The effect of parabens on DNA, RNA and protein synthesis in Escherichia coli and Bacillus subtilis

The effect of methyl, propyl and butyl esters of p -hydroxybenzoic acid on DNA and RNA synthesis has been tested in toluenized cells of Escherichia coli and Bacillus subtilis. Both RNA and DNA synthesis of these bacteria were inhibited. The inhibitory concentrations were higher than those previously reported for growth inhibition. Protein synthesis in cell-free extracts (S-30 fraction) of B. subtilis was even more sensitive to parabens than DNA and RNA synthesis, while protein synthesis in Esch. coli was largely unaffected.     

Conserved portion in bacterial ribosomal RNA

The conserved portion in bacterial ribosomal RNA was studied by the DNA-RNA hybridization method. The hybridization percentages were as follows: Bacillus subtilis DNA and B. subtilis 23S rRNA, 0.16; Escherichia coli DNA and E. coli 23S rRNA, 0.15; B. subtilis DNA and E. coli 23S rRNA, 0.03; E. coli DNA and B. subtilis 23S rRNA, 0.04. The RNA's extracted from the heterologous hybrids could be rehybridized with DNA's of B. subtilis and E. coli. The average chain lengths of the RNA's were estimated by sucrose density gradient centrifugation and Sephadex gel filtration. The results suggested that the size might be larger than 30 nucleotides. Nucleotide compositions of the RNA's in the hybrids were also studied. Both RNA's contained higher molar percentages of guanylic acid and cytidylic acid than the whole rRNA's.     

Initiation of Bacillus subtilis Ribonucleic Acid Polymerase on Deoxyribonucleic Acid from Bacteriophages 2C, φ29, T4, and Lambda

Ribonucleic acid (RNA) synthesis primed by bacteriophage T4 or lambda deoxyribonucleic acid (DNA) with Bacillus subtilis RNA polymerase is severely inhibited by high ionic strength. In contrast, RNA synthesis on B. subtilis bacteriophage 2C, SPO1, or phi29 DNA is only moderately affected under similar conditions. The basis of this inhibition lies in the inability of the enzyme to initiate RNA chains with adenosine triphosphate or guanosine triphosphate (ATP, GTP). Binding to templates and the rate of catalysis in high salt after initiation do not seem to be affected. Incorporation of gamma-(32)P-ATP and GTP under a variety of conditions suggests that the specificity of B. subtilis RNA polymerase is different from that of the Escherichia coli enzyme and that it recognizes few promoters on T4 and lambda DNA. Although B. subtilis RNA polymerase initiates RNA chains primarily with ATP or GTP, initiations with pyrimidines can occur on DNA molecules in which hydroxymethyluracil replaces thymine. RNA synthesis on denatured DNA does not seem to be inhibited by high ionic strength, and on native T4 or lambda DNA the inhibition of initiation at constant ionic strength is inversely but not linearly proportional to the ionic radii of cations used to stabilize bihelical DNA to denaturation.     

Actinomycin Analogues Containing Pipecolic Acid: Relationship of Structure to Biological Activity

Streptomyces antibioticus synthesizes a mixture of actinomycins which differ at the "imino acid" site of the peptide chains. In the presence of exogenous pipecolic acid, several new actinomycins were synthesized and 70% of the proline in the antibiotic mixture was replaced by the analogue. Three new antibiotics (designated Pip 1alpha, Pip 1beta, and Pip 2) were isolated from culture filtrates, purified, and crystallized. The molar ratio of pipecolic acid to proline was: Pip 1alpha, 1:0; Pip 1beta, 1:1; Pip 2, 2:0. These compounds inhibited the growth and cell division of gram-positive, but not gram-negative, bacteria. The relative inhibitory activity against bacteria, Escherichia coli deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase in vitro, and RNA synthesis in Bacillus subtilis and mouse L-929 cells was: actinomycin IV = Pip 1beta > Pip 2 > Pip 1alpha. Protein synthesis in B. subtilis was less affected, and DNA synthesis was inhibited only at higher concentrations of antibiotic tested. In L cells, DNA formation was reduced less than RNA synthesis, whereas protein synthesis was not blocked under the experimental conditions employed. Kinetic studies with B. subtilis revealed that RNA synthesis was inhibited rapidly followed by an inhibition of protein synthesis. All four antibiotics markedly inhibited the replication of vaccinia virus and reovirus in tissue culture cells, but the production of poliovirus was resistant to the antibiotics. These actinomycins bind to DNA, resulting in an elevation of its T(m) and a decrease in the peak extinction of the actinomycins. The mode of action, as well as the structure-activity relationships among the actinomycins, are discussed relative to a previously proposed model of binding.     

Small cytoplasmic RNA of Bacillus subtilis: functional relationship with human signal recognition particle 7S RNA and Escherichia coli 4.5S RNA.

Small cytoplasmic RNA (scRNA; 271 nucleotides) is an abundant and stable RNA of the gram-positive bacterium Bacillus subtilis. To investigate the function of scRNA in B. subtilis cells, we developed a strain that is dependent on isopropyl-beta-D-thiogalactopyranoside for scRNA synthesis by fusing the chromosomal scr locus with the spac-1 promoter by homologous recombination. Depletion of the inducer leads to a loss of scRNA synthesis, defects in protein synthesis and production of alpha-amylase and beta-lactamase, and eventual cell death. The loss of the scRNA gene in B. subtilis can be complemented by the introduction of human signal recognition particle 7S RNA, which is considered to be involved in protein transport, or Escherichia coli 4.5S RNA. These results provide further evidence for a functional relationship between B. subtilis scRNA, human signal recognition particle 7S RNA, and E. coli 4.5S RNA.     

Purification and properties of a new bacillus subtilis RNA processing enzyme. Cleavage of phage SP82 mRNA and Bacillus subtilis precursor rRNA

An RNA processing activity capable of cleaving Bacillus subtilis phage SP82 early mRNA has been purified to apparent homogeneity from crude extracts of uninfected B. subtilis. The enzyme, a functional monomer of Mr approximately 27,000, cleaves only at the 5' side of adenosine residues at processing sites and is competitively inhibited by double-stranded synthetic RNA polymers. Processed SP82 mRNAs were translated in an Escherichia coli cell-free system and no qualitative or quantitative effects of processing on the synthesis of polypeptides was observed. The processing enzyme does not cleave T7 mRNA, E. coli precursor rRNA, or double-stranded poly(AU). A recombinant plasmid containing portions of two B. subtilis rRNA gene sets was transcribed in vitro and the resulting RNA was cleaved in the spacer region between the 16 S and 23 S rRNA genes. The ability of the B. subtilis processing enzyme to cleave SP82 mRNA and B. subtilis precursor rRNA and the fact that the enzyme has high affinity for double-stranded RNA suggest that it is the functional analog of E. coli RNase III.     

Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spore DNA.

Small, acid-soluble proteins (SASP) of the alpha/beta-type are associated with DNA in spores of Bacillus subtilis. Induction of synthesis of alpha/beta-type SASP in Escherichia coli resulted in rapid cessation of DNA synthesis, followed by a halt in RNA and then protein accumulation, although significant mRNA and protein synthesis continued. There was a significant loss in viability associated with SASP synthesis in E. coli: recA+ cells became extremely long filaments, whereas recA mutant cells became less filamentous. The nucleoids of cells with alpha/beta-type SASP were extremely condensed, as viewed in both light and electron microscopes, and immunoelectron microscopy showed that the alpha/beta-type SASP were associated with the cell DNA. Induction of alpha/beta-type SASP synthesis in E. coli increased the negative superhelical density of plasmid DNA by approximately 20%; UV irradiation of E. coli with alpha/beta-type SASP gave reduced yields of thymine dimers but significant amounts of the spore photoproduct. These changes in E. coli DNA topology and photochemistry due to alpha/beta-type SASP are similar to the effects of alpha/beta-type SASP on the DNA in Bacillus spores, further suggesting that alpha/beta-type SASP are a major factor determining DNA properties in bacterial spores.     

Regulation of 6S RNA by pRNA synthesis is required for efficient recovery from stationary phase in E. coli and B. subtilis

6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ(70) during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ(70) to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.     

Organization of transfer and ribosomal ribonucleic acid genes in Bacillus subtilis.

The structural relationship between the transfer ribonucleic acid (tRNA) and the ribosomal RNA (rRNA) genes of Bacillus subtilis has been studied by restriction endonuclease analysis of total chromosomal deoxyribonucleic acid (DNA) and characterization of DNA fragments cloned in Escherichia coli. The DNA sequences encoding rRNA and tRNA were assayed by hybridization to radioactive RNA. The results support the conclusion that the tRNA genes are interspersed between and closely linked to the rRNA genes of B. subtilis. They probably do not appear between the 16S and 23S rRNA genes as in E. coli.     

Role of curved DNA in binding of Escherichia coli RNA polymerase to promoters.

The ability of curved DNA upstream of the -35 region to affect the interaction of Escherichia coli RNA polymerase and promoter DNA was examined through the use of hybrid promoters. These promoters were constructed by substituting the curved DNA from two Bacillus subtilis bacteriophage SP82 promoters for the comparable DNA of the bacteriophage lambda promoters lambda pR and lambda pL. The SP82 promoters possessed intrinsic DNA curvature upstream of their -35 regions, as characterized by runs of adenines in phase with the helical repeat. In vitro, the relative affinities of purified sigma 70-RNA polymerase for the promoters were determined in a competition binding assay. Hybrid promoters derived from lambda pR that contained curved DNA were bound by E. coli RNA polymerase more efficiently than was the original lambda pR. Binding of E. coli RNA polymerase to these hybrid promoters was favored on superhelical DNA templates according to gel retardation analysis. Both the supercoiled and relaxed forms of the hybrid lambda pL series were better competitors for E. coli RNA polymerase binding than was the original lambda pL. The results of DNase I footprinting analysis provided evidence for the wrapping of the upstream curved DNA of the hybrid lambda pR promoters around the E. coli RNA polymerase in a tight, nucleosomal-like fashion. The tight wrapping of the upstream DNA around the polymerase may facilitate the subsequent steps of DNA untwisting and strand separation.     

Molecular cloning of promoter-containing fragments from Bacillus stearothermophilus and their expression in Escherichia coli and Bacillus subtilis

Abstract Bacillus stearothermophilus DNA fragments containing a promoter were isolated in Escherichia coli using a shuttle promoter-probe vector. The molecular sizes of the isolated fragments ranged from 0.78 to 10 kb. The 0.78 and 1.1 kb fragments were selected and examined in some detail for promoter activity in both E. coli and Bacillus subtilis by analysis of expression of erythromycin-resistance (Em^r) and β-galactosidase. The results showed that the two fragments exhibit a high promoter activity in both bacteria. In vitro promoter activity of the 1.1 kb fragment was also shown by RNA syntheses catalyzed by RNA polymerases prepared from E. coli, B. subtilis and B. stearothermophilus .