bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of Saccharomyces cerevisiae mutants deficient in S-adenosylmethionine decarboxylase, spermidine, and spermine
Authors: Cohn M S , Tabor C W , Tabor H
Published: January 2020

Four mutants were isolated from Saccharomyces cerevisiae that are deficient in S-adenosylmethionine decarboxylase (spe2). All four mutants are chromosomal and fall into a single complementation group tightly linked to arg1. Since one of the mutants contained a temperature-sensitive activity, this complementation group defines the structural gene. Mutants totally lacking enzymic activity did not contain spermidine or spermine and had a greatly increased doubling time when grown in the absence of these two polyamines. Addition of 10(-6) M spermidine or 10(-5) M spermine, but not putrescine or cadaverine, restored the doubling time to that of the wild type. Diploids formed from a cross of two mutants completely deficient in spermidine and spermine were unable to sporulate in the absence of added spermidine or spermine. We obtained evidence that arg1 was not located on any of the 17 known chromosomes, and therefore we postulate that arg1 and spe2 are located on a new 18th chromosome.
Ribosomal DNA magnification in Saccharomyces cerevisiae
Authors: Kaback D B , Halvorson H O
Published: January 2020

Strains monosomic for chromosome I of Saccharomyces cerevisiae contain 25 to 35% fewer rRNA genes than do normal diploid strains. When these strains are repeatedly subcultured, colonies are isolated that have magnified their number of rRNA genes to the diploid amount while remaining monosomic for chromosome I. We have determined the amount of DNA complementary to rRNA in viable haploid spores derived from a magnified monosomic strain. Some of these haploids contained 24 to 48% more rRNA genes than a normal euploid strain. These extra genes may be responsible for the increased number of rRNA genes in the strain monosomic for chromosome I. Genetic analysis of the haploids containing extra rRNA genes suggested that these genes are linked to chromosomal DNA and are heterozygous. They were not closely linked to any centromere and were not located on chromosome I. Furthermore, all the DNA complementary to rRNA in one of these haploid strains with magnified rRNA genes sedimented at a chromosomal molecular weight, consistent with chromosomal linkage. In addition, several new mutations mapping on chromosome I were used to show that ribosomal DNA magnification was not due to a chromosome I duplication.
Characterization of two types of yeast ribosomal DNA genes
Authors: Petes T D , Hereford L M , Skryabin K G
Published: January 2020

The intragenic organization of ribosomal DNA from a diploid strain of Saccharomyces cerevisiae was analyzed by using recombinant DNA molecules constructed in vitro. Restriction analysis of the yeast ribosomal DNA with the EcoRI restriction enzyme indicated that eight restriction fragments were present in the ribosomal DNA of this strain: X' (1.87 X 10(6) daltons), A (1.77 X 10(6) daltons), B (1.48 X 10(6) daltons), C (1.22 X 10(6) daltons), D (0.39 X 10(6) daltons), E (0.36 X 10(6) daltons), F (0.22 X 10(6) daltons), and G (0.17 X 10(6) daltons). These fragments were distributed between two different types of ribosomal DNA genes, which had the restriction maps: (formula: see text) in which the underlined region shows the repeating unit. The diploid yeast strain contained approximately equal amounts of each of these two types of genes. The analysis of the recombinant DNA molecules also indicated that the yeast ribosomal genes are homogeneous and extensively clustered.
Location of the 5.8S rRNA gene of Saccharomyces cerevisiae
Authors: Skryabin K G , Maxam A M , Petes T D , Hereford L
Published: January 2020

Direct DNA sequence analysis of Saccharomyces cerevisiae ribosomal DNA cloned in an Escherichia coli plasmid revealed part of the structural gene for 5.8S rRNA at one end of a 700-base-pair EcoRI fragment. Taken with the previously established EcoRI restriction map of the ribosomal repeat unit, this sequence establishes that the yeast 5.8S RNA segment is located between the 18S and 28S segments in the 42S rRNA precursor and in the DNA which codes for it.
Tryptophan biosynthesis in Saccharomyces cerevisiae: control of the flux through the pathway
Authors: Miozzari G , Niederberger P , Hütter R
Published: January 2020

Enzyme derepression and feedback inhibition of the first enzyme are the regulatory mechanisms demonstrated for the tryptophan pathway in Saccharomyces cerevisiae. The relative contributions of the two mechanisms to the control of the flux through the pathway in vivo were analyzed by (i) measuring feedback inhibition of anthranilate synthase in vivo, (ii) determining the effect of regulatory mutations on the level of the tryptophan pool and the flux through the pathway, and (iii) varying the gene dose of individual enzymes of the pathway at the tetraploid level. We conclude that the flux through the pathway is adjusted to the rate of protein synthesis by means of feedback inhibition of the first enzyme by the end product, tryptophan. The synthesis of the tryptophan enzymes could not be repressed below a basal level by tryptophan supplementation of the media. The enzymes are present in excess. Increasing or lowering the concentration of individual enzymes had no noticeable influencing on the overall flux to tryptophan. The uninhibited capacity of the pathway could be observed both upon relieving feedback inhibition by tryptophan limitation and in feedback-insensitive mutants. It exceeded the rate of consumption of the amino acid on minimal medium by a factor of three. Tryptophan limitation caused derepression of four of the five tryptophan enzymes and, as a consequence, led to a further increase in the capacity of the pathway. However, because of the large reserve capacity of the "repressed" pathway, tryptophan limitation could not be imposed on wild-type cells without resorting to the use of analogs. Our results, therefore, suggest that derepression does not serve as an instrument for the specific regulation of the flux through the tryptophan pathway.
alpha-amylase from five strains of Bacillus amyloliquefaciens: evidence for identical primary structures
Authors: Borgia P T , Campbell L L
Published: January 2020

The alpha-amylases from five strains of Bacillus amyloliquefaciens were compared to determine whether differences in primary structure are responsible for variations in catalytic properties previously reported among the enzymes. Amino acid analysis established virtually identical compositions for the proteins. Reaction with dimethylaminoaphthylene sulfonylchloride indicated the amino-terminal amino acid of each amylase to be valine. Carboxyl termini of the enzymes have been determined by digestion with carboxypeptidase A. The resulting kinetic data indicate tyrosine as the carboxyl terminus and leucine as the penultimate residue for all five proteins. Isoelectric focusing of the enzymes yielded isoelectric points in the pH range of 5.09 to 5.18. Tryptic digests of the enzymes chromatographed on a cation-exchange column showed identical elution patterns. It is concluded that the primary structure of the amylase from the five strains is identical or exhibits only conservative substitutions.
Repression of Escherichia coli pyridine nucleotide transhydrogenase by leucine
Authors: Gerolimatos B , Hanson R L
Published: January 2020

Addition of 0.1% casein hydrolysate to a minimal growth medium decreased membrane-bound transhydrogenase activity in Escherichia coli by about 80%. Of the amino acids added individually to the growth medium, only leucine and, to a lesser extent, methionine and alanine were effective, alpha-Ketoisocaproate- and leucine-containing peptides repressed the activity, and leucine also repressed activity in adenyl cyclase-deficient and relaxed strains. Derepression of transhydrogenase followed the removal of leucine from the growth medium and was sensitive to rifampin and chloramphenicol. A phosphoglucoisomerase-deficient strain that was forced to use the hexose monophosphate shunt exclusively had normal levels of transhydrogenase, which was repressed by leucine. Transhydrogenase activity doubled in mutants lacking either of the shunt dehydrogenases but was still repressed by leucine. In strains constitutive for the leucine biosynthetic operon, transhydrogenase was repressed by leucine but in strains livR and lst R, with leucine transport resistant to leucine repression, transhydrogenase was not repressed by leucine. These data suggest that transhydrogenase may have a function in the transport of branched-chain amino acids. In a hisT strain (which has altered leucyl-tRNA), transhydrogeanse was at a repressed level without the addition of leucine, suggesting that leucyl-tRNA may be involved in the regulation.
Biosynthesis of exopolysaccharide by Pseudomonas aeruginosa
Authors: Mian F A , Jarman T R , Righelato R C
Published: January 2020

In batch cultures of Pseudomonas aeruginosa, the maximum rate of exopolysaccharide synthesis occurred during exponential growth. In nitrogen-limited continuous culture, the specific rate of exopolysaccharide synthesis increased from 0.27 g g of cell-1 h-1 at a dilution rate (D) of 0.05 h-1 to 0.44 g g of cells h-1 at D=0.1 H-1. The yield of exopolysaccharide on the basis of glucose used was in the range of 56 to 64%. Exopolysaccharide was also synthesized in carbon-limited cultures at 0.19 g g of cell-1 h-1 at D=0.05 h-1 in a 33% yield. Nonmucoid variants appeared after seven generations in continuous culture and rapidly increased in proportion to the total number of organisms present.
Molybdenum independence of nitrogenase component synthesis in the non-heterocystous cyanobacterium Plectonema
Authors: Nagatani H H , Haselkorn R
Published: January 2020

The cyanobacterium Plectonema boryanum (IU 594-UTEX 594) fixes N2 only in the absence of combined N and of O2. We induced nitrogenase by transfer to anaerobic N-free medium and studied the effect of Mo starvation on nitrogenase activity and synthesis. Activity was first detected within 3 h after transfer by the acetylene reduction assay in controls, increasing for at least 25 h. Cells grown on nitrate and Mo and then transferred to N-free, Mo-free medium produced 8% of the control nitrogenase activity. Addition of W to the Mo-free medium reduced the activity to 0.5%. Under both Mo starvation conditions, nitrogenase protein components were synthesized. Component II of the cyanobacterial enzyme was detected by in vitro complementation with Mo-containing component I from Klebsiella pneumoniae or Azotobacter vinelandii but not Clostridium pasteurianum. Component I activity was restored by addition of Mo to cultures in which new enzyme synthesis was blocked by chloramphenicol. Acidified extracts of Plectonema induced in Mo-containing medium contained the Fe-Mo cofactor required to activate extracts of the Azotobacter mutant UW45 in vitro, but they did not activate extracts of Mo-starved Plectonema. Analysis of 35SO4(2-)-labeled proteins by polyacrylamide gel electrophoresis suggested that Mo is required for the conversion of a high-molecular-weight precursor to component I in Plectonema.
Characterization of a Salmonella typhimurium hisU mutant defective in tRNA precursor processing
Authors: Bossi L , Ciampi M S , Cortese R
Published: January 2020

The DA11 mutant of Salmonella typhimurium, originally isolated as derepressed for the histidine operon, carries a temperature-dependent alteration in a nucleolytic enzyme specifically involved in the maturation of tRNA. As a consequence of this alteration, no detectable synthesis of any mature tRNA species occurs in DA11 upon shift at 43 degrees C, whereas many tRNA precursors, whose sizes range between 80 and 750 nucleotides, do accumulate. Kinetic studies on the synthesis and processing of these maturation intermediates show that these molecules represent different stages in the maturation pathway, most of them being the products of previous nucleolytic events. These RNA molecules are in vivo substrates of methylation and thiolation enzymes and can be cleaved in vitro to 4S RNA by wild-type but not by DA11 cell-free extract. Evidence is presented that DA11 is very probably a ribonuclease P mutant.
Intracytoplasmic membrane, phospholipid, and sterol content of Methylobacterium organophilum cells grown under different conditions
Authors: Patt T E , Hanson R S
Published: January 2020

Intracytoplasmic membranes were present in Methylobacterium organophilum when cells were grown with methane, but not methanol or glucose, as the sole carbon and energy source. Cells grown with methane as the carbon and energy source and low levels of dissolved oxygen had the greatest amount of intracytoplasmic membrane. Cells grown with increased levels of dissolved oxygen had less intracytoplasmic membrane. The amount of total lipid correlated with the amount of membrane material observed in thin sections. The individual phospholipids varied in amount, but the same four were present in M. organophilum grown with different substrates and oxygen levels. Phosphatidyl choline was present as a major component of the phospholipids. Sterols were present, and they differed from those in the type I methylotroph Methylococcus capsulatus. The relative amounts of different sterols and squalene changed with the substrate provided for growth. The greatest amounts of sterols were found in methane-grown cells grown at low levels of dissolved oxygen. None of the unusual or usual membrane components assayed was uniquely present in the intracytoplasmic membranes.
Chemoresponses of Chlamydomonas reinhardtii
Authors: Hirschberg R , Rodgers S
Published: January 2020

Cells of Chlamydomonas reinhardtii have been found to respond to chemicals in two ways: chemokinesis and chemotaxis. Several amino acids, fatty acids, and inorganic salts can stimulate these responses.
Escherichia coli K-12 mutants deficient in uracil-DNA glycosylase
Authors: Duncan B K , Rockstroh P A , Warner H R
Published: January 2020

A new assay specific for uracil-DNA glycosylase is described, Escherichia coli mutants partially and totally deficient in uracil-DNA glycosylase activity have been isolated by using this assay in mass-screening procedures. These have been designated ung mutants. The ung gene maps between tyrA and nadB on the E. coli chromosome. T4 phage containing uracil in their DNA grow on the most glycosylase-deficient hosts but are unable to grow on wild-type bacteria. This provides a simple spot test for the ung genotype. The ung mutants show slightly higher rates of spontaneous mutation to antibiotic resistance. Taken together, these results suggest a central role for uracil-DNA glycosylase in the initiation of an excision repair pathway for the exclusion of uracil from DNA.
Some Properties of
Streptomyces viridochromogenes
Spores
Authors: Hirsch C. F. , Ensign J. C.
Published: January 2020

Dry spores of
Streptomyces viridochromogenes
exhibited no endogenous metabolism when tested by the sensitive radiorespirometric technique. Wetting the spores resulted in a sharp increase in endogenous respiration followed by a gradual decrease to a constant level, whereby 0.02% of the spore carbon was respired to CO
2
per h. The rate of endogenous metabolism increased slowly as unactivated and heat-activated spores germinated in a defined germination medium, reaching rapid rates only after germination was completed. Components of the defined germination medium, adenosine,
l
-alanine, and
l
-glutamic acid, were oxidized to CO
2
at appreciable rates only after germination was complete. The QO
2
(microliters of O
2
uptake per hour per milligram [dry weight] of spores) values for endogenously respiring spores were 3.9 for unactivated and 7.8 for activated spores. Various sugars, amino acids, and organic acids were oxidized only slowly or not at all by unactivated and activated spores. The dry spores contained 5.2 × 10
−2
μmol of ATP per g (dry weight). The ATP content of spores increased approximately 4-fold after suspension in buffer and approximately 11-fold after heat activation. During germination, the ATP level increased to a level of 1 μmol of ATP per g (dry weight) and remained constant. Germination was accompanied by excretion from the spores of approximately 8 and 12% of the total spore carbon from unactivated and activated spores, respectively. A potent germination inhibitor was released from the germinated spores. The germination inhibitor had no effect on heat-activated spores or spores which had begun germination for as short a time as 5 min.
Association of the Folded Chromosome with the Cell Envelope of
Escherichia coli
: Nature of the Membrane-Associated DNA
Authors: Drlica Karl , Burgi Elizabeth , Worcel Abraham
Published: January 2020

Membrane-associated folded chromosomes isolated from
Escherichia coli
in the presence of spermidine sedimented at about 5,800S. The folded chromosome and the membrane fragment were each stable in the absence of the other; a 1,700S folded chromosome was obtained after removal of the membrane by a Sarkosyl treatment, and a 4,000S membrane fragment remained after digestion of the chromosomal DNA with deoxyribonuclease I. The interaction between the folded chromosome and the membrane fragment was stable, and, even when the DNA was unfolded, both components remained associated and cosedimented. The large frictional effect of the unfolded DNA reduced the sedimentation rate of the complex to about 2,000S. Partial removal of this unfolded DNA with restriction endonucleases caused the membrane fragments and the remaining associated DNA to sediment faster, at about 3,500S. The DNA remaining associated with the membrane fragments after restriction endonuclease treatment, about 4.5% of the total DNA when
Eco
RI was used, was indistinguishable from the DNA released from the membranes by three criteria: (i) DNA size distribution in agarose gels after electrophoresis, (ii) reassociation kinetics, and (iii) thermal elution from hydroxylapatite. This finding, that random DNA sequences rather than specific ones were responsible for the majority of the DNA-membrane interactions, argues against the folded chromosome's being a static structure with specific DNA sequences interacting with the cell envelope.
Apparent molecular weights of a heat-modifiable protein from the outer membrane of Escherichia coli in gels with different acrylamide concentrations
Authors: Heller K B
Published: January 2020

The apparent molecular weights of the two forms of a heat-modifiable protein from the outer membrane of Escherichia coli K-12, estimated in gels with different concentrations of acrylamide, indicate that the protein binds excess amounts of sodium dodecyl sulfate, possibly due to large beta structures before boiling.
Approximation of the cell cycle in synchronized populations of Streptococcus faecium
Authors: Hinks R P , Daneo-Moore L , Shockman G D
Published: January 2020

Slowly growing populations (TD = 70 to 80 min) of Streptococcus faecium (S. faecalis ATCC 9790) were synchronized by selection after sucrose gradient fractionation. The cell cycle was approximated by correlating the patterns of DNA accumulation and cell division. More specifically, the beginning of cell cycle was equated with the beginning of a rapid linear increase in DNA accumulation. The DNA content of the culture approximately doubled during the period of accumulation, which lasted about 51 min. The period of rapid DNA accumulation, was followed by a period of reduced accumulation that lasted about 24 min. During synchronized growth, cell numbers increased rapidly in coordination with the period of rapid DNA accumulation and exhibited a plateau during the period of reduced DNA accumulation. In contrast, RNA and protein appeared to accumulate exponentially throughout the cell cycle at the same rate as culture mass.
Mu-induced polarity in the unc operon of Escherichia coli
Authors: Gibson F , Downie J A , Cox G B , Radik J
Published: January 2020

Mutant strains of Escherichia coli were isolated in which mutator (Mu) phage was inserted into various unc genes. Partial diploid strains were prepared from each of the Mu-induced unc mutants by using F-plasmids carrying mutations in one of the known unc genes (uncA, uncB, uncC, or uncD). The partial diploid strains and the corresponding segregant strains were examined for their ability to grow on succinate. The aerobic growth yields on limiting concentrations of glucose were also determined. Magnesium-stimulated adenosine triphosphatase activities, ATP-dependent transhydrogenase activities, and Atebrin fluorescence quenching activities were determined by using membrane preparations from each strain. Genetic complementation was assessed from the results obtained, and it was concluded that the four unc genes examined are part of a single transcriptional unit and that they are transcribed in the order uncBADC.
Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli: isolation of a new F-prime factor, F80, and its implication for the mechanism of F integration into the chromosome
Authors: Ohtsubo E , Hsu M T
Published: January 2020

A new F-prime factor, F80, was isolated from an Escherichia coli strain harboring the F-prime factor F8 by selecting for transfer of the supE marker to a RecA- recipient. Genetic analysis shows that F80 carries a segment of the chromosomal DNA between lip and suc in addition to the tol-gal region normally in F8. Physical analysis by the electron microscope heteroduplex method suggests that the formation of F80 from F8 involves recombination between the alphabeta segment of F, which is present in F8, and the homologous sequence of F present in the E. coli chromosome at the site where F is supposed to integrate to form HfrP3. The implications of this result for the general mechanisms of F integration to form Hfr's are discussed.
Complementary functioning of the component proteins of nitrogenase from several bacteria
Authors: Emerich D W , Burris R H
Published: January 2020

The nitrogenase proteins from eight organisms have been highly purified, and a survey of their cross-reactions shows that the nitrogenase proteins from a wide variety of organisms can interact with one another. An active cross-reaction is the complementary functioning of the MoFe protein and the Fe protein from different organisms. Of 64 possible combinations of component proteins, 8 yielded homologous nitrogenases (components from the same organism); 45 of the 56 possible heterologous crosses generated active hybrid nitrogenases; 4 heterologous crosses yielded no measurable nitrogenase activity but did form inactive tight-binding complexes; 6 crosses did not give measurable activity; and 1 cross was not made. All these crosses were assayed for acetylene reduction, and some also were assayed for ammonia formation, hydrogen evolution, and ATP hydrolysis activity. The activity generated by combining two complementary heterologous nitrogenase components depended on pH, component ratio, and protein concentration, the same factors that determine the activity of homologous nitrogenases. However, several crosses showed an unusual dependency on component ratio and protein concentration, and some cross-reactions showed interesting ATP hydrolysis activity.
3-hydroxypyruvate substitutes for pyridoxine in serC mutants of Escherichia coli K-12
Authors: Shimizu S , Dempsey W B
Published: January 2020

Escherichia coli K-12 mutants with serC genotype required pyridoxine and serine for normal growth, as do E. coli B mutants of this type. Mutants of the K-12 strain, however, reverted easily to pyridoxine independence without regaining activity in the 3-phosphoserine oxoglutarate transaminase coded for by the serC gene. Both these revertants and the parental type synthesized pyridoxine in normal amounts when 3-hydroxypyruvate was used as a supplement, although neither of these mutants could use this compound to satisfy their serine requirement. Since serine alone was inadequate to provide the nutritional requirement of serC mutants, these mutants must have been unable to synthesize 3-hydroxypyruvate from serine. We suggest that 3-phosphoserine oxoglutarate transaminase in normal E. coli serves as a catalyst for transaminating small amounts of serine to 3-hydroxypyruvate, which is then used in pyridoxine biosynthesis. In serC mutants, this activity is blocked, and these mutants then show a double requirement for serine and pyridoxine.
F'-plasmid transfer from Escherichia coli to Pseudomonas fluorescens
Authors: Mergeay M , Gerits J
Published: January 2020

Various F' plasmids of Escherichia coli K-12 could be transferred into mutants of the soil strain 6.2, classified herein as a Pseudomonas fluorescens biotype IV. This strain was previously found to receive Flac plasmid (N. Datta and R.W. Hedges, J. Gen Microbiol. 70:453-460, 1972). ilv, leu, met, arg, and his auxotrophs were complemented by plasmids carrying isofunctional genes; trp mutants were not complemented or were very poorly complemented. The frequency of transfer was 10(-5). Subsequent transfer into other P. fluorescens recipients was of the same order of magnitude. Some transconjugants were unable to act as donors, and these did not lose the received information if subcultured on nonselective media. Use of F' plasmids helped to discriminate metabolic blocks in P. fluorescens. In particular, metA, metB, and argH mutants were so distinguished. In addition, F131 plasmid carrying the his operon and a supD mutation could partially relieve the auxotrophy of thr, ilv, and metA13 mutants, suggesting functional expression of E. coli tRNA in P. fluorescens. In P. fluorescens metA Rifr mutants carrying the F110 plasmid, which carried the E. coli metA gene and the E. coli rifs allele, sensitivity to rifampin was found to be dominant at least temporarily over resistance. This suggests interaction of E. coli and P. fluorescens subunits of RNA polymerase. his mutations were also complemented by composite P plasmids containing the his-nif region of Klebsiella pneumoniae (plasmids FN68 and RP41). nif expression could be detected by acetylene reduction in some his+ transconjugants. The frequency of transfer of these P plasmids was 5 X 10(-4).
Ultrastructural Study of
Salmonella typhimurium
Treated with Membrane-Active Agents: Specific Reaction of Dansylchloride with Cell Envelope Components
Authors: Schindler Peter R. G. , Teuber Michael
Published: January 2020

Amino groups of cell envelope proteins, lipids, and lipopolysaccharides cannot be labeled in intact cells of
Salmonella typhimurium
G 30 by using 5-dimethylaminonaphthalene-1-sulfonylchloride incorporated in lecithin-cholesterol vesicles. However, application of membrane-interacting agents like tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, ethylenediaminetetraacetate (Na salt) (EDTA), divalent cations, and sublethal doses of the cationic antibacterial agents polymyxin B and chlorhexidine induced specific fluorescent labeling of envelope proteins and lipids but not of cytoplasmic compounds, with the exception of a soluble protein with a molecular weight of 46,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with Tris-hydrochloride buffer produced labeling of the heat-modifiable protein B/B
+
and of proteins with molecular weights of 26,000, 22,000, and below 17,000. A combination of Tris-hydrochloride and EDTA induced additional dansylation of the major protein A and of proteins of molecular weights 80,000, 60,000, and 44,000. Polymyxin B and chlorhexidine caused similar labeling patterns. In every case, except with divalent cation treatment, protein B/B
+
was the most prominently labeled species. Phosphatidylethanolamine was dansylated up to 30%. Lipopolysaccharide was not reactive under any condition or treatment. In addition, the peptidoglycan-bound lipoprotein did not react with dansylchloride in either intact or Tris-hydrochloride-treated cells. The results are discussed with regard to a possible localization of labeled and unlabeled compounds of the cell envelope on the basis of a model placing cell envelope amino groups into ion-ion interactions with anionic components of other envelope compounds like phosphate and carboxyl groups.
Specificity and Regulation of γ-Aminobutyrate Transport in
Escherichia coli
Authors: Kahane S. , Levitz R. , Halpern Y. S.
Published: January 2020

A specific γ-aminobutyrate (GABA) transport system in
Escherichia coli
K-12 cells with a

K
m

of 12 μM and a

V
max

of 278 nmol/ml of intracellular water per min is described. Membrane vesicles contained
d
-lactate-dependent activity of the system. Mutants defective in GABA transport were isolated; they lost the ability to utilize GABA as a nitrogen source, although the activities of glutamate-succinylsemialdehyde transaminase (GSST) (EC 2.6.1.19) and succinylsemialdehyde dehydrogenase (SSDH) (EC 1.2.1.16), the enzymes that catalyze GABA utilization, remained as high as in the parental CS101B strain. The ability to utilize
l
-ornithine,
l
-arginine, putrescine,
l
-proline, and glycine as a nitrogen source was preserved in the mutants. The genetic lesions resulting in the loss of GABA transport,
gabP5
and
gabP9
, mapped in the
gab
gene cluster in close linkage to
gabT
and
gabD
, the structural genes of GSST and SSDH, and to
gabC
, a gene controlling the utilization of GABA, arginine, putrescine, and ornithine. The synthesis of the GABA transport carrier is subject to dual physiological control by (i) catabolite repression and (ii) nitrogen availability. Experiments with glutamine synthetase (EC 6.3.1.2)-negative and with glutamine synthetase-constitutive strains strongly indicate that this enzyme is the effector in the regulation of GABA carrier synthesis by route (ii).
Effect of cultural conditions on the concentrations of metabolic intermediates during growth and sporulation of Bacillus licheniformis
Authors: Donohue T J , Bernlohr R W
Published: January 2020

Intracellular concentrations of adenine nucleotides and intermediates of the Embden-Meyerhof pathway and the tricarboxylic acid cycle have been determined during growth and sporulation of Bacillus licheniformis in a variety of different media. The ATP pool was independent of growth rate and nitrogen source, but the use of glucose as a carbon source resulted in a twofold elevation in the ATP pool during exponential growth. The intracellular phosphoenolpyruvate pool was at least twofold higher during gluconeogenesis than during glycolysis. The finding that the use of glutamate as the sole nitrogen source resulted in at least a fivefold elevation of the alpha-ketoglutarate pool suggests a role for alpha-ketoglutarate in the repression of the enzymes of the tricarboxylic acid cycle responsible for alpha-ketoglutarate synthesis. Not one of the metabolites assayed appears to function as a signal of the nutrient deprivation which accompanies the initiation of sporulation.
Aerobic growth thermograms of Streptococcus lactis obtained with a complex medium containing glucose
Authors: Monk P R
Published: January 2020

With different culturing methods both simple and complex thermograms were obtained with Streptococcus lactis grown aerobically in a complex medium containing growth-limiting concentrations of glucose. The thermogram profiles have been interpreted in relation to growth rate, glucose degradation, and molar growth yields calculated for different time intervals during growth.
Positive regulation of amidase synthesis in Pseudomonas aeruginosa
Authors: Farin F , Clarke P H
Published: January 2020

Mutants of Pseudomonas aeruginosa were isolated that were acetamide-negative in growth phenotype at 41 degrees C and constitutive for amidase synthesis at 28 degrees C. Two mutants were derived from the magno-constitutive amidase mutant PAC111 (C11), and a third from a mutant that had enhanced inducibility by formamide, PAC153 (F6). The three temperature-sensitive mutants produced amidases with the same thermal stabilities as the wild-type enzyme. Cultures growing exponentially at 28 degrees C, synthesizing amidase constitutively, ceased amidase synthesis almost immediately on transfer to 41 degrees C. Cultures growing at 41 degrees C were transferred to 28 degrees C and had a lag of about 0.5 of a generation before amidase synthesis became detectable. Pulse-heating for 10 min at 45 degrees C of a culture growing exponentially at 28 degrees C resulted in a lag of about 0.5 of a generation before amidase synthesis recommenced after returning to 28 degrees C. Acetamide-negative mutants that were unable to synthesize amidase at any growth temperature were isolated from an inducible strain producing the mutant B amidase PAC398 (IB10). Two mutants were examined that gave revertants producing B amidase but with novel regulatory phenotypes. It is suggested that amidase synthesis is regulated by positive control exerted by gene amiR.
Membrane phospholipid asymmetry in Bacillus amyloliquefaciens
Authors: Paton J C , May B K , Elliott W H
Published: January 2020

The phospholipid distribution in the membrane of Bacillus amyloliquefaciens was studied by using phospholipase C (B. cereus), phospholipase A2 (Crotalus), and the nonpenetrating chemical probe trinitrobenzenesulfonic acid. After treatment of intact protoplasts of B. amyloliquefaciens with either phospholipase, about 70% of total membrane phospholipid was hydrolyzed; specifically, about 90, 90, and 30% of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, respectively. Under these conditions, protoplasts remained intact and sealed. However, when protoplasts that were permeabilized by cold-shock treatment were incubated with either of the phospholipases, up to 80% of cardiolipin was hydrolyzed and phosphatidylglycerol and phosphatidylethanolamine were hydrolyzed virtually to completion. In intact cells, 92% of the phosphatidylethanolamine could be labeled with trinitrobenzenesulfonic acid under conditions in which the reagent did not penetrate the membrane to any significant extent. These results indicate that 70% of total phospholipid of this bacillus exists in the outer half of the bilayer. The distribution of phosphatidylethanolamine in this bilayer is highly asymmetric with it being located predominantly in the outer half. The results with phospholipases suggest that the distributions of cardiolipin and phosphatidylglycerol are also asymmetric but independent confirmation of this is required.
Inducible reactivation and mutagenesis of UV-irradiated bacteriophage P22 in Salmonella typhimurium LT2 containing the plasmid pKM101
Authors: Walker G C
Published: January 2020

The inducible (Weigle) reactivation of UV-irradiated bacteriophage P22 has been examined on strains of Salmonella typhimurium with and without the mutagenesis-enhancing plasmid pKM101. A large inducible reactivation was observed in the plasmid-containing strain, but only a small response was observed in the strain lacking the plasmid. An increased frequency of clear-plaque mutants was detected among the survivors. The efficiencies of the plasmid-mediated and cellular repair processes have been determined. The kinetics of induction of the phage reactivation have been investigated. The relationship of the observed results to the inducible reactivation of UV-irradiated lambda in Escherichia coli and to error-prone repair is discussed.
Replication of bromodeoxyuridylate-substituted mitochondrial DNA in yeast
Authors: Leff J , Eccleshall T R
Published: January 2020

The DNA of several strains of Saccharomyces cerevisiae was labeled by growing the culture in medium supplemented with thymidylate and bromodeoxyuridylate. It was thus possible to follow the course of mitochondrial DNA replication in density shift experiments by determining the buoyant density distribution of unreplicated and replicated DNAs in analytical CsCl gradients. DNA replication was followed for three generations after transfer of cultures from light medium to heavy medium and heavy medium to light medium. Under both conditions, the density shifts observed for mitochondrial DNA were those expected for semiconservative, nondispersive replication. This was further confirmed by analysis of the buoyant density of alkali-denatured hybrid mitochondrial DNA. With this method, no significant recombination between replicated and unreplicated DNA was detected after three generations of growth.
Compact Escherichia coli nucleoids in a highly supercoiled conformation
Authors: Materman E C , Van Gool A P
Published: January 2020

Escherichia coli nucleoids were visualized in a compact, highly supercoiled conformation at 1 M NaCl. After the salt concentration was lowered to 0.15 M NaCl, the nucleoids uncoiled in beaded fibers in which RNA-mediated structural domains could be distinguished.
Cell-cell interactions in conjugating Escherichia coli: role of F pili and fate of mating aggregates
Authors: Achtman M , Morelli G , Schwuchow S
Published: January 2020

Bacterial conjugation between Escherichia coli cells was investigated by a combination of physical and genetic techniques, using Hfr, F', or R+ donors and F- recipients. DNA transfer occurred in mating aggregates of up to 50 cells. Multiple interactions between donor and recipient cells occurred, and both F- pilus connections and wall-to-wall contacts were detectable. The detectable F- pilus contacts could be destroyed without either disrupting the mating aggregates or preventing DNA transfer. Hfr X F- mating aggregates did not disaggregate even though recombinant frequencies were inversely proportional to the distance from the origin of DNA transfer. F' or R+ donors formed mating aggregates with F- cells which disaggregated soon after transfer of the autonomous sex factor DNA.
Caulobacter flagellar organelle: synthesis, compartmentation, and assembly
Authors: Lagenaur C , Agabian N
Published: January 2020

In Caulobacter crescentus biogenesis of the flagellar organelle occurs during one stage of its complex life cycle. Thus in synchronous cultures it is possible to assay the sequential synthesis and assembly of the flagellum and hook in vivo with a combination of biochemical and radioimmunological techniques. The periodicity of synthesis and the subcellular compartmentation of the basal hook and filament subunits were determined by radioimmune assay procedures. Unassembled 27,000-dalton (27K) flagellin was preferentially located in isolated membrane fractions, whereas the 25K flagellin was distributed between the membrane and cytoplasm. The synthesis of hook began before that of flagellin, although appreciable overlap of the two processes occurred. Initiation of filament assembly coincided with the association of newly synthesized hook and flagellin subunits. Caulobacter flagella are unusual in that they contain two different flagellin subunits. Data are presented which suggest that the ratio of the two flagellin subunits changes along the length of the filament. Only the newly synthesized 25K flagellin subunit is detected in filaments assembled during the swarmer cell stage. By monitoring the appearance of flagellar hooks in the culture medium, the time at which flagella are released was determined.
Ligninolytic enzyme system of Phanaerochaete chrysosporium: synthesized in the absence of lignin in response to nitrogen starvation
Authors: Keyser P , Kirk T K , Zeikus J G
Published: January 2020

The relationship between growth, nutrient nitrogen assimilation, and the appearance of ligninolytic activity was examined in stationary batch cultures of the wood-destroying hymenomycete Phanerochaete chrysosporium Burds. grown under conditions optimized for lignin metabolism. A reproducible sequence of events followed inoculation: 0 to 24 h, germination, linear growth, and depletion of nutrient nitrogen; 24 to 48 h, cessation of linear growth and derepression of ammonium permease activity (demonstrating nitrogen starvation); 72 to 96 h, appearance of ligninolytic activity (synthetic 14C-lignin leads to 14CO2). Experiments with cycloheximide demonstrated that appearance of ligninolytic activity occurs irrespective of the presence of lignin; lignin did not induce additional activity. Addition of NH4+ to cultures immediately prior to the time of appearance of the ligninolytic system delayed its appearance, suggesting that the NH4+ led to interference with synthesis of the enzyme system. Addition of NH4+ to ligninolytic cultures resulted in an eventual, temporary decrease in ligninolytic activity. The results suggest that all or essential protein components of the ligninolytic enzyme system are synthesized as part of a series of physiological ("secondary metabolic") events that are initiated by nutrient nitrogen starvation.
Relationship of large and small invertases in Saccharomyces: mutant selectively deficient in small invertase
Authors: Abrams B B , Hackel R , Mizunaga T , Lampen J O
Published: January 2020

A mutant strain of Saccharomyces cerevisiae (D10-ER1) has been isolated after a two-step mutagenesis of strain 4059-358D (SUC 1) using ethyl methane sulfonate. Cells of this new strain produced a level of total invertase equaling that of 4059 but contained only trace amounts of the small, internal, aglycan form of the enzyme (less than 0.1% of total in D10-ER1 compared with 6% in 4059). When D10.ER1 was crossed with an invertase-hyperproducing strain dgr3 (SUC3), progeny were isolated (HZ400-5A and HZ400-2C) in which levels of total invertase had at least quadrupled. The percentage of small invertase, however, remained insignificant. Levels of small invertase in strain HZ400-5A were determined by affinity chromatography on conconavalin A-Sepharose, gel permeation chromatography, and isopycnic centrifugation in CsCl. The large invertase of the SUC1 yeasts described here was found to contain a form apparently greater in size than the large invertase of the SUC2 strain FH4C; this probably reflects a higher content of carbohydrate. The overall results of this study do not support a direct structural relationship between large and small invertases. The implications on invertase biosynthesis and structure are discussed.
Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae
Authors: Roberts G P , MacNeil T , MacNeil D , Brill W J
Published: January 2020

Two hundred and thirty-five Nif- strains of Klebsiella pneumoniae were characterized by two-dimensional polyacrylamide gel electrophoresis. Forty-two of these strains were tested further by in vitro acetylene reduction assays. By these techniques, nine nif-coded polypeptides were identified, and eight of these were assigned to specific nif genes. Nitrogenase component I required nifK and nifD, which coded for the beta and alpha subunits, and nifB, -E, and -N were required for the iron-molybdenum cofactor, which is a part of the active site of nitrogenase. nifH coded for the structural protein of component II, and nifM and nifS products seemed to be necessary for the synthesis of an active component II. There were two genes, nifF and nifJ, that were required for N2 fixation in vivo but not for N2 fixation in vitro. There were at least two cases (nifE and nifN, nifK and nifD) of two proteins that seemed to require each other for stability in vivo. Regulation of N2 fixation is apparently complex, and this is reflected by the assignment of regulatory functions to the gene products of nifA, nifL, nifK, nifD, nifH, and NIFJ.
Cloned DNA fragment specifying major outer membrane protein a in Escherichia coli K-12
Authors: Gayda R C , Markovitz A
Published: January 2020

Plasmid pMC44 is a recombinant plasmid that contains a 2-megadalton EcoRI fragment of Escherichia coli K-12 DNA joined to the cloning vehicle, pSC101. The polypeptides specified by plasmid pMC44 were identified and compared with those specified by pSC101 to determine those that are unique to pMC44. Three polypeptides specified by plasmid pMC44 were localized in the cell envelope fraction of minicells: a Sarkosyl-insoluble outer membrane polypeptide (designated M2), specified by the cloned 2-megadalton DNA fragment, and two Sarkosyl-soluble membrane polypeptides specified by the cloning plasmid pSC101. Bacteria containing plasmid pMC44 synthesized quantities of M2 approximately equal to the most abundant E. coli K-12 outer membrane protein. Evidence is presented that outer membrane polypeptide M2, specified by the recombinant plasmid pMC44, is the normal E. coli outer membrane protein designated protein a by Lugtenberg and 3b by Schnaitman.
Bacteriophage Mu-induced modification of DNA is dependent upon a host function
Authors: Khatoon H , Bukhari A I
Published: January 2020

The DNA of bacteriophage Mu, extracted from induced lysates, is partially resistant to digestion by the endonuclease BalI. This modification of DNA is controlled by the Mu modification function (mom), which acts in conjunction with the dam (DNA-adenine methylation) function of Escherichia coli. Since the BalI recognition site is apparently different from the dam recognition site, these results imply that either the specificity of the dam function is changed by the mom function or the mom function requires the dam function for its activity.
Degradation of 4-hydroxyphenylacetic acid by Trichosporon cutaneum
Authors: Sparnins V L , Anderson J J , Omans J , Dagley S
Published: January 2020

Trichosporon cutaneum degraded 4-hydroxyphenylacetic acid to acetoacetic and malic acids. 3.4-Dihydroxyphenylacetic acid, an intermediate in the reaction sequence, underwent hydroxylation before the benzene ring was cleaved.
One-carbon metabolism in methanogenic bacteria: analysis of short-term fixation products of 14CO2 and 14CH3OH incorporated into whole cells
Authors: Daniels L , Zeikus J G
Published: January 2020

Methanobacterium thermoautotrophicum, M. ruminantium, and Methanosarcina barkeri were labeled with 14CO2 (14CO2 + H14CO3- + 14CO32-) for from 2 to 45 s. Radioactivity was recovered in coenzyme M derivatives, alanine, aspartate, glutamate, and several unidentified compounds. The properties of one important structurally unidentified intermediate (yellow fluorescent compound) displayed UV absorbance maxima at pH 1 of 290 and 335 nm, no absorbance in the visible region, and a fluorescence maximum at 460 nm. Label did not appear in organic phosphates until after 1 min. 14CH3OH was converted by M. barkeri primarily into coenzyme M derivatives at 25 s. [2-14C]acetate was assimilated by M. thermoautotrophicum mainly into alanine and succinate during 2 to 240 s, but not into coenzyme M derivatives or yellow fluorescent compound. Cell-free extracts of M. thermoautotrophicum lacked ribulose 1,5-bisphosphate carboxylase activity. The data indicated the absence of the Calvin, serine, and hexulose phosphate paths of C1 assimilation in the methanogens examined and indicated that pyruvate was an early intermediate product of net CO2 fixation. The in vivo importance of coenzyme M derivatives in methanogenesis was demonstrated.
Orotic acid excretion in some wild-type strains of Escherichia coli K-12
Authors: Womack J E , O'Donovan G A
Published: January 2020

During rapid growth, the excretion of pyrimidines, predominantly uracil, is a common phenomenon in procaryotes and eucaryotes. In Escherichia coli, some K-12 strains excrete orotic acid and not uracil. This is caused by a mutation in the pyrF gene.
dnaT, dominant conditional-lethal mutation affecting DNA replication in Escherichia coli
Authors: Lark C A , Riazi J , Lark K G
Published: January 2020

Normally, bacteria cease DNA replication in the absence of protein synthesis. A variety of treatments, such as thymine starvation or a shift-up to rich medium, lead to continued DNA replication in the absence of protein synthesis. Mutants are described which always terminate replication under these conditions. These conditional lethal mutants, dnaT1 and dnaT2, contransduce with serB and dnaC. The mutation also affects cell division. All aspects of the mutant phenotype (obligatory termination of replication, temperature sensitivity of DNA replication and growth, and aberrant cell division at permissive growth temperatures) were transdominant to the wild-type phenotype. Episomes carrying the dnaT mutation appeared to be unstable. The existence of such a dominant mutation was predicted by a model of chromosome termination proposed by Kogoma and Lark (J. Mol. Biol. 94:243-256, 1975).