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Studies on the rate of synthesis of the beta and beta' subunits of RNA polymerase in haploid strains of Escherichia coli K12 containing poorly-suppressed rif degrees am mutations provide conclusive evidence that synthesis of at least these two subunits is regulated.
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A suppressor mutation of E. coli mut T has been isolated and mapped in the lue--ace(E/F) region.
That portion of the Escherichia coli chromosome carried by a number of lambda transducing phages, all of which carry the gua operon, was mapped using restriction endonucleases. The DNA from one of these transducing phages was subcloned onto pBR322. We have identified two recombinant plasmids which carry the Escherichia coli gene hisS, the structural gene for histidyl-tRNA synthetase. The two plasmids, pSE301 and pSE401, have in common a 3,540 bp fragment of E. coli DNA which is bounded by BglII and SalI restriction endonuclease recognition sites. Strains carrying these plasmids overproduce histidyl-tRNA synthetase 20 to 30 fold. The growth rate of these strains is not affected although the histidine biosynthetic enzymes are derepressed. This derepression seems to be in addition to that caused by introduction of a hisT mutation on the chromosome.
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Tn5-trp hybrid transposons have been constructed by insertion of a trpPOED Hind III fragment into an in vivo Tn5 internal deletion mutant or by substitution of trp for the internal Tn5 Hind III fragment. These hybrids are called, respectively, Tn409 and Tn410. Both Tn409 and Tn410 will transpose into lambda in the presence of a complementing Tn5 element. In the absence of a wild Tn5, lysogens carrying R1162::Tn409 and R1162::Tn410 plasmids will yield lambdatrp phages at less than six per cent of the complemented frequency. This reduction indicates that Tn409 and Tn410 lack a diffusible transposition function provided by wild Tn5 elements. However, the formation of lambdatrp phages without complementation is real. Most of these transducing particles contain Tn409 and Tn410 still linked to the carrier R1162 plasmid. This observation suggests that uncomplemented Tn409 and Tn410 elements mediate the formation of lambda-transposon-plasmid cointegrate structures. Thus, the missing transposition function may be involved in resolving these cointegrate structures to the final lambda::Tn409 or lambda::Tn410 product.
Two types of lambdatrp phages have been used as model systems to investigate ways of optimising the expression of bacterial genes from transducing phage genomes. Excellent yields of trp enzymes were achieved by infecting a trpR- host with Q- or Q-S- derivatives of lambdatrpAM1, which expresses its trp genese exclusively from the trp promoter. The five trp geneproducts constituted more than 50% of the total soluble protein of infected cells under these conditions, and an even higher proportion of the protein synthesized after infection. In a trpR+ host, phage DNA replication was easily able to override tryptophan-mediated repression by titration of the trp promoter were equally productive, while having the advantage of being much simpler to construct and propagate. lambdatrp phages lacking the trp promoter were used to investigate ways of optimising gene expression initiated at the phage promoter, PL. Though very powerful, the latter promoter is more difficult to harness then the trp promoter. Derepression of transcription from PL by the use of cro- mutations is accompanied by poor replication of transducing phage DNA. Attempts to circumvent this difficulty using virulent of cro,cII double mutants have not been successful. Nevertheless, cells infected with a lambdatrp phage expressing its trp genes exclusively from PL made up to 16 per cent of their protein as trp gene-products.
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The sfp gene is required for cells of Bacillus subtilis to become producers of the lipopeptide antibiotic surfactin. sfp was isolated and its nucleotide sequence was determined. sfp was expressed in Escherichia coli and its putative product was purified for use in antibody production and in amino acid sequence analysis. The gene was plasmid-amplified in B. subtilis, where it conferred a Srf+ phenotype on sfp0 (surfactin non-producing) cells. Overproduction of Sfp in B. subtilis did not cause production of an increased amount of surfactin and resulted in the repression of a lacZ transcriptional fusion of the srfA operon, which encodes enzymes that catalyze surfactin synthesis. We propose that sfp represents an essential component of peptide synthesis systems and also plays a role, either directly or indirectly, in the regulation of surfactin biosynthesis gene expression.
Numerous types of interaction between pro- and eucaryotes exist in nature, from the endosymbiosis of some bacteria with unicellular organisms and insects to the complex systems of bacterial flora associated with the skin and intestines of animals and man, and nitrogen-fixation and crown-gall tumor induction in plants. Until recently, such interactions were not thought to include genetic transfer, but an increasing body of evidence points to the probability of similar naturally-occurring exchanges with wide-ranging implications for evolution and genetic manipulation. Experiments to elucidate the possible effects of procaryotic genes in eucaryotic systems have included in vitro and in vivo studies with both plant and animal systems, for instance the translation of bacterial messenger RNAs in the wheat germ and rabbit reticulocyte systems and the introduction of bacterial genes into plant protoplasts, animal cells and whole organisms. In the present paper we have tried to summarize the results of experiments involving the uptake, replication, transcription, translation and integration of procaryotic genes in various eucaryotic systems and to discuss the implications of such findings for basic research as well as for possible biomedical applications. Awareness of the possibility of procaryotic-eucaryotic genetic interactions may help to elucidate unresolved questions in pathology, such as possible involvement of the intestinal flora in carcinogenesis, as well as to provide valuable probes of eucaryotic control mechanisms and new approaches in agricultural genetic engineering.
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The genome of bacteriophage P1 contains a segment which is invertible by site specific recombination between sequences near the outside ends of the inverted repeats which flank it. Immediately adjacent to this C segment is the coding sequence for cin, the enzyme catalyzing inversion. We show that multicopy plasmids carrying cin and the sequences at which it acts (cix) can form dimers in the absence of the host recA function. Further, such plasmids can be cotransduced with P1 markers at high frequency from recA lysogens, indicating cointegration with the P1 genome. It is thus demonstrated that a system whose primary role is the inversion of a specific DNA segment can also mediate intermolecular recombination.
The technique developed by Rosenfeld and Brenchley [J Bacteriol 144, 848-851 (1980)] has been used to introduce Mu d1 (Apr lac) into Salmonella typhimurium for purposes of constructing pyr::lac fusion strains. A stable pyrB::lac fusion mutant was subsequently derived and used for the genetic characterization of the pyrB gene. The direction of transcription of pyrB was determined to be counterclockwise on the S. typhimurium linkage map and argI was shown to be located clockwise of pyrB. Mutants altered in the regulation of expression of pyrB were isolated and two of the isolates chosen for further study were tentatively categorized as promoter or operator mutants.
UNLABELLED: The effect of three different types of mutations in ribosomal protein S5 of Escherichia coli on translational fidelity has been studied. Two of them, namely that conferring resistance to spectinomycin and that selected for partial suppression of a temperature-sensitive analyl-tRNA synthetase mutation, do not exhibit ribosomal ambiguity in the in vivo and in vitro test system employed. In contrast, mutations in ribosomal protein S5 selected for suppression of streptomycin dependence mutations are able to derestrict the restriction of translational ambiguity imposed by str A mutations, though to different degrees depending on the type of mutation. Mutants in which streptomycin dependence is suppressed by an alteration in protein S5 are more restrictive than mutants resistant to streptomycin. Again, the extent of restriction depends on the type of the str Ad allele. IN CONCLUSION: mutations in ribosomal protein S5 can act as ram mutations like mutations in protein S4. The part of the S5 polypeptide involved in control of translational fidelity is different from regions altered in spectinomycin resistant strains and in the alanyl-tRNA synthetase suppressor mutant.
Mutants of a specialized lambda dmet transducing phage bearing the metJBLF gene cluster of Escherichia coli K12 were constructed using transposon Tn5. Two of these mutants, lambda dmet128::Tn5, MW77 and lambda dmet128::Tn5, 3-1, were used to locate precisely as well as confirm the existence of the metF transcription unit (approximately 1,000 base pairs in size). The introduction of new restriction sites within the metJBLF gene cluster due to the Tn5 insertion events allowed the metF transcription unit to be cloned into the high copy number plasmid pBR322. Analyses of the structures of two of these recombinant plasmids, pTJ77H and pTJ13-1H, are presented. Expression of the plasmid borne metF allele in cells grown in the absence, or presence, of exogenous L methionine (0.2 mM) demonstrates that the amplification of the metF copy number does not abolish met regulon mediated control of the gene's activity.