bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycolic Acid Composition and Thermally Adaptative Changes in
Nocardia asteroides
Authors: Tomiyasu Ikuko
Published: January 2020

The nocardomycolic acid compositions of extractable and the cell wall-bound lipids from five strains of
Nocardia asteroides
(A-23007, A-23094, B-23006, B-23095, and IFO 3384) were compared by using gas chromatography-mass spectrometry. The molecular species composition of mycolic acid differed significantly among the strains of
N. asteroides
. The A-23007 strain possessed the shortest species, centering at C
44(46)
, and the A-23094 and IFO-3384 strains followed, each centering at C
52
. The B-23006 and B-23095 strains possessed the longest species, centering at C
56
or C
54
, thus indicating that
N. asteroides
strains accommodate a heterogeneous group in respect to carbon numbers of mycolic acids. The doublebond isomers of mycolic acids from the representative strain IFO 3384 were fully separated and analyzed by argentation thin-layer chromatography, followed by gas chromatography-mass spectrometry. The reference strain (IFO 3384) possessed up to four double bonds on the straight chain of mycolic acids ranging from C
46
to C
60
. All of the species possessed a C
14
alkyl branch at C-2. The more highly unsaturated subclasses consisted of the longer-chain mycolic acids. Marked changes in mycolic acid composition were induced by altering the growth temperature of strain IFO 3384. The cells grown at the higher temperature (50°C) contained more saturated mycolic acids, whereas those grown at the lower temperature (17°C) had more polyunsaturated (up to tetraenoic) mycolic acids, although a significant difference in carbon chain length was not detected. These changes in the degree of unsaturation of mycolic acids occurred shortly after shifting the growth temperature from 17 to 50°C at logarithmic stages of the bacterial growth, thus indicating that
N. asteroides
can adapt to changes in the environmental temperature by altering the structure of mycolic acids of the cell walls.
Biochemical characterization of HgCl2-inducible polypeptides encoded by the mer operon of plasmid R100
Authors: Jackson W J , Summers A O
Published: January 2020

Minicells carrying the subcloned mer operon from plasmid R100 were pulse-labeled with [35S]methionine, and the labeled polypeptides were analyzed at various subsequent times by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Hg(II) reductase monomer encoded by plasmid R100 occurred as two proteins of 69 and 66 kilodaltons (kd). The minor 66-kd protein is a modified form of the 69-kd protein. This modification occurs in vivo. Both of these mer proteins are found in the soluble fraction of the cell; however, the 66-kd protein appears to have a slight affinity for the cellular envelope. Both the 69- and 66-kd mer proteins have pI values greater (pI = 5.8) than that reported (pI = 5.3) for the analogous monomer encoded by plasmid R831. The 15.1- and 14-kd mer proteins are localized in the inner membrane and are probably elements of the mer-determined Hg(II) uptake system. These two mer membrane proteins, which are antigenically unrelated to the Hg(II) reductase monomer, are quite basic (pI values greater than 7.8). The 12-kd mer protein is also a basic polypeptide that is present in the soluble fraction of the cell. Unlike the two membrane-bound mer proteins, the 12-kd mer protein is processed from a 13-kd precursor.
Expression of a copy number control gene (copB) of plasmid R1 is constitutive and growth rate dependent
Authors: Light J , Molin S
Published: January 2020

The copy number control gene copB from plasmid R1 was fused to the lacZ gene in vitro, resulting in expression of a fused polypeptide consisting of the first 53 amino acids of the CopB polypeptide and the beta-galactosidase polypeptide minus its first 8 amino acids. Based on measurements of specific activities of this fused protein under various conditions, it was concluded that expression of copB is gene dosage dependent, unregulated by plasmid-coded functions, and proportional to growth rate between 0.4 and 2.0 doublings per h. The rate of expression of the copB gene is surprisingly high compared with other known cases of regulatory proteins.
Characterization of glutamine-requiring mutants of Pseudomonas aeruginosa
Authors: Janssen D B , Joosten H M , Herst P M , van der Drift C
Published: January 2020

Revertants were isolated from a glutamine-requiring mutant of Pseudomonas aeruginosa PAO. One strain showed thermosensitive glutamine requirement and formed thermolabile glutamine synthase, suggesting the presence of a mutation in the structural gene for glutamine synthetase. The mutation conferring glutamine auxotrophy was subsequently mapped and found to be located at about 15 min on the chromosomal map, close to and before hisII4. Furthermore, in transduction experiments, it appeared to be very closely linked to gln-2022, a suppressor mutation affecting nitrogen control. With immunological techniques, it could be demonstrated that the glutamine auxotrophs form an inactive glutamine synthetase protein which is regulated by glutamine or a product derived from it in a way similar to other nitrogen-controlled proteins.
Control of rRNA and tRNA syntheses in Escherichia coli by guanosine tetraphosphate
Authors: Ryals J , Little R , Bremer H
Published: January 2020

The expression of stable RNA (rRNA and tRNA) genes and the concentration of guanosine tetraphosphate (ppGpp) were measured in an isogenic pair of relA+ and relA derivatives of Escherichia coli B/r. The cells were either growing exponentially at different rates or subject to amino acid starvation when they were measured. The specific stable RNA gene activity (rs/rt, the rate of rRNA and tRNA synthesis relative to the total instantaneous rate of RNA synthesis) was found to decrease from 1.0 at a ppGpp concentration of 0 (extrapolated value) to 0.24 at saturating concentrations of ppGpp (above 100 pmoles per optical density at 460 nm unit of cell mass). The same relationship between the rs/rt ratio and ppGpp concentration was obtained independent of the physiological state of the bacteria (i.e., independent of the growth rate or of amino acid starvation) and independent of the relA allele. It can be concluded that ppGpp is an effector for stable RNA gene control and that stable RNA genes are not controlled by factors other than the ppGpp-mediated system. The results were shown to be qualitatively and quantitatively consistent with data on in vitro rRNA gene control by ppGpp, and they were interpreted in the light of reported ideas derived from those in vitro experiments.
Control of RNA synthesis in Escherichia coli after a shift to higher temperature
Authors: Ryals J , Little R , Bremer H
Published: January 2020

Parameters of RNA synthesis were measured after a temperature upshift in a pair of Escherichia coli B/r strains that are isogenic except for having relA and relA+ loci, to examine the cause for a reported anomaly in the correlation between guanosine tetraphosphate (ppGpp) and stable RNA (rRNA, tRNA) synthesis under such conditions. Two main results were: (i) the specific stable RNA gene activity (stable RNA per total RNA synthesis) correlated in the conventionally expected fashion with the level of ppGpp but was obscured by a nonspecific increase in the RNA chain elongation rate due to the higher temperature; (ii) the temperature upshift caused a transient reduction in the RNA polymerase activity (transcribing per total enzyme) that accounts for the previously observed oscillating RNA synthesis rate after a temperature shift.
Isolation and analysis of sacculi from Streptococcus sanguis
Authors: Reusch V M
Published: January 2020

Sacculi were prepared from Streptococcus sanguis 34 by exhaustive extraction of bacteria with hot 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol. Lyophilized residue was dissociated by brief sonication to single bodies closely resembling streptococci in phase-contrast microscopic density, staining properties, and morphology. Electron micrographs revealed bodies that contained variable amounts of cellular contents and were bounded by intact cell walls. Chemical analyses of sacculi demonstrated the presence of peptidoglycan, carbohydrate, protein, and phosphate. The hexose content of sacculi varied 10-fold depending upon the composition of the growth medium. When sacculi were subjected to treatment with 5 M LiCl, 8 M urea, 40% phenol (25 degrees C), or dimethyl sulfoxide most of the nitrogen and carbohydrate present was recovered in the insoluble fraction. These data suggest that sacculi contain the cell wall fraction of the extracted bacteria and that most of the carbohydrates and proteins of sacculi are firmly bound to the insoluble fraction, which contains the peptidoglycan matrix.
Transformation of intact yeast cells treated with alkali cations
Authors: Ito H , Fukuda Y , Murata K , Kimura A
Published: January 2020

Intact yeast cells treated with alkali cations took up plasmid DNA. Li+, Cs+, Rb+, K+, and Na+ were effective in inducing competence. Conditions for the transformation of Saccharomyces cerevisiae D13-1A with plasmid YRp7 were studied in detail with CsCl. The optimum incubation time was 1 h, and the optimum cell concentration was 5 x 10(7) cells per ml. The optimum concentration of Cs+ was 1.0 M. Transformation efficiency increased with increasing concentrations of plasmid DNA. Polyethylene glycol was absolutely required. Heat pulse and various polyamines or basic proteins stimulated the uptake of plasmid DNA. Besides circular DNA, linear plasmid DNA was also taken up by Cs+-treated yeast cells, although the uptake efficiency was considerably reduced. The transformation efficiency with Cs+ or Li+ was comparable with that of conventional protoplast methods for a plasmid containing ars1, although not for plasmids containing a 2 microns origin replication.
Mechanism of CRP-mediated cya suppression in Escherichia coli
Authors: Harman J G , Dobrogosz W J
Published: January 2020

Escherichia coli strain NCR30 contains a cya lesion and a second-site cya suppressor mutation that lies in the crp gene. NCR30 shows a pleiotropic phenotypic reversion to the wild-type state in expressing many operons that require the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex for positive control. In vivo beta-galactosidase synthesis in NCR30 was sensitive to glucose-mediated repression, which was relieved not only by cAMP but also by cyclic GMP and cyclic CMP. The CRP isolated from NCR30 differed from the protein isolated from wild-type E. coli in many respects. The mutant protein bound cAMP with four to five times greater affinity than wild-type CRP. Protease digestion studies indicated that native NCR30 CRP exists in the cAMP-CRP complex-like conformation. The protein conferred a degree of cAMP independence on the in vitro synthesis of beta-galactosidase. In addition, the inherent positive control activity of the mutant protein in vitro was enhanced by those nucleotides that stimulate in vivo beta-galactosidase synthesis in NCR30. The results of this study supported the conclusion that the crp allele of NCR30 codes for a protein having altered effector specificity yet capable of promoting positive control over catabolite-sensitive operons in the absence of an effector molecule.
Assembly In Vivo of Enterotoxin from
Escherichia coli
: Formation of the B Subunit Oligomer
Authors: Hirst Timothy R. , Hardy Simon J. S. , Randall Linda L.
Published: January 2020

An oligomer of the B subunit of heat-labile enterotoxin of
Escherichia coli
has been observed in minicells and in whole cells. There is a delay after synthesis of the B subunit before it appears in the oligomer. The delay is not due to slow processing of the precursor. A similar delay in oligomerization of the major outer membrane protein OmpF is also described.
Porin channels in Escherichia coli: studies with beta-lactams in intact cells
Authors: Nikaido H , Rosenberg E Y , Foulds J
Published: January 2020

Wild-type Escherichia coli K-12 produces two porins, OmpF (protein 1a) and OmpC (protein 1b). In mutants deficient in both of these "normal" porins, secondary mutants that produce a "new" porin, protein PhoE (protein E), are selected for. We determined the properties of the channels produced by each of these porins by measuring the rates of diffusion of various cephalosporins through the outer membrane in strains producing only one porin species. We found that all porin channels retarded the diffusion of more hydrophobic cephalosporins and that with monoanionic cephalosporins a 10-fold increase in the octanol-water partition coefficient of the solute produced a 5- to 6-fold decrease in the rate of penetration. Electrical charges of the solutes had different effects on different channels. Thus, with the normal porins (i.e., OmpF and OmpC proteins) additional negative charge drastically reduced the penetration rate through the channels, whereas additional positive charge significantly accelerated the penetration. In contrast, diffusion through the PhoE channel was unaffected by the presence of an additional negative charge. We hypothesize that the relative exclusion of hydrophobic and negatively charged solutes by normal porin channels is of ecological advantage to E. coli, which must exclude hydrophobic and anionic bile salts in its natural habitat. The properties of the PhoE porin are also consistent with the recent finding (M. Argast and W. Boos, J. Bacteriol. 143:142-150, 1980; J. Tommassen and B. Lugtenberg, J. Bacteriol. 143:151-157, 1980) that its biosynthesis is derepressed by phosphate starvation; the channel may thus act as an emergency pore primarily for the uptake of phosphate and phosphorylated compounds.
Bacillus subtilis relG
Mutant: Defect in Glucose Uptake
Authors: Price Virginia L. , Gallant Jonathan A.
Published: January 2020

A previously described
Bacillus subtilis
mutant which exhibits a relaxed phenotype after glucose limitation (
relG
) has been characterized further. Analysis of this mutant and of the downshift mechanism in
B. subtilis
has shown that the primary defect lies in glucose uptake.
Sequence and substrate specificity of isolated DNA methylases from Escherichia coli C
Authors: Urieli-Shoval S , Gruenbaum Y , Razin A
Published: January 2020

Two DNA methylase activities of Escherichia coli C, the mec (designates DNA-cytosine-methylase gene, which is also designated dcm) and dam gene products, were physically separated by DEAE-cellulose column chromatography. The sequence and substrate specificity of the two enzymes were studied in vitro. The experiments revealed that both enzymes show their expected sequence specificity under in vitro conditions, methylating symmetrically on both DNA strands. The mec enzyme methylates exclusively the internal cytosine residue of CCATGG sequences, and the dam enzyme methylates adenine residues at GATC sites. Substrate specificity experiments revealed that both enzymes methylate in vitro unmethylated duplex DNA as efficiently as hemimethylated DNA. The results of these experiments suggest that the methylation at a specific site takes place by two independent events. A methyl group in a site on one strand of the DNA does not facilitate the methylation of the same site on the opposite strand. With the dam methylase it was found that the enzyme is incapable of methylating GATC sites located at the ends of DNA molecules.
Vanadate uptake in Neurospora crassa occurs via phosphate transport system II
Authors: Bowman B J
Published: January 2020

Vanadate, a potent inhibitor of plasma membrane ATPases, is taken up by Neurospora crassa only when cells are growing in alkaline medium and starving for phosphate. The appearance of a vanadate uptake system (Km = 8.2 microM; Vmax = 0.15 mmol/min per liter of cell water) occurs under the same conditions required for derepression of a high-affinity phosphate transport system. Phosphate is a competitive inhibitor of vanadate uptake, and vanadate is a competitive inhibitor of phosphate uptake. Furthermore, mutant strains which are either partially constitutive or non-derepressible for the high-affinity phosphate transport system are also partially constitutive or non-derepressible for vanadate uptake. These data indicate that vanadate enters the cell via phosphate transport system II.
Isolation of Saccharomyces cerevisiae TRP3
Authors: Paluh J L , Zalkin H
Published: January 2020

Several plasmids, isolated from two plasmid pools, complemented a Saccharomyces cerevisiae trp3 mutant with defective indole-3-glycerol-phosphate synthase activity. Restriction mapping indicated that a 1.2-kilobase StuI segment was common to all complementing plasmids. Southern blot hybridization established that a cloned 5.2-kilobase BamHI fragment was derived intact from chromosomal DNA. A yeast trp3 mutant transformed with trp3-complementing plasmids contained approximately 40-fold elevated indole-3-glycerol-phosphate synthase activity. These plasmids also complemented an Escherichia coli trpC mutant, and transformants exhibited enzyme activity. Yeast trp3 is therefore associated with a 1.2-kilobase StuI DNA segment.
Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis
Authors: van der Plas J , Hellingwerf K J , Seijen H G , Guest J R , Weiner J H , Konings W N
Published: January 2020

Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.
In vivo packaging of cosmids in transposon-mediated mutagenesis
Authors: White F F , Klee H J , Nester E W
Published: January 2020

A technique was developed that permits the analysis of large regions of DNA by transposition mutagenesis. Large fragments of the pTiA6NC plasmid were cloned into the broad host range cosmid pHK17 and subjected to transposition mutagenesis by Tn3. Cosmids containing Tn3 insertions were selected by in vivo packaging by lambda cI857 and transduction to a new host. The insertions were localized by DNA restriction endonuclease analysis and transferred to the Ti-plasmid by marker exchange.
Internal promoters of the his operon in Salmonella typhimurium
Authors: Schmid M B , Roth J R
Published: January 2020

Two internal promoters in the his operon of Salmonella typhimurium have been precisely mapped genetically. The internal promoters are found in, or very close to, gene border regions in the his operon. The his operon was examined for the presence of additional internal promoters whose transcripts were sensitive to rho-mediated transcription termination and therefore had escaped detection. No new internal promoters were found. It is argued that the internal promoters described here are not likely to be fortuitous message start sites, but may play a physiologically important role in operon expression.
Origin of Escherichia coli K-12 Hfr B7
Authors: Virolle M J , Gélugne J P , Béjar S , Bouché J P
Published: January 2020

Several F' plasmids encoding resistance to tetracycline have been derived from a trg::Tn10 Hfr B7 strain of Escherichia coli K-12. One of these plasmids, JGF312, was analyzed by restriction endonuclease digestion and Southern blot hybridization to cloned chromosomal fragments. This analysis revealed that JGF312 was formed by Tn10-promoted deletion from the Tn10 insertion (31.4 min) to within the prophage rac at 30.1 min. Hfr B7 was shown to result from recombination between IS2 of F delta (33-43) and a chromosomal IS2 located within the rac-man region at 30.9 min on the genetic map.
Regulation of yeast trehalase by a monocyclic, cyclic AMP-dependent phosphorylation-dephosphorylation cascade system
Authors: Ortiz C H , Maia J C , Tenan M N , Braz-Padrão G R , Mattoon J R , Panek A D
Published: January 2020

Mutation at the GLC1 locus in Saccharomyces cerevisiae resulted in simultaneous deficiencies in glycogen and trehalose accumulation. Extracts of yeast cells containing the glc1 mutation exhibited an abnormally high trehalase activity. This elevated activity was associated with a defective cyclic AMP (cAMP)-dependent monocyclic cascade which, in normal cells, regulates trehalase activity by means of protein phosphorylation and dephosphorylation. Trehalase in extracts of normal cells was largely in a cryptic form which could be activated in vitro by ATP . Mg in the presence of cAMP. Normal extracts also exhibited a correlated cAMP-dependent protein kinase which catalyzed incorporation of label from [gamma-32P]ATP into protamine. In contrast, cAMP had little or no additional activating effect on trehalase or on protamine phosphorylation in extracts of glc1 cells. Similar, unregulated activation of cryptic trehalase was also found in glycogen-deficient strains bearing a second, independently isolated mutant allele, glc1-2. Since trehalase activity was not directly affected by cAMP, the results indicate that the glc1 mutation results in an abnormally active protein kinase which has lost its normal dependence on cAMP. Trehalase in extracts of either normal or mutant cells underwent conversion to a cryptic form in an Mg2+-dependent, fluoride-sensitive reaction. Rates of this reversible reduction of activity were similar in extracts of mutant and normal cells. This same, unregulated protein kinase would act on glycogen synthase, maintaining it in the phosphorylated low-activity D-form. The glc1 mutants provide a novel model system for investigating the in vivo metabolic functions of a specific, cAMP-dependent protein kinase.
Genetic transformation of obligately chemolithotrophic thiobacilli
Authors: Yankofsky S A , Gurevich R , Grimland N , Stark A A
Published: January 2020

Genetic transformation of Thiobacaillus thioparus auxotrophs to prototrophy was obtained at frequencies of up to 10(-2) when proliferating cell populations were exposed to chromosomal DNA from a nutritionally independent strain of the same bacterium. The rate at which transformation occurred depended on recipient growth rate and could be drastically reduced by depriving otherwise competent cells of either nitrogen or exogenous energy substrate. Interspecies marker transfer was also shown among several obligately chemolithotrophic members of the genus.
Multiple genes for membrane-bound phosphatases in Escherichia coli and their action on phospholipid precursors
Authors: Icho T , Raetz C R
Published: January 2020

We have devised a coupled radiochemical assay for detecting phosphatidylglycerolphosphate (PGP) phosphatase activity in Escherichia coli colonies immobilized on filter paper. There appeared to be at least two enzymes capable of dephosphorylating PGP, as judged by the characterization of mutations in two genes designated pgpA and pgpB. The former is located near min 10 and is cotransducible with proC and dnaZ. The latter is situated near min 28 and is closely linked to cysB. The available mutant alleles of pgpA reduced the specific activity of PGP phosphatase in crude extracts by about 30%, but they had no effect on phosphatidic acid (or lysophosphatidic acid) phosphatase. Mutants altered in the pgpB locus inactivated most of the residual PGP phosphatase activity present in single-step pgpA mutants, and the level of phosphatidic acid phosphatase was also reduced 20-fold. The available mutations in pgpA and pgpB elevated the cellular PGP pool by 10- to 50-fold. The maximal PGP levels never exceeded 5%, and these strains were not conditionally lethal. The simplest interpretation of our findings is that there are at least two membrane-associated phosphatases in E. coli, both distinct from alkaline phosphatase. The pgpA gene product is specific for PGP, whereas the pgpB gene product also acts on phosphatidic acid and lysophosphatidic acid.
Growth rate-dependent regulation of 6-phosphogluconate dehydrogenase level in Escherichia coli K-12: beta-galactosidase expression in gnd-lac operon fusion strains
Authors: Baker H V , Wolf R E
Published: January 2020

The level of 6-phosphogluconate dehydrogenase is positively correlated with the cellular growth rate. To determine whether growth rate-dependent regulation of expression of gnd, which encodes this enzyme, is carried out by a transcriptional mechanism, the structural genes of the lactose operon were fused to and brought under the control of the gnd promoter through the use of phage Mu d1(Apr lac). Four independent gnd::Mu d1(Apr lac) operon fusion strains were isolated. After the Mu d1 prophage was replaced with lambda p1(209), Lac+ specialized transducing phages carrying the gnd-lac fusions were prepared. These phages were used to demonstrate that the lac genes were fused to the gnd promoter by crossing them with gnd promoter deletion mutants and with polar phage Mu cts-induced gnd mutants. A genetic map of the fusion joints was deduced. The level of beta-galactosidase in each fusion strain was the same in cells growing on acetate as in cells growing on glucose (with specific growth rate constants of 0.1 and 0.55 h-1, respectively) and was unaffected by the presence of a gnd+ gene in trans. Our results suggest that a post-transcriptional mechanism mediates growth rate-dependent regulation of gnd and that this regulation is not autogenous. Models for regulation are discussed with respect to these results and the physiology and DNA sequence of gnd.
Integration of corynebacteriophages beta tox+, omega tox+, and gamma tox- into two attachment sites on the Corynebacterium diphtheriae chromosome
Authors: Rappuoli R , Michel J L , Murphy J R
Published: January 2020

The bacterial attachment sites of independently isolated Corynebacterium diphtheriae strains C7s and (belfanti)1030 lysogenic for corynebacteriophages beta tox+, omega tox+, and gamma tox- were determined by Southern blot analysis. Both corynebacterial strains contained two distinct bacterial attachment sites (attB1 and attB2). We found that infection by any of the three closely related corynebacteriophages may give rise to single, double, and triple lysogens. In the case of toxigenic C. diphtheriae strains C7s(beta tox+) and C7s(omega tox+), the final yields of diphtheria toxin produced under optimal conditions were equivalent and varied by one-, two-, or threefold depending upon the number of integrated prophage.
Regulation at the
glnL
-Operator-Promoter of the Complex
glnALG
Operon of
Escherichia coli
Authors: Ueno-Nishio Shizue , Backman Keith C. , Magasanik Boris
Published: January 2020

We identified the portion of the
glnALG
operon that contains the promoter and operator of the
glnLG
suboperon and showed that the product of
glnG
represses transcription initiated at
glnLp
.
Changes in penicillin-binding proteins during sporulation of Bacillus subtilis
Authors: Sowell M O , Buchanan C E
Published: January 2020

The penicillin-binding proteins (PBPs) of Bacillus subtilis were examined in samples collected at various times from sporulating cultures and compared with the PBPs in a presporulation sample. Large increases in vegetative PBPs 2B and 3 and the appearance of at least one new PBP (42,000 daltons) occurred at reproducible times during sporulation. In some strains a second new PBP (60,000 daltons) was also produced. By comparing the PBP activities in sporulating cells and two spo0 mutants we have classified these changes as sporulation-related events rather than the consequences of stationary-phase aging. The other vegetative PBPs (PBPs 1, 2A, 4, and 5) decreased during sporulation, but not in sufficient amount or at the appropriate time to account for the appearance of the new proteins. A possible connection between specific PBP changes and the penicillin-sensitive stages of sporulation is suggested.
Straight Mutants of
Spirillum volutans
Can Swim
Authors: Padgett P. J. , Friedman M. W. , Krieg N. R.
Published: January 2020

Nonhelical mutant cells of
Spirillum volutans
ATCC 19554 can swim as fast as the helical cells. Consequently, a helical cell shape is not required for motility of this species, and the function of the polar flagellar fascicles is not merely to cause rotation, and therefore translocation, of the corkscrew-shaped cell.
Identification of Two Distinct Lactate Dehydrogenases inRhodospirillum rubrum
Authors: Mueller Paul R. , Collins Mary Lynne Perille
Published: January 2020

The activities of pyridine nucleotide-independentd- andl-lactate dehydrogenases were detected in membranes fromRhodospirillum rubrumgrown under aerobic and phototrophic conditions. Crossed immunoelectrophoretic analysis revealed two antigenically distinct enzymes that were further distinguished by specificity ford- andl-stereoisomers of lactate and by the sensitivity of thed-lactate dehydrogenase to inhibition by oxamate and oxalate.
Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing
Authors: Gasson M J
Published: January 2020

The production and regeneration of bacterial protoplasts promoted the loss of three different plasmid-specified traits in Streptococcus lactis subsp. diacetylactis strains. The loss of five different plasmids, including small multicopy molecules, was readily detected in Streptococcus lactis 712 by screening lysates of random protoplast regenerants on agarose gels. In this strain sequential rounds of protoplast regeneration were used to produce a plasmid-free strain and derivatives carrying only single molecules from the plasmid complement. During these experiments a 33-megadalton plasmid, pLP712, was found to encode genes for lactose and protein utilization. Only this plasmid was required for normal growth and acid production in milk; the remaining four plasmids appeared to be cryptic. Lactose-defective derivatives of a strain carrying only pLP712 were readily isolated. Although these derivatives included instances of plasmid loss, deletions of pLP712 were frequently found. Many different deleted derivatives of pLP712, including some in which the lactose or protein utilization determinant or both were lost, were isolated. The molecular instability of pLP712 largely accounted for previous observations of plasmid complements in S. lactis 712 after lactose determinant curing or transfer by conjugation and transduction. Curing of cryptic molecules from multiple plasmid complements by protoplast regeneration may prove to be generally valuable in lactic streptococci and other gram-positive species.
Purine and glycine metabolism by purinolytic clostridia
Authors: Dürre P , Andreesen J R
Published: January 2020

Cell extracts of Clostridium acidiurici, C. cylindrosporum, and C. purinolyticum converted purine, hypoxanthine, 2-hydroxypurine, 6,8-dihydroxypurine, and uric acid into xanthine by the shortest possible route. Adenine was transformed to xanthine only by C. purinolyticum, whereas the other two species formed 6-amino-8-hydroxypurine, which was neither deaminated nor hydroxylated further. 8-Hydroxypurine was formed from purine by all three species. Xanthine dehydrogenase activity was constitutively expressed by C. purinolyticum. Due to the lability of the enzyme activity, comparative studies could not be done with a purified preparation. All enzymes reported to be involved in formiminoglycine metabolism of C. acidiurici and C. cylindrosporum were present in C. purinolyticum. However, glycine was reduced directly to acetate in all three species, as indicated by radiochemical data and by the detection of glycine reductase in cell extracts of C. cylindrosporum and C. purinolyticum. The expression of glycine reductase and the high ratio of glycine fermented to uric acid present points to an energetic advantage for the glycine reductase system, which is expressed when selenium compounds are added to the growth media.
Regulation of toxinogenesis in Corynebacterium diphtheriae: mutations in the bacterial genome that alter the effects of iron on toxin production
Authors: Cryz S J , Russell L M , Holmes R K
Published: January 2020

Mutants of Corynebacterium diphtheriae C7(beta) that are resistant to the inhibitory effects of iron on toxinogenesis were identified by their ability to form colonies surrounded by toxin-antitoxin halos on agar medium containing both antitoxin and a high concentration of iron. Chromosomal mutations were essential for the altered phenotypes of four independently isolated mutant strains. During growth in deferrated liquid medium containing various amounts of added iron, these mutants differed from wild-type C. diphtheriae C7(beta) in several ways. Their growth rates were slower under low-iron conditions and were stimulated to various degrees under high-iron conditions. The concentrations of iron at which optimal toxin production occurred were higher for the mutants than for wild-type C. diphtheriae C7(beta). Toxin production by the mutants during growth in low-iron medium occurred throughout the period of exponential growth at nearly constant rates that were proportional to the bacterial growth rates. In contrast, toxin production by wild-type C. diphtheriae C7(beta) in similar low-iron cultures occurred predominantly during the late exponential phase, when iron was a growth-limiting nutrient. Additional studies demonstrated that these mutants had severe defects in their transport systems for ferric iron. We propose that the altered regulation of toxinogenesis by iron in our mutants was caused by the severe defects in their iron transport systems. As a consequence, the mutants exhibited a low-iron phenotype during growth under conditions that permitted wild-type C. diphtheriae C7(beta) to exhibit a high-iron phenotype.
Locus of the Pseudomonas aeruginosa toxin A gene
Authors: Hanne L F , Howe T R , Iglewski B H
Published: January 2020

The gene for Pseudomonas aeruginosa toxin A has been mapped in the late region of the chromosome of strain PAO. Strain PAO-PR1, which produces parental levels of toxin A antigen that is enzymatically inactive and nontoxic, was used as the donor for R68.45 plasmid-mediated genetic exchange. Strain PAO-PR1 (toxA1) was mated with toxin A-producing strains, and exconjugates for selected prototrophic markers were tested for the transfer of toxA1. The toxA1 gene was located between cnu-9001 and pur-67 at approximately 85 min on the PAO chromosome.
Insertional inactivation of staphylococcal methicillin resistance by Tn551
Authors: Berger-Bächi B
Published: January 2020

Transposon Tn551 was translocated into the chromosome of a methicillin-resistant (mec) strain of Staphylococcus aureus by heat inactivation of a thermo-sensitive plasmid carrying Tn551 and selection for erythromycin-resistant (Emr) survivors. Two independent chromosomal insertions of Tn551 were obtained which reduced the level of the methicillin resistance by a factor of 50 to 100, making the strains phenotypically methicillin sensitive (Mecs). Each of the Tn551 insertions was on the largest fragment produced by EcoRI digestion of the chromosomal DNA of these strains. The integration sites lie about 1 kilobase apart. These Mecs strains reverted to Mecr at frequencies of 2.4 X 10(-8) and 3.6 X 10(-5), respectively. The majority of Mecr revertants still were Emr; only a few lost the Emr phenotype concomitantly with reversion to the Mecr phenotype. Hybridization data with labeled Tn551 showed complex rearrangements and deletions in the region of the insertion. These two Tn551 insertions do not lie on the same linkage group, II, as the mec determinant. The phenotypic expression of methicillin resistance, therefore, is also dependent upon a chromosomal genetic marker not physically linked to the mec determinant.
The Product of
glnL
Is Not Essential for Regulation of Bacterial Nitrogen Assimilation
Authors: Backman Keith C. , Chen Yu-Mei , Ueno-Nishio Shizue , Magasanik Boris
Published: January 2020

The
glnL
product is not necessary for the control of expression of
glnA
and other nitrogen-regulated genes, but it presumably communicates reduntant information on the availability of ammonia from an ammonia-sensitive system consisting of the products of
glnB
and
glnD
to the regulatory products of
glnF
and
glnG
.
Cloning and orientation of the gene encoding polynucleotide phosphorylase in Escherichia coli
Authors: Crofton S , Dennis P P
Published: January 2020

Mutations which affect the activity of polynucleotide phosphorylase (PNPase) map near 69 min on the bacterial chromosome. This region of the chromosome has been cloned by inserting the kanamycin-resistant transposon Tn5 near the argG and mtr loci at 68.5 min. Large SalI fragments of chromosomal DNA containing the Tn5 element were inserted into pBR322, and selection was made for kanamycin-resistant recombinant plasmids. Two of these plasmids were found to produce high levels of PNPase activity in both wild-type and host strains lacking PNPase activity. The pnp gene was further localized and subcloned on a 4.8 kilobase HindIII-EcoRI fragment. This fragment was shown to encode an 84,000-molecular weight protein which comigrated with purified PNPase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The orientation of the pnp gene was determined by insertion of Tn5 into the 4.8 kilobase fragment cloned in pBR322. Some of the insertions had lost the ability to elevate the level of PNPase activity in the host bacterium. Restriction mapping of the positions of the Tn5 insertions and analysis of plasmid-encoded polypeptides in UV-irradiated maxi-cells indicated that the pnp gene is oriented in the counterclockwise direction on the bacterial chromosome.
Proton Electrochemical Gradients in Washed Cells of
Nitrosomonas europaea
and
Nitrobacter agilis
Authors: Kumar Sharad , Nicholas D. J. D.
Published: January 2020

The components of the proton motive force (Δp), namely, membrane potential (Δψ) and transmembrane pH gradient (ΔpH), were determined in the nitrifying bacteria
Nitrosomonas europaea
and
Nitrobacter agilis
. In these bacteria both Δψ and ΔpH were dependent on external pH. Thus at pH 8.0,
Nitrosomonas europaea
and
Nitrobacter agilis
had Δψ values of 173 mV and 125 mV (inside negative), respectively, as determined by the distribution of the lipophilic cation [
3
H]tetraphenyl phosphonium. Intracellular pH was determined by the distribution of two weak acids,
14
C-benzoic and
14
C-acetyl salicylic, and the weak base [
14
C]methylamine.
Nitrosomonas europaea
accumulated
14
C-benzoic acid and
14
C-acetyl salicylic acid when the external pH was below 7.0 and [
14
C]methylamine at alkaline pH. Similarly,
Nitrobacter agilis
accumulated the two weak acids below an external pH of about 7.5 and [
14
C]methylamine above this pH. As these bacteria grow best between pH 7.5 and 8.0, they do not appear to have a ΔpH (inside alkaline). Thus, above pH 7.0 for
Nitrosomonas europaea
and pH 7.5 for
Nitrobacter agilis
, Δψ only contributed to Δp. In
Nitrosomonas europaea
the total Δp remained almost constant (145 to 135 mV) when the external pH was varied from 6 to 8.5. In
Nitrobacter agilis
, Δp decreased from 178 mV (inside negative) at pH 6.0 to 95 mV at pH 8.5. Intracellular pH in
Nitrosomonas europaea
varied from 6.3 at an external pH of 6.0 to 7.8 at external pH 8.5. In
Nitrobacter agilis
, however, intracellular pH was relatively constant (7.3 to 7.8) over an external pH range of 6 to 8.5. In
Nitrosomonas europaea
, Δp and its components (Δψ and ΔpH) remained constant in cells at various stages of growth, so that the metabolic state of cells did not affect Δp. Such an experiment was not possible with
Nitrobacter agilis
because of low cell yields. The effects of protonophores and ATPase inhibitors on ΔpH and Δψ in the two nitrifying bacteria are considered.
Analysis of ColE1 expression in vitro after chromosome fragmentation
Authors: Chen H Z , Zubay G
Published: January 2020

The RNA and protein products synthesized from ColE1 DNA were observed before and after cutting the DNA with different restriction enzymes. Synthesis was carried out in the DNA-directed coupled transcription translation system. The S-30 extracts used to catalyze synthesis were prepared from a recB mutant in which the linear DNA fragments resulting from restriction enzyme cleavage were spared from the usual degradation by exonucleolytic attack. By correlating the observed in vitro synthesized products with the location of the cleavage sites in the plasmid chromosome, it was possible to identify specific gene products. The col gene catalyzes the synthesis of numerous peptides in addition to the 56-kilodalton colicin protein encoded by this gene. Most of the subsidiary products appear to arise as the result of premature termination by a mechanism(s) which remains to be determined. A unique RNA and protein were characterized as products of the imm gene. The RNA has an estimated mass of 150 kilodaltons, and the protein has an estimated mass of 13 kilodaltons. From the DNA sequence of the chromosome, it was concluded that the transcripts from the imm and col genes must crisscross each other over a region of about 75 base pairs. Such a pattern of transcription might lead to interference of transcription of one gene by the other gene. Consistent with this hypothesis, it was found that imm gene transcription increased severalfold in vitro when the chromosome was cleaved in a way that eliminated transcription originating at the col gene promoter. Surprisingly, the increase in transcription by this mechanism did not result in a significant increase in the synthesis of the imm gene-encoded protein.
Multiple pathways for isoleucine biosynthesis in the spirochete Leptospira
Authors: Westfall H N , Charon N W , Peterson D E
Published: January 2020

Spirochetes of the genus Leptospira have previously been shown to use an unusual pathway to synthesize isoleucine. For reasons of convenience, we assume that only one unusual pathway is found in the genus, and we refer to it as the pyruvate pathway. We determined the distribution of this pyruvate pathway in representatives of the seven Leptospira DNA hybridization groups. Our method included labeling the representative strains with radioactive carbon dioxide and other radioactive precursors, fractionating the cells, and determining the specific activities (counts detected per nanomole) of the amino acids found in the protein fractions. On the basis of isoleucine biosynthesis, we found that the genus can be classified as follows: class I primarily, if not exclusively, uses the well-known threonine pathway; class II uses mostly the pyruvate pathway, with a minor amount of isoleucine being synthesized via the threonine pathway; and class III uses the pyruvate pathway exclusively. No relationship appears to exist between the degree of DNA hybridization and the classes of isoleucine biosynthesis. Although the precise intermediates on the pyruvate pathway are unknown, the origin of the carbon skeleton of isoleucine synthesized by this pathway is consistent with a borrowing of the leucine biosynthetic enzymes. However, we found that the pyruvate pathway is not controlled by leucine and that the two isoleucine pathways are independently regulated. Finding major and highly evolved multiple biosynthetic pathways of a specific amino acid within one genus is unique, and, conceivably, represents phylogenetic diversity within Leptospira.
Growth pattern of Rickettsia tsutsugamushi in irradiated L cells
Authors: Hase T
Published: January 2020

Irradiated L cells infected with Rickettsia tsutsugamushi were studied under the electron microscope to define the morphological growth pattern of the organism. For 2 days after inoculation, no rickettsiae were found either extra- or intracellularly; after 2 days multiple rickettsiae appeared within the host cells without morphological evidence of their entry. These observations showed that the rickettsiae within the cell were assembled in situ by segregation of portions of the granular cytoplasm and subsequent internal differentiation and surface membrane assembly of the segregated bodies. The protoplasmic (P) bodies, which seemed to be formed by shedding infected-cell granular cytoplasm, consistently appeared on the surface and within the phagosomes of the host cells. Rickettsiae were occasionally seen entering host cells in the later phase of infection; these were apparently the ones assembled within the P bodies. This suggested that the P bodies, and not the rickettsiae, were the major infectious particles that transmitted the rickettsial genetic substance among the host cells. On the basis of the present morphological observations, viral-type multiplication for R. tsutsugamushi is proposed.
Extracellular slime associated with Proteus mirabilis during swarming
Authors: Stahl S J , Stewart K R , Williams F D
Published: January 2020

Light microscopy, transmission electron microscopy, and scanning electron microscopy were used to visualize the extracellular slime of Proteus mirabilis swarm cells. Slime was observed with phase-contrast microscopy after fixation in hot sulfuric acid-sodium borate. Ruthenium red was used to stain slime for transmission electron microscopy. Copious quantities of extracellular slime were observed surrounding swarm cells; the slime appeared to provide a matrix through which the cells could migrate. Swarm cells were always found embedded in slime. These observations support the argument that swarming of P. mirabilis is associated with the production of large quantities of extracellular slime. Examination of nonswarming mutants of P. mirabilis revealed that a number of morphological changes, including cell elongation and increased flagellum synthesis, were required for swarm cell migration. It is still unclear whether extracellular slime production also is required for migration.
Assembly of Rickettsia tsutsugamushi progeny in irradiated L cells
Authors: Hase T
Published: January 2020

In the assembly of Rickettsia tsutsugamushi progeny in irradiated L cells, nascent forms first appear as undemarcated foci in the host cell granular cytoplasm, in which electron-lucent filamentous (f) and electron-dense granular (g) areas differentiate. Morphological observations indicated that the assembly involves formation of a filamentous network in the f area, manufacture of rickettsial ribosomes in the g area, and formation of mildly electron-dense fuzzy zones, along which a double membrane assembles.
Isolation, by affinity chromatography, of mutant escherichia coli cells with novel regulation of lamB expression
Authors: Ferenci T , Lee K S
Published: January 2020

Affinity chromatography was used as a positive genetic selection technique for the isolation of cells exhibiting high levels of surface receptor expression. Starting from a large population of Escherichia coli with no maltodextrin receptor due to a deletion of malT, the positive regulator gene required for receptor synthesis, cells were chromatographically enriched that could bind to starch-Sepharose, an immobilized ligand of the receptor. One such isolate showed over 25% of wild-type-induced levels of receptor in the absence of malT and levels higher than that of the wild type in a malT+ background. In contrast to wild-type cells, receptor expression in the isolate was insensitive to control by cAMP. The maltodextrin receptor synthesized by the mutant was identical to wild-type protein in terms of ligand affinity and electrophoretic mobility and was dependent on lamB, the structural gene for the receptor. The directed evolution of this novel form of lamB expression was dependent on at least two mutations in the isolate.