Isolation and structure of the replicon of the promiscuous plasmid pCU1.

Evidence is presented to indicate that a PvuII fragment of approx. 2 kb isolated from the 39-kb IncN-group plasmid pCU-1 contains all plasmid-borne determinants for stable maintenance as an extrachromosomal element in Escherichia coli K-12. The fragment was sequenced. The features of this sequence include a group of 13 direct tandem repeats of 37 bp and a second group of two other direct repeats of 30 bp flanking a third partial member of this group. In addition, for a 19-bp sequence that overlaps a member of this second group, there are inverted repeats that straddle the members of the first group. There are three open reading frames within the fragment. We compare features of this sequence with that of other plasmid replicons and draw attention to similar and to dissimilar features.

Relationships of the Col plasmids E2, E3, E4, E5, E6, and E7: restriction mapping and colicin gene fusions.

Thirteen ColE plasmids representing the E2-E7 types have been compared by restriction mapping. Over 80% of their restriction sites were found to be similarly positioned, indicating that these plasmids share a common structure. Three variants are ColE2-CA42 and ColE7-K317, both of which contain 1.8-kb DNA segments in place of a 2.5-kb segment common to the other plasmids, and ColE6-CT14, which has an additional 5.0-kb DNA segment compared to the other plasmids. The colicin (col), immunity (imm), and colicin release (hic) genes of these plasmids have been localized to regions corresponding to those known for ColE3-CA38 and ColE2-P9, with the imm and hic genes adjacent to the 3' end of the col gene. Active colicin is produced from hybrid col genes containing 5' and 3' ends from different E-type plasmids. The 3'-termini of the fused col genes specify the colicin type.

Stable maintenance in chemostat-grown Escherichia coli of pBR322 and pACYC184 by disruption of the tetracycline resistance gene.

Plasmid stability was studied in antibiotic-free chemostat cultures. Disruption, either by deletion or insertion, of the tetracycline resistance gene in the EcoR1/EcoRV region of the cloning vector pBR322 or in the HindIII/BamH1 region of pACYC184 yields plasmids markedly more stable than the parent plasmids. Thus, at least for these two instances, cloning of a partitioning (par) locus is not prerequisite for plasmid maintenance.

A direct-selection vector derived from pColE3-CA38 and adapted for foreign gene expression.

The construction of a plasmid vector, pVT25, which allows an efficient and direct selection for transformed cells carrying recombinant plasmids is described. In this vector, the replicon and ApR gene from plasmid pBR327 are fused to the colE3 gene of pColE3-CA38, whereby positive selection is based on the inactivation of the lethal colicin E3 by the insertion of a foreign DNA fragment. However, pVT25 can be maintained within the Escherichia coli cells when complemented with another plasmid, pVT26, which expresses the colicin E3 immunity (imm) and the TcR phenotypes. Furthermore, pVT25 was used to regulate the expression of the synthetic human proinsulin gene fused to the colE3 gene at the single ClaI site. The production of the characteristic C-peptide of proinsulin, monitored by radioimmunoassay, was shown to be under the control of the inducible promoter of the colE3 gene.

Comparative nucleotide sequences encoding the immunity proteins and the carboxyl-terminal peptides of colicins E2 and E3.

Using the M13 dideoxy sequencing technique, we have established the DNA sequences of colicins E2 and E3 which encompass the receptor-binding and the catalytic domains of each of the nucleases, and their immunity (imm) genes. The imm gene of plasmid ColE2-P9 is 255 bp long and is separated from the end of the col gene by a dinucleotide. This gene pair is arranged similarly in plasmid ColE3-CA38 except that the intergenic space is 9 bp and the E3 imm gene is one codon shorter than its E2 counterpart. Comparisons of the E2 and E3 imm sequences indicate considerable divergence whereas the receptor-binding domains of both colicins are highly conserved. The two nuclease domains appear to share some sequence homology. A possible evolutionary relationship between colicin E3 and other microbial extracellular ribonucleases is also suggested from the sequence alignment analysis.

The immunity genes of colicins E2 and E8 are closely related.

We have determined the nucleotide sequence of the newly characterized colicin E8 imm gene which exists in tandem with the colicin E3 imm gene in the ColE3-CA38 plasmid. Comparison of these immunity structures reveals considerable sequence divergence, but the ColE8 imm gene is markedly homologous to the colicin E2 imm gene from the ColE2-P9 plasmid.

Characterization and nucleotide sequence of a colicin-release gene in the hic region of plasmid ColE3-CA38.

Downstream from its colicin and immunity genes (col. imm), Escherichia coli plasmid ColE3-CA38 contains a 0.81-kb DNA segment, the hic region, which is required for high colicin production. Characterization of derived plasmids, carrying the col-imm operon but varying in the hic region, showed that the latter functions in lacuna production, colicin release, cell death, and lysis. The hic gene expression after induction was shown to be dependent on the col gene promoter. The nucleotide sequence of the 0.81-kb region was determined and the hic gene localized to its imm-distal portion following an open reading frame (ORF) with no known function. There are two overlapping ORFs in that portion of the sequence, one of which was identified as the hic gene by its partial homology to lysis gene H of CloDF13. The 3' half of the hic gene is non-essential and contains a terminator-like DNA sequence. Preceding the gene, there are also inverted repeats which may attenuate its transcription.

Comparison of plasmids Co1E2-P9 and Co1E2-CA42 and their immunity proteins.

Plasmids ColE2-CA42 (6.1 kilobases) and ColE2-P9 (6.8 kilobases) were found to have homologous (4.3-kilobase) DNA segments which contain their colicin and immunity genes. The relatedness of their immunity proteins was verified by determining their amino acid compositions and N-terminal sequences. These characteristics were compared with those of ColE3-CA38.

Cloning of the ColE3-CA38 colicin and immunity genes and identification of a plasmid region which enhances colicin production.

The colicin and immunity genes of plasmid ColE3-CA38 have been localized by characterization of bacteria carrying its cloned restriction fragments. They are within a 3.14-kb EcoRI segment, such that the immunity gene contains the KpnI site, and the colicin gene is adjacent to it within a 2.1-kb KpnI-HincII segment. The immunity gene and one end of the colicin gene are in the region of ColE3-CA38 which is not homologous to the closely related plasmid ColE2-P9. A 0.64-kb PvuI-EcoRI segment of the plasmid adjacent to that containing the colicin and immunity genes was found to augment colicin production on solid media, and also affected the morphology of clearing zones produced by the cells when used as indicators in overlays of stabs of colicin E2 or E7 producers. The 0.64-kb segment was required in its native orientation relative to the 3.14-kb EcoRI segment to cause its effects.

Identification and characterization of Col plasmids from classical colicin E-producing strains.

A series of transformants have been derived which carry the Col plasmids from 11 E-colicin-producing strains isolated by Fredericq or Hamon. The ColE plasmids identified included four of type E1, five of type E2, and two of a type designated E4 by Horak (Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 233:58-63, 1975). Strain K317 was shown to carry a ColE7 plasmid and a 2.7-megadalton (Md) ColE2imm plasmid which confers immunity to colicin E2 but not the ability to produce colicin. Other plasmids identified in the colicinogenic isolates were a 3.4-Md ColN plasmid and a 1.3-Md Col plasmid of unknown type. The ColE1 plasmids all continued replicating in cultures treated with chloramphenicol. Strains carrying the ColE4 or ColE3-CA38 plasmid exhibited partial sensitivity to their own colicins and exhibited an unusual clearing-zone morphology when overlaid on stabs of colicin E2- or E7-producing strains. Except for ColE1-K53, ColE1-K47, ColE2-CA42, and ColE7-K317, all of the ColE plasmids were found to be about 4.3 Md in size. ColE2-CA42 and ColE7-K317 are both about 3.9 Md and are the only two plasmids of the E2, E3, E4, and E7 types that did not yield a 0.39-Md deoxyribonucleic acid fragment as an EcoRI digestion product. The comparisons of the ColE plasmids suggest several structural and functional relationships among them.

Restriction endonuclease mapping of ColE2-P9 and ColE3-CA38 plasmids.

Using single and double restriction-endonuclease digestions, 16 and 17 cleavage sites have been mapped for the ColE2-P9 and ColE3-CA38 plasmids, respectively. One or more sites for AvaI, BglI, EcoRI, HincII, PvuI, PvuII, SmaI and XhoI endonucleases were found in both plasmids, two BglII sites were found only in ColE2-P9, and one KpnI site was unique to ColE3-CA38. ColE2-P9 was found to be slightly smaller than ColE3-CA38, 4.4 Md compared to 4.6 Md. Eleven restriction sites are common to both plasmids in that they are identically placed relative to each other. These sites define a continuous DNA segment equal to over 60% of each plasmid. The remaining portions of the plasmids, which contain the non-homologous regions identified by Inselburg and Johns (1975) have no restriction sites in common, and differ in size by about 0.2 Md.

Structural homologies in alanine-rich acidic ribosomal proteins from procaryotes and eucaryotes.

The amino acid composition and amino-terminal sequence have been determined for the alanine-rich, acidic ribosomal 'A' protein (equivalent to Escherichia coli L7/L12) from three procaryotic cell types that live under extreme environmental conditions (Arthrobacter glacialis, Clostridium pasteurianum, and Bacillus stearothermophilus) as well as from wheat germ, a eucaryote source. These data are compared with previously published 'A' protein sequences from other procaryotes and eucaryotes. All the procaryotic 'A' proteins, with the exception of the very acidic 'A' protein from Halobacterium cutirubrum, show similar charge, size, and amino acid composition, as well as an extensive sequence homology in the N-terminal region. Some differences are observed between gram-negative and gram-positive bacteria. The 'A' proteins from eucaryotes contain two tyrosine molecules, an amino acid absent in procaryotic 'A' proteins, as well as a reduced number of valine residues and an increased amount of aspartic acid. The N-terminal sequence of wheat germ 'A' protein shows considerable homology with other eucaryotic 'A' proteins and also with H. cutirubrum. It also shows some sequence homology with E. coli 'A' proteins.

Topography of Escherichia coli ribosomal protein L12 in situ.

The ionization constants of each individual free amino group of Escherichia coli ribosomal protein L12 as it exists on the native intact 50-S subunit has been determined. All these amino groups are exposed and have pK values varying from 9.7 to 11.0. The amino terminus, on the other hand, is unreactive and therefore appears to be buried. In addition, the reactivities indicate that the aminoterminal region of residues 1--59 undergoes a pH-dependent conformational change on the 50-S subunit.

Mitogenic activity of Neisseria gonorrhoeae surface antigens in mouse splenic lymphocyte culture.

The mitogenic effects of Neisseria gonorrhoeae endotoxin, fractionated envelope componenents, and intact cells were examined on unsensitized mouse splenic lymphocytes in vitro. The stimulatory effect of these substances was measured by increased [3H]thymidine incorporation in spleen cell cultures. Intact cells, purified lipopolysaccharide (LPS), and cell envelope preparations were highly stimulatory and the stimulation index was dose dependent. Fractionated components of the envelope demonstrated variable stimulation when tested at identical LPS concentrations, reflecting the mitogenic activity of the protein moieties. The stimulatory dose responses for purified N. gonorrhoeae and Escherichia coli LPS were compared and mitogenicity was higher with gonococcal LPS at all concentrations tested. Alkaline detoxification or succinylation of N. gonorrhoeae LPS results in loss of ability to induce blast transformation. The mitogenicity of cell-surface components of N. gonorrhoeae is discussed in terms of LPS and protein content.