bluuebunny/crossref_metadata_2025 huggingface dataset:
delta1-piperideine-2-carboxylate reductase of Pseudomonas putida
Authors: Payton C W , Chang Y F
Published: January 2020

Pseudomonas putida metabolizes D-lysine to delta 1-piperideine-2-carboxylate and L-pipecolate. The second step of this catabolic pathway is catalyzed by delta 1-piperideine-2-carboxylate reductase. This enzyme was isolated and purified from cells grown on DL-lysine as substrate. The enzyme was very unstable, resulting in low recovery of activity and low purity after a six-step purification procedure. The enzyme had a pH optimum of 8.0 to 8.3. The Km values for delta 1-piperideine-2-carboxylate and NADPH were 0.23 and 0.13 mM, respectively. NADPH at concentrations above 0.15 mM was inhibitory to the enzyme. Delta 1-pyrroline-5-carboxylate, pyroglutamate, and NADH were poor substrates or coenzyme for delta 1-piperideine-2-carboxylate reductase. The enzyme reaction from delta 1-piperideine-2-carboxylate to L-pipecolate was irreversible. EDTA, sodium pyrophosphate, and dithiothreitol at concentrations of 1 mM protected the enzyme during storage. The enzyme was inhibited almost totally by Zn2+, Mn2+, Hg2+ Co2+, and p-chloromercuribenzoate at concentrations of 0.1 mM. The enzyme had a molecular weight of about 200,000. Both D-lysine and L-lysine were good inducers for the enzyme. Neither delta1-piperideine-2-carboxylate nor L-pipecolate was an effective inducer for the enzyme. P. putida cells grew on D-lysine only after a 5- to 8-h lag, which could be abolished by adding a supplement of 0.01% alpha-ketoglutarate or other readily metabolizable compounds. Such a supplement also converted the noncoordinate induction of this enzyme and pipecolate oxidase, both of the D-lysine pathway, to coordinacy. However, this effect was not observed if the enzyme pair was from different pathways of lysine metabolism in this organism (i.e., the D- and L-lysine pathways).
L-histidine utilization in Aspergillus nidulans
Authors: Polkinghorne M A , Hynes M J
Published: January 2020

Histidase activity rather than uptake of L-histidine is the limiting factor for the utilization of histidine as the sole nitrogen source for Aspergillus nidulans. Histidine cannot act as the sole carbon source, and evidence is presented indicating that this is attributable to an inability to convert histidine to L-glutamate in vivo. It has been shown that this fungus lacks an active urocanase enzyme and that histidine is quantitatively converted to urocanate, which accumulates in the extracellular medium. The use of histidine as a nitrogen source is regulated by nitrogen metabolite repression control of histidase synthesis. In addition, evidence for a requirement for a carbon source for histidase synthesis and for a minor form of control by nitrate is presented. The activity of the histidase enzyme is inhibited by micromolar concentrations of the product urocanate and by physiological levels of L-glutamate and L-glutamine.
Tetracycline-inducible transfer of tetracycline resistance in Bacteroides fragilis in the absence of detectable plasmid DNA
Authors: Rashtchian A , Dubes G R , Booth S J
Published: January 2020

Tetracycline resistance of three Bacteroides fragilis strains was shown to be inducible by subinhibitory concentrations of tetracycline. Tetracycline resistance markers could be transferred to another B. fragilis strain by filter mating. The transferability was inducible by subinhibitory concentrations of tetracycline and did not take place in the absence of tetracycline. The optimum concentration of tetracycline for induction of transfer was about 2 microgram/ml. The transfer was shown to be a conjugation-like process requiring cell-to-cell contact between donor and recipient. Screening of parental donor strains for the presence of plasmid DNA did not demonstrate any detectable plasmids in two of the strains. A 3.0-megadalton plasmid, designated pBY5, was present in the third donor strain. Mobilization of pBY5 by another plasmid (pBF4) showed that pBY5 did not carry the genes responsible for tetracycline resistance. It appears that the genes responsible for resistance to tetracycline as well as those responsible for conjugal transfer may be carried on the chromosome in all three donor strains.
Comparison of transcription of beta-lactamase genes specified by various ampicillin transposons
Authors: Yamamoto T , Yamagata S , Horii K , Yamagishi S
Published: January 2020

The beta-lactamase gene from four kinds of ampicillin transposons, Tn2601, Tn3, Tn2602 and Tn1, specifying the type I (or TEM type, alternatively) beta-lactamase was cloned onto plasmid pACYC184, and the level of in vivo transcription from each beta-lactamase gene was determined by DNA-RNA hybridization. Type I beta-lactamase is very uniform enzymologically, but heterogeneous in absolute levels of enzyme activity. The results demonstrated that the heterogeneity can be explained by the efficiency of transcription of each beta-lactamase gene, suggesting a difference in its promoter efficiency. A comparison of the levels of transcription of the beta-lactamase gene and the whole ampicillin transposon suggested that the beta-lactamase gene has the strongest promoter all of the genes in the ampicillin transposon.
Complementation analysis of Agrobacterium tumefaciens Ti plasmid mutations affecting oncogenicity
Authors: Klee H J , Gordon M P , Nester E W
Published: January 2020

A wide host range cosmid vector has been constructed by insertion of the lambda cos site into the plasmid pRK2501. This cosmid, which is maintained in Agrobacterium tumefaciens and is compatible with the Ti plasmid, has been used to make a clone bank of the A. tumefaciens pTiA6 plasmid. Several pTiA6 cosmids have been used to complement Tn5-induced Ti plasmid mutations. Five avirulent mutations which map outside of the region of the plasmid maintained in plant tumours (T-DNA) could be complemented in a trans orientation. Two mutations which are located on a single HpaI restriction fragment outside of the T-DNA, as well as three mutations which map within the T-DNA region, could not be complemented in a trans orientation in a REC- host.
Electron microscopic observations of surface projections on Chlamydia psittaci reticulate bodies
Authors: Matsumoto A
Published: January 2020

Electron microscopic observations were carried out to confirm the presence of surface projections on Chlamydia psittaci reticulate bodies (RBs). The morphology of the projections on RBs was identical with that on elementary bodies (EBs); one end of each projection was connected with the cytoplasmic membrane, but the other end of the projection protruded beyond the cell wall through a fine hole or rosette in the cell wall. The results demonstrated that the rosettes seen in RB cell walls were morphological markers indicating the presence of the surface projections. A statistical anaylsis of the number of projections on EBs and the number of rosettes in RB cell walls prepared at 10, 15, and 20 h after infection demonstrated that all RBs had the projections and that the number of projections was maximal by 10 h after infection and then decreased gradually to approximately the same number of projections on EBs.
Isolation in high frequency of rough variants of Mycobacterium intracellulare lacking C-mycoside glycopeptidolipid antigens
Authors: Barrow W W , Brennan P J
Published: January 2020

Rough variants of serovars from the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex were isolated in high frequency from pellicle growth of the wild-type strains. Rough morphology could be correlated with the lack of an outer cell wall sheath and its constituent C-mycoside glycopeptidolipids of both the serologically active and inactive types.
Occurrence of
l
-Lysine ε-Dehydrogenase in
Agrobacterium tumefaciens
Authors: Misono Haruo , Nagasaki Susumu
Published: January 2020

A novel amino acid dehydrogenase catalyzing the oxidative deamination of the ε-amino group of
l
-lysine was found in the crude extract of
Agrobacterium tumefaciens
ICR 1660. The enzyme required NAD
+
and was specific for
l
-lysine. The enzyme was optimally active at about pH 9.7.
Myo-inositol transport in Saccharomyces cerevisiae
Authors: Nikawa J , Nagumo T , Yamashita S
Published: January 2020

myo-Inositol uptake in Saccharomyces cerevisiae was dependent on temperature, time, and substrate concentration. The transport obeyed saturation kinetics with an apparent Km for myo-inositol of 0.1 mM, myo-Inositol analogs, such as scyllo-inositol, 2-inosose, mannitol, and 1,2-cyclohexanediol, had no effect on myo-inositol uptake, myo-Inositol uptake required metabolic energy. Removal of D-glucose resulted in a loss of activity, and azide and cyanide ions were inhibitory. In the presence of D-glucose, myo-inositol was accumulated in the cells against a concentration gradient. A myo-inositol transport mutant was isolated from UV-mutagenized S. cerevisiae cells using the replica-printing technique. The defect in myo-inositol uptake was due to a single nuclear gene mutation. The activities of L-serine and D-glucose transport were not affected by the mutation. Thus it was shown that S. cerevisiae grown under the present culture conditions possessed a single and specific myo-inositol transport system. myo-Inositol transport activity was reduced by the addition of myo-inositol to the culture medium. The activity was reversibly restored by the removal of myo-inositol from the medium. This restoration of activity was completely abolished by cycloheximide.
Demonstration of hydrogenase in extracts of the homoacetate-fermenting bacterium Clostridium thermoaceticum
Authors: Drake H L
Published: January 2020

Cell-free extracts of the homoacetate-fermenting bacterium Clostridium thermoaceticum were shown to catalyze the hydrogen-dependent reduction of various artificial electron acceptors. The activity of the hydrogenase was optimal at pH 8.5 to 9 and was extremely sensitive to aeration. EDTA did not significantly reduce the liability of the enzymic activity to oxidation (aeration). At 50 degrees C, when both methyl viologen and hydrogen were at saturating concentrations with respect to hydrogenase, the specific activity of cell-free extracts approximated 4 mumol of H2 oxidized per min per mg of protein; fourfold higher specific activities were obtained when benzyl viologen was utilized as an electron acceptor. Activity stains of polyacrylamide gels demonstrated the presence of a single hydrogenase band, suggesting that the catalytic activity in cell extracts was due to a single enzyme. The activity was stable for at least 32 min at 55 degrees C but was slowly inactivated at 70 degrees C. NAD, NADP, flavin adenine dinucleotide, flavin mononucleotide, and ferredoxin were not significantly reduced, but possible reduction of the particulate b-type cytochrome of C. thermoaceticum was observed. NaCl, sodium dodecyl sulfate, iodoacetamide, and CO were shown to inhibit catalysis. A kinetic study is presented, and the possible physiologic roles for hydrogenase in C. thermoaceticum ar discussed.
Scanning electron microscope observation of the swarming phenomenon of Vibrio parahaemolyticus
Authors: Belas M R , Colwell R R
Published: January 2020

Scanning electron microscopy was used to study the production of lateral flagella and the swarming phenomenon in Vibrio parahaemolyticus. Differences in the size and diameter of the sheathed, polar flagellum and lateral flagella were apparent in these preparations. Swarming of V. parahaemolyticus was found to be similar to the swarming of Proteus spp. in that swarm cells which are heavily flagellated and elongated are formed.
d
-(–)-Tartrate Dehydratase of
Rhodopseudomonas sphaeroides
: Purification, Characterization, and Application to Enzymatic Determination of
d
-(–)-Tartrate
Authors: Rode Hergo , Giffhorn Friedrich
Published: January 2020

An isolate of
Rhodopseudomonas sphaeroides
was capable of growing phototrophically and chemotrophically (μ = 0.15 h
−1
for either condition) with
d
-(–)-tartrate as the carbon source. A
d
-(–)-tartrate dehydratase, (
d
-(–)-tartrate hydrolyase, EC 4.1.2.70) was induced in the presence of
d
-(–)-tartrate. The enzyme was purified 30-fold from cell extracts of
R. sphaeroides
to a specific activity of 7.5 U/mg of protein and was subsequently crystallized in the presence of 1 M KCl. The enzyme was homogeneous upon analytical electrophoresis in 5% polyacrylamide gels and by criteria of ultracentrifugation. The native enzyme had a molecular weight of 158,000 ± 1,000 as determined by gel filtration and ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single polypeptide chain with an estimated molecular weight of 39,500 ± 500, indicating that
d
-(–)-tartrate dehydratase was a tetramer. The isoelectric point of the native enzyme was at pH 5.5. The enzyme catalyzed irreversibly the conversion of
d
-(–)-tartrate to oxaloacetate and water, and the turnover number was calculated to be 1,185. The reaction followed Michaelis-Menten kinetics, and a

K
m

value of 1.8 × 10
−4
M was determined.
d
-(–)-Tartrate dehydratase required Mg
2+
for activity. The pH optimum was within a range from 6.2 to 7.2, and the activation energy of the reaction (Δ H
0
) was 63.2 kJ/mol. The enzyme was specific for
d
-(–)-tartrate; it did not react with
l
-(+)-tartrate,
meso
-tartrate, and other hydroxycarboxylic acids.
d
-(–)-Tartrate dehydratase was strongly inhibited by
meso
-tartrate (50% at 0.6 mM).
l
-(+)-Tartrate and a variety of hydroxycarboxylic acids caused 50% inhibition at concentrations of >30 mM.
Energy coupling of facilitated transport of inorganic ions in Rhodopseudomonas sphaeroides
Authors: Hellingwerf K J , Friedberg I , Lolkema J S , Michels P A , Konings W N
Published: January 2020

Within the scope of a study on the effects of changes in medium composition on the proton motive force in Rhodopseudomonas sphaeroides, the energy coupling of sodium, phosphate, and potassium (rubidium) transport was investigated. Sodium was transported via an electroneutral exchange system against protons. The system functioned optimally at pH 8 and was inactive below pH 7. The driving force for the phosphate transport varied with the external pH. At pH 8, Pi transport was dependent exclusively on delta psi (transmembrane electrical potential), whereas at pH 6 only the delta pH (transmembrane pH gradient) component of the proton motive force was a driving force. Potassium (rubidium) transport was facilitated by a transport system which catalyzed the electrogenic transfer of potassium (rubidium) ions. However, in several aspects the properties of this transport system were different from those of a simple electrogenic potassium ionophore such as valinomycin: (i) accumulated potassium leaked very slowly out of cells in the dark; and (ii) the transport system displayed a threshold in the delta psi, below which potassium (rubidium) transport did not occur.
Ligase-deficient yeast cells exhibit defective DNA rejoining and enhanced gamma ray sensitivity
Authors: Moore C W
Published: January 2020

Yeast cells deficient in DNA ligase were also deficient in their capacity to rejoin single-strand scissions in prelabeled nuclear DNA. After high-dose-rate gamma irradiation (10 and 25 krads), cdc9-9 mutant cells failed to rejoin single-strand scissions at the restrictive temperature of 37 degrees C. In contrast, parental (CDC9) cells (incubated with mutant cells both during and after irradiation) exhibited rapid medium-independent DNA rejoining after 10 min of post-irradiation incubation and slower rates of rejoining after longer incubation. Parental cells were also more resistant than mutant cells to killing by gamma irradiation. Approximately 2.5 +/- 0.07 and 5.7 +/- 0.6 single-strand breaks per 10(8) daltons were detected in DNAs from either CDC9 or cdc9-9 cells converted to spheroplasts immediately after 10 and 25 krads of irradiation, respectively. At the permissive temperature of 23 degrees C, the cdc9-9 cells contained 2 to 3 times the number of DNA single-strand breaks as parental cells after 10 min to 4 h of incubation after 10 krads of irradiation, and two- to eightfold more breaks after 10 min to 2.5 h of incubation after 25 krads of irradiation. Rejoining of single-strand scissions was faster in medium. After only 10 min in buffered growth medium and after 10 krads of irradiation, the number of DNA single-strand breaks was reduced to 0.32 +/- 0.3 (at 23 degrees C) or 0.21 +/- 0.05 (at 37 degrees C) per 10(8) daltons in parental cells, but remained at 2.1 +/- 0.06 (at 23 degrees C) or 2.3 +/- 0.07 (at 37 degrees C) per 10(8) daltons in mutant cells. After 10 or 25 krads of irradiation plus 1 h of incubation in medium at 37 degrees C, only DNA from CDC9 cells was rejoined to the size of DNA from unirradiated cells, whereas at 23 degrees C, DNAs in both strains were completely rejoined.
Fine-structure deletion map and complementation analysis of the glnA-glnL-glnG region in Escherichia coli
Authors: MacNeil T , MacNeil D , Tyler B
Published: January 2020

A total of 399 independent mutants of Escherichia coli were obtained which have point and insertion mutations in the glnA region. Mutants isolated included Gln- and Reg- strains (unable to utilize arginine as a nitrogen source). Mutations were mapped with 73 deletion-containing derivatives of a lambda gln phage. Complementation analysis was performed with lambda gln derivatives containing point mutations which conferred a Gln- or Reg- phenotype. Deletion mapping and complementation analysis assigned 104 mutations in 24 deletion intervals to glnA. Mutations in Reg- strains were assigned to two genes, glnL and glnG. glnL contained 131 mutations in 12 deletion intervals, and glnG contained 164 mutations in 10 deletion intervals. The gene order is glnA-glnL-glnG, transcribed from left to right. Polarity of insertion mutations indicates that glnL and glnG form from left to right. Polarity of insertion mutations indicates that glnL and glnG form an operon. Complementation analysis of glnA insertion mutations with glnL and glnG mutations showed polarity of glnA onto most glnL and glnG alleles, suggesting that transcription of glnA may proceed into the glnL-glnG operon. All mutations analyzed in glnA conferred a Gln- phenotype. However, we also found that over half of the Gln- strains isolated ater chemical mutagenesis contained point mutations in glnG. Mutants which synthesized a high level of glutamine synthetase in the presence of ammonia (GlnC phenotype) were selected as revertants of a strain with a Tn10 insertion in glnD and were mapped with chromosomal deletions. Results indicate that mutations in 12 and 15 examined strains clearly map outside of glnA, probably in glnL.
13C nuclear magnetic resonance study of trehalose mobilization in yeast spores
Authors: Barton J K , Den Hollander J A , Hopfield J J , Shulman R G
Published: January 2020

Using high-resolution 13C nuclear magnetic resonance, we examined the mobilization of endogenous trehalose in suspensions of yeast asci. Sporulation of yeast cells in [1-13C]acetate resulted in incorporation of label into the C-3 and C-4 positions of trehalose within the asci. During germination of these asci with [1-13C]glucose, the consumption of both endogenous trehalose and exogenous glucose were followed simultaneously by 13C nuclear magnetic resonance, as was the formation of glycerol and ethanol, their glycolytic and products. Time courses for carbohydrate consumption indicated that trehalose, although it decreased to 25% of its initial value upon germination, was not preferentially catabolized and did not provide the primary energy supply for germination with glucose. The ratio of trehalose to glucose catabolized was 0.09. Exogenous glucose levels appeared to regulate trehalose mobilization since trehalose was only consumed when sufficiently high levels (more than 2 mM) of glucose were present. Upon glucose depletion newly synthesized [1-13C]trehalose was observed. Nuclear magnetic resonance spectra of extracts confirmed the trehalose peak assignments and showed products of [1-13C]glucose catabolism. In addition by quantitating trehalose consumption and 2-deoxyglucose incorporation in dormant yeast asci, we found that 3.8 +/- 0.l4 molecules of 2-deoxyglucose were incorporated for each trehalose molecule consumed. Trehalose can therefore function as a carbohydrate source for ATP formation during dormancy.
Iron- and molybdenum-repressible outer membrane proteins in competent Azotobacter vinelandii
Authors: Page W J , von Tigerstrom M
Published: January 2020

Azotobacter vinelandii produced three major proteins of 93,000, 85,000, and 81,000 daltons and a minor 77,000-dalton protein in the outer membrane of Fe-limited cells, and these cells were competent for transformation by DNA. The synthesis of these proteins was repressed in Fe-sufficient medium. Mo limitation of nitrogen-fixing cells resulted in the hyperproduction of a 44,000-dalton protein and the production of a minor 77,000-dalton protein in the outer membrane. Mo limitation enhanced competence in Fe-limited medium and induced competence in Fe-sufficient medium. The 44,000-dalton protein was replaced by a 45,000-dalton protein when Fe-sufficient medium also contained NH4+, but the cells were noncompetent. The synthesis of these proteins was repressed in Mo-sufficient medium and by NH4+ in Fe-limited medium. All of the culture supernatants contained a blue-white fluorescent material (absorbance maximum, 214 nm) which appeared to coordinate Fe3+, Fe2+, MoO4(2-), WO3(2-), and VO3(-).
Arginine and pyrimidine biosynthetic defects in Neisseria gonorrhoeae strains isolated from patients
Authors: Shinners E N , Catlin B W
Published: January 2020

Neisseria gonorrhoeae strains with nutritional requirements that include arginine (Arg-), uracil (Ura-), and hypoxanthine have attracted attention because of their tendency to cause disseminated infections, as a basis for genetic studies of arginine and pyrimidine biosynthesis, we examined the activities of four enzymes of these pathways in cell-free extracts of both prototrophic and Arg- Ura- strains. Activities of glutamate acetyltransferase, aspartate transcarbamylase, and orotate phosphoribosyltransferase, encoded respectively by argE, pyrB, and pyrE, were absent in some Arg- Ura- isolates. Gonococci that were unable to utilize ornithine for growth in place of citrulline lacked activity of carbamyl phosphate synthetase (encoded by car). Defects of car imposed requirements for both citrulline (or arginine) and a pyrimidine because of the dual role of carbamyl phosphate in the two pathways. Defects of argE, car, pyrB, and pyrE were separately introduced by genetic transformation into representatives of a gonococcal strain which initially was prototrophic. Results of enzyme assays of these isogenic auxotrophic transformants confirmed the gene-enzyme relationships.
Three-dimensional structure of the crystalloid in the microbody of Kloeckera sp.: composite crystal model
Authors: Osumi M , Nagano M , Yamada N , Hosoi J , Yanagida M
Published: January 2020

Electron microscopic investigations using the cryosectioning technique, together with electron diffraction, optical diffraction, and computer simulation, were carried out for the determination of the intrinsic structure of the crystalloid in the microbody of Kloeckera sp. The lattice images seen in the cryosections could be changed from one to another by tilting the specimen at an appropriate angle, the images obtained being well consistent with those obtained by computer simulation. The electron diffraction patterns also agreed with those obtained by optical diffraction. The results demonstrated that the crystalloid was composed of two different types of particles, large and small ones, arranged alternately and making up the composite crystal of rock salt structure. Large particles seemed to consist of alcohol oxidase molecules, while the small particles were presumably made up of catalase molecules.
Plasmid-mediated uptake and metabolism of sucrose by Escherichia coli K-12
Authors: Schmid K , Schupfner M , Schmitt R
Published: January 2020

The conjugative plasmid pUR400 determines tetracycline resistance and enables cells of Escherichia coli K-12 to utilize sucrose as the sole carbon source. Three types of mutants affecting sucrose metabolism were derived from pUR400. One type lacked a specific transport system (srcA); another lacked sucrose-6-phosphate hydrolase (scrB); and the third, a regulatory mutant, expressed both of these functions constitutively (scrR). In a strain harboring pUR400, both transport and sucrose-6-phosphate hydrolase were inducible by fructose, sucrose, and raffinose; if a scrB mutant was used, fructose was the only inducer. These data suggested that fructose or a derivative acted as an endogenous inducer. Sucrose transport and sucrose-6-phosphate hydrolase were subject to catabolite repression; these two functions were not expressed in an E. coli host (of pUR400) deficient in the adenosine 3-,5'-phosphate receptor protein. Sucrose uptake (apparent Km = 10 microM) was dependent on the scrA gene product and on the phosphoenolpyruvate-dependent sugar:phosphotransferase system (PTS) of the host. The product of sucrose uptake (via group translocation) was identified as sucrose-6-phosphate, phosphorylated at C6 of the glucose moiety. Intracellular sucrose-6-phosphate hydrolase catalyzed the hydrolysis of sucrose-6-phosphate (Km = 0.17 mM), sucrose (Km = 60 mM), and raffinose (Km = 150 mM). The active enzyme was shown to be a dimer of Mr 110,000.
Suppression of Escherichia coli dnaA46 mutations by integration of plasmid R100.1. derivatives: constraints imposed by the replication terminus
Authors: Louarn J , Patte J , Louarn J M
Published: January 2020

We have studies the phenotypic suppression of a dnaA46 mutation by plasmid integration at preselected chromosomal sites after introducing homologous sequences (Mu prophages) onto both the chromosomes and the suppressive plasmid. The plasmids used were all derived from plasmid R100.1. We found that the conditions required to get viable suppressive integration varied as the plasmid integration site moved from the origin to the terminus of chromosome replication. Two constraints were observed. Both appeared to be linked to the new characteristics acquired by chromosome replication from the integrated plasmid. One constraint was that strains with integrative suppression near the terminus terC were viable only in minimal medium. The rich medium sensitivity of these strains was correlated with a loss of regulation of initiation. The other constraint was a requirement for a specific orientation in certain regions of the chromosome. The two branches defined by normally initiated replication, between oriC and terC, were also symmetrical with respect to these plasmid orientation constraints. In studying the possible reasons for a plasmid orientation constraint, we found that, of the two forks initiated in bidirectional replication from the integrated plasmid, one was capable of moving across the terC region with a higher movability than the other.
Transcription initiation at the tryptophanase promoter of Escherichia coli K-12
Authors: Deeley M C , Yanofsky C
Published: January 2020

Restriction fragments containing the region preceding the tryptophanase structural gene, tnaA, were used as templates for in vitro transcription experiments. A transcription initiation site was detected that was dependent on the catabolite gene activator protein (CAP) plus cyclic AMP (cAMP). The mRNA produced in vitro was fingerprinted, and the nucleotide at which transcription was initiated was localized to the vicinity of two guanine residues 316 and 318 base pairs upstream of tnaA. A region exhibiting extensive difold symmetry and homology to the CAP binding site adjacent to the lactose operon promoter exists approximately 60 base pairs preceding the site of transcription initiation. Two HinfI restriction sites are located in this region. Restriction enzyme cleavage at these sites was prevented when DNA containing the promoter region was preincubated with CAP and cAMP. RNA polymerase was incapable of protecting these sites against this cleavage. CAP and cAMP addition did not protect against cleavage at a DdeI restriction site located in the -20 region of the promoter. RNA polymerase did protect against DdeI cleavage but only in the presence of CAP and cAMP. Thus, transcription initiation at the tryptophanase promoter involves cAMP-dependent, CAP-facilitated binding of RNA polymerase to the DNA.
Characteristic lipids of Bordetella pertussis: simple fatty acid composition, hydroxy fatty acids, and an ornithine-containing lipid
Authors: Kawai Y , Moribayashi A
Published: January 2020

The lipids and fatty acids of Bordetella pertussis (phases I to IV) were analyzed by thin-layer chromatography, gas-liquid chromatography, and mass spectrometry and compared with those of B. parapertussis and B. bronchiseptica. The major lipid components of the three species were phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, lysophosphatidylethanolamine, and an ornithine-containing lipid. The ornithine-containing lipid was characteristic of the genus Bordetella. The fatty acid composition of the total extractable cellular lipids of B. pertussis was mostly hexadecanoic and hexadecenoic acids (90%) in a ratio of about 1:1. The hexadecenoic acid of B. pertussis was in the cis-9 form. The fatty acid composition of the residual bound lipids was distinctly different from that of the extractable lipids, and residual bound lipids being mainly 3-hydroxytetradecanoic, tetradecanoic, and 3-hydroxydecanoic acids, with 3-hydroxydodecanoic acid occurring in some strains. It was determined that the 3-hydroxy fatty acids were derived from lipid A. The fatty acid composition of the total extractable cellular lipids of B. parapertussis and B. bronchiseptica, mainly composed of hexadecanoic and heptadecacyclopropanoic acid, differed from that of B. pertussis. Although the fatty acid composition of the residual bound lipids of B. parapertussis was similar to that of the residual bound lipids of B. pertussis, 2-hydroxydodecanoic acid was detected only in the bound lipids of B. bronchiseptica.
Nickel transport in Methanobacterium bryantii
Authors: Jarrell K F , Sprott G D
Published: January 2020

Methanobacterium bryantii, grown autotrophically on H2-CO2, transported nickel against a concentration gradient by a high-affinity system (Km = 3.1 microM). The system had a pH optimum of 4.9 and a temperature optimum of 49 degrees C with an energy of activation of 7.8 kcal/mol (ca. 32.6 kJ/mol). A headspace of H2-CO2 (4:1, vol/vol) was required for maximum rate of transport. The system was highly specific for nickel and was unaffected by high levels of all monovalent and divalent ions tested (including Mg2+) with the sole exception of Co2+. Kinetic experiments indicated that accumulated nickel became increasingly incorporated into cofactor F430 and protein. Nickel transport was inhibited by nigericin, monensin, and gramicidin but not by carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone, carbonyl cyanide-m-chlorophenyl hydrazone, N,N'-dicyclohexylcarbodiimide, valinomycin plus potassium, or acetylene. The ineffectiveness of carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone, carbonyl cyanide-m-chlorophenyl hydrazone, and N,N'-dicyclohexylcarbodiimide may be related to difficulties in the penetration of these compounds through the outer cell barriers. Nickel uptake was greatly stimulated by an artificially imposed pH gradient (inside alkaline). The data suggest that nickel transport is not dependent on the membrane potential or on intracellular ATP, but is coupled to proton movement.
Mercuric reductase enzyme from a mercury-volatilizing strain of Thiobacillus ferrooxidans
Authors: Olson G J , Porter F D , Rubinstein J , Silver S
Published: January 2020

Cell-free mercury volatilization activity (mercuric reductase) was obtained from a mercury-volatilizing Thiobacillus ferrooxidans strain, and the properties of intact-cell and cell-free activities were compared with those determined by plasmid R100 in Escherichia coli. Intact cells of T. ferrooxidans volatilized mercury at pH 2.5, whereas cells of E. coli did not. Cell-free enzyme preparations from both bacteria functioned best at or above neutral pH and not at all at pH 2.5. The T. ferrooxidans mercuric reductase was a soluble enzyme that was dependent upon added NAD(P)H. The enzyme activity was stable at 80 degrees C, required an added thiol compound, and was stimulated by EDTA. Antisera against purified mercuric reductases from transposon Tn501 and plasmid R831 (which inactivated mercuric reductases from a wide range of enteric and pseudomonad strains) did not inactivate the enzyme from T. ferrooxidans.
A gene coding for a periplasmic protein is located near the locus for termination of chromosome replication in Escherichia coli
Authors: Harayama S , Hazelbauer G L
Published: January 2020

Hybrid plasmids carrying trg, the genetic locus in closest proximity to terC, coded for several polypeptides in addition to the Trg protein. Polypeptides of 59,000 and 61,000 apparent molecular weight were the most prominent products synthesized in minicells containing the hybrid plasmids. Analysis of the effects of deletions generated by a restriction endonuclease identified a region of DNA immediately adjacent to trg as the putative gene coding for the two polypeptides. Studies with whole cells and minicells showed that the 59,000-dalton polypeptide is a periplasmic protein. Analysis by limited proteolysis indicated that the two polypeptides are related, and a number of observations support the notion that the 61,000-dalton protein is a precursor form of the 59,000-dalton mature exported protein. The identification and characterization of a protein, in addition to Trg, which is produced by a gene in close proximity to terC emphasizes the fact that the region does contain intact and active genes.
Analysis of btuB receptor function by use of nonsense suppression
Authors: Hunter M G , Glass R E
Published: January 2020

Informational suppression of btuB nonsense mutants allows the study of the effect of known, single amino acid substitutions on receptor function. We found that ligand uptake is largely unaffected by such amino acid changes. The few instances in which certain substitutions destroyed sensitivity to the two lethal agents (phage BF23, colicin E3) without affecting vitamin B12 uptake suggest a common region on the btuB receptor involved in the binding of these proteinaceous agents.
Membrane Proteins of
Mycoplasma gallisepticum
Authors: Archer David B. , Rodwell Alan W.
Published: January 2020

Mycoplasma gallisepticum
membrane proteins were separated by two-dimensional gel electrophoresis. We found no evidence for the presence of a membrane glycoprotein.
Regulation of nitrogenase in the photosynthetic bacterium Rhodopseudomonas capsulata as studied by two-dimensional gel electrophoresis
Authors: Hallenbeck P C , Meyer C M , Vignais P M
Published: January 2020

By using two-dimensional electrophoresis, five putative soluble nif gene products were identified, and the regulation of nif gene expression in Rhodopseudomonas capsulata was investigated. Expression of nif was repressed by ammonia and atmospheric concentrations of oxygen. Deprivation of molybdenum caused an interesting pattern of partial repression of nif gene expression that was not relieved by tungsten. These results are discussed in relation to the better understood system of nif regulation in Klebsiella pneumoniae.
Escherichia coli mutants defective in the uncH gene
Authors: Humbert R , Brusilow W S , Gunsalus R P , Klionsky D J , Simoni R D
Published: January 2020

Plasmids carrying cloned segments of the unc operon of Escherichia coli have been used in genetic complementation analyses to identify three independent mutants defective in the uncH gene, which codes for the delta subunit of the ATP synthetase. Mutations in other unc genes have also been mapped by this technique. ATPase activity was present in extracts of the uncH mutants, but the enzyme was not as tightly bound to the membrane as it was in the parental strain. ATP-dependent membrane energization was absent in membranes isolated from the uncH mutants and could not be restored by adding normal F1 ATPase from the wild-type strain. F1 ATPase prepared from uncH mutants could not restore ATP-dependent membrane energization when added to wild-type membranes depleted of F1. Membranes of the uncH mutants were not rendered proton permeable as a result of washing with low-ionic-strength buffer.
Derepression of the synthesis of D-ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum
Authors: Sarles L S , Tabita F R
Published: January 2020

The synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodospirillum rubrum was greatly influenced by the conditions of culture. When grown photolithotrophically in an atmosphere containing low levels of CO2 (1.5 to 2%), enzyme synthesis was derepressed, with the result that the enzyme comprised up to 50% of the soluble protein of the cells as determined by immunological quantitation. This response was not observed when R. rubrum was grown photolithotrophically in an atmosphere of 5% CO2 in hydrogen. Similarly, the derepression of ribulose 1,5-bisphosphate carboxylase/oxygenase was observed in photoheterotrophically (butyrate)-grown cultures only after the HCO3- supply was nearly exhausted. The increase in enzyme activity observed in derepressed cultures was not paralleled by an increase in the in vivo CO2 fixation rate. Apparently, R. rubrum derepresses the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase when exposed to low CO2 concentrations to scavenge the limited CO2 available to such cultures.
Regulatory Citrate Lyase Mutants of
Salmonella typhimurium
Authors: Kulla Hans G.
Published: January 2020

Citrate lyase, the key enzyme of anaerobic citrate catabolism, could not be deleted from
Salmonella typhimurium
. The only class of mutants found had a mode of covalent regulation that strongly resembled the
Escherichia coli
system: citrate lyase was only active, i.e., acetylated, when a cosubstrate was present.
Orientation of succinate dehydrogenase and cytochrome b558 in the Bacillus subtilis cytoplasmic membrane
Authors: Hederstedt L , Rutberg L
Published: January 2020

The orientation of the three subunits of the membrane-bound succinate dehydrogenase (SDH)-cytochrome b558 complex in Bacillus subtilis was studied in protoplasts ("right side out") and isolated membranes (random orientation), using immunoadsorption and surface labeling with [35S]diazobenzenesulfonate. Anti-SDH antibodies were adsorbed by isolated membranes but not by protoplasts. The SDH Mr 65,000 flavoprotein subunit was labeled with [35S]diazobenzenesulfonate in isolated membranes but not in protoplasts. The flavoprotein subunit is thus located on the cytoplasmic side of the membrane. The location of the SDH Mr 28,000 iron-protein subunit was not definitely established, but most probably the iron-protein subunit also is located on the cytoplasmic side of the membrane. Antibodies were not obtained to the hydrophobic cytochrome b558. The cytochrome was strongly labeled with [35S]diazobenzenesulfonate in protoplasts, and labeling was also obtained with isolated membranes. Cytochrome b558 is thus exposed on the outside of the membrane. In B. subtilis SDH binds specifically to cytochrome b558, which suggests that the cytochrome is exposed also on the cytoplasmic side of the membrane. The results obtained suggest that the B. subtilis SDH is exclusively located on the cytoplasmic side of the membrane where it is bound to cytochrome b558, which spans the membrane.
Bacterial Oxidation of Naphthalene and Phenanthrene
Authors: Barnsley E. A.
Published: January 2020

An organism grown on both of the hydrocarbons naphthalene and phenanthrene induces a separate enzyme for the initial oxidation of each.
Plasmids and Bacteriocins in
Caulobacter
Species
Authors: Schoenlein Patricia V. , Ely Bert
Published: January 2020

A survey of wild-type
Caulobacter
strains revealed naturally occurring plasmids in three species. Further analysis showed instances of naturally occurring antibiotic resistance and bacteriocin production.
pH-sensitive CDP-diglyceride synthetase mutants of Escherichia coli: phenotypic suppression by mutations at a second site
Authors: Ganong B R , Raetz C R
Published: January 2020

In Escherichia coli, mutations which lower the level of CDP-diglyceride synthetase are designated cds and map at min 4. The cds-8 mutation resulted in strikingly defective enzyme activity and also rendered cells pH sensitive for growth. Both the inhibition of growth and the massive accumulation of phosphatidic acid which occur in a cds-8 mutant at pH 8 were suppressed by mutations at a second locus, designated cdsS, which mapped between argG and gltB near min 68. The cdsS3 mutation by itself did not affect CDP-diglyceride synthetase activity in wild-type cells, but it caused a twofold stimulation of the residual activity present in strains harboring cds-8. Both the insensitivity to pH and the twofold stimulation of residual activity were lost by introduction of an F' strain carrying cdsS+ into a recA1 cds-8 cdsS3 host. When a culture of a cds-8 cdsS+ strain was shifted to pH 8, the residual specific activity of synthetase dropped by 75% within 100 min. In a cds-8 cdsS3 double mutant under the same conditions, the activity declined appreciably less, about to the level found in the cds-8 cdsS+ strain under permissive conditions (pH 6). Thus, it appears that mutations in the cdsS gene suppress the pH sensitivity of cds mutants by inhibiting the decay of residual CDP-diglyceride synthetase activity at the nonpermissive pH. The cdsS locus appears to be distinct from any known nonsense or missense suppressor.
Isolation and characterization of P1 minireplicons, lambda-P1:5R and lambda-P1:5L
Authors: Sternberg N , Austin S
Published: January 2020

We have isolated two new classes of P1 miniplasmids, called lambda-P1:5R and lambda-P1:5L, by the in vivo extension of a cloned P1 fragment, EcoRI-5, which by itself is not capable of plasmid replication. The lambda-P1:5R plasmids contain EcoRI-5 plus a variable portion of the adjacent P1 EcoRI fragment 8. They have a copy number like that of P1 (about 1 per host chromosome), are faithfully segregated at cell division, and are subject to incompatibility exerted by either a single copy of P1 or a single copy of EcoRI-5. In contrast, the lambda-P1:5L plasmids contain EcoRI-5 and a portion of adjacent P1 DNA that includes at least P1 EcoRI fragments 15, 18, and 23 and a part of fragment 17. These plasmids have a copy number of about 15 per cell chromosome. Despite this they are segregated to daughter cells somewhat less faithfully than are lambda-P1:5R plasmids. They are sensitive to incompatibility exerted by a single copy of P1, but not to incompatibility exerted by a single copy of EcoRI-5. lambda-P1:5L plasmids are, however, sensitive to incompatibility exerted by multiple copies of EcoRI-5. These results show that the relative copy numbers of exerting and responding elements are important for the incompatibility phenotype and strongly suggest that lambda-P1:5L plasmids lack a repressor of replication that can be supplied in trans from P1 but not from EcoRI fragment 5. We suggest that P1 normally uses the 5R replicon and that the 5L replicon may be a backup system that ensures plasmid maintenance should the primary replication event fail to initiate.
Genetic map of the opp (Oligopeptide permease) locus of Salmonella typhimurium
Authors: Higgins C F , Hardie M M , Jamieson D , Powell L M
Published: January 2020

The uptake of peptides by Salmonella typhimurium is mediated by three apparently independent transport systems. One of these systems, the oligopeptide permease, is encoded by a genetic locus (opp) which has been mapped at 34 min on the S. typhimurium chromosomal map. We accurately mapped the location of opp by cotransduction frequencies and by deletion analysis and show that the gene order for this region of the chromosome is cysB-trp-tonB-opp-galU-tdk. All opp mutants, independently isolated by a variety of means, mapped at this one locus, between tonB and galU. Spontaneous and transposon Tn10-generated deletions were used to construct a fine-structure genetic map of opp. Evidence is presented which indicates that opp covers a 5- to 6-kb segment of DNA and is therefore likely to consist of more than one gene.
A voltage clamp inhibits chemotaxis of Spirochaeta aurantia
Authors: Goulbourne E A , Greenberg E P
Published: January 2020

Anaerobic conditions were employed to study the relationship between membrane potential and chemotaxis in Spirochaeta aurantia. When cells were grown anaerobically and suspended in anaerobic potassium phosphate buffer (pH 5.5), membranes did not appear to be polarized. Nevertheless, motility was supported by a transmembrane pH gradient, and the anaerobic cells exhibited D-xylose taxis. Introduction of trace amounts of air into anaerobic cell suspensions resulted in a transient membrane polarization. The addition of valinomycin to cells suspended under anaerobic conditions did not alter the steady-state value of membrane potential appreciably but served to clamp membrane potential at the preset level. Although there was no detectable effect of valinomycin on the motility of anaerobic cells in potassium phosphate buffer, D-xylose taxis was completely inhibited by this treatment. These data indicate the the action of valinomycin as a voltage clamp serves to inhibit the chemotaxis of S. aurantia and provide evidence to support the suggestion that the mechanism of chemotaxis in this organism involves the transduction of sensory signals in the form of membrane potential fluctuations.
Mutations affecting lipoamide dehydrogenases of Pseudomonas putida
Authors: Sokatch J R , McCully V , Sahm J G , Reyes-Maguire M
Published: January 2020

Pseudomonas putida grown on valine produces two lipoamide dehydrogenases, LPD-glu (Mr, 56,000 and LPD-val (Mr, 49,000). The 49,000-dalton protein is used by P. putida for branched-chain keto acid dehydrogenase, whereas the 56,000-dalton protein is presumably used for pyruvate and 2-ketoglutarate dehydrogenases. The objective of this study was to isolate and characterize mutants of P. putida with mutations affecting lipoamide dehydrogenases in order to study the relationship of these two proteins. Mutant JS287 lacked LPD-val, the lipoamide dehydrogenase which is induced by growth on valine and is specific for branched-chain keto acid dehydrogenase, and had normal amounts of LPD-glu, the lipoamide dehydrogenase which is formed during growth on glucose and which is probably used by both pyruvate and 2-ketoglutarate dehydrogenases. Mutant JS94 was a pleiotropic mutant with defects in 2-ketoglutarate, branched-chain, and lipoamide dehydrogenases. Proteolysis of LPD-glu and LPD-val produced completely different digestion products, suggesting that these two proteins are products of separate structural genes. Antisera prepared against LPD-glu reacted only with LPD-glu, whereas antisera prepared against LPD-val reacted with LPD-val and cross-reacted with LPD-glu. Although mutant JS94 did not produce active lipoamide dehydrogenase, cell-free extracts of this mutant contained a protein which cross-reacted with anti-LPD-val.