Characterization of P1 argF derivatives from Escherichia coli K12 transduction III. P1Cm13argF derivatives

We have found that coliphage P1 can be used to transduce cloned DNA from Escherichia coli to Myxococcus xanthus. Transduction occurred at a high efficiency, and no evidence for DNA restriction was observed. The analysis of the transductants showed that they fall into three general categories: (i) haploid cells which contain portions of the cloned DNA substituted for homologous chromosomal DNA; (ii) heterozygous merodiploids which contain the recombinant plasmid integrated into the chromosome at a region of homology; and (iii) homozygous merodiploids which contain two copies of a portion of the cloned DNA with the loss of the chromosomal copy of the genes. The merodiploids, once formed, are relatively stable. They were used to analyze two genes necessary for aggregation and thus fruiting body formation. P1 transduction also permits the reintroduction and substitution of mutated regions of cloned DNA into M. xanthus for the analysis of the role of the DNA in cellular physiology and development.

Section: Discussionmentioning

confidence: 99%

Coliphage P1-mediated transduction of cloned DNA from Escherichia coli to Myxococcus xanthus: use for complementation and recombinational analyses

O’Connor¹,

Zusman²

1983

“…Each reaction started with 2.25 p.g of pABN20-6 DNA. Shown are the products without S1 nuclease treatment (lane A) and with S1 nuclease at 0.4 U/,Lg ( (21,56) and the plasmid-coded rafoperon in E. coli (27) is IS] flanked. Several other genes involved in virulence or substrate utilization have in the last few years been shown to be coded within transposon-like structures, such as the E. coli genes for citrate utilization (22), raffinose utilization (27), heat-stable enterotoxin (45), heat-labile enterotoxin (55), and the Vibrio cholerae toxin genes (29).…”

mentioning

confidence: 99%

Aerobactin genes in clinical isolates of Escherichia coli

Bindereif¹,

Neilands²

1985

The location of the aerobactin gene complex on either the chromosome or plasmid was determined in eight aerobactin-positive clinical isolates of Escherichia coli by Southern hybridization analysis, using as probes the cloned aerobactin genes from the ColV-K30 plasmid. The aerobactin genes were in two cases detected on large plasmids, whereas in the other strains the aerobactin genes are most likely located on the chromosome. Restriction mapping revealed only slight variations in the structural genes and an at least 3.4-kilobase-long upstream region conserved in all three plasmid-coded systems. A 7.7-kilobase Hindlll fragment upstream and adjacent to the 16.3-kilobase HindlIl fragment carrying the complete aerobactin system was cloned from the ColV-K30 plasmid. Fine-structure restriction mapping identified the left insertion sequence in the upstream region as IS], in inverted orientation to the IS] element downstream from the aerobactin operon. The upstream and downstream sequences of IS] appear to have perfect homology, as indicated by S1 nuclease resistance of a 760-base-pair DNA duplex formed by both IS) elements. * Corresponding author.Recently we reported a survey of a large number of E. coli clinical isolates for aerobactin synthesis (J. Z. Montgomerie, A. Bindereif, J. B. Neilands, G. Kalmanson, and L. B. Guze, submitted for publication). A high incidence of aerobactin-positive strains (39%) was found. In this paper we determined the genetic location, viz., whether plasmid coded or chromosomal, of the aerobactin biosynthesis and transport determinants in eight aerobactin-positive clinical isolates of E. coli and compare the particular organization of the aerobactin gene complex in these strains. MATERIALS AND METHODSClinical isolates of E. coli. A collection of 54 clinical isolates of E. coli was obtained from J. Z. Montgomerie, Rancho Los Amigos Hospital, Downey, Calif. In addition, the survey included E. coli 0111, a clinical isolate from the Centers for Disease Control, Atlanta, Ga. Recombinant DNA. Restriction enzymes, SI nuclease, DNA polymerase I, and T4 DNA ligase were purchased from Bethesda Research Laboratories (Bethesda, Md.). The large fragment of DNA polymerase I was from New England Biolabs (Beverly, Mass.) The conditions recommended by the supplier were used for restriction enzyme digestions and ligations. The protocols given by Maniatis et al. (26) were followed for most standard recombinant DNA procedures.The cloning vector for the construction of pABN20 was pUC8, 2.7 kilobases (kb) (49). E. coli JM83 [ara A(lac-pro) thi rpsL 4)80dlacZzAM15I was the host for pUC8-derived recombinant plasmids. E. coli JM83 was screened for plasmids by a quick minilysate procedure, which was also used for large-scale isolations of small plasmids (20). For largescale isolations of large plasmids (pColV-K30 and total plasmid DNA from E. coli isolates) the Portnoy procedure was employed (38). Cells from 11 cultures were harvested after 2 to 3 days of growth in Tris-buffered medium (41) with sodium succinate (30 ...

“…One of the loops is about 12 to 13 kb of DNA, including the lac operon, that has no counterpart in the S. typhimurium genome (15). Another loop is about 12 kb of DNA that includes the argF gene and two copies of the insertion sequence IS] (12,35). Other nonhomologous segments are present in this region.…”

mentioning

confidence: 99%

“…The newD and supQ genes of S. typhimurium have no counterparts in the corresponding parts of the E. coli genome (3,8,24,26), and the codA and phoA genes of E. coli have no counterparts in the S. typhimurium genome (3,26,29). The size and extent of both the lac and argF loops have been determined previously (12,15,35). The dimensions of the other loops have not been established.…”

mentioning

confidence: 99%

Location and analysis of nucleotide sequences at one end of a putative lac transposon in the Escherichia coli chromosome

Buvinger¹,

Lampel²,

Bojanowski³

et al. 1984

A segment of Escherichia coli DNA that contained a discontinuity of homology with Salmonella typhimurium DNA was isolated. The segment, 1,430 base pairs long, was derived from one end of the lac "loop," a region of about 12 kilobase pairs of E. coli DNA, including the lac operon which has no detectable homology with S. typhimurium DNA (K. Lampel and M. Riley, Mol. Gen. Genet. 186:82-86, 1982). The nucleotide sequence of the 1,430-base-pair segment of DNA was determined. The location of the junction of discontinuity of homology within the segment was established by hybridization experiments. Nucleotide sequences at or near the junction were determined to be similar to sequences that are involved in site-specific inversion in S. typhimurium, E. coli, phage P1, and phage Mu. Similar sequences are also present within the terminal inverted repeat sequences of transposon TnS and at the V-D-J joining sequences of eucaryotic immunoglobulin genes. Therefore, the lac operon, together with flanking DNA, may have been inserted into the E. coli chromosome at one time via a sitespecific recombination event. Rearrangement events of this kind undoubtedly have played a significant role in the evolutionary divergence of chromosomal DNAs.