bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of additional low-molecular-weight basic proteins degraded during germination of Bacillus megaterium spores
Authors: Setlow P
Published: January 2020

Dormant spores Bacillus megaterium contained a group of low-molecular-weight (5,000 to 11,000) basic (pI greater than 9.4) proteins (termed D, E, F, and G proteins) which could be extracted from disrupted spores with strong acids. These proteins were distinct from the previously described A, B, and C proteins which are degraded during spore germination. However, the D, E, F, and G proteins were also rapidly degraded during spore germination, accounting for 10 to 15% of the protein degraded. Proteins similar to the D, E, F, and G species were also present in spores of other bacterial species. In B. megaterium, the D, E, F, and G proteins were low or absent (less than 15% of the spore level) in vegetative and young sporulating cells and appeared only late in sporulation. The D, E, F, and G proteins were purified to homogeneity, and all contained a high percentage of hydrophilic amino acids; one protein (G) contained 31% basic amino acids and also contained tryptophan. All four proteins were rapidly degraded in vitro by dormant spore extracts. Two proteins (D and F) were degraded in vitro by the previously described spore protease which initiates degradation of the A, B, and C proteins in vivo; the spore enzyme (s) degrading proteins E and G have not been identified.
Iron in Neisseria meningitidis: minimum requirements, effects of limitation, and characteristics of uptake
Authors: Archibald F S , DeVoe I W
Published: January 2020

A simple defined medium (neisseria defined medium) was devised that does not require iron extraction to produce iron-limited growth of Neisseria meningitidis (SDIC). Comparison of this medium to Mueller-Hinton broth and agar showed nearly identical growth rates and yields. The defined medium was used in batch cultures to determine the disappearance of iron from the medium and its uptake by cells. To avoid a number of problems inherent in batch culture, continuous culture, in which iron and dissolved oxygen were varied independently, was used. Most of the cellular iron was found to be nonheme and associated with the particulate fraction in sonically disrupted cells. Nonheme and catalase-heme iron were reduced by iron starvation far more than cytochromes b and c and N,N,N',N'-tetramethylphenylenediamine-oxidase. The respiration rate and efficiency also decreased under iron limitation, whereas generation times increased. The iron-starved meningococcus took up iron by an energy-independent system operating in the first minute after an iron pulse and a slower energy-dependent system inhibited by respiratory poisons and an uncoupler. The energy-dependent system showed saturation kinetics and was stimulated nearly fourfold by iron privation. In addition, to determine the availability to the meningococcus of the iron in selected compounds, a sensitive assay was devised in which an iron-limited continuous culture was pulsed with the iron-containing compound.
Proline transport carrier-defective mutants of Escherichia coli K-12: properties and mapping
Authors: Motojima K , Yamato I , Anraku Y
Published: January 2020

A series of mutants of Escherichia coli K-12 requiring a high concentration of L-proline for growth were isolated from a proline auxotroph strain, JE2133. Genetic studies of the mutants, PT19, PT21, and PT22, showed that all the mutations (proT) were point mutations, and these were mapped at 82 min on the E. coli genetic map. Intact cells and cytoplasmic membrane vesicles of these mutants were specifically defective in L-proline transport activity. Strain PT21 had no detectable activity of the L-proline transport carrier at all, and strains PT19 and PT22 had only 1/35 and 1/70, respectively, of the transport activity of the parental strain. The mutants were also shown to have a defect in proline-binding function of the carrier by measuring specific binding of proline to sonically disrupted membranes. These results indicate that the gene proT determines the function of proline carrier in the cytoplasmic membrane.
Genetic mapping of tyramine oxidase and arylsulfatase genes and their regulation in intergeneric hybrids of enteric bacteria
Authors: Murooka Y , Higashiura T , Harada T
Published: January 2020

The genes for arylsulfatase (atsA) and tyramine oxidase (tynA) have been mapped in Klebsiella aerogenes by P1 transduction. They are linked to gdhD and trp in the order atsA-tynA-gdhD-trp-pyrF. Complementation analysis using F' episomes from Escherichia coli suggested an analogous location of these genes in E. coli, although arylsulfatase activity was not detected in E. coli. P1 phage and F' episomes were used to create intergeneric hybrid strains of enteric bacteria by transfer of the ats and tyn genes between K. aerogenes, E. coli, and Salmonella typhimurium. Intergeneric transduction of the tynK gene from K. aerogenes to an E. coli restrictionless strain was one to two orders less frequent than that of the leuK gene. The tyramine oxidase of E. coli and S. typhimurium in regulatory activity resemble very closely the enzyme of K. aerogenes. The atsE gene from E. coli was expressed, and latent arylsulfatase protein was formed in K. aerogenes and S typhimurium. The results of tyramine oxidase and arylsulfatase synthesis in intergeneric hybrids of enteric bacteria suggest that the system for regulation of enzyme synthesis is conserved more than the structure or function of enzyme protein during evolution.
Heat-modifiable outer membrane proteins of Neisseria meningitidis and their organization within the membrane
Authors: Frasch C E , Mocca L F
Published: January 2020

Neisseria meningitidis group B serotype 2 strain M986 contains two predominant outer membrane proteins, with apparent molecular weights of 41,000 (protein b) and 28,000 (protein e). Heating of outer membrane vesicles at 56 degrees C for 20 min caused much of b** to disaggregate and denature into b (41,000 daltons). In contrast, protein e could be rapidly solubilized by SDS at room temperature into its monomeric state (e*), but it was not converted to its final higher apparent molecular weight of 28,000 (e) unless heated at 100 degrees C for 2 min. We propose that protein b exists in the membrane as trimers or tetramers in a transmembrane configuration and that protein e exists as subunits on the exterior surface of the outer membrane and has a highly ordered tertiary structure.
Tumor-inducing (Ti) plasmids of Agrobacterium share extensive regions of DNA homology
Authors: Drummond M H , Chilton M D
Published: January 2020

Labeled Ti plasmid DNAs from diverse Agrobacterium strains were hybridized to Southern blots of pTi-B6-806 plasmid DNA digest fragments of known map order. The map location of DNA sequences common to all Ti plasmids was found to be extensive, consistent with the view that Ti plasmids have evolved from a common ancestral plasmid.
Energy Supply for Active Transport in Anaerobically Grown
Escherichia coli
Authors: Boonstra Johannes , Downie J. Allan , Konings Wil N.
Published: January 2020

Escherichia coli
K-12, grown under anaerobic conditions with glucose as the sole source of carbon and energy without any terminal electron acceptor added, contains a fumarate reductase system in which electrons are transferred from formate or reduced nicotinamide adenine dinucleotide via menaquinone and cytochromes to fumarate reductase. This fumarate reductase system plays an important role in the metabolic energy supply of
E. coli
, grown under so-called “glycolytic conditions,” as is indicated by the growth yields and maximal growth rates of mutants impaired in electron transfer or adenosine triphosphatase (
uncB
). In mutants deficient in menaquinone, cytochromes, or fumarate reductase, these values are considerably lower than in mutants deficient in ubiquinone or a functional adenosine triphosphatase. Electron transfer in this fumarate reductase system leads to the generation of a membrane potential, as is indicated by the uptake of the lipophilic cation triphenylmethylphosphonium by membrane vesicles prepared from cytochrome-sufficient and
uncB
cells. The generation of a proton-motive force by the fumarate reductase system was also demonstrated by the uptake of amino acids under anaerobic conditions in membrane vesicles of cytochrome containing and
uncB
cells grown under glycolytic conditions. Membrane vesicles of cytochrome-deficient cells failed to accumulate triphenyl-methylphosphonium and amino acids under these conditions, indicating that cytochromes are essential for the generation of a proton-motive force. Using glutamine uptake as an indication of the generation of ATP and proline uptake as an indication of the generation of a proton-motive force, it was demonstrated in whole cells that the proton-motive force is formed by ATP hydrolysis in cytochrome-deficient cells and by electron transfer in the
uncB
cells. In cytochrome-containing cells it was not possible to distinguish between these two possibilities, but the growth parameters suggest that, under glycolytic conditions, the proton-motive force is generated via electron transfer in the fumarate reductase system rather than via ATP hydrolysis.
Dissimilatory reduction of bisulfite by Desulfovibrio vulgaris
Authors: Drake H L , Akagi J M
Published: January 2020

The reduction of bisulfite by Desulfovibrio vulgaris was investigated. Crude extracts reduced bisulfite to sulfide without the formation (detection) of any intermediates such as trithionate or thiosulfate. When the particulate fractions was removed from crude extracts by high-speed centrifugation, the soluble supernatant fraction reduced bisulfite sequentially to trithionate, thiosulfate, and sulfide. Addition of particles or purified membranes to the soluble fraction restored the original activity demonstrated by crude extracts, i.e., reduction of bisulfite to sulfide without the formation of trithionate and/or thiosulfate. By using antiserum directed against bisulfite reductase, the reduction of bisulfite by crude extracts was inhibited. This finding, in addition to several recycling studies of thiosulfate reduction, provided evidence that bisulfite reduction by D. vulgaris operated through the pathway involving trithionate and thiosulfate as intermediates. The role of membranes in this process is discussed.
Regulation of
lac
Operon Expression: Reappraisal of the Theory of Catabolite Repression
Authors: Wanner Barry L. , Kodaira Ryoji , Neidhardt Frederick C.
Published: January 2020

The physiological state of
Escherichia coli
with respect to (permanent) catabolite repression was assessed by measuring the steady-state level of β-galactosidase in induced or in constitutive cells under a variety of growth conditions. Four results were obtained. (i) Catabolite repression had a major effect on fully induced or constitutive expression of the
lac
gene, and the magnitude of this effect was found to be dependent on the promoter structure; cells with a wild-type
lac
promoter showed an 18-fold variation in
lac
expression, and cells with the
lacP37
(formerly
lac-L37
) promoter exhibited several hundred-fold variation. (ii) Exogenous adenosine cyclic 3′,5′-monophosphoric acid (cAMP) could not abolish catabolite repression, even though several controls demonstrated that cAMP was entering the cells in significant amounts. (Rapid intracellular degradation of cAMP could not be ruled out.) (iii) Neither the growth rate nor the presence of biosynthetic products altered the degree of catabolite repression; all variation could be related to the catabolites present in the growth medium. (iv) Slowing by imposing an amino acid restriction decreased the differential rate of β-galactosidase synthesis from the wild-type
lac
promoter when bacteria were cultured in either the absence or presence of cAMP; this decreased
lac
expression also occurred when the bacteria harbored the catabolite-insensitive
lacP5
(formerly
lacUV5
) promoter mutation. These findings support the idea that (permanent) catabolite repression is set by the catabolites in the growth medium and may not be related to an imbalance between catabolism and anabolism.
Inactivation of the chemoattractant folic acid by cellular slime molds and identification of the reaction product
Authors: Pan P , Wurster B
Published: January 2020

Enzymes inactivating the chemoattractants folic acid and pterin were detected in extracellular, intracellular, and particulate fractions obtained from Dictyostelium discoideum strains NC4 and AX-2 and Polysphondylium violaceum. The products of the inactivation reaction were analyzed by thin-layer chromatography and UV spectroscopy. Results obtained indicate that folic acid and pterin were deaminated to 2-deamino folic acid and lumazine, respectively.
Further Characterization of the Reduced Nicotinamide Adenine Dinucleotide Phosphate:Nitrate Oxidoreductase in
Aspergillus nidulans
Authors: Downey Ronald J. , Steiner Francis X.
Published: January 2020

The reduced nicotinamide adenine dinucleotide phosphate (NADPH):nitrate oxidoreductase (EC 1.6.6.2) from
Aspergillus nidulans
wild-type
bi
-1 was purified by means of salt fractionation, gel filtration, affinity chromatography, and polyacrylamide gel electrophoresis. Enzyme which was adsorbed on Cibacron blue agarose could be eluted with 2 mM NADPH or 2 mM oxidized NADP (NADP
+
), the former being about three times more effective than the latter. About half the total NADPH:nitrate reductase activity adsorbed on agarose required elution with 1 M NaCl. This salt-elutable form remained active with NADPH and was not converted to the NADPH-elutable form after readsorption on Cibacron blue agarose. The NADPH-eluted enzyme exhibited a markedly different electrophoretic mobility than the enzyme eluted with NADP
+
or NaCl. After electrophoresis on polyacrylamide gels, the NADPH-eluted NADPH:nitrate reductase was separated into four proteins, two of which contained nonheme iron and exhibited reduced methyl viologen-nitrate reductase activity. None of these proteins, singly or in combination, reduced nitrate with NADPH as substrate. Difference spectra analyses and specific heme iron stains revealed the presence of cytochrome
b
557
in the largest of the proteins. The molecular weights of the four proteins, which were determined from the relationship of their mobilities on varied concentrations of acrylamide gel, were 360,000, 300,000, 240,000, and 118,000. The subunit molecular weights of these, which are determined via sodium dodecyl sulfate slab gel electrophoresis, were 49,000, 50,000, and 75,000. The key role of NADPH in maintenance of the active form of the heteromultimer is further substantiated.
Intracellular cyclic GMP-binding proteins in cellular slime molds
Authors: Mato J M , Woelders H , Konijn T M
Published: January 2020

The complexity of cyclic GMP-binding activity in the 48,000 X g supernatant of three species of the cellular slime molds (Dictyostelium discoideum, Dictyostelium rosarium, and Polysphondylium violaceum) was studied by gel filtration chromatography on AcA 34 Ultrogel. All these species have in common a cyclic GMP-binding protein of molecular weight of about 2.5 X 10(5) which specifically binds this nucleotide. In addition, Scatchard plots of assays carried out with the 48,000 X g supernatant of these species exhibit cyclic GMP-binding activity with an apparent dissociation constant of about 1 nM. None of the cyclic GMP-binding proteins separated by chromatography on AcA 34 Ultrogel was associated to protein kinase activity stimulation. In view of the cyclic GMP function during chemotactic transduction in the cellular slime molds, the possible molecular function for this 2.5 X 10(5)-dalton cyclic GMP-binding protein is discussed.
Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae
Authors: Klein H P , Jahnke L
Published: January 2020

A method is shown to be effective over a wide range of enzyme ratios for the simultaneous detection of the two isoenzymes of acetyl coenzyme A synthetase [acetate:coenzyme A ligase (AMP-forming); EC 6.2.1.1] in homogenates and cellular fractions of Saccharomyces cerevisiae. When this method was used, it was found that cells grown under anaerobic conditions contained only one variety of this enzyme, designated the nonaerobic synthetase, whereas cells grown with vigorous aeration contained principally the other, aerobic, synthetase. In cells grown as standing cultures (i.e., semi-aerobically), both enzymes were present and were found mainly in the extramitochondrial material of homogenates. When anaerobic cultures were aerated, the amount of aerobic enzyme increased steadily over a 24-h period, so that at the end of this time, aerated cells contained predominantly aerobic enzyme. During this same period, the amount of nonaerobic enzyme decreased. The percentage of aerobic enzyme that sedimented with the mitochondria increased steadily during this period of aeration, so that, at the end of 24 h of aeration, essentially all of the aerobic enzyme sedimented with the mitochondria. The nonaerobic enzyme was never found in this cellular compartment.
Tumor Inhibitory and Non-Tumor Inhibitory
l
-Asparaginases from
Pseudomonas geniculata
Authors: Kitto G. Barrie , Smith Gale , Thiet Tran-Quang , Mason Mark , Davidson Lois
Published: January 2020

Two enzymes that catalyze the hydrolysis of
l
-asparagine have been isolated from extracts of
Pseudomonas geniculata
. After initial salt fractionation, the enzymes were separated by chromatography on diethylaminoethyl-Sephadex and purified to homogeneity by gel filtration, ion-exchange chromatography, and preparative polyacrylamide electrophoresis. The enzymes differ markedly in physicochemical properties. One enzyme, termed asparaginase A, has a molecular weight of approximately 96,000 whereas the other, termed asparaginase AG, has a molecular weight of approximately 135,000. Both enzymes are tetrameric. The asparaginase A shows activity only with
l
-asparagine as substrate, whereas the asparaginase AG hydrolyzes
l
-asparagine and
l
-glutamine at approximately equal rates and it is also active with
d
-asparagine and
d
-glutamine as substrates. The asparaginase A was found to be devoid of antitumor activity in mice, whereas the asparaginase AG was effective in increasing the mean survival times of both C3H mice carrying the asparagine-requiring Gardner 6C3HED tumor line and Swiss mice bearing the glutamine-requiring Ehrlich ascites tumor line. These differences in antitumor activity were related to differences in the

K
m

values for
l
-asparagine for the two enzymes. The asparaginase A has a

K
m

value of 1 × 10
−3
M for this substrate whereas the corresponding value for the AG enzyme is 1.5 × 10
−5
M. Thus the concentration of asparagine necessary for maximal activity of the asparaginase A is very high compared with that of the normal plasma level of asparagine, which is approximately 50 μM.
Direct determination of the properties of peptide transport systems in Escherichia coli, using a fluorescent-labeling procedure
Authors: Payne J W , Bell G
Published: January 2020

A direct study of peptide uptake by Escherichia coli was made using a fluorescent procedure. After incubation with the bacteria, peptides remaining in the medium were dansylated, separated chromatographically, and quantitated from their fluorescent intensities and/or from their incorporated radioactivity when tritiated dansyl derivatives were prepared. Peptide uptake was apparently not regulated and proceeded continuously until complete, with the absorbed peptides undergoing rapid intracellular hydrolysis and the excess amino acid residues leaving the cell. Thus, peptide uptake and amino acid exodus occur concurrently. However, peptidase-resistant substrates, e.g. triornithine and glycylsarcosine, which can be similarly estimated in cell extracts, were accumulated about 1,000-fold. The influence of amino acid composition and chain length on rates of transport was assessed. Different strains of E. coli showed variability in their rates of di- and oligopeptide transport. With respect to energy coupling, both the di- and oligopeptide permeases behaved like shock-sensitive transport systems.
Homology of the Gene Coding for Outer Membrane Lipoprotein Within Various Gram-Negative Bacteria
Authors: Nakamura Kenzo , Pirtle Robert M. , Inouye Masayori
Published: January 2020

The mRNA for a major outer membrane lipoprotein from
Escherichia coli
was found to hybridize specifically with one of the
Eco
RI and one of the
Hin
dIII restriction endonuclease-generated fragments of total DNA from nine bacteria in the family
Enterobacteriaceae: E. coli, Shigella dysenteriae, Salmonella typhimurium, Citrobacter freundii, Klebsiella aerogenes, Enterobacter aerogenes, Edwardsiella tarda, Serratia marcescens
, and
Erwinia amylovora
. However, among the
Enterobacteriaceae
, DNA from two species of
Proteus
(
P. mirabilis
and
P. morganii
) did not contain any restriction endonuclease fragments that hybridized with the
E. coli
lipoprotein mRNA. Furthermore, no hybrid bands were detected in four other gram-negative bacteria outside the family
Enterobacteriaceae: Pseudomonas aeruginosa, Acinetobacter
sp. HO1-N,
Caulobacter crescentus
, and
Myxococcus xanthus
. Envelope fractions from all bacteria in the family
Enterobacteriaceae
tested above cross-reacted with antiserum against the purified
E. coli
free-form lipoprotein in the Ouchterlony immunodiffusion test. Both species of
Proteus
, however, gave considerably weaker precipitation lines, in comparison with the intense lines produced by the other members of the family. All of the above four bacteria outside the family
Enterobacteriaceae
did not cross-react with anti-
E. coli
lipoprotein serum. From these results, the rate of evolutionary changes in the lipoprotein gene seems to be closely related to that observed for various soluble enzymes of the
Enterobacteriaceae
.
Derepression of beta-1,3-glucanases in Penicillium italicum: localization of the various enzymes and correlation with cell wall glucan mobilization and autolysis
Authors: Santos T , Sánchez M , Villanueva J R , Nombela C
Published: January 2020

The localization of the derepressible beta-1,3-glucanases of Penicillium italicum and the cell wall autolysis under conditions of beta-1,3-glucanase derepression (24 h in a low-glucose medium) were studied. About 15% of the total activity was secreted into the culture medium during the 24-h period and consisted of similar amounts of each of the three beta-1,3-glucanases (I, II, III) produced by this species. Treatment of derepressed mycelia with periplasmic enzyme-inactivating agents resulted in a loss of 45% of the mycelium-bound beta-1,3-glucanase. Analysis of periplasmic enzymes solubilized by 2 M NaCl or by autolysis of isolated cell walls revealed that only beta-1,3-glucanases II and III were bound to the cell wall. These two enzymes were capable of releasing in vitro reducing sugars from cell walls, whereas beta-1,3-glucanase I was not. In addition, the autolytic activity of cell walls isolated from derepressed mycelium was greater than that of cell walls isolated from repressed mycelium. The incubation of the fungus in the low-glucose medium also resulted in the in vivo mobilization of 34% of the cell wall beta-1,3-glucan, and this mobilization was fully prevented by cycloheximide, which also blocked derepression of beta-1,3-glucanases. Derepression of beta-1,3-glucanase seems to be coupled to the mobilization of cell wall glucan.
Sodium-dependent transport of L-leucine in membrane vesicles prepared from Pseudomonas aeruginosa
Authors: Hoshino T , Kageyama M
Published: January 2020

Membrane vesicles were prepared by osmotic lysis of spheroplasts of Pseudomonas aeruginosa strain P14, and the active transport of amino acids was studied. D-Glucose, gluconate, and L-malate supported active transport of various L-amino acids. The respiration-dependent leucine transport was markedly stimulated by Na+. Moreover, without any respiratory substrate, leucine was also transported transiently by the addition of Na+ alone. This transient uptake of leucine was not inhibited either by carbonyl cyanide p-trifluoromethyoxyphenylhydrazone or by valinomycin, but was completely abolished by gramicidin D. Increase in the concentration of Na+ of the medium resulted in a decrease of the Km for L-leucine transport, whereas the Vmax was not significnatly affected. Active transport of leucine was inhibited competitively by isoleucine or by valine, whose transport was also stimulated by Na+. On the other hand, Na+ was not required for the uptake of other L-amino acids tested, but rather was inhibitory for some of them. These results show (i) that a common transport system for branched-chain amino acids exists in membrane vesicles, (ii) that the system requires Na+ for its activity, and (iii) that an Na+ gradient can drive the system.
The relA locus specifies a positive effector in branched-chain amino acid transport regulation
Authors: Quay S C , Oxender D L
Published: January 2020

The regulation of branched-chain amino acid transport and periplasmic binding proteins was studied in Escherichia coli strains which were isogenic except for the relA locus, the gene for the "stringent factor," which is responsible for guanosine tetraphosphate synthesis. The strain containing the relA mutation could not be derepressed for the synthesis of leucine transport or binding proteins when shifted from a medium containing all 20 amino acids in excess to one in which leucine was limiting. The relA+ strain showed normal derepression under these conditions.
In Vitro Incorporation of Molybdate into Demolybdoproteins in
Escherichia coli
Authors: Scott Randolph H. , Sperl George T. , DeMoss John A.
Published: January 2020

When
Escherichia coli
was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [
99
Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated
99
Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate,
99
Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of
99
Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [
99
Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.
Two Forms of Nitrogenase from the Photosynthetic Bacterium
Rhodospirillum rubrum
Authors: Carithers Robert P. , Yoch Duane C. , Arnon Daniel I.
Published: January 2020

Acetylene reduction by nitrogenase from
Rhodospirillum rubrum
, unlike that by other nitrogenases, was recently found by other investigators to require an activation of the iron protein of nitrogenase by an activating system comprising a chromatophore membrane component, adenosine 5′-triphosphate (ATP), and divalent metal ions. In an extension of this work, we observed that the same activating system was also required for nitrogenase-linked H
2
evolution. However, we found that, depending on their nitrogen nutrition regime,
R. rubrum
cells produced two forms of nitrogenase that differed in their Fe protein components. Cells whose nitrogen supply was totally exhausted before harvest yielded predominantly a form of nitrogenase (A) whose enzymatic activity was not governed by the activating system, whereas cells supplied up to harvest time with N
2
or glutamate yielded predominantly a form of nitrogenase (R) whose enzymatic activity was regulated by the activating system. An unexpected finding was the rapid (less than 10 min in some cases) intracellular conversion of nitrogenase A to nitrogenase R brought about by the addition to nitrogen-starved cells of glutamine, asparagine, or, particularly, ammonia. This finding suggests that mechanisms other than de novo protein synthesis were involved in the conversion of nitrogenase A to the R form. The molecular weights of the Fe protein and Mo-Fe protein components from nitrogenases A and R were the same. However, nitrogenase A appeared to be larger in size, because it had more Fe protein units per Mo-Fe protein than did nitrogenase R. A distinguishing property of the Fe protein from nitrogenase R was its ATP requirement. When combined with the Mo-Fe protein (from either nitrogenase A or nitrogenase R), the R form of Fe protein required a lower ATP concentration but bound or utilized more ATP molecules during acetylene reduction than did the A form of Fe protein. No differences between the Fe proteins from the two forms of nitrogenase were found in the electron paramagnetic resonance spectrum, midpoint oxidation-reduction potential, or sensitivity to iron chelators.
Influence of the Lactose Plasmid on the Metabolism of Galactose by
Streptococcus lactis
Authors: LeBlanc Donald J. , Crow Vaughan L. , Lee Linda N. , Garon Claude F.
Published: January 2020

Streptococcus lactis
strain DR1251 was capable of growth on lactose and galactose with generation times, at 30°C, of 42 and 52 min, respectively. Phosphoenolpyruvate-dependent phosphotransferase activity for lactose and galactose was induced during growth on either substrate. This activity had an apparent

K
m

of 5 × 10
−5
M for lactose and 2 × 10
−2
M for galactose. β-
d
-Phosphogalactoside galactohydrolase activity was synthesized constitutively by these cells. Strain DR1251 lost the ability to grow on lactose at a high frequency when incubated at 37°C with glucose as the growth substrate. Loss of ability to metabolize lactose was accompanied by the loss of a 32-megadalton plasmid, pDR
1
, and Lac
−
isolates did not revert to a Lac
+
phenotype. Lac
−
strains were able to grow on galactose but with a longer generation time. Galactose-grown Lac
−
strains were deficient in β-
d
-phosphogalactoside galactohydrolase activity and phosphoenolpyruvate phosphotransferase activity for both lactose and galactose. There was also a shift from a predominantly homolactic to a heterolactic fermentation and a fivefold increase in galactokinase activity, relative to the Lac
+
parent strain grown on galactose. These results suggest that
S. lactis
strain DR1251 metabolizes galactose primarily via the tagatose-6-phosphate pathway, using a lactose phosphoenolpyruvate phosphotransferase activity to transport this substrate into the cell. Lac
−
derivatives of strain DR1251, deficient in the lactose phosphoenolpyruvate phosphotransferase activity, appeared to utilize galactose via the Leloir pathway.
Enzymes of Pyrimidine Metabolism in
Mycoplasma mycoides
subsp.
mycoides
Authors: Mitchell Alana , Finch Lloyd R.
Published: January 2020

The major pathways of ribonucleotide biosynthesis in
Mycoplasma mycoides
subsp.
mycoides
have been proposed from studies on its use of radioactive purines and pyrimidines. To interpret more fully the observed pattern of pyrimidine usage, cell extracts of this organism have been assayed for several enzymes associated with the salvage synthesis of pyrimidine nucleotides.
M. mycoides
possessed uracil phosphoribosyltransferase, uridine phosphorylase, uridine (cytidine) kinase, uridine 5′-monophosphate kinase, and cytidine 5′-triphosphate synthetase. No activity for phosphorolysis of cytidine was detected, and no in vitro conditions were found to give measurable deamination of cytidine. Of the two potential pathways for incorporation of uridine, our data suggest that this precursor would largely undergo initial phosphorolysis to uracil and ribose-1-phosphate. Conversely, cytidine is phosphorylated directly to cytidine 5′-monophosphate in its major utilization, although conversion of cytidine to uracil, uridine, and uridine nucleotide has been observed in vivo, at least when uracil is provided in the growth medium. Measurements of intracellular nucleotide contents and their changes on additions of pyrimidine precursors have allowed suggestions as to the operation of regulatory mechanisms on pyrimidine nucleotide biosynthesis in
M. mycoides
in vivo. With uracil alone or uracil plus uridine as precursors of pyrimidine ribonucleotides, the regulation of uracil phosphoribosyltransferase and cytidine 5′-triphosphate synthetase is probably most important in determining the rate of pyrimidine nucleotide synthesis. When cytidine supplements uracil in the growth medium, control of cytidine kinase activity would also be important in this regard.
Enzymatic deacylation of lipoteichoic acid by protoplasts of Streptococcus faecium (Streptococcus faecalis ATCC 9790)
Authors: Kessler R E , Shockman G D
Published: January 2020

High-molecular-weight, micellar lipoteichoic acid (LTA) was converted to a lower-molecular-weight, apparently deacylated polymer when the former was incubated in the presence of growing protoplasts of Streptococcus faecium (S. faecalis ATCC 9790), but not when incubated in fresh or spent protoplast medium. The mobility of the low-molecular-weight polymer upon agarose gel electrophoresis was indistinguishable from that of native extracellular lipoteichoic acid LTA(X) from this organism or from chemically deacylated LTA. Native LTA(X) was shown to contain less than one fatty acid equivalent per 18 LTA(X) molecules, in contrast to the 4:1 ratio of fatty acids to polyglycerolphosphate chains in micellar LTA.
Chloramphenicol resistance mutation in Escherichia coli which maps in the major ribosomal protein gene cluster
Authors: Baughman G A , Fahnestock S R
Published: January 2020

Localized mutagenesis and selection for streptomycin resistance were utilized to isolate a chloramphenicol resistance mutation in Escherichia coli K-12 linked to the strA (rpsL) locus. Bacteriophage P1 transduction verified the map position of the new resistance mutation at 72 min, placing it within a dense cluster of ribosomal protein genes. The map position differs from that of known cmlA and cmlB mutations, which map at 18 and 21 min, respectively. Ribosomes prepared from chloramphenicol-resistant and -sensitive isogenic transductants were analyzed in vitro for activity in formation of N-formylmethionyl-puromycin, polyphenylalanine, and polylysine in the presence of inhibitory concentrations of chloramphenicol. Comparisons were also made of 14C-chloramphenicol binding to 70S ribosomes and of the two-dimensional polyacrylamide gel electrophoresis pattern of ribosomal proteins from each strain. There was no detectable difference between ribosomes from sensitive and resistant strains as measured by these assays. Enzymatic modification by chloramphenicol acetyltransferase is not responsible for the observed phenotype.
23S ribosomal ribonucleic acid of macrolide-producing streptomycetes contains methylated adenine
Authors: Graham M Y , Weisblum B
Published: January 2020

Coresistance to macrolide, lincosamide, and streptogramin B-type (MLS) antibiotics by a common biochemical mechanism characterizes clinically resistant pathogens. Of 10 streptomycetes tested for resistance to macrolide, lincosamide, and streptogramin B-type antibiotics, only 1, Streptomyces erythreus, the organism used for production of erythromycin, was found resistant to all three classes; moreover, it was the only streptomycete in the series tested found to contain N6-dimethyladenine (m62A) in 23S ribosomal ribonucleic acid, the structural alteration of ribosomal ribonucleic acid associated with clinical resistance. Of the seven streptomycetes tested for the presence of m62A and N6-methyladenine (m6A), two, S. fradiae and S. cirratus, which produce the macrolide antibiotics tylosin and cirramycin, respectively, were found to contain m6A, but not m62A. The remaining strains tested, including strains which produce lincomycin and streptogramins, contained neither m6A nor m62A.
Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli
Authors: Yamato I , Ohki M , Anraku Y
Published: January 2020

Mutants of Escherichia coli K-12 requiring high concentrations of branched-chain amino acids for growth were isolated. One of the mutants was shown to be defective in transport activity for branched-chain amino acids. The locus of the mutation (hrbA) was mapped at 8.9 min on the E. coli genetic map by conjugational and transductional crosses. The gene order of this region is proC-hrbA-tsx. The hrbA system was responsible for the uptake activity of cytoplasmic membrane vesicles. It was not repressed by leucine. The substrate specificities and kinetics of the uptake activities were studied using cytoplasmic membrane vesicles and intact cells of the mutants grown in the presence or absence of leucine. Results showed that there are three transport systems for branched-chain amino acids, LIV-1, -2, and -3. The LIV-2 and -3 transport systems are low-affinity systems, the activities of which are detectable in cytoplasmic membrane vesicles. The systems are inhibited by norleucine but not by threonine. The LIV-2 system is also repressed by leucine. The LIV-1 transport system is a high-affinity system that is sensitive to osmotic shock. When the leucine-isoleucine-valine-threonine-binding protein is derepressed, the high-affinity system can be inhibited by threonine.
Relationship between Deoxyribonucleoside Triphosphate Pools and Deoxyribonucleic Acid Synthesis in an
nrdA
Mutant of
Escherichia coli
Authors: Manwaring John D. , Fuchs James A.
Published: January 2020

A shift to 42°C in an
nrdA
mutant causes a decrease in deoxyribonucleic acid synthesis without a concomitant decrease in deoxynucleotide triphosphate pools.
Catabolite and transient repression in Escherichia coli do not require enzyme I of the phosphotransferase system
Authors: Yang J K , Bloom R W , Epstein W
Published: January 2020

Transient and catabolite repression with changes in intracellular concentrations of cyclic adenosine 3',5-monophosphate is produced by glycerol and by glucose-6-phosphate in a strain with a partial deletion of the structural gene for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.
Receptor for bacteriophage lambda of Escherichia coli forms larger pores in black lipid membranes than the matrix protein (porin)
Authors: Boehler-Kohler B A , Boos W , Dieterle R , Benz R
Published: January 2020

The receptor for phage lambda in Escherichia coli was isolated by cholate extraction and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands corresponding to the monomer and the dimer were eluted from the gel and tested for their activity to inactivate phage lambda and to form pores in black lipid membranes. It was found that only the dimer inactivated phage lambda, whereas both the monomer and the dimer were active in forming pores. The pore characteristics were similar to those exhibited by the matrix protein (porin) (R. Benz, K. Janko, W. Boos, and P. Läuger, Biochim. Biophys. Acta 511:305--319, 1978). In comparison, the lambda receptor showed a somewhat higher degree of cation specificity, and its pore size was larger. Assuming that the thickness of the outer membrane is 7.5 nm and that the pore is an ideal hydrophilic channel, the pore diameter in vivo was estimated to be 1.6 nm for the lambda receptor and 1.2 nm for the matrix protein.
Induction and catabolite repression of L-rhamnose dehydrogenase in Pullularia pullulans
Authors: Vieira M M , Rigo L U , Maréchal L R , Veiga L A
Published: January 2020

The growth of Pullularia pullulans on L-rhamnose (6-deoxy-L-mannose) as the sole carbon source induces the synthesis of L-rhamnose dehydrogenase, a nicotinamide adenine dinucleotide-dependent enzyme that catalyzes the oxidation of the deoxy sugar to L-rhamnonolactone. The enzyme induction is inhibited by cycloheximide, suggesting de novo synthesis. The presence of d-glucose (0.2%) or D-galactose (0.2%) simultaneously with the inducer in the induction medium produced 50% repression of dehydrogenase synthesis, but no effect was detected with D-fructose and D-mannose at the same concentration. High levels of D-glucose (2%), under maximal catabolite repression conditions, produced a complete inhibition of enzyme synthesis.
Export of extracellular levansucrase by Bacillus subtilis: inhibition by cerulenin and quinacrine
Authors: Caulfield M P , Berkeley R C , Pepper E A , Melling J
Published: January 2020

Bacillus subtilis B secretes an inducible, extracellular enzyme, levansucrase. Inhibition studies were undertaken to investigate the possible mechanism of release of this enzyme. The antibiotic cerulenin, at a concentration of 10 micrograms/ml, totally inhibited de novo lipid synthesis in B. subtilis B for at least 1 h, while only slightly reducing protein and RNA synthesis. At this concentration cerulenin, added concomitantly with the inducer sucrose, prevented the release of levansucrase for at least 150 min. This was not due to the prevention of inducer uptake by the cells. The release of the enzyme was also independent of cell division. In B. subtilis 1007 the induction of beta-galactosidase by 5 mM lactose was not prevented by cerulenin. Preliminary evidence indicated the association of a lipid moiety with the enzyme as it passes through the cytoplasmic membrane. Quinacrine (0.2 mM), which inhibits the penicillinase-releasing protease of Bacillus licheniformis, inhibited levansucrase release from B. subtilis B, but had no effect on lipid synthesis.
Control of cyclic adenosine 3',5'-monophosphate levels by depolarizing agents in fungi
Authors: Trevillyan J M , Pall M L
Published: January 2020

It has been reported that diverse treatments which depolarize the plasma membrane of Neurospora crassa produce rapid increases in cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels. In the current study, membrane active antibiotics, which are known or putative depolarizing agents, were found to produce similar cyclic AMP increases, not only in N. crassa, but also in the distantly related fungi Saccharomyces cerevisiae and Mucor racemosus. Uncouplers of oxidative phosphorylation, which have been found to depolarize Neurospora, also produced cyclic AMP increases in all three fungi. The time course of the cyclic AMP response to these various treatments was similar in all three fungi. The fungal studies and studies on depolarized central nervous tissue suggest that cyclic AMP increases may be produced in response to plasma membrane depolarization in diverse eucaryotic cells. A model is proposed for eucaryotic microorganisms in which membrane depolarization serves as a signal of breakdown of the plasma membrane integrity. The subsequent cyclic AMP increase, in turn, may mediate cellular response to help protect the plasma membrane from chemical and mechanical threats to its integrity.
Ionizing Radiation Damage to the Folded Chromosome of
Escherichia coli
K-12: Repair of Double-Strand Breaks in Deoxyribonucleic Acid
Authors: Ulmer Kevin M. , Gomez Reinaldo F. , Sinskey Anthony J.
Published: January 2020

The extremely gentle lysis and unfolding procedures that have been developed for the isolation of nucleoid deoxyribonucleic acid (DNA; K. M. Ulmer et al., J. Bacteriol.
138:
475–485, 1979) yield undamaged, replicating genomes, thus permitting direct measurement of the formation and repair of DNA double-strand breaks at biologically significant doses of ionizing radiation. Repair of ionizing radiation damage to folded chromosomes of
Escherichia coli
K-12 strain AB2497 was observed within 2 to 3 h of post-irradiation incubation in growth medium. Such behavior was not observed after post-irradiation incubation in growth medium of a
recA13
strain (strain AB2487). A model based on recombinational repair is proposed to explain the formation of 2,200 to 2,300S material during early stages of incubation and to explain subsequent changes in the gradient profiles. Association of unrepaired DNA with the plasma membrane is proposed to explain the formation of a peak of rapidly sedimenting material (greater than 3,100S) during the later stages of repair. Direct evidence of repair of double-strand breaks during post-irradiation incubation in growth medium was obtained from gradient profiles of DNA from ribonuclease-digested chromosomes. The sedimentation coefficient of broken molecules was restored to the value of unirradiated DNA after 2 to 3 h of incubation, and the fraction of the DNA repaired in this fashion was equal to the fraction of cells that survived at the same dose. An average of 2.7 double-strand breaks per genome per lethal event was observed, suggesting that one to two double-strand breaks per genome are repairable in
E. coli
K-12 strain AB2497.
Transformation-derived Neisseria gonorrhoeae plasmids with altered structure and function
Authors: Sox T E , Mohammed W , Sparling P F
Published: January 2020

Plasmid deoxyribonucleic acid from Neisseria gonorrhoeae containing a 7.1-kilobase (kb) (4.7-megadalton) penicillinase (Pcr) plasmid transformed homogenic gonococci to penicillinase production at a low frequency. About 25% of the penicillinase-producing gonococcal transformants contained Pcr plasmids which were either larger or smaller than the 7.1 kb donor plasmid; these Pcr plasmids varied in size from 3.45 to 42 kb. Some of these altered plasmids differed from the donor plasmid in stability or in frequency of mobilization by a 36-kb (24-megadalton) conjugative plasmid. A restriction endonuclease cleavage map of the 7.1-kilobase Pcr plasmid and several of the smaller deleted plasmids was constructed. The most common size of altered Pcr plasmid was 5.1 kb (3.4 megadaltons). A Pcr plasmid isolated from a gonococcus in London, England, was identical with these 5.1-kb transformant plasmids in both size and restriction endonuclease cleavage profiles, suggesting that the 5.1-kb Pcr plasmid could have arisen from a 7.1-kb Pcr plasmid by a transformation-associated deletion in nature.
Effects of 8-substituted analogs of cyclic adenosine 3',5'-monophosphate on in vivo and in vitro syntheses of beta-galactosidase in Escherichia coli
Authors: Ito T , Yokota T , Sasaki Y , Suzuki N , Sowa T
Published: January 2020

Several 8-substituted alkylthio and alkylamino cyclic adenosine 3',5'-monophosphate (cAMP) derivatives were tested for their ability to stimulate beta-galactosidase synthesis in Estherichia coli in vivo and in vitro and to inhibit the cAMP phosphodiesterase activity of E. coli. Stimulation of beta-galactosidease synthesis in vivo by cAMP derivatives decreased with increasing length of the unbranched carbon chain of the substituent. On the other hand, the stimulation in vitro was increased as the carbon chain elongated. The 8-decylthio- and 8-dodecylthio-cAMP compounds stimulated beta-galactosidase synthesis almost eight-fold compared with cAMP, whereas 8-undecyl-, 8-dodectyl-, and 8-tridecylamino-cAMP stimulated beta-galactosidase synthesis about threefold. However, in in vitro experiments with a phosphodiesterase-deficient strain of E. coli, the Crooks strain, the stimulatory effects of the derivatives disappeared, except for 8-dodecylthio cAMP which stimulated beta-galactosidase about 1.4- to 1.6-fold. All derivatives were quite resistant to hydrolysis by phosphodiesterase. Most derivatives competitively inhibited the hydrolysis of cAMP by phosphodiesterase.