bluuebunny/crossref_metadata_2025 huggingface dataset:
Occurrence of alpha-tocopherolquinone and alpha-tocopherolquinol in microorganisms
Authors: Hughes P E , Tove S B
Published: January 2020

Both alpha-tocopherolquinol and alpha-tocopherolquinone were found in 56 of 93 strains of microorganisms examined. Organisms that contained these compounds included the single example of a eucaryotic alga, a Euglena, and a cyanobacterium (blue-green alga), 22 of 32 genera of bacteria, and 9 genera of yeasts. In the bacteria and yeasts the levels of quinone and hydroquinone were nearly equal and averaged about 3 nmol of each compound g-1 of packed cells. Included among the bacteria that contained these compounds were three examples from the newly proposed kingdom of Archaebacteriae. Those microorganisms that did not contain alpha-tocopherolquinol or alpha-tocopherolquinone tended to fall into two groups. One group consisted of gram-positive, anaerobic or facultative bacteria with a low content of guanine and cytosine, and the second group encompassed all of the filamentous microorganisms studied. No metabolic function is known for alpha-tocopherolquinol or its quinone other than as a cofactor in the biohydrogenation of unsaturated fatty acids that can be carried out by only a few organisms.
Carbamate kinase from Pseudomonas aeruginosa: purification, characterization, physiological role, and regulation
Authors: Abdelal A T , Bibb W F , Nainan O
Published: January 2020

Pseudomonas aeruginosa PAO1 possessed a carbamate kinase (CKase) distinct from carbamoylphosphate synthetase as well as from a constitutive acetate kinase which also catalyzes the phosphorylation of ADP by carbamoylphosphate. CKase was purified to homogeneity. Polyacrylamide gel electrophoresis of cross-linked CKase in the presence of sodium dodecyl sulfate showed that the enzyme consists of two subunits with identical molecular weights (37,000). The optimal pH of enzyme activity is 7.0. The double-reciprocal plot for carbamoylphosphate was linear at 2 mM ADP, yielding an apparent Km of 5 mM. However, at 0.25 mM ADP, the plot was concave upward, and a Hill plot of the data yielded a coefficient of 1.4. This apparent cooperativity at low ADP concentrations might serve to reduce the extent of catabolism of carbamoylphosphate under growth conditions yielding high energy charge. Experiments on the regulation of synthesis under various growth conditions showed a response to three regulatory signals: CKase was induced to high levels by anaerobiosis, induced to moderate levels by arginine, and repressed by ammonia. Thus, CKase expression is regulated in a manner that allows the enzyme to function as a provider of ammonia under aerobic conditions and of ATP under anaerobic conditions. ATP was an effective inhibitor of CKase activity; this inhibition provides the cell with an effective mechanism for avoiding a futile cycle resulting from the simultaneous operation of CKase and carbamoylphosphate synthetase when cells are grown in the presence of exogenous arginine.
Effects of Lysozyme and Inorganic Anions on the Morphology of
Streptococcus mutans
BHT: Electron Microscopic Examination
Authors: Cho Moon-Il , Holt Stanley C. , Iacono Vincent J. , Pollock Jerry J.
Published: January 2020

The effects of hen egg white lysozyme and the inorganic salt sodium thiocyanate on the integrity of
Streptococcus mutans
BHT were studied by transmission electron microscopy. Both control cells and cells exposed to NaSCN possessed thick outer cell walls and densely staining inner cell walls juxtaposed to the plasma membranes. In the presence of NaSCN, however, the
S. mutans
BHT nucleoid was coagulated into thick electron-dense filaments. Exposure of
S. mutans
BHT to 150 μg of hen egg white lysozyme per ml resulted in the progressive destruction of both the cell walls and the plasma membranes. The enzyme appeared to affect the region of the cell wall septum, and exposure to 150 μg of hen egg white lysozyme per ml for as short a time as 10 min resulted in visible morphological cell wall alterations. At 30 min, ultrastructural observations revealed that the majority of the cells were in the process of expelling a portion of their cytoplasmic contents from the septal and other regions of the cells at the time of fixation. After 3 h of incubation in the presence of this high lysozyme concentration, gelled protoplasmic masses, which were free from the cells, were evident. In addition, extensive damage to the outer and inner cell walls and to the plasma membranes was apparent, although the cells maintained their shape. On some areas of the cell surface, the outer cell wall and plasma membrane were completely absent, whereas at other locations the outer cell wall was either split away from the inner cell wall and plasma membrane or distended from an area free of inner cell wall and plasma membrane. Upon addition of NaSCN to the hen egg white lysozyme-treated cells, both the gelled protoplasmic masses and the damaged cells exhibited an exploded appearance and existed as membrane ghosts, cell wall fragments, or dense aggregates of cytoplasmic components. The effects of a low lysozyme concentration (22.5 μg/ml) on
S. mutans
morphology were less pronounced at short incubation times (i.e., 10 and 30 min) than those that were observed with a high enzyme concentration; however, breaks in the cell walls and dissolution of the plasma membranes with resulting cell lysis were visible after a prolonged (3-h) incubation and after subsequent addition of NaSCN.
Choline-containing bacteriophage receptors in Streptococcus pneumoniae
Authors: Lopez R , Garcia E , Garcia P , Ronda C , Tomasz A
Published: January 2020

Choline-containing teichoic acid seems to be essential for the adsorption of bacteriophage Dp-1 to pneumococci. This conclusion is based on the following observations: In contrast to pneumococci grown in choline-containing medium, cells grown in medium containing ethanolamine or other submethylated aminoalcohols instead of choline were found to be resistant to infection by Dp-1. Live choline-grown bacteria and heat- or UV-inactivated cells and purified cell walls prepared from these cells were capable of adsorbing phage Dp-1; ethanolamine-grown pneumococci or cell wall preparations were unable to do so. Adsorption of Dp-1 to choline-containing cell walls was competitively inhibited by phosphorylcholine and by several choline-containing soluble cell surface components, such as the Forssman antigen and the teichoic acid-glycan complexes formed by autolytic cell wall degradation. Cell walls prepared from pneumococci grown in ethanolamine or phosphorylethanolamine were inactive. Electron microscopic studies with pneumococci that had segments of choline-containing cell wall material amid ethanolamine-containing regions indicated that the Dp-1 phage particles adsorbed exclusively to the choline-containing surface areas. We suggest that the choline residues of the pneumococcal teichoic acid are essential components of the Dp-1 phage receptors in this bacterium.
Fructan from Erwinia herbicola
Authors: Blake J D , Clarke M L , Jansson P E , McNeil K E
Published: January 2020

Levan production by strains of Erwinia herbicola is common, and this property has some taxonomic significance for species differentiation within the "herbicola" group. The extracellular polysaccharide elaborated by strain 403 was characterized by nuclear magnetic resonance spectroscopy and methylation analysis. Results showed it to be a typical bacterial levan.
Effect of starvation on cytoplasmic pH, proton motive force, and viability of an acidophilic bacterium, Thiobacillus acidophilus
Authors: Zychlinsky E , Matin A
Published: January 2020

The question of whether Thiobacillus acidophilus maintains its cytoplasmic pH at values close to neutrality by active or passive means was explored by subjecting the organism to long-term starvation (up to 22 days). Starving cells maintained a delta pH of 2 to 3 U throughout starvation, although cellular poly-beta-hydroxybutyric acid and ATP, the proton motive force, and culture viability were low or not detectable after 200 h. Cells exposed to azide or azide plus N,N'-dicyclohexylcarbodiimide immediately exhibited characteristics of cells starved for more than 200 h. Thus, a large delta pH in T. acidophilus was maintained in the absence of ATP, ATPase activity, respiration, significant levels of proton motive force, and cell viability and was therefore not dependent on chemiosmotic ionic pumping. The transition from a metabolically active to an inactive state was accompanied by a large increase in the positive membrane potential, which nearly completely compensated for the delta pH in the inactive cells. The longevity of the acidophile during starvation was comparable to that reported previously for neutrophiles, and the loss of viability occurred not because of the acidification of the cytoplasm but apparently because of energy depletion.
Enzymatic activity of precursors of Bacillus megaterium spore protease
Authors: Hackett R H , Setlow P
Published: January 2020

The protease that initiates rapid proteolysis during germination of Bacillus megaterium spores is synthesized during sporulation as a 46,000-molecular-weight polypeptide (P46) and is processed later in sporulation to a 41,000-molecular-weight polypeptide (P41), which is converted to a 40,000-molecular-weight polypeptide (P40) early in spore germination. P40 is known to be both tetrameric and enzymatically active. In this work, we show that P46 and P41 are both tetrameric, but that only P41 is enzymatically active. The identification of a zymogen form (P46) of this protease explains in part the regulation of the activity of this enzyme.
Assessment of a futile cycle involving reconversion of fructose 6-phosphate to fructose 1,6-bisphosphate during gluconeogenic growth of Escherichia coli
Authors: Daldal F , Fraenkel D G
Published: January 2020

In gluconeogenesis, fructose 6-phosphate is formed from fructose 1,6-bisphosphate, and if fructose 1,6-bisphosphate were reformed by the phosphofructokinase reaction there would be a "gluconeogenic futile cycle." We assessed the extent of this cycling in Escherichia coli growing on glycerol 3-phosphate, using a medium containing 32Pi. Fructose 1,6-bisphosphate coming from glycerol 3-phosphate should be unlabeled, but any coming from fructose 6-phosphate should contain label from the gamma-position of ATP. The amount of labeling of the 1-position of fructose 1,6-bisphosphate was only 2 to 10% of that of the gamma-position of ATP in a series of isogenic strains differing in phosphofructokinases (Pfk-1, Pfk-2, or Pfk-2). In control experiments with glucose 6-phosphate instead of glycerol 3-phosphate, the two positions were equally labeled. Thus, although the presence of Pfk-2 causes gluconeogenic impairment (Daldal et al., Eur. J. Biochem., 126:373-379, 1982), gluconeogenic futile cycling cannot be the reason.
Regulation of the aromatic pathway in the cyanobacterium Synechococcus sp. strain Pcc6301 (Anacystis nidulans)
Authors: Hall G C , Flick M B , Jensen R A
Published: January 2020

A pattern of allosteric control for aromatic biosynthesis in cyanobacteria relies upon early-pathway regulation as the major control point for the entire branched pathway. In Synechococcus sp. strain PCC6301 (Anacystis nidulans), two enzymes which form precursors for L-phenylalanine biosynthesis are subject to control by feedback inhibition. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (first pathway enzyme) is feedback inhibited by L-tyrosine, whereas prephenate dehydratase (enzyme step 9) is feedback inhibited by L-phenylalanine and allosterically activated by L-tyrosine. Mutants lacking feedback inhibition of prephenate dehydratase excreted relatively modest quantities of L-phenylalanine. In contrast, mutants deregulated in allosteric control of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase excreted large quantities of L-phenylalanine (in addition to even greater quantities of L-tyrosine). Clearly, in the latter mutants, the elevated levels of prephenate must overwhelm the inhibition of prephenate dehydratase by L-phenylalanine, an effect assisted by increased intracellular L-tyrosine, an allosteric activator. The results show that early-pathway flow regulated in vivo by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase is the dominating influence upon metabolite flow-through to L-phenylalanine. L-Tyrosine biosynthesis exemplifies such early-pathway control even more simply, since 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase is the sole regulatory enzyme subject to end-product control by L-tyrosine.
Effects of culture age on symbiotic infectivity of Rhizobium japonicum
Authors: Bhuvaneswari T V , Mills K K , Crist D K , Evans W R , Bauer W D
Published: January 2020

The infectivity of the soybean symbiont Rhizobium japonicum changed two- to fivefold with culture age for strains 110 ARS, 138 Str Spc, and 123 Spc, whereas culture age had relatively little effect on the infectivity of strains 83 Str and 61A76 Str. Infectivity was measured by determining the number of nodules which developed on soybean primary roots in the zone which contained developing and preemergent root hairs at the time of inoculation. Root cells in this region of the host root are susceptible to Rhizobium infection, but this susceptibility is lost during acropetal development and maturation of the root cells within a period of 4 to 6 h (T. V. Bhuvaneswari, B. G. Turgeon, and W. D. Bauer, Plant Physiol. 66:1027-1031, 1980). Profiles of nodulation frequency at different locations on the root were not affected by the age of the R. japonicum cultures, indicating that culture age affected the efficiency of Rhizobium infection rather than how soon infections were initiated after inoculation. Inoculum dose-response experiments also indicated that culture age affected the efficiency of infection. Two strains, 61A76 Str and 83 Str, were relatively inefficient at all culture ages, particularly at low inoculum doses. Changes in infectivity with culture age were reasonably well correlated with changes in the proportion of cells in a culture capable of binding soybean lectin. Suspensions of R. japonicum in water were found to retain their viability and infectivity.
Phenotypic reversal in dam mutants of Escherichia coli K-12 by a recombinant plasmid containing the dam+ gene
Authors: Arraj J A , Marinus M G
Published: January 2020

A recombinant plasmid, pMQ3, carrying the dam gene of Escherichia coli K-12, was constructed and transformed into dam+ and dam- strains. Both dam- and dam+ strains containing pMQ3 showed a wild phenotype for all traits, including mutation rate, except for a 10-fold increase in DNA adenine methylase activity.
Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism
Authors: Crow V L , Davey G P , Pearce L E , Thomas T D
Published: January 2020

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis C10, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-beta-galactosidase activities were also absent in Lac- derivatives of strains H1 and 133 and were low (possibly absent) in C10 Lac-. In all three Lac- derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac- derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac- derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lac- mutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway.
ompT: Escherichia coli
K-12 Structural Gene for Protein
a
(3b)
Authors: Rupprecht K. R. , Gordon G. , Lundrigan M. , Gayda R. C. , Markovitz A. , Earhart C.
Published: January 2020

Chromosomal DNA from strain UT400, a previously described deletion mutant of
Escherichia coli
K-12 that lacks outer membrane protein
a
, failed to hybridize with plasmid DNA (pGGC110) containing the structural gene for protein
a
. We designate the genetic locus for protein
a
, located at approximately 12.5 min of the
E. coli
chromosome,
ompT
.
Cloning of the aerobactin-mediated iron assimilation system of plasmid ColV
Authors: Bindereif A , Neilands J B
Published: January 2020

The high-affinity iron assimilation system of plasmid ColV-K30 was cloned on the vector plasmid pPlac. Plasmid pABN1 was isolated by means of sensitivity to cloacin, a bacteriocin using the same outer membrane receptor as ferric aerobactin. Restriction maps were determined for this plasmid and for a subclone, pABN5. Plasmid pABN1 codes for the complete gene complex, whereas plasmid pABN5 encodes only the biosynthetic genes for aerobactin. Regulation of the uptake system by iron is retained in cloned sequences of pABN1.
Cloning, mapping, and expression of the fumarase gene of Escherichia coli K-12
Authors: Guest J R , Roberts R E
Published: January 2020

Two classes of fumarase-transducing phages, lambda fumA and lambda fumB, were isolated from populations of recombinant phages containing HindIII fragments of Escherichia coli DNA; they were isolated by virtue of their ability to complement the metabolic lesion of a fumarase-negative mutant. The strongly complementing lambda fumA phages contained a 6.2-kilobase HindIII fragment encoding: the fumA gene, located at 35.5 min in the E. coli linkage map and expressing the major fumarase activity; the mannosephosphate isomerase gene, manA; and an unidentified gene, g48. The three genes were located relative to the restriction map of the cloned fragment and the genetic linkage map (terC-g48-fumA-manA-uidAoR), their transcription polarities were defined as anticlockwise in the chromosome, and the molecular weights of the corresponding gene products were established: fumA, 61,500; manA, 42,000; g48, 48,000. Organisms containing the fumA gene sub-cloned in multicopy plasmids overproduced fumarase up to 50-fold. The weakly complementing class of transducing phages, lambda fumB, contained several genes in an 8.2-kilobase HindIII fragment, including one (fumB) that determines a minor fumarase activity. Complementation by fumB was only observed in high-copy situations such as transduction plaques and in strains containing a multicopy plasmid in which 40% of normal fumarase activity was detected. The basis for the complementation by fumB was not defined.
Yeast mutant defective in phosphatidylcholine synthesis
Authors: Greenberg M L , Klig L S , Letts V A , Loewy B S , Henry S A
Published: January 2020

The Saccharomyces cerevisiae opi3-3 mutant was shown to be defective in the synthesis of phosphatidylcholine via methylation of phosphatidylethanolamine. The opi3-3 mutant was isolated on the basis of an inositol excretion phenotype and was not auxotrophic for choline. Inositol, but not choline, stimulated growth of the mutant. The opi3-3 mutation was recessive and was genetically linked to the ino4 locus. When grown in the absence of exogenous choline, the opi3-3 mutant had a phospholipid composition consisting of 2 to 3% phosphatidylcholine compared with 40 to 50% in wild-type strains. In addition, the mutant accumulated elevated amounts of two intermediates, phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine. The incorporation of label from [methyl-14C]methionine into phosphatidylcholine was reduced 80 to 90% in the mutant compared with wild-type strains. However, label was recovered in the intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine. The mutant is believed to be defective in the third and possibly the second methylation reaction in the formation of phosphatidylcholine from phosphatidylethanolamine. The first methylation reaction appeared to be occurring at normal or even elevated levels. Based upon incorporation of choline into phosphatidylcholine, it is concluded that the opi3-3 mutant has no defect in the synthesis of phosphatidylcholine from exogenous choline. Furthermore, phosphatidylcholine represents over 25% of the phospholipid composition of the mutant when it is grown in the presence of exogenous choline.
Cloning, and expression in Escherichia coli K-12, of the chromosomal hemolysin (phospholipase C) determinant of Pseudomonas aeruginosa
Authors: Coleman K , Dougan G , Arbuthnott J P
Published: January 2020

A hemolysin determinant was cloned from Pseudomonas aeruginosa PA103 by inserting Sau3a-generated DNA fragments between the BamHI sites of the lambda replacement vector WL47.1. A 9.5-kilobase HindIII fragment encoding the hemolysin was subcloned from this phage and inserted into the plasmid vector pHC79 to generate the recombinant plasmid pKC95. Escherichia coli K-12 strains harboring pKC95 exhibited zones of hemolysis after several days of growth on blood agar plates. Hemolysis was shown to be due to phospholipase C activity by using the chromogenic substrate p-nitrophenylphosphorylcholine. Deletion mutants of pKC95 were isolated, and polypeptides expressed from these plasmids were examined by using the E. coli minicell system. A polypeptide of 78,000 daltons was associated with phospholipase C activity. The hemolytic activity was cell associated when expressed in E. coli.
Physiology of F-Pilin Synthesis and Utilization
Authors: Sowa Blair A. , Moore Deanna , Ippen-Ihler Karin
Published: January 2020

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to study the synthesis and turnover of F-pilin in membrane preparations of
Escherichia coli
K-12 under conditions which have been reported to affect the production of F-pili. Incorporation of [
35
S]methionine into membrane F-pilin by cells in log phase was barely detectable at 25°C, but increased with temperature. The labeled pilin band was prominent in membranes from 37°C cultures and even more prominent if the growth temperature was raised to 42°C. The appearance of other
tra
products in the membranes was similarly temperature dependent. In cultures grown in glucose minimal medium at 37°C, the relative amount of membrane pilin and
traT
product synthesis remained unchanged from early log phase through early stationary phase; provision of glycerol or arabinose as a substitute carbon source had no obvious effect. Turnover of
traT
product and membrane F-pilin, as assessed in an
Flac tra
mutant strain which is incapable of elaborating pili, was not rapid. Both
traT
product and pilin subunits labeled in mid-log phase cells were still apparent in the membranes after growth of the cells to stationary phase. The relative amount of labeled pilin decreased with prolonged incubation in stationary phase, but the relative amount of
traT
product did not decrease even after the culture was incubated for 24 h. When wild-type F
lac
piliated cells were used, a similar result was obtained, but in this case, loss of F-pilin from the preparations could be acclerated by blending the cells. Although intermittent blending during culture growth caused a slow depletion of the labeled pilin pool, continuous blending resulted in the rapid disappearance of this pool from our preparations. Loss of other membrane polypeptides was not accelerated by our blending procedure, and blending did not affect the turnover of the pilin pool of the F
lac tra
mutant. Our data are consistent with a model in which pilin subunits are assembled transiently into pili, conserved by retraction, and made available for subsequent reassembly. Growth in 0.01% sodium dodecyl sulfate did not accelerate loss of pilin from the F
lac
strain compared with the F
lac tra
strain, and we suggest that in the presence of sodium dodecyl sulfate at this concentration, F-pili are not elaborated from cell surfaces.
Mitochondrial translation products during release from glucose repression in Saccharomyces cerevisiae
Authors: Falcone C , Agostinelli M , Frontali L
Published: January 2020

Mitochondrial protein synthesis was studied during release from glucose repression in Saccharomyces cerevisiae cells bearing different mitochondrial genomes. The increase in the rate of the synthesis of mitochondrial translation products was analyzed during respiratory induction. Different kinetic patterns were found for strains having a different structure of mitochondrial mosaic genes, even when the nuclear background was the same. A very limited response of the synthesis of the var1 ribosomal protein to inducing conditions was observed.
Iron-binding compounds of Mycobacterium avium, M. intracellulare, M. scrofulaceum, and mycobactin-dependent M. paratuberculosis and M. avium
Authors: Barclay R , Ratledge C
Published: January 2020

Fifty-three strains of M. avium and related species all produced one or more exochelins, the extracellular iron-binding compounds of the mycobacteria, when grown iron deficiently. Only those strains which could grow without the addition of mycobactin (i.e., mycobactin independent) produced mycobactin, the intracellular iron-binding compound of the mycobacteria. Exochelins varied from 20 to 2,000 micrograms per g of cell dry weight; mycobactins were between 1 and 10 mg per g of cell dry weight. M. paratuberculosis (13 strains) and 13 strains of M. avium, both species dependent upon mycobactin for growth, failed to produce spectrophotometrically detectable amounts of mycobactin (less than 0.2 microgram per g of cell dry weight), although mycobactin could be recognized in one strain of M. avium grown with an additional supply of salicylate and examined by a radiolabeling technique. On repeated subculture three of the mycobactin-dependent strains of M. avium, but none of those of M. paratuberculosis, lost their mycobactin dependence and on reexamination were found to produce their own mycobactin at 0.3 mg per g of cell dry weight. It is concluded that mycobactin biosynthesis is probably strongly repressed in the mycobactin-dependent strains rather than being a genetic deletion. The exochelins, when examined by high-pressure thin-layer chromatography were revealed as being multiples of similar compounds, with up to 20 individual iron-binding compounds being recognizable with some strains. It is argued that the exochelins represent the single most important means of iron acquisition in mycobacteria growing in vitro and in vivo, and their elaboration by the fastidious M. paratuberculosis and related species explains how these organisms are able to grow in vivo in the absence of an external supply of mycobactin.
Development of Salt-Resistant Active Transport in a Moderately Halophilic Bacterium
Authors: Kushner D. J. , Hamaide Francette , MacLeod R. A.
Published: January 2020

The moderately halophilic bacterium
Vibrio costicola
accumulates α-aminoisobutyric acid (AIB) by active transport. Substantial amounts of Na
+
ions are needed for this transport. This is not due to an ionic requirement for respiration; cells respire as well as KCl as in NaCl but do not transport AIB in KCl. In cells grown in the presence of 1.0 or 2.0 M NaCl, AIB transport took place in higher NaCl concentrations than in cells grown in the presence of 0.5 M NaCl. The latter cells developed salt-resistant transport when they were exposed to 1.0 M NaCl in the presence of chloramphenicol and other antibiotics that inhibit protein synthesis. Two levels of salt-resistant transport were observed. One level (resistance to 3.0 M NaCl) developed in 1.0 M NaCl without the addition of nutrients, did not seem to require an increase in internal solute concentration, and was not lost when cells grown in 1.0 M NaCl were suspended in 0.5 M NaCl. The second level (resistance to 4.0 M NaCl) developed in 1.0 M NaCl only when nutrients were added, may have required an increased internal solute concentration, and was lost when 1.0 M NaCl-grown cells were suspended in 0.5 M NaCl or KCl. Among the substances that stimulated the development of salt-resistant AIB transport, betaine was especially active. Furthermore, direct addition of betaine permitted cells to transport AIB at higher NaCl concentrations. High salt concentrations inhibited endogenous respiration to a lesser extent than AIB transport, especially in 0.5 M NaCl-grown cells. Thus, these concentrations of salt did not inhibit AIB transport by inhibiting respiration. However, oxidation of glucose and oxidation of succinate were at least as sensitive to high salt concentrations as AIB transport, suggesting that a salt-sensitive transport step(s) is involved in the oxidation of these substrates.
Evidence for formation of superoxide and formate radicals in Methanobacterium formicicum
Authors: Barber M J , Rosen G M , Siegel L M , Rauckman E J
Published: January 2020

Using spin labeling and spin trapping techniques in combination with electron paramagnetic resonance spectrometry, we have detected the formation of superoxide by whole cells of Methanobacterium formicicum under aerobic conditions in the presence and absence of sodium formate. Rates of superoxide generation have been estimated. The formation of additional free radical species, including formate, was observed. Production of these and other free radicals resulted in lipid peroxidation and concomitant cell damage.
Labeling pattern of major penicillin-binding proteins of Escherichia coli during the division cycle
Authors: Wientjes F B , Olijhoek T J , Schwarz U , Nanninga N
Published: January 2020

Escherichia coli cells were synchronized by the elutriation technique. The pattern of penicillin-binding proteins (PBPs) in synchronously growing cells was determined with an iodinated derivative of ampicillin in intact cells as well as in isolated membranes. This was done under nonsaturating conditions as well as under conditions in which the PBPs were saturated with [125I]ampicillin. No evidence was found for fluctuations in the PBP pattern: the PBPs seem to be present in a constant ratio throughout the division cycle. The E. coli cells exert their control on shape maintenance and cell wall growth apparently not on the level of concentration of PBPs in the cell but rather on activation of existing components.
A new gene (alkB) of Escherichia coli that controls sensitivity to methyl methane sulfonate
Authors: Kataoka H , Yamamoto Y , Sekiguchi M
Published: January 2020

Seven mutants of Escherichia coli were isolated that are sensitive to methyl methane sulfonate but not to UV light. They exhibited decreased host cell reactivation capacity for methyl methane sulfonate-treated phage lambda. Five of the mutations were mapped in the same region as alkA (previously called alk) and may indeed be identical to known mutations. Another mutation was found near nalA, and the gene responsible was named alkB. Its phenotype was different from that of ada, since the alkB mutant exhibited a normal adaptive response to N-methyl-N'-nitro-N-nitrosoguanidine. A third type of mutation was mapped near polA, but this mutant contained an almost normal level of DNA polymerase I activity.
Recessive mutations conferring resistance to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae
Authors: Matsumoto K , Yoshimatsu T , Oshima Y
Published: January 2020

A total of 37 recessive mutations showing enhanced resistance to the glucose repression of galactokinase synthesis have been isolated by a selection procedure with a GAL81 gal7 double mutant. These mutations were grouped into three different complementation classes. One class, reg1, contains mutants arising from mutations at a site close to, but complementing, the gal3 locus. The reg1 mutant also showed resistance to the glucose repression of invertase synthesis but not to that of alpha-D-glucosidase. The two other classes were identified as arising from recessive mutations at the GAL82 locus and the GAL83 locus, respectively, at which various dominant mutations were isolated previously. When in a constitutive background due to the GAL81 or gal80 mutation, the GAL82 and GAL83 mutations did not show a mutually additive effect on the resistance to glucose repression of galactokinase synthesis, while the reg1 and GAL82 (or GAL83) mutations did. Based upon the specific behavior of cells with various genotypes for the above genes in response to the concentration of galactose and glucose in the medium, we propose a model involving three independent circuits for glucose signals in the regulation of the structural genes for the galactose pathway enzymes.
Transposon-facilitated chromosome mobilization in Agrobacterium tumefaciens
Authors: Pischl D L , Farrand S K
Published: January 2020

We improved chromosomal gene transfer in Agrobacterium tumefaciens strain 15955 by constructing donors containing homologous transposons on both the sex factor plasmid and chromosome. First, we constructed plasmid pDP35, a kanamycin-sensitive derivative of R68.45. We then constructed derivatives of pDP35 that contained insertions of the kanamycin resistance transposon Tn5. By restriction endonuclease analysis, we identified two plasmids, pDP37 and pDP38, in which Tn5 was inserted in the same region of the plasmid but in opposite orientations. We also constructed isolates of A. tumefaciens containing an insertion of Tn5 in the chromosome. We transferred pDP37 or pDP38 into these chromosomal Tn5 strains and tested their ability to mobilize chromosomal markers to a series of auxotrophic recipients. Mobilization was observed at frequencies ranging from 10(-4) to 10(-7) recombinants per input donor for most markers tested. Both the plasmid and the chromosomal Tn5 elements were found to be required for mobilization at these higher frequencies. Donors were shown to transfer chromosomal markers in a polarized fashion. Recombinants coinherited unselected markers at frequencies of from 100 to 0.3 percent. The improved transfer frequencies and the observed polarity in chromosome transfer suggest that with this method we can genetically characterize A. tumefaciens chromosomal functions.
Relationship between phosphorylation potential and electrochemical H+ gradient during glycolysis in Streptococcus lactis
Authors: Maloney P C
Published: January 2020

Assays of intracellular ATP, ADP, and inorganic phosphate allowed calculation of the phosphorylation potential (delta G'ATP/F) maintained during glycolysis by Streptococcus lactis. At the same time, the electrochemical H+ gradient (delta mu-H+/F) was evaluated by distribution methods, using radioactive tetraphenylphosphonium bromide as a probe for the membrane potential and salicylic acid as a probe for the pH gradient. Detailed comparisons were made at pH 5, when the reaction mediated by the proton-translocating ATPase (BF0F1) was likely to have been poised near equilibrium; for those conditions, the ratio delta G'ATP/delta mu-H+ was used to estimate stoichiometry for BF0F1 during ATP hydrolysis. At an external pH of 5, in the presence or absence of valinomycin, this ratio was close to 3, over a range of 370 to 510 mV (8.5 to 11.7 kcal/mol) for delta G'ATP/F and a range of 128 to 167 mV for delta mu-H+/F. Other work suggested that delta G'ATP/delta mu-H+ increased from its minimum value of 3 to 4.3 as the external pH changed from pH 5 to 7.
Integration of the overproduced bacteriophage T5 receptor protein in the outer membrane of Escherichia coli
Authors: Menichi B , Buu A
Published: January 2020

The tonA gene codes for an outer membrane protein, a receptor of phage T5, the TonA protein. Strains harboring pLG513, a multicopy plasmid in which the tonA gene has been cloned, overproduced TonA protein, which appeared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell envelope proteins as a 78,000-molecular-weight protein. Identical results have been observed by Plastow et al. (FEBS Lett. 131:262-264, 1981) with plasmid pLC19-19, in which the tonA gene has also been cloned. The activity of the TonA protein, measured by its capacity to inactivate phage T5, increased by five- to sixfold in purified envelopes of cells harboring pLG513 compared with cells lacking the plasmid. Solubilization of the cytoplasmic membrane by Triton-Mg2+ treatment did not increase this activity. However, partial solubilization of outer membrane proteins by Triton-EDTA unmasked further T5 receptor activity, resulting in a final increase of around 50-fold, a value more consistent with the expected gene dosage effect. Treatment of whole cells by trypsin in conditions in which trypsin is allowed to enter the outer membrane revealed that part of the overproduced T5 receptors were embedded in the outer membrane and masked by a trypsin-sensitive protein. In addition, no T5 receptor activity was detected in either the periplasmic space or the cytoplasm. These results suggest that all of the overproduced TonA molecules were synthesized in an active form and integrated in the outer membrane, but only a small fraction could be reached or recognized by phage T5 in vivo.
Cell Wall Receptor for Yeast Killer Toxin: Involvement of (1 → 6)-β-
d
-Glucan
Authors: Hutchins Kendrick , Bussey Howard
Published: January 2020

The linear (1 → 6)-β-
d
-glucans pustulan and luteose were effective competitive inhibitors of killer toxin action. Affinity chromatography of killer toxin on a pustulan-Sepharose column showed that toxin bound directly to a (1 → 6)-β-linked polysaccharide. Other polysaccharides found in yeast cell walls, including (1 → 3)-β-
d
-glucan, mannan, chitin, and glycogen, were not effective as inhibitors of toxin. Fractionation of yeast cell walls was attempted to identify the toxin receptor in sensitive
Saccharomyces cerevisiae
. The receptor activity was retained among the insoluble glucans in alkali-washed cells; yeast mannan and alkali-soluble glucan had little receptor activity. A minor fraction of receptor activity was removed from alkali-washed cells by hot acetic acid extraction, a procedure which solubilized some (1 → 6)-β-
d
-glucan and glycogen. The major fraction (>70%) of receptor activity remained with the acid-insoluble (1 → 6)-β-and (1 → 3)-β-glucans. Zymolyase, an endo-(1 → 3)-β-
d
-glucanase, solubilized a substantial fraction of the receptor activity in the acid-insoluble glucans. The receptor activity in yeast cell walls was periodate and (1 → 6)-β-
d
-glucanase sensitive, but was resistant to (1 → 3)-β-
d
-glucanase and α-amylase. The acid-soluble glucan fractions of a sensitive strain and a
krel-l
receptor-defective toxin-resistant mutant were examined. The
krel-l
strain had a reduced amount (ca. 50%) of (1 → 6)-β-
d
-glucan compared with the sensitive parent strain. A sensitive revertant of the
krel-l
strain regained the parental level of glucan. These results implicate (1 → 6)-β-
d
-glucan as a component of the yeast cell wall receptor for killer toxin.
Genetic and Biochemical Analyses ofEscherichia coliMutants Altered in the Temperature-Dependent Regulation of Membrane Lipid Composition
Authors: Ulrich Amy Klages , de Mendoza Diego , Garwin Jeffrey L. , Cronan John E.
Published: January 2020

We have previously studied two mutants ofEscherichia colialtered in the regulation of membrane lipid composition by temperature. One class (represented by thefabFlallele) fails to regulate upon temperature shift and is defective incis-vaccenic acid synthesis owing to the lack of the fatty acid elongation enzyme β-ketoacyl-acyl carrier protein synthase II(EC 2.3.1.41). A second class of mutant, given the phenotypic designation Vtr, overproducescis-vaccenic acid at all temperatures and hence is altered in temperature regulation. In this paper we report evidence for the following conclusions. (i) The Vtr andfabFlmutations show very tight genetic linkage. (ii) The Vtr lesion is allelic to thefabFlmutation since the presence of thefabFlmutation in merodiploid strains carrying the Vtr orfabF+alleles results in fatty acid compositions intermediate between those of the two monoploid strains. Merodiploids carrying both thefabF+and Vtr alleles likewise show an intermediate composition. These results indicate intra-allelic complementation. (iii) The twoE. coliproteins recently discovered by Rock (J. Bacteriol.152:1298-1300, 1982) that form mixed disulfide cross-links to acyl carrier protein are directly demonstrated to be β-ketoacyl-acyl carrier protein synthases I and II. (iv) ThefabFlstrains produce a synthase II band of altered electrophoretic mobility, indicating that thefabFlocus is the structural gene for synthase II. (v) The synthase II of Vtr strains is abnormally sensitive to cerulenin, an antibiotic that specifically inhibits synthases I and II. This increased sensitivity is readily demonstrated in vivo, but in vitro we failed to detect an increased sensitivity of the Vtr synthase II to cerulenin, nor have we detected any other kinetic or structural alteration in the enzyme. We interpret these results in terms of specific interactions of synthase II with other cellular components which occur in vivo but are not duplicated in vitro.
Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels
Authors: Hitchcock P J , Brown T M
Published: January 2020

The morphological heterogeneity of lipopolysaccharides (LPSs) among salmonella mutants with different LPS chemotypes was analyzed in silver-stained polyacrylamide gels. The biochemical differences in the LPS chemotypes were reflected in the unique profiles of the purified LPSs. The LPS profiles in the whole-cell lysates were also unique for each chemotype. (Whole-cell lysates were assessed by a method which preferentially silver stains LPS and by a proteinase K digest of whole-cell lysates. The silver-stained LPS profiles of proteinase K-digested lysates were similar to the homologous purified LPS and could be used to preliminarily characterize the LPS chemotype before purification.) In summary, biochemical variation in LPS composition can be detected in silver-stained polyacrylamide gels.
Adaptation kinetics in bacterial chemotaxis
Authors: Block S M , Segall J E , Berg H C
Published: January 2020

Cells of Escherichia coli, tethered to glass by a single flagellum, were subjected to constant flow of a medium containing the attractant alpha-methyl-DL-aspartate. The concentration of this chemical was varied with a programmable mixing apparatus over a range spanning the dissociation constant of the chemoreceptor at rates comparable to those experienced by cells swimming in spatial gradients. When an exponentially increasing ramp was turned on (a ramp that increases the chemoreceptor occupancy linearly), the rotational bias of the cells (the fraction of time spent spinning counterclockwise) changed rapidly to a higher stable level, which persisted for the duration of the ramp. The change in bias increased with ramp rate, i.e., with the time rate of change of chemoreceptor occupancy. This behavior can be accounted for by a model for adaptation involving proportional control, in which the flagellar motors respond to an error signal proportional to the difference between the current occupancy and the occupancy averaged over the recent past. Distributions of clockwise and counterclockwise rotation intervals were found to be exponential. This result cannot be explained by a response regular model in which transitions between rotational states are generated by threshold crossings of a regular subject to statistical fluctuation; this mechanism generates distributions with far too many long events. However, the data can be fit by a model in which transitions between rotational states are governed by first-order rate constants. The error signal acts as a bias regulator, controlling the values of these constants.
Confirmation of protoplast fusion-derived linkages in Staphylococcus aureus by transformation with protoplast DNA
Authors: Stahl M L , Pattee P A
Published: January 2020

Transformation provided definitive evidence for linkage between tyrB282::Tn551 ermB321 and omega (Chr::Tn551)34, and thus between the separate large linkage groups containing these markers, in Staphylococcus aureus NCTC 8325. Transformation also defined the chromosomal loci for the purC193::Tn551 and omega (Chr::Tn551)42 markers and the linkage of a tetracycline resistance marker (tet-3490) with a fusidic acid resistance marker (fus-149). The use of DNA isolated from protoplasts under conditions that reduced hydrodynamic shear greatly facilitated the demonstration of most of these linkages. These results provide direct evidence confirming several of the linkages predicted by a microcomputer-assisted protoplast fusion analysis in a previous study (M. L. Stahl and P. A. Pattee, J. Bacteriol. 154:395-405, 1983); those markers whose predicted linkages were not confirmed by transformation are probably separated by chromosomal distances that exceed the limits of detection by transformation, even with protoplast DNA.
Replication control mutations of plasmid R6-5 and their effects on interactions of the RNA-I control element with its target
Authors: Brady G , Frey J , Danbara H , Timmis K N
Published: January 2020

Nine high copy number mutations of plasmid R6-5, representing five phenotypically distinct groups, have been identified by DNA sequencing. In each mutant plasmid examined, a single nucleotide change was found. The effects of the mutations on possible gene products, and DNA-RNA secondary structure, were analyzed and compared with the observed phenotypes. The results of this study exclude the possibility that the primary plasmid replication control element, the product of the copA gene, is a polypeptide, and they are consistent with a model of plasmid replication control by the copA product which has the following features: (i) RNA-I, a short untranslated RNA molecule, is the product of the copA gene and regulates the frequency of initiation of plasmid replication, (ii) the hexanucleotide single-strand loop of the major hairpin of RNA-I is its active site, (iii) this active site functions by base pair interactions with its "target," its DNA template strand, or its complementary sequence on RNA-II, a transcript of opposite polarity that is the message of the repA gene, and (iv) the sequence and size of the loop, and the stability of the stem of the hairpin, are all critical factors that govern the functioning of RNA-I.
Conditional change of DNA replication control in an RNA polymerase mutant of Escherichia coli
Authors: Rasmussen K V , Atlung T , Kerszman G , Hansen G E , Hansen F G
Published: January 2020

A temperature-sensitive mutant of Escherichia coli with a temperature-dependent change in the control of initiation of DNA replication was isolated. The phenotype of the mutant was dependent on a mutation in the RNA polymerase gene rpoC. In vitro RNA polymerase activity was temperature sensitive. The mutant grew and synthesized DNA at 30 degrees C as did the wild type. After a shift to 39 degrees C, a temperature still permissive for growth, the mutant increased its origin concentration more than twofold. After a shift from 39 to 30 degrees C, initiation of DNA replication was inhibited until the normal origin concentration was reestablished.