bluuebunny/crossref_metadata_2025 huggingface dataset:
Blue ghosts: a new method for isolating amber mutants defective in essential genes of Escherichia coli
Authors: Brown S , Brickman E R , Beckwith J
Published: January 2020

We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli. Using this approach, we have isolated three amber mutants defective in the rho gene. An extension of the technique allows the detection of ochre mutants and transposon insertions in essential genes.
Cloning of sporulation gene spoOB of Bacillus subtilis and its genetic and biochemical analysis
Authors: Hirochika H , Kobayashi Y , Kawamura F , Saito H
Published: January 2020

A specialized transducing phage carrying a sporulation gene (spoOB) was constructed from Bacillus subtilis temperate phage rho 11 by in vitro and in vivo recombinations. Transformation experiments showed that the spoOB gene resides on a 1.4-megadalton fragment generated by EcoRI endonuclease treatment of the phage deoxyribonucleic acid (DNA). Mutants of this phage which lost transducing activity were isolated and used for genetic complementation tests and the analysis of protein(s) coded by the 1.4-megadalton fragment. The spoOB locus was shown to be composed of one cistron. Sodium dodecyl sulfate-polyacrylamide gel analysis of proteins synthesized in ultraviolet-irradiated cells infected with these phages showed that the 1.4-megadalton fragment codes at least one protein, of molecular weight 39,000, which is synthesized in both vegetative and sporulating cells. A cleavage map of the phage DNA was constructed by use of restriction endonucleases, EcoRI, BamHI, and SalI, and the site of integration of the 1.4-megadalton fragment was determined. Expression and function of the spoOB gene are discussed.
Effect of cerulenin on cellular autolytic activity and lipid metabolism during inhibition of protein synthesis in Streptococcus faecalis
Authors: Carson D D , Pieringer R A , Daneo-Moore L
Published: January 2020

Cellular autolytic activity as well as lipid and lipoteichoic acid metabolism have been studied in cultures of Streptococcus faecalis receiving various combinations of the following treatments: chloramphenicol addition, starvation for an essential amino acid (valine), and cerulenin treatment. Lipoteichoic acid initially accumulated in chloramphenicol-treated and amino acid-starved cells and decreased relative to the cellular mass in cerulenin-treated cells. The relative phosphatidylglycerol content of amino acid-starved cultures or of cultures treated with either antibiotic rapidly decreased upon initiation of each treatment. In all cases, cerulenin initially stimulated diphosphatidylglycerol synthesis. Pretreatment of cultures with cerulenin prevented the inhibition of cellular synthesis autolysis normally observed during chloramphenicol treatment, but did not affect amino acid starvation-induced inhibition of autolytic activity. Variations in the levels of the nonionic lipid fraction, predominantly diglycerides, correlated best with the patterns of autolytic activity observed during chloramphenicol treatment, whereas variations in the levels of diphosphatidylglycerol and lipoteichoic acid correlated best with the patterns of autolytic activity observed during amino acid starvation. Components of the nonionic lipid fraction were demonstrated to inhibit autolytic activity 50% in whole cell and in cell wall assays at 60 and 120 nmol/mg (dry weight), respectively.
Metabolism of myo-inositol during sporulation of myo-inositol-requiring Saccharomyces cerevisiae
Authors: Schroeder R , Breitenbach M
Published: January 2020

We investigated the sporulation properties of a series of diploid Saccharomyces cerevisiae strains homozygous for inositol auxotrophic markers. The strains required different amounts of inositol for the completion of sporulation. Shift experiments revealed two phases of inositol requirement during sporulation which coincided with the two phases of lipid synthesis found by earlier workers. Phase I was at the beginning and during premeiotic deoxyribonucleic acid synthesis; phase II immediately preceded the appearance of mature asci. Of the inositol taken up by sporulating cells, 90% was incorporated into inositol phospholipids. By two-dimensional thin-layer chromatography, eight compounds were resolved, one of which was sporulation specific. The majority of the inositol phospholipids were, however, identical to those found in vegetatively growing cells. In the absence of inositol, the cells did not sporulate but, after a certain time, were unable to return to vegetative growth. These nonsporulating cells did, however, incorporate acetate into lipids and double their deoxyribonucleic acid content in the premeiotic phase. We believe that it is this lack of coordination of biosynthetic events which causes inositol-less death on sporulation media without inositol.
Nucleoid structure in freeze fractures of Streptococcus faecalis: effects of filtration and chilling
Authors: Edelstein E , Parks L , Tsien H C , Daneo-Moore L , Higgins M L
Published: January 2020

With the techniques used in this study, the nucleoid of Streptococcus faecalis could not be seen in freeze-etch preparations unless glutaraldehyde had been added to cultures of cells before they were frozen. With time, the nucleoid became visible as a network of fibers, apparently as a result of the aggregation of individual chromosomal elements in the presence of glutaraldehyde. When glutaraldehyde was added to undisturbed cultures, the fibers that became visible were observed in small patches that were seemingly scattered throughout the cytoplasm. However, if cells were chilled or placed on filters before glutaraldehyde was added, the fibers which then developed were seen in large central areas. The appearance of centralized nucleoids in freeze fractures of cells that had been chilled or filtered could be correlated with a decrease in the central density of the cytoplasm, as seen by light microscopy, in cells embedded in gelatin or bovine serum albumin. These observations are discussed in relation to a model for the normal structure of the nucleoid which suggests that the treatments routinely used to study the morphology-physiology of cells (chilling, filtration, and fixation) result in a reorganization of the cytoplasm, leading to an increase in the centralization of nuclear material.
Genetic mapping of a mutation conferring sensitivity to bacteriophage Mu in Salmonella typhimurium LT2
Authors: Faelen M , Mergeay M , Gerits J , Toussaint A , Lefèbvre N
Published: January 2020

Two strains of Salmonella typhimurium LT2, SA1475 and MA411, were fortuitously found to be sensitive to bacteriophage Mu. The Mu-sensitivity allele of SA1475 was called musA1 and shown to be linked to the histidine operon both in conjugation and transduction experiments. The Mus allele of MA411 was unlinked to the his region and was tentatively designated musB2. Strains carrying large deletions of the his operon were also tested for Mu sensitivity; those of which the his-rib region is deleted were also sensitive to Mu. Transduction data led to the order zee-2 hisOGDCBAHFIE gnd musA. An Hfr injecting the his operon early (HfrK9) an carrying hisG9424::Tn10 delta 4 delta 11 and musA1 was isolated; this Hfr made it possible to introduce the Mus character into most derivatives of S. typhimurium LT2. Since strain SA1475 is resistant to bacteriophage P1, it could be used to select a new P1-Mu hybrid which has the host range of Mu and the transduction properties of P1.
Selective enrichment of Pseudomonas spp. defective in catabolism after exposure to halogenated substrates
Authors: Wigmore G J , Ribbons D W
Published: January 2020

Significant selective enrichments of mutants defective in catabolic pathways can be achieved by exposure of pseudomonad cells to halogenated analogs of growth substrates. Between 3 and 95% of viable clones rescued from such enrichments have been defective in specific catabolic pathways. This has been demonstrated for eight different catabolic pathways for aromatic compounds in pseudomonads, in which the genes are located on plasmids or on the chromosome. The plasmid-encoded pathways studied include those for the catabolism of p-cymene (CYM), m- and p-xylenes (TOL), naphthalene (NAH), salicylate (SAL), and 4-methylphthalate (MOP), and the chromosome-encoded pathways include those for p-hydroxybenzoate, monohydric phenols, and p-anisate utilization. The recalcitrance of halogenated compounds may, in part, be explained by these observations, which introduce an as yet not widely recognized factor in assessment of biodegradability of halogenated compounds and their effects on the transformation of the natural substrates.
Biosynthesis of bacterial glycogen: purification and structural properties of Rhodospirillum tenue adenosine diphosphate glucose synthetase
Authors: Yung S G , Preiss J
Published: January 2020

Adenosine diphosphate glucose synthetase from the photosynthetic bacterium Rhodospirillum tenue has been purified greater than 95%. The molecular weight of the enzyme is approximately 215,000, with a subunit molecular weight of about 51,000. The enzyme appears to be composed of four similar if not identical subunits. Although the amino acid composition of the enzyme is similar to that of Escherichia coli and Salmonella typhimurium, no apparent homology has been observed between their N-terminal amino acid sequences. Antisera prepared against the R. tenue enzyme can partially inhibit the activities of adenosine diphosphate glucose synthetases from other photosynthetic bacteria.
Sensory adaptation and deadaptation by Bacillus subtilis
Authors: Goldman D J , Ordal G W
Published: January 2020

Cells of Bacillus subtilis, when tethered by using antiflagellar antibody, rotate briefly counterclockwise (swimming behavior) or clockwise (tumbling behavior) when amino acids are added or removed, respectively. "Dissociation constants" for attractant-binding site interactions, calculated from duration of the rotational response to addition of amino acids, agreed with those calculated for their removal and with previous values calculated from sensitivity capillary assays. The ratio of adaptation times for addition versus removal of attractant averaged 1.7, which differs greatly from the value of 50 for Escherichia coli.
Biochemical genetics of tryptophan synthesis in Pseudomonas acidovorans
Authors: Buvinger W E , Stone L C , Heath H E
Published: January 2020

Sixty independent tryptophan auxotrophs of Pseudomonas acidovorans were isolated and characterized for nutritional response to intermediates of the pathway, accumulation of intermediates, and levels of tryptophan-synthetic enzymes. Mutants for each of the seven proteins catalyzing the five steps of tryptophan synthesis were obtained. Transductional analysis established three unlinked chromosomal regions: trpE, trpGDC, and trpFBA. The order of the genes within the two clusters was not determined. The levels and enzymatic activities of wild-type and mutant strains indicated that trpE and trpGDC were repressed by tryptophan. In contrast, trpFBA was not derepressed significantly by starvation for tryptophan. The trpG mutants had an additional requirement for p-aminobenzoate, which suggested that anthranilate synthase subunit II also served as glutamine-binding protein in the analogous reaction catalyzed by p-aminobenzoate synthase. In addition, trpD mutants revealed the ability of P. acidovorans to degrade anthranilate via the beta-ketoadipate pathway.
Localization of an Amikacin 3′-Phosphotransferase in
Escherichia coli
Authors: Perlin Michael H. , Lerner Stephen A.
Published: January 2020

A plasmid-encoded enzyme reported by us to phosphorylate amikacin in a laboratory strain of
Escherichia coli
has been localized in the bacterial cell. More than 88% of this amikacin phosphotransferase (APH) activity was retained in spheroplasts formed by ethylenediaminetetraacetate-lysozyme treatment of an APH-containing
E. coli
transconguant known to form spheroplasts readily. By comparison, the spheroplasts retained 94% of deoxyribonucleic acid polymerase I and 98% of glutamyl-transfer ribonucleic acid synthetase, two internal markers, whereas less than 10% of the activity of a periplasmic marker, acid phosphatase, was present in spheroplasts. Treatment of whole cells of the transconjugant with chemical probes incapable of crossing the plasma membrane obliterated acid phosphatase activity, whereas the internal markers deoxyribonucleic acid polymerase I, glutamyl-transfer ribonucleic acid synthetase, and β-galactosidase were virtually unaffected after treatment for 5 min; more than 60% of the APH activity remained. As a control, similar chemical treatment of sonic extracts, in which enzymes were not protected by bacterial compartmentalization, produced more extensive reduction in the activities of all test enzymes, including APH. Spheroplasts preincubated with adenosine triphosphatase were shown by thin-layer chromatography to phosphorylate amikacin. Spheroplasts of cells grown in the presence of H
3
32
PO
4
were shown to utilize internally generated adenosine 5′-triphosphate in the phosphorylation of amikacin. The absence of
32
P-phosphorylated amikacin after incubation of [γ-
32
P]adenosine 5′-triphosphate with spheroplasts confirmed that exogenous adenosine 5′-triphosphate was not used in the reaction. These results suggest an internal location for APH. This conclusion has implications for the role of such enzymes in aminoglycoside resistance of gram-negative bacteria.
Characterization of a membrane-associated cytidine diphosphate-diacylglycerol-dependent phosphatidylserine synthase in bacilli
Authors: Dutt A , Dowhan W
Published: January 2020

The synthesis of phosphatidylserine in two gram-positive aerobic bacteria has been partially characterized. We have located a cytidine 5'-diphospho-diacylglycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase) activity in the membrane fraction of Bacillus licheniformis and Bacillus subtilis. The activity was demonstrated to be membrane associated by differential centrifugation, sucrose gradient centrifugation, and detergent solubilization. The direct involvement of cytidine 5'-diphospho-diacylglycerol in the reaction was demonstrated by the conversion of the liponucleotide phosphatidyl moiety to phosphatidylserine. This activity is dependent on divalent metal ion (manganese being optimal) and is stimulated by nonionic detergent and its product phosphatidylserine. Based on studies with various combinations of products and substrates, the reaction appears to follow a sequential BiBi kinetic mechanism.
Cloning of an early sporulation gene in Bacillus subtilis
Authors: Dubnau E , Ramakrishna N , Cabane K , Smith I
Published: January 2020

A 0.8-megadalton BglII restriction fragment of Bacillus licheniformis cloned into the BglII site of plasmid pBD64 can complement spo0H mutations of Bacillus subtilis. The clone was isolated by selecting for the Spo+ phenotype and antibiotic resistance, using the helper system described by Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980). The insert is functional in both orientations and thus presumably has its own promoter. A deletion generated within the 0.8-megadalton insert by HindIII restriction and subsequent religation eliminates the ability of the cloned fragment to complement spo0H mutations. The cloned B. licheniformis deoxyribonucleic acid segment specifies the synthesis, in minicells, of a polypeptide of approximately 27,000 daltons. This protein is observed with both orientations, but not when the HindIII deletion is present in the cloned B. licheniformis chromosomal fragment. We have also demonstrated that ribonucleic acid complementary to the cloned B. licheniformis sporulation gene is transcribed in B. licheniformis both during vegetative growth and sporulation.
Position of the lacZX90 mutation and hybridization between complete and incomplete beta-galactosidase
Authors: Mandecki W , Fowler A V , Zabin I
Published: January 2020

The position of the termination codon in lacZX90 was determined by isolation of a lac+ revertant. Lysine was found to replace tyrosine at position 1,012 of beta-galactosidase, indicating that X90 protein lacked the carboxyl-terminal 10 residues. A heat- and urea-sensitive hybrid enzyme was formed in vivo when supC, which supplies tyrosine to the position in the polypeptide corresponding to the nonsense codon, was used to suppress lacZX90. This result shows that suppression that adds back the original amino acid may not lead to the production of the wild-type enzyme if the latter is multimeric, because incomplete chains can be incorporated into the oligomer.
Molecular mechanisms of pyrimidine dimer excision in Saccharomyces cerevisiae: excision of dimers in cell extracts
Authors: Reynolds R J , Love J D , Friedberg E C
Published: January 2020

Cell-free extracts prepared from rad1-19, rd2-2, rad3-1, rad4-3, rad7-1, rad10-1, rd14-1, rad16-1, and cyc1-1 (rad7) mutants of Saccharomyces cerevisiae all catalyze the preferential excision of thymine-containing pyrimidine dimers from ultraviolet-irradiated DNA specifically incised with M. luteus ultraviolet deoxyribonucleic acid incising activity.
Effects of lipophilic cations on motility and other physiological properties of Bacillus subtilis
Authors: Zaritsky A , Macnab R M
Published: January 2020

Lipophilic cations (tetraphenylarsonium, tetraphenylphosphonium, and triphenylmethylphosphonium) caused a number of major changes in the physiology of Bacillus subtilis. Macromolecular synthesis was inhibited, adenosine 5'-triphosphate concentration increased, swimming speed was reduced, tumbling was suppressed, and the capacity to take up the cations was greatly enhanced; respiration was not significantly altered. The effects occurred at lipophilic cation concentrations in the range commonly employed for measurement of membrane potential. Neither the enhancement of cation uptake nor the motility inhibition was a consequence of alteration of membrane potential, since both effects were still seen in the presence of valinomycin, with the extent of 86Rb+ uptake indicating a constant potential. Because suppression of tumbling accompanied speed reduction, as has also been found when protonmotive force is reduced, it is likely that lipophilic cations are perturbing the process of conversion of proton energy into work, rather than simply causing structural damage.
Cloning of the genes for penicillinase, penP and penI, of Bacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis, and Bacillus licheniformis
Authors: Imanaka T , Tanaka T , Tsunekawa H , Aiba S
Published: January 2020

By using plasmid pMB9, penicillinase genes (penP and penI) from both the wild-type and constitutive strains of Bacillus licheniformis 9945A were cloned in EScherichia coli. When a low-copy-number plasmid was used, both wild-type and constitutive penicillinase genes could be transferred into Bacillus subtilis. However, when a high-copy-number plasmid was used, only the genes of the wild type could be transferred. These recombinant plasmids in B. subtilis could all be transferred by the protoplast transformation procedure into B. licheniformis. Transformants of E. coli were resistant to ampicillin (20 micrograms/ml) in spite of the low penicillinase activities (7 U/mg of cells). However, transformants of B. subtilis and B. licheniformis were sensitive to ampicillin (20 micrograms/ml) even in high penicillinase activities (more than 10,000 U/mg of cells). The secretion of penicillinase was rarely observed in E. coli. In contrast, penicillinases secreted from transformants of B. subtilis and B. licheniformis were around 30 and 60% of the total activities, respectively. We took advantage of the plasmids to permit the construction of hetero- and mero-polyploid structures in host cells, and we discuss a regulatory mechanism of penicillinase synthesis in B. licheniformis.
Physical characterization of the ilvHI operon of Escherichia coli K-12
Authors: Squires C H , De Felice M , Wessler S R , Calvo J M
Published: January 2020

The ilvHI and leu genes of Escherichia coli K-12 are contained on a single 10.9-kilobase EcoRI fragment of deoxyribonucleic acid derived from the leu transducing phage lambda G4. Since the expression of all of these genes is controlled by leucine, we investigated whether they are part of single operon or whether they constitute separate but adjacent operons controlled from a common site. Both cloning and hybridization studies indicated that ilvHI and leu are distinct operons. They are transcribed in opposite directions and are separated by approximately 1,500 base pairs of deoxyribonucleic acid. Hybridization experiments showed that the expression of ilvHI is regulated chiefly at the level of transcription. The size of the ilvHI messenger ribonucleic acid is estimated to be 2,550 bases.
Mechanism of thiamine-induced respiratory deficiency in Saccharomyces carlsbergensis
Authors: Nakamura I , Isobe N , Nakamura N , Kamihara T , Fukui S
Published: January 2020

Cells of Saccharomyces carlsbergensis 4228 grown aerobically with added thiamine (1 microgram . ml-1) in a vitamin B6-free medium contained no detectable heme precursors, such as delta-aminolevulinate, coproporphyrin III, or protoporphyrin IX. The deficiency in heme precursors in the thiamine-grown cells was accompanied by previously reported phenomena, i.e., growth depression, vitamin B6 deficiency, and respiratory deficiency due to a marked decrease in the activities of heme-containing enzymes and cytochrome level (I. Nakamura et al., FEBS Lett. 62: 354-358, 1976). It has been reported that all of the effects of thiamine are abolished by adding pyridoxine to the medium. delta-Aminolevulinate was found to have quite similar effects to those of pyridoxine, except that growth was partially improved by delta-aminolevulinate, whereas it was fully restored by pyridoxine. Incubation of the thiamine-grown cells with delta-aminolevulinate resulted in the appearance of the heme precursors and the heme-containing enzymes. Consistent with the lowered amount of vitamin B6, the thiamine-grown cells had a lowered activity of delta-aminolevulinate synthase, a pyridoxal phosphate-dependent enzyme. Not only the holoenzyme activity but also the apoenzyme activity was very low in these cells. These results indicate that the thiamine-induced vitamin B6 deficiency brings about the decrease in delta-aminolevulinate synthase activity, which leads to heme deficiency and therefore to respiratory deficiency.
Regulation of the activity of the Bacillus licheniformis A5 glutamine synthetase
Authors: Donohue T J , Bernlohr R W
Published: January 2020

The regulation of glutamine synthetase activity by positive and negative effectors of enzyme activity singularly and in combinations was studied by using a homogeneous enzyme preparation from Bacillus licheniformis A5. Phosphorylribosyl pyrophosphate at concentrations greater than 2mM stimulated glutamine synthetase activity by approximately 70%. The concentration of phosphorylribosyl pyrophosphate required for half-maximal stimulation of enzyme activity was 0.4 mM. Results obtained from studies of fractional inhibition of glutamine synthetase activity were consistent with the presence of one allosteric site for glutamine binding (apparent I0.5, 2.2mM) per active enzyme unit at a glutamate concentration of 50 mM. At a glutamate concentration of 30 mM or less, the data were consistent with the enzyme containing two binding sites for glutamine (one of which was an allosteric site with an apparent I0.5 of 0.4 mM). Bases on an analysis of the response of glutamine synthetase activity to positive and negative effectors in vitro and to the intracellular concentration of these effectors in vivo, the primary modulators of glutamine synthetase activity in B. licheniformis A5 appear to be glutamine and alanine (apparent I0.5, 5.2mM).
Genetic transformation in Staphylococcus aureus: isolation and characterization of a competence-conferring factor from bacteriophage 80 alpha lysates
Authors: Birmingham V A , Pattee P A
Published: January 2020

The competence-conferring activity in crude lysates of the staphylococcal bacteriophage 80 alpha was concentrated and purified by (NH4)2SO4 precipitation, differential ultracentrifugation and rate-zonal centrifugation through Ficoll. This concentrated preparation exhibited lytic activity toward assay cells of Staphylococcus aureus 8325-4 that could not be attributed to the residual 80 alpha infectious particles present. Electron microscopic examination of the concentrated competence-conferring activity revealed an occasional intact but empty virion and large numbers of free phage tails. Sodium dodecyl sulfate-polyacrylamide gel analysis of this material confirmed that the competence-conferring activity contained only some, but not all, of the major virion proteins. The competence-conferring activity exhibited single-hit kinetics when assay cells and 80 alpha transfecting deoxyribonucleic acid were present in excess. The competence-conferring activity thus seems to be a unique morphogenic precursor of the 80 alpha virion that mediates transfection and transformation in the presence of 0.1 M CaCl2.
Regulatory isozymes of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase in the cyanobacterium Anabaena sp. strain ATCC 29151
Authors: Hall G C , Jensen R A
Published: January 2020

Two regulatory isozymes of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase from the cyanobacterium Anabaena sp. strain ATCC 29151 were separated via gel filtration. One isozyme (Mr = 205,000) was sensitive to phenylalanine inhibition, and the other (Mr = 60,000) was sensitive to tyrosine inhibition. The tyrosine-sensitive isozyme from wild-type cells was unstable to gel filtration, whereas the corresponding isozyme from an analog-resistant mutant was insensitive to tyrosine inhibition and was stable.
Formation of trideuteromethane from deuterated trimethylamine or methylamine by Methanosarcina barkeri
Authors: Walther R , Fahlbusch K , Sievert R , Gottschalk G
Published: January 2020

Methanosarcina barkeri was grown on trimethylamine, methylamine, or methanol containing completely deuterated methyl groups. Methane was collected and analyzed in a mass spectrometer. It contained 79 to 83% CD3H and 14 to 18% CD2H2. This demonstrated that the methyl groups of the above compounds served primarily as direct precursors of methane.
Overproduction of the Escherichia coli recA protein without stimulation of its proteolytic activity
Authors: Uhlin B E , Clark A J
Published: January 2020

Plasmid pBEU14, which carries the Escherichia coli recA+ gene and which can be amplified by manipulation of growth temperature, was constructed. When pBEU14 deoxyribonucleic acid was amplified, a high rate of synthesis and accumulation of recA protein resulted. Amplification of the recA gene and protein did not cause induction of prophage lambda, indicating that the proteolytic activity of the recA protein was not stimulated.
Role of gene fadR in Escherichia coli acetate metabolism
Authors: Maloy S R , Nunn W D
Published: January 2020

Mutants of Escherichia coli K-12 constitutive for fatty acid degradation (fadR) showed an increased rate of utilization of exogenous acetate. Acetate transport, oxidation, and incorporation into macromolecules was approximately fivefold greater in fadR mutants than fadR+ strains during growth on succinate as a carbon source. This effect was due to the elevated levels of glyoxylate shunt enzymes in fadR mutants, since (i) similar results were seen with mutants constitutive for the glyoxylate shunt enzymes (iclR), (ii) induction of the glyoxylate shunt in fadR+ strains by growth on acetate or oleate increased the rate of acetate utilization to levels comparable to those in fadR mutants, and (iii) fadR and fadR+ derivatives of mutants defective for the glyoxylate shunt enzymes showed equivalent rates of acetate utilization under these conditions. These results suggest that the operation of the glyoxylate shunt may play a significant role in the utilization of exogenous acetate by fadR mutants.
Cloning of immunity and structural genes for colicin V
Authors: Frick K K , Quackenbush R L , Konisky J
Published: January 2020

The colicin V immunity and structural genes of plasmid pColV-B188 were cloned into the vectors pMB9, pBR322, and pMK16. Both genes are closely linked and can be isolated on a 900-base-pair deoxyribonucleic acid fragment. Insertion of the transposon Tn5 into this cloned sequence led to the construction of a mutant plasmid which conferred colicin V immunity, but not the ability to produce this colicin. Analysis of the products determined by these cloned genes in cells has led to the conclusion that the polypeptide involved in immunity has a molecular weight of about 6,500, whereas the colicin has a molecular weight of approximately 4,000.
Effects of growth substrate and respiratory chain composition on bioenergetics in Acinetobacter sp. strain HO1-N
Authors: Ensley B D , Irwin R M , Carreira L A , Hoffman P S , Morgan T V , Finnerty W R
Published: January 2020

The relationship between respiratory chain composition and efficiency of coupling phosphorylation to electron transport was examined in Acinetobacter sp. strain HO1-N. Cells containing only cytochrome o as a terminal oxidase displayed the same stoichiometries of adenosine 5'-triphosphate synthesis and proton extrusion as cells which contained both cytochromes o and d as terminal oxidases. In addition, CO inhibition and photo-relief of cytochromes o or d did not alter the efficiency of energy coupling. These findings indicate that adenosine 5'-triphosphate synthesis is coupled to electron transport through both cytochromes o and d in Acinetobacter.
Heterotrophic Carbon Metabolism by
Beggiatoa alba
Authors: Strohl William R. , Cannon Gordon C. , Shively Jessup M. , Güde Hans , Hook Leonard A. , Lane Candace M. , Larkin John M.
Published: January 2020

The assimilation and metabolism of CO
2
and acetate by
Beggiatoa alba
strain B18LD was investigated. Although
B. alba
was shown to require CO
2
for growth, the addition of excess CO
2
(as NaHCO
3
) to the medium in a closed system did not stimulate growth. Approximately 24 to 31% of the methyl-labeled acetate and 38 to 46% of the carboxyl-labeled acetate were oxidized to
14
CO
2
by
B. alba
. The apparent
V
max
values for combined assimilation and oxidation of [2-
14
C]acetate by
B. alba
were 126 to 202 nmol min
−1
mg of protein
−1
under differing growth conditions. The
V
max
values for CO
2
assimilation by heterotrophic and mixotrophic cells were 106 and 131 pmol min
−1
mg of protein
−1
, respectively. The low
V
max
values for CO
2
assimilation, coupled with the high
V
max
values for acetate oxidation, suggested that the required CO
2
was endogenously produced from acetate. Moreover, exogenously supplied acetate was required by
B. alba
for the fixation of CO
2
. From 61 to 73% of the [
14
C]acetate assimilated by washed trichomes was incorporated into lipid. Fifty-five percent of the assimilated [2-
14
C]acetate was incorporated into poly-β-hydroxybutyric acid. This was consistent with chemical data showing that 56% of the heterotrophic cell dry weight was poly-β-hydroxybutyric acid. Succinate and CO
2
were incorporated into cell wall material, proteins, lipids, nucleic acids, and amino and organic acids, but not into poly-β-hydroxybutyric acid. Glutamate and succinate were the major stable products after short-term [1-
14
C]acetate assimilation. Glutamate and aspartate were the first stable
14
CO
2
fixation products, whereas glutamate, a phosphorylated compound, succinate, and aspartate were the major stable
14
CO
2
fixation products over a 30-min period. The CO
2
fixation enzymes isocitrate dehydrogenase (nicotinamide adenine dinucleotide phosphate; reversed) and malate dehydrogenase (nicotinamide adenine dinucleotide phosphate; decarboxylating) were found in cell-free extracts of both mixotrophically grown and heterotrophically grown cells. The data indicate that the typical autotrophic CO
2
fixation mechanisms are absent from
B. alba
B18LD and that the CO
2
and acetate metabolism pathways are probably linked.
Growth-phase-dependent immunodeterminants of Rhizobium trifolii lipopolysaccharide which bind trifoliin A, a white clover lectin
Authors: Hrabak E M , Urbano M R , Dazzo F B
Published: January 2020

The lipopolysaccharide (LPS) from Rhizobium trifolii 0403 was isolated at different stages of growth and was examined for its (i) ability to bind a white clover lectin (trifoliin A), (ii) immunochemical properties, and (iii) composition. There was significantly more binding of trifoliin A to purified LPS and cells in the early stationary phase than to cells in the exponential phase. Immunofluorescence and enzyme-linked immunosorbent assays indicated that new antigenic determinants of the LPS appeared for brief periods on cells at the end of the lag phase and again at the beginning of the stationary phase. These new antigens were not detected on cells in midexponential or late stationary phase. Monovalent fragments of immunoglobulin G antibodies raised against the unique antigenic determinants in the LPS competitively blocked the binding of trifoliin A to cells in the early stationary phase. Gas chromatographic analysis showed that the relative quantity of several glycosyl components in the LPS increased as the culture advanced from the midexponential to the early stationary phase. In addition, LPS from cells in the early stationary phase had a higher aggregate molecular weight. Quinovosamine (2-amino-2,6-dideoxyglucose) was identified by combined gas chromatography-mass spectrometry as a sugar component of the LPS which had not been previously reported. D-Quinovosamine, N-acetyl-D-quinovosamine, and its n-propyl-beta-glycoside were effective hapten sugars which inhibited the binding of trifoliin A, anti-clover root antibody, and homologous antibody to these new determinants in the LPS. White clover plants had more infected root hairs after incubation with an inoculum of cells in the early stationary phase than after incubation with cells in the midexponential phase. The profound influence of the growth phase on the composition of lectin-binding polysaccharides of Rhizobium may be a major underlying cause of conflicting data among laboratories testing the lectin-recognition hypothesis. In addition, these chemical modifications may reflect mechanisms which regulate Rhizobium-root hair recognition in this nitrogen-fixing symbiosis.
Deoxyribonucleic acid methylation in mycobacteria
Authors: Srivastava R , Gopinathan K P , Ramakrishnan T
Published: January 2020

Deoxyribonucleic acid modification in six strains of mycobacteria was investigated. The presence of 5-methylcytosine in the virulent strain Mycobacterium tuberculosis H37Rv and its absence in the avirulent strain M. tuberculosis H37Ra and other saprophytic, fast-growing mycobacteria appear to be the salient features. However, deoxyribonucleic acid from M. smegmatis SN2 lysogenized with the temperature phage I3 showed the presence of 5-methylcytosine. All of the strains had N6-methyladenine.
Transformation of Haemophilus influenzae by plasmid RSF0885 containing a cloned segment of chromosomal deoxyribonucleic acid
Authors: Setlow J K , Notani N K , McCarthy D , Clayton N L
Published: January 2020

A plasmid containing a single cloned insertion of Haemophilus influenzae chromosomal deoxyribonucleic acid that carried a novobiocin resistance marker was 2.6 times larger than the parent plasmid, RSF0885, which conferred ampicillin resistance. The most frequent type of transformation by this plasmid (designated pNov1) was the transfer of novobiocin resistance to the chromosome, with the loss of the plasmid from the recipient. In accord with this observation, after radioactively labeled pNov1 entered a competent cell, it lost acid-insoluble counts, as well as biological activity. The level of ampicillin transformation, which involved establishment of the plasmid, was almost two orders of magnitude lower than the level of novobiocin transformation. Both types of transformation were depressed profoundly in rec-1 and rec-2 mutants. Ampicillin transformants of wild-type cells always contained plasmids that were the same size as pNov1, although most of these transformants were not novobiocin resistant. Plasmid pNov1 in wild-type cells but not in rec-1 or rec-2 cells often recombined with the chromosome, causing a homologous region of the chromosome to be substituted for part of the plasmid, as shown by restriction and genetic analyses. Our data suggested that plasmid-chromosome recombination took place only around the time when the plasmid entered a cell, rather than after it became established.
Inactivation of cell-associated fructosyltransferase in Streptococcus salivarius
Authors: Jacques N A , Wittenberger C L
Published: January 2020

In stationary phase, 95% of the fructosyltransferase (FTase) activity of Streptococcus salivarius ATCC 25975 was found associated with the cells. Within the first 15 min after inoculation into fresh medium, the specific activity of cell-associated FTase decreased by 92% of its initial value. After this period of initial loss, the enzyme was synthesized during exponential growth until a maximum level equivalent to that present before inoculation was obtained. The inactivation of FTase was also demonstrated in a nongrowing system. Washed cell suspensions incubated at 37 degrees C in 200 mM potassium phosphate buffer (pH 6.5) containing 10 microM Cu2+ lost 80 to 95% of their FRase activity after 30 min. This loss could be prevented by the addition of histidine, cysteine, or Ca2+ to the suspension mixture. A factor(s) essential for the inactivation of cell-associated FTase could itself be preferentially inactivated by heating cells at 40 degrees C for periods of up to 3 h, or by storage of cells at 0 to 4 degrees C for several days in a low-ionic-strength, low-pH, potassium phosphate buffer. Treatment of cells with the N-acetylmuramidase enzyme M-1, in the presence of 0.5 M melezitose, resulted in the release of FTase from the cell. The released enzyme was recovered in the supernatant fraction after centrifugation at 160,000 x g for 90 min. Comparison of solubilized active and inactivated FTase preparations by polyacrylamide gel electrophoresis demonstrated that the inactivation of cell-associated FTase activity was associated with the loss of specific protein bands.
Acyl coenzyme A carboxylase of Propionibacterium shermanii: detection and properties
Authors: Stirling L A , Ahmad P M , Ahmad F
Published: January 2020

An acyl coenzyme A (CoA) carboxylase, which catalyzes the adenosine triphosphate-dependent fixation of CO2 into acetyl-, propionyl-, and butyryl-CoA, was detected in fractionated cell extracts of Propionibacterium shermanii. Catalytic activity was inhibited by avidin but was unaffected by avidin pretreated with excess biotin. The carboxylase levels detected were relatively small and were related to cellular growth. Maximal carboxylase activity was detected in cells grown for about 96 h. Thereafter, the activity declined rapidly. Optimal CO2 fixation occurred at pH 7.5. Other parameters of the assay system were optimized, and the apparent Km values for substrates were determined. The end product of the reaction (with acetyl-CoA as the substrate) was identified as malonyl-CoA. The stoichiometry of the reaction was such that, for every mole of acetyl-CoA and adenosine triphosphate consumed, 1 mol each of malonyl-CoA, adenosine diphosphate, and orthophosphate was formed. These data provide the first evidence for the presence of another biotin-containing enzyme, an acyl-CoA carboxylase, in these bacteria in addition to the well-characterized methylmalonyl-CoA carboxyltransferase.
Electron transport system of an aerobic carbon monoxide-oxidizing bacterium
Authors: Kim Y M , Hegeman G D
Published: January 2020

Experiments with crude extracts of Pseudomonas carboxydohydrogena revealed that a quinone is necessary for CO oxidation, and that cytochromes of the a, b, and c types and functional terminal oxidase(s) are found in cells grown on CO as the sole source of carbon and energy. CO dehydrogenase delivers electrons to the electron transport system at the level of quinone, and nicotinamide adenine dinucleotide (phosphate) is not involved in CO oxidation.
Valine-Resistant
Escherichia coli
K-12 Strains with Mutations in the
ilvB
Operon
Authors: Sutton Ann , Newman Thomas , Francis Marilyn , Freundlich Martin
Published: January 2020

Escherichia coli
K-12 mutants resistant to growth inhibition by valine were isolated. These strains contained mutations in the
ilvB
operon effecting either the regulation of acetohydroxy acid synthase I or the sensitivity of the enzyme to end product inhibition by valine.
Physical and genetic analysis of deletion mutants of plasmid R91-5 and the cloning of transfer genes in Pseudomonas aeruginosa
Authors: Moore R J , Krishnapillai V
Published: January 2020

We isolated deletion mutants of Pseudomonas aeruginosa plasmid R91-5 by both in vitro and in vivo means. Many of the deletion mutants selected on the basis of resistance to donor-specific phages fell into a few groups of apparently identical mutants, although the mutants were nonsibs. By analyzing plasmids with large deletions, we found that the essential replication genes of R91-5 were within a 3.85-kilobase region between coordinates 45.5 and 48.9. The origin of plasmid transfer (oriT) was mapped to a 4.5-kilobase region between coordinates 1.7 and 6.2. We indirectly determined the direction of plasmid transfer from oriT. By combining the data from our analysis of the deletions with data from complementation tests between cloned R91-5 fragments and known reference mutants, we ordered and mapped the 10 known transfer (tra) cistrons of R91-5. All of the tra cistrons mapped within the Tra2 region, and their order was as follows: traX, -Y, -T, -Q, -(V, R), -U, -(S, Z), -W (the cistrons in parentheses could not be ordered with respect to each other).
Antibody analysis of relationships among methanogenic bacteria
Authors: Conway de Macario E , Wolin M J , Macario A J
Published: January 2020

A bank of antisera to the majority of methanogenic bacteria is now available. Three antibody probes, R, S, and T, were derived from each antiserum in the bank and used for analysis of antigenic relatedness among methanogens by immunofluorescence. The T probe reacted only with the immunizing (or homologous) strain, the S probe gave strong cross-reactions with strains of the same species, and the R probe revealed some interspecies relationships. The results were confirmed and extended by enzyme immunoassays and standard serological methods involving serial dilution analysis, cross-adsorptions, and the use of reference strains. The immunological methods and standardized antibody probes are useful for rapid identification of methanogens and measurements of antigenic relationships which aid in the classification of these bacteria.
Identification of cell wall subunits in bacillus subtilis and analysis of their segregation during growth
Authors: Schlaeppi J M , Pooley H M , Karamata D
Published: January 2020

Continuous as well as pulse-labeling and chase experiments with Bacillus subtilis demonstrated that the cell wall (both peptidoglycan and teichoic acid) is composed of a limited number of blocks which, once completed, segregate during subsequent growth without undergoing any mixing with newly synthesized blocks. This observation suggests that new wall material is inserted in a limited number of zones. Previously reported observations which suggested diffuse intercalation of new wall material are reinterpreted on the basis of our results. Experiments performed on different media showed that the number of segregation units per unit of cell length and thus the density of insertion zones increases with medium richness. This finding suggests analogies between the regulation of cell wall and DNA synthesis.
Molybdenum cofactor requirement for biotin sulfoxide reduction in Escherichia coli
Authors: del Campillo-Campbell A , Campbell A
Published: January 2020

The bisC gene of Escherichia coli is tentatively identified as the structural gene for biotin sulfoxide reductase by the isolation of bisC(Ts) mutants that make thermolabile enzyme. The products of four other E. coli genes (chlA, chlB, chlE and chlG) are also needed for enzymatic activity. Mutations previously assigned to the bisA, bisB, and bisD genes belong to genes chlA, chlE, and chlG, respectively. The biotin sulfoxide reductase deficiency of a chlG, mutant is partially reversed by the addition of 10 mM molybdate to the growth medium. Mutational inactivation of the chlD gene reduces the specific activity of biotin sulfoxide reductase about twofold. This effect is reversed by the addition of 1 mM molybdate to the growth medium. The specific activity of biotin sulfoxide reductase is decreased about 30-fold by the presence of tungstate in the growth medium, an effect that has been observed previously with nitrate reductase and other molybdoenzymes. The specific activity of biotin sulfoxide reductase is not elevated in a lysate prepared by derepressing a lambda cI857 chlG prophage. Whereas biotin sulfoxide reductase prepared by sonic extraction of growing cells is almost completely dependent on the presence of a small heat-stable protein resembling thioredoxin, much of the enzyme obtained from lysates of thermoinduced lambda cI857 lysogens does not require this factor.
Occurrence of nickel in carbon monoxide dehydrogenase from Clostridium pasteurianum and Clostridium thermoaceticum
Authors: Drake H L
Published: January 2020

The carbon monoxide (CO) dehydrogenase activity band from Clostridium pasteurianum was shown to contain nickel by in situ activity staining of polyacrylamide gels. However, the majority of the nickel in cell extracts was found to electrophorese independently of CO dehydrogenase. Comparative analysis with Clostridium thermoaceticum demonstrated that, although the majority of nickel was accounted for in CO dehydrogenase in anaerobic extracts, the metal dissociated from the enzyme when inactivated by oxidation.
Respiration-dependent proton translocation in Nitrosomonas europaea and its apparent absence in Nitrobacter agilis during inorganic oxidations
Authors: Hollocher T C , Kumar S , Nicholas D J
Published: January 2020

Oxygen pulse experiments were carried out with the nitrifying bacteria Nitrosomonas europaea and Nitrobacter agilis and with spheroplasts and everted vesicles prepared from Nitrobacter agilis. In addition to thiocyanate, the salting-in anions perchlorate and trichloroacetate proved to be permeant and effective in allowing respiration-dependent proton translocation with Nitrosomonas europaea. Valinomycin-K+, however, was generally ineffective in this respect with Nitrosomonas europaea. The observed leads to H+/O ratio for ammonium ion oxidation by Nitrosomonas europaea was 3.4; that for hydroxylamine and hydrazine cation oxidation was 4.4. These values, when corrected for production of stoichiometric protons and for the fact that the first step in ammonium ion oxidation (hydroxylamine production) is mediated by a monooxygenase, give effective leads to H+/O ratios of about 4 for these three substrates. This value compares favorably with those obtained with other aerobes. No convincing evidence was obtained for operation of a respiratory proton pump in Nitrobacter agilis during nitrite oxidation. Implications of this unexpected result are discussed.
Isolation of a maltase structural gene from Saccharomyces carlsbergensis
Authors: Federoff H J , Cohen J D , Eccleshall T R , Needleman R B , Buchferer B A , Giacalone J , Marmur J
Published: January 2020

The maltase structural gene MAL6 of the yeast Saccharomyces carlsbergensis has been cloned by transformation of a maltose nonfermenting recipient strain with autonomously replicating chimeric recombinant plasmids. One recombinant plasmid, pMAL26, was shown by positive hybridization translation, as well as by Southern and Northern blot experiments, to carry the MAL6 structural gene.