Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColE1 plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColE1, they were nevertheless compatible with ColE1. P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.

Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.

[This corrects the article on p. 100 in vol. 41.].

Energy conservation in chemotrophic anaerobic bacteria.

With a stepwise degradation and terminal labeling procedure the 3′-terminal sequence of E. coli 16S ribosomal RNA is shown to be Pyd-A-C-C-U-C-C-U-U-AOH. It is suggested that this region of the RNA is able to interact with mRNA and that the 3′-terminal U-U-AOH is involved in the termination of protein synthesis through base-pairing with terminator codons. The sequence A-C-C-U-C-C could recognize a conserved sequence found in the ribosome binding sites of various coliphage mRNAs; it may thus be involved in the formation of the mRNA·30S subunit complex.

https://www.pnas.org/content/pnas/71/4/1342.full.pdf

The 3′-Terminal Sequence of Escherichia coli 16S Ribosomal RNA: Complementarity to Nonsense Triplets and Ribosome Binding Sites

A new minimal medium for enterobacteria has been developed. It supports growth of Escherichia coli and Salmonella typhimurium at rates comparable to those of any of the traditional media that have high phosphate concentrations, but each of the macronutrients (phosphate, sulfate, and nitrogen) is present at a sufficiently low level to permit isotopic labeling. Buffering capacity is provided by an organic dipolar ion, morpholinopropane sulfonate, which has a desirable pK (7.2) and no apparent inhibitory effect on growth. The medium has been developed with the objectives of (i) providing reproducibility of chemical composition, (ii) meeting the experimentally determined nutritional needs of the cell, (iii) avoiding an unnecessary excess of the major ionic species, (iv) facilitating the adjustment of the levels of individual ionic species, both for isotopic labeling and for nutritional studies, (v) supplying a complete array of micronutrients, (vi) setting a particular ion as the crop-limiting factor when the carbon and energy source is in excess, and (vii) providing maximal convenience in the manufacture and storage of the medium.

Culture Medium for Enterobacteria

The penicillin method (Davis, 1948; Lederberg and Zinder, 1948) has permitted convenient isolation of auxotrophicl mutants of Escherichia coli with specific growth requirements for most of the known water-soluble vitamins, as well as amino acids, purines, and pyrimidines. Accordingly, when crystalline vitamin B12 became available, a search was made for mutants requiring this nutrilite. Several strains of the desired type were promptly recovered. In all cases methionine, but not homocysteine, could be substituted for the vitamin. This paper describes certain biochemical properties of these mutants, as well as of others responding to methionine but not to B,2.

Mutants of escherichia coli requiring methionine or vitamin b12

Mechanism of bactericidal action of aminoglycosides.

The aminoglycoside antibiotics streptomycin, neomycin, kanamycin, paromomycin, gentamicin, hygromycin B, and viomycin all disturb the fidelity of the reading of polyribonucleotides in polypeptide synthesis. This effect is seen with all four homopolymers: poly U, poly C, poly A, and poly I. The extent and the spectrum of misreading vary with the antibiotic used and with the sRNA concentration. The antibiotics also influence the incorporation of the "correct" amino acid: with poly A they cause either stimulation or inhibition of lysine incorporation, depending on the sRNA concentration; poly C-directed proline incorporation was stimulated at all sRNA concentrations tested; and with poly U-directed phenylalanine incorporation the drugs were only inhibitory with most extracts. In all systems, with increasing sRNA the inhibition by the drug of "correct" reading became more prominent, and the relative misreading less prominent. In the light of recent knowledge of coding triplets, the effects observed suggest that aminoglycosides can (a) cause misreading of only one base at a time in UUU and CCC triplets, but of all three in AAA or III; (b) cause misreading of a base in only the 59 and the internal position in the pyrimidine codons, but in all three positions in the purine codons; and (c) allow both transition misreadings (purine to purine, or pyrimidine to pyrimidine) and transversion misreadings (purine to pyrimidine, or pyrimidine to purine). Transversion misreadings would seem to be rare in pyrimidine-containing polymers.

Misreading of RNA codewords induced by aminoglycoside antibiotics.

Many secreted proteins are synthesised as a large precursor with an additional hydrophobic N-terminal signal sequence that is cleaved by a membrane-bound enzyme. The proteins are secreted as nascent chains. The work leading to the current models of protein secretion is reviewed and the value of bacterial systems in the study of protein transfer across membranes is stressed.

Bernard D. Davis

Papers

Mutants of escherichia coli requiring methionine or vitamin b12

Mechanism of bactericidal action of aminoglycosides.

Misreading of RNA codewords induced by aminoglycoside antibiotics.

The mechanism of protein secretion across membranes

Isolation of biochemically deficient mutants of bacteria by penicillin.