bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of gluconeogenesis by the glucitol enzyme III of the phosphotransferase system in Escherichia coli
Authors: Yamada M , Feucht B U , Saier M H
Published: November 2016

The gut operon was subcloned into various plasmid vectors (M. Yamada and M. H. Saier, Jr., J. Bacteriol. 169:2990-2994, 1987). Constitutive expression of the plasmid-encoded operon prevented utilization of alanine and Krebs cycle intermediates when they were provided as sole sources of carbon for growth. Expression of the gutB gene alone (encoding the glucitol enzyme III), subcloned downstream from either the lactose promoter or the tetracycline resistance promoter, inhibited utilization of the same compounds. On the other hand, overexpression of the gutA gene (encoding the glucitol enzyme II) inhibited the utilization of a variety of sugars as well as alanine and Krebs cycle intermediates by an apparently distinct mechanism. Phosphoenolpyruvate carboxykinase activity was greatly reduced in cells expressing high levels of the cloned gutB gene but was nearly normal in cells expressing high levels of the gutA gene. A chromosomal mutation in the gutR gene, which gave rise to constitutive expression of the chromosomal gut operon, also gave rise to growth inhibition on gluconeogenic substrates as well as reduced phosphoenolpyruvate carboxykinase activity. Phosphoenolpyruvate synthase activity in general varied in parallel with that of phosphoenolpyruvate carboxykinase. These results suggest that high-level expression of the glucitol enzyme III of the phosphotransferase system can negatively regulate gluconeogenesis by repression or inhibition of the two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthase.
Relationship between two major immunoreactive forms of arginase in Neurospora crassa
Authors: Borkovich K A , Weiss R L
Published: November 2016

Two major immunoreactive proteins of Mr 41,700 and 36,100 have been detected in crude mycelial extracts with polyclonal antibodies raised against arginase purified from Neurospora crassa. The latter corresponded to the protein used to obtain the antibodies. Both polypeptides were either missing or present in very low amounts in mutant strains having little or no detectable arginase activity. The relative proportion of the two species was altered in strains containing the nitrogen catabolite regulatory mutation nit-2. Peptide mapping indicated that the two species are very closely related, but several of the peptides which appeared to be identical by staining reacted differently with the antibodies. Both species were produced by in vitro translation of poly(A)+ mRNA, although the larger species was produced to a much smaller extent than was expected from its abundance in vivo. The results suggest the existence of multiple forms of arginase in N. crassa which differ in their response to nitrogen catabolite regulation.
Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae
Authors: Hottiger T , Schmutz P , Wiemken A
Published: November 2016

Heat shock resulted in rapid accumulation of large amounts of trehalose in Saccharomyces cerevisiae. In cultures growing exponentially on glucose, the trehalose content of the cells increased from 0.01 to 1 g/g of protein within 1 h after the incubation temperature was shifted from 27 to 40 degrees C. When the temperature was readjusted to 27 degrees C, the accumulated trehalose was rapidly degraded. In parallel, the activity of the trehalose-phosphate synthase, the key enzyme of trehalose biosynthesis, increased about sixfold during the heat shock and declined to the normal level after readjustment of the temperature. Surprisingly, the activity of neutral trehalase, the key enzyme of trehalose degradation, also increased about threefold during the heat shock and remained almost constant during recovery of the cells at 27 degrees C. In pulse-labeling experiments with [14C]glucose, trehalose was found to be turned over rapidly in heat-shocked cells, indicating that both anabolic and catabolic enzymes of trehalose metabolism were active in vivo. Possible functions of the heat-induced accumulation of trehalose and its rapid turnover in an apparently futile cycle during heat shock are discussed.
Processing of Escherichia coli 16S rRNA with bacteriophage lambda leader sequences
Authors: Krych M , Sirdeshmukh R , Gourse R , Schlessinger D
Published: November 2016

To test whether any specific 5' precursor sequences are required for the processing of pre-16S rRNA, constructs were studied in which large parts of the 5' leader sequence were replaced by the coliphage lambda pL promoter and adjacent sequences. Unexpectedly, few full-length transcripts of the rRNA were detected after the pL promoter was induced, implying that either transcription was poor or most of the rRNA chains with lambda leader sequences were unstable. Nevertheless, sufficient transcription occurred to permit the detection of processing by S1 nuclease analysis. RNA transcripts in which 2/3 of the normal rRNA leader was deleted (from the promoter up to the normal RNase III cleavage site) were processed to form the normal 5' terminus. Thus, most of the double-stranded stem that forms from sequences bracketing wild-type 16S pre-rRNA is apparently not required for proper processing; the expression of such modified transcripts, however, must be increased before the efficiency of processing of the 16S rRNA formed can be assessed.
Identification of two ancillary subunits of Escherichia coli type 1 fimbriae by using antibodies against synthetic oligopeptides of fim gene products
Authors: Abraham S N , Goguen J D , Sun D , Klemm P , Beachey E H
Published: November 2016

We have chemically synthesized oligopeptides corresponding to the NH2-terminal stretch of two gene products, designated FimG and FimH, of the fim gene cluster of Escherichia coli. These synthetic peptides, designated S-T1FimG(1-16) and S-T1FimH(1-25)C, evoked antibodies in rabbits that reacted with 14- and 29-kilodalton subunits, respectively, of dissociated fimbriae encoded by the recombinant plasmid pSH2 carrying the genetic information for the synthesis and expression of functional type 1 fimbriae. Neither of these fimbrial proteins was detected in dissociated fimbrial preparations from nonadhesive E. coli cells carrying the mutant plasmid pUT2002, containing a restriction site-specific deletion of fimG and fimH. Anti-S-T1FimH(1-25)C inhibited the adherence of type 1 fimbriated E. coli to epithelial cells. Immunoelectron microscopy revealed that anti-S-T1FimH(1-25)C, but not anti-S-T1FimG(1-16), bound to intact type 1 fimbriae of E. coli at the fimbrial tips and at long intervals along the fimbrial filaments. Anti-S-T1FimG(1-16) appeared to be directed at epitopes not accessible on the intact fimbriae and consequently failed to bind to intact fimbriae or to block fimbrial attachment. Our results suggest that the fimG and fimH gene products are components of type 1 fimbriae and that FimH may be the tip adhesin mediating the binding of type 1 fimbriated E. coli to D-mannose residues on mucosal surfaces.
Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid
Authors: Wise K S , Kim M F
Published: November 2016

Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125I-labeled proteins with a series of monoclonal antibodies (MAbs). Surface proteins p70, p65, p50, and p44 were shown to be integral membrane components by selective partitioning into the hydrophobic phase during Triton X-114 (TX-114)-phase fractionation, whereas p41 was concomitantly identified as a surface protein exclusively partitioning into the aqueous phase. Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [35S]methionine, 14C-amino acids, or [3H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Covalent lipid attachment was established by high-performance liquid chromatography identification of [3H]methyl palmitate after acid methanolysis of delipidated proteins. An additional, unidentified methanolysis product suggested conversion of palmitate to another form of lipid also attached to these proteins. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a larger p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11. These studies establish that a highly restricted set of distinct, lipid-modified hydrophobic membrane proteins are major surface antigens of M. hyopneumoniae and that structural variants of surface antigens occur within this species.
Three-dimensional structure of the surface protein of Desulfurococcus mobilis
Authors: Wildhaber I , Santarius U , Baumeister W
Published: November 2016

The spherical cells of the thermophilic, sulfur-dependent archaebacterium Desulfurococcus mobilis are completely covered with a relatively poorly ordered, tetragonally arrayed surface protein. The structure of this surface protein was examined by using three-dimensional electron microscopy. The protein lattice forms an open meshwork composed of cross-shaped morphological units, which are released when glycerol is added. These subunits make contact at the distal ends of their four arms. The p4 symmetry requires that each of these morphological subunits represents a tetramer. The strong interaction of the monomers within the crosses and the relatively weak interaction of the intersecting arms of the crosses within the lattice structure suggest that the tetramers are assembled before their incorporation into the lattice.
Actinomycin synthesis in Streptomyces antibioticus: enzymatic conversion of 3-hydroxyanthranilic acid to 4-methyl-3-hydroxyanthranilic acid
Authors: Jones G H
Published: November 2016

A methyltransferase which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate was identified in detergent-treated extracts of the bacterium Streptomyces antibioticus. The enzyme catalyzes the transfer of methyl groups from [14C]S-adenosylmethionine to HAA, but does not catalyze the methylation of 3-hydroxy-DL-kynurenine. Enzyme, substrate, time, and pH dependencies for the methyl transfer reaction were examined. Reaction products obtained from scaled-up reaction mixtures were fractionated by chromatography on Dowex 1, and the Dowex 1 fractions were examined by paper and thin-layer chromatography. One Dowex fraction was shown to contain a radioactive product with the chromatographic properties of 4-methyl-3-hydroxyanthranilic acid (MHA), a known intermediate in the biosynthesis of actinomycin. Available evidence indicates that the conversion of HAA to MHA is an early step in the biosynthesis of actinomycin by S. antibioticus and other actinomycin-producing streptomycetes.
Unique phenotype of opaque cells in the white-opaque transition of Candida albicans
Authors: Anderson J M , Soll D R
Published: November 2016

Select strains of Candida albicans switch reversibly and at extremely high frequency between a white and an opaque colony-forming phenotype, which has been referred to as the white-opaque transition. Cells in the white phase exhibit a cellular phenotype indistinguishable from that of most standard strains of C. albicans, but cells in the opaque phase exhibit an unusually large, elongate cellular shape. In comparing the white and opaque cellular phenotypes, the following findings are demonstrated. (i) The surface of the cell wall of maturing opaque cells when viewed by scanning electron microscopy exhibits a unique pimpled, or punctate, pattern not observed in white cells or standard strains of C. albicans. (ii) The dynamics of actin localization which accompanies opaque-cell growth first follows the pattern of budding cells during early opaque-bud growth and then the pattern of hypha-forming cells during late opaque-bud growth. (iii) A hypha-specific cell surface antigen is also expressed on the surface of opaque budding cells. (iv) An opaque-specific surface antigen is distributed in a punctate pattern.
Cloning of genes that suppress an Escherichia coli K-12 alanine auxotroph when present in multicopy plasmids
Authors: Wang M D , Buckley L , Berg C M
Published: November 2016

To facilitate molecular analyses of a previously uncharacterized gene involved in alanine synthesis, attempts were made to clone the wild-type allele of this gene, alaA, with a mini-Mu plasmid element used for in vivo cloning. Seventy-six independent Ala+ plasmids were isolated and characterized. Physiological, enzymological, and restriction endonuclease analyses indicated that three different genes, none of them alaA, were cloned. These genes were avtA+, which encodes the alanine-valine transaminase (transaminase C); tyrB+, which encodes the tyrosine-repressible transaminase (transaminase D); and a previously undescribed gene, called alaB, which encodes an alanine-glutamate transaminase.
Genetic studies of avermectin biosynthesis in Streptomyces avermitilis
Authors: Ikeda H , Kotaki H , Omura S
Published: November 2016

A genetic recombination study of an industrial strain of Streptomyces avermitilis which produces avermectin is described. A genetic map has been constructed by analysis of haploid recombinants and linkage relationships of 16 marker loci. Fifteen avermectin-nonproducing mutants, produced by mutagenesis, were classified into two phenotypically different groups, of which one produced avermectin aglycon and the other was able to convert avermectin aglycon to avermectins. Two different mutants were found to map closely to each other.
Production of heat shock protein is independent of cell cycle blockage in the yeast Saccharomyces cerevisiae
Authors: Barnes C A , Singer R A , Johnston G C
Published: November 2016

In response to certain environmental stresses, cells display a response characterized by the production of heat shock proteins. In this study we showed that blockage of cells of the yeast Saccharomyces cerevisiae at specific points in the mitotic cell cycle was not in itself a stress that induced the production of heat shock proteins. Nevertheless, cell cycle blockage did not preclude a normal heat shock response in arrested cells subjected to elevated temperatures.
Method for localization of cloned DNA fragments on the Escherichia coli chromosome
Authors: Fayet O , Prere M F
Published: November 2016

In exponentially growing cultures of Escherichia coli strains carrying the dnaC28 mutation, DNA replication can be synchronized by temperature changes (R. L. Rodriguez, M. S. Dalbey, and C. I. Davern, J. Mol. Biol. 74:599-604, 1973). We used this synchronization procedure and DNA-DNA hybridization to develop a technique for the localization of cloned chromosomal fragments on the genetic map. Because of the bidirectional nature of replication in E. coli, our method gave two possible positions (one on each replication arm). However because of the precision obtained for each position (+/- 1 map unit), the final mapping with various genetic techniques was greatly facilitated. Using this technique and a simple chromosomal mobilization test, we located at 93.2 +/- 1 min a cloned DNA fragment carrying an extragenic suppressor of dnaA46, a thermosensitive mutation in the dnaA initiation gene. Further analysis showed that the groES (mopA) and groEL (mopB) genes, both located at 94.2 min on the standard map, were indeed carried by the cloned suppressor fragment.
Glyceraldehyde-3-phosphate dehydrogenase gene from Zymomonas mobilis: cloning, sequencing, and identification of promoter region
Authors: Conway T , Sewell G W , Ingram L O
Published: November 2016

The gene encoding glyceraldehyde-3-phosphate dehydrogenase was isolated from a library of Zymomonas mobilis DNA fragments by complementing a deficient strain of Escherichia coli. It contained tandem promoters which were recognized by E. coli but appeared to function less efficiently than the enteric lac promoter in E. coli. The open reading frame for this gene encoded 337 amino acids with an aggregate molecular weight of 36,099 (including the N-terminal methionine). The primary amino acid sequence for this gene had considerable functional homology and amino acid identity with other eucaryotic and bacterial genes. Based on this comparison, the gap gene from Z. mobilis appeared to be most closely related to that of the thermophilic bacteria and to the chloroplast isozymes. Comparison of this gene with other glycolytic enzymes from Z. mobilis revealed a conserved pattern of codon bias and several common features of gene structure. A tentative transcriptional consensus sequence is proposed for Z. mobilis based on comparison of the five known promoters for three glycolytic enzymes.
In vivo mutagenesis of bacteriophage Mu transposase
Authors: Toussaint A , Desmet L , Faelen M , Alazard R , Chandler M , Pato M
Published: November 2016

We devised a method for isolating mutations in the bacteriophage Mu A gene which encodes the phage transposase. Nine new conditional defective A mutations were isolated. These, as well as eight previously isolated mutations, were mapped with a set of defined deletions which divided the gene into 13 100- to 200-base-pair segments. Phages carrying these mutations were analyzed for their ability to lysogenize and to transpose in nonpermissive hosts. One Aam mutation, Aam7110, known to retain the capacity to support lysogenization of a sup0 host (M. M. Howe, K. J. O'Day, and D. W. Shultz, Virology 93:303-319, 1979) and to map 91 base pairs from the 3' end of the gene (R. M. Harshey and S. D. Cuneo, J. Genet. 65:159-174, 1987) was shown to be able to complement other A mutations for lysogenization, although it was incapable of catalyzing either the replication of Mu DNA or the massive conservative integration required for phage growth. Four Ats mutations which map at different positions in the gene were able to catalyze lysogenization but not phage growth at the nonpermissive temperature. Phages carrying mutations located at different positions in the Mu B gene (which encodes a product necessary for efficient integration and lytic replication) were all able to lysogenize at the same frequency. These results suggest that the ability of Mu to lysogenize is not strictly correlated with its ability to perform massive conservative and replicative transposition.
Reiterated DNA sequences in Rhizobium and Agrobacterium spp
Authors: Flores M , González V , Brom S , Martínez E , Piñero D , Romero D , Dávila G , Palacios R
Published: November 2016

Repeated DNA sequences are a general characteristic of eucaryotic genomes. Although several examples of DNA reiteration have been found in procaryotic organisms, only in the case of the archaebacteria Halobacterium halobium and Halobacterium volcanii [C. Sapienza and W. F. Doolittle, Nature (London) 295:384-389, 1982], has DNA reiteration been reported as a common genomic feature. The genomes of two Rhizobium phaseoli strains, one Rhizobium meliloti strain, and one Agrobacterium tumefaciens strain were analyzed for the presence of repetitive DNA. Rhizobium and Agrobacterium spp. are closely related soil bacteria that interact with plants and that belong to the taxonomical family Rhizobiaceae. Rhizobium species establish a nitrogen-fixing symbiosis in the roots of legumes, whereas Agrobacterium species is a pathogen in different plants. The four strains revealed a large number of repeated DNA sequences. The family size was usually small, from 2 to 5 elements, but some presented more than 10 elements. Rhizobium and Agrobacterium spp. contain large plasmids in addition to the chromosomes. Analysis of the two Rhizobium strains indicated that DNA reiteration is not confined to the chromosome or to some plasmids but is a property of the whole genome.
Carbon monoxide-driven electron transport in Clostridium thermoautotrophicum membranes
Authors: Hugenholtz J , Ivey D M , Ljungdahl L G
Published: November 2016

Membrane vesicles of Clostridium thermoautotrophicum prepared by osmotic lysis after lysozyme treatment contained carbon monoxide dehydrogenase and methylenetetrahydrofolate dehydrogenase with specific activities three- to fourfold higher than the specific activity of the cytoplasm. The membrane-associated carbon monoxide dehydrogenase mediated the reduction with CO or the oxidation with CO2 of b-type cytochromes and other electron carriers in the membrane.
Genetic mapping of katA, a locus that affects catalase 1 level in Bacillus subtilis
Authors: Loewen P C , Switala J
Published: November 2016

Several mutants of Bacillus subtilis deficient in catalase synthesis generated by nitrosoguanidine mutagenesis have been used to map a locus affecting catalase activity. Two- and three-factor bacteriophage PBS1 transductional crosses were used to locate the locus, named katA, between recH and thiA with 98% linkage to thiA at 70 degrees on the B. subtilis genome. The synthesis of catalase 1, found only in vegetative cells, was affected by katA.
Differential transcription of the two glutamine synthetase genes of Bradyrhizobium japonicum
Authors: Carlson T A , Martin G B , Chelm B K
Published: November 2016

Bradyrhizobium japonicum induces the formation of nitrogen-fixing symbiotic root nodules on soybean plants. The B. japonicum genome encodes two isoforms of glutamine synthetase (GS). One form, GSI, encoded by the gene glnA, is similar in structure and activity to the enzyme found in all other bacteria. The second form, GSII, encoded by glnII, is structurally related to the eucaryotic enzyme. Genetic analyses indicate that glnA or glnII alone is sufficient to provide glutamine prototrophy, whereas the double mutation glnA glnII produces glutamine auxotrophy. The glnA gene is transcribed from a single promoter that has a structure most similar to that of the bacterial consensus promoter. The level of transcription of glnA is not specifically affected by nitrogen limitation of growth. The glnII gene is also transcribed from a single promoter; however, this promoter has structural features characteristic of promoters controlled by the nitrogen regulation system. In contrast to glnA, physiological studies indicate that glnII transcription is regulated in response to nitrogen source availability. Under aerobic growth conditions, expression of glnII is induced when growth is limited by nitrogen source depletion as expected for regulation by the nitrogen regulation system.
An open reading frame in the Escherichia coli bacteriophage lambda genome encodes a protein that functions in assembly of the long tail fibers of bacteriophage T4
Authors: Montag D , Henning U
Published: November 2016

Assembly of the long tail fibers of the Escherichia coli bacteriophage T4 requires the catalytic action of two auxiliary proteins. It was found that a gene of the entirely unrelated phage lambda codes for a protein which can substitute for one of these T4 polypeptides, protein 38. The lambda gene was designated tfa (tail fiber assembly). Protein 38 consists of 183 residues, and the Tfa protein consists of 194 residues; the two polypeptides are about 40% homologous. Although the tfa gene is dispensable for the growth of phage lambda, these results indicate that it may have a function in lambda morphogenesis.
Control of glycolysis by glyceraldehyde-3-phosphate dehydrogenase in Streptococcus cremoris and Streptococcus lactis
Authors: Poolman B , Bosman B , Kiers J , Konings W N
Published: November 2016

The decreased response of the energy metabolism of lactose-starved Streptococcus cremoris upon readdition of lactose is caused by a decrease of the glycolytic activity (B. Poolman, E. J. Smid, and W. N. Konings, J. Bacteriol. 169:1460-1468, 1987). The decrease in glycolysis is accompanied by a decrease in the activities of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase. The steady-state levels of pathway intermediates upon refeeding with lactose after various periods of starvation indicate that the decreased glycolysis is primarily due to diminished glyceraldehyde-3-phosphate dehydrogenase activity. Furthermore, quantification of the control strength exerted by glyceraldehyde-3-phosphate dehydrogenase on the overall activity of the glycolytic pathway shows that this enzyme can be significantly rate limiting in nongrowing cells.
Podospora anserina does not senesce when serially passaged in liquid culture
Authors: Turker M S , Cummings D J
Published: November 2016

A procedure was developed for the prolonged growth of the ascomycete fungus Podospora anserina in liquid culture to determine the effects of such growth on the senescence phenotype. Senescence in P. anserina, which is maternally inherited and associated with the excision and amplification of specific mitochondrial plasmids, occurs when this species is grown on solid medium. In two independent experiments no evidence of senescence was observed as mycelia were serially passaged in liquid culture. Further, when separable mycelial masses, termed puff balls, from the liquid cultures were plated on solid medium, a significant increase in their average longevity was observed. The apparent immortality of P. anserina in liquid culture was not dependent upon mitochondrial DNA rearrangements, nor was it affected by the presence of a previously described senescence plasmid, alpha senDNA. Evidence was obtained indicating that growth in liquid culture exerts selective pressure to maintain the wild-type mitochondrial genome.
Involvement of glutamate in the respiratory metabolism of Bradyrhizobium japonicum bacteroids
Authors: Salminen S O , Streeter J G
Published: November 2016

Bradyrhizobium japonicum bacteroids were isolated anaerobically and supplied with 14C-labeled succinate, malate, aspartate, or glutamate for periods of up to 60 min in the presence of myoglobin to control the O2 concentration. Succinate and malate were absorbed about twice as rapidly as glutamate and aspartate. Conversion of substrate to CO2 was most rapid for malate, followed by succinate, glutamate, and aspartate. When CO2 production was expressed as a proportion of total carbon taken up, malate was still the most rapidly respired substrate, with 68% of the label absorbed converted to CO2. The comparable values for succinate, glutamate, and aspartate were 37, 50, and 38%, respectively. Considering the fate of labeled substrate not respired, greater than 95% of absorbed glutamate remained as glutamate in the bacteroids. In contrast, from 39 to 66% of the absorbed succinate, malate, or aspartate was converted to glutamate. An increase in the rate of CO2 formation from labeled substrates after 20 min appeared to coincide with a maximum accumulation of label in glutamate. The results indicate the presence of a substantial glutamate pool in bacteroids and the involvement of glutamate in the respiratory metabolism of bacteroids.
Divergent transcription of the sn-glycerol-3-phosphate active transport (glpT) and anaerobic sn-glycerol-3-phosphate dehydrogenase (glpA glpC glpB) genes of Escherichia coli K-12
Authors: Ehrmann M , Boos W , Ormseth E , Schweizer H , Larson T J
Published: November 2016

The glpTQ operon and the glpA and glpB genes are located adjacent to one another near min 49 of the linkage map of Escherichia coli K-12. The positions and directions of transcription of the glpA and glpB genes with respect to the glpTQ operon were determined in the present work. Strains harboring Mu d1(Ap lac) fusions in either glpA or glpB were converted to the respective lambda p1(209) lysogens. Induction of these lysogens with mitomycin C resulted in production of Lac+ phage progeny which carried adjacent chromosomal DNA. Genetic crosses with a collection of glpT mutant strains were performed with several such phage lines. A fine-structure deletion map of the glpT gene was thus constructed. All phages used for this mapping carried DNA starting with the promoter-proximal end of glpT. This indicated that the glpTQ operon and the glpA and glpB genes are transcribed divergently. Additional evidence supporting this conclusion was obtained by physical mapping of restriction endonuclease cleavage sites in plasmids carrying these genes and in plasmids carrying glpA-lacZ or glpB-lacZ fusions. A new designation (glpC) for the gene encoding the 41,000-Mr subunit of the anaerobic sn-glycerol-3-phosphate dehydrogenase was proposed to distinguish it from the glpA gene, which encodes the 62,000-Mr subunit of the dehydrogenase, and the glpB gene, which encodes a membrane anchor subunit of the dehydrogenase. These three genes were present in an operon transcribed in the order glpA glpC glpB in the clockwise direction on the linkage map of E. coli.
Effect of growth phase on phospholipid biosynthesis in Saccharomyces cerevisiae
Authors: Homann M J , Poole M A , Gaynor P M , Ho C T , Carman G M
Published: November 2016

The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDP-diacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined. Maximum activities were found in the exponential phase of cells grown in complete synthetic medium. As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5- to 5-fold. The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase. When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline. Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline. The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were depressed. Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture. The phospholipid composition of cells in the exponential and stationary phase of growth was also examined. The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells. The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.
Effects of light deprivation on RNA synthesis, accumulation of guanosine 3'(2')-diphosphate 5'-diphosphate, and protein synthesis in heat-shocked Synechococcus sp. strain PCC 6301, a cyanobacterium
Authors: Surányi G , Korcz A , Pálfi Z , Borbély G
Published: November 2016

The rate of total RNA synthesis, the extent of guanosine 3'(2')-diphosphate 5'-diphosphate (ppGpp) accumulation, and the pattern of protein synthesis were studied in light-deprived and heat-shocked Synechococcus sp. strain PCC 6301 cells. There was an inverse correlation between the rate of total RNA synthesis and the pool of ppGpp, except immediately after a temperature shift up, when a parallel increase in the rate of RNA synthesis and accumulation of ppGpp was observed. The inverse correlation between RNA synthesis and ppGpp accumulation was more pronounced when cells were grown in the dark. Heat shock treatment (47 degrees C) had an unexpected effect on ppGpp accumulation; there was a fairly stable level of ppGpp under heat shock conditions, which coincided with a stable steady-state rate of RNA synthesis even in the dark. We found that the pattern of dark-specific proteins was altered in response to heat shock. The transient synthesis of several dark-specific proteins was abolished by an elevated temperature (47 degrees C) in the dark; moreover, the main heat shock proteins were synthesized even in the dark. This phenomenon might be of aid in the study of cyanobacterial gene expression.
Characterization of bromoethanesulfonate-resistant mutants of Methanococcus voltae: evidence of a coenzyme M transport system
Authors: Santoro N , Konisky J
Published: November 2016

Mutants of Methanococcus voltae were isolated that were resistant to the coenzyme M (CoM; 2-mercaptoethanesulfonic acid) analog 2-bromoethanesulfonic acid (BES). The mutants displayed a reduced ability to accumulate [35S]BES relative to the sensitive parental strain. BES inhibited methane production from CH3-S-CoM in cell extracts prepared from wild-type sensitive or resistant strains. BES uptake required the presence of both CO2 and H2 and was inhibited by N-ethylmaleimide and several reagents that are known to disrupt energy metabolism. The mutants showed normal uptake of isoleucine and were not cross-resistant to either azaserine or 5-methyltryptophan and, thus, were neither defective in general energy-dependent substrate transport nor envelope permeability. Both HS-CoM and CH3-S-CoM prevented the uptake of BES and protected cells from inhibition by it. We propose that M. voltae has an energy-dependent, carrier-mediated uptake system for HS-CoM and CH3-S-CoM which can also mediate uptake of BES.
Operator-constitutive mutations of the Escherichia coli metF gene
Authors: Belfaiza J , Guillou Y , Margarita D , Perrin D , Saint Girons I
Published: November 2016

The Escherichia coli metF gene codes for 5,10-methylene-tetrahydrofolate reductase, the enzyme that leads to the formation of N-methyltetrahydrofolate, supplying the methyl group of methionine. Transcription of metF, as well as most of the methionine genes, is repressed by the metJ gene product complexed with S-adenosylmethionine. A metF'-'lacZ gene fusion was used to isolate mutants that have altered expression from the metF promoter. The nucleotide sequences of the metF regulatory region from five such mutants were determined. The mutations were located in the region previously defined as the potential target of the methionine repressor by its similarity to other binding sites. The mutationally defined metF operator thus consists of a 40-base-pair-long region, with five 8-base-pair imperfect palindromes spanning the metF transcription start. The altered operators do not recognize the purified repressor in an in vitro transcription-translation system, although the repressor binds efficiently to the metF wild-type operator.
Dienelactone hydrolase from Pseudomonas sp. strain B13
Authors: Ngai K L , Schlömann M , Knackmuss H J , Ornston L N
Published: November 2016

Dienelactone hydrolase (EC 3.1.1.45) catalyzes the conversion of cis- or trans-4-carboxymethylenebut-2-en-4-olide (dienelactone) to maleylacetate. An approximately 24-fold purification from extracts of 3-chlorobenzoate-grown Pseudomonas sp. strain B13 yielded a homogeneous preparation of the enzyme. The purified enzyme crystallized readily and proved to be a monomer with a molecular weight of about 30,000. Each dienelactone hydrolase molecule contains two cysteinyl side chains. One of these was readily titrated by stoichiometric amounts of p-chloromercuribenzoate, resulting in inactivation of the enzyme; the inactivation could be reversed by the addition of dithiothreitol. The other cysteinyl side chain appeared to be protected in the native protein against chemical reaction with p-chloromercuribenzoate. The properties of sulfhydryl side chains in dienelactone hydrolase resembled those that have been characterized for bacterial 4-carboxymethylbut-3-en-4-olide (enol-lactone) hydrolases (EC 3.1.1.24), which also are monomers with molecular weights of about 30,000. The amino acid composition of the dienelactone hydrolase resembled the amino acid composition of enol-lactone hydrolase from Pseudomonas putida, and alignment of the NH2-terminal amino acid sequence of the dienelactone hydrolase with the corresponding sequence of an Acinetobacter calcoaceticus enol-lactone hydrolase revealed sequence identity at 8 of the 28 positions. These observations foster the hypothesis that the lactone hydrolases share a common ancestor. The lactone hydrolases differed in one significant property: the kcat of dienelactone hydrolase was 1,800 min-1, an order of magnitude below the kcat observed with enol-lactone hydrolases. The relatively low catalytic activity of dienelactone hydrolase may demand its production at the high levels observed for induced cultures of Pseudomonas sp. strain B13.
Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp. strain B13
Authors: Frantz B , Ngai K L , Chatterjee D K , Ornston L N , Chakrabarty A M
Published: November 2016

The clcD structural gene encodes dienelactone hydrolase (EC 3.1.1.45), an enzyme that catalyzes the conversion of dienelactones to maleylacetate. The gene is part of the clc gene cluster involved in the utilization of chlorocatechol and is carried on a 4.3-kilobase-pair BglII fragment subcloned from the Pseudomonas degradative plasmid pAC27. A 1.9-kilobase-pair PstI-EcoRI segment subcloned from the BglII fragment was shown to carry the clcD gene, which was expressed inducibly under the tac promoter at levels similar to those found in 3-chlorobenzoate-grown Pseudomonas cells carrying the plasmid pAC27. In this study, we present the complete nucleotide sequence of the clcD gene and the amino acid sequence of dienelactone hydrolase deduced from the DNA sequence. The NH2-terminal amino acid sequence encoded by the clcD gene from plasmid pAC27 corresponds to a 33-residue sequence established for dienelactone hydrolase encoded by the Pseudomonas sp. strain B13 plasmid pWR1. A possible relationship between the clcD gene and pcaD, a Pseudomonas putida chromosomal gene encoding enol-lactone hydrolase (EC 3.1.1.24) is suggested by the fact that the gene products contain an apparently conserved pentapeptide neighboring a cysteinyl side chain that presumably lies at or near the active sites; the cysteinyl residue occupies position 60 in the predicted amino acid sequence of dienelactone hydrolase.
Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure
Authors: Ben-Bassat A , Bauer K , Chang S Y , Myambo K , Boosman A , Chang S
Published: November 2016

Methionine aminopeptidase (MAP) catalyzes the removal of amino-terminal methionine from proteins. The Escherichia coli map gene encoding this enzyme was cloned; it consists of 264 codons and encodes a monomeric enzyme of 29,333 daltons. In vitro analyses with purified enzyme indicated that MAP is a metallo-oligopeptidase with absolute specificity for the amino-terminal methionine. The methionine residues from the amino-terminal end of the recombinant proteins interleukin-2 (Met-Ala-Pro-IL-2) and ricin A (Met-Ile-Phe-ricin A) could be removed either in vitro with purified MAP enzyme or in vivo in MAP-hyperproducing strains of E. coli. In vitro analyses of the substrate preference of the E. coli MAP indicated that the residues adjacent to the initiation methionine could significantly influence the methionine cleavage process. This conclusion is consistent, in general, with the deduced specificity of the enzyme based on the analysis of known amino-terminal sequences of intracellular proteins (S. Tsunasawa, J. W. Stewart, and F. Sherman, J. Biol. Chem. 260:5382-5391, 1985).
Gene encoding the 37,000-dalton minor sigma factor of Bacillus subtilis RNA polymerase: isolation, nucleotide sequence, chromosomal locus, and cryptic function
Authors: Duncan M L , Kalman S S , Thomas S M , Price C W
Published: November 2016

We began an analysis of rpoF, the gene encoding the cryptic, 37,000-dalton minor sigma factor (sigma-37) of Bacillus subtilis RNA polymerase. Using antibody raised against sigma-37 holoenzyme to probe a lambda gt11 expression vector library, we isolated a 901-base-pair EcoRI fragment that expressed the COOH-terminal half of sigma-37 fused to lacZ. We used this fragment as a hybridization probe to isolate the entire rpoF gene and additional flanking sequences. Identity of the cloned gene was confirmed by the size and immunological reaction of its product expressed in Escherichia coli and, after DNA sequencing, by the homology of its predicted product (264 residues; 30,143 daltons) with other sigma factors. The DNA sequence also suggested that rpoF may lie in a gene cluster. Upstream of rpoF was an open reading frame that would encode a protein of 17,992 daltons; this frame overlapped the rpoF-coding sequence by 41 base pairs. Immediately following rpoF was a reading frame that would encode a protein of at least 20,000 daltons; expression of this region may be translationally coupled to that of rpoF. By plasmid integration and PBS1 transduction, we found the chromosomal locus of rpoF linked to ddl and dal at 40 degrees on the B. subtilis map and near no known lesions affecting growth regulation or development. Further, an rpoF null mutation resulting from gene disruption had no effect on cell growth or sporulation in rich medium, suggesting that sigma-37 may partly control a regulon not directly involved in the sporulation process.
Lipid acyl chain-dependent effects of sterols in Acholeplasma laidlawii membranes
Authors: Rilfors L , Wikander G , Wieslander A
Published: November 2016

Acholeplasma laidlawii was grown with different fatty acids for membrane lipid synthesis (saturated straight- and branched-chain acids and mono- and di-unsaturated acids). The ability of 12 different sterols to affect cell growth, lipid head group composition, the order parameter of the acyl chains, and the phase equilibria of in vivo lipid mixtures was studied. The following two effects were observed with respect to cell growth: with a given acyl chain composition of the membrane lipids, growth was stimulated, unaffected, reduced, or completely inhibited (lysis), depending on the sterol structure; and the effect of a certain sterol depended on the acyl chain composition (most striking for epicoprostanol, cholest-4-en-3-one, and cholest-5-en-3-one, which stimulated growth with saturated acyl chains but caused lysis with unsaturated chains). The three lytic sterols were the only sterols that caused a marked decrease in the ratio between the major lipids monoglucosyldiglyceride and diglucosyldiglyceride and hence a decrease in bilayer stability when the membranes were enriched in saturated (palmitoyl) chains. With these chains correlations were found for several sterols between the glucolipid ratio and the order parameter of the acyl chains, as well as the lamellar-reversed hexagonal phase transition, in model systems. A shaft experiment revealed a marked decrease in the ratio of monoglucosyldiglyceride to diglucosyldiglyceride with the lytic sterols in unsaturated (oleoyl) membranes. The two cholestenes induced nonlamellar phases in in vivo mixtures of oleoyl A. laidlawii lipids. The order parameters of the oleoyl chains were almost unaffected by the sterols. Generally, the observed effects cannot be explained by an influence of the sterols on the gel-to-liquid crystalline phase transition.
Lipopolysaccharide size and distribution determine serum resistance in Salmonella montevideo
Authors: Grossman N , Schmetz M A , Foulds J , Klima E N , Jimenez-Lucho V E , Leive L L , Joiner K A , Jiminez V
Published: November 2016

The survival of Salmonella montevideo during serum treatment depends on the presence of an O antigen (O-Ag) associated with the lipopolysaccharide molecule. In this organism, the O antigen is a polysaccharide composed of 0 to more than 55 subunits, each containing 4 mannose residues together with glucose and n-acetylglucosamine. We used a mutant strain of S. montevideo that requires exogenous mannose for the synthesis of O-Ag. Lipopolysaccharide (LPS) was prepared from these cells grown under three different conditions where the availability of exogenous mannose was regulated such that the average number of O-Ag units per LPS molecule, the percentage of LPS molecules bearing long O-Ag side chains, and the percentage of lipid A cores bearing O-Ag were all varied. These changes in LPS profiles were monitored on sodium dodecyl sulfate-polyacrylamide gels, and cells with different LPS profiles were tested for their ability to survive treatment with pooled normal human serum. Survival in serum was associated with LPS that contained an average of 4 to 5 O-Ag units per LPS molecule, and 20 to 23% of the LPS molecules had more than 14 O-Ag units per LPS molecule. Serum survival was less clearly associated with the percentage of lipid A cores covered with O-Ag. We propose, based on these data and on previous work, that the O-Ag polysaccharide provides the cell protection from serum killing by sterically hindering access of the C5b-9 complex to the outer membrane and that a critical density of long O-Ag polysaccharide is necessary to provide protection.
Mechanism of nitrogenase switch-off by oxygen
Authors: Goldberg I , Nadler V , Hochman A
Published: November 2016

Oxygen caused a reversible inhibition (switch-off) of nitrogenase activity in whole cells of four strains of diazotrophs, the facultative anaerobe Klebsiella pneumoniae and three strains of photosynthetic bacteria (Rhodopseudomonas sphaeroides f. sp. denitrificans and Rhodopseudomonas capsulata strains AD2 and BK5). In K. pneumoniae 50% inhibition of acetylene reduction was attained at an O2 concentration of 0.37 microM. Cyanide (90 microM), which did not affect acetylene reduction but inhibited whole-cell respiration by 60 to 70%, shifted the O2 concentration that caused 50% inhibition of nitrogenase activity to 2.9 microM. A mutant strain of K. pneumoniae, strain AH11, has a respiration rate that is 65 to 75% higher than that of the wild type, but its nitrogenase activity is similar to wild-type activity. Acetylene reduction by whole cells of this mutant was inhibited 50% by 0.20 microM O2. Inhibition by CN- of 40 to 50% of the O2 uptake in the mutant shifted the O2 concentration that caused 50% inhibition of nitrogenase to 1.58 microM. Thus, when the respiration rates were lower, higher oxygen concentrations were required to inhibit nitrogenase. Reversible inhibition of nitrogenase activity in vivo was caused under anaerobic conditions by other electron acceptors. Addition of 2 mM sulfite to cell suspensions of R. capsulata B10 and R. sphaeroides inhibited nitrogenase activity. Nitrite also inhibited acetylene reduction in whole cells of the photodenitrifier R. sphaeroides but not in R. capsulata B10, which is not capable of enzymatic reduction of NO2-. Lower concentrations of NO2- were required to inhibit the activity in NO3- -grown cells, which have higher activities of nitrite reductase.(ABSTRACT TRUNCATED AT 250 WORDS)
A Rhizobium meliloti mutant that forms ineffective pseudonodules in alfalfa produces exopolysaccharide but fails to form beta-(1----2) glucan
Authors: Geremia R A , Cavaignac S , Zorreguieta A , Toro N , Olivares J , Ugalde R A
Published: November 2016

A mutant of Rhizobium meliloti that elicited the formation of inactive nodules in alfalfa was found not to form beta-(1----2) glucan in vivo or in vitro. It was nonmotile because it lacks flagella. The 235-kilodalton protein which acts as an intermediate in beta-(1----2) glucan synthesis was undetectable in the mutant. These properties of the mutant are common to those of chvB mutants of Agrobacterium tumefaciens. Exopolysaccharide formation by the R. meliloti mutant was about double that by the wild type.