DNa extending upstream to -131 directed a very low level of ops-lacZ fusion expression, but the inclusion of DNA to -208 greatly increased the amount of developmentally regulated expression. M. xanthus DNA upstream from -108 in the tps gene and -311 in the ops gene was required for maximal expression of gene fusion activity. The upstream regulatory regions of both the tps and ops genes seem to be involved in positive transcriptional regulation. Two mutations, a deletion of 1 bp at -8 in the tps gene and a 3-bp substitution at -27 to -29 in the ops gene, greatly increased the level of vegetative expression of gene fusion activity, suggesting that both genes may also be subject to negative regulation in M. xanthus.
Tn5-mediated transposition of plasmid DNA after transduction to Myxococcus xanthus
Authors: Downard J S
Published: November 2016

After coliphage P1-mediated transfer of Tn5-containing plasmid DNA from Escherichia coli to Myxococcus xanthus, transductants were identified which contained plasmid sequences integrated at many sites on the bacterial chromosome. The unaltered plasmid DNA sequences in these transductants were apparently flanked by intact Tn5 or IS50 sequences. These results suggest that Tn5-mediated transposition has occurred and provide a method for integrating plasmid DNA into the M. xanthus chromosome without the requirement for homologous recombination.
RNA polymerase as a repressor of transcription in the hut(P) region of mutant Klebsiella aerogenes histidine utilization operons
Authors: Nieuwkoop A J , Bender R A
Published: November 2016

Mutants of Klebsiella aerogenes able to express the hutUH operon in the absence of positive effectors were isolated and characterized. These mutations improve the hutUH promoter (PUH) by changing the -10 region to match the consensus sequence more closely. These mutations also affect another, oppositely oriented promoter in this region, PC. Although the mutations lie far outside PC, they cause PC to be inactive, apparently because binding of RNA polymerase to the PUH promoter blocks the overlapping PC site. Thus, in the mutants, RNA polymerase bound at the strong (mutant) PUH site effectively repress the PC promoter.
Induction of the autolytic system of Escherichia coli by specific insertion of bacteriophage MS2 lysis protein into the bacterial cell envelope
Authors: Walderich B , Ursinus-Wössner A , van Duin J , Höltje J V
Published: November 2016

Bacterial lysis induced by the expression of the cloned lysis gene of the RNA bacteriophage MS2 in Escherichia coli was shown to be under the same regulatory control mechanisms as penicillin-induced lysis. It was controlled by the stringent response and showed the phenomenon of tolerance when E. coli was grown at pH 5. Changes in the fine structure of the murein were found to be the earliest physiological changes in the cell, taking place 10 min before the onset of cellular lysis and inhibition of murein synthesis. Both the average length of the glycan strands and, with a time lag, the degree of cross-linkage were altered, indicating that a lytic transglycosylase and a DD-endopeptidase had been triggered. After extensive separation of the membranes by isopycnic sucrose gradient centrifugation, the lysis protein was present predominantly in the cytoplasmic membrane and in a fraction of intermediate density and, to a lesser degree, in the outer membrane, irrespective of the conditions of growth. However, only under lysis-permissive conditions could a 17% increase in the number of adhesion sites between the inner and outer membranes be observed. Thus, a casual relationship between lysis and the formation of lysis protein-induced adhesion sites seems to exist.
Membrane-bound phosphatases in Escherichia coli: sequence of the pgpB gene and dual subcellular localization of the pgpB product
Authors: Icho T
Published: November 2016

The phosphatidyl glycerophosphate B phosphatase of Escherichia coli has a multiple substrate specificity and a peculiar dual subcellular localization in the envelope. Its phosphatidyl glycerophosphate phosphatase activity is higher in the cytoplasmic membrane, while phosphatidic acid and lysophosphatidic acid phosphatase activities are higher in the outer membrane. The DNA sequencing of the pgpB gene revealed a protein of 251 amino acids which had at least five hydrophobic membrane-spanning regions. About 37 hydrophilic residues in the middle of the sequence had considerable homology with the C-terminal conserved region of the ras family genes in eucaryotes. A protein of 28,000 daltons was expressed from the pgpB gene under a tac promoter in a runaway replication plasmid. This overproduced protein also revealed the dual subcellular localization.
Peptidoglycan biosynthesis in stationary-phase cells of Escherichia coli
Authors: Blasco B , Pisabarro A G , de Pedro M A
Published: November 2016

The ability of stationary-phase cells of Escherichia coli W7 to incorporate radioactive precursors into macromolecular murein has been studied. During the initial 6 h of the stationary phase, resting cells incorporated meso-[3H]diaminopimelic acid at a rate corresponding to the insertion of 1.3 X 10(4) disaccharide units min-1 cell-1. Afterwards, the rate of incorporation dropped drastically (90%) to a low but still detectable level. Incorporation during stationary phase did not result in an increased amount of total murein in the culture, suggesting that it was related to a turnover process. Analysis of the effects of a number of beta-lactam antibiotics indicated that incorporation of murein precursors in stationary-phase cells was mediated by penicillin-binding proteins, suggesting that the activity of penicillin-binding protein 2 was particularly relevant to this process.
Purification and properties of the gamma-butyrobetaine-binding protein from an Agrobacterium sp
Authors: Nobile S , Baccino D , Takagi T , Deshusses J
Published: November 2016

A binding protein for gamma-butyrobetaine was purified from osmotic shock fluid of an Agrobacterium sp. It was a monomeric protein with an apparent molecular weight of 52,000 or 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The isoelectric point was 4.3, as determined by isoelectric focusing. Amino acid analysis of the protein showed that Asx and Glx were predominant components and that the protein contained no cysteine. The dissociation constant of this protein for gamma-butyrobetaine was found to be 0.7 microM by equilibrium dialysis. Attempts to sequence the amino-terminal end with the Edman method failed, suggesting that this region of the protein is blocked.
Mutagenic nucleoside analog N4-aminocytidine: metabolism, incorporation into DNA, and mutagenesis in Escherichia coli
Authors: Negishi K , Tamanoi K , Ishii M , Kawakami M , Yamashita Y , Hayatsu H
Published: November 2016

N4-Aminocytidine, a nucleoside analog, is strongly mutagenic to various organisms including Escherichia coli. Using E. coli WP2 (trp), we measured the incorporation of [5-3H]N4-aminocytidine into DNA and at the same time measured the frequency of reversion of the wild type, thereby attempting to correlate the incorporation with mutation induction. First, we observed that N4-aminocytidine uptake by the E. coli cells was as efficient as cytidine uptake. High-pressure liquid chromatographic analysis of nucleoside mixtures obtained by enzymatic digestion of isolated cellular DNA showed that the DNA contained [3H]N4-aminodeoxycytidine, corresponding to 0.01 to 0.07% of the total nucleoside; the content was dependent on the dose of N4-aminocytidine. There was a linear relationship between the N4-aminocytosine content in DNA and the mutation frequency observed. These results constitute strong evidence for the view that the N4-aminocytidine-induced mutation in E. coli is caused by the incorporation of this agent into DNA as N4-aminodeoxycytidine. We also found that the major portion of radioactivity in DNA of cells that had been treated with [5-3H]N4-aminocytidine was in the deoxycytidine fraction. We propose a metabolic pathway for N4-aminocytidine in cells of E. coli. This pathway involves the formation of both N4-aminodeoxycytidine 5'-triphosphate and deoxycytidine 5'-triphosphate; the deoxycytidine 5'-triphosphate formation is initiated by conversion of N4-aminocytidine into uridine. In support of this proposed scheme, a cytidine deaminase preparation obtained from E. coli catalyzed the decomposition of N4-aminocytidine into uridine and hydrazine.
Chromosomal genes essential for stable maintenance of the mini-F plasmid in Escherichia coli
Authors: Niki H , Ichinose C , Ogura T , Mori H , Morita M , Hasegawa M , Kusukawa N , Hiraga S
Published: November 2016

We have isolated mutants of Escherichia coli which do not support stable maintenance of mini-F plasmids (delta ccd rep+ sop+). These host mutations, named hop, were classified into five linkage groups on the E. coli chromosome. Genetic analyses of these hop mutations by Hfr mating and P1 transduction showed their loci on the E. coli genetic map to be as follows: hopA in the gyrB-tnaA region, hopB in the bglB-oriC region, hopD between 8 and 15 min, and hopE in the argA-thyA region. Kinetics of stability of the sop+ and delta sop mini-F plasmids in these hop mutants suggest that the hopA mutants are defective in partitioning of mini-F rather than in plasmid replication. The hopB, hopC, and hopD mutants were partially defective in replication of mini-F. The physical structure of the plasmid DNA was normal in hopA, B, C, and D mutants. Large amounts of linear multimers of plasmid DNA accumulated in mutants of the fifth linkage group (hopE). None of the hop mutations in any linkage group affected the normal growth of cells.
Identification and characterization of the Myxococcus xanthus bsgA gene product
Authors: Gill R E , Bornemann M C
Published: November 2016

The bsgA mutants of Myxococcus xanthus are blocked at a very early stage of the developmental program. They fail to produce fruiting bodies or to sporulate under normal conditions but can be rescued by extracellular complementation in mixtures with wild-type cells. A bsgA-lacZ gene fusion was constructed and expressed in Escherichia coli. The resulting fusion protein, which has beta-galactosidase enzyme activity, was partially purified by affinity chromatography and preparative polyacrylamide gel electrophoresis. The protein was used to immunize mice, which produced a hybridoma secreting monoclonal antibody that was specific for the bsgA gene product. The monoclonal antibody was used in Western blot (immunoblot) experiments to determine the apparent cellular location of the bsgA protein in M. xanthus and to compare the level of this protein at various times in the Myxococcus life cycle.
Pathway and sites for energy conservation in the metabolism of glucose by Selenomonas ruminantium
Authors: Melville S B , Michel T A , Macy J M
Published: November 2016

On the basis of enzyme activities detected in extracts of Selenomonas ruminantium HD4 grown in glucose-limited continuous culture, at a slow (0.11 h-1) and a fast (0.52 h-1) dilution rate, a pathway of glucose catabolism to lactate, acetate, succinate, and propionate was constructed. Glucose was catabolized to phosphoenol pyruvate (PEP) via the Emden-Meyerhoff-Parnas pathway. PEP was converted to either pyruvate (via pyruvate kinase) or oxalacetate (via PEP carboxykinase). Pyruvate was reduced to L-lactate via a NAD-dependent lactate dehydrogenase or oxidatively decarboxylated to acetyl coenzyme A (acetyl-CoA) and CO2 by pyruvate:ferredoxin oxidoreductase. Acetyl-CoA was apparently converted in a single enzymatic step to acetate and CoA, with concomitant formation of 1 molecule of ATP; since acetyl-phosphate was not an intermediate, the enzyme catalyzing this reaction was identified as acetate thiokinase. Oxalacetate was converted to succinate via the activities of malate dehydrogenase, fumarase and a membrane-bound fumarate reductase. Succinate was then excreted or decarboxylated to propionate via a membrane-bound methylmalonyl-CoA decarboxylase. Pyruvate kinase was inhibited by Pi and activated by fructose 1,6-bisphosphate. PEP carboxykinase activity was found to be 0.054 mumol min-1 mg of protein-1 at a dilution rate of 0.11 h-1 but could not be detected in extracts of cells grown at a dilution rate of 0.52 h-1. Several potential sites for energy conservation exist in S. ruminantium HD4, including pyruvate kinase, acetate thiokinase, PEP carboxykinase, fumarate reductase, and methylmalonyl-CoA decarboxylase. Possession of these five sites for energy conservation may explain the high yields reported here (56 to 78 mg of cells [dry weight] mol of glucose-1) for S. ruminantium HD4 grown in glucose-limited continuous culture.
Regulation of carbon flow in Selenomonas ruminantium grown in glucose-limited continuous culture
Authors: Melville S B , Michel T A , Macy J M
Published: November 2016

We have applied a model that permits the estimation of the sensitivity of flux through branch point enzymes (D. C. LaPorte, K. Walsh, and D. E. Koshland, J. Biol. Chem. 259:14068-14075, 1984) in order to analyze the control of flux through the lactate-acetate branch point of Selenomonas ruminantium grown in glucose-limited continuous culture. At this branch point, pyruvate is the substrate of both the NAD-dependent L-(+)-lactate dehydrogenase (LDH) and the pyruvate:ferredoxin oxidoreductase (PFOR). The LDH was purified, and it exhibited positive cooperativity for the binding of pyruvate. The LDH had an [S].5 for pyruvate of 0.43 mM, a Hill coefficient of 2.4, and a K' equal to 0.13 mM. The PFOR, assayed in cell extracts, exhibited Michaelis-Menten kinetics for pyruvate, with a Km of 0.49 mM. Carbon flux through the LDH and the PFOR increased 80-fold and 3-fold, respectively, as the dilution rate was increased from 0.07 to 0.52 h-1 in glucose-limited continuous culture. There was nearly a twofold increase, from 6.5 to 11.2 mumol min-1 mg of protein-1 in the specific activity (i.e., maximum velocity) of the LDH at dilution rates of 0.11 and 0.52 h-1, respectively. A flux equation was used to calculate the intracellular concentration of pyruvate; a fourfold increase in pyruvate, from 0.023 to 0.093 mM, was thereby predicted as the dilution rate was increased from 0.07 to 0.52 h-1. When these calculated values of intracellular pyruvate concentration were inserted into the flux equation, the predicted values of flux through the LDH and the PFOR were found to match closely the flux actually measured in the chemostat-grown cells. Thus, the 80-fold increase in flux through the LDH was due to a twofold increase in the maximum velocity of the LDH and a fourfold increase in the intracellular pyruvate concentration. In addition, the flux through the LDH exhibited ultrasensitivity to changes in both the maximum velocity of the LDH and the intracellular concentration of pyruvate. The flux through the PFOR exhibited ultrasensitivity to changes in the maximum velocity of the LDH and hyperbolic sensitivity to changes in the intracellular concentration of pyruvate.
Localization of the kdsA gene with the aid of the physical map of the Escherichia coli chromosome
Authors: Woisetschläger M , Hödl-Neuhofer A , Högenauer G
Published: November 2016

The isolation and analysis of two recombinant plasmids containing the kdsA gene from Escherichia coli chromosomal gene libraries is reported. The subfragments obtained from the inserts correspond to the fragment pattern around coordinate 1,282 kilobases of the physical map of the E. coli chromosome (Kohara et al. Cell 50:495-508, 1987). The kdsA gene has been located at coordinates 1,282 through 1,283 kilobases, corresponding to min 26.7 in the classical map coordinates. The kdsA gene is transcribed from this position toward the nearby nar gene.
Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein
Authors: Dooley J S , Trust T J
Published: November 2016

The surface protein composition of members of a serogroup of Aeromonas hydrophila which exhibit high virulence for fish was examined. Treatment of whole cells of representative strain A. hydrophila TF7 with 0.2 M glycine buffer (pH 4.0) resulted in the release of sheets of a tetragonal surface protein array. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis showed that this sheet material was composed primarily of a protein of apparent molecular weight 52,000 (52K protein). A 52K protein was also the predominant protein in glycine extracts of other members of the high-virulence serogroup. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed the 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase 125I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size was required for A. hydrophila S-layer anchoring. The 52K S-layer protein exhibited hear-dependent SDS-solubilization behavior when associated with OM, but was fully solubilized at all temperatures after removal from the OM, indicating a strong interaction of the S layer with the underlying OM. The native S layer was permeable to 125I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behaviour characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein.
Rapid response to osmotic upshift by osmoregulated genes in Escherichia coli and Salmonella typhimurium
Authors: Jovanovich S B , Martinell M , Record M T , Burgess R R
Published: November 2016

The rapid in vivo response of both Escherichia coli and Salmonella typhimurium osmoregulated genes to an osmotic upshift was analyzed in detail by using chromosomal operon fusions. Within 10 min after the addition of 0.3 M NaCl to the culture medium, the differential rates of expression of both an S. typhimurium proU-lac fusion and a proP-lac fusion increased by 180- and 17-fold respectively, while an E. coli ompC-lac fusion increased by 3.4-fold. For all three stimulated promoters, the increased rate of expression was maintained until about 40 min after the osmotic upshift. Thereafter, proU expression continued at a steady-state rate that was 27-fold higher than that of the control, while proP and ompC expression fell to 1.4- and 2-fold of the control rates, respectively. In contrast, expression of an E. coli ompF-lac fusion decreased twofold within 2.5 min. For proU, the length of the lag phase, which preceded the onset of the rapid response, increased with the degree of osmotic upshift, above a threshold of 0.2 M NaCl; the onset of the rapid proU response also preceded the resumption of growth. The rapid response phase, which was first quantitated for proU, proP, ompC, and ompF in this study, is an important component of the osmoregulation of these promoters. The addition of the osmoprotectant glycine betaine at the time of osmotic upshift decreased both the length of the rapid response and the subsequent steady-state of expression of proU.
Characterization of a DNA uptake reaction through the nuclear membrane of isolated yeast nuclei
Authors: Tsuchiya E , Shakuto S , Miyakawa T , Fukui S
Published: November 2016

Isolated yeast nuclei were able to incorporate 3H-labeled pJDB219 DNA in vitro in the presence of ATP and Mg2+. The number of plasmid molecules incorporated into each nucleus was calculated to be 60 under the conditions we used. Enzyme-histochemical staining of the incorporated biotinylated pJDB219 with streptavidin-biotinylated-peroxidase complex indicated a uniform distribution of the incorporated plasmids within each nucleus. After intranuclear incorporation, substrate pJDB219 DNAs (open and closed circular forms) were changed to the linear form and were weakly digested over the longer incubation period (over 60 min). Facile release of the once-incorporated plasmid DNA was never observable; discharge of the incorporated [3H]pJDB219 during a 60-min incubation was less than 5%. The addition of adenylyl-imidodiphosphate, N,N'-dicyclohexylcarbodiimide (DCCD), or quercetin inhibited in vitro DNA uptake reaction. DCCD and quercetin inhibited the nuclear ATPase and apparent protein kinase, respectively; hence, the involvement of these enzymes in the nuclear DNA transport system was suggested.
Regulation of nitrogenase gene expression in anaerobic cultures of Anabaena variabilis
Authors: Helber J T , Johnson T R , Yarbrough L R , Hirschberg R
Published: November 2016

Derepression of nitrogenase gene expression was studied at the mRNA and enzyme activity levels in anaerobic cultures of Anabaena variabilis 29413. Cells, previously grown with ammonium chloride, were incubated in the absence of fixed nitrogen compounds under an Ar atmosphere with dichlorophenyldimethyl-urea present to inhibit oxygen evolution. The appearance of nitrogenase mRNA (measured by dot blot hybridization analysis) and nitrogenase activity (measured as acetylene-reducing activity) was followed, and the cells were also observed by phase-contrast microscopy. Nitrogenase mRNA could be detected after 1.5 to 2.0 h of nitrogen starvation; enzyme activity appeared about 1 h later. Although enzyme activity increased for many hours, mRNA levels reached a steady state rapidly. Neither heterocysts nor proheterocysts formed under these conditions; however, the cells were observed to shrink and become chlorotic. When anaerobic, derepressed cultures were exposed to oxygen, nitrogenase mRNA levels decreased very rapidly.
Characterization of the Pseudomonas aeruginosa recA gene: the Les- phenotype
Authors: Kokjohn T A , Miller R V
Published: November 2016

The Les- phenotype (lysogeny establishment deficient) is a pleiotropic effect of the lesB908 mutation of Pseudomonas aeruginosa PAO. lesB908-containing strains are also (i) deficient in general recombination, (ii) sensitive to UV irradiation, and (iii) deficient in UV-stimulated induction of prophages. The P. aeruginosa recA-containing plasmid pKML3001 complemented each of these pleiotropic characteristics of the lesB908 mutation, supporting the hypothesis that lesB908 is an allele of the P. aeruginosa recA gene. The phenotypic effects of the lesB908 mutation may be best explained by the hypothesis that the lesB908 gene product is altered in such a way that it has lost synaptase activity but possesses intrinsic protease activity in the absence of DNA damage. The Les- phenotype is a result of the rapid destruction of newly synthesized phage repressor, resulting in lytic growth of the infecting virus. This hypothesis is consistent with the observations that increasing the number of copies of the phage repressor gene by increasing the multiplicity of infection (i.e., average number of phage genomes per cell) or by introducing the cloned phage repressor gene into a lesB908 mutant will also suppress the Les- phenotype in a phage-specific fashion.
The frdR gene of Escherichia coli globally regulates several operons involved in anaerobic growth in response to nitrate
Authors: Kalman L V , Gunsalus R P
Published: November 2016

Fumarate reductase catalyzes the terminal step of anaerobic electron transport with fumarate as a terminal electron acceptor. Transcription of the fumarate reductase (frdABCD) operon in Escherichia coli is repressed in the presence of the preferred terminal electron acceptors, oxygen and nitrate. To identify trans-acting genes involved in regulation by nitrate, a number of E. coli mutants were generated in which expression of a frdA'-'lacZ protein fusion was no longer fully repressed by nitrate. One of these mutants, strain LK23R35, exhibited 17-fold higher beta-galactosidase activity than the wild-type strain when grown anaerobically in the presence of nitrate. When grown aerobically in the presence of nitrate, it contained three- to fourfold more beta-galactosidase activity than the wild-type strain did. Oxygen regulation of frd expression, however, was unaffected by the mutation, since the level of beta-galactosidase activity in both strains was nearly identical when they were grown in the absence of nitrate either aerobically or anaerobically. To confirm that the mutation acts in trans to frdABCD, we measured fumarate reductase levels and found them to parallel FrdA'-beta-galactosidase activity under all growth conditions tested. The effect of the mutation is pleiotropic, since the levels of nitrate reductase in LK23R35 were not induced by the addition of nitrate. The frdR mutant was also derepressed for nitrate control of the trimethylamine-N-oxide reductase and alcohol dehydrogenase enzymes. The mutation maps in a region between trp and hemA at 27 min on the E. coli chromosome. This gene, where we call frdR, is involved in both positive and negative regulation of electron transport and fermentation associated genes. A cloned 4.9-kilobase fragment of chromosomal DNA was found to complement the frdR mutation; both repression of fumarate reductase gene expression and activation of nitrate reductase gene expression were restored.
Genetic transformation in Streptococcus pneumoniae: molecular cloning and characterization of recP, a gene required for genetic recombination
Authors: Rhee D K , Morrison D A
Published: November 2016

A 225-base-pair fragment of a recombination gene was identified by insertion-duplication mutagenesis and used as a radioactive probe to clone the corresponding rec locus from Streptococcus pneumoniae in Escherichia coli plasmid vectors. Attempts to clone large pieces of this locus were unsuccessful, but small pieces of DNA from this region were cloned in the E. coli transcriptional terminator vectors pKK232-8 and pJDC9. The extent of the rec region, 2.1 to 2.2 kilobases, was defined by determining the competence phenotype of insertion mutations constructed in vitro. A deletion of the rec locus showed it to be necessary for chromosomal integration but not for plasmid establishment. A plasmid carrying the entire locus encoded a 72-kilodalton polypeptide in a cell-free E. coli transcription-translation system.
Metabolism of periplasmic membrane-derived oligosaccharides by the predatory bacterium Bdellovibrio bacteriovorus 109J
Authors: Ruby E G , McCabe J B
Published: November 2016

Membrane-derived oligosaccharides (MDO), a class of osmotically active carbohydrates, are the major organic solutes present in the periplasm of Escherichia coli and many other gram-negative bacteria when cells are grown in a medium of low osmolarity. Analyses of growing cells of Bdellovibrio bacteriovorus, a gram-negative predator of other bacteria, have confirmed that they also synthesize a characteristic MDO-like class of oligosaccharides. The natural growth environment of bdellovibrios is the periplasm of other gram-negative bacteria. Because of this location, prey cell MDO constitute a potential source of organic nutrients for growing bdellovibrios. Using cells of E. coli whose MDO were 3H labeled, we examined the extent to which B. bacteriovorus 109J metabolizes these prey cell components. Interestingly, there was neither significant degradation nor incorporation of prey cell MDO by bdellovibrios during the course of their intracellular growth. In fact, bdellovibrios had little capability either to degrade extracellular MDO that was made available to them or to transport glucose, the major monomeric constituent of prey cell MDO. Instead, periplasmic MDO were irreversibly lost to the extracellular environment during the period of bdellovibrio attack and penetration. Thus, although prey cell periplasmic proteins are retained, other important periplasmic components are released early in the bdellovibrio growth cycle. The loss of these MDO may aid in the destabilization of the prey cell plasma membrane, increasing the availability of cytoplasmic constituents to the periplasmic bdellovibrio.
Metabolic control and autogenous regulation of nit-3, the nitrate reductase structural gene of Neurospora crassa
Authors: Fu Y H , Marzluf G A
Published: November 2016

In Neurospora crassa, the expression of nit-3, the structural gene which encodes nitrate reductase, is highly regulated and requires both nitrate induction and nitrogen catabolite derepression. The major nitrogen regulatory gene, nit-2, acts in a positive fashion to turn on the expression of nit-3 and other nitrogen-related genes during nitrogen derepression. A second regulatory gene, designated nmr, acts in a negative fashion to repress the expression of nitrate reductase and related enzymes, and nmr mutants are partially insensitive to nitrogen repression. Using cloned genes as specific hybridization probes, we demonstrated that nmr does not affect the transcription of nit-2 but does appear to control nit-3 gene expression. Unlike nmr+ expression, nit-3 expression occurred to some degree even under nitrogen repression conditions in nmr mutant cells. In wild-type cells, nitrate reductase gene expression was dependent upon the presence of nitrate as an inducer. In sharp contrast, nit-3 mRNA expression occurred to a full extent in three different nit-3 mutants, even in the complete absence of any added inducer. Similarly, a nit-1 mutant which was devoid of nitrate reductase activity because it lacked an essential molybdenum cofactor expressed nit-3 without a requirement for induction by nitrate. These results suggest that nitrate reductase autogenously regulates its own expression and that this control is exerted at the transcriptional level.
Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium
Authors: Kramer G F , Ames B N
Published: November 2016

Selenium is a constituent in Escherichia coli of the anaerobic enzyme formate dehydrogenase in the form of selenocysteine. Selenium is also present in the tRNA of E. coli in the modified base 5-methylaminomethyl-2-selenouracil (mnm5Se2U). The pathways of bacterial selenium metabolism are largely uncharacterized, and it is unclear whether nonspecific reactions in the sulfur metabolic pathways may be involved. We demonstrated that sulfur metabolic pathway mutants retain a wild-type pattern of selenium incorporation, indicating that selenite (SeO32-) is metabolized entirely via selenium-specific pathways. To investigate the function of mnm5Se2U, we isolated a mutant which is unable to incorporate selenium into tRNA. This strain was obtained by isolating mutants lacking formate dehydrogenase activity and then screening for the inability to metabolize selenium. This phenotype is the result of a recessive mutation which appears to map in the general region of 21 min on the Salmonella typhimurium chromosome. A mutation in this gene, selA, thus has a pleiotropic effect of eliminating selenium incorporation into both protein and tRNA. The selA mutant appears to be blocked in a step of selenium metabolism after reduction, such as in the actual selenium insertion process. We showed that the absence of selenium incorporation into suppressor tRNA reduces the efficiency of suppression of nonsense codons in certain contexts and when wobble base pairing is required. Thus, one function of mnm5Se2U in tRNA may be in codon-anticodon interactions.
Disruption of the Escherichia coli cls gene responsible for cardiolipin synthesis
Authors: Nishijima S , Asami Y , Uetake N , Yamagoe S , Ohta A , Shibuya I
Published: November 2016

The cls gene of Escherichia coli is responsible for the synthesis of a major membrane phospholipid, cardiolipin, and has been proposed to encode cardiolipin synthase. This gene cloned on a pBR322 derivative was disrupted by either insertion of or replacement with a kanamycin-resistant gene followed by exchange with the homologous chromosomal region. The proper genomic disruptions were confirmed by Southern blot hybridization and a transductional linkage analysis. Both types of disruptants had essentially the same properties; cardiolipin synthase activity was not detectable, but the strains grew well, although their growth rates and final culture densities were lower than those of the corresponding wild-type strains and strains with the classical cls-1 mutation. A disruptant harboring a plasmid that carried the intact cls gene grew normally. The results indicate that the cls gene and probably the cardiolipin synthase are dispensable for E. coli but may confer growth or survival advantages. Low but definite levels of cardiolipin were synthesized by all the disruptants. Cardiolipin content of the cls mutants depended on the dosage of the pss gene, and attempts to transfer a null allele of the cls gene into a pss-1 mutant were unsuccessful. We point out the possibilities of minor cardiolipin formation by phosphatidylserine synthase and of the essential nature of cardiolipin for the survival of E. coli cells.
Expression of an Agrobacterium Ti plasmid gene involved in cytokinin biosynthesis is regulated by virulence loci and induced by plant phenolic compounds
Authors: John M C , Amasino R M
Published: November 2016

The nopaline-type Ti plasmid T37 of Agrobacterium tumefaciens carries two distinct genes that encode enzymes involved in cytokinin biosynthesis. In this report, we show that the level of expression of one of these genes was increased dramatically by culture conditions that increased the expression of Ti plasmid virulence genes, including coculture with plant cells or treatment with acetosyringone, a plant phenolic compound. When this nopaline-type Ti plasmid gene was introduced into Agrobacterium strains containing an octopine-type Ti plasmid, similar induction of expression by culture conditions was observed, and analysis of virulence region mutants demonstrated that this induction was under the control of the virA and virG regulatory loci. We further show that induction was strongly pH dependent in octopine strains but, under the conditions examined, pH independent in nopaline strains.
Cloning and dependence pattern of the sporulation operon spoVH
Authors: Cutting S M , Mandelstam J
Published: November 2016

The spoVH locus, involved in the sporulation of Bacillus subtilis, was cloned in derivatives of the temperate bacteriophage luminal diameter 105. Two recombinant phages were obtained which contained 4.2 kilobases of chromosomal DNA. Both phages only partially complemented a mutation in the spoVH operon, spoVH516. Nevertheless, analysis of the cloned locus with integrational plasmids showed that the complete operon had been cloned. A spoVH'-lacZ transcriptional fusion was constructed, and this indicated that the spoVH operon was expressed 2.25 h after the start of sporulation. The distribution of beta-galactosidase in sporulating cells containing a spoVH'-lacZ fusion showed that spoVH was expressed in the spore compartment; lac fusion experiments were also used to study spoVH expression in the presence of other sporulation mutations. Expression of spoVH was prevented by mutations in any of the stage 0 or stage II loci and also by mutations in spoIIIA, spoIIIB, and spoIIIE. A similar pattern of dependence was found previously for the expression of spoVA, which is also expressed in the spore compartment.
Regulation of phosphatidylinositol kinase activity in Saccharomyces cerevisiae
Authors: Holland K M , Homann M J , Belunis C J , Carman G M
Published: November 2016

The effects of growth phase and carbon source on membrane-associated phosphatidylinositol kinase in cell extracts of Saccharomyces cerevisiae were examined. Phosphatidylinositol kinase activity increased 2- and 2.5-fold in glucose- and glycerol-grown cells, respectively, in the stationary phase as compared with the exponential phase of growth. The increase in phosphatidylinositol kinase activity in the stationary phase of growth correlated with an increase in the relative amounts of phosphatidylinositol 4-phosphate, the product of the reaction. The increase in phosphatidylinositol kinase activity was not due to the presence of water-soluble effector molecules in cell extracts as indicated by mixing experiments. Phosphatidylinositol kinase activity decreased in cell extracts of exponential-phase cells preincubated under phosphorylation conditions which favor cyclic AMP-dependent protein kinase activity. Phosphatidylinositol kinase activity was not affected in cell extracts of stationary-phase cells preincubated under phosphorylation conditions.
beta-Alanine auxotrophy associated with dfp, a locus affecting DNA synthesis in Escherichia coli
Authors: Spitzer E D , Jimenez-Billini H E , Weiss B
Published: November 2016

Strains containing the conditional-lethal dfp-707 mutation, which have a defect in DNA synthesis at 42 degrees C, were found to require either pantothenate or its precursor, beta-alanine, for growth at 30 degrees C. The auxotrophy and conditional lethality were corevertible. Through localized mutagenesis of the dfp-pyrE region of Escherichia coli, another mutation, dfp-1, was obtained. It conferred the auxotrophy but not the conditional lethality of dfp-707. Complementation analysis, performed with a set of plasmid-borne deletion and insertion mutations, revealed a correspondence between the complementation of each mutant phenotype and the production of the dfp gene product, previously identified as a 45-kilodalton flavoprotein. The dfp mutants had a normal level of aspartate-1-decarboxylase, which is the only enzyme known to produce beta-alanine in E. coli and which is specified by the distant panD gene. A prototrophic pseudorevertant of a dfp-1 strain was found to have retained the dfp mutation, to be genetically unstable, and to have an elevated level of aspartate-1-decarboxylase, suggesting that it had acquired a duplication of panD. It is not known what steps in pantothenate or DNA metabolism are affected by the mutant dfp product or how its flavin moiety may be involved.
Temporal control of transposition in Tn5
Authors: McCommas S A , Syvanen M
Published: November 2016

IS50R is an insertion sequence associated with the transposon Tn5. IS50R carries the structural genes for two proteins; one (P1) is the Tn5 transposase, and the other (P2) is an inhibitor of transposition. These two proteins are translated from two different transcripts, m1 and m2. When bacteriophage lambda::IS50R DNA was introduced into a bacterial cell, m1 and m2 were initially at relative levels of about 1 to 2. As time progressed the amount of m1 fell, whereas the amount of m2 continued to increase, until after about 3 h the ratio of m1 to m2 was about 1 to 80. The temporal changes in the levels of these transcripts correlated with temporal changes in P1 and P2 levels and Tn5 transposition that have been documented in other studies. We measured the stability of the messages and showed that the differences in the levels of m1 and m2 must reflect real differences in the strengths of their promoters and that the changes in transcription kinetics are mediated by the dam methylation system of the cell and are not determined by IS50R products. Our results show that the 5' end of m2 is about twice as stable as that of m1, which raises the possibility that differential message stability does, in part, influence the ratio of inhibitor to transposase.
nusA amber mutation that causes temperature-sensitive growth of Escherichia coli
Authors: Tsugawa A , Saito M , Court D L , Nakamura Y
Published: November 2016

The nusA134 mutation was isolated from a sup0 strain as a temperature-sensitive mutant which grew at 32 degrees C but not at 42 degrees C. Immunoblot analysis showed that this mutant produced a 31,000-dalton nusA-encoded protein instead of the full-size 54,500-dalton product. Sequence and genetic analyses of the mutant nusA gene revealed a substitution of T for C at the PstI site (i.e., CTGCAG to CTGTAG), thereby creating a nonsense UAG codon. These results indicate that nusA134 is an amber mutation and that the 31,000-dalton amber fragment is active for Escherichia coli growth at 32 degrees C but not at 42 degrees C. Most lambda bacteriophage variants tested grew normally on the nusA134 mutant both at permissive and at nonpermissive temperatures. However, lambda r32, which carries an IS2 insertion beyond the tR1 terminator, was restricted at 42 degrees C. Defects in the transcriptional antitermination process, but not in transcription termination, were observed. A comparative study of nusA134 protein and a PstI-truncated protein suggests that truncation of the peptide chain at the PstI site by the amber mutation, rather than the loss of the glutamine residue, is primarily responsible for the defect in antitermination. The mode of the involvement of mutant nusA proteins in the N-mediated antitermination reaction is discussed.
Regulation of expression of the cytochrome d terminal oxidase in Escherichia coli is transcriptional
Authors: Georgiou C D , Dueweke T J , Gennis R B
Published: November 2016

The cytochrome d complex is one of the two terminal oxidases in the aerobic respiratory system of Escherichia coli. This enzyme is not present in cells grown with high levels of dissolved oxygen in the culture medium but accumulates after mid-exponential growth, reaching high levels in stationary-phase cells. In this study, the transcriptional activity of the cyd operon, encoding the two subunits of the enzyme, was examined under a variety of growth conditions. This was accomplished by the use of a chromosomal operon fusion, cyd-lacZ, generated in vivo by a lambda plac-Mu hopper bacteriophage and also by the use of a cyd-lacZ protein fusion created in vitro on a plasmid, transferred onto a lambda transducing phage, and examined as a single-copy lysogen. Transcription of the gene fusions was monitored by determination of beta-galactosidase activity. The data clearly show that cyd is transcriptionally regulated and that induction is observed when the culture reaches a sufficient cell density so as to substantially reduce the steady-state levels of dissolved oxygen. The transcriptional activity is also regulated by other growth conditions, including the carbon source. The turn-on of cyd under semianaerobic conditions does not require the fnr gene product, cyclic AMP, or the cyclic AMP-binding protein.
The dnaK gene of Escherichia coli functions in initiation of chromosome replication
Authors: Sakakibara Y
Published: November 2016

A newly isolated dnaK mutant of Escherichia coli, which contains the mutation dnaK111, has been found to be conditionally defective in initiation of DNA replication. Mutant cells that were transferred to high temperature exhibited residual DNA synthesis before the synthesis stopped completely. Analysis of the DNA synthesized at high temperature by hybridization with probe DNAs for detection of DNA replicated in the origin (oriC) and terminal (terC) regions has revealed that this mutant is unable to initiate a new round of DNA replication at high temperature after termination of the round in progress. The cells exposed to high temperature were subsequently capable of initiating DNA replication at low temperature in a synchronous manner. DNA synthesis of this mutant became temperature resistant upon inactivation of the rnh gene, similar to that of dnaA mutants, although cell growth of the dnaK mutant with the inactive rnh gene remained temperature sensitive. The dnaK mutation prevented DNA synthesis of lambda bacteriophage at high temperature even in the absence of the rnh gene function.
Rheotactic behavior of a gliding mycoplasma
Authors: Rosengarten R , Klein-Struckmeier A , Kirchhoff H
Published: November 2016

Mycoplasma mobile, a new gliding mycoplasma isolated from the gills of a fish, was capable of positive rheotaxis; the cells glided upstream in a moving fluid. To our knowledge this is the first demonstration of rheotactic behavior among the procaryotes.