Complete amino acid sequence of the coat protein of the Pseudomonas, aeruginosa RNA bacteriophage PP7

As part of a project intending to assess the evolutionary kinship between the RNA coliphages and RNA bacteriophages of other bacterial genera, we have sequenced the coat protein of $\text{Pseudomonas}\text{,}\text{aeruginosa}$ RNA phage PP7. Like the coat proteins of coliphages MS2 and Qβ and of the broad host range RNA phage PRR1, PP7 coat protein (127 residues) is highly hydrophobic, and contains a cluster of basic residues between positions 40 to 60. Minimal mutation distance values were calculated for comparison of PP7 coat protein with each MS2, Qβ and PRR1 coat proteins. Application of the Moore-Goodman criterion to those values, shows that these four RNA bacteriophage coat proteins very likely descent from a common ancestor.     

A mutation affecting the valine sensitivity of the acetohydroxyacid synthase III isoenzyme in E. coli K-12

The $\text{ilv-751}$ mutation (obtained by $\text{mu}$ phage mediated mutagenesis) affects the sensitivity to valine inhibition of the acetohydroxy acid synthase III isoenzyme of $\text{E. coli}$ K-12, as shown by constructing multiple mutants containing the $\text{ilv-751}$ mutation and only one of the genes for the expression of the three acetohydroxy acid synthase isoenzymes, at once. The mutation is dominant. This suggests that the phenotype of $\text{ilv-751}$ mutation is not caused by inactivation of a gene concerned with the expression of the AHASIII enzyme, consequent to prophage insertion into that locus.     

Hyperproductivity of extracellular α-amylase by a tunicamycin resistant mutant of Bacillussubtilis

Several tunicamycin resistant mutants were obtained from $\text{Bacillus}\text{subtilis}$ NA64. One of them, B7 strain produced a 5-fold larger amount of α-amylase than NA64 did. Only the amount of α-amylase, among excreted proteins, was enhanced. Genetic analyses by transformation suggested that a single mutation in B7 induced both resistance to tunicamycin and hyperproductivity of extracellular α-amylase.     

Repair of deaminated cytosine residues of DNA: biological significance of the absence of uracil from DNA.

Uracil-DNA glycosylase of $\text{Bacillus}\text{subtilis}$ is involved in repair of deaminated cytosine residues of DNA. Survivals of SPO2 phage after treatment with bisulfite and weak alkali are considerably higher in wild type strains than in $\text{urg}$ mutants, which are deficient in the enzyme activity, whereas survivals of bisulfite/alkali-treated PBS1 phage in the two types of cells are essentially the same. The spontaneous mutation frequency of a $\text{urg}$ mutant is three fold higher than is that of a wild type strain.     

Prophage P2 does not kill recB bacteria.

$\text{Escherichia}\text{coli}$ carrying a temperature-sensitive $\text{recB}$ mutation and lysogenic for phage P2 was able to grow normally even at 42°C, at which temperature the bacteria are phenotypically $\text{recB}{}^{\mathrm{?}}$. At this temperature, the bacteria were, however, unable to support the growth of λspi^? phages.     

Identification of the sid outer membrane receptor protein in Salmonella typhimurium SL1027.

Summary A protein of molecular weight 78,000 daltons, missing in albomycin and phage ES18 resistant mutants, has been identified in the outer membrane of Salmonella typhimurium SL1027. Mutants with a tonB like resistance and overproduction of outer membrane proteins due to iron shortage were also isolated. The mutation which leads to the protein deficiency maps in the sid gene region, the mutation related to overproduction of proteins maps near trp. Although the S. typhimurium and the E. coli protein mediate translocation of the iron complex ferrichrome and the structurally analogous antibiotic albomycin through the outer membrane no cross-reactivity exists in binding the phages T5, T1 and ES18 or colicin M.     

Transduction in Bacillus megaterium.

A bacteriophage, MP13, isolated from the soil on $\text{B. megaterium}$ QM B1551 has been found to transduce several auxotrophic markers. Transduction required inactivation of the phage to approximately 0.01% survival with UV light and it was enhanced by the absence of salts that are probably necessary for phage readsorption.     

Membrane mutation related to the production of extracellular -amylase and protease in bacillus subtilis

Mutants which had a genetic character to increase the production of both α-amylase and protease simultaneously, were isolated from a transformable strain of $\text{Bacillus}$$\text{subtilis}$ Marburg by NTG treatment. This mutation seems to have occurred at a single gene of the bacterial chromosome and was not linked to $\text{aro}$₁₁₆ which was closely linked to the α-amylase gene. When this mutation and an α-amylase regulator gene ($\text{amyR}$^h) coexisted in one strain, their synergistic effect on extracellular α-amylase production ws observed. The introduction of this mutation resulted in a loss of competence for the transformation. The SDS disc gel electrophoretic profiles of the membrane proteins from the original strain, the mutants and transformants with this mutation showed a remarkable difference in one component.     

Saccharomyces cerevisiae killer expression mutant kex2 has altered secretory proteins and glycoproteins.

The extracellular proteins and glycoproteins of a yeast mutant $\text{kex}\text{2–15}$ defective in killer toxin expression were separated by one and two dimensional polyacyylamide gel electrophoresis. Many mutant extracellular proteins and glycoproteins show both altered electrophoretic mobility and isoelectric points when compared with the parent strain. Altered proteins and glycoproteins from $\text{kex}\text{2–15}$ were identified with their parental counterparts by peptide mapping. The observed alterations co-segregated with the $\text{kex}\text{2}$ nuclear mutation in genetic crosses.     

Template specificity changes of DNA-dependent RNA polymerase in B. subtilis during sporulation

Previous work has indicated that loss of ability of DNA dependent RNA polymerase, from stationary phase cultures of $\text{B. subtilis}$, to transcribe phage øe DNA was a $\text{sine qua non}$ for sporulation. To ascertain if this change in template specificity was sporulation-specific, we repeated these experiments using a defined sporulation medium. The changes observed previously did not occur in the defined medium although sporulation was normal. The ability of the enzyme to transcribe other DNA templates was also examined. Similar studies were carried out using a polymerase from a rifamycin-resistant, sporulation conditional mutant. The significance of these findings with regard to the regulation of sporulation in $\text{B. subtilis}$ is discussed.     

The role of the lysines in the alkaline heme-linked ionization of ferric cytochrome c.

Phage ?¹⁵ adsorbed at a low temperature (or by short-time incubation) to the outer surface of $\text{Salmonella}\text{anatum}$ gathers on further incubation at a high temperature to a certain region where the inner and outer membranes may join. This was demonstrated by separating the inner and outer membranes of the cells in sucrose gradient after addition of ³⁵S-labeled ?¹⁵ to the cells. Radioactivity adsorbed at 4° was first recovered mainly with the dense outer membrane but disappeared by further incubation at 35° within 5 min. Instead, the radioactivity was recovered with the membrane fraction which had intermediate density. Such phage translocation was not observed when phage ?¹⁵ was added to a $\text{pep}\text{mutant of}\text{S.}\text{anatum}$ to which the phage can adsorb but fail to infect. A host range mutant phage which can infect the $\text{pep}$ mutant migrated to the intermediate dense region.     

The separation of phage promoter from bacterial lac promoter for beta-galactosidase expression in transducing phage lambda plac5.

The replication defective transducing phage λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 carries a portion of the $\text{E.}\text{coli}\text{lac}$ operon in the $\text{b}$2 region of the lambda phage. This $\text{lac}$ operon segment contains the $\text{lac}$ promoter, the $\text{lac}$ operator, and the β-galactosidase $\text{z}$ gene, but does not contain the $\text{lac}$ repressor $\text{i}$ gene. The $\text{z}$ gene can be expressed from both the inserted $\text{lac}$ promoter and the phage promoter. When $\text{E.}\text{coli}$ strain 594 ($\text{z}$^?, $\text{i}$⁺) or JC6256 (Δ$\text{lac}$) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the absence of additional cyclic AMP, β-galactosidase synthesis is shown to be expressed from the phage promoter. When 594 (λ⁺) or JC6256 (λ⁺) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the presence of additional cyclic AMP and IPTG, β-galactosidase synthesis is shown to be expressed from the inserted $\text{lac}$ promoter.The ability to separate the phage promoter from the inserted $\text{lac}$ promoter for β-galactosidase expression will simplify the interpretation whenever λp$\text{lac}$5 is used.     

Properties and structure of a gene 24-controlled T4 giant phage

Giant T4 bacteriophage were found by Doermann et al. (1973a) with point mutants in gene 23 and by Cummings et al. (1973) after l-canavanine induction followed by an arginine chase. We now find T4 giant phage with 14 out of 15 tested temperature-sensitive mutants in gene 24 grown at intermediate temperatures between 33 °C and 37 °C.For one of these mutants, T4,24(tsB86), we found that (a) the optimum temperature for giant phage production is 34.8 °C, (b) the head-length distribution peaks sharply between 10 and 12 normal T4 phage head lengths, (c) about 75% of our giant phage have two tails, (d) the buoyant density in CsCl is greater than that of normal phage, (e) they are infectious and show an increased u.v. resistance, (f) their sodium dodecyl sulphate gel electrophoresis pattern is qualitatively similar to that of normal T4 phage, although the relative intensities of some of the bands are different, showing for example, a decreased $\text{P24}{}^1\text{P23}{}^1$² ratio, (g) optical diffraction and filtering of the flattened cylindrical part of the giant heads show a p6 surface net with a lattice constant of approximately 130 Å, a unique $\text{u}\text{v}$ ratio of $\text{15}\text{5}$ and a capsomer morphology of the type 1 + 6 + 6.Mixed infections with T4 wild type and T4.24(amN65) also yield giant phage. These are produced in highest amounts with a multiplicity of infection ratio of 5:5; no giants are observed at ratios of 1:9 or 9:1, suggesting that their formation may be caused by a dosage effect of P24.     

The argECBH-bearing EcoRI segment of the Escherichia coli chromosome

The transducing phage λd$\text{arg}\text{14}$, carrying a portion of the $\text{E. coli}$ chromosome including $\text{argECBH}$, is derived from the heat-inducible, lysis-defective strain λy199, which has the $\text{b}\text{519}$ and $\text{b}\text{515}$ deletions. Cleavage of λy199 DNA by $\text{Eco}$RI endonuclease, followed by agarose slab gel electrophoresis, results in bands corresponding to the known C, D, E, and F segments of λ, and a segment A′ (A plus B minus $\text{b}\text{519}$ minus $\text{b}\text{515}$, the cleavage site between A and B being eliminated). Cleavage of λd$\text{arg}\text{14}$ DNA by $\text{Eco}$RI yields the expected D, E, and F segments of λ and four other segments, termed 14-1 through 14-4, whose length is 17.5, 6.2, 3.0, and 2.0 kilobases, respectively, as determined by electron microscopy and corroborated by electrophoretic mobility. Heteroduplex analysis shows that the $\text{E. coli argECBH}$ cluster is on the 14-1 segment.     

Affinity chromatography of thermolysin and of neutral proteases from B. subtilis

Affinity chromatographic systems are described for the purification of neutral metalloendopeptidases on columns of acetyl-$\text{D}$-phenylalanine or succinyl-$\text{D}$-leucine covalently linked to Sepharose by spacers of various lengths. The neutral proteases of $\text{B. subtilis}$ are separated in a single chromatographic procedure from all other proteins of the culture filtrates and subfractionated into two active species. An analogous chromatographic system is effective in the purification of thermolysin of $\text{B. thermoproteolyticus}$.     

Diffusion of tetracycline across the outer membrane of Escherichia coli K-12: involvement of protein Ia.

The possibility that passage of tetracycline across the outer membrane of $\text{E.}\text{coli}$ K-12 is controlled by one or more of the proteins I_a, I_b and II¹ (Henning's nomenclature) was investigated. A mutant lacking protein I_a (obtained by selection for resistance to phage TuI_a) was more resistant to tetracycline than wild-type strains or those lacking only proteins I_b or II¹. The envelope protein composition of a tetracycline-resistant mutant ($\text{cmlB}$) was altered in several respects, but the major change involved loss of protein I_a. These data support our previous suggestion [12] that tetracycline diffuses across the outer membrane through hydrophilic regions. Furthermore, they imply that only protein I_a plays a significant role in the passage of this antibiotic across the outer membrane.     

Synthesis of quinolinate from D-aspartate in the mammalian liver-Escherichia coli quinolinate synthetase system.

Two proteins (A and B) from $\text{Escherichia coli}$ are required for the synthesis of the NAD precursor quinolinate from aspartate and dihydroxyacetone phosphate. Mammalian liver contains a FAD linked protein which replaces $\text{E. coli}$ B protein for quinolinate synthesis. D-aspartic acid but not L-aspartic acid is a substrate for quinolinic acid synthesis in a system composed of the B protein replacing activity of mammalian liver and $\text{E. coli}$ A protein. In contrast the $\text{E. coli}$ B protein-$\text{E. coli}$ A protein quinolinate synthetase system requires L-aspartic acid as substrate. The previous report that L-aspartate was a substrate in the liver-$\text{E. coli}$ system was due to contamination of commercially available [¹⁴C]L-aspartate with [¹⁴C]D-aspartate. These and other observations suggest that liver B protein is D-aspartate oxidase and $\text{E. coli}$ B protein is L-aspartate oxidase.     

Carbon monoxide and hydroxymercuribenzoate sensitivity of a fatty acid (omega-2) hydroxylase from Bacillus megaterium.

DNA-free minicells of $\text{Escherichia coli}$ will not allow growth of phage T-7, nor is RNA synthesis stimulated by phage infection. Thus, these miniature cells seem not to contain $\text{in vivo}$ RNA polymerase activity. However, DNA-dependent RNA polymerase activity can be unmasked in extracts with poly(dA-T) and Mn²⁺. This activity may represent a cytoplasmic pool of inactive RNA polymerase in normal cells.     

Identification of a phi X174 coded protein involved in the inhibition of beta-galactosidase synthesis in Escherichia coli

The synthesis of β-galactosidase (EC 3.2.1.23: β-D-galactoside galactohydrolase) in $\text{Escherichia}\text{coli}$ is repressed as a result of infection with single-stranded DNA phage ØX174. An $\text{amber}$ mutant in ØX174 cistron A, which codes for two proteins, does not inhibit the enzyme synthesis while $\text{amber}$ mutants in all other genes do cause repression. A mutant near the amino-terminal end of cistron A, which produces the small 35,000 molecular weight cistron A polypeptide, also inhibits the synthesis of β-galactosidase. Inhibition is also observed in an $\text{Escherichia}\text{coli}\text{rep}$ mutant which does not support the replication of replicative-form DNA. Exogenous nucleotide bases and cyclic 3′,5′-adenosine monophosphate (cyclic AMP) do not have any effect on the degree of repression.