bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and localization of two membrane-bound esterases from Escherichia coli
Authors: Pacaud M
Published: January 2020

Hydrolytic activities of isolated membrane fractions of Escherichia coli against chromogenic substrates, p-nitrophenyl ester and beta-naphthyl ester derivatives of N-substituted amino acids, were investigated by spectrophotometric and electrophoretic methods. Although detergents were absolutely necessary for the solubilization of enzymes, the amount of solubilized activities was increased by adding salt, such as NaCl or KCl. Two esterases were identified and separated by PAGE and by chromatography of the solubilized proteins in the presence of detergent. One hydrolyzed the alanine derivatives preferentially, whereas the other was mainly active on phenylalanine derivatives. Only the first was inactivated by diisopropyl fluorophosphate, a serine hydrolase inhibitor. Whereas the chymotrypsin-like enzyme was equally distributed between the inner and the outer membrane, the alanine activity was only detected in the inner membrane. They were both resistant to extraction with high salt concentrations, indicating their integral association with membranes. A study of the accessibility of these enzymes to their substrate in membrane vesicles with known polarity suggests that both alanine and phenylalanine activities are localized near the external surface of the cytoplasmic (inner) membrane. However, the phenylalanine activity (chymotrypsin-like enzyme) appears to be deeply buried inside the outer membrane. Because of its insensitivity to diisopropyl fluorophosphate, this last esterase seems to be distinct from the previously isolated periplasmic endopeptidase, protease I, which is also a chymotrypsin-like enzyme.
Dansyl chloride labeling of Pseudomonas aeruginosa treated with pyocin R1: change in permeability of the cell envelope
Authors: Uratani Y
Published: January 2020

Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, caused an increase in binding of fluorescent label, 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride), to sensitive cells. In pyocin R1-treated cells, cytoplasmic soluble proteins and crude ribosomes as well as cell envelopes were labeled by dansyl chloride. The amount of bound dye was proportional to the multiplicity of pyocin R1 and reached a maximal level at high multiplicity. In addition, pyocin R1 rapidly caused an increase in fluorescence intensity of the hydrophobic probes N-phenyl-1-naphthylamine, pyrene, and perylene, which were mixed with cells. These results show that pyocin R1 damages locally a cell envelope barrier to hydrophobic solutes and allows dyes to penetrate into the intracellular space across the barrier.
Salmonella typhimurium mutants defective in the formate dehydrogenase linked to nitrate reductase
Authors: Barrett E L , Riggs D L
Published: January 2020

Six fdn mutants of Salmonella typhimurium defective in the formation of nitrate reductase-linked formate dehydrogenase (FDHN) but capable of producing both the hydrogenase-linked formate dehydrogenase (FDHH) and nitrate reductase were characterized. Results of phage P22 transduction experiments indicated that there may be three fdn genes located on the metE-metB chromosomal segment and distinct from all previously identified fdh and chl loci. All six FDHH+ FDHN- mutants were found to make FDHN enzyme protein which was indistinguishable from that of the wild type in electrophoretic studies. However, the results of the spectral studies indicated that all six mutants were defective in the anaerobic cytochrome b559 associated with FDHN. All contained the cytochrome b559 associated with nitrate reductase in amounts equal to or greater than the wild type. The results of the transduction experiments also indicated that the metE- metB segment of the Salmonella chromosome resembles that of Escherichia coli more than was originally thought.
Microcalorimetric studies of the growth of sulfate-reducing bacteria: comparison of the growth parameters of some Desulfovibrio species
Authors: Traore A S , Hatchikian C E , Le Gall J , Belaich J P
Published: January 2020

We performed a comparative study of the growth energetics of some species of Desulfovibrio by measuring microcalorimetric and molar growth yield values. Lactate and pyruvate were used as energy sources for sulfate reduction. On lactate-sulfate media Desulfovibrio desulfuricans Norway, Desulfovibrio gigas, and Desulfovibrio africanus exhibited molar growth yields of 4.1 +/- 0.6, 3.7 +/- 1.7, and 1.8 +/- 0.1 g/mol, respectively, whereas on pyruvate-sulfate media the molar growth yields were higher (8.5 +/- 0.8, 7.7 +/- 1.6, and 3.5 +/- 0.5 g/mol, respectively). Thus, we found that D. africanus was the least efficient species in converting energy into cell material. The uncoupling of energy in this strain was obvious since its catabolic activities were high compared with those of the two other strains. The enthalpy changes associated with lactate and pyruvate metabolism were -49 +/- 0.7 and -70.2 +/- 6.0 jK/mol, respectively, for D. desulfuricans, -76.6 +/- 1.8 and -91.2 +/- 1.1 kJ/mol, respectively, for D. gigas, and -78.8 +/- 7.2 and -88.0 +/- 6.2 kJ/mol, respectively, for D. africanus. D. gigas and D. africanus produced only acetate, CO2 and hydrogen sulfide as metabolic end products. In addition to these normal end products, D. desulfuricans Norway produced a small amount of butanol. This butanol production was interpreted as reflecting a regulatory system of electron flow during the catabolism of both substrates. Such metabolism was comparable to that reported for D. vulgaris, which lost part of the reducing power of its energy sources through hydrogen evolution.
Purification of pyruvate formate-lyase from Streptococcus mutans and its regulatory properties
Authors: Takahashi S , Abbe K , Yamada T
Published: January 2020

Pyruvate formate-lyase (EC 2.3.1.54) from Streptococcus mutans strain JC2 was purified in an anaerobic glove box, giving a single band on disk and sodium dodecyl sulfate electrophoresis. This enzyme was immediately inactivated by exposure to the air. Enzyme activity was unstable even when stored anaerobically, but the activity was restored by preincubating the inactivated crude enzyme with S-adenosyl-L-methionine, oxamate, and reduced for ferredoxin or methylviologen. On the other hand, the purified enzyme was not reactivated. Either D-glyceraldehyde 3-phosphate or dihydroxyacetone phosphate strongly inhibited this enzyme. The inhibitory effects of these compounds were largely influenced by enzyme concentration. The inhibition of these triose phosphates in cooperation with the reactivating effect of ferredoxin and the fluctuations of both the enzyme and the triose phosphate levels may efficiently regulate the pyruvate formate-lyase activity in S. mutans in vivo.
The B66.0 protein of Escherichia coli is the product of the dnaK+ gene
Authors: Georgopoulos C , Tilly K , Drahos D , Hendrix R
Published: January 2020

B66.0 is one of the most abundant proteins of Escherichia coli. Its relative rate of synthesis is highly regulated depending on temperature and the growth rate of the culture. We identified the B66.0 protein to be the dnaK+ structural gene product since dnaK756 mutant bacteria synthesized a B66.0 protein with a more acidic isoelectric point.
Identification of citrate utilization transposon Tn3411 from a naturally occurring citrate utilization plasmid
Authors: Ishiguro N , Sato G , Sasakawa C , Danbara H , Yoshikawa M
Published: January 2020

We have isolated a new transposon, Tn3411, encoding citrate-utilizing ability, from a naturally occurring citrate utilization (Cit) plasmid, pOH3001. Citrate transposon Tn3411 was transposed from pOH3001 to lambda b519 b515 cI857 S7 (abbreviated lambda bb) phage, and further from the resulting lambda bb:Tn3411 to a vector plasmid, pBR322, in recA-deficient strains. The Cit+ plasmids (pOH2 and pOH3) constructed by the integration of Tn3411 into pBR322 were examined by restriction endonuclease and heteroduplex analysis. The results obtained were as follows: (i) Tn3411 was 7.4 kilobases long and flanked by small inverted repeats, and it contained one more pair of inverted repeats at the opposite orientation in the internal region, thus making alternate repeats; and (ii) the Cit+ structure gene was located on the fragment (5.5 kilobases) between two SalI cleavage sites on Tn3411.
Cell wall turnover in growing and nongrowing cultures of Bacillus subtilis
Authors: de Boer W R , Meyer P D , Jordens C G , Kruyssen F J , Wouters J T
Published: January 2020

Cell wall turnover was studied in cultures of Bacillus subtilis in which growth was inhibited by nutrient starvation or by the addition of antibiotics. Concomitantly, the synthesis of wall, as measured by the incorporation of radioactively labeled N-acetylglucosamine, was followed in some of these cultures. In potassium- or phosphate-starved cultures, growth stopped, but wall turnover continued at a rate slightly lower than that in the control cultures. Lysis of cells did not occur. In glucose-starved cultures, continued wall turnover caused lysis of cells, since wall synthesis apparently was inhibited. The same phenomenon was observed after growth arrest by the addition of wall synthesis inhibitors such as fosfomycin, cycloserine, penicillin G, and vancomycin. Growth arrest by the addition of chloramphenicol allowed the continuation of wall synthesis; therefore, the observed turnover generally did not cause cell lysis.
Electrical nature of the taxis signal in cyanobacteria
Authors: Murvanidze G V , Glagolev A N
Published: January 2020

Electrical events after a light-dark stimulus were studied in the multicellular organism Phormidium uncinatum. Normally, such a stimulus causes the gliding trichome to reverse direction. By directing a large light spot on the end of a batch of trichomes and then switching it off, we achieved synchronization of the trichomes, since the "head" is much more sensitive than the "tail." The abrupt disappearance of a uniform light produced a depolarization wave which initiated at the head, as registered by externally applied electrodes. The second stimulus produced a depolarization of the opposite direction, reflecting the reorientation of the trichomes. No electrical response was observed at Ca2+ concentrations less than or equal to 10(-8) M. Factors causing oscillatory reversals, i.e., a combination of Ca2+ and A23187, or a viscous environment also abolished the electrical signal. Changes in an externally applied electrical field (4 V/cm2) had little effect on the motile behavior of P. uncinatum or Oscillatoria princeps. However, in the presence of 5 microM Ca2+-1 microM A23187, all the trichomes reversed synchronously to the anode after a change in polarity of an externally applied electrical field. We suggest that an increased Ca2+ concentration together with a change in delta psi (or delta mu H+) represents the taxis signal in cyanobacteria.
The catabolite-sensitive promoter for the chloramphenicol acetyl transferase gene is preceded by two binding sites for the catabolite gene activator protein
Authors: Le Grice S F , Matzura H , Marcoli R , Iida S , Bickle T A
Published: January 2020

DNase I protection experiments have indicated that the cyclic AMP-catabolite gene activator protein complex binds to two regions preceding the chloramphenicol acetyl transferase (cat) gene in Escherichia coli. One of these lies adjacent to the RNA polymerase binding site, whereas the second lies approximately 130 base pairs upstream from the starting point of transcription. Additional DNase protection experiments and in vitro transcription experiments with modified templates indicate that the catabolite gene activator protein site proximal to the cat promoter functions independently of the distal site, indicating that in vitro the second of these sites is not required for transcriptional activation of the cat gene.
Resident enhanced repair: novel repair process action on plasmid DNA transformed into Escherichia coli K-12
Authors: Strike P , Roberts R J
Published: January 2020

The survival of UV-irradiated DNA of plasmid NTP16 was monitored after its transformation into recipient cells containing an essentially homologous undamaged plasmid, pLV9. The presence of pLV9 resulted in a substantial increase in the fraction of damaged NTP16 molecules which survived in the recipient cells. This enhanced survival requires the host uvrA+ and uvrB+ gene products, but not the host recA+ gene product. The requirement for both homologous DNA and the uvrA+ and uvrB+ gene products suggests that a novel repair process may act on plasmid DNA. Possible mechanisms for this process are considered.
Purification and Properties of Cellobiosidase from
Ruminococcus albus
Authors: Ohmiya Kunio , Shimizu Masatoshi , Taya Masahito , Shimizu Shoichi
Published: January 2020

An enzyme active against
p
-nitrophenyl-β-
d
-cellobioside was purified from
Ruminococcus albus
to homogeneity. The enzyme was identified as a cellobiosidase.
Transposition of the carbenicillin-hydrolyzing beta-lactamase gene
Authors: Katsu K , Inoue M , Mitsuhashi S
Published: January 2020

We isolated a new transposon Tn2101, from plasmid Rms433 in Enterobacter cloacae. Tn2101 encoded the formation of type IV (carbenicillin-hydrolyzing) beta-lactamase and multiple resistance to streptomycin, sulfanilamide, spectinomycin, and mercury in addition to ampicillin. Tn2101 was transposable between conjugative (or nonconjugative) plasmids and the host chromosome. Transposition occurred independently of the general recombination ability of the host cell. Tn2101 had a molecular size of 9.5 x 10(6) and contained short inverted repeat terminal sequences.
Adenine nucleotide and lysine transport in Chlamydia psittaci
Authors: Hatch T P , Al-Hossainy E , Silverman J A
Published: January 2020

Isolated reticulate bodies of Chlamydia psittaci were found to transport ATP and ADP by an ATP-ADP exchange mechanism. ATP uptake activity was not detected in elementary bodies. The apparent Km of transport for both ATP and ADP was approximately 5 microM, and the calculated Vmax for both was about 1 nmol of nucleotide transported per min per mg of protein. ADP competitively inhibited ATP transport with a Ki of 4.5 microM. Other nucleotides tested had no effect on the uptake of ATP. A magnesium-dependent, oligomycin-sensitive ATPase (ATP phosphohydrolase, EC 3.6.1.3) was associated with reticulate bodies, and most of the transported ATP was hydrolyzed to ADP, which was exchanged for additional, extracellular nucleotide. Some ADP was hydrolyzed to AMP, which exited the cells slowly. Lysine was transported against the electrochemical gradient by reticulate bodies in the presence of ATP. Oligomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited ATP-dependent lysine transport. Lysine exited reticulate bodies when the reticulate bodies were incubated in the presence of ADP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or a reduced concentration of ATP. The results support the concept that chlamydiae are energy parasites which are capable of drawing upon the adenine nucleotides of their hosts, hydrolyzing ATP, and establishing an energized membrane.
Osmotically induced changes in the ornithine decarboxylase activity of Dictyostelium discoideum
Authors: Harris W A , North M J
Published: January 2020

Myxamoebae of Dictyostelium discoideum from exponentially growing cultures showed altered ornithine decarboxylase activity upon external osmotic perturbation. On transfer to hypotonic NaCl solutions (20 mosmol/kg), cells showed high enzyme activity which was relatively independent of the concentration of the coenzyme pyridoxal phosphate (assay concentrations, 5 and 200 microM). In hypertonic solution (400 mosmol/kg) cells had a reduced level of ornithine decarboxylase activity which was dependent on the coenzyme concentration. The changes in activity were freely reversible in further external osmotic manipulation. The response to osmotic change occurred rapidly, within a few minutes. The changes still occurred at 7 degrees C but 2 mM sodium azide prevented the formation of the high activity form, although this effect was reversed when azide was removed. Cycloheximide had no effect on the osmotically induced changes. Addition of putrescine caused ornithine decarboxylase eventually to the converted to the low-activity form regardless of the osmolality of the solution. The characteristic cofactor concentration dependence of the high- and low-activity form were retained on storage of the cell extracts. No evidence was found for diffusible effectors which stabilized one or the other form of the activity. The enzymes responsible for the two forms were of the same molecular size as judged by gel filtration, and the activities had similar thermostabilities. The results are interpreted in terms of an osmotically induced interconversion of two forms of a single ornithine decarboxylase.
Energetics of vacuolar compartmentation of arginine in Neurospora crassa
Authors: Drainas C , Weiss R L
Published: January 2020

The energy requirements for the uptake and retention of arginine by vacuoles of Neurospora crassa have been studied. Exponentially growing mycelial cultures were treated with inhibitors of respiration or glycolysis or an uncoupler of respiration. Catabolism of arginine was monitored as urea production in urease-less strains. The rationale was that the rate and extent of such catabolism was indicative of the cytosolic arginine concentration. No catabolism was observed in cultures treated with an inhibitor or an uncoupler of respiration, but cultures treated with inhibitors of glycolysis rapidly degraded arginine. These differences could not be accounted for by alterations in the level or activity of arginase. Mycelia growing in arginine-supplemented medium and treated with an inhibitor or uncoupler of respiration degraded an amount of arginine equivalent to the cytosolic fraction of the arginine pool. The inhibitors and the uncoupler of respiration reduced the ATP pool and the energy charge. The inhibitors of glycolysis reduced the ATP pool but did not affect the energy charge. The results suggest that metabolic energy is required for the transport of arginine into the vacuoles but not for its retention. The latter is affected by inhibitors of glycolysis. The form of energy and the nature of the vacuolar transport mechanism(s) are discussed.
Conformation and Segregation of Nucleoids Accompanying Cell Length Extension After Completion of a Single Round of DNA Replication in Germinated and Outgrowing
Bacillus subtilis
Spores
Authors: Hariharan I. K. , Czolij R. , Wake R. G.
Published: January 2020

When germinating spores of the temperature-sensitive DNA initiation mutant of
Bacillus subtilis
TsB134 are shifted to the restrictive temperature at a time such that just one or two rounds of replication are accomplished, the completed, nonreplicating nucleoids that form eventually adopt a doublet conformation. This conformation has now been observed after fixation by glutaraldehyde or osmium tetroxide, as well as by Formalin as found previously. The doublet was observed in media of different degrees of richness and under both light and electron microscopes. Electron micrographs of serial sections through the doublet were consistent with its formation by the gradual pulling apart of a single mass of DNA into two lobes. A systematic study was made of the effect of the time of shifting from the permissive to the restrictive temperature and of the restrictive temperature used on the number of nucleoids segregating within the outgrowing rod. It was established that the doublet nucleoid behaved as a single unit in replication control and segregation in both rich and poor media. Measurement of the relative position of the two segregating nucleoids within the outgrowing rod after completion of just one round of replication yielded quantitative information on the segregation and cell length extension processes. Segregation was accompanied by cell length extension at approximately equal rates on both sides of each nucleoid. Furthermore, the data were consistent with an exponential increase in such an extension with time over the early and major portion of the period studied, but it was not possible to rule out other models of length extension.
Changes in the organization of the outer membrane of Proteus mirabilis during swarming: freeze-fracture structure and membrane fluidity analysis
Authors: Armitage J P
Published: January 2020

Freeze-fracture studies of short, nonswarming Proteus mirabilis revealed the characteristic gram-negative profile of fractured inner membrane with densely packed particles and sectioned outer membrane with little or no fracture plane. Long swarming cells, however, fractured easily along both the inner membrane and a second membrane, probably the outer membrane. The inner membrane had a typical profile, whereas the outer membrane had fewer but more prominent particles. Isolation and purification of the inner and outer membranes of the short and long bacteria and examination of them with electron paramagnetic resonance measurements after spinlabeling supported the above observations. The outer membrane of swarmer cells allowed higher mobility of the spin label than did the outer membrane of the nonswarming short cells, which showed a typical rigid profile. These results suggest that regions of lipid bilayer appear in the outer membrane during swarmer formation. Previous observation of the behavior and biochemistry of P. mirabilis during swarming are discussed in light of these results.
Oxygen-induced genetic changes in dry yeast cells
Authors: Hieda K
Published: January 2020

Cells of Saccharomyces cerevisiae were dried in vacuum, exposed to oxygen, nitrogen, air, and water vapor, and rehydrated with degassed medium without exposure to air. Drying per se caused few genetic changes, but the exposure of dry cells to oxygen increased the frequency of adenine-requiring colonies.
Metabolic Basis for the Isoleucine, Pantothenate or Methionine Requirement of
ilvG
Strains of
Salmonella typhimurium
Authors: Primerano Donald A. , Burns R. O.
Published: January 2020

Salmonella typhimurium
strain DU501, which was found to be deficient in acetohydroxy acid synthase II (AHAS II) and to possess elevated levels of transaminase B and biosynthetic threonine deaminase, required isoleucine, methionine, or pantothenate for growth. This strain accumulated α-ketobutyrate and, to a lesser extent, α-aminobutyrate. We found that α-ketobutyrate was a competitive substrate for ketopantoate hydroxymethyltransferase, the first enzyme in pantothenate biosynthesis. This competition with the normal substrate, α-ketoisovalerate, limited the supply of pantothenate, which resulted in a requirement for methionine. Evidence is presented to support the conclusion that the ambivalent requirement for either pantothenate or methionine is related to a decrease in succinyl coenzyme A, which is produced from pantothenate and which is an obligatory precursor of methionine biosynthesis. The autointoxification by endogenously produced α-ketobutyrate could be mimicked in wild-type
S. typhimurium
by exogenously supplied α-ketobutyrate or salicylate, a known inhibitor of pantothenate biosynthesis. The accumulation of α-ketobutyrate was initiated by the inability of the residual AHAS activity provided by AHAS I to efficiently remove the α-ketobutyrate produced by biosynthetic threonine deaminase. The accumulation of α-ketobutyrate was amplified by the action of transaminase B, which decreased the isoleucine pool by catalyzing the formation of α-keto-β-methylvalerate and aminobutyrate from isoleucine and α-ketobutyrate; this resulted in release of threonine deaminase from end product inhibition and unbridled production of α-ketobutyrate. Isoleucine satisfied the auxotrophic requirement of the AHAS II-deficient strain by curtailing the activity of threonine deaminase. Additional lines of evidence based on genetic and physiological experiments are presented to support the basis for the autointoxification of strain DU501 as well as other nonpolarigenic
ilvG
mutant strains.
Expression of an alternative nitrogen fixation system in Azotobacter vinelandii
Authors: Bishop P E , Jarlenski D M , Hetherington D R
Published: January 2020

Nitrogenase activities were determined from maximum acetylene reduction rates for mutant strains of Azotobacter vinelandii which are unable to fix N2 in the presence of molybdenum (Nif-) but undergo phenotypic reversal to Nif+ under conditions of Mo deficiency. The system responsible for N2 fixation under these conditions is thought to be an alternative N2 fixation system (Bishop et al., Proc. Natl. Acad. Sci. U.S.A. 77:7342-7346, 1980). Phenotypic reversal of Nif- strains to Nif+ strains was also observed in N-free medium without Mo but with either V or Re. Two protein patterns were found on two-dimensional gels of proteins from the extracts of wild-type cells cultured in N-free medium without Mo and with or without V or Re. The expression of each protein pattern in the wild-type strain of A. vinelandii seemed to depend upon the physiological state of the N2-fixing culture. Electron paramagnetic resonance experiments were conducted on whole cells of A. vinelandii grown under conditions of Mo deprivation in the absence of fixed N. No g = 3.65 signal (an electron paramagnetic resonance signal characteristic of the Mo-containing component of nitrogenase) was detectable in these cells, regardless of whether V or Re was present during growth of these cells, These results are discussed from the perspective that the well-known effect of V on N2 fixation by A. vinelandii may involve an alternative N2 fixation system.
Properties of oxaloacetate decarboxylase from Veillonella parvula
Authors: Ng S K , Wong M , Hamilton I R
Published: January 2020

Oxaloacetate decarboxylase was purified to 136-fold from the oral anaerobe Veillonella parvula. The purified enzyme was substantially free of contaminating enzymes or proteins. Maximum activity of the enzyme was exhibited at pH 7.0 for both carboxylation and decarboxylation. At this pH, the Km values for oxaloacetate and Mg2+ were at 0.06 and 0.17 mM, respectively, whereas the Km values for pyruvate, CO2, and Mg2+ were 3.3, 1.74, and 1.85 mM, respectively. Hyperbolic kinetics were observed with all of the aforementioned compounds. The Keq' was 2.13 X 10(-3) mM-1 favoring the decarboxylation of oxaloacetate. In the carboxylation step, avidin, acetyl coenzyme A, biotin, and coenzyme A were not required. ADP and NADH had no effect on either the carboxylation or decarboxylation step, but ATP inhibited the carboxylation step competitively and the decarboxylation step noncompetitively. These types of inhibition fitted well with the overall lactate metabolism of the non-carbohydrate-fermenting anaerobe.
Construction and behavior of strains with mutations in two chemotaxis genes
Authors: DeFranco A L , Koshland D E
Published: January 2020

Double mutants have been constructed by transducing each of the six che genes from the main che gene cluster into the cheC mutant with reversed behavior. The behavioral properties of these double-mutant strains were examined. The results are interpreted in terms of a model based on the cheC gene product being the component of the flagellar basal body that generates tumbling or smooth swimming in response to changes in the level of the response regulator. The properties of the double mutants can then be explained in ways which provide further understanding of the bacterial sensing system.
Pseudomonas cepacia mutants blocked in the Entner-Doudoroff pathway
Authors: Allenza P , Lessie T G
Published: January 2020

Pseudomonas cepacia mutants deficient in either 6-phosphogluconate (6PGA) dehydratase (Edd-) or 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (Eda-) failed to utilize glucose or gluconate despite the prominence of of 6-phosphogluconate dehydrogenase (6PGAD) ii this bacterium and the potential for utilizing the pentose shunt suggested by its growth on ribitol and xylose. The Eda- strains grew normally on glucuronic acid, indicating that in P. cepacia its degradation does not depend upon KDPG aldolase as it does in Escherichia coli. Both 6PGA dehydratase and KDPG aldolase were inducible enzymes, with 6PGA rather than gluconate the apparent inducer. Edd- as well as Eda- strains were sensitive to growth inhibition by glucose, gluconate, fructose, and related carbohydrates when these substrates were present in combination with alternate carbon sources such as citrate or phthalate, presumably as a consequence of accumulation and toxicity of 6PGA, KDPG, or both. Edd- mutants were somewhat less sensitive to such inhibition than were Eda- strains. Certain derivatives of the Edd- strains we examined were able to utilize gluconate despite their deficiency of 6PGA dehydratase. Such mutants formed higher levels of 6PGAD than did the wild type. It is likely that the elevated levels of 6PGAD in these strains prevents accumulation of toxic levels of 6PGA that would otherwise result from a block in he Entner-Doudoroff pathway. The results suggest that P. cepacia can mutate to grow slowly on gluconate utilizing only the pentose shunt.
Photopigments in Rhodopseudomonas capsulata cells grown anaerobically in darkness
Authors: Madigan M , Cox J C , Gest H
Published: January 2020

The phototrophic bacterium Rhodopseudomonas capsulata can obtain energy for dark anaerobic growth from sugar fermentations dependent on accessory oxidants such as trimethylamine-N-oxide or dimethyl sulfoxide. Cells grown for one to two subcultures in this fashion, with fructose as the energy source, showed approximately a twofold increase in bacteriochlorophyll content (per milligram of cell protein) and developed extensive intracytoplasmic membranes in comparison with cells grown photosynthetically at saturating light intensity. Cells harvested from successive anaerobic dark subcultures, however, showed progressively lower pigment contents. After ca. 20 transfers, bacteriochlorophyll and carotenoids were barely detectable, and the amount of intracytoplasmic membrane diminished considerably. Spontaneous mutants incapable of producing normal levels of photosynthetic pigments arose during prolonged anaerobic dark growth. Certain mutants of this kind appear to have a selective advantage over wild-type cells under fermentative growth conditions. Of four pigment mutants characterized (two being completely unable to produce bacteriochlorophyll), only one retained the capacity to grow photosynthetically.
Major sites of metal binding in Bacillus licheniformis walls
Authors: Beveridge T J , Forsberg C W , Doyle R J
Published: January 2020

Isolated and purified walls of Bacillus licheniformis NCTC 6346 his contained peptidoglycan, teichoic acid, and teichuronic acid (0.36 mumol of diaminopimelic acid, 0.85 mumol of organic phosphorus, and 0.43 mumol of glucuronic acid per mg [dry weight] of walls, respectively). The walls also contained a total of 0.208 mumol of metal per mg. When these walls were subjected to metal-binding conditions (T. J. Beveridge and R. G. E. Murray, J. Bacteriol. 127:1502-1518, 1976) for nine metals, the amount of bound metal above background ranged from 0.910 mumol of Na to 0.031 mumol of Au per mg of walls. Most were in the 0.500-mumol mg-1 range. Electron-scattering profiles from unstained thin sections indicated that the metal was dispersed throughout the wall fabric. Mild alkali treatment extracted teichoic acid from the walls (97% based on phosphorus) but left the peptidoglycan and teichuronic acid intact. This treatment reduced their capacity for all metals but Au. Thin sections revealed that the wall thickness had been reduced by one-third, but metal was still dispersed throughout the wall fabric. Trichloroacetic acid treatment of the teichoic acid-less walls removed 95% of the teichuronic acid (based on glucuronic acid) but left the peptidoglycan intact (based on sedimentable diaminopimelic acid). The thickness of these walls was not further reduced, but little binding capacity remained (usually less than 10% of the original binding). The staining of these walls with Au produced a 14.4-nm repeat frequency within the peptidoglycan fabric. Sedimentation velocity experiments with the extracted teichuronic acid in the presence of metal confirmed it to be a potent metal-complexing polymer. These results indicated that teichoic and teichuronic acids are the prime sites of metal binding in B. licheniformis walls.
Direct participation of lexA protein in repression of colicin E1 synthesis
Authors: Ebina Y , Kishi F , Nakazawa A
Published: January 2020

Spontaneous colicin E1 production by plasmid RSF2124 in a recA lexA(spr) strain of Escherichia coli was about 10-fold greater than that observed in a wild-type strain. The synthesis was repressed nearly to the level of a recA strain in the presence of the plasmid pMCR551, which carries the lexA gene.
Isolation and behavior of Escherichia coli deletion mutants lacking chemotaxis functions
Authors: Parkinson J S , Houts S E
Published: January 2020

Six Escherichia coli che loci (cheA,-B,-R,-W,-Y, and Z) are located in two adjacent operons that map at minute 42 on the chromosome. Point mutants defective in any of these six functions have aberrant swimming patterns and are generally nonchemotactic. Deletions within the two major che gene operons were isolated in order to examine epistatic interactions among these genes. We first constructed a specialized transducing phage (lambda che22), which carries both of the che operons and their associated promoters. Deleted lambda che22 derivatives were selected by chelating agent inactivation, and these derivatives were characterized by mapping them against a series of host strains with point mutations. Representative nonpolar deletions were then transferred into the E. coli chromosome by homologous recombination. Although the phenotype of cheR mutants (smooth swimming) was expected to be epistatic to that of cheB mutants (tumbly swimming), we found that deletion mutants lacking both of these functions exhibited frequent directional changes or tumbling episodes as they swam. An examination of larger deletions indicated that both the cheA-cheW and cheY-cheZ functions were required for the anomalous tumbling behavior observed in these mutants. Loss of the cheB function was also correlated with an inverted behavioral response to sodium acetate, a strong repellent of wild-type cells. These findings indicate that an important component of the signal transducing machinery may be altered in cheB mutants.
Mutations that alter the transport function of the LamB protein in Escherichia coli
Authors: Wandersman C , Schwartz M
Published: January 2020

Some Escherichia coli K-12 lamB mutants, those producing reduced amounts of LamB protein (one-tenth the wild type amount), grow normally on dextrins but transport maltose when present at a concentration of 1 microM at about one-tenth the normal rate. lamB Dex- mutants were found as derivatives of these strains. These Dex- mutants are considerably impaired in the transport of maltose at low concentrations (below 10 microM), and they have a structurally altered LamB protein which is impaired in its interaction with phages lambda and K10 but still interacts with a lambda host range mutant lambda hh*. The Dex- mutants are double lamB mutants carrying one mutation, already present in the parental strains, that reduces LamB synthesis and a second that alters LamB structure. The secondary mutations, present in different independent Dex- mutants, are clustered in the same region of the lamB gene. Dex+ revertants were isolated and analyzed: when the altered LamB protein is made in wild-type amount, due to a reversion of the first mutation, the phenotype reverts to Dex+. However, these Dex+ revertants are still very significantly impaired in maltose transport at low concentrations (below 10 microM).
Isolation and characterization of purine regulatory mutants of Salmonella typhimurium with an episomal purE-lac fusion
Authors: Thomulka K W , Gots J S
Published: January 2020

Expression of the purE operon of Salmonella typhimurium was analyzed by using an Escherichia coli F' episome containing a purE-lac fusion. The fusion removes the lacOP and part of the lacZ genes of the lac operon and places the intact lacY and lacA genes under control of the purE operon as shown by inhibition of growth on melibiose (lacY) and repression of thiogalactoside transacetylase (lacA) by various purines. Two classes of regulatory-deficient mutants were found among those resistant to inhibition by purines. One class was trans active (chromosomal) and corresponded to previously described purR mutants involving a deficient cytoplasmic repressor substance. These were also altered in the expression of the purF, purD, purG amd purI genes as evidenced by loss of repressibility of the synthesis of glycinamide ribotide and aminoimidazole ribotide. The other class was cis active (episomal), specific for only purE expression, and thus corresponded to an altered purE operon signal (operator or promoter). The metabolic requirements for the expression of purE were also monitored by measuring repression of the transacetylase in strains with various genetically altered metabolic backgrounds. Repression by guanine required an intact guanine phosphorbosyltransferase (gpt) and repression by adenine and all nucleosides required purine nucleoside phosphorylase (deoD). Synthesis of cyclic AMP (cya) and its receptor protein (crp) were no longer required for the expression of the lac genes under purE control.
Nitrogen catabolite repression in a glutamate auxotroph of Saccharomyces cerevisiae
Authors: Kang L , Keeler M L , Dunlop P C , Roon R J
Published: January 2020

The biosynthesis of asparaginase II in Saccharomyces cerevisiae is subject to nitrogen catabolite repression. In the present study we examined the physiological effects of glutamate auxotrophy on cellular metabolism and on the nitrogen catabolite repression of asparaginase II. Glutamate auxotrophic cells, incubated without a glutamate supplement, had a diminished internal pool of alpha-ketoglutarate and a concomitant inability to equilibrate ammonium ion with alpha-amino nitrogen. In the glutamate auxotroph, asparaginase II biosynthesis exhibited a decreased sensitivity to nitrogen catabolite repression by ammonium ion but normal sensitivity to nitrogen catabolite repression by all amino acids tested.
Effect of inositol starvation on the in vitro syntheses of mannan and N-acetylglucosaminylpyrophosphoryldolichol in Saccharomyces cerevisiae
Authors: Hanson B A , Lester R L
Published: January 2020

An early consequence of starvation for inositol in yeast is inhibition of synthesis of the major cell wall components mannan and glucan. In looking for the mechanism of this inhibition, we found that the activity of the enzyme catalyzing the synthesis of N-acetylglucosaminylpyrophosphoryldolichol was diminished in particular membrane preparations from cells starved for inositol. This loss of reactivity was observed under a variety of in vitro assay conditions and could be restored by the addition of phosphatidylinositol but not by other phosphoinositol-containing sphingolipids known to occur in yeast. When assayed in the presence of high concentrations of Triton X-100, enzyme preparations from both control and inositol-starved cells required phosphatidylinositol for maximal activity. Since this enzyme catalyzed an early step in the synthesis of mannan that is N-linked to protein, a reasonable hypothesis is that inhibition of mannan synthesis in inositol-starved cells results from the depletion of the necessary cofactor phosphatidylinositol.
Several copies of the same insertion sequence are present in alpha-hemolytic plasmids belonging to four different incompatibility groups
Authors: Zabala J C , de la Cruz F , Ortiz J M
Published: January 2020

Alpha-hemolytic plasmids belonging to four different incompatibility groups contained several copies (two to six) of the same DNA sequence. This sequence was capable of illegitimate recombination and thus behaved as an insertion sequence element. Its size (1.9 kilobases) and restriction enzyme cleavage map suggested that it was different from previously described insertion sequence elements.
Transcription of Regions within the divergent argECBH operon of Escherichia coli: evidence for lack of an attenuation mechanism
Authors: Beny G , Cunin R , Glansdorff N , Boyen A , Charlier J , Kelker N
Published: January 2020

Using in vitro and in vivo assays, we could detect no early termination of DNA transcription in the proximal part of the argCBH arm of the argECBH divergent operon. The discrepancy noted previously between the respective amplitudes of variation of messenger and enzyme synthesis must therefore be attributed to other causes than a difference in efficiency between attenuation and repression.
“Start” Mutants of
Saccharomyces cerevisiae
Are Suppressed in Carbon Catabolite-Derepressing Medium
Authors: Shuster Jeffrey R.
Published: January 2020

Temperature-sensitive cell division “start” mutants
cdc28, cdc36, cdc37
, and
cdc39
of the yeast
Saccharomyces cerevisiae
arrested cell division in the G1 phase of the cell cycle in glucose medium. I report here that
cdc28, cdc36
, and
cdc39
mutants were suppressed when grown in carbon catabolite-derepressing medium.
Mutational separation of transport systems for branched-chain amino acids in Pseudomonas aeruginosa
Authors: Hoshino T , Kageyama M
Published: January 2020

Several types of Pseudomonas aeruginosa mutants defective in the transport systems for branched-chain amino acids were isolated by selection for resistance to 5',5',5'-DL-trifluoroleucine, a leucine analog, under certain conditions. Mutants resistant to trifluoroleucine in the absence of Na+ were defective in the high-affinity system. These mutants fell into two classes. One class showed a defect in the production of a periplasmic binding protein for leucine, isoleucine, valine, alanine, and threonine, and the other showed normal production of the binding protein as determined by a binding assay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Properties of the former class of mutants have been partly described (T. Hoshino and M. Kageyama, J. Bacteriol. 141:1055-1063, 1980). Mutants selected for resistance to trifluoroleucine with Na+ and an excess amount of alanine showed a defect in the low-affinity system. Membrane vesicles prepared from such a mutant lost the transport activity for leucine. A mutant which showed increased activity of the low-affinity system with a defect in the high-affinity system was obtained from strain PML1453 (high-affinity system defective) by selecting for utilization of isoleucine as a carbon source.
Purification and characterization of three separate bacteriolytic enzymes excreted by Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus saprophyticus
Authors: Valisena S , Varaldo P E , Satta G
Published: January 2020

As a further development of previous investigations showing that different staphylococcal species display different bacteriolytic activity patterns (lyogroups), the bacteriolytic enzymes excreted by three different Staphylococcus species, Staphylococcus aureus (lyogroup I), S. simulans (lyogroup II), and S. saprophyticus (lyogroup IV); have been purified and characterized. A representative strain from each species was grown in a preselected medium made of fully dialyzable products. Culture supernatants were collected in the appropriate growth phase. Two different affinity adsorbents were used for enzyme purification. One was obtained by coupling lysozyme-digested pure peptidoglycan from Micrococcus luteus to cyanogen bromide-activated Sepharose 4B. The second affinity adsorbent used was chitin. The S. aureus bacteriolytic enzyme bound to the solubilized peptidoglycan but not to chitin, whereas the opposite was true for the S. simulans enzyme. The bacteriolytic enzyme from S. saprophyticus did not bind to either the Sepharose 4B-peptidoglycan resin or to chitin, and its purification was achieved by two ion-exchange chromatography steps combined with gel filtration. All three enzymes were purified to apparent homogeneity. Their subsequent characterization indicated that all acted as endo-beta-N-acetylglucosaminidases. However, the three glucosaminidases differed significantly in their kinetics of activity and bacteriolytic spectrum against heat-killed cells of a variety of microorganisms. Very different values also resulted from molecular weight determinations: 80,000 for the S. aureus enzyme, 45,000 for the S. simulans enzyme, and 31,000 for the S. saprophyticus enzyme. Other important differences were observed in their stability, optimal pH and ionic strength for their activity, and their responses to temperature and divalent cations. These results confirmed the previous proposal that different staphylococcal species excrete different lytic enzymes.
Isolation and characterization of a Pseudomonas aeruginosa PAO mutant defective in the structural gene for the LIVAT-binding protein
Authors: Hoshino T , Nishio K
Published: January 2020

A mutant of Pseudomonas aeruginosa PAO which has a defect in the structural gene for a binding protein for leucine, isoleucine, valine, alanine, and threonine (LIVAT-binding protein) was isolated and characterized. DL-4-azaleucine was taken up via the high-affinity branched-chain amino acid transport system (LIV-I), but not via the low affinity system (LIV-II), and then inhibited the growth of P. aeruginosa cells. This finding enabled us to select mutants defective in the LIV-I transport system alone. Among such mutants, strain PAO3530 was found to produce an altered LIVAT-binding protein. The shock fluid of this strain contained a normal level of the protein which corresponded to the wild-type LIVAT-binding protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by an immunological test. However, the shock fluid showed almost no binding activity for branched-chain amino acids, suggesting that strain PAO3530 has a defect in the structural gene for the LIVAT-binding protein. The mutation locus (bra-310) was mapped in a region between cnu-9001 and oru-325 on the chromosome of P. aeruginosa PAO by conjugation mediated by plasmid FP5 or R68.45.
Role of proton motive force in genetic transformation of Bacillus subtilis
Authors: van Nieuwenhoven M H , Hellingwerf K J , Venema G , Konings W N
Published: January 2020

This study explored the role of the proton motive force in the processes of DNA binding and DNA transport of genetic transformation of Bacillus subtilis 168 strain 8G-5 (trpC2). Transformation was severely inhibited by the ionophores valinomycin, nigericin, and 3,5-di-tert-4-hydroxybenzylidenemalononitrite (SF-6847) and by tetraphenylphosphonium. The ionophores valinomycin and nigericin also severely inhibited binding of transforming DNA to the cell envelope, whereas SF-6847 and carbonylcyanide-p-trifluoromethoxyphenylhydrazone hardly affected binding. The proton motive force, therefore, does not contribute to the process of DNA binding, and valinomycin and nigericin interact directly with the DNA binding sites at the cell envelope. The effects of ionophores, weak acids, and tetraphenylphosphonium on the components of the proton motive force and on the entry of transforming DNA after binding to the cell envelope was investigated. DNA entry, as measured by the amount of DNase I-resistant cell-associated [3H]DNA and by the formation of DNA breakdown products, was severely inhibited under conditions of a small proton motive force and also under conditions of a small delta pH and a high electrical potential. These results suggest that the proton motive force and especially the delta pH component functions as a driving force for DNA uptake in transformation.