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[Factors influencing transduction of genetic determinants of penicillinase activity and pathogenicity in staphylococcus aureua. II. Antiphage activity of acridine derivatives].

Acridine dyes examined earlier (acrichine, acridine orange, proflavine and rivanol) and newly-synthesized preparations (acridines No. No. 37--40) were studied in the capacity of nonspecific agents influencing the lytic cycle in development of staphylococcus phages. Acrichine and acridine No. 37 failed to prevent lysis of the indicator staphylococcus cultures (strains 16/160 and 8325) by bacteriophages; proflavine, rivanol, acridines No. No. 39--40 produced a marked inhibitory effect; acridine orange and acridine No. 38 inhibited the staphylococcus lysis completely. Some preparations could be used to investigate the transduction phenomenon.

Acridines

Role of a lipopolysaccharide gene for immunogenicity of the enterobacterial common antigen.

It is known that only certain strains of the family of Enterobacteriaceae, notably rough (R) mutants with the type R1 or R4 core, evoked antibodies in high titers against the common enterobacterial antigen (CA) after immunization of rabbits with heated cell suspensions. The present investigation deals with genetic and immunochemical aspects of certain R1 and R4 mutants isolated from Escherichia coli 08 and various Shigella serotypes which, unexpectedly, do not induce CA antibody formation. Immunochemical and genetical (transduction and conjugation) experiments revealed that the rough phenotype of these special mutants was evoked by a mutation of pyrE-linked rfa gene, called rfaL, which is involved in translocation of O-specific polysaccharides onto the lipopolysaccharide core. The transduction of the defective rfaL, allele into appropriate rough recipients results in transductants which have simultaneously lost the ability to evoke CA antibodies. This finding suggests that a close connection exists between the function of the rfaL gene and the expression of CA immunogenicity in R1 and R4 mutants. One of the strains synthesized neither O-hapten nor CA, suggesting a mutation in a region equivalent to the rfe genes of Salmonella.

Animals

The structure of the phi 80d3 ilv+ Su+7 transducing phage and the origin of its Su+7 tRNA-gene containing fragment.

The Bam HI, XhoI, and EcoRI sites of the transducing phage phi 80d3Su+7ilv+ are located. The 1.2 x 10(6) MD EcoRI fragment which, when cloned, contains tRNAAsp and expresses the mutant tRNATry gene, Su+7, and which also relaxes control of stable RNA synthesis is found immediately adjacent to the rrnC region. Its tRNA genes, tRNAAsp and tRNATry, are transcribed in the same direction as the ribosomal RNA genes, though no mature rRNA subsequences are on the fragment. This fragment also exists as such in another F-prime factor derived from the same Hfr host, and therefore presumably also in the Hfr chromosome itself. It is composed of about half ordinary chromosomal and half F DNA sequences, the latter from the gamma-delta region of F. The advantages of a novel mapping method used are discussed.

Chromosome Mapping

Genetic organization of the Salmonella typhimurium ilv gene cluster.

A number of Salmonella typhimurium ilv::Tn10 insertion strains were used to analyze the Salmonella ilv gene cluster. Tn10 generated ilv deletion mutants were employed in mapping experiments to conclusively define the gene order as ilvG-E-D-A-C. Examination of ilv enzyme levels confirms that the direction of transcription of ilvGEDA is from ilvG to ilvA. The major control locus, designated ilvO, is located before ilvG forming an ilvOGEDA transcriptional unit that is multivalently repressed by isoleucine, valine and leucine. Two internal promoters, one before ilvE and anonother before ilvD, are identified and are shown to provide repressed levels of the ilvE, D and A gene products. Possible regulation of transcription from these promoters in response to isoleucine limitation is discussed in terms of attenuation.

Chromosome Mapping

The isolation and characterisation of a plaque-forming derivative of bacteriophage Mu carrying a fragment of Tn3 conferring ampicillin resistance.

We have isolated a plaque-forming derivative of phage Mu which carries a determinant for ApR. The biological properties of this MuAp phage are similar to those of normal Mu. Its genome contains a 1.1 kb substitution where Mu DNA from the right end of the G region has been replaced by a similar length of DNA from the transposon Tn3. This fragment of Tn3 DNA carries the ApR gene, but is no longer capable of independent transposition.

Ampicillin

Overproducing araC protein with lambda-arabinose transducing phage.

Escherichia coli infected with bacteriophage lambda-arabinose transducing phage were tested as sources of araC protein. Infection of cells with such phage produces an intracellular concentration of araC protein up to 100 times that present in wild-type E. coli, apparently resulting from fusion of the araC gene to bacteriophage lambda promoters. Lysates from these phage-infected cells may be fractionated to yield another 100-fold enrichment in araC activity so that the total enrichment is 10,000-fold. A nonsense mutation in araC provided proof of the identification on gel electrophoresis of a band in the purified material. Biologically active araC protein is a dimer with 28,000 M.W. subunits. The araC gene in these phage replaces the int-xis genes but is oriented in the opposite direction. Nonetheless, it appears to be transcribed in this position by the phage promoter pr via transcription the long way around. Furthermore, because araC gene is in this position, we were able to isolate phage on which the araC gene was under phage late gene control by deletion of the late gene transcription stop signals in the b2 region.

Arabinose

Transposition of DNA inserted into deletions of the Tn5 kanamycin resistance element.

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.

Chromosome Mapping

Transposition of TnA does not generate deletions.

We have examined the incidence of loss of the TnA unit, Tn801, from RP1 under conditions where transposition of Tn801 to another replicon. R388, was readily detected. We found that the frequency of transposition of Tn801 from RP1 to R388 exceeded, by at least a factor of one hundred, the frequency at which it was deleted from RP1. We conclude that, in general, transposition of Tn801 does not generate derivatives of the donor plasmid which specifically lack Tn801. The relevance of these findings to the mechanism of transposition is discussed.

Chromosome Aberrations

Phage Mu-1 mediated transposition: a tool to study the organization of ribosomal protein genes in Escherichia coli.

Phage Mu-1 mediated transposition has been used to map genes coding for ribosomal proteins and elongation factor G inside transcriptional units. The data indicate that 1) the str A and fusA genes belong to the same operon, 2) the spcA and strA genes are expressed independently, 3) the spcA gene is located in a different transcriptional unit to that of the eryA and eryB genes.

Coliphages

Origin of replication, oriC, or the Escherichia coli chromosome on specialized transducing phages lambda asn.

Specialized transducing phages lambda asn harboring chromosomal DNA and genetic markers on either side of the asn gene were isolated. Phages carrying chromosomal DNA counterclockwise of the asn gene can upon infection establish themselves as self-replicating plasmids in asn, recA hosts lysogenic for lambda. It is concluded that this bypassing of normal lambda immunity is due to the presence of the chromosomal replication origin, oriC, in this class of phages. Genetic analysis and the determination of restriction endonuclease cleavage patterns of the different lambda asn lead to the allocation of oriC within 1.5 megadaltons of the asn gene towards the uncA, uncB genes at 82 min on the genetic map of E. coli. The clockwise order of genes on the chromosomes is found to be: bglB, (pst, glmS), (uncA, uncB), oriC, asn, trkD, rbs, rrnC, ilv.

Chromosome Mapping

Cloning the trpR gene.

In Escherichia coli, the structural gene for purine nucleoside phosphorylase, deoD, is subject to insertional inactivation by prophage lambda. From one such secondary site lambda lysogen, strain SP265, one may isolate deletions that remove all or part of the trpR gene and other genes in the deo-thr sector of the E. coli chromosome. Specialized transducing phages harboring serB+ and trpR+ were liberated following induction of SP265. All such phages were N-defective, bio-type pseudolysogens whose DNA persisted in the form of plasmids. A collection of transducing phages, differing in their complement of bacterial DNA, was used to locate cleavage sites for BamHI, SalI, and PvuI within the deoD-trpR region of the E. coli genome. The trpR gene lies within a specific 950 base pair BamHI-PvuI segment. A 1250 base pair BamHI fragment carrying a functional trpR gene was cloned into the amplifiable plasmid pBR322. A single SalI site in this fragment was shown to lie within the TrpR gene. In two situations where increased gene dosage might generate elevated amounts of Trp repressor (N-defective trpR+ pseudolysogens and strains harboring pBR322 trpR+ plasmids) neither tryptophan auxotrophy, enhanced sensitivity to DL-5-methyl-tryptophan, nor super repression of the tryptophan biosynthetic enzymes was observed.

Bacteriophage lambda

A new host gene (groPC) necessary for lambda DNA replication.

The isolation of a bacterial mutation in a gene, designated groPC, which affects the growth of phages lambda and P2 is described. Lambda replication is severely limited in the strain, and some lambda pi mutations, which map in (or near) the P gene, allow growth. The gro mutation, groPC259, is recessive to wild type and maps between threonine (thr) and diaminopimelate (dapB) on the E. coli chromosome. The possibility that the groPC gene is concerned with host DNA replication is discussed.

Chromosome Mapping