bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence of the structural gene for tryptophanase of Escherichia coli K-12
Authors: Deeley M C , Yanofsky C
Published: January 2020

The tryptophanase structural gene, tnaA, of Escherichia coli K-12 was cloned and sequenced. The size, amino acid composition, and sequence of the protein predicted from the nucleotide sequence agree with protein structure data previously acquired by others for the tryptophanase of E. coli B. Physiological data indicated that the region controlling expression of tnaA was present in the cloned segment. Sequence data suggested that a second structural gene of unknown function was located distal to tnaA and may be in the same operon. The pattern of codon usage in tnaA was intermediate between codon usage in four of the ribosomal protein structural genes and the structural genes for three of the tryptophan biosynthetic proteins.
Control of meta-cleavage degradation of 4-hydroxyphenylacetate in Pseudomonas putida
Authors: Barbour M G , Bayly R C
Published: January 2020

Synthesis of enzymes of the 4-hydroxyphenylacetate meta-cleavage pathway was studied in Pseudomonas putida wild-type strain P23X1 (NCIB 9865) and mutant strains which had either structural or regulatory gene mutations. Induction studies with mutant strains each defective in an enzyme of the pathway showed that 4-hydroxyphenylacetate induced the hydroxylase and that 3,4-dihydroxyphenylacetate induced the 2,3-oxygenase, aldehyde dehydrogenase, isomerase, decarboxylase, and hydratase. This showed that the hydroxylase structural gene does not exist in an operon that contains any other structural gene of this meta pathway. Studies of mutant strains that synthesized constitutively the 2,3-oxygenase and subsequent enzymes suggested that the regulation of synthesis of these enzymes was coincident, and, in such strains, the hydroxylase was inducible only. Observations made with a putative polarity mutant that lacked 2,3-oxygenase activity suggested that the structural genes encoding this enzyme and subsequent enzymes of the pathway exist in the same operon. Studies of a regulatory mutant strain that was defective in the induction of the 2,3-oxygenase and subsequent enzymes suggest that the 2,3-oxygenase operon is under positive control.
Proposed pathway of triterpenoid carotenoid biosynthesis in Staphylococcus aureus: evidence from a study of mutants
Authors: Marshall J H , Wilmoth G J
Published: January 2020

Mutants of Staphylococcus aureus were isolated which showed changes in pigment composition compared with the parent strain. On the basis of differences in their triterpenoid carotenoid composition they were classified into seven types. In five of these types, there appeared to be a blockage in the biosynthetic pathway which resulted in the absence of some products and accumulation of others. The changes in the other two types appeared to be a consequence of some change in regulation. A scheme for the biosynthesis of triterpenoid carotenoids is presented in which the first C30 intermediate, 4,4'-diapophytoene, is converted via 4,4'-diapophytofluene, 4,4'-diapo-zeta-carotene, 4,4'-diaponeurosporene, 4,4'-diaponeurosporenal, 4,4'-diaponeurosporenoic acid, and glucosyl-diaponeurosporenoate to the major pigment staphyloxanthin.
Integration of the bacteriophage phi 3T-coded thymidylate synthetase gene into the Bacillus subtilis chromosome
Authors: Stroynowski I T
Published: January 2020

Transformation of Bacillus subtilis 168 Thy- auxotrophs with phi 3T deoxyribonucleic acid (DNA) to thymine independence was found to involve site-specific recombination of phi 3T DNA sequences with their homologous counterparts in the bacterial chromosome. During the transformation, the phage phi 3T-encoded thymidylate synthetase gene, thyP3, was shown to integrate at two genetically distinct sites in the B. Subtilis 168 chromosome. The first site was identified to be in the bacterial thymidylate synthetase gene, thyA. The second site was in a prophage (SPB) known to be carried in the host genome. The frequency of the integration of the thyP3 gene at each of the two loci and some of the parameters affecting this frequency were studied. The common origin of the thyP3 and thyA genes and their molecular evolution are also reported.
Genetic Elements Novel for
Corynebacterium diphtheriae
: Specialized Transducing Elements and Transposons
Authors: Buck Gregory A. , Groman Neal B.
Published: January 2020

It was shown in an accompanying paper (Buck and Groman, J. Bacteriol.
148:
131-142, 1981) that γ-
tsr
-1 phage stocks produced by heat induction of lysogens are a mixture of two phages which differ in the content of their deoxyribonucleic acid (DNA). This difference is evidenced by the appearance of “heterogeneous” (HET) fragments in restriction enzyme digests of γ-
tsr
-1 phage DNA. It was estimated that 20 to 80% of the phage in these lysates produced HET fragments. The appearance of HET fragments correlated with the appearance of a DNA insertion (DI-1) in the γ phage genome as revealed in heteroduplexes of DNA from γ-
tsr
-1 and β corynebacteriophages. The HET fragments were seen in DNA from heat-induced lysates, but not in DNA from phage stocks produced by lytic infection. By DNA-DNA hybridization analysis it was shown that a fraction of γ-
tsr
-1 phages from heat-induced lysates carried an insertion of bacterial DNA in the vegetative phage attachment site (
att
P), and that this insertion was responsible for the formation of HET fragments. Since the phage produced by this event carried a complete phage genome plus a small segment of bacterial DNA, they were called transducing elements. On the basis of these facts it was concluded that heat-induced γ-
tsr
-1 prophage was excised at an abnormal site at a very high frequency. Abnormal excision was highly specific, and the change in excision specificity occurred simultaneously with the spontaneous mutation of the phage to heat inducibility. From this and other data it was postulated that a mutation in the immune repressor was reponsible for an alteration in the specificity of the normal excision process. This distinguishes the mechanism of formation of γ-
tsr
-1 transducing elements from that employed by other phages. A second DNA insertion (DI-2) in the
tox
(diphtheria toxin) gene of γ-
tsr
-1 and γ-
tsr
-2 was also identified as an insertion of bacterial DNA. The DI-2 insertion had a stem-and-loop structure similar to that seen in heteroduplexes visualizing transposons or insertion elements. It seems likely that γ wild-type phage, which is mutant for
tox
, was originally
tox
+
, but that transposition of bacterial DNA into the gene inactivated it.
Regulation of polypeptide synthesis during Caulobacter development: two-dimensional gel analysis
Authors: Milhausen M , Agabian N
Published: January 2020

The gram-negative bacterium Caulobacter crescentus progresses through three distinct morphological transitions, including both motile and nonmotile cell types, during its cell cycle. Assessment of the extent of regulation of polypeptide synthesis during these transitions was carried out with two-dimensional gel electrophoresis of whole-cell extracts. Synchronous cells were pulse-labeled with 14C-amino acids for 10-min intervals throughout the entire 2-h cell cycle. The radioactively labeled polypeptides were analyzed by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms resulting from fluorography of the second dimension provided the detection of approximately 1,000 unique spots. The 600 predominant polypeptide spots, representing approximately 40% of the coding capacity of Caulobacter deoxyribonucleic acid, were analyzed for major changes in their synthetic rates. Quantitation by densitometric scanning of individual polypeptide spots represented on the sequential fluorograms demonstrated significant changes in the temporal synthesis of 6% of the polypeptides. Extracts from asynchronous cells were fractionated to obtain total-membrane and deoxyribonucleic acid-binding polypeptide fractions. Subsequent electrophoresis of these cellular fractions revealed approximately 100 membrane polypeptides and 25 deoxyribonucleic acid-binding polypeptides. Eight of the regulated polypeptides were identified as membrane or deoxyribonucleic acid-binding proteins. The regulated polypeptides can be grouped into three main categories based on their interval of synthesis. The three categories are in direct correlation with the three distinct cell cycle stages. This analysis has also revealed a unique transition period in the cell cycle in which a significant proportion of gene expression is regulated.
Identification of the molybdenum cofactor in chlorate-resistant mutants of Escherichia coli
Authors: Amy N K
Published: January 2020

Experiments were performed to determine whether defects in molybdenum cofactor metabolism were responsible for the pleiotropic loss of the molybdoenzymes nitrate reductase and formate dehydrogenase in chl mutants of Escherichia coli. In wild-type E. coli, molybdenum cofactor activity was present in both the soluble and membrane-associated fractions when the cells were grown either aerobically or anaerobically, with and without nitrate. Molybdenum cofactor in the soluble fraction decreased when the membrane-bound nitrate reductase and formate dehydrogenase were induced. In the chl mutants, molybdenum cofactor activity was found in the soluble fraction of chlA, chlB, chlC, chlD, chlE, and chlG, but only chlB, chlC, chlD, and chlG expressed cofactor activity in the membrane fraction. The defect in the chlA mutants which prevented incorporation of the soluble cofactor into the membrane also caused the soluble cofactor to be defective in its ability to bind molybdenum. This cofactor was not active in the absence of molybdate, and it required at least threefold more molybdate than did the wild type in the Neurospora crassa nit-1 complementation assay. However, the cofactor from the chlA strain mediated the dimerization of the nit-1 subunits in the presence and absence of molybdate to yield the 7.9S dimer. Growth of chlA mutants in medium with increased molybdate did not repair the defect in the chlA cofactor nor restore the molybdoenzyme activities. Thus, molybdenum cofactor was synthesized in all the chl mutants, but additional processing steps may be missing in chlA and chlE mutants for proper insertion of cofactor in the membrane.
Dissociation and reassembly of Escherichia coli type 1 pili
Authors: Eshdat Y , Silverblatt F J , Sharon N
Published: January 2020

Escherichia coli type 1 pili, which mediate the mannose-sensitive adherence of the bacterium to eucaryotic cells, are comprised of very stable arrays of pilin protein subunits (molecular weight, approximately 17,000). Previous methods for the dissociation of pili caused their irreversible denaturation. We have found that incubation of pili in saturated guanidine hydrochloride at 37 degrees C led to their complete dissociation, as evidenced by nephelometry and electron microscopy. Gel chromatography of the dissociated pili on a Sepharose CL-6B column in the presence of saturated guanidine hydrochloride yielded a single protein peak with a molecular weight corresponding to that of pilin. Dialysis of this peak against 5 mM tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0) and rechromatography in the same buffer afforded a major protein peak, probably consisting of pilin dimers. About 25% of the protein in this peak bound to a mannan-sepharose column and could be eluted with methyl alpha-D-mannoside. The pilin dimer gave a single protein band upon polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate (molecular weight, 16,600) or 10 M urea and penetrated completely into 7% gels in the absence of denaturants. Reassembly of the pilin dimers into pili was achieved upon dialysis against the tris(hydroxymethyl)aminomethane buffer containing 5 mM MgCl2, as observed by electron microscopy. Thus, the conditions used allow renaturation of the dissociated subunits and may aid in further studies of the structure-function relationship of pili.
Role of adherence in growth of Acinetobacter calcoaceticus RAG-1 on hexadecane
Authors: Rosenberg M , Rosenberg E
Published: January 2020

The high affinity of Acinetobacter calcoaceticus RAG-1 for liquid hydrocarbons permitted the isolation of a spontaneous nonadherent mutant, MR-481. Strain MR-481 exhibited no significant affinity for three test hydrocarbons, yet resembled the wild type in many properties, including production of the extracellular emulsifying agent emulsan. To study the role of adherence in growth on hydrocarbons, RAG-1 and MR-481 were compared for growth on hexadecane under conditions of limited agitation and at low initial cell densities. Adherent RAG-1 cells were able to grow rapidly under these conditions, whereas nonadherent MR-481 cells failed to grow for at least 54 h. However, the addition of emulsan either initially or at various times after inoculation enabled the nonadherent MR-481 cells to grow on hexadecane. Growth was not the result of reversion of MR-481 from nonadherent to adherent cells. The data demonstrate that adherence is a crucial factor in the growth of A. calcoaceticus RAG-1 on hexadecane in the absence of extracellular emulsification of the substrate.
Stability of the Adenosine 5′-Triphosphate Pool in
Coxiella burnetii
: Influence of pH and Substrate
Authors: Hackstadt Ted , Williams Jim C.
Published: January 2020

The ability of
Coxiella burnetii
to couple oxidation of metabolic substrates to adenosine 5′-triphosphate (ATP) synthesis in axenic reaction buffers was examined. Pyruvate, succinate, and glutamate were catabolized and incorporated at the highest rates of 11 substrates tested. Glutamate oxidation, however, resulted in the greatest stability of the ATP pool and highest intracellular ATP levels over a 48-h period. At pH 4.5, the optimum for metabolism by
C. burnetii
, glutamate oxidation resulted in maintenance of the ATP pool at a concentration of approximately 0.7 nmol of ATP per mg of dry weight over a 96-h period. In the absence of substrate, ATP declined by 96 h to less than 0.01 nmol/mg of dry weight. When cells were maintained at pH 7.0 in the presence or absence of glutamate, ATP pools were considerably more stable, presumably due to the minimal metabolic activity displayed by
C. burnetii
at pH 7. The stability of the ATP pool reflected viability as there was greater than an 8-log decrease in viable
C. burnetii
after incubation for 7 days at pH 4.5 in the absence of glutamate. Viability was retained in the presence of glutamate at pH 4.5 or 7.0 in the absence of any added substrate. The stability of the ATP pool was due to endogenous synthesis of ATP coupled to substrate oxidation as shown by depression of ATP levels in the presence of inhibitors of electron transport or oxidative phosphorylation. In addition, the adenylate energy charge increased from an initial value of 0.57 to 0.73 during glutamate oxidation with a concomitant rise in the total adenylate pool size.
C. burnetii
therefore appears able to regulate endogenous ATP levels in response to substrate availability and pH, thus effecting a conservation of metabolic energy in neutral or alkaline environments. Such a mechanism has been proposed to play a role in the resistance of
C. burnetii
to environmental conditions and subsequent activation upon entry into the phagolysosome in which this organism replicates.
Incision and postincision steps of pyrimidine dimer removal in excision-defective mutants of Saccharomyces cerevisiae
Authors: Wilcox D R , Prakash L
Published: January 2020

cdc9, a temperature-sensitive mutant defective in polynucleotide deoxyribonucleic acid (DNA) ligase activity, accumulates low-molecular-weight DNA fragments (as measured by sedimentation of DNA in alkaline sucrose gradients) at the nonpermissive temperature after irradiation with ultraviolet light. This phenotype of cdc9 is a sensitive indicator of successful incision during excision repair of dimers. In strains containing excision-defective mutations in any of nine genes in combination with the cdc9 mutation, the absence of low-molecular-weight DNA at the nonpermissive temperature after ultraviolet treatment suggests that these mutants are incision defective, whereas the presence of low-molecular-weight DNA indicates that the mutants are defective in a step after incision. With rad1, rad2, rad3, rad4, and rad10 mutants, the molecular weight of the DNA remained unchanged after ultraviolet irradiation and incubation at the restrictive temperature, despite the presence of the cdc9 mutation; these mutants are therefore incision defective. Low-molecular-weight DNA was observed in rad14 cdc9 and rad16 cdc9 strains. With the rad16 strain, the accumulation of low-molecular-weight DNA correlated with the amount of excision taking place, whereas in the rad14 mutant strain, no evidence of dimer removal was obtained. Therefore, rad14 is likely to be defective in a step after incision.
Temperature-sensitive mutation affecting synthesis of phosphoenolpyruvate carboxykinase in Escherichia coli
Authors: Goldie A H , Sanwal B D
Published: January 2020

A mutation has been characterized in Escherichia coli which results in temperature-sensitive expression of phosphoenolpyruvate carboxykinase activity and antigen. The enzyme produced by the mutant strain at a permissive temperature or by cells treated with chloramphenicol at nonpermissive temperatures had normal activity and stability in extracts. Since phosphoenolpyruvate carboxykinase had a monomeric structure, the mutation probably affects the synthesis, rather than the structure or assembly, of the enzyme.
Bilinear cell growth of Escherichia coli
Authors: Kubitschek H E
Published: January 2020

Recent electron micrograph measurements of bacterial dimensions in exponentially growing cultures of Escherichia coli support a model of bilinear increase in cell surface area and volume, with a sharp doubling in growth rate at a discrete age during the cell cycle. The results also indicate coordinate regulation of increase of surface area and volume.
Effects of recB21, recF143, and uvrD152 on recombination in lambda bacteriophage-prophage and Hfr by F- crosses
Authors: Howard-Flanders P , Bardwell E
Published: January 2020

The effects of the mutation pairs recB21 recF143 and recB21 uvrD152 on the frequency of genetic recombination were investigated in lambda phage-prophage crosses under homoimmune conditions. To prevent recombinants from being formed by the phage red system, these experiments were performed with phages and prophages carrying red and gam mutations. Both spontaneous and damage-induced recombination was measured, the phages being either undamaged or treated with trimethylpsoralen and 360-nm light to cross-link the phage DNA. Control and damaged phages were allowed to infect lysogenic host cells under conditions in which phage gene expression was repressed and phage DNA replication was blocked by lambda immunity. Although the double mutations recB21 recF143 and recB21 uvrD152 reduced recombination in Hfr by F- crosses to 0.3 to 0.02% of the wild-type controls, the presence of these pairs of mutations in the host lysogens had relatively little effect on the results of the phage-prophage crosses. In the latter system, recB21 recF143 reduced spontaneous and damaged-induced recombination by less than threefold whereas recB21 uvrD152 increased it to three times the wild-type level, the increase being attributable to the uvrD mutation. Evidently, the gene products of recB,C uvrD, and recF wee not needed for lambda phage-prophage recombination under repressed conditions.
Enhancement of ribosomal ribonucleic acid synthesis by deoxyribonucleic acid gyrase activity in Escherichia coli
Authors: Oostra B A , van Vliet A J , Ab G , Gruber M
Published: January 2020

The effect of the deoxyribonucleic acid (DNA) gyrase inhibitors coumermycin A1, novobiocin, and oxolinic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was studied in vivo and in vitro. Preferential inhibition of ribosomal RNA (rRNA) synthesis was observed. No effect of oxolinic acid and coumermycin on rRNA synthesis was seen in mutants having a DNA gyrase which is resistant to these inhibitors. In a temperature-sensitive DNA gyrase mutant rRNA synthesis was decreased at nonpermissive temperatures. Thus, a functional DNA gyrase is required for rRNA synthesis. Purified DNA gyrase had no effect on rRNA synthesis in a purified system. However, DNA gyrase does show preferential stimulation of rRNA synthesis in a system supplemented with other proteins. Apparently, DNA gyrase stimulation of rRNA synthesis requires another protein.
Tn9 and IS1 inserts in a ribosomal ribonucleic acid operon of Escherichia coli are incompletely polar
Authors: Brewster J M , Morgan E A
Published: January 2020

Transcription is known to be coupled to translation in many or all bacterial operons which code for proteins. In these operons, nonsense codons which prevent normal translation often result in premature termination of transcription (polarity). However, efficient transcription of ribosomal ribonucleic acid operons (rrn operons) occurs, although rrn transcripts are not translated. It therefore seemed possible that insertion sequences and transposable elements which are polar in protein-coding operons might not be polar in rrn operons. Previously, it has been shown (E. A. Morgan, Cell 21:257-265, 1980) that Tn10 is incompletely polar in the rrnX operon. Here we show that the transposon Tn9 and the insertion sequence IS1 also incompletely polar in rrnX. In normal cells expression of sequences distal to the insertions can be detected by genetic methods. In ultraviolet-irradiated cells expression of distal sequences is about 80% of that observed in uninterrupted rrnX operons. These observations provide evidence that ribonucleic acid polymerase molecules beginning at rrnX promoters can read through Tn9 and IS1 and that, at least in ultraviolet-irradiated cells, read-through is very efficient.
Regulation of amino acid transport in Thiobacillus thioparus
Authors: Stark A A , Yankofsky S A
Published: January 2020

Amino acid transport in amino acid auxotrophs of Thiobacillus thioparus was enhanced during growth on rate-limiting amino acid concentration. A pleiotropic mutation enhanced general amino acid transport as manifested by higher values of Vmax of amino acid transport. Affinity constants remained unaltered. Mutants with enhanced transport properties did not show changes in oxidation of thiosulfate, did not oxidize various organic compounds, and did not increase the heterotrophic potential of T. thioparus. The mutations for enhanced transport caused increased synthesis of amino acid transport system components. A method for genetic transformation of T. thioparus is described.
Relationship between temperature and growth rate of bacterial cultures
Authors: Ratkowsky D A , Olley J , McMeekin T A , Ball A
Published: January 2020

The Arrhenius Law, which was originally proposed to describe the temperature dependence of the specific reaction rate constant in chemical reactions, does not adequately describe the effect of temperature on bacterial growth. Microbiologists have attempted to apply a modified version of this law to bacterial growth by replacing the reaction rate constant by the growth rate constant, but the modified law relationship fits data poorly, as graphs of the logarithm of the growth rate constant against reciprocal absolute temperature result in curves rather than straight lines. Instead, a linear relationship between in square root of growth rate constant (r) and temperature (T), namely, square root = b (T - T0), where b is the regression coefficient and T0 is a hypothetical temperature which is an intrinsic property of the organism, is proposed and found to apply to the growth of a wide range of bacteria. The relationship is also applicable to nucleotide breakdown and to the growth of yeast and molds.
The Mistaken Identity of Colicin A
Authors: Luria S. E.
Published: January 2020

In a series of published articles, colicin A has been mistakenly labeled as colicin K.
Interaction of the maltose-binding protein with membrane vesicles of Escherichia coli
Authors: Richarme G
Published: January 2020

The interaction of the radioactively labeled purified maltose-binding protein of Escherichia coli with membrane vesicles was studied. The maltose-binding protein bound specifically to the vesicles, in the presence of maltose, on few sites. Under conditions in which a potential was imposed across the membrane, the specific binding was (i) increased, (ii) dependent on maltose, and (iii) abolished in a mutant defective in the tar gene product, one of the methyl-accepting chemotaxis proteins. At least 1,300 binding sites were present in the membrane fraction of logarithmically growing cells.
Effect of bacteriophage lambda infection on synthesis of groE protein and other Escherichia coli proteins
Authors: Drahos D J , Hendrix R W
Published: January 2020

We used two-dimensional gel electrophoresis to quantitate the changes in rates of synthesis that follow phage lambda infection for 21 Escherichia coli proteins, including groE and dnaK proteins. Although total protein synthesis and the rates of synthesis of most individual E. coli proteins decreased after infection, some proteins, including groE protein, dnaK protein, and stringent starvation protein, showed increases to rates substantially above their preinfection rates. Infection by lambda Q- affected host synthesis in the same way as infection by gamma+, whereas infection by lambda N- showed no detectable effect on host synthesis. Deletion of the early genes between att and N abolished the effect, and shorter deletions in this region gave intermediate effects. By this sort of deletion mapping, we show that a large part, though not all, of the effect of lambda infection on host protein synthesis can be ascribed to the early region that contains phage genes Ea10 and ral. We compared the changes in protein synthesis after infection with the changes that occur in uninfected cells upon heat shock or amino acid starvation. The spectrum of changes that occurred on infection was very different from that seen after heat shock but quite similar to that seen during amino acid starvation. Despite this similarity of the effects of lambda infection and starvation, we did not detect any increase in the level of guanosine tetraphosphate during infection. We show that the groE protein is the same protein as B56.5 of Lemaux et al. (Cell 13:427-434, 1978) and A protein of Subramanian et al. (Eur. J. Biochem. 67:591-601, 1976).
Nutrition and carbon metabolism of Methanococcus voltae
Authors: Whitman W B , Ankwanda E , Wolfe R S
Published: January 2020

Methanococcus voltae is a heterotrophic, H2-oxidizing methanogenic bacterium. In complex medium, this bacterium has a doubling time of 1.2 h at its temperature optimum of 38 degrees C. In defined medium, optimal growth is obtained with 0.75 mM isoleucine, 0.75 mM leucine, 2.5 mM acetate, 5 mM NH4Cl, 84 mM MgSO4, 0.4 M NaCl, 1 mM CaCl2, 10 microM Fe2O3, and 0.2 microM NiCl2. In addition, pantothenate, sodium selenate, and cobalt stimulate growth. Optimal growth is obtained between pH 6.0 and 7.0 with either H2 or formate as the electron donor. The volatile fatty acids 2-methylbutyrate and isovalerate can substitute for isoleucine and leucine, respectively. Cellular carbon is derived from acetate (31%), isoleucine (22%), leucine (25%), and carbon dioxide (23%). The amino acids and fatty acids are incorporated almost exclusively into protein. A comparison of the incorporation of U-14C-amino acids and 1-14C-fatty acids indicated that the fatty acids are degraded during incorporation into cell protein. The distribution of carbon from the amino acids suggests that acetyl coenzyme A is not a major intermediate in the degradation of these compounds. Thus, M. voltae may convert isoleucine and leucine to other amino acids by a unique mechanism. The lipid carbon is derived largely from acetate. Thus, the isoprenoid lipids are synthesized de novo from acetate rather than by degradation of leucine. The carbon in the nucleic acids is derived from carbon dioxide (45%), the C-1 of acetate (25%), the C-2 of acetate (22%), and isoleucine and leucine (7%). This labeling pattern is consistent with known biochemical pathways.
Properties of formate dehydrogenase in Methanobacterium formicicum
Authors: Schauer N L , Ferry J G
Published: January 2020

Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.
Altered expression of biodegradative threonine dehydratase in Escherichia coli mutants
Authors: Merberg D , Datta P
Published: January 2020

A number of strains of Escherichia coli K-12 failed to synthesize significant amounts of biodegradative threonine dehydratase (EC 4.2.1.16) when grown anaerobically in tryptone-yeast extract medium, a condition which is optimal for the induction of this enzyme. However, the addition of 10 mM potassium nitrate to the culture medium enabled a few of these strains, notably MB201, to induce the enzyme. An examination of the kinetic parameters, modifier sensitivity, and immunological cross-reactivity revealed that the enzyme produced by MB201 in nitrate-supplemented medium appeared indistinguishable from the dehydratase of a wild-type strain. The reduced expression of threonine dehydratase in MB201 appeared highly specific; the synthesis of two other inducible enzymes, D-serine deaminase and tryptophanase, and two "anaerobic" proteins, namely, fumarate reductase and cytochrome c551, remained unaffected. The mutation (tdcI) responsible for the altered expression of the dehydratase in MB201 was located at min 91 on the E. coli chromosome and appeared to tightly linked to if not identical with pgi, the gene encoding phosphoglucose isomerase, as judged by growth experiments on glucose and fructose, direct assay of phosphoglucose isomerase activity, spontaneous and simultaneous reversion of MB201 (tdcI) to TdcI+ and Pgi+ phenotype, and cosegregation of the two loci during transduction with P1 phage. Because not all strains lacking the dehydratase showed nitrate-dependent enzyme synthesis or had lesions at the pgi locus, it appears that mutations at multiple loci on the E. coli chromosome may influence the expression of the enzyme in vivo.
Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome
Authors: Olsen R H , DeBusscher G , McCombie W R
Published: January 2020

A host-vector system for Pseudomonas aeruginosa PAO was developed. Scattered regions of the strain PAO chromosome were cloned by direct selection for complementation of auxotrophs or from a DNA gene bank which contains over 1,000 independently isolated chromosome-vector recombinant plasmids. The use of partially digested chromosomal DNA facilitated the selection of a variety of strain PAO chromosomal markers. The progenitor of the vector was a small, multicopy plasmid, pRO1600, found in a PAO strain which had acquired RP1 in a mating experiment. The bacterial host range that could be determined by transformation of vectors produced from pRO1600 resembles that for plasmid RP1. Two derivative plasmids were formed: pRO1613, for cloning DNA cleaved with restriction endonuclease PstI, and pRO1614, which was formed by deleting part of pRO1613 and fusion with plasmid pBR322. Plasmid pRO1614 utilizes known cloning sites within the tetracycline resistance region of pBR322.
Construction and characterization of mutants impaired in the biosynthesis of the K88ab antigen
Authors: Mooi F R , Wouters C , Wijfjes A , de Graaf F K
Published: January 2020

Plasmid pFM205 contains the genetic determinant for the K88ab antigen and is composed of a 4.3-megadalton DNA fragment derived from wild-type K88ab plasmid pRI8801 and cloning vehicle pBR322. The K88 NA of pFM205 contains five genes, which code for polypeptides with apparent molecular weights of 17,000, 26,000 (the K88ab subunit), 27,000 27,500, and 81,000. All five polypeptides were synthesized as precursors approximately 2,000 daltons larger than the mature polypeptides, indicating that they are transported across the cytoplasmic membrane by means of a signal sequence. A set of deletion derivatives of pFM205 was constructed, each containing a deletion in one of the five genes. In strains harboring derivatives of pFM205 containing a deletion in the gene for the 17,000- or 81,000-dalton polypeptide, the K88ab subunit was synthesized and transported to the outside of the cell. However, these strains did not adhere to brushborders or guinea pig erythrocytes, suggesting that the K88ab subunits were not assembled into normal fimbriae. Strains harboring plasmids containing a deletion in the gene for the 27,500-dalton polypeptide still adhered to brush borders and guinea pig erythrocytes, although very little K88ab antigen could be detected with an immunological assay. In strains harboring plasmids containing a deletion in the gene for the 27,000-dalton polypeptide, the K88ab subunit was synthesized but was probably subsequently degraded rapidly.
Evidence for a Second Nitrate Reductase Activity That Is Distinct from the Respiratory Enzyme in
Salmonella typhimurium
Authors: Barrett Ericka L. , Riggs Daniel L.
Published: January 2020

Significant nitrate reductase activity was detected in mutants of
Salmonella typhimurium
which mapped at or near
chlC
and which were incapable of growth with nitrate as electron acceptor. The same mutants were sensitive to chlorate and performed sufficient nitrate reduction to permit anaerobic growth with nitrate as the sole nitrogen source in media containing glucose. The mutant nitrate-reducing protein did not migrate with the wild-type nitrate reductase in polyacrylamide electrophoretic gels. Studies of the electrophoretic mobility in gels of different polyacrylamide concentration revealed that the wild-type and mutant nitrate reductases differed significantly in both size and charge. The second enzyme also differed from the wild-type major enzyme in its response to repression by low pH and its lack of response to repression by glucose. The same mutants were found to be derepressed for nitrite reductase and for a cytochrome with a maximal reduced absorbance at 555 nm at 25°C. This cytochrome was not detected in preparations of the wild type grown under the same conditions. Extracts of these mutants contained normal amounts of the
b
-type cytochromes which, in the wild type, were associated with nitrate reductase and formate dehydrogenase, respectively, although they could not mediate the oxidation of these cytochromes with nitrate. They were capable of oxidizing the derepressed 555-nm peak cytochrome with nitrate. It is suggested that these mutants synthesize a nitrate-reducing enzyme which is distinct from the
chlC
gene product and which is repressed in the wild type during anaerobic growth with nitrate.
Control of the actinomycin biosynthetic pathway in and actinomycin resistance of Streptomyces spp
Authors: Ochi K
Published: January 2020

Using actinomycin-producing and nonproducing strains of Streptomyces antibioticus, I studied several steps in the biosynthetic pathway of this antibiotic. Actinomycin-nonproducing strains derived after acriflavine or novobiocin treatment showed activity of kynurenine formamidase and phenoxazinone synthase as high as that of the parental strain, but these nonproducing strains failed to convert 4-methyl-3-hydroxy-anthranilic acid to actinomycin. In addition, accumulation of 4-methyl-3-hydroxyanthranilic acid (in the presence of D-valine) was not detected in the nonproducing isolates. Actinomycin-nonproducing strains derived after acriflavine treatment of Streptomyces parvulus showed a drastic decrease of resistance to the antibiotic. However these strains regained resistance after preincubation with a small amount of actinomycin D.
L-serine degradation in Escherichia coli K-12: directly isolated ssd mutants and their intragenic revertants
Authors: Newman E B , Malik N , Walker C
Published: January 2020

Two methods for the direct isolation of spontaneous ssd mutants of Escherichia coli K-12 strains are described; (i) by growth with L-serine as the carbon source, and (ii) by low-level kanamycin resistance. A newly isolated mutant had the same phenotype as the mutant described previously, including inefficient use of the glucose, inability to growth with succinate, altered transport characteristics, and altered resistance to certain growth effectors. Succinate-utilizing derivatives which appear to be intragenic are characterized in detail. The relation between the mutants isolated here and mutants which are thought to have impairment in a system of coupling respiratory energy to active transport (ecfB mutants) is discussed.
Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies
Authors: Horinouchi S , Weisblum B
Published: January 2020

pE194 is a small plasmid (isolated originally in Staphylococcus aureus) which confers erythromycin-inducible resistance to macrolide, lincosamide, and streptogramin type B (MLS) antibiotics. The nucleotide sequence of pE194 contains 3,728 base pairs (bp), corresponding to a molecular mass of 2.4 million daltons. By means of site-specific cleavage with restriction endonucleases and cloning resultant fragments, determinants of the two major biological functions of p E194, i.e., inducible MLS resistance and replication, could be localized and assigned to specific sequences in the plasmid. Restriction endonuclease TaqI cut pE194 at three sites. TaqI fragment A (1,443 bp) contained the determinant for inducible MLS resistance, whereas TaqI fragment B (1,354 bp) contained a determinant necessary for plasmid replication. Regulatory mutations resulting in constitutive expression of MLS resistance mapped in TaqI fragment A, whereas a mutation associated with elevated plasmid copy number was mapped in TaqI fragment B. Also mapping in TaqI fragment B was a plasmid replication determinant comprising two sets of inverted complementary repeat sequences, one of which spanned 124 bp and was adjacent to a second smaller set which was rich in guanine and cytosine residues. pE194 contained six open reading frames which were theoretically capable of coding for proteins with maximum molecular masses as follows (in daltons): A, 48,300; B, 29,200; C, 14,000; D, 13,900; E, 12,600; and F, 2,700. Insertion of plasmid pBR322 into the single PstI site located in frame A of pE194 resulted in a composite plasmid which could replicate in both Bacillus subtilis and Escherichia coli, suggesting that an intact polypeptide A is dispensable for both replication of pE194 and for MLS resistance. Frame B specified inducible MLS resistance, whereas frame F specified the putative peptide associated with the proposed B determinant translational attenuator. The extent to which frames C, D, and E, all contained in TaqI fragment B, were translated into polypeptide products is not known; however, a base change in frame E was found in a comparison between the high-copy-number mutant, cop-6, and the wild-type strains.
Isolation and characterization of cytoplasmic membranes and chlorosomes from the green bacterium Chloroflexus aurantiacus
Authors: Feick R G , Fitzpatrick M , Fuller R C
Published: January 2020

A method was developed which allows the isolation and purification of cytoplasmic membranes and chlorosomes from cells of Chloroflexus aurantiacus grown under different light conditions. The dipolar ionic detergent Deriphat (0.08%) and a sodium iodide gradient centrifugation were used in isolating cytoplasmic membranes. Chlorosomes were prepared with 0.16% of the dipolar ionic detergent Miranol and purified by a sucrose gradient centrifugation. Cytoplasmic membrane fractions prepared from either high- (3,000 W m-2), medium-(200 W m-2) or low- (7 W m-2) light-grown cells had near infrared absorption bands at 866, 808, and 755 nm in a constant characteristic absorbance ratio of 6:3.8:1. In all cytoplasmic membrane preparations, the amount of bacteriochlorophyll a (Bchl a) per cytochrome, the amount of Bchl a per reaction center, and reaction center per milligram of cytoplasmic membrane protein was found to be constant. No Bchl c was present. Five respiratory enzyme activities have been measured in the cytoplasmic membrane fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of denatured cytoplasmic membrane showed many bands, but a major polypeptide with an apparent molecular weight of 8,000. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified chlorosomes did not contain the 8,000-molecular-weight band but revealed only three distinct protein bands with molecular weights of 15,000, 12,000, and 6,000. Isolated chlorosomes contained Bchl c and a small, yet constant, amount of Bchl a (absorbing at 790 nm) in a molar ratio of 25:1. The data indicated that the components of the photosynthetic apparatus in the cytoplasmic membrane of Chloroflexus aurantiacus remained constant and only the amount of antenna Bchl c varied with light conditions.
Genetic mapping of ada and adc mutations affecting the adaptive response of Escherichia coli to alkylating agents
Authors: Sedgwick B
Published: January 2020

The adaptive response to alkylating agents is an inducible repair system which protects Escherichia coli against the mutagenicity and toxicity of these agents. Four mutations, ada-3, ada-5, ada-6, and adc-1, which confer differing phenotypes as regards this response, were shown to be cotransducible with gyrA, and were located at 47 min on the E. coli genetic map. A mutation already shown on the map at 47 min as tag (B. J. Bachmann and K. B. Low, Microbiol. Rev. 44:1--56, 1980; Karran et al., J. Mol. Biol. 140:101--127, 1980) is now known to be an ada mutation (G. Evensen and E. Seeberg, personal communication).
Use of in vitro gene fusions to study the uxuR regulatory gene in Escherichia coli K-12: direction of transcription and regulation of its expression
Authors: Ritzenthaler P , Mata-Gilsinger M
Published: January 2020

The uxuAB operon is composed of two genes coding for enzymes involved in hexuronate degradation. This operon is negatively controlled by the uxuR and exuR regulatory gene products. Fusions that brought lac gene expression under the control of transcriptional and translational signals within the uxuR gene were used to study uxuR regulation. These fusions were formed on plasmid cloning vectors constructed by Casadaban et al. (J. Bacteriol. 143:971-980, 1980). The transcriptional direction of the uxuR gene was deduced from the restriction pattern and the phenotypic properties of the new plasmids. The gene is transcribed counterclockwise on the standard Escherichia coli map, as is the uxuAB operon. Introduction in trans of a compatible plasmid carrying a wild-type uxuR gene in the lac fusion plasmid containing strain resulted in a decrease of beta-galactosidase synthesis. It was concluded that expression of the uxuR gene itself is repressed by its own product. The two other types of regulation found in the uxuAB operon, i.e., induction by fructuronate and catabolite control, also apply to the uxuR gene, whereas repression by the exuR repressor does not seem to occur for the uxuR gene.
Protein H encoded by plasmid CloDF13 is involved in excretion of cloacin DF13
Authors: Oudega B , Stegehuis F , van Tiel-Menkveld G J , de Graaf F K
Published: January 2020

Excretion of cloacin DF13 was studied in Escherichia coli cells harboring different CloDF13 insertion and deletion mutant plasmids. Insertions of a transposon at position 9.8 or 11.5% of the CloDF13 plasmid blocked the expression of gene H and strongly reduced the specific excretion of cloacin DF13 into the culture medium, but had no effect on the production of cloacin DF13. Insertions in or deletions of regions of the CloDF13 DNA upstream the cloacin operon did not affect the excretion or production of the bacteriocin. Introduction of a CloDF13 plasmid that encodes for the gene H product in cells harboring a CloDF13 plasmid with an insertion in gene H stimulated the excretion of cloacin DF13 significantly in mitomycin C-induced and in noninduced cultures. Cloacin DF13 in cloacinogenic cells that did not produce the gene H protein was found to be about 90% located in the cytoplasm. In cells that did produce the gene H product, about 30% of the cloacin DF13 molecules were found in the cytoplasm, about 18% were found in the periplasm, about 2% were in the membranes, and about 50% were located in the culture supernatant. Cyclic AMP stimulated the production but not the excretion of cloacin DF13 in cells cultivated in the presence of glucose.
Regulation of phenylalanine biosynthesis in Escherichia coli K-12: control of transcription of the pheA operon
Authors: Gowrishankar J , Pittard J
Published: January 2020

Bacteriophage lambda ppheA-lac was used to obtain strains of Escherichia coli K-12 in which pheA and lacZ are each transcribed from a separate pheA promoter. Mutants in which both beta-galactosidase and chorismate mutase P-prephenate dehydratase (the pheA gene product) were derepressed were isolated, and a transacting gene (pheR) was identified. pheR was mapped at min 93 on the E. coli chromosome; pheR mutants acquired the wild-type phenotype when either F117 (which covers the 93-min region) or F116 (which covers min 59 to 65) was introduced into the cell. A rifampin resistance mutation, rpoB366, was found to derepress transcription of the pheA operon. pheR and rpoB366 affected two different systems for the phenylalanine-mediated control of pheA. A mutation in miaA (trpX), a gene known to be involved in attenuation in the tryptophan operon, was also shown to increase transcription of the pheA gene.
Development and growth of photosynthetic membranes of Rhodospirillum rubrum
Authors: Inamine G S , Niederman R A
Published: January 2020

In cell-free extracts from low-aeration suspensions of Rhodospirillum rubrum strain G-9, bacteriochlorophyll a was distributed in two bands after rate-zone sedimentation in sucrose density gradients. From the physicochemical properties of these fractions, it was concluded that the upper band consisted of small membrane fragments, whereas the major band was composed of fragmented vesicular intracytoplasmic membrane (chromatophores). After a pulse with L-[35S]methionine, apparent polypeptide subunits of the reaction center and light-harvesting complexes within the upper pigmented fraction were labeled more rapidly than those of chromatophores; after a chase with excess unlabeled L-methionine, radioactivity from these components within the upper band appeared to be chased into the corresponding polypeptides of chromatophores. These labeling patterns are interpreted to reflect growth initiation and maturation of the photosynthetic apparatus and may, in part, represent a general mechanism for the development of vesicular intracytoplasmic membranes.