Expression of Escherichia coli ribosomal protein and RNA polymerase genes cloned on plasmids.

Summary Fragments of drif ^d 18 DNA with different end-points within the set of structural genes of ribosomal proteins L11 (rplK), L1 (rplA), L10 (rplJ) and L12 (rplL) as well as the (rpoB) and (rpoC) subunits of RNA polymerase have been cloned on plasmids. These plasmids were transformed in host cells which were mutant for each of the genes, enabling expression of both wild-type (plasmid-borne) and mutant (chromosomal) genes to be differentiated. On the basis of these results we propose the following genetic structure for the region: rplK and rplA are in one operon; rplL, rpoB and rpoC are in a second. Our data suggest the possibility that rplJ is by itself in an operon situated between the other two.     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. VI. Mapping of F14 sequences homologous to phi 80dmetBJF and phi 80dargECBH bacteriophages

The positions of the metBJF and the argECBH sequences on F14 have been mapped by studying heteroduplexes of F14 with φ80dmet and φ80darg transducing phage DNAs. The structures of the DNAs of the transducing phage φ80d-metB isolated by Konrad (1969), of two φ80dmetB phages isolated by Press et al. (1971), and of some derived φ80darg phages, have been determined. They all have complex structures. In addition to the bacterial chromosome sequences corresponding to the met and arg genes, they contain certain F sequences, which have been recognized as active in F-related recombination events. Plausible models for the integration and excision events leading to the formation of the phage DNA molecules are proposed.     

Radiological mapping of functional transcription units of bacteriophages phiX174 and S13. The function of proximal and distal promoters.

We have found that the nearest promoter is not always the primary promoter for making translatable message. The technique of ultraviolet mapping was used to determine the location of promoter sites for translated mRNA coded for by bacteriophages φX174 and S13. The method is based on the theory that the “target size” for u.v. inactivation of expression of a gene is proportional to the distance between the promoter and the 3′ end of the gene. This method has revealed an expected and some unexpected locations for the promoters responsible for gene expression. Ultraviolet-survival curves for expression of phage genes were interpreted in the following way. The contiguous genes D, F, G and H are expressed as a unit under the control of a promoter located near gene D. However, gene B (and probably the adjacent genes K and C) are controlled by a promoter distant from gene B, possibly in the region of gene H, rather than from a promoter located just before gene B. Likewise, gene A is controlled by a promoter distant from gene A.     

Either of the chromosomal tuf genes of E. coli K-12 can be deleted without loss of cell viability

A method of λ-mediated gene replacement was used to disrupt tufA or tufB on the chromosome of the E. coli K-12 strain MG1655. Both tuf genes, which are almost identical but map in different chromosomal contexts, encode the essential peptide chain elongation factor EF-Tu, one of the most abundant cytoplasmic proteins. Southern analysis confirmed replacement of the chromosomal tufA or tufB gene by a chloramphenicol resistance marker, demonstrating that both tuf genes are individually dispensable for growth. Under conditions of rapid growth, deletion of tufB had no significant effect on growth rate, but deletion of tufA resulted in a 35% increase in generation time. In minimal medium we observed no negative effects of tufA deletion on growth rate. Strains with a single tuf gene are useful for the expression of mutant forms of EF-Tu as the sole species in cells; this was demonstrated by introducing the hybrid tufAhis gene, encoding EF-TuA extended with a C-terminal (His)₆ tag, into the chromosome of a strain lacking tufB.     

The Role of O-antigen in P1 Transduction of Shigella flexneri and Escherichia coli with its Alternative S' Tail Fibre

Enterobacteria phage P1 expresses two types of tail fibre, S and S'. Despite the wide usage of phage P1 for transduction, the host range and the receptor for its alternative S' tail fibre was never determined. Here, a ΔS-cin Δpac E. coli P1 lysogenic strain was generated to allow packaging of phagemid DNA into P1 phage having either S or S' tail fibre. P1(S') could transduce phagemid DNA into Shigella flexneri 2a 2457O, Shigella flexneri 5a M90T and Escherichia coli O3 efficiently. Mutational analysis of the O-antigen assembly genes and LPS inhibition assays indicated that P1(S') transduction requires at least one O-antigen unit. E. coli O111:B4 LPS produced a high neutralising effect against P1(S') transduction, indicating that this E. coli strain could be susceptible to P1(S')-mediated transduction. Mutations in the O-antigen modification genes of S. flexneri 2a 2457O and S. flexneri 5a M90T did not cause significant changes to P1(S’) transduction efficiency. A higher transduction efficiency of P1(S') improved the delivery of a cas9 antimicrobial phagemid into both S. flexneri 2457O and M90T. These findings provide novel insights into P1 tropism-switching, by identifying the bacterial strains which are susceptible to P1(S')-mediated transduction, as well as demonstrating its potential for delivering a DNA sequence-specific Cas9 antimicrobial into clinically relevant S. flexneri.     

Prophage lambda at unusual chromosomal locations. II. Mutations induced by bacteriophage lambda in Escherichia coli K12

An Escherichia coli strain deleted for the primary λ attachment site was lysogenized with λ at secondary sites. Some lysogens became mutants because of prophage insertion in the affected gene. Mutagenesis by phage λ is not random with respect to the gene affected: most mutants were pro, although certain other genes could be mutated at lower frequencies. In the case of several independent ilv and gal mutants, the sites of prophage insertion were in the same segment of the ilv region and galT gene respectively. The galT location may also be a preferred site for the insertion of DNAs other than prophage λ. Insertion of prophage λ within an operon can reduce the expression of operator-distal genes. A trpC λ insertion mutant expresses the operator-distal trpB function constitutively at a low level. This expression probably derives from a promoter located in the left arm of the prophage.     

Synthesis of the Escherichia coli K12 isoenzymes of ornithine transcarbamylase, performed in vitro.

Summary The in vitro synthesis of enzymaticallyactive ornithine transcarbamylase (OTCase) directed by each of the E. coli K-12 OTCase genes (argF and argI) is described. The E. coli OTCase isoenzyme subunits are not identical, whether synthesized in vivo or in vitro, the argF-coded product being about 5% smaller. The OTCase protomers are enzymatically inactive but associate in vitro to an enzymatically active multimer. The rates of subunit association of argF and argI isoenzymes are considerably different. Utilizing the facile assay protocol presented, the regulation of in vitro OTCase synthesis by the specific holorepressor of the arginine regulon is demonstrated. Calculations based upon data presented indicate that there are about 65 molecules of argR gene product per bacterium, a substantially lower estimate than previously reported.This work is dedicated to Luigi Gorini without whom none of this would have been possible. His unbounded love of science and freedom will be remembered by so many, for so long.     

A study of a mutant elongation factor properties of E. coli HAK88 and its mutant elongation factor Tu.

Summary The E. coli chromosome contains two genes for elongation factor Tu, tufA (near the fusidic acid resistance marker) and tufB (near the rifampicin resistance marker). It has been discovered that the mutant E. coli K12 strain HAK88 bears a mutation in the tufB gene, which leads to the synthesis of a protein of increased acidity. To determine whether the mutation has altered the protein's function in peptide chain elongation, we have compared the reactivities of normal tufA EF-Tu and mutant tufB EF-Tu (purified together from HAK88) with the components of the AA-tRNA binding cycle. Normal tufA EF-Tu and mutant tufB EF-Tu are indistinguishable in their affinities for GDP, EF-Ts, and phe-tRNA, and differ only slightly in their affinities for ribosomes. Coupled with the results of a separate study showing the similarity of the normal tufA and tufB gene products, these experiments demonstrate that the mutation has not altered the function of tufB EF-Tu in peptide chain elongation. Contrary to the original report (Kuwano et al., 1974; J. Mol. Biol. 86, 689–698) the HAK88 strains we have examined no longer possess a temperature-sensitive EF-Ts. The growth rates of HAK88 strains resemble the parent HAK8 strain in their lack of tRNA dependence but unlike HAK8 show varying degrees of temperature sensitivity. We conclude that HAK88 contains a physically altered but functionally intact tufB EF-Tu. The mutation in tufB should be valuable for studying in vivo the control of expression of the genes for EF-Tu.     

Restriction of lambda trp bacteriophages by Escherichia coli K

trp-transducing derivatives of phage λ have been used to study Escherichia coli K specific restriction in vivo. The expression of the trp genes from unmodified phages during infection of a rec⁺, restricting host is eliminated by restriction. In a K-restricting recB,C host, where degradation of restricted phage DNA is prevented, expression of the trp genes is little affected by the presence of a single unmodified, K-restriction recognition site, even when that site is within the trpE gene. RI restriction, in contrast to K restriction, prevents trp gene expression in a recB,C host when the restriction target is between the trp genes and the relevant promoter. The presence of two K-restriction recognition sites in a λtrp phage can have a marked effect on trp gene expression. This effect can be interpreted as the result of preferential breakage between the two restriction recognition sites. We conclude that K restriction does not break susceptible DNA at, or even preferentially near, a restriction recognition sequence.