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.     

Density Transfer Studies of DNA Isolated from BACILLUS SUBTILIS after Exposure to Phenethyl Alcohol

The aim of this study was to determine whether there are specific weak points in the Bacillus subtilis chromosome and if so whether the replication point is the site of breakage. To answer these questions, B. subtilis chromosomes were partially labeled with 5-bromodeoxyuridine (5-BUdR). Sheared or unsheared preparations of partially labeled chromosomes which may or may not contain replication forks were analyzed for the distribution of genetic markers in a CsCl density gradient. Two sets of experiments based upon the density transfer experiments of Yoshikawa and Sueoka (1963) were performed: (1) experiments in which the origin of the chromosome was labeled and (2) experiments in which the terminus of the chromosome was labeled. In the first experiment, strain 23 (thy(-), his(-)) spores were germinated in the presence of 5-BUdR for various lengths of time and then transferred to fresh medium containing phenethyl alcohol (PEA) and thymidine (TdR). The DNA was isolated before and after transfer to PEA and TdR. In the second experiment strain 23 (thy(-), his(-)) spores were germinated in the presence of TdR and then PEA was added. After various lengths of time transfer was made to fresh medium containing PEA and 5-BUdR. The DNA was extracted by an extremely gentle technique to avoid breakage and centrifuged in a CsCl density gradient. PEA was added to the germinated spores to prevent dichotomous replication, but PEA did not prevent dichotomous replication in any of these experiments. This contradicts the conclusion of others that PEA prevents the chromosome from entering a new round of replication, but allows the chromosome to complete the round of replication already begun. The following observations offer support for the hypothesis that the replication point is a weak point in the chromosome: (1) when conditions were created to obtain partially labeled chromosomes with replication points: (a) labeled markers appeared at the hybrid density, (b) unlabeled markers appeared at the light density, (c) shearing of the DNA had little effect on the CsCl density gradient, except on a small proportion of labeled markers which had not appeared at the hybrid density prior to shearing; (2) when conditions were created to obtain partially labeled chromosomes with no replication points: (a) the majority of DNA molecules appeared at an intermediate density between the hybrid and the light densities, (b) the labeled and unlabeled markers appeared in the intermediate peak with approximately the same ratio as in the DNA preparations, (c) the labeled markers were found in the intermediate peak except where dichotomous replication had occurred, (d) after shearing, the labeled markers appeared at the hybrid density and the unlabeled markers appeared at the light density. Thus it is concluded that the replication point is a weak point in the B. subtilis chromosome where breakage easily occurs.     

Identification and mapping of the replication genes of an R factor, R100-1, integrated into the chromosome of Escherichia coli K-12

A stable Hfr strain of Escherichia coli K-12 was obtained by integrative suppression by an R factor, R100-1. The R factor was integrated into the right of 81 min, and chromosome transfer occurred counterclockwise. Mating experiments revealed two linkage groups of genes on the R factor. Drug-resistant transductants of a dnaA-ts recipient from an R-factor Hfr and from an R(+) strain differ in their drug resistance patterns, temperature sensitivity, and transferability of drug resistance as well as chromosome markers. Transductants that transferred chromosome markers were further classified as to the origin and direction of chromosome transfer. For temperature-sensitive transductants, the reversion frequency to temperature resistance was determined, and these revertants were scored for transfer of drug resistance as well as chromosome markers. Two genes responsible for integrative suppression (designated as repA) and the other for autonomous replication (designated as repB) were identified and mapped. The arrangement of genes on the R factor is... (sul, str, cml)... repA... tra... (tet, repB).... The map of the autonomously replicating R factor is probably a circle connecting both sides of this linear map. Thus, a method has been established to map a plasmid that could not finely be analyzed under autonomous state by transduction. It also permits genetic analysis of genes responsible for replication of the plasmid without making use of a conditional mutant of itself but with that of the host, dnaA.     

Replication terminus of the Bacillus subtilis chromosome.

Bidirectional replication of the Bacillus subtilis chromosome terminates at a point on the circular chromosome which is symmetrically opposite to the replication origin. Since replication rates are similar in both "halves" of the chromosome, termination presumably occurs at the meeting point of the two replication forks. To investigate whether the DNA sequence of this region of the chromosome contributes to the termination event, we have determined the latest replicating region of a chromosome in which this DNA sequence is no longer symmetrically opposite to the origin. The merodiploid strain GSY1127 has a very large nontandem duplication (approximately 25% of the total chromosome length) in the left-hand half of the chromosome, so that size and symmetry of this chromosome are grossly different from those of normal strains. We have examined the replication order of genetic markers in this strain by measuring subtilis terminal marker for replication remains a terminal marker in the merodiploid, i.e., replicates later than a marker situated symmetrically opposite to the replication origin. These results were supported by replication orders determined by pulse-density transfer experiments during synchronous replication. The data obtained indicate that there is a preferred site for the termination of replication in the B. subtilis chromosome.     

Analysis of replication patterns in chromosomes of normal Chinese hamster and its cell lines

Replication patterns of the normal male Chinese hamster chromosomes and the three cell lines CHW, 1102 and 1103, were determined using fluorescent, plus Giemsa or acridine orange, techniques. The individual chromosomes or chromosomal segments were consistent in the replication patterns of normal Chinese hamster chromosomes and all the transformed cell lines. Late DNA replication was regularly identified in the long arm of the X chromosome, the entire Y chromosome, the short arms of chromosomes 6 and 7, and the paracentromeric regions of chromosomes 8, 9 and 10. A similar consistency was demonstrated in the large late replicating areas of chromosomes X and Y. Each cell line had specific marker chromosomes by which the cell line was identified and their replication patterns have been described. The chromosome analysis in cell line 1103 indicated that chromosomes 2, 3, 8 and 9 were more stable than others, of which chromosome 2 was extremely stable. The markers M4 and M5 in cell line 1103 are very interesting. The cytogenetic behaviour of marker M4 indicated a new phenomenon of translocation by simple association. The marker chromosome M5 indicated that inactivation spread to the early replicating distal region. These cell lines are very useful tools for studying replication patterns and providing a basic understanding of mammalian cytogenetics.     

Regulation of Chromosome Replication in Bacillus subtilis: Effects of Amino Acid Starvation in Strain W23

Amino acid starvation allows limited synthesis of deoxyribonucleic acid (DNA) in Bacillus subtilis strain W23. DNA synthesis increased by about 30% after leucine starvation and by about 60% after histidine starvation. Genetic analysis on the DNA synthesized after amino acid starvation showed that all genetic markers examined have replicated, regardless of which amino acid was starved for. Initially, all markers replicated equally, but upon further replication, the thr cysB and the argA to lys regions replicated ahead of their neighboring, proximal regions. This could indicate that preferred stopping sites exist in these regions or additional sites from which replication can originate reside there. The results suggest that chromosome replication continues from those sites where it had stopped during amino acid starvation.     

Genetic Mapping in Bacillus subtilis by 5-Bromouracil Sensitization to Ultraviolet Inactivation of Transforming Activities

A new method for chromosome mapping of Bacillus subtilis Marburg is presented which is based on the sensitization to ultraviolet irradiation of transforming deoxyribonucleic acid that has incorporated 5-bromouracil instead of thymine. Deoxyribonucleic acid was extracted at intervals from the outgrowing spores of a thymine-requiring mutant incubated with 5-bromodeoxyuridine and subjected to a definite dose of ultraviolet irradiation. The residual activities of various genetic markers were assayed by transformation. The marker activity of deoxyribonucleic acid that had incorporated 5-bromodeoxyuridine was approximately 10 times as sensitive to ultraviolet irradiation as that of normal deoxyribonucleic acid. The markers proximal to the replication origin were sensitized at earlier times of outgrowth than distal markers. The chromosome replication in outgrowing spores was sufficiently synchronous and allowed the definite determination of when a marker became sensitized by incorporation of 5-bromodeoxyuridine. The time, designated "sensitization time," was estimated by plotting the logarithmic values of relative residual activities versus incubation times. The map constructed with sensitization times as a measurement showed good agreement with those constructed by other methods. The replication of the chromosome under the described conditions appeared to occur in the following marker order: (purA, hisA)-(purB)-(thr, pyrA)-(metC)-(leuA)-(lys, trpC, metB).     

Transport of Donor Deoxyribonucleic Acid into the Cell Interior of Thymine-starved Bacillus subtilis with Chromosomes Arrested at the Terminus

The chromosomes of a tryptophan(-), thymine(-) double auxotroph of Bacillus subtilis were uniformly aligned at the chromosome terminus by an amino acid starvation treatment. By subsequent incubations, the starved culture was rendered competent, while its state of synchronous chromosome arrest was maintained by thymine starvation. The competent, chromosome-arrested cells were transformed for three unlinked markers, located in two different chromosome regions. Shortly after addition of deoxyribonucleic acid, the cell walls were removed with lysozyme in a medium containing deoxyribonuclease and no thymine, and the protoplasted culture was assayed for single and double transformants. It was found that markers both near and distant from the terminus entered freely into the cell interior. There was no important difference in the relative frequency of entry of different markers between synchronously arrested cells and nonsynchronized control cultures. It is concluded that entry of a given marker into the cell interior can occur even if the replication site of the chromosome is stationary at a location distant from the locus of the resident homolog of the entering marker. A mechanism of donor deoxyribonucleic acid entry involving homology at the replication fork is excluded.     

Genetic mapping of the chromosome of Rhizobium trifolii.

Cultures of the wild strain and auxotrophic mutants of Rhizobium trifolii T37 synchronized by means of phenylethanol have been mutagenized with nitrosoguanidine. Fifteen genetic markers were characterized in respect of their order and the time of replication based on the peaks of mutations of the genes. The time of R. trifolii chromosome replication was estimated using inhibitors of the initiation of DNA replication: rifampicin, chloramphenicol and phenylethanol. The replicative map of R. trifolii chromosome has been constructed. Taking into account the replicative map, linkages of the genes, and the bidirectional model of the Rhizobium chromosome replication, a circular genetic map of the chromosome of R. trifolii T37 was elaborated.     

A global response to sulfur starvation in Pseudomonas putida and its relationship to the expression of low-sulfur-content proteins

Staphylococcus xylosus is a ubiquitous bacterium frequently isolated from mammalian skin and occurring naturally on meat and dairy products. A physical and genetic map of the S. xylosus C2a chromosome was constructed by pulsed-field gel electrophoresis analysis after digestion with AscI, ApaI, I-CeuI, SfiI and SmaI enzymes and hybridization analysis. The chromosome size was estimated to be 2868+/-10 kb. Thirty-three genetic markers were mapped. The probable origin of replication (oriC) was positioned. Six rrn loci were located, and their orientation was determined. The chromosomes of six additional S. xylosus strains were also analysed by I-CeuI digestion, and an intraspecies diversity of the chromosome size and the number of rrn operons was shown.     

The Escherichia coli chromosome is organized with the left and right chromosome arms in separate cell halves

We have developed a system for the simultaneous labelling of two specific chromosomal sites using two different fluorescent ParB/parS systems. Using this, we demonstrate that the two chromosome arms are spatially arranged in newborn cells such that markers on the left arm of the chromosome lie in one half of the cell and markers on the right arm of the chromosome lie in the opposite half. This is achieved by reorganizing the chromosome arms of the two nucleoids in pre-division cells relative to the cell quarters. The spatial reorganization of the chromosome arms ensures that the two replication forks remain in opposite halves of the cell during replication. The relative orientation of the two reorganized nucleoids in pre-division cells is not random. Approximately 80% of dividing cells have their nucleoids oriented in a tandem configuration.     

Suppression of an Escherichia coli dnaA mutation by the integrated R factor R.100.1: Change of chromosome replication origin in synchronized cultures.

We have followed, by deoxyribonucleic acid-deoxyribonucleic acid hybridization, the order of replication of three chromosomal markers during a synchronous round of replication in three strains of Escherichia coli carrying a dnaAts mutation: one strain in which the F-like R factor R.100.1 was established as a plasmid and two strains in which the dnaA mutation was suppressed by the integration of R.100.1 into the chromosome. In the R+ strain at 30C, replication of the plasmid took place simultaneously with the initiation of chromosome replication at the normal origin. In the integratively suppressed Hfr strains, at 42.5 C, chromosome replication was initiated preferentially from the integrated plasmid; little or no initiation occurred at the normal origin. Similar results were obtained for the one strain tested at 30 C. For both Hfr strains at 42.5 C, the data suggest that at least part of the population replicated bidirectionally. This conclusion had been confirmed using an autoradiographic procedure. Both types of experiment indicate a wide variation in the rate of travel of individual replication forks within the population.     

Bi-Directional Chromosomal Replication in Salmonella typhimurium

Transducing frequencies of phage P22 lysates prepared from Salmonella typhimurium exponential cultures in minimal and nutrient broth media were compared. The assumption is that cells grown in a minimal medium will have one replication fork per replication unit, but cells in nutrient broth will have multiple replication forks; therefore, the frequency of genetic markers near the origin of replication will be higher in the nutrient broth culture. Analysis of transduction showed a gradient of marker frequencies from the highest (the cysG-ilv region) to the lowest (purE-trpB region) in both clockwise and counter clockwise directions. This supports our previous observation that chromosome replication proceeds bidirectionally from the origin between cysG (109 min on S. typhimurium map) and ilv (122 min) to a terminus in purE-trpB region (20 to 53 min). Since this method avoids possible artifacts of other methods, the results are assumed to reflect the sequence of chromosome replication in exponentially growing cells. Evidence for the existence of multiple replication forks in nutrient broth-grown cells was supported by the following: (i) the marker frequency data fitted the assumption of multiple replication fork formation; (ii) residual deoxyribonucleic acid increase after inhibition of protein synthesis to complete a round of chromosome synthesis which was 44% in cells grown in a minimal medium and 82% in those in nutrient broth; (iii) segregation patterns of the (3)H-thymidine-labeled chromosome strands during subsequent growth in non-radioactive medium were studied by autoradiography, and the number of replication points per chromosome per cell was estimated as 5.6 for the nutrient broth culture and 2.5 for the minimal medium culture. These data support a model of symmetrical and bidirectional chromosome replication.     

Revision of the linkage map of Bacillus subtilis 168: indications for circularity of the chromosome.

A revision of the linkage map of the Bacillus subtilis 168 chromosome has been undertaken with the use of the generalized transducing phage PBS1. The mapping of four new markers (narB1, mtlB1, aroI906, and tre-12) has allowed a determination of the relative orientation of the purB-dal segment and its linkage with the lin markers. The chromosomal segment comprised between the sacQ36 and gtaA12 markers has been linked with the narA1, ctrA1, and sacA321 markers. The recA1 marker has been mapped relative to the thyA and citB17 markers. Indications of linkage have been found between the tre-12 and catA markers and the aroG932 and sacQ36 markers. According to these results, a circular genetic map of the chromosome of B. subtilis 168 is presented. Taken together, the transduction data and the order of marker replication determined by Harford in the accompanying paper support strongly the hypothesis of a symmetrical and fully bidirectional mode of replication for the B. subtilis 168 chromosome.     

Heat Induction of Prophage φ105 in Bacillus subtilis: Replication of the Bacterial and Bacteriophage Genomes

A temperature-inducible mutant of temperate Bacillus bacteriophage phi105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in "mock-induced" wild-type phi105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome.     

Dynamics of Escherichia coli chromosome segregation during multifork replication

Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division, the chromosomes contain multiple replication forks and must be segregated while this complex pattern of replication is still ongoing. Here, we show that replication and segregation continue in step, starting at the origin and progressing to the replication terminus. Thus, early-replicated markers on the multiple-branched chromosomes continue to separate soon after replication to form separate protonucleoids, even though they are not segregated into different daughter cells until later generations. The segregation pattern follows the pattern of chromosome replication and does not follow the cell division cycle. No extensive cohesion of sister DNA regions was seen at any growth rate. We conclude that segregation is driven by the progression of the replication forks.     

Selective association of the chromosome with membrane in a stable L-form of Bacillus subtilis.

A stalbe L-form (Sal-1) of Bacillus subtilis was found to have retained a markedly modified chromosome-membrane association when compared to intact cells. The membrane-deoxyribonucleic acid complex of the L-form was similar to that of its parental strain in quantity and stability. Genetic analysis of the L-form membrane-deoxyribonucleic acid complex revealed enrichment for markers close to the replication origin, but not for internal markers, indicating preferential attachment of the origin of chromosomal replication to the membrane. These results are in close agreement with those found for the parental bacterial form. In contrast, the replication termius region was not preferentially attached to the membrane of the L-form, even though it is enriched in the bacterial form. The association of the chromosome with the membrane at the replication terminus does not appear to be necessary for cell growth and separation, but because the L-form divides aberrantly, it may be one of the factors required for normal deoxyribonucleic acid segregation and septation.     

Temperature-Sensitive Initiation of Chromosome Replication in a Mutant of Bacillus subtilis

A mutant of Bacillus subtilis Ts37 has been isolated in which deoxyribonucleic acid (DNA) synthesis is inhibited at high temperature. The results presented here indicate that the process of initiation of DNA replication is temperature sensitive in this mutant. After shifting to 45 C, DNA increases 40 to 50% before synthesis ceases; an inhibition of protein synthesis permits an equivalent amount of DNA to be synthesized. A density shift experiment coupled with a marker frequency analysis shows that DNA synthesized at 45 C is highly enriched in the markers situated at the end of the chromosome. Transforming DNA extracted from a culture which has been incubated at 45 C exhibits the relative transforming efficiency for origin and terminus markers characteristic of completed chromosomes. After a shift back from 45 C to 30 C, reinitiation appears to occur always in the same region of the bacterial chromosome; in addition, replication as well as cell division is synchronized.     

Functioning of the F-plasmid origin of replication in an Escherichia coli K12 Hfr strain during exponential growth

The pattern of chromosome replication in the Escherichia coli K12 Hfr strain KL99 was investigated during exponential growth by DNA-DNA hybridization. The levels of chromosomal markers close to the point of insertion of F (near pyrC) were raised in relation to other markers by comparison with the situation in an isogenic F- strain. The data are shown to be consistent with the proposal that the integrated F plasmid was regulating its copy number by a mass-titration mechanism.     

Replication of prophage P1 during the cell cycle of Escherichia coli.

Summary We have followed, by DNA-DNA hybridization, the variation in the number of copies of prophage P1 relative to two chromosomal markers when the doubling time of the host cells is modified by a change in carbon source. The ratio of P1/chromosome terminus undergoes a twofold decrease when the cell doubling time increases from 24 to 215 min, whereas the ratio of P1/chromosome origin increases 1.4 fold; both ratios tend towards unity at slow growth rates. This suggests that the replication of prophage P1 is not simultaneous with chromosome initiation or chromosome termination. The chromosome replication time is unaffected by the presence of P1, and remains constant over the range of doubling times studied, with a value of about 40 min. Following amino acid starvation, the P1/chromosome origin ratio increases from 0.7 to 0.9, suggesting that P1 retains the ability to replicate after chromosome initiation has stopped and in the absence of essential amino acids. The results are discussed with reference to similar studies done on F and R1.