Mutations in the CCGTTCACA DnaA box of Mycobacterium tuberculosis oriC that abolish replication of oriC plasmids are tolerated on the chromosome

The origin of replication (oriC) region in some clinical strains of Mycobacterium tuberculosis is a hot spot for IS6110 elements. To understand how clinical strains with insertions in oriC can replicate their DNA, we characterized the oriC regions of some clinical strains. Using a plasmid-based oriC-dependent replication assay, we showed that IS6110 insertions that disrupted the DnaA box sequence CCGTTCACA abolished oriC activity in M. tuberculosis. Furthermore, by using a surface plasmon resonance technique we showed that purified M. tuberculosis DnaA protein binds native but not mutant DnaA box sequence, suggesting that stable interactions of the DnaA protein with the CCGTTCACA DnaA box are crucial for replication of oriC plasmids in vivo. Replacement by homologous recombination of the CCGTTCACA DnaA box sequence of the laboratory strain M. tuberculosis H37Ra with a mutant sequence did not result in nonviability. Together, these results suggest that M. tuberculosis strains have evolved mechanisms to tolerate mutations in the oriC region and that functional requirements for M. tuberculosis oriC replication are different for chromosomes and plasmids.     

Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli.

In vivo and in vitro evidence is presented implicating a function of GATC methylation in the Escherichia coli replication origin, oriC, during initiation of DNA synthesis. Transformation frequencies of oriC plasmids into E. coli dam mutants, deficient in the GATC-specific DNA methylase, are greatly reduced compared with parental dam+ cells, particularly for plasmids that must use oriC for initiation. Mutations that suppress the mismatch repair deficiency of dam mutants do not increase these low transformation frequencies, implicating a new function for the Dam methylase. oriC DNA isolated from dam- cells functions 2- to 4-fold less well in the oriC-specific in vitro initiation system when compared with oriC DNA from dam+ cells. This decreased template activity is restored 2- to 3-fold if the DNA from dam- cells is first methylated with purified Dam methylase. Bacterial origin plasmids or M13-oriC chimeric phage DNA, isolated from either base substitution or insertion dam mutants of E. coli, exhibit some sensitivity to digestion by DpnI, a restriction endonuclease specific for methylated GATC sites, showing that these dam mutants retain some Dam methylation activity. Sites of preferred cleavage are found within the oriC region, as well as in the ColE1-type origin.     

The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome

We have developed a simple three-step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild-type chromosomal origin of a recBCsbcB host by recombination. The mutations were subsequently transferred to a wild-type host by transduction. oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function. The other mutant strains showed the same growth rates, DNA contents and cell mass as wild-type cells. Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry. In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication. We present evidence that the functionality of certain mutated oriCs is far better on the chromosome than on a minichromosome.     

Genetic mapping of the chromosomal replication origin of Salmonella typhimurium.

Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.     

Characterization of plasmids that encode streptomycin-resistance in bacterial epiphytes of apple

Streptomycin resistance in strains of Pseudomonas syringae pv. papulans, Pantoea agglomerans and a yellow-pigmented, non-fluorescent Pseudomonas sp. (Py), isolated from apple orchards in New York and Washington states, is predominantly associated with strA-strB genes carried on conjugal plasmids (R plasmids). None of 128 resistant Erwinia amylovora strains from the eastern and western USA hybridized with a strA-strB probe, SMP3. Resistant Py strains transfered R plasmids to Ps. syringae pv. papulans and to Py in vitro at frequencies of 10(-1)-10(-2) per recipient cell whereas Ps. syringae pv. papulans transferred its plasmids at frequencies of 10(-2) to below detectable levels. Transfer of R plasmids to P. agglomerans was not detected and resistant P. agglomerans did not transfer their R plasmids to any recipients. R plasmids were found to be highly diverse as measured by DNA fingerprint analysis. Transfer-deficient transposon mutants of R plasmid pCPP519 were generated, and 3.9 kb EcoRI and 3.0 kb SmaI fragments that hybridized with a Tn5 probe were cloned and sequenced. The deduced amino acid sequences of the 3.9 kb fragment were similar to proteins involved in replication, nicking at oriT, and piliation in other bacteria.     

Escherichia coli SeqA protein affects DNA topology and inhibits open complex formation at oriC.

Chromosome replication in Escherichia coli is initiated by the DnaA protein. Binding of DnaA to the origin, oriC, followed by formation of an open complex are the first steps in the initiation process. Based on in vivo studies the SeqA protein has been suggested to function negatively in the initiation of replication, possibly by inhibiting open complex formation. In vitro studies have shown that SeqA inhibits oriC-dependent replication. Here we show by KMnO(4) probing that SeqA inhibits open complex formation. The inhibition was not caused by prevention of DnaA binding to the oriC plasmids, indicating that SeqA prevented strand separation in oriC either directly, by interacting with the AT-rich region, or indirectly, by changing the topology of the oriC plasmids. SeqA was found to restrain the negative supercoils of the oriC plasmid. In comparison with the effect of HU on plasmid topology, SeqA seemed to act more cooperatively. It is likely that the inhibition of open complex formation is caused by the effect of SeqA on the topology of the plasmids. SeqA also restrained the negative supercoils of unmethylated oriC plasmids, which do not bind SeqA specifically, suggesting that the effect on topology is not dependent on binding of SeqA to a specific sequence in oriC.     

Suppression of initiation defects of chromosome replication in Bacillus subtilis dnaA and oriC-deleted mutants by integration of a plasmid replicon into the chromosomes.

We constructed Bacillus subtilis strains in which chromosome replication initiates from the minimal replicon of a plasmid isolated from Bacillus natto, independently of oriC. Integration of the replicon in either orientation at the proA locus (115 degrees on the genetic map) suppressed the temperature-sensitive phenotype caused by a mutation in dnaA, a gene required for initiation of replication from oriC. In addition, in a strain with the plasmid replicon integrated into the chromosome, we were able to delete sequences required for oriC function. These strains were viable but had a slower growth rate than the oriC+ strains. Marker frequency analysis revealed that both pyrD and metD, genes close to proA, showed the highest values among the markers (genes) measured, and those of other markers decreased symmetrically with distance from the site of the integration (proA). These results indicated that the integrated plasmid replicon operated as a new and sole origin of chromosome replication in these strains and that the mode of replication was bidirectional. Interestingly, these mutants produced anucleate cells at a high frequency (about 40% in exponential culture), and the distribution of chromosomes in the cells was irregular. A change in the site and mechanism (from oriC to a plasmid system) of initiation appears to have resulted in a drastic alteration in coordination between chromosome replication and chromosome partition or cell division.     

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.     

Involvement of DNA superhelicity in minichromosome maintenance in Escherichia coli.

Evidence is presented that Escherichia coli minichromosomes are harbored at superhelical densities which are lower than those measured for other E. coli plasmids but are comparable to that of the chromosome. When introduced into gyrB decreased-supercoiling mutants, minichromosomes were much more unstable than in strains with normal or increased supercoiling properties; in fact, certain minichromosome derivatives could not be introduced into top gyrB decreased-supercoiling mutants. These observations were unique to minichromosomes, since the maintenance of plasmids which did not replicate from oriC was not altered in these mutants. Analyses of minichromosomes of identical sizes but with different restriction fragment orientations suggested that supercoiling-dependent alterations in promoter-terminator functions, as well as direct effects of supercoiling on replication, may play a role in the observed minichromosome instability.     

Characterization of the functional replication origin of Mycobacterium tuberculosis.

The gene order in the 5kb Mycobacterium tuberculosis dnaA region is rnpA, rpmH, dnaA, dnaN and recF. We show that M. tuberculosis DNA fragment containing the dnaA-dnaN intergenic region functioned as oriC, i.e., allowed autonomous replication to otherwise nonreplicative plasmids, in M. tuberculosis H37Ra (H37Ra), avirulent strain of M. tuberculosis, and in Mycobacterium bovis BCG (BCG), a closely related, slowly growing mycobacterial strain. Removal of Escherichia coli plasmid replication origin (ColE1) from the M. tuberculosis oriC plasmids did not abolish their ability to function as oriC, confirming that the autonomous replication activity of these plasmids is due to the presence of the DNA fragment containing the dnaA-dnaN intergenic region. Deletion analyses revealed that the minimal oriC DNA fragment is 814bp. The copy number of M. tuberculosis oriC plasmids containing ColE1 ori relative to chromosomal oriC is one and the 5' flanking region of minimal oriC contains features that support stable autonomous replication. The M. tuberculosis oriC did not function in rapidly growing mycobacterial species such as M. smegmatis. M. smegmatis oriC functioned only in M. fortuitum, but not in any of the slowly growing mycobacterial species such as M. tuberculosis and BCG. Together these data suggest that the replication initiation mechanisms in the slowly growing Mycobacteria are similar and probably different from those in the rapidly growing Mycobacteria and vice versa.     

Topoisomerase I confers specificity in enzymatic replication of the Escherichia coli chromosomal origin

Crude soluble enzyme fractions that initiate bidirectional replication from the unique Escherichia coli chromosomal origin (oriC) have been fractionated further to identify the components and mechanisms of this complex system. Among the necessary factors is a class of specificity proteins that suppress initiations on plasmids which lack the oriC sequence and which do not depend on dnaA protein. One such specificity factor has been identified as RNase H (Ogawa, T., Pickett, G. G., Kogoma, T., and Kornberg, A. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1040-1044). Another, described here, has proved to be topoisomerase I. A protein was purified to near homogeneity based on assays of (i) inhibition of the replication of plasmids (and other supercoiled DNA) lacking oriC and (ii) conferral of dnaA protein dependence on the replication of an oriC plasmid. This specificity protein is indistinguishable from authentic E. coli topoisomerase I by several criteria: (i) molecular weight under denaturing conditions, (ii) relaxation activity on negatively supercoiled DNA, (iii) cleavage pattern of single-stranded DNA, (iv) specificity factor activity, and (v) neutralization of activity by antibody against topoisomerase I. One possible mechanism of the specificity action of topoisomerase I is destabilization of primers for replication except when they are preserved at an oriC sequence bound by dnaA protein and other replication proteins.     

Transfer of a gonococcal beta-lactamase plasmid to conjugation-deficient Neisseria cinerea strains by transformation

We have previously shown that some strains of Neisseria cinerea can serve as recipients in conjugation (Con+) with Neisseria gonorrhoeae while others cannot (Con-). To determine if a replication defect contributes to the inability of certain strains of N. cinerea to serve as recipients in conjugation, we attempted to introduce a naturally occurring gonococcal beta-lactamase plasmid into N. cinerea by transformation. Various methods were employed, and all proved unsuccessful. Since specific sequences are required for DNA uptake in transformation of N. gonorrhoeae, we constructed a number of hybrid plasmids containing N. cinerea chromosomal DNA inserted into the N. gonorrhoeae/Escherichia coli beta-lactamase shuttle vector, pLES2. When nine randomly selected plasmids with inserts were used to transform an N. cinerea strain which did not accept the gonococcal beta-lactamase plasmid by conjugation, transformants were observed with four of the hybrid plasmids. The presence of one of the hybrid plasmids, pCAG9, in transformants was confirmed by agarose gel electrophoresis, Southern hybridization, and beta-lactamase production. When an N. gonorrhoeae donor strain containing pCAG9 was used in conjugation with several N. cinerea strains, only those strains that were previously shown to act as recipients could accept and maintain pCAG9. The ability of pCAG9 and the other three hybrid plasmids to transform Con- strains demonstrates that the beta-lactamase plasmid can replicate in Con- strains, and, therefore, the Con- phenotype is due to a block in some other stage of the conjugation process.     

Isolation and characterization of plasmids carrying a partially defective Escherichia coli replication origin

The replication origin (oriC) of the Escherichia coli chromosome has been cloned and the region essential for chromosomal replication has been delimited to 245 base pairs. In previous studies the ability of recombinants between oriC and ColE1-type vectors, to transform E. coli polA- strains was used to determine which nucleotides in oriC are essential for replication. In this paper we have used a different approach by isolating partial defective replication mutants of a minichromosome (pCM959) that contains oriC as the single replication origin. Our results demonstrate that many mutations are allowed within oriC that do not affect oriC function as measured by the ability to transform E. coli polA- strains. In the minimal oriC region we detected 8 mutations at positions that are conserved in the sequence of six bacterial origins. The implications of these results on previous work will be discussed. Our data also demonstrate that a mutation producing an oriC- phenotype may be suppressed by secondary mutations. An E. coli strain was found that facilitates the isolation of partially defective minichromosomes. The results with this strain indicate a specific function of the sequence surrounding the base pair at position 138.     

Inhibition of DNA synthesis at the hemimethylated pBR322 origin of replication by a cell membrane fraction.

The replication of both ColE1-type plasmids and plasmids bearing the origin of replication of the Escherichia coli chromosome (oriC) has been shown to be inhibited by hemimethylation of adenine residues within GATC sequences. In the case of oriC plasmids, this inhibition was previously shown to be mediated by the specific affinity of the hemimethylated origin DNA for an outer cell membrane fraction. Here, we suggest that a similar mechanism is operating in the case of the ColE1-like plasmid pBR322 as (i) a hemimethylated DNA fragment carrying the promoter for the RNA which primes DNA synthesis (RNAII) is specifically bound by the same membrane fraction and, (ii) the addition of the membrane fraction to a soluble assay of pBR322 replication results in preferential inhibition of initiation on the hemimethylated template. We suggest that membrane sequestration of hemimethylated origin DNA and/or associated replication genes following replication may be a common element restricting DNA replication to precise moments in the cell cycle.     

Klebsiella pneumoniae origin of replication (oriC) is not active in Caulobacter crescentus, Pseudomonas putida, and Rhodobacter sphaeroides.

A DNA fragment carrying genes encoding the conjugal transfer system of the broad host range plasmid RK2 was inserted into a plasmid carrying the chromosomal origin of replication (oriC) from Klebsiella pneumoniae. The resulting plasmid, pEON1, was readily transferred between gram-negative bacteria and carried two potential origins of replication: oriC and the replication origin from pBR322 (oriPBR). Although pEON1 could be transferred to Caulobacter crescentus, Pseudomonas putida, and Rhodobacter sphaeroides, pEON1 was not maintained in these strains. However, an oriC-containing plasmid was maintained in these nonenteric bacteria when an RK2 origin of replication was present on the plasmid. Thus, the inability of pEON1 to be established in a nonenteric bacterium represents a failure of oriC to function as an origin of replication rather than a toxic effect of oriC. The initiation potential of the chromosomal origin of replication from K. pneumoniae appears to be realized only in enteric bacteria.     

The segregation of Escherichia coli minichromosomes constructed in vivo by recombineering

Circularized regions of the chromosome containing the origin of replication, oriC, can be maintained as autonomous minichromosomes, oriC plasmids. We show that oriC plasmids containing precise, pre-determined segments of the chromosome can be generated by a simple in vivo recombineering technique. We generated two such plasmids carrying fluorescent markers. These were transferred to a recipient strain with a different fluorescent marker near the chromosomal copy of oriC. Thus the fates of the oriC plasmid and chromosomal origins could be followed independently in living cells by fluorescence microscopy. In contrast to a previous report, we show that there is a strong tendency of oriC plasmid copies to accumulate at the cell center as a single or double focus at the plane of cell division. This is not simply due to exclusion from the nucleoid space but rather appears to be a specific recognition and retention of the plasmid by some central-located cell site.     

Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells.

Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome. The absence of the initiation cascade in Dam- cells is proposed to account for this observation of apparent incompatibility between plasmid and chromosomal copies of oriC. Studies using oriC-pBR322 chimeric plasmids and their deletion derivatives indicated that the incompatibility determinant is an intact and functional oriC sequence. The seqA2 mutation was found to overcome the incompatability phenotype by increasing the cellular oriC copy number 3-fold thereby allowing minichromosomes to coexist with the chromosome. The replication pattern of a wild-type strain with multiple integrated minichromosomes in the oriC region of the chromosome, led to the conclusion that initiation of DNA replication commences at a fixed cell mass, irrespective of the number of origins contained on the chromosome.     

Host controlled plasmid replication: Escherichia coli minichromosomes

Escherichia coli minichromosomes are plasmids replicating exclusively from a cloned copy of oriC, the chromosomal origin of replication. They are therefore subject to the same types of replication control as imposed on the chromosome. Unlike natural plasmid replicons, minichromosomes do not adjust their replication rate to the cellular copy number and they do not contain information for active partitioning at cell division. Analysis of mutant strains where minichromosomes cannot be established suggest that their mere existence is dependent on the factors that ensure timely once per cell cycle initiation of replication. These observations indicate that replication initiation in E. coli is normally controlled in such a way that all copies of oriC contained within the cell, chromosomal and minichromosomal, are initiated within a fairly short time interval of the cell cycle. Furthermore, both replication and segregation of the bacterial chromosome seem to be controlled by sequences outside the origin itself.     

Conversion to bidirectional replication after unidirectional initiation from R1 plasmid origin integrated at oriC in Escherichia coli

The cell division phenotypes of Escherichia coli with its chromosome replication driven by oriR (from plasmid R1) were examined by fluorescence microscopy and flow cytometry. Chromosome replication patterns in these strains were followed by marker frequency analyses. In one of the strains, the unidirectional oriR was integrated so that the replication fork moved clockwise from the oriC region, and bacterial growth and division were similar to those of the wild-type parent. The bacteria were able to convert the unidirectional initiation from oriR into bidirectional replication. The site for conversion of uni- to bidirectional replication seemed to be localized and could be mapped genetically within 6 min to the immediate right of the minimal oriC . Replication starting in the counterclockwise direction from the R1 replicon integrated at the same site in the opposite orientation could not be described as either bi- or unidirectional, as no single predominant origin could be discerned from the more or less flat marker frequency pattern. These strains also showed extensive filamentation, irregular nucleoid distribution and the presence of anucleate cells, indicative of segregation and division defects. Comparison among intR1 derivatives differing in the position of the integrated oriR relative to the chromosome origin suggested that the oriC sequence itself was dispensable for the conversion to bidirectionality. However, passage of the replication fork over the 6 min region to the right of oriC seemed important for the bidirectional replication pattern and normal cell division phenotype.     

The 245 base-pair oriC sequence of the E. coli chromosome directs bidirectional replication at an adjacent region

The replication origin of the E. coli K-12 chromosome has been isolated as autonomously replicating molecules(oriC plasmid), and the DNA region essential for replicating function(oriC) has been localized to a sequence of 232-245 base-pairs(bp) by deletion analysis. In this report, the functional role of oriC was analysed by using an in vitro replication system and various OriC+ and OriC- plasmids previously constructed. The results obtained were summarized as follows: (1) The oriC sequence contained information enough to direct bidirectional replication. (2) The actual DNA replication began at a region near, but outside, oriC and progressed bidirectionally. (3) Initiation of DNA synthesis at the specific region required the dnaA-complementing fraction from cells harboring a dnaA-carrying plasmid.