bluuebunny/crossref_metadata_2025 huggingface dataset:
Carbonic Acid from Decarboxylation by “Malic” Enzyme in Lactic Acid Bacteria
Authors: Pilone Gordon J. , Kunkee Ralph E.
Published: January 2020

Carbonic anhydrase studies were used to determine the primary form of carbonic acid produced from decarboxylation of
l
-malic acid by “malic” enzyme in malolactic strains of five different species of lactic acid bacteria. Addition of carbonic anhydrase to the reaction mixture containing crude bacterial extract and
l
-malic acid, at
p
H 7, in all five cases resulted in an increase (13 to 23%) in the rate of carbon dioxide evolution over the control. The results indicated that the primary form of carbonic acid released from “malic” enzyme was not anhydrous carbon dioxide as previously supposed and as has been shown for other decarboxylating enzymes. The standard free-energy changes of the malo-lactic reaction with the various forms of carbonic acid as the primary decarboxylation product were calculated. The reaction is less exergonic when carbonic acid, bicarbonate ion, or carbonate ion is the primary decarboxylation product compared to anhydrous carbon dioxide. The free-energy of the reaction is not biologically available to the bacteria; with carbon dioxide not the primary decarboxylation product, the potential energy lost in a malo-lactic fermentation is not as great as previously considered. Endogenous carbonic anhydrase activity was not found.
Inhibition of Vitamin K
2
and Carotenoid Synthesis in
Staphylococcus aureus
by Diphenylamine
Authors: Hammond Ray K. , White David C.
Published: January 2020

Diphenylamine at concentrations which did not effect the growth rate inhibited the synthesis of vitamin K
2
in both anaerobic and aerobic cultures by about 50%. At this concentration, diphenylamine inhibited the synthesis of the cyclic carotenoids δ-carotene and the rubixanthins 25 to 35% anaerobically and 60 to 90% aerobically. The inhibition of synthesis of cyclic carotenoids and vitamin K
2
by diphenylamine had no detectable effect on the formation of the membrane-bound electron transport system.
Galactose Transport in
Saccharomyces cerevisiae
II. Characteristics of Galactose Uptake and Exchange in Galactokinaseless Cells
Authors: Kou Shou-Chang , Christensen Michael S. , Cirillo Vincent P.
Published: January 2020

The characteristics of the inducible galactose system in
Saccharomyces cerevisiae
were studied by using the nonmetabolized galactose analogues,
l
-arabinose and
d
-fucose, and galactokinaseless and transportless mutants. Induced wild-type cells transport
l
-arabinose by facilitated diffusion. Transportless cells transport neither galactose nor
l
-arabinose above the noninduced rate, whereas galactokinaseless cells transport galactose
l
-arabinose and
d
-fucose by facilitated diffusion. Determination of unidirectional rate of
14
C-labeled galactose uptake by preloaded galactokinaseless cells, containing a large unlabeled free-galactose pool, showed that the rate of galactose uptake by facilitated diffusion is greater than the rate of galactose metabolism at similar external galactose concentrations.
Regulation of Ribonucleic Acid Synthesis by Histidine and Methionine During Recovery of
Escherichia coli
from Magnesium Starvation
Authors: Cleaves Graham R. , Cohen Paul S.
Published: January 2020

During magnesium starvation of
Escherichia coli
B, most of the ribosomes break down to low-molecular-weight components. When magnesium is restored to the medium, the cells recover. The rate of recovery can be increased greatly by supplementing the growth medium with a mixture of 21 amino acids. This increased rate of recovery is shown to be due to the effect of only two amino acids, histidine and methionine, which initially stimulate accumulation of cellular ribonucleic acid without increasing the rate of protein synthesis. In contrast, histidine and methionine supplementation to logarithmically growing
E. coli
B is not as effective in stimulating growth as is the complete amino acid mixture. Since cells recovering from magnesium starvation preferentially synthesize ribosomes, it is possible that histidine and methionine play a special role(s) in ribosomal ribonucleic acid synthesis or stability.
Extracellular Cell Wall Lytic Enzyme from
Staphylococcus aureus:
Purification and Partial Characterization
Authors: Huff Eskin , Silverman Carol S. , Adams Nancy J. , Awkard Woodruff S.
Published: January 2020

An autolysin obtained from culture fluid of
Staphylococcus aureus
strain 8507 was purified 3,000-fold. One milligram of this preparation (S-5DL) will solubilize 12 mg of cell wall in 1 hr. The major activity is
N
-acetylmuramyl-
l
-alanine amidase. Recovery of lytic activity in the purified preparation was repeatably only 20% of the starting level. This suggests that other cell wall lytic enzymes may be present in the starting material. The S-5DL enzyme has been compared to freeze-thaw extracted enzyme (AFZ). Both enzymes precipitate in 0.01
m
KPO
4
(
p
H 6.0) and dissolve in 0.1 to 0.7
m
NaCl. Fifty per cent of the AFZ activity and 66% of the S-5DL activity bind rapidly to cell walls of
S. aureus
at 0 C in the presence of magnesium ion. None of the AFZ activity and 66% of the S-5DL activity bind to cell walls at 0 C in the absence of magnesium ion. The cell walls of nine different strains of
S. aureus
were compared for level of native autolysin activity. These same walls after inactivation of the native autolysin were tested for susceptibility to the S-5DL enzyme.
Transport of Folinate and Related Compounds in
Pediococcus cerevisiae
Authors: Mandelbaum-Shavit Frederika , Grossowicz Nathan
Published: January 2020

The properties of folinate and 5-methyltetrahydrofolate (5-CH
3
-H
4
PteGlu) transport mechanism of
Pediococcus cerevisiae
were studied. The uptake was dependent on temperature,
p
H (optimum for both compounds at
p
H 6.0), and glucose. Iodoacetate, potassium fluoride, and sodium azide inhibited the uptake. 5-CH
3
-H
4
-PteGlu was apparently not metabolized but folinate was metabolized. Metabolism of folinate was reduced by preincubation of cells with fluorodeoxyuridine. The transport system for folinate and 5-CH
3
-H
4
PteGlu were specific for the
l
-isomers. Pteroylglutamate, aminopterin, and amethopterin did not interfere with the uptake. Tetrahydrofolate competed with the uptake of folinate. The transport of folinate and 5-CH
3
-H
4
PteGlu at 37 C conformed to Michaelis-Menten kinetics; apparent
K
m
for both compounds was 4.0 × 10
−7
m
, and the
V
max
for folinate was 1.0 × 10
−10
moles per min per mg (dry weight) and for 5-CH
3
-H
4
PteGlu it was 1.6 × 10
−10
moles per min per mg (dry weight). Both compounds accumulated in the intracellular pool at a concentration about 80- to 140-fold higher than that in the external medium. Folinate inhibited competitively the uptake of 5-CH
3
-H
4
PteGlu with a
K
i
of 0.4 × 10
−7
m
. Unlike 5-CH
3
-H
4
PteGlu, which accumulated only at 37 C, folinate was also taken up at 0 C by a glucose- and temperature-independent process, which was not affected by the metabolic inhibitors mentioned above. Since at 0 C the intracellular concentration of folinate was also considerably higher than the external, binding of the substrate to some cellular component is assumed. The finding of an efficient transport system for
l
-5-CH
3
-H
4
PteGlu is of special interest, since this compound has no growth-promoting activity for
P. cerevisiae
.
Characterization and Properties of the Pyruvate Phosphorylation System of
Acetobacter xylinum
Authors: Benziman Moshe , Palgi A.
Published: January 2020

The enzyme responsible for the direct phosphorylation of pyruvate during gluconeogenesis in
Acetobacter xylinum
has been purified 46-fold from ultrasonic extracts and freed from interfering enzyme activities. The enzyme was shown to catalyze the reversible Mg
2+
ion-dependent conversion of equimolar amounts of pyruvate, adenosine triphosphate (ATP), and orthophosphate (P
i
) into phosphoenolpyruvate (PEP), adenosine monophosphate (AMP), and pyrophosphate (PP). The optimal
p
H for PEP synthesis was
p
H 8.2; for the reversal it was
p
H 6.5. The ratio between the initial rates of the reaction in the forward and reverse directions was 5.1 at
p
H 8.2 and 0.45 at
p
H 6.5. The apparent
K
m
values of the components of the system in the forward reaction were: pyruvate, 0.2 m
m
; ATP, 0.4 m
m
; P
i
, 0.8 m
m
; Mg
2+
, 2.2 m
m
; and for the reverse reaction: PEP, 0.1 m
m
; AMP, 1.6 μ
m
; PP, 0.067 m
m
; Mg
2+
, 0.87 m
m
. PEP formation was inhibited by AMP and PP. The inhibition by AMP was competitive with regard to ATP (
K
i
= 0.2 m
m
). The reverse reaction was inhibited competitively by ATP and noncompetitively by pyruvate. The enzyme was strongly inhibited by
p
-hydroxymercuribenzoate. The inhibition was reversed by dithiothreitol and glutathione. The properties of the enzyme are discussed in relation to the regulation of the opposing enzymatic activities involved in the interconversion of PEP and pyruvate in
A. xylinum
.
Enzymes of the Tryptophan Pathway in
Acinetobacter calco-aceticus
Authors: Twarog Robert , Liggins George L.
Published: January 2020

All enzymes of the tryptophan synthetic pathway were detectable in extracts from wild-type
Acinetobacter calco-aceticus
. The levels of these enzymes were determined in extracts from a number of auxotrophs grown under limiting tryptophan. In each case only anthranilate synthetase was found to be present in increased amounts, whereas the specific activities of the remaining enzymes remained unchanged and unaffected by the tryptophan concentration. Derepression of anthranilate synthetase was found to occur as the concentration of tryptophan became limiting. Anthranilate synthetase and phosphoribosyl transferase activities are both feedback-inhibited by tryptophan. Molecular weight determination carried out by gel filtration and zonal centrifugation in sucrose revealed that all the enzymes are less than 100,000, and no molecular aggregates of these enzymes were detected. The data indicate that tryptophan synthesis in
Acinetobacter
is regulated both by feedback inhibition of the first two enzymes of the pathway and by repression control of anthranilate synthetase.
Cytological Studies of Deoxyribonucleic Acid Replication in
Escherichia coli
15T
−
: Replication at Slow Growth Rates and After a Shift-Up into Rich Medium
Authors: Chai Ning-Chun , Lark K. G.
Published: January 2020

We examined the gross nuclear morphology of
Escherichia coli
15T
−
grown in different media with doubling times ranging from 22 to 270 min. In slowly growing cells, deoxyribonucleic acid synthesis was measured by autoradiography and shown to occur with greatest probability during the first two-thirds of the division cycle. In such cells, segregation occurred later, at the end of the division cycle rather than at the end of deoxyribonucleic acid replication. Nuclear regions in L-broth cells (22-min doubling time) cannot correspond to separate chromosomes but probably represent regions of replication activity. Segregation of template nucleotide strands was measured after a shift-up from proline M9 or glucose M9 media into L broth. A model is presented to account for the pattern of segregation observed.
Alterations of the Respiratory System of
Neurospora crassa
by the mi-1 Mutation
Authors: Eakin R. T. , Mitchell H. K.
Published: January 2020

The respiratory mechanism of the cytochrome-defective mi-1 mutant of
Neurospora crassa
was partially characterized and compared with that of the wild type. Both systems of electron transport were linked to oxidative phosphorylation and showed similar characteristics with respect to substrates and components. The system operating in the cytochrome-deficient mi-1 mutant showed no characteristic α absorption bands of cytochromes
a
or
b
and was very unstable in vitro. The possibility that this system may be an altered form of the normal cytochrome electron transport chain which occurs in the wild type is discussed.
Gamma-Ray-Induced Spore Germination of
Dictyostelium discoideum
Authors: Khoury A. T. , Deering R. A. , Levin G. , Altman G.
Published: January 2020

60
Co gamma rays can induce germination of the spores of the cellular slime mold,
Dictyostelium discoideum
, in the absence of heat shock, amino acids, or bacteria food source. About 65% amoebae emergence occurs by 13 hr after a dose of 180 krad.
Phospholipids of
Streptococcus faecalis
Authors: Mota J. M. dos Santos , Den Kamp J. A. F. Op , Verheij H. M. , Van Deenen L. L. M.
Published: January 2020

Autoradiograms of total lipid extracts from
Streptococcus faecalis
ATCC 9790, harvested in the stationary phase from a medium containing
32
P-orthophosphate, showed six major spots. The corresponding compounds were identified as diphosphatidylglycerol (possibly with a penta acyl structure); phosphatidylglycerol; a provisionally identified mixture of alanylphosphatidylglycerol and of the 2′-lysyl-derivative of phosphatidylglycerol; the 3′-lysyl-derivative of phosphatidylglycerol, probably together with some arginylphosphatidylglycerol; a diglucosyl derivative of phosphatidylglycerol; and a compound which was tentatively identified as the 2′,3′-dilysyl derivative of phosphatidylglycerol.
Time-Dependent Effects of Inosine on Competence Development in
Haemophilus influenzae
Authors: Miller Donald H. , Huang P. C.
Published: January 2020

The effect of inosine on the development of competence is dependent upon the time at which it is added to the competence medium. Competence is inhibited if inosine is added within 15 min after the development begins, but is stimulated if the addition is made at later times. Protein synthesis is stimulated by the addition of inosine, but the effect is quantitatively similar regardless of the time at which inosine is added.
Elimination of Sex Factors in
Escherichia coli
by Urea
Authors: Tomoeda Munemitsu , Kokubu Masako , Nabata Hiroyuki , Minamikawa Satoko
Published: January 2020

Eliminatory action of urea on the sex factor (F) in
Escherichia coli
K-12 strains is reported. Growth of
E. coli
harboring F or F′8 (F-
gal
) factors in Penassay Broth containing urea led to the loss of these genetic elements and yielded F
−
cells. Appearance of F
−
cells among survivors was already observed when the culture was in the very early stage of exponential phase. However, frequencies of F
−
cells formed did not increase much as a function of the incubation time. Unusual F
+
or F′8 cells which retained the ability of genetic transfer but showed resistance to M12 phage were also isolated. Addition of sucrose to broth with urea led to the favorable growth of cells in the culture and the increase, if little, of elimination frequencies of F factors by urea. These findings, coupled with other observations, suggest that urea has two separate actions in enhancing the frequency of F
−
bacteria, namely, (i) to inactivate F by direct action, such as mutation, and (ii) to select the F
−
variants by differentially inhibiting the growth of F
+
.
Examination of the Protein Composition of the Cell Envelope ofEscherichia coliby Polyacrylamide Gel Electrophoresis
Authors: Schnaitman Carl A.
Published: January 2020

An envelope preparation containing the cell wall and cytoplasmic membrane ofEscherichia coliwas obtained by breaking the cells with a French pressure cell and sedimentating the envelope fraction by ultracentrifugation. This fraction was prepared for polyacrylamide gel electrophoresis by dissolving the protein in an acidifiedN,N′-dimethylformamide, removing lipids by gel filtration in the same organic solvent and removing the solvent by dialysis against aqueous urea solutions. More than 80% of the total protein of the envelope fraction was recovered in soluble form. Electrophoresis on sodium dodecyl sulfate-containing gels yielded from 20 to 30 well-resolved bands of protein. One major protein band was observed on the gels. This protein had a molecular weight of 44,000 and accounted for as much as 40% of the total protein of the envelope fraction. A double-labeling technique was used to examine the protein composition of the envelope fraction from cells grown under different sets of conditions which result in large changes in the levels of membrane-bound oxidative enzymes. These changes in growth conditions resulted in only minor alterations in the protein profiles observed on the gels, suggesting that this organism is able to adapt to changes in growth environment with only minor modifications of the major proteins of the cell envelope.
Scanning Electron Microscopy of Intact Colonies of Microorganisms
Authors: Whittaker D. K. , Drucker D. B.
Published: January 2020

Colonies of
S. mutans
OMZ61,
Streptococcus
sp. D182,
Staphylococcus aureus
Oxford NCTC 6571, and
Candida albicans
type A, MRL 3153 were grown on various media. Cubes of agar bearing two to three colonies were excised and processed for scanning electron microscopy. The characteristic shape of the colonies was seen when examined at low magnifications. At a magnification of 2,000 diameters, the arrangement of individual organisms within the colonies was observed. Plano-convex colonies consisted of uniformly distributed organisms, whereas
S. mutans
colonies presented a more complex arrangement possibly associated with the production of extracellular polysaccharides. Certain colonies were totally or partially covered by an adherent film through which the outline of the organisms could be distinguished.
Structural Properties and Features of Parasitic
Bdellovibrio bacteriovorus
Authors: Abram Dinah , Davis Bridgett K.
Published: January 2020

The structure of five parasitic strains of
Bdellovibrio bacteriovorus
was studied by electron microscope after negative staining and in shadow-case and etched freeze-fractured preparations. Special attention was paid to the cell wall and the flagellar sheath which is continuous with the wall or part of it. These structural components reveal distinct features which are induced by certain staining substances; they are exceedingly susceptible to disruption by physical treatments, and in old cells often appear impaired. In freeze-fractured cells the wall shows characteristic fracturing tendencies not known in other microorganisms. These structural properties and features are distinct to
Bdellovibrio
wall and flagellar sheath, the structural integrity of which is a fundamental requirement for the infectivity and survival of this organism. The anterior end of
Bdellovibrio
is differentiated: 6 to 12 ring-like structures (9 to 12 nm, outer diameter) are built into its wall and several fibers (7 to 10 nm wide, up to 1.5 μm long) emerge from it. Intracellular structures, which are revealed as compact oval bodies bulging from the cell border and have internal laminated organization, are characteristic of
Bdellovibrio
after negative staining with certain compounds. These findings on the structure of parasitic
Bdellovibrio
substantiate previous observations indicating the uniqueness of this organism and add criteria for the identification of this genus.
Localization in the Cell and Extraction of Alkaline Phosphatase from
Bacillus subtilis
Authors: Wood David A. W. , Tristram H.
Published: January 2020

Study of protoplasts, lysed protoplasts, and cells treated with lysozyme in the absence of osmotic stabilizer suggested that the alkaline phosphatase (EC 3.1.3.1.) of
Bacillus subtilis
is located in the protoplasmic membrane. Cytochemical evidence in support of this view is presented. The enzyme protein was strongly bound to the membrane structure and could not be solubilized by a number of treatments known to release enzymes from membranes and other lipoprotein structures. Alkaline phosphatase was, however, solubilized by treatment of intact
B. subtilis
cells or isolated protoplasmic membranes with strong salt solutions at
p
H 7.2, suggesting that electrostatic forces are responsible for the association between membrane and enzyme protein. Dialysis of alkaline phosphatase solutions against buffer of low ionic strength resulted in precipitation of the enzyme.
Conditions Affecting Germination of
Clostridium botulinum
62A Spores in a Chemically Defined Medium
Authors: Rowley Durwood B. , Feeherry Florence
Published: January 2020

Spores of
Clostridium botulinum
type 62A were germinated in a chemically defined medium (8 m
m l
-cysteine, 11.9 m
m
sodium bicarbonate, 4.4 m
m
sodium thioglycolate; buffered with 100 m
m
TES,
p
H 7.0). The rate and extent of germination were increased when an aqueous spore suspension was heated sublethally (80 C, 60 min) before addition to the germination medium. Neither sublethal nor lethal doses of gamma radiation had any marked effect on subsequent germination. Maximum germination (>90% in 2 hr) in the defined medium occurred in the
p
H range of 6.5 to 7.5, at 30 to 37 C, with an
l
-cysteine level of 8 m
m
. Increasing
l
-cysteine to 32 m
m
increased the rate (over that with 8 m
m l
-cysteine) but not the extent of germination. The rate and extent of germination increased with NaHCO
3
addition to 8.3 m
m
, but increasing levels to 11.9 m
m
had no further effect. For maximum germination, 2.2 m
m
sodium thioglycolate was required and higher levels (to 8.8 m
m
) had no further enhancing or inhibitory effect. Under optimal conditions for germination, 97% of the spores had become heat sensitive; 98% had become sensitive to radiation; 88 and 91% had become phase dark and stainable, respectively, and the spore suspension had lost 46% of its initial optical density by 2 hr. Loss of heat resistance preceded loss of radiation resistance, acquisition of stainability, and phase darkening by about 12 min.
Carbohydrate Accumulation and Metabolism in
Escherichia coli:
Characteristics of the Reversions of
ctr
Mutations
Authors: Wang Richard J. , Morse Helvise G. , Morse M. L.
Published: January 2020

The reversion behavior of pleiotropic carbohydrate mutants, previously designated as
ctr
, was studied. The mutants revert to complete restoration of the wild-type phenotype, as well as to a spectrum of partial wild-type phenotypes. Lac
+
reversions were found in the
lac
region (11 min) and some Mal
+
reversions occurred at
malB
(79 min), at a distance from the site of the
ctr
mutations (46 to 47 min). About one-third of Lac
+
and Mal
+
revertants were constitutive for uptake of their respective substrates, and one-third modified for inducibility. The remaining third were not distinguishable from wild type. Induction of a
ctr
mutation in a
lac
constitutive strain, either operator or repressor mutant, did not affect lactose metabolism. A polar-like
ctr
mutant, deficient in both enzyme I and heat-stable protein of the phosphoenolpyruvate-dependent phosphotransferase strain was also described. Partial revertants of
ctr
were still found to lack enzyme I.
Evidence for a Magnesium Pump in
Bacillus cereus
T
Authors: Schmidt Gale B. , Rosano C. L. , Hurwitz C.
Published: January 2020

Unlike
Escherichia coli, Bacillus cereus
T appears to accumulate Mg
2+
in its cell sap against a concentration gradient. Over a range of Mg
2+
in the growth medium from 5 × 10
−5
to 1.35 × 10
−2
m
, the concentration of Mg
2+
in the cell sap of
B. cereus
T was maintained at about 6 × 10
−3
m
, and ribosome-bound Mg
2+
and spermidine, as well as the spermidine concentration in the cell sap, appear to be unaffected by the concentration of Mg
2+
in the growth medium. Inhibition of growth of
E. coli
by streptomycin is progressively reversed by increasing the concentration of Mg
2+
in the growth medium above 5 m
m
. The finding that similar increases of Mg
2+
in the growth medium did not reverse the inhibition of
B. cereus
T is also consistent with the conclusion that
B. cereus
T, unlike
E. coli
, accumulates Mg
2+
to a constant concentration in its cell sap.
Metabolic Role, Regulation of Synthesis, Cellular Localization, and Genetic Control of the Glyoxylate Cycle Enzymes in
Neurospora crassa
Authors: Flavell Richard B. , Woodward Dow O.
Published: January 2020

The glyoxylate shunt enzymes, isocitrate lyase and malate synthase, were present at high levels in mycelium grown on acetate as sole source of carbon, compared with mycelium grown on sucrose medium. The glyoxylate shunt activities were also elevated in mycelium grown on glutamate or Casamino Acids as sole source of carbon, and in amino acid-requiring auxotrophic mutants grown in sucrose medium containing limiting amounts of their required amino acid. Under conditions of enhanced catabolite repression in mutants grown in sucrose medium but starved of Krebs cycle intermediates, isocitrate lyase and malate synthase levels were derepressed compared with the levels in wild type grown on sucrose medium. This derepression did not occur in related mutants in which Krebs cycle intermediates were limiting growth but catabolite repression was not enhanced. No Krebs cycle intermediate tested produced an efficient repression of isocitrate lyase activity in acetate medium. Of the two forms of isocitrate lyase in
Neurospora
, isocitrate lyase-1 constituted over 80% of the isocitrate lyase activity in acetate-grown wild type and also in each of the cases already outlined in which the glyoxylate shunt activities were elevated on sucrose medium. On the basis of these results, it is concluded that the synthesis of isocitrate lyase-1 and malate synthase in
Neurospora
is regulated by a glycolytic intermediate or derivative. Our data suggest that isocitrate lyase-1 and isocitrate lyase-2 are the products of different structural genes. The metabolic roles of the two forms of isocitrate lyase and of the glyoxylate cycle are discussed on the basis of their metabolic control and intracellular localization.
d
-Mannitol Utilization in
Salmonella typhimurium
Authors: Berkowitz David
Published: January 2020

A biochemical and genetic analysis of
d
-mannitol metabolism in
Salmonella typhimurium
indicates that
d
-mannitol is phosphorylated by the phosphoenolpyruvate-dependent phosphotransferase system.
d
-Mannitol-1-phosphate is converted to
d
-fructose-6-phosphate by mannitol-1-phosphate dehydrogenase. Two classes of mannitol mutants are described. Both map at about 115 min on the
Salmonella
chromosome. Mutants missing mannitol-1-phosphate dehydrogenase activity are mannitol sensitive; i.e., either growth is inhibited or the cells are lysed in the presence of mannitol. In a strain missing adenyl cyclase activity, the mannitol genes require exogenous cyclic adenosine-3′,5′-monophosphate for expression.
Characterization and Regulation of Protease Synthesis and Activity in
Bacillus licheniformis
Authors: Bernlohr R. W. , Clark V.
Published: January 2020

Extracts of growing and sporulating cells contain a protease activity that has a broad
p
H optimum and an unusually broad specificity. The activity, which resides in at least two protein fractions, hydrolyzes all peptide bonds and can reduce a mixture of proteins into a mixture of free amino acids with a high efficiency. No inhibitors of the activity were found, but the protease showed a definite preference for denatured protein as substrate. The synthesis of the intracellular protease activity is under catabolite repression control, as is the extracellular activity. However, the synthesis of the two activities is not coordinate, making the relationship between the two unclear. Due to (i) the specificity of the intracellular activity, (ii) the fact that it is synthesized most rapidly under slow or nongrowing conditions, and (iii) our inability to measure in vivo protein turnover in cells containing high levels of enzyme, a scavenger role is postulated for the enzyme. The rate of protein turnover is not a function of the protease content of the cells.
Transformation in
Bacillus amyloliquefaciens
Authors: Coukoulis Helen , Campbell L. Leon
Published: January 2020

The methodology and some of the requirements for the deoxyribonucleic acid-mediated transformation of an arginine auxotroph of
Bacillus amyloliquefaciens
to prototrophy are described.
Some Effects of Nalidixic Acid on Conjugation in
Escherichia coli
K-12
Authors: Hane Michael W.
Published: January 2020

The role of deoxyribonucleic acid (DNA) synthesis in the
Escherichia coli
conjugation system has been studied using nalidixic acid (NAL) to specifically inhibit DNA synthesis in matings between reciprocal combinations of male (Hfr) and female (F
−
) mutants resistant and sensitive to NAL; the physiological action of NAL on the strains utilized was also studied. Matings between combinations of mutants resistant (Nal
r
) and sensitive (Nal
s
) to NAL allow various parental functions to be established: pair formation studies show that the female cells are responsible for the slight decrease in pair formation when NAL is present in Hfr(Nal
s
) × F
−
(Nal
s
) matings. Preformed mating pairs are stable in the presence of NAL. In matings between Hfr(Nal
s
) and F
−
(Nal
r
), the transfer gradient constant increases linearly with low NAL concentration (0.1 to 0.6 μg of NAL per ml). Higher concentrations of NAL (5 μg/ml) act on Nal
s
males to rapidly stop chromosome transfer; under these conditions, however, DNA degradation is unmeasurable as determined from single-strand nicking in the male cells. This result is consistent with a model for chromosome transfer which requires DNA synthesis in the male cell. Inhibition of DNA synthesis (by 85%) in the female has no effect on conjugal chromosome transfer. High concentrations of NAL (>20 μg/ml) produce slight inhibition in chromosome transfer for the Hfr(Nal
r
) × F
−
(Nal
r
) mating tested; this effect is probably caused by action of NAL on the male. The inhibition of chromosomal transfer by NAL appears to be irreversible in the normal sense. A pulse of NAL, applied during transfer, immediately stops the transfer which is in progress. On removal of the NAL block, the temporal appearance of recombinants is consistent with the idea that a new round of transfer has commenced from the sex factor location on the male chromosome.
Requirements ofSalmonella typhimuriumfor Recovery from Thermal Injury
Authors: Tomlins Richard I. , Ordal Z. John
Published: January 2020

The heating ofSalmonella typhimurium7136 at 48 C for 30 min produces a population of cells that are incompetent at division on Levine Eosin Methylene Blue Agar containing 2.0% NaCl (EMB-NaCl). When these injured cells were placed in fresh citrate salts medium they recovered, and regained their tolerance to the EMB-NaCl medium and grew out. The addition of the selective inhibitors rifamycin, 5-fluorouracil, 2,4-dinitrophenol, chlorotetracycline, chloramphenicol, and 5-methyl-tryptophan to the recovery medium showed that the recovery process was dependent on ribosomal ribonucleic acid (RNA) synthesis, adenosine triphosphate synthesis, and the synthesis of new protein. These results were substantiated by incorporation experiments, which demonstrated that during recovery no deoxyribonucleic acid synthesis, and hence no cell division, occurred. Ribosomal RNA was synthesized during recovery, but its synthesis was not the rate-limiting step. A small but significant amount of protein was also formed during the latter part of the recovery period.
Inhibition of Growth of
Salmonella typhimurium
and of Threonine Deaminase and Transaminase B by β-Chloroalanine
Authors: Arfin Stuart M. , Koziell David A.
Published: January 2020

β-Chloro-
l
-alanine (CA) was found to inhibit the growth of
Salmonella typhimurium
. The inhibition was overcome by isoleucine plus valine. CA inhibited the activity of threonine deaminase and transaminase B in vitro. The inhibition of threonine deaminase was reversible and was not affected by desensitization to isoleucine inhibition. Transaminase B was irreversibly inactivated. Derepression of some of the isoleucine and valine biosynthetic enzymes was achieved by growth of wild-type cells on low levels of CA.
Temperature-Sensitive Mutants of
Escherichia coli
Affecting β-Galactoside Transport
Authors: Crandall Marjorie , Koch Arthur L.
Published: January 2020

Six different temperature-sensitive (
ts
) mutants have been isolated which have parental β-galactoside permease levels at low temperatures but have decreased permease levels when grown at high temperatures. These mutants were derived from
Escherichia coli
ML308 (
lacI
−
Y
+
Z
+
A
+
). After
N
-methyl-
N
′-nitro-
N
′-nitro-soguanidine mutagenesis, ampicillin was used to select for cells unable to grow on low lactose concentrations at 42 C. Temperature-sensitive mutants were assayed for galactoside permease activity after growth in casein hydrolysate medium at 25 or 42 C by measuring both radioactive methylthio-β-
d
-galactoside uptake and in vivo
o
-nitrophenyl-β-
d
-galactoside hydrolysis. The six conditional isolates have decreased levels of galactoside permease which are correlated with decreased growth rates at elevated temperatures. The low permease levels are not due to a temperature labile
lacY
gene product but rather to a temperature labile synthesis rate of functional permease. Some of the mutants exhibit a
ts
increase in permeability as shown by the increased leakage of intracellular β-galactosidase and by the increased rate of in vivo
o
-nitrophenyl-β-
d
-galactoside hydrolysis via the nonpermease mediated entry mechanism. Preliminary evidence indicates that transport in general is decreased in these mutants, yet there is some specificity in the mutational lesion since glucoside transport is unaffected. All these observations suggest that these mutants have
ts
alterations in membrane synthesis which results in pleiotropic effects on various membrane functions.
Carotenoids in Yellow-Pigmented Enterococci
Authors: Taylor Richard F. , Ikawa Miyoshi , Chesbro William
Published: January 2020

Pigments extracted from three strains of yellow enterococci showed the spectral and solvent partition characteristics of carotenoids. An unusual C
32
carotenoid aldehyde appears to predominate.
Synonymy of
Metschnikowia pulcherrima
and
Torulopsis burgeffiana
Authors: Miller M. W. , Johnson Ellen R. , Pitt J. I.
Published: January 2020

Torulopsis burgeffiana
is placed in the genus
Metschnikowia
because it has been shown to have a perfect stage corresponding with this genus. Due to the characteristic shape of the ascus and ascospores,
T. burgeffiana
is to be considered a synonym of
M. pulcherrima
. The physiological differences which exist between strains are not sufficiently important to maintain a separate species.
Transport of Aromatic Amino Acids by
Pseudomonas aeruginosa
Authors: Kay W. W. , Gronlund Audrey F.
Published: January 2020

Kinetic studies of the transport of aromatic amino acids by
Pseudomonas aeruginosa
revealed the existence of two high-affinity transport systems which recognized the three aromatic amino acids. From competition data and studies on the exchange of preformed aromatic amino acid pools, the first transport system was found to be functional with phenylalanine, tyrosine, and tryptophan (in order of decreasing activity), whereas the second system was active with tryptophan, phenylalanine, and tyrosine. The two systems also transported a number of aromatic amino acid analogues but not other amino acids. Mutants defective in each of the two and in both transport systems were isolated and described. When the amino acids were added at low external concentrations to cells growing logarithmically in glucose minimal medium, the tryptophan pool very quickly became saturated. Under identical conditions, phenylalanine and tyrosine each accumulated in the intracellular pool of
P. aeruginosa
at a concentration which was 10 times greater than that of tryptophan.
Mechanism of Excretion of a Bacterial Proteinase: Factors Controlling Accumulation of the Extracellular Proteinase of a
Sarcina
Strain (Coccus P)
Authors: Bissell Mina J. , Tosi Roberto , Gorini Luigi
Published: January 2020

It has been known that the extracellular proteinase of Coccus P is found only in cultures grown in the presence of Ca
2+
. It is now shown that this cation is required neither for synthesis, excretion, or activation of a zymogen nor as a prosthetic factor necessary for enzymatic activity. The only function of Ca
2+
is to stabilize the active structure of the enzyme molecule, presumably by substituting for absence of
S-S
bridges. In the absence of Ca
2+
, the excreted proteinase undergoes rapid autodigestion and, instead of the active protein, its hydrolytic products are accumulated in the culture fluid. In minimal medium and under conditions of enzyme stability [presence of Ca
2+
and Ficoll (Pharmacia)], Coccus P accumulates the proteinase at a gradually reduced speed although the rate of cultural growth remains constant. It is shown that this decline in rate of accumulation is caused by the excreted proteinase itself, possibly acting on its own precursor emerging from the cell in a form susceptible to proteolytic attack and not amenable to Ca
2+
protection. A proteinase precursor is actually demonstrable in a calciumless culture at the onset of the enzyme accumulation which follows Ca
2+
addition. It is suggested that excreted proteins require an unfolded (or incompletely folded) structure to cross the cell envelope.
Composition of the Sulfur Particle ofChromatium vinosumStrain D
Authors: Schmidt Gregory L. , Nicolson Garth L. , Kamen Martin D.
Published: January 2020

Sulfur particles were isolated from the purple sulfur photosynthetic bacterium,Chromatium vinosumstrain D. The composition of these particles was determined to be 93% sulfur, 5% protein, and 0.6% lipid. Gel electrophoresis indicated the presence of a single protein species with a molecular weight of 13,500 daltons. From these results, the sulfur particle is postulated to be bounded by a membrane consisting entirely of protein.
Repression of Diaminopimelate Decarboxylase by
l
-Lysine in Different
Bacillus
Species
Authors: Grandgenett Duane P. , Stahly Donald P.
Published: January 2020

Synthesis of diaminopimelate decarboxylase in 7 of 11 different
Bacillus
species tested was subject to
l
-lysine repression.
Studies on
Escherichia coli
Sex Factors: Evidence That Covalent Circles Exist Within Cells and the General Problem of Isolation of Covalent Circles
Authors: Freifelder David , Folkmanis Atis , Kirschner Ilana
Published: January 2020

We examined in detail conditions necessary for making reproducible and for maximizing the amount of deoxyribonucleic acid obtained from a sex factor-containing cell as covalent circles. The results argue that under optimal conditions covalent circles are neither created nor lost during the isolation procedure. The causes of the culture-to-culture variation in recovery of covalent circular deoxyribonucleic acid were investigated but an understanding of this is not yet at hand. Some commonly used conditions which drastically reduce the recovery of covalent circles are described.
Binding of Deoxyribonucleic Acid by Cell Walls of Transformable and Nontransformable Streptococci
Authors: Deddish Peter , Slade Hutton D.
Published: January 2020

Cell walls isolated from competent streptococci (group H strain Challis) were shown to bind more homologous and heterologous deoxyribonucleic acid (DNA) than noncompetent walls. Heat- and alkali-denatured DNA was not bound by either wall preparation. Pretreatment of cell walls with cetyltrimethylammonium bromide sharply increased the binding of DNA but did not increase transformation of whole cells. Pretreatment of the walls with either sodium dodecylsulfate, deoxyribonuclease and ribonuclease, or with crude competence-provoking factor did not affect the binding of DNA. Antiserum prepared against whole competent cells completely blocked transformation and also inhibited DNA binding to competent cell walls. Adsorption of this antiserum with competent Challis cells removed its blocking action for both binding and transformation. Pretreatment of walls with trypsin and Pronase destroyed their ability to bind DNA. Trypsin treatment also blocked transformation in whole cells. The transforming activity of DNA bound to cell walls was found to be protected from deoxyribonuclease action. Significant differences were observed in the arginine, proline, and phenylalanine content of competent and noncompetent walls. With few exceptions, the amino acids released from competent cell walls by trypsin were several-fold greater than from noncompetent walls. The results indicate that (i) two binding sites exist, one in competent cells only and essential for subsequent transformation, and a second, present in all cells, which is not involved in transformation; (ii) both sites are protein in nature; (iii) the transformation site is blocked by antibody; and (iv) the competent cell wall possesses tryptic-sensitive protein not present in the noncompetent wall.
Regulation of Phosphatidylcholine Biosynthesis in
Saccharomyces cerevisiae
Authors: Waechter Charles J. , Lester Robert L.
Published: January 2020

Evidence is presented which indicates that the biosynthesis of phosphatidylcholine by the methylation pathway in growing cultures of
Saccharomyces cerevisiae
is repressed by the presence of choline in the growth medium. This result, obtained previously for glucose-grown cells, was also observed for lactate-grown cells, of which half of the phosphatidylcholine is mitochondrial. A respiration-deficient mutant of the parent wild-type strain has been studied, and its inability to form functional mitochondria cannot be due to an impaired methylation pathway, as it has been shown to incorporate
14
C-CH
3
-methionine into all of the methylated glycerophosphatides. The incorporation rate is depressed by the inclusion of 1 m
m
choline in the growth medium, suggesting a regulatory effect similar to that demonstrated for the wild-type strain. The effects of choline on the glycerophospholipid composition of lactate and glucose-grown cells is presented. The repressive effects of the two related bases, mono- and dimethylethanolamine, were examined, and reduced levels of
14
C-CH
3
-methionine incorporation were found for cells grown in the presence of these bases. The effect of choline on the methylation rates is reversible and glucosegrown cells regain the nonrepressed level of methylation activity in 60 to 80 min after removal of choline from the growth medium.
Chromosome Transfer and Recombinant Formation with Deoxyribonucleic Acid Temperature-Sensitive Strains of
Escherichia coli
Authors: Stallions Donald R. , Curtiss Roy
Published: January 2020

Haploid recombinant yield is reduced in matings conducted at 42.5 C between deoxyribonucleic acid (DNA) temperature-sensitive [
dna
−
(TS)] recipients unable to synthesize DNA at 42.5 C and any of the three major donor types (Hfr, F
+
, F′) of
Escherichia coli
. No such reduction is observed in matings conducted at 42.5 C when the
dna
−
(TS) mutation is in the donor parent. Evidence is presented which indicates that chromosome transfer from donors to recipients unable to replicate DNA at 42.5 C during vegetative growth occurs at normal frequencies when the mating is conducted at 42.5 C. It is concluded that some stage in haploid recombinant formation is adversely affected in
dna
−
(TS) recipients mated at the temperature restrictive for DNA synthesis.
Saturated Fatty Acid Requirer of
Neurospora crassa
Authors: Henry Susan A. , Keith Alec D.
Published: January 2020

Dietary saturated fatty acids containing 12- to 18-carbon atoms satisfy growth requirements of
Neurospora crassa
mutant
cel
(previously named
ol
; Perkins et al., reference 11); unsaturated fatty acids are synthesized by direct desaturation when an appropriate saturate is available. Odd-chain saturates, 15 carbons and 17 carbons long, satisfy the requirement, and elaidic acid (18:1 Δ
9
trans
) results in slow growth. Oleic acid and other
cis
-unsaturated fatty acids do not satisfy growth requirements; however, oleic acid plus elaidic acid result in growth at a faster rate than elaidate alone. The use of a spin-label fatty acid reveals that hyphae produced by
cel
during a slow basal level of growth have lipids that reflect a relatively rigid state of viscosity compared to wild type.
cel
Supplemented with fatty acids and wild type supplemented in the same way have lipids of the same viscosities as reflected by electron spin resonance.
Effect of Temperature on the Fatty Acid Composition of
Thermus aquaticus
Authors: Ray Paul H. , White David C. , Brock Thomas D.
Published: January 2020

Thermus aquaticus
contains four major fatty acids, iso-C
15
(28%), iso-C
16
(9%), normal-C
16
(13%), and iso-C
17
(48%), when grown at 70 C, as determined by gas chromatography and mass spectrometry. Small amounts of iso-C
12
, normal-C
12:1
, iso-C
13
, normal-C
14
, iso-C
14
, and normal-C
15:1
were also detected. A change in growth temperature (50 to 75 C at 5-C intervals) affects a shift in the proportions of some of the fatty acids. The proportions of the monoenoic and branched-C
17
fatty acids decreased and the proportions of the higher-melting iso-C
16
and normal-C
16
fatty acids increased. Cells grown at 75 C contained 70% more total fatty acids than cells grown at 50 C. The largest increases, in absolute amounts, were in the content of iso-C
16
and normal-C
16
fatty acids, with only a 1.6-fold increase in the major iso-C
15
and iso-C
17
fatty acids. There was a 2.5-fold decrease in normal-C
15:1
and at least a 24-fold decrease in anteiso-C
17
, which is present at 50 and 55 C but not at higher temperatures. There was no difference in proportion or amount of fatty acids between exponential and stationary-phase cells grown at 70 C. When cells were grown on glutamate instead of yeast-extract and tryptone at 70 C, the total fatty acid content remained constant, but there was an increase in the proportions of iso-C
16
and normal-C
16
fatty acids concomitant with a decrease in the proportions of the iso-C
15
and iso-C
17
fatty acids.
Autolytic Activity Associated with Competent Group H Streptococci
Authors: Ranhand Jon M. , Leonard C. Gomez , Cole Roger M.
Published: January 2020

Competent cells of group H streptococci strains Wicky and Challis autolyzed markedly when placed at 37 C in 0.05mtris(hydroxymethyl)methyl-amino-propane sulfonic acid buffer (pH 9.0 to 9.1) containing 0.02m2-mercaptoethanol, whereas noncompetent cells autolyzed slightly. Autolysis of competent Wicky cells did not occur at 0 C or after the cells were heated at 100 C for 5 min. Culture fluids derived from strain Challis that contained competence factor (CF) activity did not contain lytic activity. Addition of native deoxyribonucleic acid (DNA) to competent Wicky cells caused a retardation in the rate of autolysis; ribonucleic acid and alkali-denatured DNA had less of an effect. Supernantant fluids derived from competent cell lysates lysed noncompetent Wicky cells but were inactive against cells ofHydrogenomonas eutropha, a group AStreptococcus, and against a commercial lysozyme substrate (Micrococcus lysodeikticus). This lytic activity was inactivated by heat (5 min at 100 C). Electron microscopic observations of autolyzed cells showed that autolysis occurs only at the site of cross-wall formation. A close relationship between the development of competence and autolysis is suggested by the fact that certain conditions that prevent the establishment of the competent state in Wicky populations (such as no CF, addition of CF simultaneously with chloramphenicol, and addition of trypsin-inactivated CF) also prevent autolysis. This observation emphasizes the indirect or inductive nature of CF on these processes.
Biosynthesis of the Iron-Transport Compound Enterochelin: Mutants of
Escherichia coli
Unable to Synthesize 2,3-Dihydroxybenzoate
Authors: Young I. G. , Langman L. , Luke R. K. J. , Gibson F.
Published: January 2020

Mutants of
Escherichia coli
K-12 blocked in each of the three enzymatic reactions between chorismate and 2,3-dihydroxybenzoate, in the pathway leading to the iron-sequestering compound enterochelin, have been isolated and biochemically characterized. The three genes concerned (designated
entA, entB
and
entC
) have been shown to be clustered on the chromosome between
purE
and
gal
and to be located near minute 14 by cotransduction with the
purE, lip
, and
fep
genes.
entA, entB
, and
entC
were shown to be the structural genes for 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, 2,3-dihydro-2,3-dihydroxybenzoate synthetase, and isochorismate synthetase, respectively.