bluuebunny/crossref_metadata_2025 huggingface dataset:
Regular segregation of composite plasmid Rms201
Authors: Ike Y , Hashimoto H , Mitsuhashi S
Published: January 2020

Copy number mutants Rms201ts15 and Rms201ts16 were isolated at 30 degrees C from a temperature-sensitive replication mutant (Rms201ts14) of the conjugative plasmid Rms201. The numbers of plasmids per chromosome of ML1410(Rms201ts14), ML1410(Rms201ts15), and ML1410(Rms201ts16) grown at 30 degrees C were 2.2, 7.4, and 20, respectively. The synthesis of covalently closed circular plasmid deoxyribonucleic acid stopped in Rms201ts14, Rms201ts15, and Rms201ts16 immediately after a "shift-up" in temperature (42 degrees C). At 42 degrees C, antibiotic-sensitive derivatives appeared after a certain lag time: the lag times of ML1410(Rms201ts14), ML1410(Rms201ts15), and ML1410(Rms201ts16) were 2.5, 5, and 6.8 generations, respectively. After these times, plasmid-positive cells in the populations decreased at a rate of about 50% per generation in all of the mutants. From these results we conclude that plasmid segregation (partition) of Rms201 occurs by regular segregation (partition).
Characterization of a macrolide, lincosamide, and streptogramin resistance plasmid in Staphylococcus epidermidis
Authors: Parisi J T , Robbins J , Lampson B C , Hecht D W
Published: January 2020

A strain of Staphylococcus epidermidis was transduced to erythromycin resistance, and all of the transductants exhibited the macrolide, lincosamide, streptogramin B resistance phenotype. Curing and antibiotic disk studies also indicated that these resistances were controlled by a single plasmid determinant and were constitutive. Agarose gel electrophoresis of plasmid deoxyribonucleic acid (DNA) from donor, cured, and transduced strains showed that a single plasmid was responsible. This plasmid, designated pNE131, was examined for sequence homology to two other plasmids, pE194 and p1258, from Staphylococcus aureus, which also code for erythromycin resistance. DNA from plasmids pNE131 and pE194 hybridized with one another, but no extensive homology to pI258 with either pNE131 or pE194 was found. Restriction endonuclease digests of pNE131 and pE194 showed no common fragments. However, sequence homology was localized to the nucleotides in pE194 that code for the 29,000-dalton protein responsible for erythromycin resistance. pNE131 was calculated to have 2,220 base pairs and is the smallest naturally occurring plasmid with a known function yet reported in S. epidermidis.
Biosynthetic and Bioenergetic Functions of Citric Acid Cycle Reactions in
Rhodopseudomonas capsulata
Authors: Beatty J. Thomas , Gest Howard
Published: January 2020

Rhodopseudomonas capsulata
can grow in a number of alternative modes, including (i) photosynthetic, defined here as anaerobic growth with light as the energy source, and (ii) heterotrophic, referring to aerobic heterotrophic growth in darkness. The functions of citric acid cycle sequences in these growth modes were investigated using wild-type and appropriate mutant strains. Results of growth tests and O
2
utilization experiments showed that in the heterotrophic mode, energy conversion is dependent on operation of the classical citric acid cycle. Alpha-ketoglutarate dehydrogenase (KGD) activity in wild-type strain B10 is substantially higher in cells grown heterotrophically than in cells grown photosynthetically. Molecular oxygen, even at low concentration, appears to be important in regulation of KGD synthesis and, thus, in expression of citric acid cycle activity. Extracts of (photosynthetically grown) mutant strain KGD11 lack demonstrable KGD activity, and in contrast to the wild type, KGD11 is unable to grow heterotrophically on succinate, malate, or pyruvate owing to failure of the energy conversion function of the citric acid cycle. KGD11, however, grows well photosynthetically on malate or on CO
2
+ H
2
. The KGD activity level required to support the bioenergetic function of the citric acid cycle is evidently much higher than that necessary to satisfy biosynthetic demands; thus, a very low rate of succinyl-coenzyme A formation (needed for biosynthesis) in the mutant would suffice for growth under photosynthetic conditions. In wild-type
R. capsulata
, the α-ketoglutarate required for glutamate synthesis is ordinarily generated via citric acid cycle reactions, which include the conversions catalyzed by citrate synthase and isocitrate dehydrogenase. Mutants blocked in the former or both of these enzymes can grow photosynthetically if provided with an exogenous source of α-ketoglutarate or glutamate, but grow very poorly (if at all) as heterotrophs since the energy supply under these conditions depends on operation of the complete citric acid cycle.
Purification of a branched-chain keto acid dehydrogenase from Pseudomonas putida
Authors: Sokatch J R , McCully V , Roberts C M
Published: January 2020

We purified branched-chain keto acid dehydrogenase to a specific activity of 10 mumol/min per mg of protein from Pseudomonas putida grown on valine. The purified enzyme was active with 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate in a ratio of 1.0:0.8:0.7 but showed no activity with either pyruvate or 2-ketoglutarate. There were four polypeptides in the purified enzyme (molecular weights, 49,000, 46,000, 39,000, and 37,000). The purified enzyme was deficient in the specific lipoamide dehydrogenase produced during growth on valine (molecular weight, 49,000). Branched-chain keto acid dehydrogenase required L-valine, oxidized nicotinamide adenine dinucleotide, coenzyme A, thiamine pyrophosphate, and magnesium chloride. A partially purified preparation catalyzed the oxidation of 2-keto-[1-14C]isovalerate to [14C]carbon dioxide, isobutyryl-coenzyme A, and reduced nicotinamide adenine dinucleotide in equimolar amounts. Both the Km and the Vmax for 2-ketoisovalerate were affected by the addition of L-valine to the assay mixture. However, only the Vmax values for oxidized nicotinamide adenine dinucleotide and coenzyme A were affected when L-valine was present. This suggested that valine acted by affecting the binding of branched-chain keto acids to subunit E1 of the complex.
Virulence-associated plasmids from Yersinia enterocolitica and Yersinia pestis
Authors: Portnoy D A , Falkow S
Published: January 2020

A 44-megadalton plasmid associated with virulence and Ca2+ dependence from Yersinia enterocolitica 8081 was compared at the molecular level with a 47-megadalton plasmid associated with Ca2+ dependence from Yersinia pestis EV76. The plasmids were found to share 55% deoxyribonucleic acid sequence homology distributed over approximately 80% of the plasmid genomes. One region in which the plasmids differed was found to contain sequences concerned with essential plasmid functions. Forty-five mutants of Y. pestis were isolated which had spontaneously acquired the ability to grow on calcium-free medium (Ca2+ independence). Of these mutants, 21 were cured of their 47-megadalton plasmid, whereas the remaining had either suffered a major deletion of the plasmid or had a 2.2-kilobase insertion located in one of two adjacent BamHI restriction fragments encompassing approximately 9 kilobases. The inserted sequence was found at numerous sites on the Y. pestis chromosome and on all three plasmids in the strain and may represent a Y. pestis insertion sequence element.
Detection of Glutamate Synthase in Heterocysts of
Anabaena
sp. Strain 7120
Authors: Gupta Manju , Carr Noel G.
Published: January 2020

The formation of [
14
C]glutamate from [
14
C]glutamine in the presence of α-oxoglutarate was observed in extracts of heterocysts of
Anabaena
sp. strain 7120 under conditions that indicate the operation of glutamate synthase.
Physiological characteristics of Thiomicrospira sp. Strain L-12 isolated from deep-sea hydrothermal vents
Authors: Ruby E G , Jannasch H W
Published: January 2020

Growth of the obligately chemolithotrophic Thiomicrospira sp. strain L-12, isolated from a hydrothermal vent at a depth of 2,550 m in the Galapagos Rift region, was optimal at pH 8 and required 200 mM Na+ and divalent ions (Ca2+ and Mg2+). The organism was microaerophilic and tolerated 300 microM sulfide without a decrease in the rate of CO2 incorporation. Growth and CO2 incorporation occurred within the temperature range of 10 to 35 degrees C, with both optimal at 25 degrees C. At the in situ pressure of 250 atm. the rate of CO2 incorporation was reduced by 25% relative to that measured at 1 atm: it was entirely suppressed at 500 atm. The results of this physiological characterization suggest that Thiomicrospira sp. strain L-12 can be an active autotroph in the hydrothermal environment.
Regulation of fatty acid composition in Escherichia coli: a proposed common mechanism for changes induced by ethanol, chaotropic agents, and a reduction of growth temperature
Authors: Ingram L O
Published: January 2020

Growth of Escherichia coli in the presence of ethanol and chaotropic salts resulted in the synthesis of lipids containing elevated levels of unsaturated fatty acids analogous to the effect of a reduction in growth temperature. Both ethanol and chaotropic agents acted at the level of fatty acid biosynthesis and altered lipid composition by decreasing the proportion of saturated acyl chains available for the synthesis of phospholipids. A reduction in temperature causes similar effects on fatty acid biosynthesis in vivo and in vitro. Ethanol, chaotropic salts, and a decrease in temperature all weaken hydrophobic interactions. Antichaotropic salts antagonized and effects of these treatments on fatty acid synthesis in vitro. These results are consistent with a common mechanism for the effects of chaotropic agents, temperature, and ethanol on fatty acid synthesis. The biosynthesis of saturated and unsaturated acyl chains may be regulated by the strength of hydrophobic interactions. Changes in the strength of hydrophobic interactions could alter enzyme structure, substrate structure, or the equilibrium between the soluble enzymes of fatty acid synthesis and their respective acyl carrier protein substrates.
Tn7 and Tn501 Insertions into Pseudomonas aeruginosa plasmid R91-5: mapping of two transfer regions
Authors: Moore R J , Krishnapillai V
Published: January 2020

We constructed a restriction endonuclease map of the Pseudomonas aeruginosa narrow-host-range plasmid R91-5. Insertions of transposons Tn7 and Tn501 into the plasmid DNA were characterized physically and genetically. The distribution of sites of insertion showed some regional specificity for the insertion of these transposons, especially TN501. The insertion of Tn7 was unusual in that all 42 of 43 insertions were in the same orientation. By relating phenotypic changes to the site of insertion, the Tn1 transposon that was already present on R91-5 and coded for carbenicillin resistance was mapped, and its orientation was determined. Two major transfer regions were identified. We believe that Tra1 is involved in conjugal DNA metabolism, whereas Tra2 is involved mainly in production of the sex pili.
Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis
Authors: McDonald K O , Burke W F
Published: January 2020

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.
Differential effects of antibiotics inhibiting gyrase
Authors: Engle E C , Manes S H , Drlica K
Published: January 2020

Both oxolinic acid and coumermycin A1, inhibitors of DNA gyrase, block DNA synthesis in Escherichia coli. At low concentrations of oxolinic acid, the rate of bacterial DNA synthesis first declines rapidly but then gradually increases. This gradual increase in synthesis rate depended on the presence of wild-type recA and lexA genes; mutations in either gene blocked the increase in synthesis rate. In such mutants, oxolinic acid caused a rapid decline, followed by a slow, further decrease in DNA synthesis rate. Coumermycin A1, however, produced a more gradual decline in synthesis rate which is unaffected by defects in the recA or lexA genes. An additional difference between the two drugs was observed in a dnaA mutant, in which initiation of replication is temperature sensitive. Low concentrations of oxolinic acid, but not coumermycin A1, reduced thermal inhibition of DNA synthesis rate.
Effects of Growth Temperature on Fatty Acid and Alk-1-Enyl Group Compositions of
Veillonella parvula
and
Megasphaera elsdenii
Phospholipids
Authors: Johnston Norah C. , Goldfine Howard
Published: January 2020

Veillonella parvula
ATCC 10790, an anaerobic gram-negative coccus, contains diacyl and alk-1-enyl acyl (plasmalogen) forms of phosphatidylethanolamine and phosphatidylserine. We studied the effect of growth temperature on the lipid composition of this strain. There was a small increase in the phosphatidylethanolamine content but no change in the content of plasmalogens at the lower growth temperatures tested. The total acyl chains and the plasmalogen acyl chains contained between 73 and 80% mono-unsaturated fatty acids at all growth temperatures. The plasmalogen alk-1-enyl chains were significantly more unsaturated in cells grown at 30 and 25°C than in cells grown at 37°C. Differential scanning calorimetry of the hydrated phospholipids showed lower phase transition temperatures for the lipids from the cells grown at the lower temperatures. In
Megasphaera elsdenii
lipids, which are similar in composition to the lipids of
V. parvula
, the proportion of phosphatidylethanolamine also increased slightly at lower growth temperatures, with no significant change in the content of plasmalogens.
M. elsdenii
contained cyclopropane fatty acyl and alk-1-enyl chains in addition to the mono-unsaturated and saturated chains previously reported. As cells entered the stationary phase of growth at 30 and 42.5°C, there was a reciprocal increase in the proportion of cyclopropane acyl chains and decrease in the unsaturated moieties. The total proportion of cyclopropane and unsaturated acyl and alk-1-enyl chains was more than 65% at all growth temperatures studied, and there was no discernible increase in the sum of these moieties at the lower growth temperatures.
Mobilization of nonconjugative antibiotic resistance plasmids in Haemophilus ducreyi
Authors: Deneer H G , Slaney L , Maclean I W , Albritton W L
Published: January 2020

A clinical isolate of Haemophilus ducreyi was found to harbor three plasmids: a 23.5-megadalton (Mdal) phenotypically cryptic plasmid, a 7.0-Mdal ampicillin resistance plasmid, and a 4.0-Mdal sulfonamide resistance plasmid. The two smaller plasmids were transferable by conjugation to Haemophilus recipients, but only if the donor cell harbored the 23.5-Mdal plasmid as well, indicating that this large plasmid had mobilizing capabilities. Transfer was also possible to Escherichia coli recipients. Haemophilus influenzae transconjugants which had acquired both the 23.5-Mdal plasmid and one of the R-plasmids could subsequently retransfer the R-plasmid to other Haemophilus recipients at higher frequencies. A derivative of the 23.5 Mdal plasmid was isolated which was shown by restriction endonuclease analysis to contain an ampicillin resistance transposon and to have retained its conjugative ability.
Isolation of mycoplasma membranes by dicyclohexylcarbodiimide-induced lysis
Authors: Shirvan M H , Rottem S , Ne'eman Z , Bittman R
Published: January 2020

A simple procedure was devised to prepared membranes from Mycoplasma gallisepticum cells. The cells were lysed in an isosmotic NaCl solution by dicyclohexylcarbodiimide, which blocks ATPase activity and interferes with the regulation of cell volume. The procedure can be used to isolate membranes of other osmotically resistant mycoplasmas.
Cellular levels, excretion, and synthesis rates of cyclic AMP in Escherichia coli grown in continuous culture
Authors: Matin A , Matin M K
Published: January 2020

Changes in dilution rate did not elicit large and systematic changes in cellular cyclic AMP levels in Escherichia coli grown in a chemostat under carbon or phosphate limitation. However, the technical difficulties of measuring low levels of cellular cyclic AMP in the presence of a large background of extracellular cyclic AMP precluded firm conclusions in this point. The net rate of cyclic AMP synthesis increased exponentially with increasing dilution rate through either the entire range of dilution rates examined (phosphate limitation) or a substantial part of the range (lactose and glucose limitations). Thus, it is probable that growth rate regulates the synthesis of adenylate cyclase. The maximum rate of net cyclic AMP synthesis was greater under lactose than under glucose limitation, which is consistent with the notion that the uptake of phosphotransferase sugars is more inhibitory to adenylate cyclase than the uptake of other carbon substrates. Phosphate-limited cultures exhibited the lowest rate of net cyclic AMP synthesis, which could be due to the role of phosphorylated metabolites in the regulation of adenylate cyclase activity. Under all growth conditions examined, greater than 99.9% of the cyclic AMP synthesized was found in the culture medium. The function of this excretion, which consumed up to 9% of the total energy available to the cell and which evidently resulted from elaborate regulatory mechanisms, remains entirely unknown.
Heterogeneity of tetracycline resistance determinants in Streptococcus
Authors: Burdett V , Inamine J , Rajagopalan S
Published: January 2020

We found that naturally occurring tetracycline resistance in streptococci is encoded by more than one genetic determinant. Two of these distinct determinants were cloned, and the regions that are necessary and sufficient for expression of tetracycline resistance were defined by deletion analysis. These cloned determinants were further characterized by DNA-DNA hybridization experiments which also identified a third genetically unrelated tetracycline resistance determinant. Some of these genetic differences appear to represent mechanistic differences. The tetL determinant was associated with small nonconjugative plasmids and mediated resistance to tetracycline. The tetM determinant was most often "nonplasmid" associated and mediated resistance to minocycline as well as tetracycline. The tetN determinant was represented on a large conjugative plasmid and was genetically distinct from tetL and tetM, although phenotypically it resembled tetM.
Rhizobium japonicum mutants that are hypersensitive to repression of H2 uptake by oxygen
Authors: Maier R J , Merberg D M
Published: January 2020

The synthesis of an H2 oxidation system in free-living Rhizobium japonicum wild-type strain SR is repressed by oxygen. Maximal H2 uptake rates were obtained in strain SR after derepression in 11 microM or less dissolved oxygen. Oxygen levels above 45 microM completely repressed H2 uptake in strain SR. Five R. japonicum mutant strains that are hypersensitive to repression or H2 oxidation by oxygen were derived from strain SR. The mutants were obtained by screening H2 uptake-negative mutants that retained the ability to oxidize H2 as bacteroids from soybean nodules. As bacteroids, the five mutant strains were capable of H2 oxidation rates comparable to that of the wild type. The mutants did not take up H2 when derepressed in 22 microM dissolved oxygen, whereas strain SR had substantial activity at this oxygen concentration. The O2 repression of H2 uptake in both the wild-type and two mutant strains, SR174 and SR200, was rapid and was similar to the effect of inhibiting synthesis of H2 uptake system components with rifampin. None of the mutant strains was able to oxidize H2 when the artificial electron acceptors methylene blue or phenazine methosulfate were provided. The mutant strains were not sensitive to killing by oxygen, they took up O2 at rates similar to strain SR, and they did not produce an H2 uptake system that was oxygen labile. Cyclic AMP levels were comparable in strain SR and the five mutant strains after subjection of the cultures to the derepression conditions.
relA-dependent RNA polymerase activity in Escherichia coli
Authors: Ryals J , Bremer H
Published: January 2020

Parameters relating to RNA synthesis were measured after a temperature shift from 30 to 42 degrees C, in a relA+ and relA- isogenic pair of Escherichia coli strains containing a temperature-sensitive valyl tRNA synthetase. The following results were obtained: (i) the rRNA chain growth rate increased 2-fold in both strains; (ii) newly synthesized rRNA became unstable in both strains; (iii) the stable RNA gene activity (rRNA and tRNA, measured as stable RNA synthesis rate relative to the total instantaneous rate of RNA synthesis) decreased 1.7-fold in the relA+ strain and increased 1.9-fold in the relA mutant; and (iv) the RNA polymerase activity (measured by the percentage of total RNA polymerase enzyme active in transcription an any instant) decreased from 20 to 3.6% in the relA+ strain and remained unchanged (or increased at most to 22%) in the relA mutant. It is suggested that both rRNA gene activity and the RNA polymerase activity depend on the intracellular concentration of guanosine tetraphosphate, whereas the altered chain elongation rate and stability of rRNA are temperature or amino acid starvation effects, respectively, without involvement of relA function.
Cyclic AMP may not be involved in catabolite repression in Saccharomyes cerevisiae: evidence from mutants capable of utilizing it as an adenine source
Authors: Matsumoto K , Uno I , Toh-E A , Ishikawa T , Oshima Y
Published: January 2020

Mutants able to utilize 5'-AMP or cyclic AMP as the adenine source were isolated from an ade6 ade10 double mutant by ethyl methane sulfonate mutagenesis. A single amp1 mutation, primarily selected on 5'-AMP medium, confers the phenotype for utilization of exogenous 5'-AMP as the adenine source. From the ade6 ade10 amp1 triple mutant, a mutant able to utilize cyclic AMP was isolated, and the mutant phenotype was proven to be due to the simultaneous occurrence of triple mutations designated as cam1, cam2, and cam3. The cam3 mutation, but not cam1 or cam2, also confers the phenotype for utilizing 5'-AMP, the same phenotype as the amp1 mutation. All of these mutations are recessive to the respective wild-type counterparts. Cells having the ade6 ade10 amp1 cam1 cam2 cam3 genotype showed significant ability to take up exogenous cyclic AMP, whereas no differences were observed in cyclic AMP phosphodiesterase activity in comparison with that of the original strains used in the mutant isolation. Since glucose severely repressed galactokinase synthesis in the constitutive GAL81 mutant having the ade6 ade10 amp1 cam1 cam2 cam3 genotype, irrespective of the presence or absence of cyclic AMP in the medium, it was suggested that cyclic AMP is not involved in the mechanism of catabolite repression in Saccharomyces cerevisiae. It does, however, have a stimulative effect on the galactokinase synthesis in the GAL81 mutant in the absence of glucose.
dnaC-dependent reconstitution of replication forks in Escherichia coli lysates
Authors: Nüsslein-Crystalla V , Niedenhof I , Rein R
Published: January 2020

Lysates of Escherichia coli exhibit a DNA-synthesizing activity that depends on the presence of replication forks and of replication proteins. Replicative activity was reconstituted in vitro by mixing lysates prepared from temperature-sensitive dnaB mutants with wild-type dnaB protein. Lysates of double mutants deficient in both dnaB and dnaC genes could only be complemented by the addition of both dnaB and dnaC proteins, whereas lysates deficient in dnaC protein did not require the addition of any exogenous factor. This shows that the replication machinery, once it is running along the chromosome, is independent of dnaC protein, dnaC activity, however, is required for the replacement of defective dnaB protein at running replication forks.
Intragenic complementation by the nifJ-coded protein of Klebsiella pneumoniae
Authors: Stacey G , Zhu J , Shah V K , Shen S C , Brill W J
Published: January 2020

A single mutation, nifC1005 (Jin et al. Sci. Sin. 23:108-118, 1980), located between nifH and nifJ in the nif cluster of Klebsiella pneumoniae, genetically complemented mutations in each of the 17 known nif genes. This suggested that the mutation is located in a new nif gene. We showed by complementation analyses that only 3 of 12 nifJ mutations tested were complemented by nifC1005. Nitrogenase activity in cell extracts of the mutant with nifC1005 as well as NifJ- mutants was stimulated by the addition of the iron-molybdenum cofactor or nitrogenase component I. The molecular weight of the native NifJ protein is approximately 257,000--a dimer of identical subunits. Some nifC-/nifJ- or nifJ-/nifJ- merodiploids produced active but unstable nifJ proteins. Fine-structure mapping placed the nifC1005 allele within the nifJ gene bounded on both sides by well-characterized nifJ mutations. This indicates that the nifC1005 does not define a separate gene from nifJ. The data are consistent with the occurrence of intragenic complementation between two defective nifJ polypeptides. This explains the isolated examples of genetic complementation between the nifC1005 mutation and certain nifJ mutations.
Molecular weight and amino acid composition of a thermostable lytic endopeptidase
Authors: Turkington E O , Welker N E
Published: January 2020

A thermostable lytic endopeptidase from Bacillus stearothermophilus 1503-4R was purified 14,500-fold, with a 34% recovery of lytic activity. The enzyme is a basic protein (pI, 9.7) with a molecular weight of 15,100 and is composed of approximately 129 amino acid residues.
Analysis of the promoter-distal region of the tra operon of the F sex factor of Escherichia coli K-12 encoded by EcoRI restriction fragments f17, f19, and f2
Authors: Manning P A , Kusecek B , Morelli G , Fisseau C , Achtman M
Published: January 2020

The promoter-distal region of the tra operon of the F sex factor Escherichia coli K-12 was analyzed, using the chimeric plasmid pRS31, which contains the F EcoRI restriction fragments f17, f19, and f2 cloned into the EcoRI site of pSC101. A series of deletion plasmids of pRS31, extending increasing distances from a site in f17 through f19 and ending in f2, were isolated. These plasmids were examined by heteroduplex analysis with the parent DNA, and a restriction map of this region of DNA was constructed. A series of Tn5 insertion derivatives of pRS31 were also isolated and mapped, using both heteroduplex analysis and restriction mapping. Both the insertion and deletion mutants were tested in minicells for the synthesis of radioactively labeled proteins. This allowed the identification of the individual gene products and mapping of the genes. The result is a saturated physical map of this region of DNA from fragment f17 through to the IS3 insertion sequence near the promoter-distal end of f2.
Elongation of teichuronic acid chains by a wall-membrane preparation from Micrococcus luteus
Authors: Traxler C I , Goustin A S , Anderson J S
Published: January 2020

A wall-plus-membrane preparation from Micrococcus luteus catalyzes the incorporation of [14C]glucose from UDP-[14C]glucose, into two fractions of teichuronic acid, which is the cell wall polysaccharide consisting of alternating residues of glucose and N-acetylmannosaminuronic acid (ManNAcUA). Membrane-associated teichuronic acid was extracted from the wall-membrane fraction of reaction mixtures by sodium dodecyl sulfate. The synthesis of membrane-associated teichuronic acid required UDP-glucose, UDP-ManNAcUA, and UDP-N-acetylglucosamine and was inhibited by tunicamycin. Glucose incorporated into wall-bound teichuronic acid remained in wall fragments after extraction with sodium dodecyl sulfate, and its incorporation required UDP-glucose and UDP-ManNAcUA (but not UDP-N-acetylglucosamine) and was insensitive to tunicamycin. Radioactive material incorporated into wall-bound teichuronic acid could be released by treatment with mild acid or by digestion with lysozyme, indicating that the wall-bound teichuronic acid was covalently linked to peptidoglycan. There were about 600 pmol of wall-bound teichuronic acid acceptor sites for glucose per mg of protein as measured in incorporation reaction mixtures lacking UDP-ManNAcUA. In the presence of both UDP-glucose and UDP-ManNAcUA, elongation of teichuronic acid acceptor sites occurred, with the addition of six to eight disaccharide units to each acceptor site.
Xylitol-mediated transient inhibition of ribitol utilization by Lactobacillus casei
Authors: London J , Hausman S
Published: January 2020

The growth of Lactobacillus casei strain Cl-16 at the expense or ribitol was inhibited if the non-metabolizable substrate xylitol was included in the medium at concentrations of 6 mM or greater. At these concentrations, xylitol, did not competitively inhibit ribitol transport. The cessation of growth was caused by the intracellular accumulation of xylitol-5-phosphate, which occurred because growth on ribitol had gratuitously induced a functional xylitol-specific phosphotransferase system but not the enzymes necessary for the further metabolism of xylitol-5-phosphate. Eventually, the cells overcame the xylitol-mediated inhibition by repressing the synthesis of enzyme II of the xylitol phosphotransferase system so that xylitol-5-phosphate would no longer be accumulated within the cell.
Characterization of two tetracycline resistance determinants in Streptococcus faecalis JH1
Authors: LeBlanc D J , Lee L N
Published: January 2020

Streptococcus faecalis strain JH1 harbors two conjugative plasmids: pJH1, an R plasmid mediating resistance to kanamycin, streptomycin, gentamicin, erythromycin, and tetracycline, and pJH2, a hemolysin-bacteriocin plasmid. Studies of plasmid-cured derivatives of strain JH1 and of transconjugates obtained after mixed incubation of JH1 with the plasmid-free S. faecalis strain JH2-2 revealed the presence of two tetracycline resistance determinants in strain JH1. One determinant mediated constitutive resistance to 40 micrograms of tetracycline per ml and was associated with plasmid pJH1. The second determinant, either on the chromosome of strain JH1 or on an undetectable plasmid, was inducible by tetracycline and enabled the host strain, in the absence of pJH1, to grow in the presence of 80 micrograms of tetracycline per ml. One transconjugant, strain DL172, was resistant to 80 micrograms of tetracycline per ml, sensitive to kanamycin, streptomycin, and erythromycin, and hemolytic in the presence, but not in the absence, of tetracycline. A single plasmid, pDL172, from this strain consisted of plasmid pJH2 and a 17.8-kilobase segment of DNA homologous to total cell DNA from strain JH1 but did not contain plasmid pJH1. Whether the addition of heterologous DNA to plasmid pJH2 occurred by translocation of a 17.8-kilobase tetracycline resistance transposon or by classical recombination with pJH2 has not been determined.
Bacterial spore heat resistance correlated with water content, wet density, and protoplast/sporoplast volume ratio
Authors: Beaman T C , Greenamyre J T , Corner T R , Pankratz H S , Gerhardt P
Published: January 2020

Five types of dormant Bacillus spores, between and within species, were selected representing a 600-fold range in moist-heat resistance determined as a D100 value. The wet and dry density and the solids and water content of the entire spore and isolated integument of each type were determined directly from gram masses of material, with correction for interstitial water. The ratio between the volume occupied by the protoplast (the structures bounded by the inner pericytoplasm membrane) and the volume occupied by the sporoplast (the structures bounded by the outer pericortex membrane) was calculated from measurements made on electron micrographs of medially thin-sectioned spores. Among the various spore types, an exponential increase in the heat resistance correlated directly with the wet density and inversely with the water content and with the protoplast/sporoplast volume ratio. Altogether with results supported a hypothesis that the extent of heat resistance is based in whole or in part on the extent of dehydration and diminution of the protoplast in the dormant spore, without implications about physiological mechanisms for attaining this state.
Metabolism of cyclohexaneacetic acid and cyclohexanebutyric acid by Arthrobacter sp. strain CA1
Authors: Ougham H J , Trudgill P W
Published: January 2020

A strain of Arthrobacter was isolated by enrichment culture with cyclohexaneacetate as the sole source of carbon and grew with a doubling time of 4.2 h. In addition to growing with cyclohexaneacetate, the organism also grew with cyclohexanebutyrate at concentrations not above 0.05%, and with a variety of alicyclic ketones and alcohols. Oxidation of cyclohexaneacetate proceeded through formation of the coenzyme A (CoA) ester followed by initiation of a beta-oxidation cycle. beta-Oxidation was blocked before the second dehydrogenation step due to the formation of a tertiary alcohol, and the side chain was eliminated as acetyl-CoA by the action of (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase. The cyclohexanone thus formed was degraded by a well-described route that involves ring-oxygen insertion by a biological Baeyer-Villiger oxygenase. All enzymes of the proposed metabolic sequence were demonstrated in cell-free extracts. Arthrobacter sp. strain CA1 synthesized constitutive beta-oxidative enzymes, but further induction of enzymes active toward cyclohexaneacetate and its metabolites could occur during growth with the alicyclic acid. Other enzymes of the sequence, (1-hydroxycyclohexan-1-yl)acetyl-CoA lyase and enzymes of cyclohexanone oxidation, were present at negligible levels in succinate-grown cells but induced by growth with cyclohexaneacetate. The oxidation of cyclohexanebutyrate was integrated into the pathway for cyclohexaneacetate oxidation by a single beta-oxidation cycle. Oxidation of the compound could be divided into two phases. Initial oxidation to (1-hydroxycyclohexan-1-yl)acetate could be catalyzed by constitutive enzymes, whereas the further degradation of (1-hydroxycyclohexan-1-yl)acetate was dependent on induced enzyme synthesis which could be inhibited by chloramphenicol with the consequent accumulation of cyclohexaneacetate and (1-hydroxycyclohexan-1-yl)acetate.
Biochemical studies of phenoloxidase and utilization of catecholamines in Cryptococcus neoformans
Authors: Polacheck I , Hearing V J , Kwon-Chung K J
Published: January 2020

Protoplasts of Cryptococcus neoformans contain phenoloxidase as a membrane-bound enzyme. The enzyme appeared to be attached on the inner side of cytoplasmic membranes. Synthesis of the enzyme was derepressed by low levels of glucose but was not affected by the level of ammonium. Copper chelators which inhibited the phenoloxidase of other organisms did not affect cryptococcal enzymes. However, cyanide- or iron-chelating agents such as hydroximide derivates or 8-hydroxyquinoline were effective inhibitors, suggesting that cryptococcal phenoloxidase is an iron-containing enzyme. Phenoloxidase of C. neoformans catalyzed the oxidation of various diphenols via dopachrome and labile intermediates to melanin polymers. The kinetic constants (Km) of the phenoloxidase and the permease for dopamine and norepinephrine were low. The correlation between phenoloxidase and the preferential growth of C. neoformans in the host brain is discussed.
Origins of the mycoplasmas: sterol-nonrequiring mycoplasmas evolved from streptococci
Authors: Neimark H , London J
Published: January 2020

We report the establishment of a phylogenetic relationship between the sterol-nonrequiring mycoplasmas (Acholeplasma species) and streptococci. Three specific antisera prepared against purified Streptococcus faecalis fructose diphosphate aldolase and glyceraldehyde-3-phosphate dehydrogenase and Pediococcus cerevisiae glyceraldehyde-3-phosphate dehydrogenase were used for comparative enzyme immunological studies; the Ouchterlony double-diffusion technique and the quantitative microcomplement fixation procedure were employed. The reactions obtained provide evidence showing that all seven ACholeplasma species studied (A. laidlawii, A. granularum, A. modicum, A. oculi, A. axanthum. A. hippikon, and A. equifetale) are phylogenetically related to streptococci and that they evolved from streptococci. The data strongly suggest that the acholeplasmas comprise a distinct evolutionary group that has diverged from streptococci belonging to Lancefield group D or N. No reactions were observed between these enzyme antisera and cell extracts from six fermentative Mycoplasma species. These results support the view that mycoplasmas are derived from various bacteria.
Differential metabolism of cellobiose and glucose by Clostridium thermocellum and Clostridium thermohydrosulfuricum
Authors: Ng T K , Zeikus J G
Published: January 2020

Clostridium thermohydrosulfuricum consumed glucose in preference to cellobiose as an energy source for growth. The rates of substrate uptake in glucose- and cellobiose-grown cell suspensions were 45 and 24 nmol/min per mg (dry weight), respectively, at 65 degrees C. The molar growth yields (i.e., grams of cells per mole of glucose equivalents) were similar on cellobiose and glucose (19 and 16, respectively). Both glucose- and cellobiose-grown cells contained a glucose permease activity and high levels of hexokinase (greater 0.34 mumol/min per mg of protein at 40 degrees C). Growth on cellobiose was associated with induction of a cellobiose permease activity. In contrast, Clostridium thermocellum metabolized cellobiose in preference to glucose as an energy source and displayed lower growth rates on both substrates. The substrate uptake rates in cellobiose- and glucose-grown cell suspensions were 18 and 17 nmol/min per mg (dry weight), respectively. The molar yields were 38 on cellobiose and 20 on glucose. Extracts of glucose- and cellobiose-grown cells both contained cellobiose phosphorylase and phosphoglucomutase activities, whereas only glucose-grown cells contained detectable levels of glucose permease and hexokinase activities. The general catalytic and kinetic properties of the glucose- and cellobiose-catabolizing enzymes in the two species are described, and a model is proposed to distinguish differential saccharide metabolism by these thermophilic ethanologens.
Effects of the ssb-1 and ssb-113 mutations on survival and DNA repair in UV-irradiated delta uvrB strains of Escherichia coli K-12
Authors: Wang T C , Smith K C
Published: January 2020

The molecular defect in DNA repair caused by ssb mutations (single-strand binding protein) was studied by analyzing DNA synthesis and DNA double-strand break production in UV-irradiated Escherichia coli delta uvrB strains. The presence of the ssb-113 mutation produced a large inhibition of DNA synthesis and led to the formation of double-strand breaks, whereas the ssb-1 mutation produced much less inhibition of DNA synthesis and fewer double-strand breaks. We suggest that the single-strand binding protein plays an important role in the replication of damaged DNA, and that it functions by protecting single-stranded parental DNa opposite daughter-strand gaps from nuclease attack.
Characterization of the beta-lactamases of six species of Legionella
Authors: Marre R , Medeiros A A , Pasculle A W
Published: January 2020

The beta-lactamases of six Legionella species were characterized by isoelectric focusing, gel filtration, and substrate profiles. Fifteen strains of L. bozemanii, L. dumoffii, L. gormanii, L. longbeachae, and L. pneumophila produced beta-lactamases active against nitrocefin. L. micdadei enzymes previously reported to be beta-lactamase negative caused a very slow pH-dependent breakdown of nitrocefin and degraded penicillin G at high substrate concentrations. The bioassay revealed predominantly penicillinase activity for all species except L. micdadei, which had no activity in this assay. The apparent molecular weights of enzymes of L. bozemanii, L. gormanii, and L. pneumophila were in the range of 15,000 to 32,000, and those of L. micdadei and L. longbeachae were greater than 250,000. The isoelectric focusing of extracts of Legionella strains in polyacrylamide gels showed beta-lactamase types specific for species (L. bozemanii, L. gormanii, and L. pneumophila) and serotype (L. pneumophila). It demonstrated four different beta-lactamase types in L. pneumophila and revealed close relationships among L. pneumophila serotypes 1, 3, and 6. L. pneumophila enzymes formed band patterns only in polyacrylamide gels containing 6 M urea, whereas L. dumoffii and L. longbeachae enzymes did not form bands in any of the gels. None of the band patterns resembled those of known plasmid-mediated beta-lactamases. These experiments suggest that isoelectric focusing of chromosomal beta-lactamases may be a valuable tool for taxonomic studies.
Amino acid uptake and energy coupling dependent on photosynthesis in Anacystis nidulans
Authors: Lee-Kaden J , Simonis W
Published: January 2020

The photoautotrophic cyanobacterium Anacystis nidulans was used to investigate the membrane transport of branched-chain, neutral amino acids and its dependence on photosynthetic reactions. The uptake of alpha-amino [1-14C]isobutyric acid and L-[1-14C]leucine followed Michaelis, Menten kinetics and resulted in an energy-dependent accumulation. As in bacteria, different uptake systems for neutral amino acids were present: two DAG (D-alanine, aminoisobutyric acid, and glycine) systems responsible for uptake of alpha-amino [1-14C]isobutyric acid, and one LIV (leucine, isoleucine, and valine) system, responsible for uptake of leucine. The low-affinity DAG system seemed to be dependent on the presence of Na+ ions. Uptake was enhanced by white light and by monochromatic light of 630 nm. In far red light (717 nm) with and without nitrogen flushing, considerable uptake dependent on light intensity and inhibition by dibromothymoquinone and by high concentrations of KCN were observed. Therefore, the energy generated by photosystem I reactions only could perform this membrane transport. The proton translocator carbonylcyanide m-chlorophenylhydrazone and N,N-dicyclohexylcarbodiimide as an ATPase inhibitor reduced amino acid uptake to a high degree. A pH dependence of aminoisobutyric acid and leucine uptake was obvious, with a maximum at pH 6 to 7 and some at a pH as high as 9.5. At higher pH, increasing concentrations of Na+ K+ and also of triphenylmethylphosphonium ions inhibited the transport of aminoisobutyric acid. These findings are consistent with the assumption that ATP from photosynthetic reactions drives a membrane-bound proton-translocating ATPase producing a proton motive force, consisting at higher pH chiefly in a delta psi amount, which promotes a secondary active H+ or Na+/amino acid symport carrier.
lac Transcription in Escherichia coli cells treated with chloramphenicol
Authors: Graham M Y , Tal M , Schlessinger D
Published: January 2020

When protein synthesis was blocked by chloramphenicol in vivo, transcription initiation of lac mRNA was severely inhibited. In a promoter mutant (L8-UV5) or in wild-type cells supplemented with adenosine 3',5'-phosphate (greater than or equal to 5 mM), nearly normal initiation could be achieved, and when the mRNA chains formed were extracted, they coded for the 5'-terminal alpha-peptide of the lacZ gene in vitro. However, even under such conditions, only a fraction of RNA polymerases proceeded to the end of the Z gene in the presence of chloramphenicol; as a consequence, a wide range of sizes of mRNA was produced, and very few transcripts were formed all the way to the natural termination site of the operon. In other words, premature transcription termination occurred in chloramphenicol-treated cells, as current models predict, but terminations occurred to variable extents at several intragenic sites and apparently at least one intergenic site. Termination at intragenic sites occurred far less in cells bearing a mutation in the transcription termination factor rho.
Mutations releasing mitochondrial biogenesis from glucose repression in Saccharomyces cerevisiae
Authors: Böker-Schmitt E , Francisci S , Schweyen R J
Published: January 2020

Mutants which exhibit a constitutive glucose-insensitive expression of respiratory activity were selected by use of a triphenyltetrazolium staining technique. These mutants lack carbon catabolite repression, as was demonstrated by measuring cytochromes, the activity of succinate cytochrome c reduction, total cellular respiration, mitochondrial protein, and DNA synthesis. High growth rates of mutant cells in glucose medium and normal fermentative CO2 production exclude the possibility that this carbon catabolite insensitivity of mitochondrial functions is merely due to a decreased utilization of glucose. Accordingly, the activities of the two cytoplasmic enzymes measured, maltase and malate synthase, were glucose repressible to the same extent in the mutants as in the wild type. The mutations are dominant and showed nuclear inheritance. The results are discussed in terms of carbon catabolite-regulated expression of genes involved in the biogenesis of mitochondria.
Inversion of helix orientation in Bacillus subtilis macrofibers
Authors: Mendelson N H , Karamata D
Published: January 2020

The ability of helical macrofibers of Bacillus subtilis to convert from left- to right-handed structures or vice versa has been known to be controlled by the nutritional environment (N. H. Mendelson, Proc. Natl. Acad. Sci. U.S.A., 75:2478-2482, 1978). lyt mutants (Ni15, FJ3, FJ6, and FJ7) and also lyt phenocopies of wild-type strain FJ8 were able to undergo helix hand inversion as a function of temperature. The transition between right- and left-handed structures was in a very narrow range (about 2.5 degrees C) in the low to mid-40 degrees C. The helix orientation of these strains was also influenced by the concentration of divalent ions. Macrofiber handedness is governed, therefore, by at least four factors: genetic composition, temperature, and nutritional and ionic environments. Conditions normally used for growth fall, within this matrix, in the region favoring right-handed structures. Inhibition studies suggest that cell growth must occur for helix hand inversion.
Novel selection for tetracycline- or chloramphenicol-sensitive Escherichia coli
Authors: Craine B L
Published: January 2020

A method for selecting tetracycline- or chloramphenicol-sensitive Escherichia coli cells from a population of predominantly resistant cells is described. This method depends on the inability of drug-sensitive cells to induce lambda receptors in the presence of chloramphenicol or tetracycline, protein synthesis-inhibiting drugs. The addition of bacteriophage lambda vir to a mixture of drug-sensitive and drug-resistant cells, induced for lambda receptors in the presence of tetracycline or chloramphenicol, preferentially kills the drug-resistant cells (which are capable of inducing lambda receptors). The result is a culture enriched for the sensitive cells. Several common strains used for transformation were compared for their ability to be selected. E. coli 294 was found to be superior.
Utilization by Saccharomyces cerevisiae of 5'-methylthioadenosine as a source of both purine and methionine
Authors: Cone M C , Marchitto K , Zehfus B , Ferro A J
Published: January 2020

Cells of the yeast Saccharomyces cerevisiae are normally impermeable to the purine nucleosides adenosine and 5'-deoxy-5'-methylthioadenosine (MTA), a product of polyamine biosynthesis. cordycepin-sensitive, adenosine-utilizing strains of S. cerevisiae were able to use MTA to fulfill an auxotrophic requirement for purine. Cordycepin-sensitive strains carrying a met5 mutation were also able to use MTA as a source of methionine. These MTA-utilizing strains of S. cerevisiae should be useful for metabolic studies of the fate of MTA.