Chromosome replication in an Hfr strain of Escherichia coli

The pattern of chromosome replication in an exponentially growing culture of an Hfr strain of Escherichia coli has been compared to that obtained with the same Hfr following a procedure which synchronizes rounds of DNA replication. The results indicate that there is significant replication from the integrated plasmid following the synchronization procedure, whereas in the exponentially growing culture replication starts most frequently from the normal origin with little, if any, replication from the sex factor, F.     

Tagging of Bacillus subtilis soil isolates through illegitimate recombination for PCR detection

Although there are numerous bacteria of the genus Bacillus of great importance for biological control, little is known about their ecology in the soil. We wanted to test illegitimate recombination as a tagging system that would allow us to study selected or genetically engineered Bacillus soil isolates. Strains carrying the plasmid integrated into the chromosome were obtained by growing at a non-permissive temperature after transformation with a plasmid carrying a thermo-sensitive replication origin with selection for erythromycin. A laboratory strain, a commercial strain (Kodiak), and four other soil isolates were generated through this procedure and analysed. In all of these strains the integrated plasmid was maintained in multicopy. The erythromycin resistance gene (ermB) placed on the plasmid was used as a target for polymerase chain reaction (PCR). The tagged strains could be then detected when inoculated into microcosms prepared with non-sterile soil.     

IS21 insertion in the trfA replication control gene of chromosomally integrated plasmid RP1: a property of stable Pseudomonas aeruginosa Hfr strains

Summary Broad host range IncP-1 plasmids are able to integrate into the chromosome of gram-negative bacteria. Strains carrying an integrated plasmid can be obtained when the markers of a temperature-sensitive (ts) plasmid derivative are selected at non-permissive temperature; in this way Hfr (high frequency) donor strains can be formed. The integrated plasmids, however, tend to be unstable in the absence of continuous selective pressure. In order to obtain stable Hfr donor strains of Pseudomonas aeruginosa PAO, we constructed a derivative of an RP1 (ts) plasmid, pME134, which was defective in the resolvase gene (tnpR) of transposon Tn801. Chromosomal integration of pME134 was selected in a recombination-deficient (rec-102) PAO strain at 43°C. Plasmid integration occurred at different sites resulting in a useful set of Hfr strains that transferred chromosomal markers unidirectionally. The tnpR and rec-102 mutations prevented plasmid excision from the chromosome. In several (but not all) Hfr strains that grew well and retained the integrated plasmid at temperatures below 43°C, the insertion element IS21 of RP1 was found to be inserted into the trfA locus (specifying an essential trans-acting replication funtion) of the integrated plasmid. One such Hfr strain was rendered rec ⁺; from its chromosome the pME134::IS21 plasmid (=pME14) was excised and transferred by conjugation to Escherichia coli where pME14 could replicate autonomously only when a helper plasmid provided the trfA ⁺ function in trans. Thus, it appears that trfA inactivation favours the stability of chromosomally integrated RP1 in P. aeruginosa.     

Replication of minichromosomes in a host in which chromosome replication is random

Minichromosomes are plasmids with the origin of chromosome replication, oriC, as their only origin of replication. In Escherichia coli, minichromosomes are compatible with the chromosome and replicate in a cell-cycle-specific manner at the same time as oriC located on the chromosome initiates replication. In int strains, oriC has been inactivated and replaced by a plasmid origin. Because plasmids control their own replication, chromosome replication is uncoupled from the normal cell-cycle control and is random with respect to the cell cycle in the int strains. We have used an intP1 strain to address the question of whether minicromosome replication is coupled to the replication of the chromosome or is governed by cell-cycle-specific signals. Minichromosome replication was analysed by density-shift experiments and found not to be random in the randomly replicating intP1 host. This suggests that the cell-cycle-specific control functions of oriC replication are operating also in the intP1 strain.     

Establishment of Escherichia coli cells with an integrated high copy number plasmid

Summary The dnaA46 cells can grow at high temperature when a high copy number plasmid pKY31, a derivative of pBR322 carrying a segment of the E. coli chromosome, integrates into the bacterial chromosome. In contrast, the dnaA46 polA ^- cells with the integrated plasmid can not grow at high temperature. Therefore, integration of the plasmid can suppress the dnaA mutation and this suppression requires DNA polymerase I which has been known to be required for plasmid replication. Full reversion of polA or lysogenization of polA ⁺ is lethal for the dnaA46polA ^- bacteria that carry the plasmid only in integrated state. Partial reversion of polA allows these cells to grow at both low and high temperatures. Introduction of the plasmid pBR322 into cytoplasm of these bacteria suppresses the lethal effect caused by full reversion of polA or lysogenization of polA ⁺. This lethal effect expresses independent of the presence or absence of the dnaA mutation. In partial revertants of polA which have only integrated plasmid, the number of copies of a region near the replication origin of integrated plasmid increases. The number is reduced by the presence of extrachromosomal pBR322. It is suggested that the lethal effect of normal levels of DNA polymerase I in strains that carry only the integrated plasmid is due to excessive initiation of replication of the bacterial chromosome from the plasmid origin and high potential of initiation can be absorbed in many copies of cytoplasmic plasmid, probably, in their replication origins.Abbreviations Amp^r ampicillin resistant (resistance) - Tet^s tetracycline sensitive - Tet^r tetracycline resistant - MMS^r methyl methane sulfonate resistant (resistance) - ts temperature sensitive - Kb kilobase pairs     

Integrative suppression of dnaA46 by broad host-range plasmid R1162

Summary Replication of plasmid R1162 DNA does not require the product of the dnaA gene. An integrated copy of the plasmid can suppress the temperature-sensitive dnaA46 allele when (1) additional plasmid copies are present in the cytoplasm and (2) an inactive replication origin, generated by deletion, is also present in the chromosome. We propose that the inactive origin sets the rate of initiation of chromosome replication at a level compatible with cell viability, possibly by providing additional binding sites for an R1162-encoded protein that is rate-limiting for plasmid replication.     

Tn501 insertion mutagenesis in Pseudomonas aeruginosa PAO

Summary Transposon insertion mutagenesis of the Pseudomonas aeruginosa PAO chromosome with Tn1 and Tn501 was carried out using a mutant plasmid of R68::Tn501 temperature-sensitive for replication and maintenance. This method consists of three steps. Firstly, the temperature-independent, drug-resistant clones were selected from the strain carrying this plasmid. In the temperature-indepent clones, the plasmid was integrated into the chromosome by Tn1- or Tn501-mediated cointegrate formation. Secondly, such clones were cultivated at a permissive temperature to provoke the excision of the integrated plasmid from the chromosome. Excision occurred by the reciprocal recombination between the two copies of Tn1 or Tn501 flanking the integrated plasmid, leaving one Tn1 or Tn501 insertion on the chromosome. Thirdly, the excised plasmid was cured by cultivating these isolates at a non-permissive temperature without selection for the drug resistance. Using this method, we isolated 1 Tn1-induced and 43 Tn501-induced auxotropic mutations in this organism. Genetic mapping allowed us to identify two new genes, pur-8001 and met-8003. The Tn501-induced auxotrophic mutations were distributed non-randomly among auxotrophic genes, and the reversion of the mutations by precise excision of the Tn501 insertion occurred very rarely.     

Selection and timing of replication of plasmids R1drd-19 and F'lac in Escherichia coli.

The selection and timing of plasmid replication was studied in exponentially growing cultures of Escherichia coli K-12 carrying the plasmid R1drd-19 and E. coli strains B/r A and B/r F carrying the plasmid F′lac. In all cases plasmid replication was studied by analysis of covalently closed circular (CCC) DNA. The turnover time of replicating plasmid DNA into CCC-DNA was found to be less than 4 min. Density shift experiments (from ¹⁵NH₄⁺, D₂O to ¹⁴NH₄⁺, H₂O) showed that plasmids R1drd-19 and F′lac are selected randomly for replication. This means that one of the plasmid copies in a cell is selected and replicated. There is no further plasmid replication in the cell until all plasmid copies, including the newly formed ones, have the same probability of being selected for replication. The early kinetics of the appearance of light plasmid DNA after the density shift showed that the time interval between successive replications of plasmids R1drd-19 and F′lac is $\text{τ}\text{n}$, where τ is the generation time and n is the average number of plasmid replications per cell and cell cycle. In a second type of experiment, exponentially growing cells were separated into a series of size classes by low-speed centrifugation in sucrose step gradients. Replication of plasmids R1drd-19 and F′lac was equally frequent in all size classes. This result is in accordance with the results of the density shift experiment. It can therefore be concluded that replication of plasmids R1drd-19 and F′lac is evenly spread over the whole cell cycle, which means that one plasmid replication occurs every time the cell volume has increased by one initiation mass.     

Mapping of the in vivo start site for leading strand DNA synthesis in plasmid R1.

We have previously constructed Escherichia coli strains in which an R1 plasmid is integrated into the origin of chromosome replication, oriC. In such intR1 strains, oriC is inactive and initiation of chromosome replication instead takes place at the integrated R1 origin. Due to the large size of the chromosome, replication intermediates generated at the R1 origin in these strains are considerably more long-lived than those in unintegrated R1 plasmids. We have taken advantage of this and performed primer extensions on total DNA isolated from intR1 strains, and mapped the free 5' DNA ends that were generated as replication intermediates during R1 replication in vivo. The sensitivity of the mapping was considerably improved by the use of a repeated primer extension method (RPE). The free DNA ends were assumed to represent normal in vivo start sites for leading strand DNA synthesis in plasmid R1. The ends were mapped to a short region approximately 380 bp away from the R1 minimal origin, and the positions agreed well with previous in vitro mappings. The same start positions were also utilized in the absence of the DnaA protein, indicating that DnaA is not required for determination of the position at which DNA synthesis starts during initiation of replication at the R1 origin.     

Isolation and characterization of Rhodobacter capsulatus strains lacking endogenous plasmids

Phodobacter capsulatus (formerly Rhodopseudomonas capsulata) strain B10 was found to contain a single plasmid of molecular weight 86×10⁶. Strains lacking this plasmids were isolated by various methods from strains containing the mutant R plasmid, pTH10. With the exception of two strains, which were found to contain chromosomal insertions of R plasmid DNA, strains lacking the endogenous plasmid appeared to be unaffected in any of the following metabolic or genetic functions: photosynthetic, autotrophic, diazotrophic, and dark, anaerobic growth; the production of bacteriocin; homologous recombination; the restriction of foreign DNA; and the production of gene transfer agent. DNA-DNA hybridization experiments confirmed that the plasmid had been eliminated from these strains and not become integrated into the chromose. However, sequences homologous to those of the endogenous plasmid were found to be present in the chromosome of R. capsulatus B10. This suggests, among other possibilities, that the endogenous plasmid may have originated in the chromosome, and might serve to duplicate certain chromosomal functions.Abbreviations kb kilobase-pair - GTA gene transfer agent - Cma chromosome mobilizing ability     

Isolation of transfer-negative nif-plasmids (pCE1) and their integration into the chromosome of Escherichia coli with the help of phage Mu

Summary Strain JC5466 of Escherichia coli K12 harbouring the nitrogen fixation plasmid pCE1 was lysogenized with bacteriophage Mu cts, followed by partial induction and infection with bacteriophage PRD1. This made it possible to obtain transfer-defective derivatives of pCE1, carrying Mu prophage. These derivatives could be mobilized by using the helper plasmid pME400 and it was possible to segregate the helper plasmid from the donor plasmid in the transconjugants.By incubating the strains 302 and 328 at 42°C, for induction of Mu prophage, derivatives with different plasmid contents could be obtained such as strains without plasmids, some with smaller or larger plasmids and others possessing plasmids without any visible alteration in size. Integration of the nitrogen-fixation (nif) genes into the chromosomes of the strains without plasmids and those containing a smaller plasmid, was confirmed by Southern hybridization using radioactive nifKDH DNA. Conjugation assays have shown that the plasmid is integrated into the chromosome as a unit but that it can also be excised.     

Plasmids in different strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2

Summary Five strains of Streptomyces ambofaciens were examined for their plasmid content. Among these strains, four belong to the same lineage (strains B) and the other was isolated independently (strain A). A large plasmid (ca. 80 kb), called pSAM1 in this paper and already described, was present in all B strains, and absent in strain A. A second plasmid, not described before, was found as covalently closed circular DNA in two of the four B strains. This plasmid with a size 11.1 kb was called pSAM2. A restriction map for 14 enzymes was established. Hybridization experiments showed that a unique sequence homologous to this plasmid is integrated in a larger replicon, which is not pSAM1 and is probably the chromosome, in all B strains and not in strain A. It seems probable that the integrated se1uence is the origin of the free plasmid found in two strains of the B family. It is noteworthy that the integrated form and the free plasmid may be found together. Transformation experiments proved that pSAM2 may be maintained autonomously in S. ambofaciens strain A and in S. lividans. pSAM2 is a self-transmissible plasmid, able to elicit the lethal zygosis reaction. pSAM2 was compared to the plasmids SLP1, pIJ110 and pIJ408, which all come from integrated sequences in three Streptomyces species and are found as autonomous plasmids after transfer to S. lividans. If pSAM2 resembles these plasmids in its origin, it does not appear to be related directly to them. Concerning their plasmid content, the two isolates of S. ambofaciens are very different. One of them contains neither pSAM1 not pSAM2. As this isolate produces spiramycin, these plasmids probably do not play an important role in spiramycin production. Apart from its intrinsic biological interest, pSAM2 may be useful in the construction of cloning vectors for S. ambofaciens. Very stable transformants might be obtained in certain strains of S. ambofaciens, because of the possibility of integration of the pSAM2 derivative vector.     

A mitochondrial plasmid from the plant pathogenic fungus Cochliobolus heterostrophus

Summary Twenty-three strains of Cochliobolus heterostrophus were examined for the presence of plasmid DNA. One isolate, T40, contained a 1.9 kb sequence which occurred as a series of circular head-to-tail multimers with from 1 to 17 or more monomers per plasmid molecule. The plasmid was cloned in pBR322 to facilitate analysis. It was homologous to the mitochondrial chromosome of isolate T40 as well as to the mitochondrial DNAs of C. heterostrophus isolates that did not contain the plasmid; each isolate, including T40, had only one copy of the plasmid sequence integrated into the mitochondrial chromosome and the sequence mapped at the same location in all isolates tested. In the T40 isolate there were about 30 excised copies per chromosome in addition to the single integrated sequence. Presence of the plasmid had no apparent effect on the structural integrity of the mitochondrial chromosome. There was no detectable homology between the plasmid and either C. heterostrophus nuclear DNA or plasmids that have been isolated from mitochondria of Neurospora or Podospora. A circular map was constructed which has 6 sites for hexan-ucleotide-recognizing enzymes and the region of the splice site; no sites were detected in the plasmid for an additional 17 restriction enzymes. The plasmid functioned as an ARS (autonomously replicating sequence) in yeast, although it was highly unstable compared to other ARSs.     

Location and characterisation of a new replication origin in the E. coli K12 chromosome.

Summary A segment of DNA located in the region of the E. coli K12 chromosome previously identified by the Rac phenotype can function as a self-replicating plasmid. Evidence is presented that this plasmid, the oriJ plasmid, contains the origin of replication of a defective prophage postulated to be located in this chromosomal region by Low (1973). The plasmid can only be maintained in strains in which this postulated prophage has been deleted. In strains which possess the prophage selection for plasmid maintenance permits the isolation of clones containing new deletions which we postulate are the result of prophage excision.     

Construction of a food-grade multiple-copy integration system for Lactococcus lactis

A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori⁺) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori⁺ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-cross-over integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5–15) × 10⁻² copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest. Received: 23 September 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

F plasmid replication and the division cycle of Escherichia coli B/r

A terminal stage in the duplication of many bacterial plasmids involves the transient formation of catenated molecules containing two interlocked monomeric plasmid units. This property of plasmid replication was exploited to examine the relationship between F replication and the division cycle of Escherichia coli B/r cells growing in undisturbed, exponential-phase cultures. Various cultures of F′lac- or FKm^r-containing cells were briefly exposed to [³H]thymidine, and then the transfer of radioactivity into, and out of, a catenated dimer consisting of two closed circular monomers was measured during a chase period. The fraction of plasmid molecules present in this dimer form was determined by separating cellular DNA in alkaline sucrose gradients. In addition, plasmid replication was studied in synchronously growing cultures by measuring both [³H]thymidine incorporation into covalently closed circular DNA and β-galactosidase inducibility. The results suggest that replication of F plasmids can take place throughout the cell division cycle, with the probability of replication increasing toward the end of the cycle. The presence of DNA homologous to the chromosome on the F′lac did not alter the replication pattern of the plasmid during the division cycle.     

Unbalanced growth as a normal feature of development of Bdellovibrio bacteriovorus

In this study we have investigated the rates and spatial patterns of chromosome replication and cell elongation during the growth phase of wild-type and facultatively prey-independent mutant strains of Bdellovibrio bacteriovorus. For the facultatively prey-independent mutants, the total DNA content of synchronously growing cultures was found to increase exponentially, as the multiple chromosomes within each filamentous cell replicated simultaneously. Cell mass, measured as total cellular protein, also increased exponentially during this period, apparently by means of multiple elongation sites along the filament wall. The relative rates of DNA and protein synthesis were unbalanced during growth, however, with the cellular concentration of DNA increasing slightly faster than that of protein. The original cellular DNA: protein ratio was restored in the progeny cells by continued protein synthesis during the septation period that follows the termination of DNA replication. Because of technical problems, these experiments could not be conducted on the wild-type cells, but similar results are assumed. This unusual pattern of unbalanced growth may represent an adaptation by bdellovibrios to maximize their progeny yield from the determinate amount of substrate available within a given prey cell.     

Autonomous plasmid‐like replication of Bacillus ICEBs1: a general feature of integrative conjugative elements?

Integrative conjugative elements (ICEs) occur frequently in Gram‐positive and Gram‐negative bacteria. In contrast to plasmids, they are stably integrated in the bacterial genome, often inserted in a tRNA gene. They are excised from the host chromosome upon induction in order to be transferred to a recipient cell. When conjugative transfer is completed, they stably reintegrate in the chromosome. It is generally thought that ICEs are incapable of autonomous replication, instead relying on replication and segregation along with the host chromosome. In this issue of Molecular Microbiology Lee and co‐workers demonstrate that ICEBs1 from Bacillus subtilis is capable of autonomous plasmid‐like replication in its circular form after excision. The authors show that ICEBs1 replication is unidirectional; it initiates at oriT_ICEBs1 and requires the ICEBs1‐encoded conjugative relaxase NicK. Replication also requires the catalytic subunit of the host DNA polymerase PolC, the host processivity clamp DnaN and the host‐encoded alternative helicase PcrA. Autonomous replication of ICEBs1 appears to be important for its stable maintenance, but not for horizontal transfer of the element. Lee and co‐workers argue that plasmid‐like replication is likely a common property of ICEs, probably contributing to stability and maintenance of ICEs in bacterial populations. I discuss these findings in context with data on other ICEs from Gram‐positive and Gram‐negative bacteria and with respect to possible consequences of the findings for basic research on mobile genetic elements from Gram‐positive bacteria and their applications in biotechnology.     

Suppression of an Escherichia coli dnaA mutation by the integrated R factor R100.1: origin of chromosome replication during exponential growth.

We have investigated the behavior, during exponential growth, of strains of Escherichia coli carrying a dnaA(Ts) mutation that has been suppressed by the integration of the F-like R plasmid R100.1. We present evidence showing that replication in these strains proceeds largely from the normal chromosome origin at 30 degrees C, a permissive temperature for the dnaA(Ts) gene product, whereas, at 42 degrees C, replication proceeds largely from the integrated plasmid. These conclusions are based on measurements made by deoxyribonucleic acid:deoxyribonucleic acid hybridization of the relative frequencies of the prophages Mu-1 and lambdaind- and R100.1 integrated at known locations on the E. coli chromosome in these Hfr strains.     

Molecular analysis of plasmid pAP4 from Acetobacter pasteurianus

The complete nucleotide sequence of plasmid pAP4 isolated from Acetobacter pasteurianus 2374^T has been determined. Plasmid pAP4 was analysed and found to be 3,870 bp in size with a G+C content of 50.1%. Computer assisted analysis of sequence data revealed 2 possible ORFs with typical promoter regions. ORF1 codes for a protein responsible for kanamycin resistance similar with Tn5 transposone, ORF2 encodes a resistance to ampicillin identical with Tn3 transposone. Plasmid has in A. pasteurianus five copies and in E. coli DH1 about 30 copies per chromosome and it segregation stability in both strains is very high. Based on the data on replication region, plasmid does not code for a replication protein and origin region is similar with ColE1-like plasmid.