bluuebunny/crossref_metadata_2025 huggingface dataset:
Ability of Bacillus subtilis protoplasts to repair irradiated bacteriophage deoxyribonucleic acid via acquired and natural enzymatic systems
Authors: Yasbin R E , Andersen B J , Sutherland B M
Published: January 2020

A novel form of "enzyme therapy" was achieved by utilizing protoplasts of Bacillus subtilis. Photoreactivating enzyme of Escherichia coli was successfully inserted into the protoplasts of B. subtilis treated with polyethylene glycol. This enzyme was used to photoreactivate ultraviolet-damaged bacteriophage deoxyribonucleic acid (DNA). Furthermore, in polyethylene glycol-treated protoplasts, ultraviolet-irradiated transfecting bacteriophage DNA was shown to be a functional substrate for the host DNA excision repair system. Previous results (R. E. Yasbin, J. D. Fernwalt, and P. I. Fields, J. Bacteriol. 137:391-396, 1979) showed that ultraviolet-irradiated bacteriophage DNA could not be repaired via the excision repair system of competent cells. Therefore, the processing of bacteriophage DNA by protoplasts and by competent cells must be different. This sensitive protoplast assay can be used to identify and to isolate various types of DNA repair enzymes.
Influence of lipopolysaccharide and protein in the cell envelope on recipient capacity in conjugation of Salmonella typhimurium
Authors: Sanderson K E , Janzer J , Head J
Published: January 2020

In crosses of Salmonella typhimurium FfinP301 lac+ to F- strains of S. typhimurium in broth, recipient strains which were rough mutants affected in the outer core region of the lipopolysaccharide gave an average of 1.4 Lac+ transconjugants per donor cell and over 50% of the donor and recipient cells in mating aggregates, whereas smooth recipient strains gave 0.08 Lac+ transconjugants and few cells in mating aggregates. Strains with mutations affecting the inner core of the lipopolysaccharide were usually poor recipients. When cells were mated on Millipore membrane filters, both smooth and rough strains gave ca. 1.0 Lac+ transconjugants per donor cell. Plasmids in Inc groups FI, FII, M, J, and I beta gave more transconjugants with rough than smooth strains, but there were no difference in crosses with plasmids in Inc groups T, L, P, N, and W. Strains with mutations in the ompA gene (deficient in Omp Ap = 33K = II* = conjugation protein) yielded only 0.02 Lac+ transconjugants per donor cell and few cells in mating aggregates. There was no indication of a deficiency of Omp Ap in smooth strains compared with rough strains. Reduced fertility of smooth recipients may occur because the O side chains of the lipopolysaccharide shield the recipient and reduce the frequency of stabilization of mating aggregates. However, gradient-of-transmission experiments indicated that once these mating aggregates are stabilized, they are equally stable in both smooth and rough recipients. Fertility was high in crosses of S. typhimurium Flac+ to Escherichia coli K-12 F- (0.75 Lac+ transconjugants per donor cell; over 50% of the cells in mating aggregates). In crosses of E. coli K-12 Flac+ to S. typhimurium smooth F-, ca. 10(-5) Lac+ transconjugants per donor cell were obtained; in crosses to rough recipient strains, fertility was increased 14-fold, and when the recipient was defective in the SA and LT host restriction systems, fertility was increased in additional 100-fold. Thus, both the lipopolysaccharide and the protein in the cell envelope of S. typhimurium were shown to be important in the recipient function in F-mediated conjugation.
Lack of Chemotactic Response to Tricarboxylic Acids by
Escherichia coli
Carrying a Plasmid Determining Citrate Utilization
Authors: Ishigura Naotaka , Sato Gihei , Yoshikawa Masanosuke
Published: January 2020

Salmonella typhimurium
but not citrate-utilizing
Escherichia coli
carrying the Cit plasmid showed positive chemotaxis to tricarboxylic acids, indicating that the Cit plasmid encodes a transport system but not a chemoreceptor for citrate.
Comparison among patterns of macromolecular synthesis in Escherichia coli B/r at growth rates of less and more than one doubling per hour at 37 degrees C
Authors: Pierucci O , Melzer M , Querini C , Rickert M , Krajewski C
Published: January 2020

In Escherichia coli B/r, the relationship between the patterns of chromosome replication and of synthesis of envelope components differs at various growth rates. At growth rates greater than 1.0 doubling per h at 37 degrees C, the average mass and age at initiation of rounds of chromosome replication are similar to those at increase in incorporation of precursors into a major outer membrane protein and phosphatidylethanolamine. At growth rates less than 1.0 doubling per h at 37 degrees C the average mass and age at increase in the synthesis of these envelope components differ from those at initiation of chromosome replication. The average cell mass per chromosomal origin at initiation of rounds of chromosome replication is not a constant and varies between growth rates greater and less than 1.0 doubling per h.
Molecular nature of a plasmid specifying beta-lactamase production in Haemophilus ducreyi
Authors: Brunton J , Bennett P , Grinsted J
Published: January 2020

We characterized pJB1, the plasmid previously reported to mediate beta-lactamase production in Haemophilus ducreyi. We studied its relationship to pMR0360 and RSF0885, the plasmids responsible for beta-lactamase production in Neisseria gonorrhoeae and Haemophilus parainfluenzae, respectively. Although pJB1 was maintained as a multicopy pool in Escherichia coli, it was not stably maintained in the absence of antibiotic selection. Electron microscope heteroduplex studies showed that it carried 100% of the transposable ampicillin resistance sequence TnA. This sequence was transposed to plasmid pUB307 at a low rate. Heteroduplexes between pMR0360 and pJB1 showed that they contained 3.3 megadaltons of homologous sequences. Two sets of nonhomologous sequences, one a TnA sequence and the other a non-TnA sequence, took the form of insertion loops. For plasmids pMR0360 and RSF0885, previously shown to be highly related, the nonhomologous sequences took the form of a substitution loop. We concluded that all three plasmids shared major portions of their sequences but differed in discrete segments. pJB1 was the first such plasmid to have a physically and functionally intact TnA sequence.
Plasmid-determined immunity of Escherichia coli K-12 to colicin Ia Is mediated by a plasmid-encoded membrane protein
Authors: Weaver C A , Redborg A H , Konisky J
Published: January 2020

The colicin Ia structural (cia) and immunity (iia) genes of plasmid pColIa-CA53 have been cloned into the cloning vector pBR322. These two genes are closely linked, and both of them can be isolated on a deoxyribonucleic acid fragment approximately 4,800 base pairs long. An analysis of the polypeptides synthesized in ultraviolet-irradiated cells containing these cloned genes led to the conclusion that the iia gene product is a polypeptide with a molecular weight of approximately 14,500. Insertion of transposon Tn5 into the iia gene led to a concomitant loss of the immune phenotype and the ability to produce this protein. Fractionation of ultraviolet-irradiated cells harboring a plasmid carrying the iia gene showed that the immunity protein is a component of the inner (cytoplasmic) membrane. Furthermore, the mechanism of immunity to colicin Ia appears to operate at the level of the cytoplasmic membrane. This conclusion is based on our finding that membrane vesicles prepared from colicin Ia-immune cells could be depolarized by colicins E1 and Ib but not by colicin Ia.
Adenosine 3',5'-phosphate phosphodiesterase and pheromone response in the yeast Saccharomyces cerevisiae
Authors: Liao H H , Thorner J
Published: January 2020

Theophylline, aminophylline, and isobutylmethylxanthine, compounds reported to be inhibitors of adenosine 3',5'-phosphate (cAMP) phosphodiesterase, prevented the alpha-factor-induced cell cycle arrest of Saccharomyces cerevisiae a cells. To determine whether the in vivo effect of these methylxanthines on yeast pheromone response was related to their known biochemical mode of action, two assays for cAMP phosphodiesterase based on affinity of the product of the reaction (5'-AMP) for boronate groups were developed and were used to monitor the activity of the low Km cAMP phosphodiesterase present in yeast extracts. It was found that the relative efficacy of the methylxanthines as inhibitors of this enzyme in vitro was correlated with the degree to which they antagonized alpha-factor action in vivo. These results were consistent with our previous proposal that pheromone action involves a lowering of cAMP level in the target cell.
Insertion of transposons through the major cotransduction gap of Escherichia coli K-12
Authors: Fouts K E , Barbour S D
Published: January 2020

The major cotransduction gap of the Escherichia coli chromosome extends from mini 31 to 34. We have inserted transposons through this gap which, by sequential transduction, link sbcA (min 29.8) with manA (min 35.7) and thus eliminate the gap. These results indicate that the length of DNA in the region, as measured by transduction, is not significantly different from the length obtained by conjugational time of entry. Since this segment of the E. coli chromosome has few known genes, these transposon insertions will be useful for genetic manipulations in the region of the gap. We describe the usefulness of these markers for rapidly mapping mutations which may be isolated in the region from min 27 to 37.
Biochemical diversity for biosynthesis of aromatic amino acids among the cyanobacteria
Authors: Hall G C , Flick M B , Gherna R L , Jensen R A
Published: January 2020

We examined the enzymology and regulatory patterns of the aromatic amino acid pathway in 48 strains of cyanobacteria including representatives from each of the five major grouping. Extensive diversity was found in allosteric inhibition patterns of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, not only between the major groupings but also within several of the generic groupings. Unimetabolite inhibition by phenylalanine occurred in approximately half of the strains examined; in the other strains unimetabolite inhibition by tyrosine and cumulative, concerted, and additive patterns were found. The additive patterns suggest the presence of regulatory isozymes. Even though both arogenate and prephenate dehydrogenase activities were found in some strains, it seems clear that the arogenate pathway to tyrosine is a common trait that has been highly conserved among cyanobacteria. No arogenate dehydratase activities were found. In general, prephenate dehydratase activities were activated by tyrosine and inhibited by phenylalanine. Chorismate mutase, arogenate dehydrogenase, and shikimate dehydrogenase were nearly always unregulated. Most strains preferred NADP as the cofactor for the dehydrogenase activities. The diversity in the allosteric inhibition patterns for 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, cofactor specificities, and the presence or absence of prephenate dehydrogenase activity allowed the separation of subgroupings within several of the form genera, namely, Synechococcus, Synechocystis, Anabaena, Nostoc, and Calothrix.
Fractionation and characterization of the phosphoenolpyruvate: fructose 1-phosphotransferase system from Pseudomonas aeruginosa
Authors: Durham D R , Phibbs P V
Published: January 2020

The initial reactions involved in the catabolism of fructose in Pseudomonas aeruginosa include the participation of a phosphoenolpyruvate:fructose 1-phosphotransferase system (F-PTS). Fractionation of crude extracts of fructose-grown cells revealed that both membrane-associated and soluble components were essential for F-PTS activity. Further resolution of the soluble fraction by both size exclusion and ion-exchange chromatography revealed the presence of only one component, functionally analogous to enzyme I. Enzyme I exhibited a relative molecular weight of 72,000, catalyzed the pyruvate-stimulated hydrolysis of phosphoenolpyruvate to pyruvate, and mediated the phosphorylation of fructose when combined with a source of enzyme II (washed membranes). No evidence for the requirement of a phosphate carrier protein, such as HPr, could be demonstrated. Thus, the F-PTS requires a minimum of two components, a soluble enzyme I and a membrane-associated enzyme II complex, and both were shown to be inducible. Reconstituted F-PTS activity was specific for phosphoenolpyruvate as a phosphate donor (Km, approximately -0.6 mM) and fructose as the sugar substrate (Km, approximately 18 microM). Components of the Pseudomonas F-PTS did not restore activity to extracts of deletion mutants of Salmonella typhimurium deficient in individual proteins of the PTS or to fractionated membrane and soluble components of the F-PTS of Escherichia coli. Similarly, membrane and soluble components of E. coli and S. typhimurium would not cross-complement the F-PTS components from P. aeruginosa.
Properties of mitochondria as a function of the growth stages of Neurospora crassa
Authors: Schwitzguébel J P , Palmer J M
Published: January 2020

The oxidative and phosphorylative properties of mitochondria isolated from Neurospora crassa were investigated as a function of growth stage. The rates of oxidation of exogenous NADH and NADPH varied independently of each other, thus ruling out the existence of only one unspecific dehydrogenase. Two different pathways were involved in the oxidation of NAD-linked substrates, as indicated by changes in the rate of oxygen uptake, the sensitivity to rotenone, and the efficiency of phosphorylation. One pathway was sensitive to rotenone and involved three energy-coupling sites, whereas the other was resistant to rotenone and bypassed complex I. Our results indicated that the activity of complex I of the respiratory chain increased markedly in the late exponential phase of growth, remained high in the stationary phase, and then decreased when conidiae were formed. In contrast, the activity of the rotenone-resistant bypass was maximal in the early exponential phase. With malate (plus glutamate) as a substrate, the sensitivity to rotenone and the ADP/O ratios were always lower than those observed with other NAD-linked substrates, suggesting a possible cooperation between malate dehydrogenase and the rotenone-resistant pathway. The rate of oxygen uptake measured in the presence of rotenone was significantly increased by the addition of exogenous NAD+, suggesting that added NAD+ could interact with the rotenone-resistant bypass.
Transposon mutagenesis in Caulobacter crescentus
Authors: Ely B , Croft R H
Published: January 2020

Transposons Tn5 (Km) and Tn7 (Tp and Sm) were transferred to Caulobacter crescentus via P-type antibiotic resistance factors. Transposition was demonstrated by the isolation of chromosomal insertions of each transposon. With C. crescentus strains harboring RP4 aphA::Tn7, the introduction of a wild-type RP4 resulted in the loss of the resident plasmid. Simultaneous selection for Kmr and Smr yielded colonies with chromosomal insertions of Tn7. Examination of over 10,000 chromosomal insertions of Tn7 indicated no auxotrophic or motility mutants. Thus, Tn7 appears to have a high specificity of insertion in C. crescentus. The Mu-containing plasmid pJB4JI transferred Tn5 to C. crescentus, but the plasmid was not maintained. Control experiments showed that recovery of Mu-containing plasmids occurred at very low frequencies in C. crescentus and that the plasmids which were recovered had undergone extensive deletion of plasmid DNA. Presumably, some part of the Mu genome was not tolerated by C. crescentus. The instability of the Mu-containing plasmids makes them excellent vectors for the introduction of transposons, and we have used pJB4JI to isolated chromosomal insertions of Tn5. When several thousand of these insertion mutants were examined, we found auxotrophic and motility mutants at frequencies of 1 and 2%, respectively. These results indicate that Tn5 had a low specificity of insertion in C. crescentus and therefore would be a useful mutagen for obtaining a variety of mutant phenotypes.
An exo-poly-alpha-D-galacturonosidase implicated in the regulation of extracellular pectate lyase production in Erwinia chrysanthemi
Authors: Collmer A , Whalen C H , Beer S V , Bateman D F
Published: January 2020

Pectic enzymes in the supernatants of Erwinia chrysanthemi cultures in late-logarithmic-phase growth on D-galacturonan were resolved into three components: two pectate lyase isozymes and an exo-poly-alpha-D-galacturonosidase previously unreported in this organism. The hydrolytic enzyme was purified to homogeneity by ammonium sulfate fractionation, preparative electrofocusing in Ultrodex gel, and gel filtration through Ultrogel AcA54. The enzyme had a specific activity of 591 mumol/min per mg of protein, a pI of 8.3, a molecular weight of 67,000, a pH optimum of 6.0, and a Km of 0.05 mM for D-galacturonan. Analyses of reaction mixtures by paper chromatography revealed that the enzyme released only digalacturonic acid from D-galacturonan. The action of the hydrolytic enzyme on D-galacturonan labeled at the nonreducing end by partial digestion with pectate lyase revealed that it rapidly released 4,5-unsaturated digalacturonic acid from 4,5-unsaturated pectic polymers. The production of extracellular exo-poly-alpha-D-galacturonosidase was coordinately regulated with pectate lyase production. The action patterns of the two enzymes appeared complementary in the degradation of pectic polymers to disaccharides that stimulated pectic enzyme production and supported bacterial growth.
Mapping of the arginine deiminase gene in Pseudomonas aeruginosa
Authors: Mercenier A , Stalon V , Simon J P , Haas D
Published: January 2020

A mutant of Pseudomonas aeruginosa PAO lacking arginine deiminase activity (arcA) was isolated by screening for a derivative of an arcB mutant (deficient in catabolic ornithine carbamoyltransferase) that did not excrete citrulline under conditions of limited aeration. The arcA mutation was highly cotransducible with arcB.
Regulation of formation of aerial mycelia and spores of Streptomyces viridochromogenes
Authors: Coleman R H , Ensign J C
Published: January 2020

Growth of Streptomyces viridochromogenes on a solid glycerol-NH4NO3 salts medium was accompanied by the formation of aerial mycelia and spores. Adding 0.5% or more casein hydrolysate to the medium stimulated growth while completely repressing the formation of aerial mycelia and spores. This repression was temporary, as evidenced by the fact that transfer of the organisms to media not containing casein hydrolysate resulted in the appearance of aerial mycelia and spores. The effects of individual amino acids were tested. Glycine retarded growth and repressed formation of both aerial mycelia and spores. L-Aspartic acid, L-glutamic acid, and L-histidine stimulated or had little effect on growth and repressed formation of spores but not aerial mycelia. Repression by casein hydrolysate could not be attributed to the carbon/nitrogen ratio or the pH of the medium. Adding 1.25 to 2.5 mM adenine to the medium caused a reversal of the casein hydrolysate repression of aerial mycelium formation but did not reverse repression of sporulation. Dimethyladenine and 8-azaguanine had an effect similar to that of adenine, but a variety of other purine or pyrimidine derivatives had no effect on casein hydrolysate repression. The repression of aerial mycelium and spore formation by casein hydrolysate occurred only in media containing 15 mM or more phosphate. Aerial mycelia and spores were formed in media containing casein hydrolysate and 3 mM or less phosphate.
DNA homology of the promoter-distal regions of the tra operons of sex factors F and R100 in Escherichia coli K-12
Authors: Manning P A , Morelli G
Published: January 2020

The promoter-distal regions of the tra operons of F and R100-1 were analyzed by heteroduplex analysis, and the regions of nonhomology were identified. A common EcoRI restriction site was shown to be present, and this has allowed the physical maps to be aligned.
Megaplasmids in the plant-associated bacteria Rhizobium meliloti and Pseudomonas solanacearum
Authors: Rosenberg C , Casse-Delbart F , Dusha I , David M , Boucher C
Published: January 2020

Using analytical and preparative methods, we demonstrated the presence of megaplasmids with molecular weight larger than 450 x 10(6) in the two plant-associated bacteria Rhizobium meliloti and Pseudomonas solanacearum. Such giant plasmids were found in 8 of 9 P. solanacearum strains tested and in all of the 18 R. meliloti strains tested.
The product of the lexC gene of Escherichia coli is single-stranded DNA-binding protein
Authors: Meyer R R , Rein D C , Glassberg J
Published: January 2020

Extracts from lexC113 cells could not support phage G4 DNA-dependent replication unless supplemented with single-stranded DNA-binding protein. Purified lexC113 binding protein supported synthesis in a reconstituted replication assay, using purified proteins at 30 but not at 42 degrees C, indicating that the product of the lexC113 gene is an altered single-stranded DNA-binding protein.
Production by Aspergillus awamori of homologous oligosaccharides, each with an uneven number of glucosyl units and a reducing terminal (alpha , 1 leads to 4) linkage
Authors: Nordin J H , Bobbitt T F
Published: January 2020

Homologous oligosaccharides with degrees of polymerization of 7 to 15 have been isolated from the nigeran-protein complex of Aspergillus awamori Nakazawa (QM873). Each contains an uneven number of glucose units, differs from its nearest homolog by two residues, and contains the sequence Glc(alpha , 1 leads to 3)Glc- (alpha , 1 leads to 4)Glc at its reducing terminus.
Autoregulation of the tyrR gene
Authors: Camakaris H , Pittard J
Published: January 2020

Strains of Escherichia coli K-12 in which the transcription of lacZ is initiated from the tyrR promoter have been constructed by use of the Mu d (Apr lac) phage of Casadaban and Cohen (Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533, 1979). These strains have been used to examine the regulation of expression from the tyrR promoter, with the synthesis of beta-galactosidase used as an index of expression. The specific activity of beta-galactosidase fell to 51% upon introduction of lambda (Tn10) tyrR+; to 39% upon introduction of F123, an F-prime carrying tyrR+; to 29% upon introduction of pMU309, a derivative of the plasmid RP4 carrying tyrR+; and to 13.6% upon introduction of pMU352, a derivative of the multicopy plasmid pBR322 carrying tyrR+. These results indicate that the tyrR gene product interacts with its own promoter-operator region, decreasing synthesis of beta galactosidase in the tyrR::Mu d (Apr lac) strains. The increasing extent of repression of beta-galactosidase synthesis with increasing tyrR+ gene dosage was accompanied by increasing repression of the synthesis of tyrosine- and phenylalanine-repressible 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetases. The interaction of the repressor with tyrRo appears unusual in the sense that aporepressor alone is probably one of the repressing species. The levels of beta-galactosidase synthesized in the tyrR::Mu d (Apr lac) strains indicate that tyrR has a relatively efficient promoter, the maximum levels representing on the order of a relatively efficient promoter, the maximum levels representing on the order of 1,000 monomers of beta-galactosidase per cell in the tyrR strain and about 500 monomers in the tyrR+ haploid strain.
Proton motive force and the physiological basis of delta pH maintenance in thiobacillus acidophilus
Authors: Matin A , Wilson B , Zychlinsky E , Matin M
Published: January 2020

At optimal growth pH (3.0) Thiobacillus acidophilus maintained an internal pH of 5.6 (delta pH of 2.6 units) and a membrane potential (delta psi) of some +73 mV, corresponding to a proton motive force (delta p) of -83 mV. The internal pH remained poised at this value through external pH values of 1 to 5, so that the delta pH increased with decreasing external pH. The positive delta psi increased linearly with delta pH: above a delta pH of 0.6 units, some 60% of the increase in delta pH was compensated for by an opposing increase in delta psi. The highest magnitude of delta pH occurred at an external pH of 1.0, where the cells could not respire. Inhibiting respiration by CN- or azide in cells at optimal pH decreased delta pH by only 0.4 to 0.5 units and caused a corresponding opposite increase in delta psi. Thus, a sizable delta pH could be maintained in the complete absence of respiration. Treatment of cells with thiocyanate to abolish the delta psi resulted in a time-dependent collapse of delta pH, which was augmented by protonophores. We postulate that T. acidophilus possesses unusual resistance to ionic movements. In the presence of a large delta pH (greater than 0.6 pH units), limited diffusion of H+ into the cell is permitted, which generates a positive delta psi because of resistance to compensatory ionic movements. This delta psi, by undergoing fluctuations, regulates the further entry of H+ into the cell in accordance with the metabolic state of the organism. The effect of protonophores was anomalous: the delta p was only partially collapsed, and respiration was strongly inhibited. Possible reasons for this are discussed.
A multifunctional gene (tetR) controls Tn10-encoded tetracycline resistance
Authors: Beck C F , Mutzel R , Barbé J , Müller W
Published: January 2020

The tetracycline resistance regulatory gene (tetR) of transposon Tn10 was analyzed by a combination of methods involving gene fusion and cloning. This gene is located on a 695-base pair HincII DNA fragment near the center of Tn10. The direction of transcription is opposite to that of neighboring gene tetA, which encodes the TetA protein. The gene product of the tetR gene (the TetR protein) has a molecular weight of 23,000. tet-R-lacZ gene fusions encode fusion beta-galactosidases that are membrane bound, indicating that the TetR protein itself is membrane associated. Mutants defective in tetR result in constitutive tetracycline resistance, but the level of resistance is reduced. Expression of the tetR gene is induced by tetracycline; in the absence of tetracycline, the TetR protein turns off its own synthesis.
Photometric immersion refractometry of bacterial spores
Authors: Gerhardt P , Beaman T C , Corner T R , Greenamyre J T , Tisa L S
Published: January 2020

Photometric immersion refractometry was used to determine the average apparent refractive index (n) of five types of dormant Bacillus spores representing a 600-fold range in moist-heat resistance determined as a D100 value. The n of a spore type increased as the molecular size of various immersion solutes decreased. For comparison of the spore types, the n of the entire spore and of the isolated integument was determined by use of bovine serum albumin, which is excluded from permeating into them. The n of the sporoplast (the structures bounded by the outer pericortex membrane) was determined by use of glucose, which was shown to permeate into the spore only as deeply as the pericortex membrane. Among the various spore types, an exponential increase in the heat resistance correlated with the n of the entire spore and of the sporoplast, but not of the isolated perisporoplast integument. Correlation of the n with the solids content of the entire spore provided a method of experimentally obtaining the refractive index increment (dn/dc), which was constant for the various spore types and enables the calculation of solids and water content from an n. Altogether, the results showed that the total water content is distributed unequally within the dormant spore, with less water in the sporoplast than in the perisporoplast integument, and that the sporoplast becomes more refractile and therefore more dehydrated as the heat resistance becomes greater among the various spore types.
Trimethoprim-Produced F-Specific Insertion Mutations in
Escherichia coli
K-12
Authors: Mukhopadhyay Pradip , Palchaudhuri Sunil
Published: January 2020

Besides producing thymine-requiring mutants (
thy
), trimethoprim (TMP) cured the mini-ColE1 replicon pML21 at an appreciable frequency. The cured
Escherichia coli
K-12 cells behaved like
polA
mutants by failing to support the stable maintenance of the ColE1 plasmid. The mini-F replicon pSC138, which was lacking all three insertion sequences (IS
3
, γδ, and IS
2
) normally used for F-specific integration and excision, was not cured by TMP. Instead, it integrated into specific regions of the
E. coli
chromosome and thus caused auxotrophic mutations in operons which were always localized on either side of
oriC
(origin of chromosomal replication). The incompatibility and replication functions of the integrated plasmid in auxotrophs were retained, and the plasmid DNAs recovered from spontaneously occurring revertants did not show any alterations in their contour lengths as determined by electron microscopy. The F replicon (fragment 5) contained in plasmid pSC138 carried two origins of replication, the primary origin,
oriV
1
at 42.6F and the secondary origin,
oriV
2
, at 44.1F. Another mini-F plasmid pMF21, deleted of the primary origin of replication (
oriV
1
), was still capable of autonomous replication but failed to integrate onto the chromosome after TMP treatment. Furthermore, the composite plasmid pRS5, which normally uses only the replication origin and functions of the pSC101 component, was also insensitive to TMP. On the basis of these results, we propose a new scheme of F integration via the functional
oriV
1
and suggest the involvement of a similar mechanism in the formation of Hfr strains by integrative suppression.
Roles of cell surface components of Escherichia coli K-12 in bacteriophage T4 infection: interaction of tail core with phospholipids
Authors: Furukawa H , Mizushima S
Published: January 2020

The cell surface of Escherichia coli K-12, reconstituted from the OmpC protein, lipopolysaccharide, and the peptidoglycan layer, was active as a receptor for phage T4, resulting in the contraction of the tail sheath and the penetration of the core through the cell surface (Furukawa et al., J. Bacteriol. 140:1071--1080, 1979). In the present work the process of DNA ejection from the contracted T4 phage particle was studied. Contracted phage particles were adsorbed to phospholipid liposomes by the core tip. This adsorption resulted in ejection of phage DNA. Either phosphatidylglycerol or cardiolipin was active for the DNA ejection. A proton motive force across the liposome membrane was not required for these processes. The process of DNA ejection, however, was temperature dependent, whereas the adsorption of the core tip to liposomes took place at 4 degrees C. Based on these observations together with those in the previous paper, the process of T4 infection of E. coli K-12 cells is discussed with special reference to the roles of cell surface components.
A Comparative Structural Analysis of the Flagellin Monomers ofCaulobacter crescentusIndicates that These Proteins Are Encoded by Two Genes
Authors: Gill Paul R. , Agabian Nina
Published: January 2020

The flagellum ofCaulobacter crescentusis composed of two flagellin polypeptide monomers which are distinguished by molecular weight and are closely related by biochemical and immunological criteria (C. Lagenaur and N. Agabian, J. Bacteriol.132:731-733, 1977). The synthesis and assembly of these two flagellin proteins are developmentally regulated, and the periodicity of expression for each is distinct (C. Lagenaur and N. Agabian, J. Bacteriol.135:1062-1069, 1978; M. A. Osley, M. Sheffery, and A. Newton, Cell12:393-400, 1977). To understand the genetic and functional relationship between the 25,000- and 27,500-molecular-weight flagellins ofC. crescentusCB15, a detailed comparative analysis of their protein structure was made, using a number of techniques, including one- and two-dimensional peptide mapping, a novel procedure of peptide alignment, and amino terminal amino acid sequence analysis. The tryptic peptides generated by each of the flagellins were compared by two-dimensional thin-layer chromatography. This peptide map analysis indicated that approximately 36% of the peptides generated from these two proteins had similar migration properties. Together with biochemical and immunological criteria, the two-dimensional peptide map suggested some structural relatedness between the monomers. However, a comparison of peptide fragments generated during partial protease digestion of each protein by a method of one-dimensional mapping indicated that the two proteins are structurally unique. A peptide alignment technique was developed to directly compare the primary structure of these proteins. In the peptide alignment procedure the amino terminus of each protein is radioactively labeled. After partial enzymatic digestion, the peptides are fractionated by polyacrylamide gel electrophoresis: those labeled at the amino terminus are then resolved by subsequent autoradiography. Each digest contains a family of amino-terminal-labeled fragments, the sizes of which reflect the sequential alignment of cleavage sites in the protein. A comparison of the alignment of specific cleavage sites of the two flagellins by this technique further established that each flagellin is structurally unique, particularly in the carboxyl terminal region. Finally, comparison of the amino terminal amino acid sequences indicated that the amino terminal region of both flagellins is highly conserved, but that the two polypeptides are clearly not identical. These findings strongly indicate that the two flagellins are encoded by distinct genetic loci and are not the product of novel processing of a single larger precursor.
Obligate methylotrophy: evaluation of dimethyl ether as a C1 compound
Authors: Meyers A J
Published: January 2020

The suitability of dimethyl ether as a C1 compound was examined with the obligate methylobacterium Methylococcus capsulatus (Texas). The ether did not support growth and was not formed during growth on methane; it was an inhibitor of growth and oxidation of methane and a poor oxidation substrate for cell suspensions. NADH stimulation of methane, but not dimethyl ether, oxidation occurred in cell extracts.
Arginine metabolism in lactic streptococci
Authors: Crow V L , Thomas T D
Published: January 2020

Streptococcus lactis metabolizes arginine via the arginine deiminase pathway producing ornithine, ammonia, carbon dioxide, and ATP. In the four strains of S. lactis examined, the specific activities of arginine deiminase and ornithine transcarbamylase were 5- to 10-fold higher in galactose-grown cells compared with glucose- or lactose-grown cells. The addition of arginine increased the specific activities of these two enzymes with all growth sugars. The specific activity of the third enzyme involved in arginine metabolism (carbamate kinase) was not altered by the composition of the growth medium. In continuous cultures arginine deiminase was not induced, and arginine was not metabolized, until glucose limitation occurred. In batch cultures the metabolism of glucose and arginine was sequential, whereas galactose and arginine were metabolized concurrently, and the energy derived from arginine metabolism was efficiently coupled to growth. No arginine deiminase activity was detected in the nine Streptococcus cremoris strains examined, thus accounting for their inability to metabolize arginine. All nine strains of S. cremoris had specific activities of carbamate kinase similar to those found in S. lactis, but only five S. cremoris strains had ornithine transcarbamylase activity.
Construction from Mu d1 (lac Apr) lysogens of lambda bacteriophage bearing promoter-lac fusions: isolation of lambda ppheA-lac
Authors: Gowrishankar J , Pittard J
Published: January 2020

Bacteriophage Mu d1 (lac Aprr) was used to obtain strains of Escherichia coli K-12 in which the lac genes are expressed from the promoter of pheA, the structural gene for the enzyme chorismate mutase P-prephenate-dehydratase. A derivative of bacteriophage lambda which carries the pheA-lac fusion was prepared; the method used is generally applicable for the construction, from Mu dl lysogens, of specialized transducing lambda phage carrying the promoter-lac fusions. A restriction enzyme cleavage map of lambda ppheA-lac for the enzymes HindIII and PstI is presented.
Characteristics of a Binding Protein-Dependent Transport System for
sn
-Glycerol-3-Phosphate in
Escherichia coli
That Is Part of the
pho
Regulon
Authors: Schweizer Herbert , Argast Manfred , Boos Winfried
Published: January 2020

The
ugp
-dependent transport system for
sn
-glycerol-3-phosphate has been characterized. The system is induced under conditions of phosphate starvation and in mutants that are constitutive for the
pho
regulon. The system does not operate in membrane vesicles and is highly sensitive toward osmotic shock. The participation of a periplasmic binding protein in the transport process can be deduced from the isolation of transport mutants that lack the binding protein. As with other binding protein-dependent transport systems, this protein appears to be necessary but not sufficient for transport activity. The isolation of mutants has become possible by selection for resistance against the toxic analog 3,4-dihydroxybutyl-1-phosphonate that is transported by the system.
sn
-Glycerol-3-phosphate transported via
ugp
cannot be used as the sole carbon source. Strains have been constructed that lack alkaline phosphatase and glycerol kinase. In addition, they are constitutive for the
glp
regulon and contain high levels of glycerol-3-phosphate dehydrogenase. Despite the fact that these strains exhibit high
ugp
-dependent transport activity for
sn
-glycerol-3-phosphate they are unable to grow on it as a sole source of carbon. However, when cells are grown on an alternate carbon source,
14
C label from [
14
C]
sn
-glycerol-3-phosphate appears in phospholipids as well as in trichloroacetic acid-precipitable material. The incorporation of
14
C label is strongly reduced when
sn
-glycerol-3-phosphate is the only carbon source. In the presence of an alternate carbon source, this inhibition is relieved, and
sn
-glycerol-3-phosphate transported by
ugp
can be used as the sole source of phosphate.
Polarity in the
glnA
Operon: Suppression of the Reg
−
Phenotype by
rho
Mutations
Authors: Guterman Sonia K. , Roberts Gary , Tyler Bonnie
Published: January 2020

To determine the ability of mutations in
glnA
, the gene for glutamine synthetase (GS), to regulate nitrogen assimilatory enzymes, we assayed histidase and GS in 34
glnA
(Gln
−
) strains. Twenty-five
glnA
mutants were RegC, synthesizing high levels of histidase regardless of the availability of nitrogen, and nine were Reg
−
, synthesizing low levels of histidase in medium containing either limiting or excess ammonia.
rho
mutations were introduced into strains containing
glnA
point mutations or insertions in
glnA, glnL, glnG
, or
glnF
. The Reg
−
phenotype of strains with
glnA
point mutations, but not those with
glnA
or
glnF
insertions, was altered by the presence of
rho
, suggesting that
glnA
(Reg
−
) mutations are polar and exert their phenotype by decreasing expression of
glnL
and
glnG
. Consistent with this view, no GS protein was detected by two-dimensional gel electrophoresis in
glnA
(Reg
−
)
rho
+
or
glnA
(Reg
−
)
rho
double mutants, whereas GS protein was detected in cells of 10 of 11
glnA
(RegC) strains. Since
glnA
(Reg
−
)
rho
double mutants synthesize constitutive levels of histidase, GS protein is not necessary for full expression of histidase. Mu d1 insertions in
glnL
, but not those in
glnG
, responded to the presence of a
rho
allele, presumably owing to elevated transcription into
glnG
from the Mu d1 prophage. Our results suggest that
glnA
(Reg
−
) alleles are polar mutations, and a rho-dependent termination site down-stream is postulated as the basis for the polar phenomenon. The data also indicate that, under some circumstances, a significant portion of
glnL
and
glnG
transcription is initiated at the
glnA
promoter.
Interaction of
125
I-Labeled Colicin E1 with
Escherichia coli
Authors: Farid-Sabet Sohair
Published: January 2020

Colicin E1 protein was labeled with
125
I to specific activities of up to 2 × 10
8
cpm/mg of protein and with no loss of the colicin biological activity. The labeled colicin bound to colicin E1-sensitive, tolerant, and immune E1-colicinogenic
Escherichia coli
. An
E. coli
mutant resistant to colicin E1 exhibited a much lower colicin-binding capacity. The average number of bound colicin molecules per sensitive cell increased as a function of the colicin concentration in the colicin cell interaction mixture and continued to increase even after loss of viability of the entire culture. Up to 2,400 colicin E1 molecules bound per cell, but saturation was not reached. Binding kinetics showed that maximum binding occurred within 2 to 5 min of colicin addition. Survival and binding assays indicated that one colicin killing unit corresponded to an average of about 100 colicin molecules bound per bacterial cell. This number, however, decreased to about 8 in more extensively washed cells. Trypsin digestion of the colicin-treated cells removed the majority of the cell-bound colicin, but in general provided little rescue from colicin killing. At low colicin concentrations, a linear relationship existed between survival and the number of trypsin-inaccessible colicin molecules. Under these circumstances and in agreement with single-hit kinetics, the relationship between the number of colicin killing units and the number of trypsin-inaccessible colicin molecules was close to 1. After trypsin digestion, cells that were nearly saturated with colicin retained about 200 trypsin-inaccessible colicin molecules per cell. The trypsin-inaccessible colicin might represent those colicin molecules that bound to the specific E colicin receptors of
E. coli
cells.
Primary structure of the chromosomal origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae: comparisons and evolutionary relationships
Authors: Cleary J M , Smith D W , Harding N E , Zyskind J W
Published: January 2020

The nucleotide sequences of the Enterobacter aerogenes and Klebsiella pneumoniae DNA replication origins (oriC) were determined and compared with those of Escherichia coli and Salmonella typhimurium. Four interrelated, 9-base-pair repeats were identified from the conserved regions within the minimal origin. Evolutionary rates calculated from the minimal origin sequences yielded a quantitative phylogenic tree which agreed with the taxonomic classification of these genera.
Release of heat-labile enterotoxin subunits by Escherichia coli
Authors: Yamamoto T , Yokota T
Published: January 2020

Most of the heat-labile enterotoxin (LT) synthesized by Escherichia coli is cell associated; however, a small portion of LT (approximately 10%) is released by bacterial cells into the culture supernatant. The LT subunit B (LT-B) produced by a cloned LT-B gene (tox B) was released in amounts equal to the parent LT release. In contrast, no release of LT subunit A (LT-A) or its smaller derivatives was observed in strains containing cloned toxA genes. The data suggest that LT-B is necessary for the release of LT-A across the bacterial membrane.
Evidence for two genetically distinct DNA primase activities specified by plasmids of the B and I incompatibility groups
Authors: Dalrymple B P , Boulnois G J , Wilkins B M , Orr E , Williams P H
Published: January 2020

Plasmid ColIb-P9 of the I alpha incompatibility group is known to encode a DNA primase that acts in the conjugal transfer of the plasmid and can substitute for mutant dnaG gene product in vegetative replication of the Escherichia coli chromosome. The relevant genetic determinant (sog) has previously been cloned into a small multicopy vector plasmid. Prototype IncB plasmid R16 also suppresses host dnaG mutations. The equivalent gene(s) (pri) of R16 was cloned into plasmid pBR325 and shown by filter hybridization studies to be different from the ColI primase gene(s). The cloned fragment carrying the pri determinant encoded an enzyme which could initiate DNA synthesis in vitro on single-stranded phage M13 DNA template, but which was antigenically distinct from ColI primase. We used the cloned primase genes as probes in colony hybridization screening of strains carrying plasmids of the IncI complex and IncB group. Plasmids R64, R144 (I alpha), R621a (I gamma), RIP72, and R864a (B) contained nucleotide sequences homologous with the cloned ColI sog genes. Plasmids R805 (I xi), R724, (I delta), TP125, and PLG101 (B) showed sequence homology with the R16 pri determinant.
Phosphorylation and methylation of proteins during Myxococcus xanthus spore formation
Authors: Komano T , Brown N , Inouye S , Inouye M
Published: January 2020

Post-translational modification of proteins was examined during the life cycle of Myxococcus xanthus. A specific pattern of protein phosphorylation was observed in vegetative cells. When spore formation was induced by glycerol, significant changes in the pattern of protein phosphorylation were observed, including the phosphorylation of two membrane proteins. In in vitro experiments, the same membrane proteins were phosphorylated by ATP when the membrane preparation from cells treated with glycerol was used. Changes in the pattern of protein methylation were also observed during spore formation induced by glycerol or fruiting body formation. These results suggest that post-translational protein modification may be required for spore formation or fruiting body formation.
Cloning of the ilvA538 gene coding for feedback-hypersensitive threonine deaminase from Escherichia coli K-12
Authors: Calhoun D H , Gray J E
Published: January 2020

A variety of experimental results implicate the ilvA gene product, threonine deaminase, as an autoregulatory protein that affects the expression of its own gene and those coding for some related proteins. Some of the most direct evidence comes from the analysis of mutations in the ilvA gene with pleiotropic genetic regulatory effects. The most extensively documented mutation, ilvA538, lowers the expression of and abolishes repression control of the ilvGEDA transcription unit. A pleiotropic effect of the ilvA538 mutation, which may be either incidental or mechanistically related to the loss of repression control, renders threonine deaminase feedback hypersensitive to the inhibition of catalytic activity by the pathway end product, isoleucine. We transferred this mutation to lambda dilv phage and pBR322 derivatives. Direct enzyme assay of the plasmid- and phage-coded ilvA538 gene product in delta ilv hosts confirmed the feedback hypersensitivity of the enzyme product. In conjunction with the ilvG671 (phenotype, ILvG+ Valr; previously designated ilvO671) allele located in cis, high levels of the plasmid and lambda dilv phage-coded mutant enzyme suitable for protein purification were observed. Deletion mapping experiments with lambda dilv phage confirmed that the ilvA538 mutation, and not mutations promoter proximal to ilvD (transcription is from ilvG to ilvA), confer a loss of repression control. These genetic mapping studies indicate, however, that an additional mutation(s) may be present that contributes, at least in part, to the reduced enzyme levels in strains with the ilvA538 mutation.