Synthetic ColE1 plasmids carrying genes for penicillin-binding proteins in Escherichia coli

Clarke and Carbon's collection of 2000 Escherichia coli strains which harbor ColE1 plasmids carrying small random segments of the E. coli chromosome was screened for the correction of mutational defects in penicillin-binding proteins (PBPs): ponA (PBP-1a), ponB (PBP-1b), dacB (PBP-4), and pfv (PBP-5). We found plasmids carrying chromosomal segments containing ponA⁺-aroB⁺ (pLC29-47), ponB⁺-tonA⁺ (pLC4-43, pLC4-44, and pLC19-19), and argG⁺-dacB⁺ (pLC10-46 and pLC18-38). Characters of these plasmids were analyzed. Two other plasmids (pLC26-6 and pLC4-14) previously found to correct ftsI mutation (Y. Nishimura, Y. Takeda, A. Nishimura, H. Suzuki, M. Inouye, and Y. Hirota (1977)Plasmid1, 67–77) were also investigated further. Restriction maps of chromosomal DNAs carried by pLC29-47, pLC4-44, pLC19-19, pLC18-38, pLC26-6, and pLC4-14 were constructed. The regions of ponB-tonA on pLC4-44 and pLC19-19, and of leuA-ftsI-murE and F on pLC26-6 were located on the restriction maps. Although both pLC26-6 and pLC4-14 corrected a thermosensitive mutation, ftsI, which causes a defect in cell division due to abnormal PBP-3, only pLC26-6 led to restoration of PBP-3 production by an ftsI mutant, while pLC4-14 did not. Restriction and heteroduplex analyses of pLC26-6 and pLC4-14 have shown the absence of nucleotide sequence homology between them. The plasmids, pLC29-47 carrying ponA⁺ and pLC4-43, pLC4-44, and pLC19-19 carrying ponB⁺ led the host cell to overproduce the respective PBP.     

Synthetic ColE1 plasmids carrying genes for cell division in Escherichia coli.

Clarke and Carbon's collection of 2000 E. coli strains, which harbor ColE1 plasmids carrying small random segments of the E. coli chromosome, was screened for the correction of thermosensitive defects in the processes of cell division and in the synthesis of murein-lipoprotein. The genetic defects examined in this screening were those in partition of daughter nuclei (par), cleavage of cells (fts), determination of a cell shape (rod), and synthesis of murein-lipoprotein (lpo). We found plasmids carrying E. coli chromosomal segments containing ftsB⁺, ftsE⁺,ftsI⁺,ftsM⁺, and parA⁺. However, none was found to transfer ftsA⁺, ftsC⁺, ftsF⁺, ftsG⁺, ftsJ⁺, ftsK⁺, ftsL⁺, parB⁺, rod⁺, and lpo⁺. One of the donor strains transferring a gene that corrected thermosensitive cell cleavage in the ftsI^? mutant overproduced the penicillin-binding protein 3 by ca. 10-fold.     

Isolation and study of hybrid plasmids carrying Escherichia coli threonine operon genes

A fragment of Escherichia coli chromosome containing the intact threonine operon or its distinct genes has been cloned on the pBR322 plasmid. This fragment has been mapped using some restriction endonucleases. Cloning results in an increased level of appropriate enzyme activity in cells containing hybrid plasmids. Those carrying the complete threonine operon are capable of accumulating threonine up to 5 g/l in culture medium during 48 h. When multi-copy plasmids are used for gene cloning, interpretation of experiments aimed at transformation of auxotrophic bacterial strains, might be complicated. For example, transformation of appropriate threonine auxotrophs by a hybrid plasmid carrying mutation in the threonine gene, might result in prototrophic phenotype. It is possible that the great amount of mutant enzyme molecules compensated their low activity. On the contrary, the presence of a gene within the plasmid, as shown by restriction and biochemical analysis, did not always ensure the growth on a minimal medium of auxotrophs transformed by this plasmid.     

Application of hybrid plasmids carrying glycolysis genes to ATP production by Escherichia coli

The closely linked structural genes of phosphofructokinase (pfkA) and triosephosphate isomerase (tpi) of Escherichia coli were separately cloned onto plasmid pBR322. By gene dosage effects, transformed cells of E. coli C600 with these pBR322 hybrid plasmids showed 7- and 16-fold increases in the specific activities of phosphofructokinase and triosephosphate isomerase, respectively, over the specific activities in C600. Dried preparations of E. coli cells dosed with these genes showed appreciably high ATP-regenerating activity.     

Restriction mapping of recombinant plasmids carrying the genes for arginine biosynthesis in Escherichia coli K-12

ArgA and argECBH genes of Escherichia coli K-12 were cloned on the pBR322 vector. Restriction maps of the recombinant plasmids were constructed. Deletion mutants of these recombinant plasmids, retaining the functional argA and argE genes, were obtained using different restriction enzymes. All of the recombinant derivatives have the replication properties of the pBR322 vector.     

Escherichia coli host G668 stabilizes plasmids that are unstable in otherEscherichia coli strains

CloDF13 copy mutants that have their resolution site (crl) deleted accumulate as multimeric plasmid molecules in their host cells and are lost from severalEscherichia coli stains within 60 generations. Here we demonstrate that CloDF13cop3crl ^– mutants are stably maintained in theE.coli strain G668, although the plasmid copy number is not affected. Furthermore, we show that the stable maintenance of those plasmids is achieved even in the presence of multimeric molecules. Therefore, we conclude that a complete monomerization of multimeric molecules appears not to be a prerequisite for accurate partition of the plasmid molecules over daughter cells. The G668 strain may be applied as host for the stabilization of resolution-negative, unstable CloDF13 or related replicons.     

Restoration of mutability in non-mutable Escherichia coli carrying different plasmids.

Summary N and I group plasmids, which increase methylmethane sulfonate (MMS) mutagenesis in lexA ⁺ strains of E. coli WP2 may be divided into two classes: those restoring part of the mutability of lexA ^- strains (class I) and those leaving lexA ^- strains non-mutable (class II). Almost complete restoration of MMS mutability is obtained by class I plasmids in a partially suppressed lexA rnm strain, while class II plasmids cause far fewer MMS revertants in this strain than in lexA ⁺. A pair of class I and II plasmids in lexA ^- shows a synergistic effect on mutability. These two classes do not coincide with plasmid division into incompatibility groups.     

Fosfomycin causes transient lysis in Escherichia coli strains carrying fosfomycin-resistance plasmids

Escherichia coli cells carrying fosfomycin-resistance plasmids show high levels of resistance towards this drug. However, the plasmid-carrying strains exhibited a transient lytic phase induced by fosfomycin when grown in rich liquid media. This lytic phase was not observed if the cells were grown in liquid minimal media. Fosfomycin-induced lysis depended on the accumulation of drug inside the bacteria, presumably as a result of the saturation of the fosfomycin modification system. Growth recovery after lysis was not due to drug inactivation in the culture medium and could be explained by selection of mutants showing impaired fosfomycin transport when high concentrations of fosfomycin were used. However, there was no selection of mutants with low drug concentrations.     

Metabolic genes on conjugative plasmids are highly prevalent in Escherichia coli and can protect against antibiotic treatment

Conjugative plasmids often encode antibiotic resistance genes that provide selective advantages to their bacterial hosts during antibiotic treatment. Previous studies have predominantly considered these established genes as the primary benefit of antibiotic-mediated plasmid dissemination. However, many genes involved in cellular metabolic processes may also protect against antibiotic treatment and provide selective advantages. Despite the diversity of such metabolic genes and their potential ecological impact, their plasmid-borne prevalence, co-occurrence with canonical antibiotic resistance genes, and phenotypic effects remain widely understudied. To address this gap, we focused on Escherichia coli, which can often act as a pathogen, and is known to spread antibiotic resistance genes via conjugation. We characterized the presence of metabolic genes on 1,775 transferrable plasmids and compared their distribution to that of known antibiotic resistance genes. We found high abundance of genes involved in cellular metabolism and stress response. Several of these genes demonstrated statistically significant associations or disassociations with known antibiotic resistance genes at the strain level, indicating that each gene type may impact the spread of the other across hosts. Indeed, in vitro characterization of 13 statistically relevant metabolic genes confirmed that their phenotypic impact on antibiotic susceptibility was largely consistent with in situ relationships. These results emphasize the ecological importance of metabolic genes on conjugal plasmids, and that selection dynamics of E. coli pathogens arises as a complex consequence of both canonical mechanisms and their interactions with metabolic pathways.Subject terms: Antimicrobials, Microbial ecology, Microbiology     

Physical mapping and expression of hybrid plasmids carrying chromosomal beta-lactamase genes of Escherichia coli K-12.

Hybrid plasmids carrying the ampC gene of Escherichia coli K-12 that codes for the chromosomal beta-lactamase were physically studied. The ampC gene was mapped to a deoxyribonucleic acid segment encompassing 1,370 base pairs. The mapping was facilitated by the isolation of a plasmid carrying an insertion of the transposable element gamma delta (gamma delta) close to ampC. The ampA1 mutation, which increases the expression of ampC by a factor of about 20, was localized to a 370-base pair segment of the 1,370-base pair deoxyribonucleic acid segment that contains the ampC gene. Using a minicell protein labeling system, it was seen that plasmids carrying either ampA+, ampC, or ampA1 and ampC coded for a 36,000-dalton protein which comigrated with purified chromosomal beta-lactamase. In cells carrying plasmids that bore the ampA1 allele, the production of this protein was greater. In addition, a protein with a slightly higher molecular weight (38,000) was expressed by both ampA+ ampC and ampA1 ampC plasmids in this protein labeling system. This protein might represent a precursor form of chromosomal beta-lactamasee. From E. coli K-12 strains carrying the ampA1 allele, second-step mutants were isolated that hyperproduced chromosomal beta-lactamase. By reciprocal recombination, plasmid derivatives were isolated that carried these mutations. Two second-step regulatory mutations mapped within the same 370-base pair region as ampA1. This piece of deoxyribonucleic acid therefore contains ampA, a control sequence region for ampC.     

Complementation analysis of derepressed mutants of F-like plasmids of Escherichia coli

Complementation analysis of a number of conjugative transfer functions was performed in derepressed (drd) mutants of E. coli F-like plasmids. The major part of double plasmid complexes investigated has revealed the formation of complementation transfer inhibitor of Fin V-type, or less frequently--the formation of Fin U-type inhibitor. An additional complementation analysis of drd plasmids defective at Fin V region genes has demonstrated at least three genes (denoted A, B, C) in the structure of this region.     

Use of gene replacement to construct Escherichia coli strains carrying mutations in two genes required for stability of multicopy plasmids.

Escherichia coli mutants completely defective in ColE1 cer-mediated site-specific recombination have been mapped to two genes, xerA and xerB. In this study, xerA xerB double mutants were constructed by gene replacement with a lambda dv plasmid and were shown to be both viable and defective in ColE1 site-specific recombination.     

Replication of plasmids in mutants of Escherichia coli temperature-sensitive for initiation of deoxyribonucleic acid synthesis

Plasmid deoxyribonucleic acid (DNA) replication was studied in Escherichia coli hosts carrying temperature-sensitive (ts) initiation mutations. The replication of the R plasmid NR1 continues at the nonpermissive temperature in a ts dnaA mutant host but at a decreasing rate in proportion to the residual chromosome synthesis. The replication of NR1, as well as of the F plasmid F′lac, ceases immediately at the nonpermissive temperature in a ts dnaC mutant host. The ability to reinitiate R plasmid replication in the absence of protein or ribonucleic acid synthesis is accumulated at the nonpermissive temperature in a dnaC mutant host.     

Partition of P1 plasmids in Escherichia coli mukB chromosomal partition mutants.

The partition system of the low-copy-number plasmid/prophage of bacteriophage P1 encodes two proteins, ParA and ParB, and contains a DNA site called parS. ParB and the Escherichia coli protein IHF bind to parS to form the partition complex, in which parS is wrapped around ParB and IHF in a precise three-dimensional conformation. Partition can be thought of as a positioning reaction; the plasmid-encoded components ensure that at least one copy of the plasmid is positioned within each new daughter cell. We have used an E. coli chromosomal partition mutant to test whether this positioning is mediated by direct plasmid-chromosomal attachment, for example, by pairing of the partition complex that forms at parS with a bacterial attachment site. The E. coli MukB protein is required for proper chromosomal positioning, so that mukB mutants generate some cells without chromosomes (anucleate cells) at each cell division. We analyzed the plasmid distribution in nucleate and anucleate mukB cells. We found that P1 plasmids are stable in mukB mutants and that they partition into both nucleate and anucleate cells. This indicates that the P1 partition complex is not used to pair plasmids with the host chromosome and that P1 plasmids must be responsible for their own proper cellular localization, presumably through host-plasmid protein-protein interactions.     

Dwarf colony mutants of Escherichia coli: plasmids resistant to antibiotics]

Deficient dwarf colony (DDC) mutants of E. coli K 12, harboring or no resistance plasmids, were obtained in vitro. The R plasmids of parental strains and to DDC mutants were transfered by conjugation to normal colony, and to DDC mutants of E. coli K 12; the frequencies of transfer were similar for all strains studied.