Molecular mechanisms of deletion formation in Escherichia coli plasmids. I. Deletion formation mediated by long direct repeats.

Summary Derivatives of plasmid pBR327 with the tet gene interrupted by 165 pb or 401 by direct repeats were constructed. In cells harboring these plasmids, deletions which restored the wild-type tet gene gave rise to tetracycline-resistant colonies, thereby allowing a simple phenotypic test for deletion formation. The frequencies of deletions in these plasmids were measured in Escherichia coli strains proficient or deficient in general recombination. The structure of plasmid DNA isolated from tetracycline-resistant transformants was analyzed by agarose gel electrophoresis, restriction mapping and sequencing. The data presented here demonstrate that deletion formation is always associated with dimerization of plasmid DNA. Dimeric plasmids were of two types. Those which carried both a deletion and a compensating duplication were the major type in a Rec⁺ background and were rare in recA, recF, recJ and recO backgrounds. Dimers of the second type contained deletions, but no compensating duplications, and their formation was RecA-independent. The data presented demonstrate that deletion formation mediated by long direct repeats is mainly the result of unequal crossing-over between two plasmid molecules.     

Inhibition of the recBCD-dependent activation of Chi recombinational hot spots in SOS-induced cells of Escherichia coli.

Nucleotide sequences called Chi (5'-GCTGGTGG-3') enhance homologous recombination near their location by the RecBCD enzyme in Escherichia coli (Chi activation). A partial inhibition of Chi activation measured in lambda red gam mutant crosses was observed after treatment of wild-type cells with DNA-damaging agents including UV, mitomycin, and nalidixic acid. Inhibition of Chi activation was not accompanied by an overall decrease of recombination. A lexA3 mutation which blocks induction of the SOS system prevented the inhibition of Chi activation, indicating that an SOS function could be responsible for the inhibition. Overproduction of the RecD subunit of the RecBCD enzyme from a multicopy plasmid carrying the recD gene prevented the induced inhibition of Chi activation, whereas overproduction of RecB or RecC subunits did not. It is proposed that in SOS-induced cells the RecBCD enzyme is modified into a Chi-independent recombination enzyme, with the RecD subunit being the regulatory switch key.     

Cointegrate formation between homologous plasmids in Escherichia coli.

Conjugation experiments were performed in which the donor was Escherichia coli K-12 strain KP245 containing either R plasmid NR1 plus an ampicillin-resistant derivative of ColE1 (*ColE1::Tn3, called RSF2124) or NR1 plus RSF2124 carrying a cloned EcoRI fragment of NR1. The recipient was the polA amber mutant JG112, in which RSF2124 cannot replicate. Ampicillin-resistant transconjugants can arise only when the genes for ampicillin resistance are linked to NR1 or are transposed to the host chromosome. When EcoRI fragment A of NR1 (20.5 kilobases) was cloned to RSF2124, the frequency of cotransfer of ampicillin resistance with tetracycline resistance was 25 to 60%. Plasmid DNA from these ampicillin-resistant transconjugant cells was analyzed by gel electrophoresis and was shown to be a cointegrate of NR1 and the RSF2124 derivative. Analysis of plasmid DNA isolated from donor cultures showed that the cointegrates were present before conjugation, which indicates that the mating does not stimulate cointegrate formation. When the cloned fragment was EcoRI fragment H of NR1 (4.8 kilobases), the frequency of cotransfer of ampicillin resistance with tetracycline resistance was about 4%, and the majority of the ampicillin-resistant transconjugants were found to contain cointegrate plasmids. When the donor contained NR1 and RSF2124, the frequency of cotransfer of ampicillin resistance was less than 0.1%, and analysis of plasmid DNA from the ampicillin-resistant transconjugants showed that Tn3 had been transposed onto NR1. These data suggest that plasmids which share homology may exist in cointegrate form to a high degree within a host cell.     

Shuttle plasmids for Escherichia coli and Clostridium perfringens.

Small plasmids which replicate in both Escherichia coli and Clostridium perfringens were made by recombining E. coli plasmid pBR322 with three different small (less than 4 kilobases) plasmids native to C. perfringens. Subsequently, two homologous, though distinct, tetracycline resistance determinants (tet) from other C. perfringens plasmids were cloned into them. Both tet systems made E. coli resistant to at least 5 micrograms of tetracycline per ml when resident on the shuttle plasmids. The shuttle vectors have been used to transform L-phase variants and autoplasts of C. perfringens. In the latter case, the intact transforming plasmid could be isolated from walled cells after cell wall regeneration. Reciprocal transformation experiments in which plasmid DNAs derived from E. coli or C. perfringens were used suggest that restriction barriers exist between these two organisms. The plasmids contain restriction enzyme recognition sites in locations which are useful for cloning experiments.     

Copy numbers of coexisting plasmids in Escherichia coli K-12.

A strain of Escherichia coli K-12 carrying eight compatible and distinguishable plasmids was constructed. The amounts of plasmid DNA (measured as supercoiled molecules) per chromosome in this strain was about equal to the sum of the plasmid DNAs, extracted under controlled conditions, from strains each carrying one of the eight plasmids. Analysis of these DNA preparations showed that each plasmid in the multiplasmid strain was present in the same proportion per chromosome as in the single-plasmid strains. Also the level of phenotypic expression of each plasmid in the multiplasmid strain was the same as in the single-plasmid strains. Each plasmid, therefore, appears to control its own copy number irrespective of the presence of other compatible plasmids.     

Characterization of plasmids in a sucrose-fermenting strain of Escherichia coli.

A multiply drug-resistant strain of Escherichia coli isolated from a patient in Bangladesh was shown to carry four types of plasmids based on size differences. One type carries a gene or genes for sucrose fermentation.     

Mobilization of Thiobacillus ferrooxidans plasmids among Escherichia coli strains.

Nonconjugative Thiobacillus ferrooxidans plasmids were mobilized at high frequencies among Escherichia coli strains by the IncP plasmid RP4 and at low frequencies by the IncN plasmid R46, but not by the IncW plasmid pSa. The mobilization region of a nonconjugative T. ferrooxidans plasmid was located on a 5.3-kilobase T. ferrooxidans DNA fragment.     

Deletion of a ribosomal ribonucleic acid operon in Escherichia coli.

One (rrnE) of the seven operons which codes for ribosomal ribonucleic acid in Escherichia coli was deleted. No significant change in phenotype was observed even under maximum laboratory growth conditions.     

Deletion of the Escherichia coli crp Gene

Spontaneous crp mutants Escherichia coli were selected from a strain that does not require 3',5'-cyclic adenosine monophosphate for CAP activity. Several deletions of the crp gene were characterized. The crp gene was not essential for growth of E. coli. crp mutations reduced the donor ability of Hfr strains.     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. V. ilv+ Deletion mutants of F14

The structure of a number of F′ilv episomes derived from F14 by bacteriophage P1-mediated transduction have been determined by the electron microscope heteroduplex method. F16, F25, F310 and F312 are all simple deletion mutants of F14. F316 is essentially the same but contains a small insertion (0.8 kilobase) of DNA of unknown origin within the F sequences at 78.6 F. The length of these plasmids are all about the same as that of phage P1 DNA itself. The sequences of F and the sequences of bacterial DNA that are present on the episomes are contiguous on the parental F14. Thus, their structures are consistent with the usual model for the mechanism of P1 transduction. The physical order of ilv genes is also consistent with previous genetic mapping. From this order one can determine the polarity of the Escherichia coli K12 chromosomal sequences on F14 and its F′ilv derivatives relative to the F sequences. This order is consistent with the known counterclockwise transfer order of the parental Hfr AB313. F′ilv episomes carry only one copy of the 2.8 to 8.5 F sequence, which is present as a direct duplication on F14. The F′ilv episomes are genetically stable, whereas F14 is unstable because of reciprocal recombination between the two duplicate sequences. The strain F316/AB2070 is different in several respects. All of the bacteria carry P1 phage DNA. As noted above, F316 itself carries a small insertion. Two transfer-defective deletion mutants, F316Δ(65.4-78.6) and F316Δ-(78.6-0.6) are also present in the population of F316/AB2070 cells. In each case, the deletion borders on one of the junctions of inserted DNA and F14 DNA in F316. Thus, these junctions appear to be hot spots for deletion formation.     

Hfr formation by I pilus-determining plasmids in Escherichia coli K-12.

R483, an atypical, I pilus-determining plasmid, and also R144, a typical one, were shown to suppress the DnaA phenotype by integration into the Escherichia coli chromosome.     

High-molecular-weight linear multimer formation by single-stranded DNA plasmids in Escherichia coli.

We inserted foreign DNA segments into plasmids which replicate by a rolling-circle mechanism in Escherichia coli and observed the appearance of high-molecular-weight plasmid multimers (HMW). This phenomenon, which occurs more frequently with GC-rich segments, depends on the mode of replication of the plasmid and on host homologous recombination functions. We found that (i) HMW are formed upon insertion of a foreign DNA segment into a single-stranded DNA plasmid, whereas the same DNA insert has no such effect on a theta replicon, and (ii) HMW are not present in a recA mutant strain but are found in a lexA (Ind-) mutant. Enzymatic studies allowed us to define the HMW structure as linear double-stranded tandem head-to-tail plasmid repeats. Use of heteroplasmid strains showed that HMW production by one plasmid does not affect another resident plasmid, indicating that no host functions are phenotypically inactivated. This distinguishes our system from the HMW observed with various replicons in the absence of RecBCD enzyme activity. We propose that the role of the foreign insert is to protect the DNA from RecBCD exonuclease attack.     

IncH plasmids in Escherichia coli strains isolated from broiler chicken carcasses.

Plasmids conferring tellurite resistance were transferred at low temperature (27 degrees C) from Escherichia coli strains isolated from chicken carcasses at the time of slaughter and after storage. They belonged to group IncH, as evidenced by their large molecular weight and incompatibility with plasmid pIP233. E. coli strains contaminating chickens meat can thus represent a source of IncH plasmids in the food chain of humans.     

Deletion of the penicillin-binding protein 5 gene of Escherichia coli.

A strain of Escherichia coli that has a deletion of the entire dacA gene has been constructed. The complete lack of penicillin-binding protein 5 in this strain establishes that the activity of this protein is not essential for the growth of E. coli.     

Impact of rpoS Deletion on Escherichia coli Biofilms

Slow growth has been hypothesized to be an essential aspect of bacterial physiology within biofilms. In order to test this hypothesis, we employed two strains of Escherichia coli, ZK126 (ΔlacZ rpoS⁺) and its isogenic ΔrpoS derivative, ZK1000. These strains were grown at two rates (0.033 and 0.0083 h⁻¹) in a glucose-limited chemostat which was coupled either to a modified Robbins device containing plugs of silicone rubber urinary catheter material or to a glass flow cell. The presence or absence of rpoS did not significantly affect planktonic growth of E. coli. In contrast, biofilm cell density in the rpoS mutant strain (ZK1000), as measured by determining the number of CFU per square centimeter, was reduced by 50% (P < 0.05). Deletion of rpoS caused differences in biofilm cell arrangement, as seen by scanning confocal laser microscopy. In reporter gene experiments, similar levels of rpoS expression were seen in chemostat-grown planktonic and biofilm populations at a growth rate of 0.033 h⁻¹. Overall, these studies suggest that rpoS is important for biofilm physiology.