bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemical Composition and Ultrastructure of Native and Reaggregated Membranes from Protoplasts of
Bacillus cereus
Authors: Beaman Teofila Cabrera , Pankratz H. Stuart , Gerhardt Philipp
Published: January 2020

Conditions were defined for producing protoplasts with lysozyme and isolating the protoplast membranes from cells of
Bacillus cereus
T harvested late in the exponential growth phase just before sporogenesis. The membranes contained approximately 60% protein, 30% lipid, 6% carbohydrate, and 1% ribonucleic acid. Seventeen proteins were distinguished by molecular size in the membrane solubilized with sodium dodecyl sulfate, and 12 in that with phenol and acetic acid. The lipid fraction consisted of neutral lipids (28%) and phospholipids (72%). Four phospholipids were identified: diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl glycerol, and lysophosphatidyl ethanolamine. Eighteen fatty acids were identified, with a predominance of branched C
15
and C
17
and of normal C
16
acids. The carbohydrate fraction consisted of neutral hexoses. A clear supernatant solution from the solubilized preparation became reaggregated into membrane by dialysis in the presence of MgCl
2
. The reaggregated membrane had the same main components as the native membrane, but the amount and ratio of protein and lipid depended on the buffer and the MgCl
2
concentration. By electron microscopy, the reaggregated membranes appeared as vesicles or sheets, depending on the MgCl
2
concentration. Hexagonal lattices were occasionally detected in the negatively stained ultrastructure of both native and reaggregated membrane fragments.
Regulation of Leucine Catabolism in
Pseudomonas putida
Authors: Massey Linda K. , Conrad Robert S. , Sokatch John R.
Published: January 2020

The generation time of
Pseudomonas putida
with
l
-leucine was 20 h in synthetic media but only 3 h with
d
-leucine. Slow growth in the presence of
l
-leucine was partially overcome by addition of 0.1 mM amounts of either
d
-valine,
l
-valine, or 2-ketoisovalerate. The activities of five enzymes which take part in the oxidation of leucine by
P. putida
were measured under various conditions of growth. Four enzymes were induced by growth with
dl
-leucine as sole source of carbon:
d
-amino acid dehydrogenase, branched-chain keto acid dehydrogenase, 3-methylcrotonyl-coenzyme A carboxylase, and 3-hydroxy-3-methylglutaryl-coenzyme A lyase. The segment of the pathway required for oxidation of 3-methylcrotonate was induced by growth on isovalerate or 3-methylcrotonate without formation of the preceding enzymes. The synthesis of carboxylase and lyase appeared to have been repressed by the addition of
l
-glutamate or glucose to cells growing on
dl
-leucine as the sole carbon source. Mutants unable to grow at the expense of isovalerate had reduced levels of carboxylase and lyase, whereas the levels of three enzymes common to the catabolism of all three branched-chain amino acids and those of two isoleucine catabolic enzymes were normal.
Direct Enzymatic Repair of Deoxyribonucleic Acid Single-Strand Breaks in Dormant Spores
Authors: Durban E. , Grecz N. , Farkas J.
Published: January 2020

With the alkaline sucrose gradient centrifugation method, it was found that dormant spores ofClostridium botulinumsubjected to 300 krads of gamma radiation showed a distinct decrease in deoxyribonucleic acid (DNA) fragment size, indicating induction of single-strand breaks (SSB). A two- to threefold difference in radiation resistance of spores of two strains ofC. botulinum, 33A (37% survival dose [D37] = 110 krads) and 51B (D37= 47 krads), was accompanied by relatively larger DNA fragments (molecular weight 7.9 × 107) obtained during extraction from the radiation-resistant strain 33A and smaller DNA fragments (molecular weight 1.8 × 107) obtained under identical conditions from radiation-sensitive strain 51B. The apparent number of DNA SSB produced by 300 krads in strains 33A and 51B was 0.37 and 3.50, respectively, per 108daltons of DNA. Addition of 0.02 M ethylenediaminetetraacetic acid (EDTA) to spore suspensions during irradiation doubled the apparent number of SSB in strain 33A but had no effect on strain 51B. In vivo, 0.02 M EDTA present during irradiation to 100 to 300 krads decreased survival of spores of 33A by about 30% but had little or no effect on 51B. Survival of 33A was also reduced by about 45% when the spores were irradiated while frozen in dry ice (−75 C) and, after irradiation, immediately exposed to 0.03 M EDTA for 1 h to inhibit repair in the dormant spores. These results suggest that the highly radiation-resistant strain 33A may be able to accomplish repair of SSB during irradiation or after irradiation under nonphysiological conditions, i.e., in the dormant state. This repair can be inhibited by EDTA. Sedimentation patterns show that DNA from spores of both strains 33A and 51B did not show any postirradiation repair during the first 6 h of germination, as opposed toBacillus subtilisspores, which exhibit repair immediately after germination. These observations suggest the existence of direct repair in physiological dormant spores of strain 33A in the cryptobiotic resting state in the absence of germination. The repair seems to be similar to that of polynucleotide ligase activity shown to be operative in some vegetative cells. Apparently radiation-sensitive strains such as 51B andB. subtilisare generally poor in DNA repair enzyme activity under conditions of spore dormancy, which may account for the approximately threefold difference in radiation sensitivity or DNA fragility of different strains, or both.
Factors Affecting Competence for Transformation in
Staphylococcus aureus
Authors: Rudin Lars , Sjöström Jan-Eric , Lindberg Martin , Philipson Lennart
Published: January 2020

A chemically defined medium has been developed for isolation of amino acid-requiring mutants of
Staphylococcus aureus
strain 8325, and for use as a selective medium in transformation assays. Variables affecting transformation of both plasmid and chromosomal markers have been studied. The optimal pH and temperature for transformation are 6.75 to 7.0 and 30 C, respectively. Ca ions are required for transformation, and only cells lysogenic for the phage φ11 can be transformed. Superinfection of competent cells with φ11 does not increase the transformation frequency. Maximal number of transformants is obtained after 20 min of contact between cells and deoxyribonucleic acid. The transformation frequencies for the plasmid marker erythromycin resistance (
ero
) and the chromosomal markers
trp, thy
, and
cyt
are of the same order of magnitude, whereas the frequency for the chromosomal marker
tyr
is approximately one order of magnitude lower.
Evolution of
l
-1,2-Propanediol Catabolism in
Escherichia coli
by Recruitment of Enzymes for
l
-Fucose and
l
-Lactate Metabolism
Authors: Cocks G. T. , Aguilar J. , Lin E. C. C.
Published: January 2020

A mutant strain of
Escherichia coli
capable of growth on
l
-1,2-propanediol was isolated previously. The mutant is characterized by constitutive production of a propanediol:nicotinamide adenenine dinucleotide (NAD) oxidoreductase which is essential for the new growth property. In the present study, it is shown that phage P1 cotransduces the genetic locus conferring this property and the genes for the utilization of
l
-fucose. A further indication of a relationship between these two growth properties is provided by the observation that wild-type
E. coli
excretes propanediol during fermentation of
l
-fucose. Under these conditions, a propanediol dehydrogenase (lactaldehyde reductase) is induced. This enzyme migrates on diethylaminoethyl-cellulose with the propanediol dehydrogenase produced constitutively by the mutant strain. A key event in the establishment of the ability to grow on propanediol is evidently a shift in the expression and function of propanediol dehydrogenase; an enzyme catalyzing formation of a reduced fermentation product anaerobically in wild-type cells functions aerobically to oxidize this same product in the mutant.
l
-Lactaldehyde, which is thus derived from propanediol, is converted to
l
-lactate by another dehydrogenase (
l
-lactaldehyde:NAD oxidoreductase) which is constitutively produced by both wild-type and mutant cells. The normal function of this enzyme is not yet established.
l
-Lactate is converted to pyruvate by an inducible NAD-independent
l
-lactate dehydrogenase. Thus, the carbons of propanediol are brought into the central metabolic network of the cell.
An Unusual Mutant Affecting the Frequency of Organelle Mutation
Authors: Hershberger Charles L. , Morgan Donna , Weaver Fred A. , Pienkos Phil
Published: January 2020

A mutant of
Euglena gracilis
Z is described that spontaneously produces irreversibly bleached mutants at a high frequency. Bleaching only occurs during exponential growth under highly aerobic conditions. The kinetics for bleaching and growth of bleached and nonbleached cells suggest that mutation is induced by segregation of a cellular component that becomes limiting after several culture generations. The properties of spontaneous bleaching closely resemble the properties of induced organelle mutagenesis in
E. gracilis
and yeast.
Deoxyribonucleic Acid-Envelope Complexes Isolated from
Escherichia coli
by Free-Flow Electrophoresis: Biochemical and Electron Microscope Characterization
Authors: Olsen William L. , Heidrich Hans-G. , Hannig Kurt , Hofschneider Peter Hans
Published: January 2020

A procedure for the preparative isolation of
Escherichia coli
cell wall, membrane, and deoxyribonucleic acid (DNA)-envelope complex fragments has been developed. The envelope fragments were produced by controlled mechanical cell breakage and isolated by density gradient centrifugation and subsequent preparative free-flow electrophoresis. The DNA-envelope complex fragments were shown to contain biochemical markers of both the cell wall and the membrane and by electron microscopy to be cell envelope fragments containing wall/membrane adhesion zones.
Characteristics and Relationships of Mercury-Resistant Mutants and Methionine Auxotrophs of Yeast
Authors: Singh Arjun , Sherman Fred
Published: January 2020

Approximately one-half of the mutants of
Saccharomyces cerevisiae
that are selected as resistant to methyl mercury are also found to require methionine. Eighty-four percent of these
met
mutations occur at the
met
15 locus, and the remaining 16% occur at the
met
2 locus. Surprisingly, the methionine-requiring mutants are recovered at a much higher frequency on methionineless media than on media supplemented with methionine. Growth patterns of the
met
mutants on media having a continuous concentration gradient of methionine and mercury compounds indicate that, at a critical concentration of the mercury compounds, the methionine requirement of certain
met
mutants is partially or completely alleviated. This was found for
met
2,
met
15, and to a lesser extent for
met
6, but not for any other methionine mutants. This loss of methionine requirement is produced with methyl mercury, phenyl mercury, and mercuric chloride although
met
2 and
met
15 strains can be shown to be resistant only to methyl mercury. Other methionine auxotrophs are not resistant to any of the three mercury compounds. The
met
2 and
met
15 mutants, but not the other methionine auxotrophs, develop a sheen of an unidentified product when grown on media with mercuric chloride but not with methyl mercury or phenyl mercury. It is suggested that
met
2 and
met
15 mutants produce a simple diffusible substance, which detoxifies methyl mercury, which reacts with mercuric chloride to produce a sheen, and which is the cause of the methionine requirement.
Multiple Transport Components for Putrescine in
Escherichia coli
Authors: Munro George F. , Bell Carol A. , Lederman Muriel
Published: January 2020

Putrescine uptake was studied in cultures of
Escherichia coli
K-12 grown in media of high or low osmolarity. When grown in high osmolarity medium, a transport system of low

K
m

and low

V
max

was found. For cultures grown in a medium of low osmolarity, the kinetics of putrescine uptake was more complex and consistent with the existence of an additional transport system of higher

K
m

and

V
max

. This conclusion is supported by the isolation of mutants in which one or the other system appears to be defective and by the ability of chloramphenicol to block the expression of the second transport system. Both systems appear to prefer putrescine over other compounds, since several basic amino acids and other polyamines competed only weakly for transport. The action of both uptake systems was shown to cause significant displacement of intracellular putrescine. Both systems also are at least partially energy dependent.
Ribitol Catabolic Pathway in
Klebsiella aerogenes
Authors: Charnetzky W. T. , Mortlock R. P.
Published: January 2020

In
Klebsiella aerogenes
W70, there is an inducible pathway for the catabolism of ribitol consisting of at least two enzymes, ribitol dehydrogenase (RDH) and
d
-ribulokinase (DRK). These two enzymes are coordinately controlled and induced in response to
d
-ribulose, an intermediate of the pathway. Whereas wild-type
K. aerogenes
W70 are unable to utilize xylitol as a carbon and energy source, mutants constitutive for the ribitol pathway are able to utilize RDH to oxidize the unusual pentitol, xylitol, to
d
-xylulose. These mutants are able to grow on xylitol, presumably by utilization of the
d
-xylulose produced. Mutants constitutive for
l
-fucose isomerase can utilize the isomerase to convert
d
-arabinose to
d
-ribulose. In the presence of
d
-ribulose, RDH and DRK are induced, and such mutants are thus able to phosphorylate the
d
-ribulose by using the DRK of the ribitol pathway. Derivatives of an
l
-fucose isomerase-constitutive mutant were plated on
d
-arabinose, ribitol, and xylitol to select and identify mutations in the ribitol pathway. Using the transducing phage PW52, we were able to demonstrate genetic linkage of the loci involved. Three-point crosses, using constitutive mutants as donors and RDH
−
, DRK
−
double mutants as recipients and selecting for DRK
+
transductants on
d
-arabinose, resulted in DRK
+
RDH
+
-constitutive, DRK
+
RDH
+
-inducible, and DRK
+
RDH
−
-inducible transductants but no detectable DRK
+
RDH
−
constitutive transductants, data consistent with the order
rbtC-rbtD-rbtK
, where
rbtC
is a control site and
rbtD
and
rbtK
correspond to the sites for the sites for the enzymes RDH and DRK, respectively.
Effect of Inhibitors of Ribonucleic Acid and Protein Synthesis on the Cyclic Adenosine Monophosphate Stimulation of Plasmid ColE1 Replication
Authors: Katz Leonard , Helinski D. R.
Published: January 2020

Addition of cyclic adenosine 3′-5′-monophosphate (c-AMP) to growing
Escherichia coli
cells, colicinogenic for the plasmid ColE1, results in a fourfold stimulation in the rate of synthesis of the plasmid deoxyribonucleic acid (DNA). The stimulation is transient (30 min) and is succeeded by a brief period (30 min) of cessation of plasmid DNA replication. The stimulation of ColE1 DNA replication also occurs in chloramphenicol-treated cells. Rifampin inhibits ColE1 DNA replication in the presence or absence of c-AMP. Employing thymine starvation conditions to stop ColE1 DNA synthesis, it was found that c-AMP, added during the period of thymine starvation, effected a stimulation in the amount of subsequent replication which took place when replicating conditions were restored. The stimulatory effect of c-AMP under these conditions was not prevented by chloramphenicol but was completely eliminated when rifampin was present. Under these conditions, when rifampin was added after the effect of c-AMP was allowed to occur, subsequent replication of the plasmid could take place, but only one round of replication occurred. A model to account for the c-AMP effects is presented.
Effects of a Mutator Mutation of
Salmonella typhimurium
on P22 and R Factor Genes
Authors: Guterman Sonia K. , Wright Andrew
Published: January 2020

A mutator mutation of
Salmonella typhimurium
, observed in a strain resistant to P22 and rifampin, affects mutation rates of bacterial, R factor, and prophage P22 genes.
Purification and Properties of Pyridine Nucleotide-Independent
l
-Lactate Dehydrogenase from
Polyporus circinatus
Authors: Funayama Shigehiro , Zancan Glaci T.
Published: January 2020

Cell extracts of
Polyporus circinatus
grown on lactate catalyze the reduction of 2,6-dichlorophenolindophenol by
l
-lactate without the participation of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. The enzyme has been purified 78-fold and was homogenous by disc gel electrophoresis. The optimal pH was found to be 6.7. The Michaelis constant for
l
-lactate was 5.9 × 10
−4
M and the oxalate inhibition constant was 1.5 × 10
−4
M. The nature of the prosthetic group is discussed.
Thymineless Mutagenesis in
Escherichia coli
Authors: Deutch Charles E. , Pauling Crellin
Published: January 2020

To clarify the relationship between thymineless death and thymineless mutagenesis, the induction of arginine revertants of
Escherichia coli
TAU-bar by thymine starvation was examined in physiological terms. Induced revertants were detectable both on minimal medium lacking arginine and minimal medium supplemented with 1 μg of arginine per ml. Substantial thymineless mutagenesis occurred during the period before the onset of thymineless death. Mutagenesis and loss of viability were observed upon incubation in medium lacking thymine and arginine, and both were inhibited upon incubation in medium lacking thymine and uracil. Mutagenesis also occurred during thymine starvation at 25 C, where there was relatively little loss of viability. At 37 C thymineless mutagenesis did not require complete thymine starvation, and the induction of revertants appeared to be initiated at the same suboptimal thymine concentration at which lethality was first detectable. Mutagenesis was found not to occur preferentially at the growing point of deoxyribonucleic acid replication. These results suggest that thymineless mutagenesis does not involve simply errors in base pairing due to the absence of thymine. The data also suggest that the induction of mutations and thymineless death are due to the same primary event but that mutagenesis is the more sensitive response.
Evolution of a New Gene Substituting for the
leuD
Gene of
Salmonella typhimurium
: Characterization of
supQ
Mutations
Authors: Kemper Jost
Published: January 2020

Alpha-isopropylmalate isomerase, the second specific enzyme in the biosynthesis of leucine, is coded for by two genes,
leuC
and
leuD
. Leucine auxotrophs, harboring
leuD
mutations including a deletion of the entire
leuD
gene, revert to leucine prototrophy owing to mutations at a locus,
supQ
, substantially distant to the leucine operon. A large number of independently isolated
supQ
mutations were characterized. A significant increase in the spontaneous frequency of
supQ
mutations was found after mutagenesis with 2-aminopurine,
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine, diethyl sulfate, and nitrous acid. The
supQ
function in most of these strains is temperature sensitive, resulting in more efficient suppression with decreasing temperature. At higher temperatures, the
supQ
limits the growth rate of
leuD supQ
mutant strains. All
supQ
mutations are co-transducible with
proA
and
proB
, with co-transduction frequencies ranging from 5.4 to 99.9% for different
supQ
mutations. Many
supQ
mutations were isolated, especially after nitrous acid mutagenesis, that had acquired a simultaneous proline requirement. The data support the idea of two genes,
supQ
and
newD
, whose protein products form a complex. The
newD
gene product, without any genetic alteration, is capable of substituting for the missing
leuD
protein. However, mutations in the
supQ
gene (point mutations or deletions) are necessary to make the
newD
protein available, which is normally tied up in a complex with the
supQ
protein.
Altered Proteins with Triosephosphate Isomerase Activity in Suppressor-containing Strains of
Bacillus subtilis
Authors: Baptist James N. , Tevethia M. J. , Mandel Manley , Shaw Charles R.
Published: January 2020

Suppressor mutations in
Bacillus subtilis
cause the synthesis of a new protein with the enzymatic activity of
l
-leucine dehydrogenase and two groups of new proteins with the activity of triosephosphate isomerase. The new isoenzymes of triosephosphate isomerase are separable by zone electrophoresis and differ among themselves in elution behavior upon gel permeation chromatography. One group has an apparent average molecular weight of 120,000 to 135,000, which is more than twice that of the wild-type enzyme. Another group appears to be even higher in molecular weight. These data are consistent with the working hypothesis that the new isoenzymes are produced by extension of growing polypeptide chains through one or more chain-terminating triplets, although other mechanisms resulting in alteration of shapes, charges, or associations of the enzymes are not excluded.
Electron Microscopy of Young
Candida albicans
Chlamydospores
Authors: Miller Sara E. , Spurlock Ben O. , Michaels G. E.
Published: January 2020

One- to three-day-old cultures of
Candida albicans
bearing chlamydospores were grown and harvested by a special technique, free of agar, and prepared for ultramicrotomy and electron microscopy. These young chlamydospores exhibited a subcellular structure similar to that of the yeast phase, e.g., cytoplasmic membrane, ribosomes, and mitochondria. Other structural characteristics unique to chlamydospores were a very thick, layered cell wall, the outer layer of which was continuous with the outer layer of the suspensor cell wall and was covered by hair-like projections; membrane bound organelles; and large lipoid inclusions. Only young chlamydospores less than 3 to 4 days old exhibited these ultrastructural characteristics.
Inhibition of Ribonucleic Acid Synthesis by Nalidixic Acid in
Escherichia coli
Authors: Javor George T.
Published: January 2020

The effect of low concentrations of nalidixic acid on ribonucleic acid (RNA) synthesis in
Escherichia coli
was examined. It was observed that RNA synthesis in exponentially growing cells was not significantly affected, in harmony with previous studies. However, RNA synthesis was markedly depressed by nalidixic acid during starvation for an amino acid or during chloramphenicol treatment. This effect was not caused by increased killing or inhibition of nucleoside triphosphate synthesis by nalidixic acid. The pattern of radioactive uracil incorporation into transfer RNA or ribosomes was not changed by the drug. The sensitivity of RNA synthesis to nalidixic acid in the absence of protein production may be useful in probing the amino acid control of RNA synthesis.
Electron Microscope Studies of Deoxyribonucleic Acid Synthesis in
Escherichia coli:
Localization of [
3
H]Thymidine in Deoxyribonucleic Acid-Membrane Complexes
Authors: Niveleau A.
Published: January 2020

The attachment of deoxyribonucleic acid to the membrane in
Escherichia coli
15 T
−
cells incubated with [
3
H]thymidine was studied by electron microscopy. Isolated deoxyribonucleic acid-membrane complexes were prepared from synchronized and unsynchronized cells during the exponential or stationary phase of growth and were examined by autoradiography. After short pulses with [
3
H]thymidine, a specific enrichment in radioactivity was observed in areas of membranous structures in exponentially growing cells. In contrast, the grain tracks produced in autoradiographs of chromosomes from cells in stationary phase were randomly distributed. The autoradiographic patterns are, therefore, evidence that deoxyribonucleic acid replication is closely related to the bacterial membrane.
Adenosine 5′-Triphosphate Release and Membrane Collapse in Glycerol-Requiring Mutants of
Bacillus subtilis
Authors: Freese Elisabeth B. , Oh Yong K.
Published: January 2020

Glycerol-requiring mutants of
Bacillus subtilis
could not sporulate in nutrient sporulation medium even when additional glycerol was added from the beginning of growth. Sporulation could be partially restored either by the frequent addition of small amounts of glycerol during the developmental period or by the single addition of both 10 mM glycerol and 10 mM malate. But sporulation could be completely restored by the addition of 50 mM glycerol-phosphate from the beginning. At the end of growth of the glycerol mutants in nutrient sporulation medium, the cell membrane collapsed and separated from the cell wall, and much of the cellular adenosine 5′-triphosphate was released into the medium. These observations were made in two glycerol mutants, one derived from strain 168 containing glycerol-teichoic acid in the cell wall and the other derived from strain W23 containing ribitol-teichoic acid.
Completed Chromosomes in Thymine-Requiring
Bacillus subtilis
Spores
Authors: Callister Heather , Wake R. G.
Published: January 2020

Origin:terminus genetic marker ratios (both
purA: metB
and
purA:ilvA
) were measured in extracts of spores of
Bacillus subtilis
strains W23
thy his
and 168
thy
. For strain W23
thy his
, normalized to W23 spore deoxyribonucleic acid, both ratios were equal to unity and were consistent with the presence of only completed chromosomes in the spores. The same ratios in extracts of spores of 168
thy
, normalized to strain 168 or the prototroph SB19, were abnormal, i.e., 2.26 ± 0.10 and 0.71 ± 0.06 for
purA:metB
and
purA:ilvA
, respectively. These values were unaffected by the extent of extraction of the spore deoxyribonucleic acid, the richness of the medium on which they are formed, and the thymine phenotype. The high ratio for
purA:metB
is in agreement with the results of earlier workers but, because of the low
purA:ilvA
ratio, cannot be explained simply by the presence of partially replicated chromosomes in spores of strain 168
thy
. Furthermore,
purA:leuA
in such extracts is 1.01 ± 0.06, consistent with the presence of only completed chromosomes. It is concluded that the abnormal origin:terminus marker ratios are only apparent and result from non-isogenicity between strains 168
thy
and 168 in the
metB thyB ilvA
chromosome region introduced during construction of 168
thy
by transformation of strain 168 with W23
thy
deoxyribonucleic acid. It is concluded further that the chromosomes of strain 168
thy
spores are in a completed form.
Synthesis of Branched-Chain Aminoacyl-Transfer Ribonucleic Acid Synthetases in a
Salmonella typhimurium
Mutant with an Altered Biosynthetic
l
-Threonine Deaminase
Authors: Arfin Stuart M. , Miner Twyla , Hatfield G. Wesley
Published: January 2020

The differential rates of synthesis of the three branched-chain aminoacyl-transfer ribonucleic acid synthetases were measured in
Salmonella typhimurium
LT-2 and a mutant,
ilvA504
. The mutant produced an
l
-threonine deaminase with a decreased affinity for its cofactor, pyridoxal-5′-monophosphate. The addition of pyridoxal-5′-monophosphate to cultures of strain
ilvA504
growing in excess isoleucine, valine, and leucine resulted in an increased rate of growth and repression of the synthesis of the isoleucine and valine biosynthetic enzymes. No differences in the rate of synthesis of the branched-chain aminoacyl-transfer ribonucleic acid synthetases were observed in cultures of
ilvA504
growing with or without added pyridoxal-5′-monophosphate. The differential rates of synthesis of all three enzymes were similar to the rates measured in strain LT-2. These experiments suggest that different forms of the
ilvA
gene product are involved in the regulation of the branched-chain amino acid biosynthetic enzymes and the branched-chain aminoacyl-transfer ribonucleic acid synthetases.
Structural Genes for a Newly Recognized Acetolactate Synthase in
Escherichia coli
K-12
Authors: Felice Maurilio De , Guardiola John , Esposito Bruno , Iaccarino Maurizio
Published: January 2020

Evidence is reported that shows the presence in
Escherichia coli
K-12 of a newly found acetolactate synthase. This enzyme is the product of two genes,
ilvH
and
ilvI
, both located very close to
leu
. Amber mutations have been found in both genes and therefore their products are polypeptides. Mutations in the
ilvH
gene cause the appearance of an acetolactate synthase activity which is relatively resistant to valine inhibition and can be separated by adsorption on hydroxylapatite from another activity present in the extract and more sensitive to valine inhibition than the former. A mutant altered in the
ilvI
gene was isolated among the revertants sensitive to valine inhibition of an
ilvH
mutant. Such a mutant lacks the resistant acetolactate synthase. A temperature-sensitive revertant of the
ilvI
mutant contained a temperature-sensitive acetolactate synthase. Thus
ilvI
is the structural gene for a specific acetolactate synthase. The activity of the
ilvH
gene product has been measured by adding an extract containing it to a purified
ilvI
acetolactate synthase, which, upon incubation, became more sensitive to valine inhibition. Conversely, a valine-sensitive acetolactate synthase (the product of the
ilvH
and the
ilvI
genes) became more resistant to valine inhibition upon incubation with an extract of a strain containing a missense
ilvH
gene product.
Stalk Formation and Its Inhibition in
Caulobacter crescentus
Authors: Haars Ellen G. , Schmidt Jean M.
Published: January 2020

Estimates of average rates of stalk formation over several generations of growth in
Caulobacter crescentus
showed that long-stalked Sk1 mutant and phosphate-starved wild-type cultures produce stalk material at about twice the rate of wild-type
C. crescentus
grown with adequate nutrients. Thus, the long stalks of Sk1 or phosphate-starved caulobacters are not merely a function of their longer doubling times. Inhibition of cell division of Sk1 418 with mitomycin C (MC) caused production of cellular filaments and resulted in inhibition of stalk formation. There was no appreciable decrease in total cell mass or in rates of ribonucleic acid and protein synthesis in the MC-treated cultures as compared with controls, but stalk formation, which is normally dependent on these processes, was severely retarded. Average stalk lengths in MC-treated Sk1 cultures were 30% of those found in control cultures. MC-produced cellular filaments were also subjected to deoxyribonucleic acid analysis and ultrastructural examination. The deoxyribonucleic acid content of MC-treated bacteria was about 50 to 60% that of untreated bacteria. Hydroxyurea also was found to produce some cellular filaments and shorter stalks, but with accompanying decreases in growth rate and yield.
Effect of pH on the Proportions of Polar Lipids, in Chemostat Cultures of
Bacillus subtilis
Authors: Minnikin D. E. , Abdolrahimzadeh H.
Published: January 2020

Significant changes in the relative proportions of the individual polar lipids of two strains of
Bacillus subtilis
were observed when the pH of their chemostat cultures was varied. In phosphate- and magnesium-limited cultures of
B. subtilis
var.
niger
NCIB 8058. lysylphosphatidylglycerol was present in higher proportions at low pH (5.1) than at neutral pH. With magnesium-limited cultures of this strain harvested at pH 8.0, lysylphosphatidylglycerol and phosphatidylethanolamine were not detected. Phosphate-limited cultures of
B. subtilis
NCIB 3610 contained no phosphatidylethanolamine or lysylphosphatidylglycerol at neutral pH, but at low pH (5.1) both these lipids were present in substantial proportions. The proportions of phosphatidylglycerol in actively dividing cells of chemostat cultures of bacilli were always greater than those of lysylphosphatidylglycerol. The reverse is commonly found in batch cultures of bacilli and staphylococci harvested at low pH. Changes in the proportions of the other polar lipids present in these bacilli (diphosphatidylglycerol and diglucosyl diacylglycerol) with pH were also noted. Certain cultures of both strains of
B. subtilis
contained small proportions of a peptidolipid.
Synchronous germination and outgrowth of fractionated Bacillus subtilis spores: tool for the analysis of differentiation and division of bacterial cells
Authors: Siccardi A G , Galizzi A , Mazza G , Clivio A , Albertini A M
Published: January 2020

A fraction of Bacillus subtilis 168 spores, purified by Urografin isopycnic density centrifugation, heat activated, and inoculated in nutrient broth plus glucose, germinated and outgrew very synchronously. Synchrony was documented by nuclear staining and fluorescence microscopy, and by determining the variation of the buoyant density of the cells during outgrowth. Cell mass increased at constant rates and the passage from one rate to the next was dependent upon deoxyribonucleic acid (DNA) synthesis. DNA synthesis inhibitors induced the formation of anucleated sister cells in a vast majority of the population, indicating that septation was programmed and became independent from DNA synthesis very early in the cell cycle.
Effect of lysogeny on transfection and transfection enhancement in Bacillus subtilis
Authors: Yasbin R E , Wilson G A , Young F E
Published: January 2020

Strains of Bacillus subtilis 168 lysogenic for bacteriophage phi105 transfer with deoxyribonucleic acid (DNA) isolated from bacteriophage SPO2 at a higher efficiency than non-lysogenic strains. This enhancement of transfection was not the result of recombination between bacteriophages SPO2 and phi105. Superinfection marker rescue increased transfection with DNA from bacteriophage phi105 occurred simultaneously with the addition of the transfecting DNA. Again, this enhancement of transfection was not the result of recombination but rather a protection of the transfecting DNA by the superinfecting bacteriophage. The ability of the superinfecting bacteriophage to protect the transfecting DNA from inactivation was maximal when the bacteria were just becoming competent. Bacteriophage phi1 cannot replicate after the transfection of competent bacteria lacking a functional DNA replication system, whereas bacteriophage phi1 was able to replicate after infection of competent bacteria grown under comparable conditions. These observations support the hypothesis that GAPase and an inducible repair system play an important role in the development of competence.
Tryptophan catabolism in Bacillus megaterium
Authors: Bouknight R R , Sadoff H L
Published: January 2020

Bacillus megaterium grows in a medium containing L-tryptophan as the sole carbon, nitrogen, and energy source. Kynurenine, anthranilic acid, and catechol are metabolic intermediates, suggesting that this organism used the anthranilic acid pathway for tryptophan degradation. Cells that grow on L-tryptophan oxidize kynurenine, alanine, and anthranilic acid and the presence of tryptophan oxygenase (EC 1.13.1.12), kynureninase (EC 3.7.1.3), and catechol oxygenase (EC 1.13.1.1) in cell extracts provide additional evidence for the degradative pathway in B. megaterium. Tryptophan oxygenase is inhibited by sodium azide, potassium cyanide, and hydroxylamine, indicating that the enzyme has a functional heme group. D-Tryptophan is not a substrate for tryptophan oxygenase, and the D-isomer does not inhibit this enzyme. Formamidase (EC 3.5.1.9) and anthranilate hydroxylase are not detectable in extracts. Tryptophan catabolism is inducible in B megaterium and is subject to catabolite repression by glucose and glutamate. Arginine does not cause repression, and kynurenine induces both tryptophan oxygenase and kynureninase.
Vinylglycolate resistance in Escherichia coli
Authors: Shaw L , Grau F , Kaback H R , Hong J S , Walsh C
Published: January 2020

Escherichia coli K-12 vinylglycolate-resistant mutants have been isolated and characterized. Two of the mutants, JSH 150 and JSH 151, have been determined to be double mutants, lacking both membrane-bound L-and D-lactate dehydrogenases. The lactate transport system is intact in all strains; both radioactive lactate and vinylglycolate are actively taken up. Likewise, the phosphoenolypyruvate-dependent phosphotransferase system for hexose uptake is active. Vinylglycolate, previously shown to inhibit the phosphoenolpyruvate-dependent phosphotransferase system, has very little effect in the double mutants. The extent of vinylglycolate inhibition in other mutants seems directly related to the activity of the lactate dehydrogenases. This indicates that vinylglycolate is oxidized to 2-keto-3-butenoate before inactivating the phosphoenolpyruvate-dependent phosphotransferase system. These results were found in whole cells and confirmed in isolated membrane vesicles.
Escherichia coli K-12 mutant forming a temperature-sensitive D-serine deaminase
Authors: McFall E
Published: January 2020

A single-site mutant of Escherichia coli K-12 able to grow in minimal medium in the presence of D-serine at 30 C but not at 42 C was isolated. The mutant forms a D-serine deaminase that is much more sensitive to thermal denaturation in vitro at temperatures above but not below 47 C than that of the wild type. No detectable enzyme is formed by the mutant at 42 C, however, and very little is formed at 37 C. The mutant enzyme is probably more sensitive to intracellular inactivation at high temperatures than the wild-type enzyme. The mutation lies in the dsdA region. The mutant also contains a dsdO mutation, which does not permit hyperinduction of D-serine deaminase synthesis.
Adenylate energy charge in Saccharomyces cerevisiae during starvation
Authors: Ball W J , Atkinson D E
Published: January 2020

Bakers' yeast cells, Saccharomyces cerevisiae, if grown aerobically on ethanol or if grown aerobically on glucose and allowed to pass into stationary phase, with utilization of accumulated ethanol, maintain a normal value (0.8 to 0.9) of the adenylate energy charge during prolonged starvation. In contrast, cells grown anaerobically on glucose and cells in the early stages of aerobic growth on glucose exhibit a rapid decrease of energy charge if transferred to medium lacking on energy source. These results suggest that functional mitochondria or enzymes of balance of adenine nucleotides during starvation. Yeast cells remain viable at energy charge values below 0.1, in marked contrast to results previously obtained with Escherichia coli. In other respects, the engery charge responses of yeast to starvation and refeeding are generally similar to those previously reported for E. coli.
Distribution of teichoic acid in the cell wall of Bacillus subtilis
Authors: Doyle R J , McDannel M L , Helman J R , Streips U N
Published: January 2020

Hydrolysis of the cell wall of Bacillus subtilis 168 by autolysins or lysozyme resulted in the exposure of glucosylated teichoic acid molecules as evidenced by increased precipitation of [14C] concanavalin A. The number of concanavalin A-reactive sites increased significantly after only limited enzymatic digestion of the walls. Quantitative analyses of [14C] concanavalin A-treated wall or wall hydrolysate complexes indicate that approximately one-half of the teichoic acid molecules are surface-exposed, whereas the remainder are probably embedded within the peptidoglycan matrix. Treatment of the cell walls with sodium dodecyl sulfate or Triton X-100 did not result in new concanavalin A-reactive sites. Partial autolysis diminished the ability of the cell walls to adsorb bacteriophage phi25. Fluorescein-labeled concanavalin A bound intensely over the entire surface of growing B. subtilis 168 cells, suggesting that teichoic acid molecules are located on the total solvent-exposed surface area of the bacteria.
Characterization of Neurospora crassa mutants deficient in glucosephosphate isomerase
Authors: Murayama T , Ishikawa T
Published: January 2020

Two independent mutants of Neurospora crassa lacking glucosphosphate isomerase activity (gpi) were isolated. These mutants were obtained as double mutants containing the pp or T9 mutation in addition to the gpi mutation located on linkage group IV; the pp mutation caused the inability to form protoperithecium and the loss of ascospore germination, and the T9 mutation caused the alteration in glucoamylase and several growth characteristics. The gpi mutants did not grow on fructose but grew on glucose or sucrose. Growth of these mutants on glucose was stimulated by addition of fructose. The gpi mutants showed restricted colonial growth on agar media containing glucose in contrast to the normal filamentous growth of the wild-type stain.
Methylamine and ammonia transport in Saccharomyces cerevisiae
Authors: Roon R J , Even H L , Dunlop P , Larimore F L
Published: January 2020

Methylamine (methylammonium ion) entered Saccharomyces cerevisiae X2180-A by means of a specific active transport system. Methylamine uptake was pH dependent (maximum rate between pH 6.0 and 6.5) and temperature dependent (increasing up to 35 C) and required the presence of a fermentable or oxidizable energy source in the growth medium. At 23 C the vmax for methylamine transport was similar 17 nmol/min per mg of cells (dry weight) and the apparent Km was 220 muM. The transport system exhibited maximal activity in ammonia-grown cells and was repressed 60 to 70 percent when glutamine or asparagine was added to the growth medium. There was no significant derepression of the transport system during nitrogen starvation. Ammonia (ammonium ion) was a strong competitive inhibitor of methylamine uptake, whereas other amines inhibited to a much lesser extent. Mutants selected on the basis of their reduced ability to transport methylamine (Mea-R) simultaneously exhibited a decreased ability to transport ammonia.
Electron microscope heteroduplex studies of sequence relations among bacterial plasmids: identification and mapping of the insertion sequences IS1 and IS2 in F and R plasmids
Authors: Hu S , Otsubo E , Davidson N , Saedler H
Published: January 2020

Heteroduplex experiments between the plasmid R6 and one strand of the deoxyribonucleic acid (DNA) of a lambda phage carrying the insertion sequence IS1 show that IS1 occurs on R6 at the two previously mapped junctions of resistance transfer factor (RTF) DNA with R-determinant DNA. From previous heteroduplex experiments, it then follows that IS1 occurs at the same junctions in R6-5, R100-1, and R1 plasmids. Heteroduplex experiments with the DNA from a lambda phage carrying the insertion sequence IS2 show that one copy of IS2 occurs in R6, R6-5, and R100-1 (but not R1) at a point within the RTF with coordinates 67.5 TO 68.9 kilobase units (kb). In an accompanying paper, Ptashne and Cohen (1975) show that the insertion sequence IS3 occurs on R6 and R6-5. R100-25, a traC mutant, differs from its parent R100-1 only in that it contains an additional copy of IS1 inserted within the tra gene region of 82.1 kb. R100-31, atraX, TC-s mutant of R100-1, is deleted in R100-1 sequences starting at one of the IS3 termini (46.9 kb) and extending with RTF to 61.0 kb. Heteroduplex studies of F plasmids with the DNA of a lambda phage bearing insertion sequence IS2 show that the sequence of F with coordinates 16.3-17.6F is IS2. The occurrence of IS1 at the two junctions of R-determinant DNA and RTF DNA in R plasmids provides a structural basis to explain the mechanism of the previously observed formation of molecules containing one RTF unit and several tandem copies of the R-determinant unit, when R plasmids in Proteus mirabilis are grown in the presence of antibiotics, and the segregation of an R plasmid into an RTF unit and an R-determinant unit. In general, correlation of our results with previous studies shows that insertion sequences play a role in a variety of F- and R-related intra- and intermolecular recombination phenomena.
Regulation of exocellular proteases in Neurospora crassa: metabolic requirements of the process
Authors: Drucker H
Published: January 2020

To induce exocellular proteolytic enzyme from carbon-starved exponential-phase cells of Neurospora crassa, both a protein substrate and an activating protease of certain specific properties must be present at the same time. The cells must be capable of protein synthesis, since cycloheximide inhibits the process, but cell growth, as determined by increase in cell mass, does not appear to be required. Both soluble (bovine serum albumin, myoglobin) and insoluble protein substrates (collagen, corn zein) will affect protease induction, although certain soluble, globular proteins (egg white globulin, bovine gamma globulin) will not. In most cases, rates of protease induction are proportional to protein concentration, regardless of the nature of the inducing protein. All activating proteases capable of affecting induction in a manner similar to that of N. crassa exocellular protease were of bacterial origin and were exoproteases. Mammalian proteases and peptidases had little or no effect on the induction process.
Relationship between chromosome replication and cell division in a thymineless mutant of Escherichia coli B/r
Authors: Meacock P A , Pritchard R H
Published: January 2020

The relationship between chromosome replication and cell division was investigated in a thymineless mutant of Escherichia coli B/r. Examination of the changes in average cell mass and DNA content of exponential cultures resulting from changes in the thymine concentration in the growth medium suggested that as the replication time (C) is increased there is a decrease in the period between termination of a round of replication and the subsequent cell division (D). Observations on the pattern of DNA synthesis during the division cycle were consistent with this relationship. Nevertheless, the kinetics of transition of exponential cultures moving between steady states of growth with differing replication velocities provided evidence to support the view that the time of cell division is determined by termination of rounds of replication under steady-state conditions.
Size restriction on utilization of peptides by amino acid auxotrophs of Neurospora crassa
Authors: Wolfinbarger L , Marzluf G A
Published: January 2020

Growth of an amino acid auxotroph of Neurospora crassa on oligopeptides is shown to occur by extracellular hydrolysis, with subsequent utilization of monomer amino acid residues, and by transport of peptides. Peptides with a hydrodynamic volume greater than that of trileucine are not transported, and this lack of transport is shown to be due to restriction by the oligopeptide transport system rather than the cell wall.