bluuebunny/crossref_metadata_2025 huggingface dataset:
Proton translocation coupled to trimethylamine N-oxide reduction in anaerobically grown Escherichia coli
Authors: Takagi M , Tsuchiya T , Ishimoto M
Published: January 2020

Proton translocation coupled to trimethylamine N-oxide reduction was studied in Escherichia coli grown anaerobically in the presence of trimethylamine N-oxide. Rapid acidification of the medium was observed when trimethylamine N-oxide was added to anaerobic cell suspensions of E. coli K-10. Acidification was sensitive to the proton conductor 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847). No pH change was shown in a strain deficient in trimethylamine N-oxide reductase activity. The apparent H+/trimethylamine N-oxide ratio in cells oxidizing endogenous substrates was 3 to 4 g-ions of H+ translocated per mol of trimethylamine N-oxide added. The addition of trimethylamine N-oxide and formate to ethylenediaminetetraacetic acid-treated cell suspension caused fluorescence quenching of 3,3'-dipropylthiacarbocyanine [diS-C3-(5)], indicating the generation of membrane potential. These results indicate that the reduction of trimethylamine N-oxide in E. coli is catalyzed by an anaerobic electron transfer system, resulting in formation of a proton motive force. Trimethylamine N-oxide reductase activity and proton extrusion were also examined in chlorate-resistant mutants. Reduction of trimethylamine N-oxide occurred in chlC, chlG, and chlE mutants, whereas chlA, chlB, and chlD mutants, which are deficient in the molybdenum cofactor, could not reduce it. Protons were extruded in chlC and chlG mutants, but not in chlA, chlB, and chlD mutants. Trimethylamine N-oxide reductase activity in a chlD mutant was restored to the wild-type level by the addition of 100 microM molybdate to the growth medium, indicating that the same molybdenum cofactor as used by nitrate reductase is required for the trimethylamine N-oxide reductase system.
Interaction with lectins and differential wheat germ agglutinin binding of pyocin 103-sensitive and -resistant Neisseria gonorrhoeae
Authors: Connelly M C , Stein D C , Young F E , Morse S A , Allen P Z
Published: January 2020

Strains of Neisseria gonorrhoeae were treated with pyocin 611 131 (pyocin 103) from Pseudomonas aeruginosa PA103, and isogenic resistant variants were isolated. The interaction of pyocin-sensitive and isogenic pyocin-resistant strains with wheat germ agglutinin (WGA) agglutinated all pyocin-sensitive, but not pyocin-resistant, strains. Binding of WGA to three pyocin-sensitive strains and their isogenic pyocin-resistant variants was examined quantitatively by using fluorescein-conjugated lectin. Pyocin-resistant strains maximally bound one-third to one-eighth the quantity of WGA bound by isogenic-sensitive strains. Linear Scatchard plots revealed homogeneous WGA-binding sites on three pyocin-sensitive and one pyocin-resistant strains. Biphasic Scatchard plots, obtained with two pyocin-resistant strains, show that WGA-binding sites in these strains are heterogeneous. The number of WGA-binding sites for pyocin-sensitive organisms ranged from 8 x 10(5) to 1 x 10(6) sites per coccus and from 1 x 10(5) to 3 x 10(5) sites per coccus for pyocin-resistant strains. The apparent association constant for WGA binding to pyocin-sensitive strains ranged from 3 x 10(6) to 6 x 10(6) liters/mol and from 6 x 10(6) to 1 x 10(7) liters/mol for pyocin-resistant strains. Gonococcal lipopolysaccharide was shown to serve as the pyocin 103 receptor by inhibition of pyocin activity. Lipopolysaccharide from a pyocin 103-resistant strain was not able to inhibit pyocin 103 activity. Pyocin 103 resistance was correlated with a structural alteration involving N-acetylglucosamine residues in gonococcal lipopolysaccharide. Based on interactions with wheat germ, soybean, and ricin lectins, a model of lipopolysaccharide structure in N. gonorrhoeae is presented.
Feedback inhibition of nitrogenase
Authors: Gordon J K , Shah V K , Brill W J
Published: January 2020

No inhibition of nitrogenase activity by physiological levels of NH4+ or carbamyl phosphate was observed in extracts of Azotobacter vinelandii. All of the 15N2 reduced by cultures which received no NH4+ was found in the cells. By contrast, more than 95% of the 15N2 reduced by cultures which had been given NH4+ was found in the medium. Failure to examine the culture medium would lead to the erroneous conclusion that N2 fixation is inhibited by NH4+. Nitrogenase in a derepressed mutant strain of A. vinelandii was fully active in vivo in the presence of NH4+. The addition of NH4Cl to N2-fixing cultures resulted in no decrease in the N2-reducing activity of intact cells of Klebsiella pneumoniae or Clostridium pasteurianum and only a small (15%) decrease in A. vinelandii. Therefore, no significant inhibition of nitrogenase by NH4+ or metabolites derived from NH4+ exists in A. vinelandii, K. pneumoniae, or C. pasteurianum.
Molecular organization of heat-labile enterotoxin genes originating in Escherichia coli of human origin and construction of heat-labile toxoid-producing strains
Authors: Yamamoto T , Yokota T , Kaji A
Published: January 2020

Recombinant deoxyribonucleic acid technology was employed to construct heat-labile enterotoxin (LT) toxoids. A recombinant plasmid carrying both an LT promoter region and LT subunit A (LTA) gene, lacking as much as 0.25 kilobases of the region up to the C terminus, produced a peptide possessing immunological properties of LTA but lacking the ability to construct LT activity (designated as LTA*). A cloned LT subunit B (LTB) gene produced LTB when a promoter on a vector was available for the gene. Escherichia coli producing LTA* and LTB (LT toxoids) could be useful as a vaccine.
Depression of hydrogenase during limitation of electron donors and derepression of ribulosebisphosphate carboxylase during carbon limitation of Alcaligenes eutrophus
Authors: Friedrich C G
Published: January 2020

Alcaligenes eutrophus did not form the key enzymes of autotrophic metabolism, the soluble and particulate hydrogenases and ribulosebisphosphate carboxylase (RuBPC), during heterotrophic growth on succinate in batch cultures. During succinate-limited growth in a chemostat, high activities of both hydrogenases were observed. With decreasing dilution rate (D) the steady-state hydrogenase activity (H) followed first-order kinetics, expressed as follows: H = Hmax .e-alpha.D. An identical correlation was observed when autotrophic growth in a chemostat was limited by molecular hydrogen. During autotrophic growth under oxygen or carbon dioxide limitation, the activity if the soluble hydrogenase was low. These data suggested that hydrogenase formation depended on the availability of reducing equivalents to the cells. RuBPC activities were not correlated with the hydrogenase activities. During succinate-limited growth, RuBPC appeared at intermediate activities. During autotrophic growth in a carbon dioxide-limited chemostat, RuBPC was highly derepressed. RuBPC activity was not detected in cells that suffered from energy limitation with a surplus of carbon, as in a heterotrophic oxygen-limited chemostat, nor was it detected in cells limited in carbon and energy, as in the case of complete exhaustion of a heterotrophic substrate. From these data I concluded that RuBPC formation in A. eutrophus depends on two conditions, namely, carbon starvation and an excess of reducing equivalents.
Transport of α-Aminoisobutyric Acid by
Streptococcus pyogenes
and Its Derived L-Form
Authors: Reizer Jonathan , Panos Charles
Published: January 2020

We studied the uptake of α-aminoisobutyric acid (AIB) in
Streptococcus pyogenes
and its physiologically isotonic L-form.
S. pyogenes
cells starved for glucose or treated with carbonyl cyanide-
m
-chlorophenyl hydrazone accumulated limited amounts of AIB. A high apparent

K
m

value characterized the glucose-independent transport of AIB. The rate and extent of AIB accumulation significantly increased in the presence of glucose. Two saturable transport components with distinct apparent

K
m

values characterized glycolysis-coupled transport of AIB. A biphasic Lineweaver-Burk plot was also obtained for
l
-alanine transport by glycolyzing
S. pyogenes
cells. AIB seems to share a common transport system(s) with glycine,
l
- and
d
-alanine,
l
-serine, and
l
-valine. This was shown by the competitive inhibition of AIB uptake by these compounds and their ability to induce competitive exchange efflux of accumulated AIB. About 30% of the AIB uptake was not inhibited by a saturating amount of
l
-valine, indicating the existence of more than one system for AIB transport.
p
-Chloromercuribenzoate markedly inhibited the accumulation of AIB by both glycolyzing and glucose-starved cells. In contrast, carbonyl cyanide-
m
-chlorophenyl hydrazone affected only metabolism-dependent uptake of AIB, which was also sensitive to dinitrophenol,
N
-ethylmaleimide, iodoacetate, fluoride (NaF), arsenate, and
N,N
′-dicyclohexylcarbodiimide. These results are interpreted according to the chemiosmotic theory of Mitchell, whereby a proton motive force constitutes the driving force for AIB accumulation. AIB was not accumulated by the L-form. However, a temporary accumulation of AIB by a counterflow mechanism and a saturable system with a low apparent affinity were demonstrated for AIB transport by this organism. We suggest that a deficiency in the coupling of energy to AIB transport is responsible for the apparent lack of active AIB accumulation by the L-form.
DNA sequence fine-structure analysis of ilvG (IlvG+) mutations of Escherichia coli K-12
Authors: Lawther R P , Calhoun D H , Gray J , Adams C W , Hauser C A , Hatfield G W
Published: January 2020

Six ilvG (IlvG+) mutations of Escherichia coli K-12 were transferred to recombinant plasmids, and the DNA sequence of each mutation was determined. This analysis confirmed that expression of the ilvG gene product (acetohydroxy acid synthase II) requires the deletion of a single base pair or the addition of two base pairs within ilvG to displace a frameshift site present in wild-type E. coli K-12. This system should be useful in the analysis of potential frameshift mutagens.
N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes
Authors: Kuhn H , Fietzek P P , Lampen J O
Published: January 2020

The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens. Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis.
Genetic recombination in Nocardia asteroides
Authors: Kasweck K L , Little M L
Published: January 2020

Gene recombination between strains of Nocardia asteroides of diverse origins has been demonstrated. In particular pairwise combinations, recombinants made up 0.01% of the population. All nine selectable recombinant classes were recovered from a cross KK4-47 his-10 leu-1 and KK6-119 met-3 phe-3. Recombinants with an auxotrophic marker from each parent constitute 21% of the recombinants.
Amino-terminal sequence of the Tn3 transposase protein
Authors: Ditto M D , Chou J , Hunkapiller M W , Fennewald M A , Gerrard S P , Hood L E , Cohen S N , Casadaban M J
Published: January 2020

The amino-terminal sequence of the Tn3 transposase protein was determined to be Pro-Val-Asp-Phe-Leu-Thr-Thr-Glu-Gln-Val-Glu-Ser.... This was determined both from an active transposase protein purified from a transposase overproducing mutant strain and from a hybrid transposase-beta-galactosidase fusion protein. The amino acid sequence corresponded to the DNA sequence of the transposase gene beginning at an ATG initiation codon, as previously predicted from the analysis of transposase-beta-galactosidase gene fusions.
Localization of quantitation of the ornithine lipid of Thiobacillus thiooxidans
Authors: Dees C , Shively J M
Published: January 2020

The ornithine lipid of Thiobacillus thiooxidans was found to be 1.9% of the total polar lipids. Approximately 80% of this lipid was found to be localized in the outer membrane.
Correlation between auxotrophy and plasmid alteration in mutant strains of Pseudomonas cepacia
Authors: Beckman W , Gaffney T , Lessie T G
Published: January 2020

We describe here a class of mutants of Pseudomonas cepacia strain 249 in which different types of auxotrophy are associated with alterations in the 100-megadalton plasmid present in this strain.
Initiation of Yeast Sporulation by Partial Carbon, Nitrogen, or Phosphate Deprivation
Authors: Freese Elisabeth Bautz , Chu Martha I. , Freese Ernst
Published: January 2020

In this paper we show that partial deprivation of a carbon source, a nitrogen source, or phosphate in the presence of all other nutrients needed for growth initiates meiosis and sporulation of
Saccharomyces cerevisiae
homothallic strain Y55. For carbon deprivation experiments, cells were grown in synthetic medium (pH 5.5) containing an excess of one carbon source and then transferred to the same medium containing different concentrations of the same carbon source. In the case of transfer to different acetate concentrations, the log optical density at 600 nm increased at the previous rate until the cells had used up all of the acetate, whereupon the cells entered a stationary phase and did not sporulate. The same was observed with ethanol. In contrast, at different concentrations of dihydroxy-acetone or pyruvate, cells grew at different rates and sporulated optimally at intermediate concentrations (50 to 75 mM). The response to galactose was similar but reflected the presence of a low-affinity galactose transport system and the induction of a high-affinity galactose transport system. Cells could also sporulate when a glucose medium ran out of glucose, apparently because they initiated sporulation during the subsequent lag period and then used the produced ethanol as a carbon source. For phosphate deprivation experiments, cells growing with excess ethanol or pyruvate and phosphate were transferred to the same medium containing limiting amounts of phosphate. First, they used up the intracellular phosphate reserves for rapid growth, and then they sporulated optimally when an intermediate concentration (30 μM) of phosphate had been added to the medium. For nitrogen deprivation experiments, cells grown with excess acetate, ethanol, or pyruvate and NH
4
+
were transferred to the same medium from which all nitrogen had been removed. These cells sporulated well in acetate medium but poorly in ethanol and pyruvate media. However, the sporulation frequency in the latter media could be increased greatly by adding intermediate concentrations (1 mM) of the slowly metabolizable amino acids glycine, histidine, or phenylalanine. If one assumes that the sporulation response to partial deprivation of carbon-, nitrogen-, or phosphorus-containing compounds reflects control by a single metabolite, the intracellular concentration of this metabolite may decide at the START position (G1 phase) of the cell cycle whether
a
/α cells enter mitosis or meiosis.
Direct DNA repeat in plasmid R68.45 is associated with deletion formation and concomitant loss of chromosome mobilization ability
Authors: Currier T C , Morgan M K
Published: January 2020

Plasmid R68.45 has been useful in promoting the transfer of chromosomal markers in bacteria of many genera. In donors harboring R68.45, chromosome-mobilizing ability (Cma) may be lost without the loss of other plasmid markers. However, Cma can be somewhat stabilized by maintenance of the donors in the presence of kanamycin (Km). We isolated variants of R68.45 from four bacterial species of three genera. Plasmid variants isolated included those without Cma, without transfer function (Tra) and Cma, or without Tra, Cma, and Km resistance. In Erwinia carotovora subsp. atroceptica EA153, the loss of plasmid markers is dependent on the culture medium on which the donors are maintained. Restriction endonuclease analyses of the variant plasmids revealed that most are deletion mutants of R68.45. In all cases when the uncertainty in the ends of the deletions was not too great, one end of the deletion was shown to originate within or near the direct DNA duplication in R68.45 which is required for Cma and which maps close to the Km resistance determinant. Furthermore, the types of deletions observed are consistent with what might be expected for deletions generated by tandemly repeated insertion sequences. Therefore, we suggest that the DNA duplication is the source of much of the instability observed in R68.45. However, data are presented for E. carotovora subsp. atroceptica EA153 which suggest that another region of R68.45 may also play a role in its stability in this species.
Isoelectric focusing and crossed immunoelectrophoresis of heme proteins in the Escherichia coli cytoplasmic membrane
Authors: Kranz R G , Gennis R B
Published: January 2020

Isoelectric focusing (IEF), agarose electrophoresis, and crossed immunoelectrophoresis (CIE) were used to resolve the heme-containing proteins of the Escherichia coli cytoplasmic membrane after solubilization by Triton X-100. Two bands in IEF stained for heme with pI values of 4.7 and 5.3. One of the bands, with an isoelectric point of pH 5.3, was present only when the cells were grown to late log or stationary phase and possessed N,N,N,'N'-tetramethyl-p-phenylene-diamine (TMPD) oxidase activity. The pI 4.7 band was present in cells harvested in both mid-log and stationary phases. Agarose electrophoresis, using larger samples, revealed the same two components apparent by IEF, and, in addition, a third component. The heme-containing fractions were extracted after agarose electrophoresis and subjected to further study. The component which was present in cells grown to stationary phase contained hemes b, a1, and d. The other two fractions contained only b heme. One of these corresponded to the component with pI 4.7 in IEF and had catalase activity. Antisera were raised against Triton X-100-solubilized cytoplasmic membranes and against the focused TMPD oxidase complex. With these anti-sera, CIE in the presence of Triton X-100 revealed four precipitin complexes containing heme. Three of these corresponded to the components identified by IEF and agarose electrophoresis. We demonstrate that the combined use of IEF and CIE is valuable for analysis of membrane proteins. In particular, this work represents a substantial initial step toward a structural elucidation of the E. coli aerobic respiratory chain.
Isolation and characterization of a temperature-sensitive lethal mutant of Salmonella typhimurium that is conditionally defective in 3-deoxy-D-manno-octulosonate-8-phosphate synthesis
Authors: Rick P D , Young D A
Published: January 2020

A new mutant of Salmonella typhimurium was isolated which possesses a temperature-sensitive defect in the synthesis of 3-deoxy-D-manno-octulosonic acid. The defect in 3-deoxy-D-manno-octulosonic acid synthesis is due to a temperature-sensitive 3-deoxy-D-manno-octulosonate-8-phosphate synthetase, and the mutant accumulates an incomplete lipid A under nonpermissive conditions. Evidence is presented which indicates that the incomplete lipid A molecule is most likely identical in structure to the lipid A precursor synthesized by previously characterized mutants conditionally defective in 3-deoxy-D-manno-octulosonic acid synthesis. However, unlike related mutants which undergo growth stasis under nonpermissive conditions, the accumulation of lipid A precursor in the new mutant results in cell death at elevated temperatures.
Interconversion and uptake of nucleotides, nucleosides, and purine bases by the marine bacterium MB22
Authors: Foret M , Ahlers J
Published: January 2020

Whole cells and isolated membranes of the marine bacterium MB22 converted nucleotides present in the external medium rapidly into nucleosides and then into bases. Nucleosides and purine bases formed were taken up by distinct transport systems. We found a high-affinity common transport system for adenine, guanine, and hypoxanthine, with a Km of 40 nM. This system was inhibited noncompetitively by purine nucleosides. In addition, two transport systems for nucleosides were present: one for guanosine with a Km of 0.8 microM and another one for inosine and adenosine with a Km of 1.4 microM. The nucleoside transport systems exhibited both mixed and noncompetitive inhibition by different nucleosides other than those translocated; purine and pyrimidine bases had no effect. The transport of nucleosides and purine bases was inhibited by dinitrophenol or azide, thus suggesting that transport is energy dependent. Inside the cell all of the substrates were converted mainly into guanosine, xanthine, and uric acid, but also anabolic products, such as nucleotides and nucleic acids, could be found.
Protoplast fusion permits high-frequency transfer of a Streptomyces determinant which mediates actinomycin synthesis
Authors: Ochi K
Published: January 2020

Prototrophic recombinants and heterocaryotic colonies developed at high frequency when protoplasts of nutritionally complementary actinomycin-producing and nonproducing strains of Streptomyces antibioticus were fused in the presence of polyethylene glycol and plated on minimal regeneration medium. Of the spores obtained from aerial hyphae of a single heterocaryotic colony, 99% carried the act+ character regardless of whether the nutritional markers of the spore were derived from the act+ or the act parent. Similarly, a high-frequency transfer (68% in S. antibioticus, 48% in Streptomyces parvulus) of act+ determinant(s) to act was achieved by protoplast fusion. Protoplasts of a doubly auxotrophic act strain of S. parvulus were efficiently transformed in the presence of polyethylene glycol with respect to the auxotrophic markers by DNA of an act+ auxotrophic strain with complementary nutritional requirements. The transformation frequency of the nutritional (chromosomal) markers was 17%. In contrast, the transformation frequency for actinomycin synthesis was less than 1%.
Physiological and enzymatic properties of a thymidine-requiring Pediococcus cerevisiae mutant
Authors: Ariel M , Lavi H , Holtzer E , Grossowicz N
Published: January 2020

We describe the isolation and characterization of a Pediococcus cerevisiae thymidine-requiring mutant and its thymidine-independent revertant. The mutant strain lacked thymidylate synthetase activity and had an absolute requirement for low concentrations (2 micrograms/ml) of thymidine in addition to a requirement for N-5-formyl tetrahydrofolic acid (folinate). Even at high concentrations (up to 500 micrograms/ml), thymine could not replace thymidine. In contrast to its wild-type parent, which grows only on folinate, the thymidine-requiring mutant (Thy- Fol+) was able to take up and grow on picogram quantities of unreduced folic acid. When both strains were grown on folinate, the Thy- Fol+ strain was at least 10(3)-fold more resistant to the folic acid analogs aminopterin and methotrexate than the wild-type strain. On the other hand, when grown on folic acid, the Thy- Fol+ strain was as sensitive to the folic acid analogs as the Thy+ Fol+ strain and was 10(2)-fold more sensitive than the wild-type strain grown on folinate. The thymidine-independent revertant (Thy+ Fol+) regained the wild-type level of thymidylate synthetase activity, but maintained the ability to take up and grow on unreduced folic acid like its Thy- Fol+ parent.
Genetic characterization of the glutamate dehydrogenase gene (gdhA) of Salmonella typhimurium
Authors: Rosenfeld S A , Dendinger S M , Murphy C H , Brenchley J E
Published: January 2020

Salmonella typhimurium mutants, either devoid or glutamate dehydrogenase activity or having a thermolabile glutamate dehydrogenase protein, were used to identify the structural gene (gdhA) for this enzyme. Transductions showed that the mutations producing these phenotypes were linked to both the pncA and nit genes, placing the gdhA locus between 23 and 30 U on the S. typhimurium chromosome. Additional transductions with several Tn10 insertions established the gene order as pncA-gdhA-nit. Since few genetic markers exist in this region of the chromosome, Hfr strains were constructed to orient the pncA-gdhA-nit cluster with outside genes. Conjugation experiments provided evidence for the gene order pyrD-pncA-gdhA-nit-trp. To further characterize gdhA, we used Mu cts d1 (Apr lac) insertions in this gene to select numerous strains containing deletions with various endpoints. Transductions of these deletions with strains containing different gdh mutations and with a mutant having a thermolabile glutamate dehydrogenase protein permitted us to construct a deletion map of the gdhA region.
Membrane-associated alkaline phosphatase from Bacillus licheniformis that requires detergent for solubilization: lactoperoxidase 125I localization and molecular weight determination
Authors: Spencer D B , Hansa J G , Stuckmann K V , Hulett F M
Published: January 2020

When membranes of Bacillus licheniformis MC14 were extracted exhaustively with 1 M magnesium, approximately 80% of the membrane-associated alkaline phosphatase (orthophosphoric-monoester phosphohydrolase [alkaline optimum], E.C. 3.1.3.1) was solubilized. The remaining activity could be extracted with a cationic detergent, hexadecylpyridinium chloride, without loss of enzymatic activity. The detergent-extractable alkaline phosphatase was immunoprecipitable with antibody to the salt-extractable alkaline phosphatase or the secreted alkaline phosphatase, had an approximate molecular weight of 60,000, and was localized 100% on the outer surface of the cytoplasmic membrane.
Ultrastructure of square bacteria from a brine pool in Southern Sinai
Authors: Kessel M , Cohen Y
Published: January 2020

The square bacterium discovered by Walsby (Nature [London] 283:69-71, 1980) has been shown to possess the ultrastructural features of a typical halophile. The cell wall is comprised of regularly arranged subunits demonstrated by thin sectioning, shadowed replicas, and negative staining. Optical diffraction confirms the existence of both hexagonal and tetragonal arrangements of the cell wall subunits and also of different lattice constants and suggests a mixed population of bacteria.
Interchangeability of delta subunits of RNA polymerase from different species of the genus Bacillus
Authors: Achberger E C , Tahara M , Whiteley H R
Published: January 2020

RNA polymerase was purified from five species of Bacillus, including Bacillus subtilis. Each polymerase had a subunit composition analogous to that reported for B. subtilis, i.e., beta beta '2 alpha sigma delta omega 1 omega 2. The delta subunits from the B. subtilis and Bacillus thuringiensis polymerases were interchangeable, as judged from their effects on promoter selection in the polymerase binding assay.
Generality of the growth kinetics of the average individual cell in different bacterial populations
Authors: Trueba F J , Neijssel O M , Woldringh C L
Published: January 2020

The kinetics of growth of all the cells in a population is reflected in the shape of the size distribution of the population. To ascertain whether the kinetics of growth of the average individual cell is similar for different strains or growth conditions, we compared the shape of normalized size distributions obtained from steady-state populations. Significant differences in the size distributions were found, but these could be ascribed either to the precision achieved at division or to a constriction period which is long relative to the total cell cycle time. The remaining difference is quite small. Thus, without establishing the pattern itself, it is concluded that the basic course of growth is very similar for the various Escherichia coli strains examined and probably also for other rod-shaped bacteria. The effects of differences in culture technique (batch or chemostat culture), growth rate, and differences among strains were not found to influence the shape of the size distributions and hence the growth kinetics in a direct manner; small differences were found, but only when the precision at division or the fraction of constricted cells (long constriction period) were different as well.
Stored mRNA in sporangiospores of the fungus Mucor racemosus
Authors: Linz J E , Orlowski M
Published: January 2020

When introduced into nutrient medium under air, the asexual sporangiospores of Mucor racemosus germinated with 5 to 8 h, culminating with the emergence of germ tubes. We found that sporangiospores increased 20% in dry weight during the first 60 min of germination, indicating a high degree of synthetic activity. Sucrose density gradient analysis of spore extracts revealed that the percentage of ribosomes associated with mRNA increased from 22.5% in dormant spores to 85% within 10 min after the addition of medium and remained at this level for at least 3 h. L-[14C]leucine was immediately incorporated at a rapid rate into protein of a leucine auxotroph, whereas [3H]uracil or [32P]phosphate was incorporated into RNA at a significant rate only 20 min after the addition of medium. This newly synthesized RNA occurred in polysomes only after 30 min had passed. Pool synthesized RNA occurred in polysomes only after 30 min had passed. Pool equilibration of the radioactive precursors was not limiting to these measurements. Polyadenylated RNA was isolated from dormant spores by oligodeoxythymidylic acid-cellulose chromatography and was found to comprise 3.3% of the total cellular RNA. Sucrose density gradient centrifugation revealed the polyadenylated RNA to be heterodisperse in size, ranging from 6S to 20S. It was concluded that M. racemosus sporangiospores contain preformed mRNA which is translated commencing immediately upon the addition of nutrient medium.
Mapping of two ugp genes coding for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli
Authors: Schweizer H , Grussenmeyer T , Boos W
Published: January 2020

Two genes, ugpA and ugpB, coding for a binding protein-dependent sn-glycerol-3-phosphate transport system, were mapped at 75.3 min on the Escherichia coli chromosome. A Tn10 insertion in ugpA resulted in loss of transport activity but still allowed the synthesis of the sn-glycerol-3-phosphate-binding protein. This Tn10 insertion was found to be linked by P1 transduction to pit, aroB, malA, asd, and livH with 2.5, 2.8, 25, 63.5, and 83% cotransduction frequency. An insertion of Mud (Ampr lac) in ugpB resulted in the loss of the binding protein. ugpB is closely linked to ugpA. It is either the structural gene for the binding protein or located proximal to it. The analysis of the crosses allowed the ordering of the markers in the clockwise direction as follows: aroB, malA, asd, ugpA, ugpB, livH, pit.
Polar lipids in phototrophic bacteria of the Rhodospirillaceae and Chromatiaceae families
Authors: Imhoff J F , Kushner D J , Kushwaha S C , Kates M
Published: January 2020

The polar lipids of photosynthetic purple bacteria of the genera Chromatium, Thiocapsa, Thiocystis, Ectothiorhodospira, Rhodopseudomonas, Rhodospirillum, and Rhodomicrobium were analyzed. Characteristic compositions of the polar lipids were found for most of the Rhodospirillaceae and Chromatiaceae species. Phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin were the major phospholipids in most species. Phosphatidylcholine was present as a major component in all species of the genus Ectothiorhodospira, but was not detected in the remaining Chromatiaceae. It was also present in most of the Rhodospirillaceae species. No glycolipids were found in any of the Ectothiorhodospira species. In the Rhodospirillaceae, the glycolipids mono- and digalactosyl diglycerides were generally absent. Sulfoquinovosyl diglyceride was present in significant amounts in at least three species of the Rhodospirillaceae and may have been present in most of them, but only in traces. All of the Chromatiaceae species contained several glycolipids, one of which was similar to monogalactosyl diglyceride. Ornithine lipids were found in large amounts in most Rhodospirillaceae, but were absent in Ectothiorhodospira and in the other Chromatiaceae. The species examined could be divided into three groups on the basis of their lipid composition: (i) the genus Ectothiorhodospira; (ii) the remaining Chromatiaceae; and (iii) the Rhodospirillaceae. The data presented are compared with those available in the literature, and differences from other phototrophic organisms are discussed.
Melanin-lacking mutants of Cryptococcus neoformans and their virulence for mice
Authors: Kwon-Chung K J , Polacheck I , Popkin T J
Published: January 2020

A double mutant of Cryptococcus neoformans which lacked the ability to produce melanin (Mel-) on media containing diphenols and failed to grow at 37 degrees C (temperature sensitive, Tem-) was obtained by UV irradiation and subsequent cloning. The mutant showed two lesions in melanogenesis in that it lacked the active transport system for diphenolic compounds and also lacked phenoloxidase. Ultrastructures of the mutant and wild-type cells grown on a medium with or without L-dopa showed that only the wild-type cells grown on L-dopa medium formed a dark cell wall layer, presumably containing melanin. The mutant was crossed with a wild type, and the phenotypes of the progeny were analyzed. The analysis showed no linkage between the mating type and either Mel or Tem loci, but loose linkage was seen between Mel and Tem loci. The progeny, Mel+ Tem+, Mel+ Tem-, Mel- Tem+, and Mel- Tem-, were studied for their virulence in mice. Only Mel+ Tem+ types killed mice with an inoculum of 5 X 10(5) cells within 50 days.
Absence of significant membrane localization of the proteins coded by the ilvGEDAC genes of Escherichia coli K-12
Authors: Gray J E , Calhoun D H
Published: January 2020

We previously characterized a set of lambda dilv phages by genetic, restriction enzyme, and heteroduplex analyses and tentatively correlated isoleucine-valine gene products with specific ilv DNA segments by using cloned ilv segments in maxicells and lambda dilv phage infection of UV-irradiated cells. In this work, the identity of the ilvC gene product, alpha-acetohydroxy acid isomeroreductase, was confirmed by demonstrating its induction by the physiological inducers alpha-acetolactate and alpha-acetohydroxybutyrate. The identity of the ilvE gene product, transaminase, B, was confirmed by antibody precipitation of the purified enzyme. Phage derivatives with ilv regulatory mutations were found to have the predicted effect upon the ilvGEDA and ilvC protein products. The distribution of the ilvGEDA and ilvC gene products in the soluble, periplasmic, inner membrane, and outer membrane fractions was examined, and no significant membrane association was observed. The expression of the ilv genes in the lambda dilv phage from ilv and phage lambda promoters was compared in order to determine the fractional contribution of each to ilv gene expression. An additional protein of 54,000 daltons that was not detected in the previous analysis was observed to be coded by a bacterial gene but was produced only by readthrough from phage promoters.
Identification of a protein of 15,000 daltons related to isoleucine-valine biosynthesis in Escherichia coli K-12
Authors: Gray J E , Wallen J W , Calhoun D H
Published: January 2020

The effect of the ilvG671, ilvG468, and ilvG603 mutations (phenotype, IlvG+ Valr; formerly ilvO) upon proteins synthesized was determined by infection of irradiated Escherichia coli K-12 cells, using specifically constructed derivatives of lambda dilv phage. These ilvG alleles are similar to the previously studied ilvG2096(Valr) allele in that they activate the latent ilvG gene which is present in the wild-type strain, leading to the synthesis of a 62,000-dalton protein. In addition, all of these ilvG (Valr) alleles increase the synthesis of a 15,000-dalton protein. To localize the gene coding for the 15,000-dalton protein, the proteins produced in maxicells containing plasmids with specific deletions of ilv and rrnX DNA segments were analyzed. The gene coding for the 15,000-dalton protein was located within a region about 1,000 base pairs long between ilv and trpT. The function of the 15,000-dalton protein is not known.
A Regulatory Gene (
use
) Affecting the Expression of
pyrA
and Certain Other Pyrimidine Genes
Authors: Bussey Lee B. , Ingraham John L.
Published: January 2020

The
use-1
mutation in
Salmonella typhimurium
confers a complex and pleiotrophic phenotype which is primarily characterized as a temperature-dependent sensitivity to uracil. This sensitivity can be reversed by arginine or citrulline, but not by ornithine, suggesting that the
use-1
mutation affects the synthesis or the activity (or both) of carbamoylphosphate synthetase or ornithine carbamoyltransferase (or both). Activity measurements showed that
use-1
caused superrepression of both of these enzymes, especially when uracil was present in the medium. Dihydro-orotase and dihydro-orotate oxidase were also superrepressed, but aspartate carbamoyltransferase and orotate phosphoribosyltransferase were not. Lowered nucleotide triphosphate and guanosine tetra- and pentaphosphate pools in
use-1
strains indicated that the mutation affected synthesis or breakdown of all of these phosphorylated compounds, but the UTP pool increased by a larger relative factor in
use-1
strains in the presence of uracil. The uracil-sensitive phenotype of the
use-1
mutation is a complex response to several environmental factors: temperature, aerobiosis, carbon sources, and uracil concentration. Uracil sensitivity was eliminated by alteration of one or more of these factors. Uracil sensitivity was suppressed by several genetic alterations. These include introduction into
use-1
strains of a multi-copy ColE1 derivative which carries the structural gene(s) for carbamoylphosphate synthetase, episomes that carry
use
, mutations including
argR
and
pyrH
, and various unclassified intergenic suppressor mutations. These genetic changes increased significantly the expression of carbamoylphosphate synthetase or ornithine carbamoyltransferase (or both). The activity of
use-1
is not known, but the facts that it altered expression of at least four unlinked genes (
pyrA, pyrC, pyrD
, and
argI
) and that the
Escherichia coli
F′133 complemented it establish it as a
trans
-acting regulatory factor.
Identification and mobilization by cointegrate formation of a nodulation plasmid in Rhizobium trifolii
Authors: Scott D B , Ronson C W
Published: January 2020

A nodulation plasmid, pRtr-514a, of molecular size 180 megadaltons (Mdal) was identified in Rhizobium trifolii strain NZP514. This plasmid was absent in both spontaneous and heat-cured Nod- derivatives of NZP514, and these strains were unable to induce root hair curling. The ability to nodulate clover was transferred from the wild-type strain to a Nod- derivatives, PN104, with the broad-host-range plasmid R68.45 (39 megadaltons) at a cotransfer frequency of about 4 X 10(-3). Most of the Nod+ transconjugants were resistant to kanamycin, tetracycline, and carbenicillin and had received a plasmid approximately 36 or 70 Mdal larger than pRtr514a but did not contain a plasmid of the size of R68.45, indicating that pRtr-514a was mobilized as a cointegrate plasmid containing either one or possibly two copies of R68.45. Use of these cointegrate-containing strains as donors in further crosses with the Nod- derivative strain PN118 resulted in high-frequency transfer of Nod+ (10(-3) to 10(-4), with cotransfer frequencies with kanamycin of up to 100%. Introduction of R68.45 into a derivative of NZP514 containing the broad-host-range plasmid pJP4 (52 Mdal) resulted in a high frequency of transconjugants carrying a cointegrate plasmid composed of pRtr-514a and pJP4. When used as donors to Nod- derivatives, such strains cotransferred Nod+ with kanamycin plus mercury at a frequency of 67%. The identification of stable cointegrates between pRtr-514a and the broad-host-range plasmids R68.45 and pJP4 should enable several genetic manipulations to be carried out with this nodulation plasmid, including the transfer of the plasmid to most gram-negative bacterial genera.
Clockwise and counterclockwise pinwheel colony morphologies of Bacillus subtilis are correlated with the helix hand of the strain
Authors: Mendelson N H , Keener S L
Published: January 2020

Helical macrofiber-producing strains of Bacillus subtilis grown on fresh complex medium semisolid surfaces formed "pinwheel"-shaped colonies. Clockwise pinwheel projections arose from colonies of strains that produce right-handed helical macrofibers in fluid cultures. Most strains able to make left-handed helical macrofibers in fluid grew as disorganized wavy colonies without directed projections. A phage-resistant left-handed mutant was found that produces very tight colonies with pinwheel projections that lie counterclockwise relative to the colony. The pinwheel colony morphology is interpreted therefore in terms of the cell surface organization and helical growth.
Promoter mapping and selection of operator mutants by using insertion of bacteriophage Mu in the argECBH divergent operon of Escherichia coli K-12
Authors: Beny G , Boyen A , Charlier D , Lissens W , Feller A , Glansdorff N
Published: January 2020

The analysis of a large number of Arg mutants obtained by inserting phage Mu in the argECBH cluster of genes confirmed the "facing" arrangement proposed earlier for the promoters of argE (argEp) and argCBH (argCBHp) and clarified remaining ambiguities regarding the localization of argEp. Casadaban and Cohen's Mu d lac phages (M. Casadaban and S. N. Cohen, Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533, 1979) were used to construct strains where either an intact or a truncated lacZ gene was fused to argC or argB. Several operator-constitutive mutations could be selected for in such strains; the mutations affected both arms of the cluster, thereby defining one common operator region for both directions of transcription.
Aminoacyl-tRNAs from Physarum polycephalum: patterns of codon recognition
Authors: Hatfield D , Rice M , Hession C A , Melera P W
Published: January 2020

Isoacceptors of Physarum polycephalum Ala-, Arg-, Glu-, Gln-, Gly-, Ile-, Leu-, Lys-, Ser-, Thr-, and Val-tRNAs were resolved by reverse-phase chromatography and isolated, and their codon recognition properties were determined in a ribosomal binding assay. Codon assignments were made to most isoacceptors, and they are summarized along with those determined in other studies from Escherichia coli, yeasts, wheat germ, hymenoptera, Xenopus, and mammals. The patterns of codon recognition by isoacceptors from P. polycephalum are more similar to those of animals than to those of plants or lower fungi.
Size at Bud Initiation for Wild-Type and Petite Yeast Cells
Authors: Bugeja V. C. , Carter B. L. A.
Published: January 2020

Cell size at bud initiation shows the same relationship to growth rate in wild-type and petite yeast cells. The nutrient-modulated size control is related to growth rate rather than a change from fermentative to oxidative metabolism.
Isolation and characterization of plasma membranes from the fungus Podospora anserina
Authors: Labarère J , Bonneu M
Published: January 2020

This paper describes a method for separating and isolating plasma membranes from the septated fungus Podospora anserina. Plasma membranes were isolated from protoplasts (young cell plasma membranes) and mycelia (both young and aged cell plasma membranes). The procedure of fractionation consisted of a combination of differential and isopycnic centrifugations. Characterization of cellular membranes and enrichment of the fractions with plasmalemma were carried out by assays on enzymatic activities. A plasma membrane fraction was isolated in a buoyant density peak of 1.087 g/cm3, where three enzymatic activities bound to plasma membrane, adenylate cyclase, chitin synthase, and beta-glucan synthase at low affinity for UDP-Glc, peaked together. Good purity of this fraction was determined by the absence or the very low level of other enzymatic activities used as markers for intracellular membranes, i.e., succinate dehydrogenase, alpha-mannosidase, NADPH cytochrome c reductase, and beta-glucan synthase at high affinity for UDP-Glc activities.
Characterization of pBFTM10, a clindamycin-erythromycin resistance transfer factor from Bacteroides fragilis
Authors: Tally F P , Snydman D R , Shimell M J , Malamy M H
Published: January 2020

Bacteroides fragilis TMP10, which is clindamycin-erythromycin resistant (Clnr) and tetracycline resistant (Tetr), contains several plasmids and is capable of transferring drug resistance markers to suitable recipients. We were able to separate a 14.6-kilobase self-transmissible Clnr plasmid, pBFTM10, from the other plasmids of TMP10 in a tetracycline-sensitive recipient strain, B. fragilis TM4000. All Clnr transconjugants acquired an unaltered pBFTM10 and became plasmid donor strains. Transfer is proposed to occur by conjugation since it required to cell-to-cell contact of filter matings and was insensitive to DNase, but sensitive to chloroform treatment of donor cells. The efficiency of transfer of pBFTM10 in a Tets background (TM4003) was not affected by pretreatment of donor cells with clindamycin. A spontaneously occurring Clns derivative, pBFTM10 delta 1, suffered a deletion of DNA, which included a 4.4-kilobase EcoRI fragment. A complex interaction between the autonomous plasmid pBFTM10 and a tetracycline transfer element also present in strain TMP10 was observed since pretreatment of this donor with tetracycline or clindamycin resulted in a marked increase in transfer of both tetracycline and clindamycin resistance.
Plasmid loss and changes within the chromosomal DNA of Streptomyces reticuli
Authors: Schrempf H
Published: January 2020

The sporulating wild-type strain of Streptomyces reticuli, which produces a melanin pigment and the macrolide leucomycin, contains plasmid DNA of 48 to 49 megadaltons. Plasmidless variants had an altered secondary metabolism and a changed antibiotic resistance pattern. By using a new colony hybridization technique developed for streptomycetes, it could be shown that plasmidless variants could be transformed with the wild-type plasmid DNA, which, however, is quickly lost from regenerated mycelium. In contrast to the wild-type strain, the plasmidless variants contain amplified nucleotide sequences within the chromosomal DNA. The number and size of these sequences vary with the strain tested. Hybridization studies revealed that the reiterated sequences are neither amplified ribosomal nor plasmid genes, but are present in small concentrations within the wild-type chromosome. Some of them share extensive homologies with each other and are located at different positions within the chromosome. It is assumed that alterations in secondary metabolism are due to changes within both the chromosomal and the extrachromosomal DNAs of S. reticuli.
Roles of lipopolysaccharide and outer membrane protein OmpC of Escherichia coli K-12 in the receptor function for bacteriophage T4
Authors: Yu F , Mizushima S
Published: January 2020

The roles of lipopolysaccharide and OmpC, a major outer membrane protein, in the receptor function for bacteriophage T4 were studied by using Escherichia coli K-12 strains having mutations in the ompC gene or in genes controlling different stages of lipopolysaccharide synthesis. The receptor activity for T4 was monitored by (i) T4 sensitivity of intact cells, (ii) phage inactivation activity of cell envelopes, and (iii) phage inactivation activity of specimens reconstituted from purified OmpC and lipopolysaccharide. It was found that (i) in the presence of the OmpC protein, the essential region of the lipopolysaccharide for the receptor activity was the core-lipid A region that includes the heptose region, whereas the glucose region was not necessarily required for the receptor function; (ii) the OmpC protein was not required at all when the distal end of the lipopolysaccharide was removed to expose a glucose residue at the distal end; and (iii) when cells lacked both the OmpC protein and the glucose region, they became extremely resistant to T4. Based on these findings, the roles of the OmpC protein and lipopolysaccharide in T4 infection are discussed.