bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of vacuole permeability and protein degradation by the cell cycle arrest signal in Saccharomyces cerevisiae
Authors: Sumrada R , Cooper T G
Published: January 2020

Saccharomyces cerevisiae responds to deperivation of nutrients by arresting cell division at the unbudded G1 stage. Cells situated outside of G1 at the time of deperivation complete the cell cycle before arresting. This prompted an investigation of the source of nutrients used by these cells to complete division and the mechanisms controlling their availability. We found a close correlation between accumulation of unbudded cells and loss of previously formed allophanate hydrolase activity after nutrient starvation. These losses were not specific to the allantoin, system since they have been observed for a number of other enzymes and also when cellular protein levels were monitored with [3H]leucine. Loss of hydrolase activity was also observed when protein synthesis was inhibited either by addition of inhibitors or loss of the prtl gene product. We found that onset of nutrient starvation brought about release of large quantities of arginine and allantoin normally sequestered in the cell vacuole. Treatment of a cells with alpha-factor resulted in both the release of allantoin and arginine from the cell vacuole and the onset of intracellular protein degradation. These effects were not observed when either alpha cells or a/alpha diploid strains were treated with alpha-factor. These data suggest that release of vacuolar constitutents and protein turnover may be regulated by the G1 arrest signal.
Cell envelope and shape of Escherichia coli: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins
Authors: Sonntag I , Schwarz H , Hirota Y , Henning U
Published: January 2020

Starting with an Escherichia coli strain missing the outer membrane lipoprotein, multiple mutants were constructed than in addition to this defect miss the outer membrane proteins II, Ia and Ib, or Ia, Ib, and II. In contrast to all single mutants or strains missing the lipoprotein and polypeptides Ia and Ib, drastic influences on the integrity of the outer membrane and cell morphology were observed in mutants without lipoprotein and protein II. Such strains exhibited spherical morphology. They required increased concentrations of electrolytes for optimal growth, and Mg2+ or Ca2+ were the most efficient. These mutants were sensitive to hydrophobic antibiotics and detergents. Electron microscopy revealed abundant blebbing of the outer membrane, and it could clearly be seen that the murein layer was no longer associated with the outer membrane.
Enchancement of streptococcal transformation yield by proteolytic enzymes
Authors: Fuchs P G , Dobrzański W T
Published: January 2020

Trypsin and other proteolytic enzymes, added together with transforming DNA or during cell-DNA contact to competent cultures of several streptococcal strains, enchanced (10 to 600%) the yield of genetic transformation (stimulation). With few exceptions, the level of stimulation was high (over 100%) when competence was low (below 2%). Stimulation was caused by the action of an enzyme on competent cells and not on any other component of transformation mixture. The phenomenon occurred when the enzyme was added to the culture not earlier than 7 min before and not later than 5 min after the period of cell-DNA contact. The presence of trypsin during cell-DNA contact caused: (i) the alterations at cell surface, demonstrated by electron microscopy, increased release of 3H-amino acid-labeled material, and higher cell susceptibility to autolysis; (ii) the increase of both total and irreversible binding of DNA by the cells; and (iii) the decrease of early nucleolytic degradation of DNA by cells. These and other data point to the importance of a delicate balance of recipient cell's surface nuclease activities in the effectiveness of transformation process. It is also possible that trypsin eliminates an unknown cellular factor which obstructs DNA-cell receptors interaction.
Characterization of succinic dehydrogenase mutants of Bacillus subtilis by crossed immunoelectrophoresis
Authors: Rutberg B , Hederstedt L , Holmgren E , Rutberg L
Published: January 2020

Eleven succinic dehydrogenase (SDH) mutants in Bacillus subtilis were analyzed by crossed immunoelectrophoresis with antiserum prepared against wild-type B. subtilis cytoplasmic membrane. A precipitate which stained for SDH was found in Triton X-100-solubilized wild-type membranes and in membranes from two of the SDH mutants. The remaining nine mutants did not show an SDH-staining precipitate. The respective mutations in these nine mutants all map in one locus, citF (Ohné et al., J. Bacteriol. 115:738-745, 1973). An SDH-specific antiserum was prepared by immunizing rabbits with the SDH precipitate obtained in crossed immunoelectrophoresis with solubilized wild-type membrane. Using this antiserum, it was shown that all of the nine citF mutants lack an SDH-specific antigen in the membrane but five of the citF mutants have a soluble SDH-specific antigen. No major differences were found in sodium dodecyl sulfatepolyacrylamide gels of membrane proteins from wild-type B. subtilis and from SDH mutants. A model for the organization of SDH in B. subtilis is proposed.
Coordination chemistry of microbial iron transport compounds: rhodotorulic acid and iron uptake in Rhodotorula pilimanae
Authors: Carrano C J , Raymond K N
Published: January 2020

The mechanism by which iron uptake is facilitated by the siderophore rhodotorulic acid (RA) in the yeast Rhodotorula pilimanae was investigated with radioactively labeled Fe and RA and kinetically inert, chromic-substituted RA complexes. The weight of the evidence supports a model in which RA mediates iron transport to the cell but does not actually transport iron into the cell. It is proposed that RA exchanges the ferric ion at the cell surface with a membrane-bound chelating agent that completes the active transport of iron into the cell. Uptake of 55Fe in ferric rhodotorulate was much more rapid than uptake of RA itself. Two exchange-inert chromic complexes of RA showed no uptake.
In vivo regulation of glycolysis and characterization of sugar: phosphotransferase systems in Streptococcus lactis
Authors: Thompson J
Published: January 2020

Two novel procedures have been used to regulate, in vivo, the formation of phosphoenolpyruvate (PEP) from glycolysis in Streptococcus lactis ML3. In the first procedure, glucose metabolism was specifically inhibited by p-chloromercuribenzoate. Autoradiographic and enzymatic analyses showed that the cells contained glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-diphosphate, and triose phosphates.Dithiothreitol reversed the p-chloromercuribenzoate inhibition, and these intermediates were rapidly and quantitatively transformed into 3- and 2-phosphoglycerates plus PEP. The three intermediates were not further metabolized and constituted the intracellular PEP potential. The second procedure simply involved starvation of the organisms. The starved cells were devoid of glucose 6-phosphate, fructose 6-phosphate, fructose- 1,6-diphosphate, and triose phosphates but contained high levels of 3- and 2-phosphoglycerates and PEP (ca. 40 mM in total). The capacity to regulate PEP formation in vivo permitted the characterization of glucose and lactose phosphotransferase systems in physiologically intact cells. Evidence has been obtained for "feed forward" activation of pyruvate kinase in vivo by phosphorylated intermediates formed before the glyceraldehyde-3-phosphate dehydrogenase reaction in the glycolytic sequence. The data suggest that pyruvate kinase (an allosteric enzyme) plays a key role in the regulation of glycolysis and phosphotransferase system functions in S. lactis ML3.
Chromosomal location of a gene (nmpA) involved in expression of a major outer membrane protein in Escherichia coli
Authors: Foulds J , Chai T J
Published: January 2020

The phenotypic expression of protein E, a recently described major outer membrane protein, is associated with a mutation at a locus on the Escherichia coli chromosome that we call nmpA. nmpA is located between rbsK and uncA at 82.7 min on the E. coli linkage map. The nmpA locus is also the site of the mutations which lead to the formation of major outer membrane proteins Ic or e. It is likely proteins E, Ic, and e are closely related or identical. The mutant nmpA allele is dominant.
Method for isolation of Escherichia coli mutants with defects in the proton-translocating sector of the membrane adenosine triphosphatase complex
Authors: Fillingame R H , Knoebel K , Wopat A E
Published: January 2020

A technique for selecting mutants of Escherichia coli in which the proton-translocating sector of the adenosine triphosphatase (ATPase) complex has been inactivated is reported. The procedure uses a strain of E. coli (NR-70) lacking the extrinsic (F1) sector of the ATPase complex and which in consequently permeable to protons (B. P. Rosen, J. Bacteriol. 116:1124--1129, 1973). After growing strain NR-70 under noninducing conditions for the lac operon, cells were mutagenized and plated on minimal medium containing low concentrations of lactose. Several mutants of strain NR-70 were isolated as large colonies on these plates, apparently because they could concentrate lactose more efficiently. A description of one of the mutants, strain KW-1, is reported here. The most distinguishing difference in growth properties of the two strains was that, when transferred to medium containing low concentrations of lactose, strain KW-1 induced the lac operon with a shorter lag time than strain NR-70. The mutation in strain KW-1 leading to more rapid growth on lactose was cotransducible with the asn and unc loci, at 83 min on the E. coli genetic map. Intact cells of strain KW-1 actively transported L-proline as well as did wild-type cells, whereas cells of strain NR-70 were markedly deficient in L-proline transport. The improvement in the transport capacity of strain KW-1 correlated with a marked decrease in proton permeability relative to that of strain NR-70. Based on an acid-base pulse technique that measured the proton conductance of the membranes of intact cells, strain NR-70 was at least 10 times more permeable to protons than was the wild type, whereas strain KW-1 was only 2 times more permeable. The transport properties and proton conductance were also compared with membrane vesicles prepared by osmotic shock. With either D-lactate or ascorbate-N-methylphenazonium methosulfate as respiratory substrates, vesicles of strain KW-1 transported L-proline much more rapidly than did vesicles of strain NR-70, but still at rates less rapid than those of the wild type. The passive proton conductance of the membrane vesicles was quantitated by measuring the rate of H+ influx into vesicles in response to a valinomycin-generated K+ diffusion potential. The proton permeability of vesicles of strain KW-1 was reduced 1.5-fold relative to vesicles of strain NR-70, but these vesicles were still four times more permeable to protons than was the wild type. Vesicles of strain KW-1 corresponded to wild-type vesicles treated with 0.5 micrometer carbonylcyanide m-chlorophenylhydrazone (CCCP) and vesicles of strain NR-70 corresponded to wild-type vesicles treated with 1.4 micrometer CCCP. Treatment of wild-type vesicles with these concentrations of CCCP caused decreases in transport comparable to those observed in the mutants. Strain KW-1 lacked ATPase activity. Cross-reacting material to F1-ATPase was not found in strain KW-1 by double immunodiffusion analysis.
Biosynthesis and regulation of fructose-1,6-bisphosphatase and phosphofructokinase in Saccharomyces cerevisiae grown in the presence of glucose and gluconeogenic carbon sources
Authors: Foy J J , Bhattacharjee J K
Published: January 2020

The mode of synthesis and the regulation of fructose-1,6-bisphosphatase (Fbpase), a gluconeogenic enzyme, and phosphofructokinase (PFK), a glycolytic enzyme, were investigated in Saccharomyces cerevisiae after growth in the presence of different concentrations of glucose or various gluconeogenic carbon sources. The activity of FBPase appeared in the cells after the complete disappearance of glucose from the growth medium with a concomitant increase of the pH and no significant change in the levels of accumulated ethanol. The appearance of FBPase activity following glucose depletion was dependent upon the synthesis of protein. The FBPase PFK were present in glucose-, ethanol-, glycerol-, lactate-, or pyruvate-grown cells; however, the time of appearance and the levels of both these enzymes varied. The FBPase activity was always higher in 1% glucose-grown cells than in cells grown in the presence of gluconeogenic carbon sources. Phosphoglucose isomerase activity did not vary significantly. Addition of glucose to an FBPase and PFK synthesizing culture resulted in a complete loss, followed by a reappearance, of PFK activity. In the presence of cycloheximide the disappearance of glucose and the changes in the levels of FBPase and PFK were decreased significantly. It is concluded that S. cerevisiae exhibits a more efficient synthesis of FBPase after the exhaustion of glucose compared to the activity present in cells grown in the presence of exogenous gluconeogenic carbon sources. Two metabolically antagonistic enzymes, FBPase and PFK, are present during the transition phase, but not during the exponential phase, of growth, and the decay or inactivation of these enzymes in vivo may be dependent upon a glucose-induced protease activity.
Potentiometric titration of the high- and low-potential 4Fe-4S* centers of Azotobacter vinelandii ferredoxin I
Authors: Yoch D C , Carithers R P
Published: January 2020

The high-potential 4Fe-4S* center ofAzotobacter vinelandii ferredoxin I has been titrated potentiometrically by a reductive procedure. The absorbance decrease at 510 nm accompanying the reduction of the high-potential center titrated with an Em of 320 mV (n = 1). The low-potential 4Fe-4S* center was titrated by using the absorbance decrease at 410 nm to monitor its reduction. This center exhibited an Em of -424 mV (n = 1).
Catabolite-repression-like phenomenon in Rhizobium meliloti
Authors: Ucker D S , Signer E R
Published: January 2020

We report a phenomenon similar to catabolite repression in Rhizobium meliloti. Succinate, which allows the highest observed rate of growth of R. meliloti, caused an immediate reduction of beta-galactosidase activity when added to cells growing in lactose. A Lac- mutant was unaltered in nodulation and nitrogen fixation capacities, but a pleiotropic mutant deficient in several catabolic properties was unable to produce effective nitrogen-fixing nodules.
Isolation and Characterization of Glycerol-3-Phosphate Dehydrogenase-Defective Mutants of
Neurospora crassa
Authors: Denor Patrick F. , Courtright James B.
Published: January 2020

Three glycerol-nonutilizing mutants deficient in the mitochondrial glycerol-3-phosphate (G3P) dehydrogenase (EC 1.1.99.5) were isolated from
inl
ts
derivatives of
Neurospora crassa
following inositolless death at elevated temperatures on minimal glycerol medium. These mutants failed to grow on glycerol as a sole carbon source, but could grow on acetate, glucose, or mannitol media and were female fertile in genetic crosses, thereby distinguishing them from the previously reported polyol-protoperithecial defective
Neurospora
mutants. In addition, these
glp
mutants exhibited a distinct morphological alteration during vegetative growth on sucrose slants and colonial growth on sorbose-containing semicomplete medium. The
glp-2
locus was assigned a location between
arg-5
and
nuc-2
on chromosome IIR on the basis of two-factor crosses and by duplication coverage by insertional translocation ALS176, but not NM177. All mutations were allelic as judged from the absence of both complementation in forced heterokaryons and genetic recombination among
glp-2
mutations. The reversion frequency of all three mutations was less than 10
10
, indicating probable deletions in these strains. No G3P dehydrogenase activity could be detected in either cytosolic or mitochondrial extracts from mutant strains grown on glycerol, glucose, or galactose media. These results suggest that the
glp-2
locus may be the structural gene for both the cytosolic and mitochondrial forms of G3P dehydrogenase or for a cytosolic precursor of the mitochondrial G3P dehydrogenase. The defect is specific for the G3P dehydrogenase since normal activities of the mitochondrial cytochrome oxidase and succinate dehydrogenase and the cytosolic glycerol dehydrogenase and dihydroxyacetone phosphate reductase are detected in mutant extracts. During attempted growth of
glp-2
mutants on glycerol media, there was an accumulation of G3P in culture filtrates, a reduction in the mycelial growth rate, and a decreased level of glycerokinase induction.
Cell Wall Composition and Associated Properties of Methicillin-Resistant
Staphylococcus aureus
Strains
Authors: Wilkinson Brian J. , Dorian Kenneth J. , Sabath L. D.
Published: January 2020

Methicillin-resistant (MR)
Staphylococcus aureus
strains have previously been reported to be deficient in surface negative charge; this has been correlated with methicillin resistance and ascribed to a deficiency of teichoic acid at the cell surface (A. W. Hill and A. M. James, Microbios
6:
157-167, 1972). Teichoic acid was present in walls of MR organisms as revealed by appreciable phosphate levels and detection of ribitol residues. Phosphate levels in walls from five MR strains (0.54 to 0.77 μmol/mg of wall) were lower than in three unrelated methicillin-sensitive (MS) strains (0.86 to 1.0 μmol/mg of wall). However, two MS strains derived from two of the MR strains had wall phosphate levels very similar to those of the MR strains. No evidence for unusual wall polymers was found. Simple deficiency of wall teichoic acid does not result in methicillin resistance since an independently isolated teichoic acid-deficient strain (0.1 μmol of phosphate per mg of wall) was not methicillin resistant. In studies of biological properties possibly related to wall teichoic acid, it was discovered that walls isolated from MR organisms grown in the presence of methicillin autolyzed more rapidly than those isolated from organisms grown in the absence of the drug. Since methicillin resistance is enhanced by NaCl and suppressed by ethylenediaminetetraacetate, the effects of these compounds on autolysis of isolated walls were studied. NaCl (1.0 M) and ethylenediaminetetraacetate (1.0 mM) inhibited the autolysis of walls isolated from MR and MS strains. An MR strain bound phage 47, 52A, and 3A only slightly less well than their respective propagating strains.
Kasugamycin-dependent mutants of Escherichia coli
Authors: Dabbs E R
Published: January 2020

Kasugamycin-dependent mutants have been isolated from Escherichia coli B. They were obtained through mutagenesis with ethyl methane sulfonate or nitrosoguanidine in conjunction with an antibiotic underlay technique. In the case of nitrosoguanidine, dependent mutants were obtained at a frequency of about 3% of survivors growing up in the selection. In the case of ethyl methane sulfonate, the corresponding value was 1%. Nineteen mutants showing a kasugamycin-dependent phenotype were studied. In terms of response to various temperatures and antibiotic concentrations, they were very heterogeneous, although most fell into two general classes. Genetic analysis indicated that in at least some cases, the kasugamycin-dependent phenotype was the product of two mutations. Two-dimensional gel electropherograms revealed alterations in the ribosomal proteins of seven mutants. One mutant had an alteration in protein S13, and one had an alteration in protein L14. Three showed changes in protein S9. Each of two mutants had changes in two proteins, S18 and L11. Three of these mutants additionally had protein S18 occurring in a partly altered, partly unaltered form.
Carbon and nitrogen repression of arginine catabolic enzymes in Bacillus subtilis
Authors: Baumberg S , Harwood C R
Published: January 2020

Specific activities of arginase and ornithine aminotransferase, inducible enzymes of arginine catabolism in Bacillus subtilis 168, were examined in cells grown with various carbon and nitrogen sources. Levels of these enzymes were similar in arginine-induced cultures whether glucose or citrate was the carbon source (in contrast to histidase), suggesting that carbon source catabolite repression has only limited effect. In media with combinations of nitrogen sources, glutamine strongly repressed induction of these enzymes by proline or arginine. Ammonium, however, only repressed induction by proline and had no effect on induction by arginine. These effects correlate with generation times in media containing these substances as sole nitrogen sources: growth rates decreased in the order glutamine-arginine-ammonium-proline. Similar phenomena were observed when glutamine or ammonium were added to arginine- or proline-grown cultures, or when arginine or proline were added to glutamine- or ammonium-grown cultures. In the latter cases, an additional feature was apparent, namely a surprisingly long transition between steady-state enzyme levels. The results are compared with those for other bacteria and for eucaryotic microorganisms.
Requirement for membrane potential in active transport of glutamine by Escherichia coli
Authors: Plate C A
Published: January 2020

The effect of reducing the membrane potential on glutamine transport in cells of Escherichia coli has been investigated. Addition of valinomycin to tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid-treated E. coli cells in the presence of 20 mM exogenous potassium reduced the membrane potential, as measured by the uptake of the lipophilic cation triphenylmethylphosphonium, and caused a complete inhibition of glutamine transport. Valinomycin plus potassium also caused a rapid decrease in the intracellular levels of ATP of normal E. coli cells, but had little if any effect on the ATP levels of two mutants of E. coli carrying lesions in the energy-transducing ATP complex (unc mutants). Yet both the membrane potential and the capacity to transport glutamine were depressed in the unc mutants by valinomycin and potassium. These findings are consistent with the hypothesis that both ATP and a membrane potential are essential to the active transport of glutamine by E. coli cells.
Tripeptide hydroxamate from Corynebacterium kutscheri
Authors: McCullough W G , Merkal R S
Published: January 2020

A tripeptide hydroxamate was isolated from cultures of Corynebacterium kutscheri grown on an iron-limiting medium. The metabolite was characterized by spectral measurements and by chemical degradation as L-alpha-aspartyl-L-alpha-N-hydroxy-aspartyl-D-cycloserine.
Mannosyl Transfer by Membranes of
Aspergillus niger
: Mannosylation of Endogenous Acceptors and Partial Analysis of the Products
Authors: Rudick Michael J.
Published: January 2020

A smooth membrane fraction of
Aspergillus niger
catalyzed the transfer of mannose from GDP-mannose to endogenous lipid and protein acceptors. The mannolipid was acidic, as judged by diethylaminoethyl-cellulose chromatography, and had a mobility similar to ficaprenyl phosphate on thin-layer chromatograms. Mannose transfer occurred optimally at pH 6.5 to 7.5 and required Mn
2+
for use of the protein as acceptor, but either Mn
2+
or Mg
2+
with the lipid as acceptor. Glycopeptides of the mannosylated protein ([
14
C]gly) and of an α-glucosidase (α-glu) secreted by the organism were produced by Pronase digestion and separation of the products on Sephadex G-25. Because ovalbumin has a carbohydrate composition similar to that of α-glu and because the carbohydrate structure of ovalbumin is known, ovalbumin glycopeptides (Ov) were similarly obtained and used as standards in determining carbohydrate structures. Oligosaccharide chains of [
14
C]gly, α-glu, and Ov were obtained by treatment of the respective glycopeptides with endo-β-
N
-acetylglucosaminidase, reduction with NaBT
4
, and concanavalin A-Sepharose chromatography. The
3
H-labeled oligosaccharides obtained were subjected to the following treatments: (i) digestion with α- and β-mannosidases, (ii) Smith degradation, and (iii) acetolysis. Subsequently, changes in paper chromatographic mobilities were detected. Also, α-glu was permethylated, and the partially methylated alditol acetates were analyzed by gas-liquid chromatography. The resultant proposed structure shows that the oligosaccharide chain of α-glu is almost identical to that of an Ov chain, while [
14
C]gly has a structure which is probably the same as that of α-glu. It is suggested that the transferase(s) involved in [
14
C]gly synthesis in vitro may be responsible for glycosylation of secreted enzymes.
Biosynthesis and Cellular Distribution of the Two Superoxide Dismutases of
Dactylium dendroides
Authors: Shatzman Allan R. , Kosman Daniel J.
Published: January 2020

The synthesis and subcellular localization of the two superoxide dismutases of
Dactylium dendroides
were studied in relation to changes in copper and manganese availability. Cultures grew normally at all medium copper concentrations used (10 nM to 1 mM). In the presence of high (10 μM) copper, manganese was poorly absorbed in comparison to the other metals in the medium. However, cells grown at 10 nM copper exhibited a 3.5-fold increase in manganese content, while the concentration of the other metals remained constant. Cultures grown at 10 nM copper or more had 80% Cu/Zn enzyme and 20% mangani enzyme; the former was entirely in the cytosol, and the latter was mitochondrial. Removal of copper from the medium resulted in decreased Cu/Zn superoxide dismutase synthesis with a concomitant increase in the mangani enzyme such that total cellular superoxide dismutase activity remained constant. The mangani enzyme in excess of the 20% was present in the non-mitochondrial fraction. The mitochondria, therefore, show no variability with respect to superoxide dismutase content, whereas the soluble fraction varies from 100 to 13% Cu/Zn superoxide dismutase. Copper-starved cells that were synthesizing predominantly mangani superoxide dismutase could be switched over to mostly Cu/Zn superoxide dismutase synthesis by supplementing the medium with copper during growth. Immunoprecipitation experiments suggest that the decrease in Cu/Zn activity at low copper concentration is a result of decreased synthesis of that protein rather than the production of an inactive apoprotein.
Genetic analysis of Staphylococcus aureus RNA polymerase mutants
Authors: Morrow T O , Harmon S A
Published: January 2020

Spontaneous mutants of Staphylococcus aureus resistant to rifampin, rifamycin SV, streptovaricin, or streptolydigin were isolated and shown to be resistant due to chromosomal rather than plasmid mutations. Based on data concerning spontaneous mutation rates, genetic cotransduction rates, and in vitro sensitivity studies, four major antibiotic cross-resistance patterns were found. The genetic markers responsible for these cross-resistance patterns were shown to be separable by transduction. Nonpurified RNA polymerase activity in lysates of mutants showed the same sensitivity to these antibiotics as shown by the mutants on solid media. A model is proposed explaining possible structure-function relationships involved in the binding of these antibiotics to the RNA polymerase molecule and the mutations resulting in resistance to these antibiotics. This model includes generally overlapping but different-sized binding sites on the RNA polymerase protein coded for by similarly arranged mutable sites on the DNA.
DNA repair in Bacillus subtilis: excision repair capacity of competent cells
Authors: Yasbin R E , Fernwalt J D , Fields P I
Published: January 2020

Competent Bacillus subtilis were investigated for their ability to support the repair of UV-irradiated bacteriophage and bacteriophage DNA. UV-irradiated bacteriophage DNA cannot be repaired to the same level as UV-irradiated bacteriophage, suggesting a deficiency in the ability of competent cells to repair UV damage. However, competent cells were as repair proficient as noncompetent cells in their ability to repair irradiated bacteriophage in marker rescue experiments. The increased sensitivity of irradiated DNA is shown to be due to the inability of excision repair to function on transfecting DNA in competent bacteria. Furthermore, competent cells show no evidence of possessing an inducible BsuR restriction system to complement their inducible BsuR modification enzyme.
Escherichia coli
Mutants Overproducing Phenylalanyl- and Threonyl-tRNA Synthetase
Authors: Grüll J. M. , Hennecke H. , Fröhler J. , Thomale J. , Nass G. , Böck A.
Published: January 2020

The structural genes for threonyl-tRNA synthetase (ThrRS) and phenylalanyl-tRNA synthetase (PheRS) are closely linked on the
Escherichia coli
chromosome. To study whether these enzymes share a common regulatory element, we have investigated their synthesis in mutants which were selected for overproduction of either ThrRS or PheRS. It was found that mutants isolated previously for overproduction of ThrRS as strains resistant to the antibiotic borrelidin (strains Bor Res 3 and Bor Res 15) did not show an elevated level of PheRS. PheRS-overproducing strains were then isolated as revertants of strains with structurally altered enzymes. Strain S1 is a temperature-resistant derivative of a temperature-sensitive PheRS mutant, and strain G118 is a prototrophic derivative of a PheRS mutant which shows phenylalanine auxotrophy as a consequence of an altered

K
m

of this enzyme for the amino acid. In both kinds of revertants, S1 and G118, the concentration of PheRS and ThrRS was increased by factors of about 2.5 and 1.8, respectively, whereas the level of other aminoacyl-tRNA synthetases was not affected by the mutations. Genetic studies showed that the simultaneous overproduction of PheRS and ThrRS in revertants G118 and S1 is based upon gene amplification, since this property was easily lost after growing the cells in the absence of the selective stimulus, and since this loss could be prevented by the presence of the
recA
allele. By similar criteria, the four- and eightfold overproduction of ThrRS in strains Bor Res 3 and Bor Res 15, respectively, was very stable genetically, indicating that it is caused by a mutational event other than gene amplification. From these results, we conclude that the concomitant increase of PheRS and ThrRS in strains G118 and S1 is an expression of gene duplication and not of a joint regulation of these two aminoacyl-tRNA synthetases. This conclusion is further supported by the result that, in mutant G118 as well as in its parental strain G1, growth in minimal medium lacking phenylalanine led to an additional twofold increase of their PheRS concentration. This increase was restricted to the PheRS, since the level of other aminoacyl-tRNA synthetases, including the ThrRS, stayed unchanged.
Bacteriophage-associated gene transfer in pneumococcus: transduction or pseudotransduction?
Authors: Porter R D , Shoemaker N B , Rampe G , Guild W R
Published: January 2020

Lysates of pneumococcal phage PG24 transferred genes from one host to another in a process with many of the properties of generalized transduction, in that the host genes were packaged in DNase-resistant particles that closely resembled infectious phage in physical properties, adsorbed to the recipient cells like phage, and were inhibited by antisera to the phage and by trypsin. However, phage processes did not complete the transfer of host DNA as they did phage DNA. Instead, gene transfer required development of competence and entry of the host DNA by the endonuclease-dependent pathway used for transforming and transfecting DNA. This process often occurred on the assay plate hours after adsorption of the particles to the cells, and the transfer was DNase sensitive if challenged at this time. Phenotypic expression was therefore also delayed. The product of entry was like that in transformation, a single strand of DNA that integrates by formation of a hex-sensitive donor-recipient heteroduplex. Whether this gene transfer process is unique to this system or is only the first one described is not clear. The term "pseudotransduction" may be useful in calling attention to its unexpected features. The DNA of PG24 phage has anomalous physical properties reflecting unusual bases.
Purification and characterization of an extracellular beta-n-acetylhexosaminidase from Paecilomyces persicinus
Authors: Eriquez L A , Pisano M A
Published: January 2020

Both beta-N-acetylglucosaminidase nad beta-N-acetylgalactosaminidase activities were detected in the culture fluids of Paecilomyces persicinus P-10 after growth in a soybean meal-corn meal medium. The active material was purified by means of protamine sulfate fractionation and ultrafiltration, followed by ion exchange and gel chromatography. The ratio of the two activities remained constant throughout the purification, and the final product was shown to migrate as a single band by using gel isoelectric focusing, disc electrophoresis, and detergent gel electrophoresis. Temperature, pH, inhibition, and kinetic studies were performed to characterize both activities. The molecular weight of the enzyme was estimated to be about 100,000 by high-resolution gel chromatography. Based on the data obtained, it is suggested that both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside in the same protein.
Effect of light on the attachment of cyanophage AS-1 to Anacystis nidulans
Authors: Cséke C S , Farkas G L
Published: January 2020

The effect of illumination on the extent and kinetics of the adsorption of cyanophage AS-1 to the blue-green alga (cyanobacterium) Anacystis nidulans was studied by using 32P-labeled phage. The initial rate of adsorption was not significantly affected by light. However, at Na+ levels used ordinarily to culture the alga ([Na+] = 11.7 mM), the total amount of phage adsorbed was doubled in the illuminated cultures, as compared with the dark-grown ones, over a wide range of multiplicities of infection (0.05 to 20). Upon a 10-fold increase in Na+ concentration in the medium ([Na+] = 0.11 M), the dark adsorption of the phage increased to the level of light adsorption found in low Na+ medium. The effects on phage adsorption of high Na+ concentration and light were not additive.
Outer membrane of Serratia marcescens: apparent molecular weights of heat-modifiable proteins in gels with different acrylamide concentrations
Authors: Heller K B
Published: January 2020

The major proteins from the outer membrane of Serratia marcescens SM-6 are heat modifiable. The analysis of their apparent molecular weights in gels with different concentrations of acrylamide and the results obtained by radioactive labeling indicate that the major proteins are covalently linked to carbohydrate moieties.
Proton translocation in cytochrome-deficient mutants of Escherichia coli
Authors: Brookman J J , Downie J A , Gibson F , Cox G B , Rosenberg H
Published: January 2020

Cytochrome-deficient cells of a strain of Escherichia coli lacking 5-amino-levulinate synthetase have been used to study proton translocation associated with the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase region of the electron transport chain. Menadione was used as electron acceptor, and mannitol was used as the substrate for the generation of intracellular NADH. The effects of iron deficiency on NADH- and D-lactate-menadione reductase activities were studied in iron-deficient cells of a mutant strain unable to synthesize the iron chelator enterochelin; both activities were reduced. The NADH- menadione reductase activity in cytochrome-deficient cells was associated with proton translocation and could be coupled to the uptake of proline. However proton translocation associated with the NADH-menadione reductase activity was prevented by a mutation in an unc gene. It was concluded that there is no proton translocation associated with the NADH-dehydrogenase region of the electron transport chain in E. coli and that the proton translocation obtained with mannitol as substrate is due to the activity of membrane-bound adenosine triphosphatase.
Peptidoglycan Synthesis in Cocci and Rods of a pH-Dependent, Morphologically Conditional Mutant of
Klebsiella pneumoniae
Authors: Satta G. , Fontana R. , Canepari P. , Botta G.
Published: January 2020

Mir M7 is a spontaneous morphologically conditional mutant of
Klebsiella pneumoniae
which grows as round cells (cocci) at pH 7 and as normal rods at pH 5.8. We studied the rates of peptidoglycan synthesis of cocci and rods growing at pH values of 7 and 5.8, respectively. It was found that exponentially growing cocci produced a reduced amount of peptidoglycan per cell, compared with rods. Moreover, a shift of cocci to the permissive pH (5.8) caused an increase in the rate of peptidoglycan synthesis, whereas the reverse shift of rods to pH 7 determined a twofold reduction in the rate of [
3
H]diaminopimelic acid incorporation. During synchronous growth at pH 7, the rate of peptidoglycan synthesis after cell division decreased with time and rose before and during the first division. The susceptibilities of rods and cocci to β-lactam antibiotics were also studied. It was found that cocci were more sensitive both to penicillin G and to cephalexin than were rods, but they showed a high level of resistance to mecillinam. The peculiar behavior of this mutant was interpreted as supporting the existence in bacterial rods of two different sites for peptidoglycan synthesis: one responsible for lateral wall elongation and one responsible for septum formation. In Mir M7, shape damage is described as dependent on the specific inhibition, at the nonpermissive pH, of the site for lateral wall extension.
Control of the receptor for galactose taxis in Salmonella typhimurium
Authors: Fahnestock M , Koshland D E
Published: January 2020

The chemotactic response to galactose in wild-type Salmonella typhimurium is not inducible by galactose, but is inducible by fucose, a non-metabolizable analog. In a galactokinase mutant, however, the galactose receptor is inducible by galactose. These data indicate that the concentration of free galactose in the cell controls the levels of the galactose receptor. The intensities of the chemotactic responses were found to vary in proportion to the concentration of galactose receptors. In bacteria with higher levels of galactose receptors, the ribose response is inhibited by galactose. This supports the model in which the ribose and galactose receptors compete for a common component of the signaling system.
High and selective resistance to mecillinam in adenylate cyclase-deficient or cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli
Authors: Aono R , Yamasaki M , Tamura G
Published: January 2020

Adenylate cyclase-deficient (cya) mutants of Escherichia coli K-12 were selectively and highly resistant to mecillinam (FL1060) among several beta-lactam antibiotics in the absence of cyclic adenosine 3',5'-monophosphate (cAMP). They became sensitive to the drug in the presence of cAMP. Also, cAMP receptor protein-negative (crp) mutants, with the exception of strain 5333, were highly resistant to mecillinam in the presence and in the absence of cAMP. Mecillinam exerted two distinct and sequential effects in both cya+ strains and cya strains supplemented with cAMP: (i) rounding of cells and (ii) cessation of cell division. The first effect was accompanied by a decrease in growth rate, whereas the second effect was accompanied by enlargement and lysis of the rounded cells. The second effect of mecillinam was dependent on inoculum size and cAMP. When the cell density was above about 10(6) cells per ml, the rounded cells stopped dividing but did not lyse. In the absence of cAMP, cya strains neither stopped dividing nor lysed; they were resistant to the second, lethal effect of mecillinam.
Simultaneous loss of multiple differentiated functions in aerial mycelium-negative isolates of streptomycetes
Authors: Redshaw P A , McCann P A , Pentella M A , Pogell B M
Published: January 2020

Germination and outgrowth of spores of Streptomyces alboniger, Streptomyces scabies, and Streptomyces violaceus-ruber in the presence of intercalating dyes resulted in a high frequency (2 to 20%) of occurrence of aerial mycelium-negative (Amy-) isolates. Coincident with the appearance of the Amy- trait was the loss of several differentiated functions, including the characteristic pigments and earthy odor of the wild types. All S. alboniger, 27% of S. scabies, and 39% of the S. violaceus-ruber Amy- isolates were arginine auxotrophs. The missing enzyme step was identified as argininosuccinate synthetase by using a sensitive microassay for estimation of enzyme activity. The remainder of the S. scabies and S. violaceus-ruber isolates were prototrophs. In addition, S. alboniger Amy- isolates failed to produce or respond to the stimulator of aerial mycelium formation isolated from the wild type. The Amy- isolates did not revert to either Amy+ of Arg+. The lack of any detectable reversion, coupled with the high frequency of curing, supports the idea that a deletion of genetic material, possibly a plasmid, has occurred.
Location of an ampicillin resistance transposon, Tn1701, in a group of small, nontransferring plasmids
Authors: Yamada Y , Calame K L , Grindley J N , Nakada D
Published: January 2020

By restriction endonuclease cleavage mapping and electron microscopic examination of heteroduplexes, we have identified an ampicillin resistance determinant transposon, designated Tn1701, in a group of small, nontransferring plasmids which confer resistance to ampicillin (Ap), sulfonamide (Su), and streptomycin (Sm). Plasmid NTP1, which mediates Ap resistance, contains Tn1701. Recombinant plasmids NTP3 (Ap Su) and NTP4 (Ap Su Sm) contain Tn1701, indicating that they were derived by transposition of Tn1701 from NTP1 to an unrelated plasmid, NTP2 (Su Sm). The transposon Tn1701 is very similar to the known ampicillin resistance transposons Tn1, Tn2, and Tn3 in its size (3.2 x 10(6) daltons), base sequence homology observed by heteroduplex formation, restriction endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. Like the other TnA elements, Tn1701 also specifies a type TEM beta-lactamase.
Effect of dna mutations on the replication of plasmid pSC101 in Escherichia coli K-12
Authors: Hasunuma K , Sekiguchi M
Published: January 2020

Escherichia coli strains with mutations in genes dnaB, dnaC, and dnaG were tested for their capacity to replicate pSC101 deoxyribonucleic acid (DNA) at a nonpermissive temperature. Only a small amount of radioactive thymine was incorporated into pSC101 DNA in the dna mutants at 42 degrees C, whereas active incorporation into plasmid DNA took place in wild-type strains under the same conditions. The effects of the dnaB and dnaC mutations were greater on plasmid DNA synthesis than on host chromosomal DNA synthesis, suggesting that these gene products are directly involved in the process of pSC101 DNA replication. In dnaG mutants, both plasmid and chromosomal DNA synthesis were blocked soon after the shift to high temperature; although the extent of inhibition of the plasmid DNA synthesis was greater during the early period of temperature shift to 42 degrees C as compared with that of the host DNA synthesis, during the later period it was less. It was found that the number of copies of pSC101 per chromosome in dnaA and dnaC strains, grown at 30 degrees C, was considerably lower than that in wildtype strains, suggesting that the replication of pSC101 in these mutant strains was partially suppressed even under the permissive conditions. No correlation was found between the number of plasmid copies and the tetracycline resistance level of the host bacterium.
Morphology-associated expression nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase in Mucorracemosus
Authors: Peters J , Sypherd P S
Published: January 2020

The in vivo regulation of glutamate dehydrogenase (GDH) was studied in Mucor racemosus as a function of nutritional conditions and morphological state. Both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP)-dependent GDH activities were found. The effect of carbon and nitrogen source on the specific activity of the NAD-dependent GDH suggests that its role is primarily catabolic. The NAD-dependent activity was generally an order of magnitude greater in mycelial cells than in yeast-phase cells grown on the same medium. During yeast-to-hyphal morphogenesis the increase in NAD-dependent activity preceded the appearance of hyphal cells both under aerobic and anaerobic conditions. Exogenous dibutyryl-cyclic AMP prevented the increase in NAD-dependent GDH concomitantly with the suppression of morphological differentiation. The NADP-dependent activity did not change appreciably during morphogenesis.
Arrangement of the genes coding for ribosomal ribonucleic acids in Neurospora crassa
Authors: Free S J , Rice P W , Metzenberg R L
Published: January 2020

We have cloned and characterized Neurospora crassa ribosomal deoxyribonucleic acid (rDNA). The rDNA is found as a tandemly repeated 6.0-megadalton sequence. We have mapped a portion of the rDNA repeat unit with respect to its sites for 13 restriction endonucleases and defined those regions coding for the 5. 8S, 17S, and 26S ribosomal ribonucleic acids (rRNA's). We have also isolated several clones containing 5S rRNA sequences. The 5S rRNA coding sequences are not found within the rDNA repeat unit. We found that the sequences surrounding the 5S rRNA coding regions are highly heterogeneous.
Electron microscopic study of Bacillus subtilis protoplast fusion
Authors: Frehel C , Lheritier A M , Sanchez-Rivas C , Schaeffer P
Published: January 2020

When protoplasts derived from sporulating cells of Bacillus subtilis were fused by exposure to polyethylene glycol (PEG) and fixed immediately thereafter, protoplasts with two enclosed prespores could be seen by electron microscope. The number of fusion events was greatly increased, and multiply fused protoplasts appeared, when the PEG-treated suspension was diluted in hypertonic broth and reincubated before fixation. This post-PEG incubation effect is taken to indicate a fusion mechanism of two steps: a short, PEG-dependent step of membrane activation, followed by a slow, metabolism-requiring step completing fusion. When prespore-bearing protoplasts from two genetically different strains were mixed and fused, the extent of fusion could also be followed by counting clones of recombinant bacteria. Maximal from the start, their number (1% of each parent type protoplast present) was unaffected by post-PEG incubation. Fusion in this case is apparently completed after plating on the wall-regeneration medium. After optimal post-PEG incubation, the majority of the protoplasts were seen to participate in fusion, and the cytological fusion observed, corrected for wall-regeneration frequency, accounted quantitatively for the prototrophic bacteria eventually recovered. These results are in good agreement with those obtained independently by Sanchez-Rivas and Garro (J. Bacteriol. 137:1340--1345, 1979).
Incorporation of Synthetic Fatty Acid Analogs into Phospholipids of
Escherichia coli
Authors: Olsen William L. , Schaechter Moselio , Khorana H. Gobind
Published: January 2020

Escherichia coli
K1060-B5 (
fadE fabB thi
) incorporates into phospholipids several new synthetic fatty acid analogs including those containing phenyl, phenoxy, and azidophenoxy groups at the ω position.