DNA and protein sequence conservation at the replication terminus in Bacillus subtilis 168 and W23.

Cloned DNA from the replication terminus region of Bacillus subtilis 168 was used to identify and construct a restriction map of the homologous region in B. subtilis W23. With this information, DNA from the terminus region of W23 was cloned and the sequence was determined for a 1,499-base-pair segment spanning the expected terC site. The position of the site was then located more precisely. Use of the cloned DNA from strain W23 as a probe for digests of DNA from exponentially growing cells of the same strain established the presence of the slowly migrating replication termination intermediate (forked DNA). The orientation and dimensions of the forked molecule were consistent with arrest of the clockwise fork at the terC site in W23, as has been shown to occur in strain 168. Thus, despite significant differences between the two strains, the same termination mechanism appears to be used. The DNA sequences spanning the terC site in strains 168 and W23 showed a high level of homology (90.2%) close to the site but very little at a distance of approximately 250 base pairs from the site in one particular direction. The overall sequence comparison emphasised the importance of the open reading frame for a 122-amino-acid protein adjacent to terC. Although there were 22 base differences in the open reading frames between the strains, the amino acid sequence of the encoded protein was completely conserved. It is suggested that the amino acid sequence conservation reflects a role for the protein in the clockwise fork arrest mechanism as proposed earlier (M.T. Smith and R.G. Wake, J. Bacteriol. 170:4083-4090, 1988).     

Expression of the rtp gene of Bacillus subtilis is required for replication fork arrest at the chromosome terminus

It was earlier proposed that clockwise replication fork arrest at the chromosome terminus in Bacillus subtilis is dependent upon expression of the rtp gene adjacent to the site of arrest, terC [Smith and Wake, J. Bacteriol. 170 (1988) 4083-4090]. A merodiploid strain of B. subtilis, in which rtp was placed under the control of the IPTG-inducible spac-1 promoter, was constructed. Replication fork arrest at terC, as monitored by the level of a forked DNA molecule of predicted dimensions, was shown to be dependent upon IPTG-induced expression of rtp in this strain. The very low concentration of IPTG needed to induce a substantial level of fork arrest suggests that relatively little RTP, the protein product of rtp, is needed for fork arrest at terC.     

Evidence of a ter specific binding protein essential for the termination reaction of DNA replication in Escherichia coli.

Activity binding specifically to the 22 bp of the DNA replication terminus (ter) sequence on plasmid R6K and the Escherichia coli genome was detected in the crude extract of E. coli cells. This activity was inactivated by heat or by protease but not by RNase treatments. Overproduction of the ter binding activity was observed when the extract was prepared from the cell carrying a plasmid with a chromosomal-derived 5.0 kb EcoRI fragment, on which one of the four terC sites, terC2, was also located. By mutagenesis of the 5.0 kb fragment on the plasmid with transposon Tn3 and subsequent replacement of the corresponding chromosomal region with the resulting mutant alleles, we isolated tau- mutants completely defective in ter binding activity. These mutants simultaneously lost the activity to block the progress of the DNA replication fork at any ter site, on the genome or the plasmid. It would thus appear that the ter binding protein plays an essential role in the termination reaction, at the ter sites.     

Insertion and deletion mutations in the repA4 region of the IncFII plasmid NR1 cause unstable inheritance.

Mutants of IncFII plasmid NR1 that have transposons inserted in the repA4 open reading frame (ORF) are not inherited stably. The repA4 ORF is located immediately downstream from the replication origin (ori). The repA4 coding region contains inverted-repeat sequences that are homologous to the terC inverted repeats located in the replication terminus of the Escherichia coli chromosome. The site of initiation of leading-strand synthesis for replication of NR1 is also located in repA4 near its 3' end. Transposon insertions between ori and the right-hand terC repeat resulted in plasmid instability, whereas transposon insertions farther downstream did not. Derivatives that contained a 35-bp frameshift insertion in the repA4 ORF were all stable, even when the frameshift was located very near the 5' end of the coding region. This finding indicates that repA4 does not specify a protein product that is essential for plasmid stability. Examination of mutants having a nest of deletions with endpoints in or near repA4 indicated that the 3' end of the repA4 coding region and the site of leading-strand initiation could be deleted without appreciable effect on plasmid stability. Deletion of the pemI and pemK genes, located farther downstream from repA4 and reported to affect plasmid stability, also had no detectable effect. In contrast, mutants from which the right-hand terC repeat, or both right- and left-hand repeats, had been deleted were unstable. None of the insertion or deletion mutations in or near repA4 affected plasmid copy number. Alteration of the terC repeats by site-directed mutagenesis had little effect on plasmid stability. Plasmid stability was not affected by a fus mutation known to inactivate the termination function. Therefore, it appears that the overall integrity of the repA4 region is more important for stable maintenance of plasmid NR1 than are any of the individual known features found in this region.     

Regularities of the location of genes having different functions and of some other nucleotide sequences in the bacterial chromosome

The review considers the results of genomic research performed over the last decade that shed light on the location in the bacterial chromosomes of genes having different functions. A tendency towards polarity of the chromosome composition is observed: vitally important genes tend to be concentrated in the region of replication origin (oriC), and their concentration decreases toward the region of replication termination (terC). An oppositely directed polarity (an increase near the terC region) is observed for the distribution of certain oligonucleotides involved in the process of chromosome recombination and segregation.     

Regularities of the location of genes having different functions and of some other nucleotide sequences in the bacterial chromosome

The review considers the results of genomic research performed over the last decade that shed light on the location in the bacterial chromosomes of genes having different functions. A tendency towards polarity of the chromosome composition is observed: vitally important genes tend to be concentrated in the region of replication origin (oriC), and their concentration decreases toward the region of replication termination (terC). An oppositely directed polarity (an increase near the terC region) is observed for the distribution of certain oligonucleotides involved in the process of chromosome recombination and segregation.     

Suppression of Escherichia coli dnaA46 mutations by integration of plasmid R100.1. derivatives: constraints imposed by the replication terminus.

We have studies the phenotypic suppression of a dnaA46 mutation by plasmid integration at preselected chromosomal sites after introducing homologous sequences (Mu prophages) onto both the chromosomes and the suppressive plasmid. The plasmids used were all derived from plasmid R100.1. We found that the conditions required to get viable suppressive integration varied as the plasmid integration site moved from the origin to the terminus of chromosome replication. Two constraints were observed. Both appeared to be linked to the new characteristics acquired by chromosome replication from the integrated plasmid. One constraint was that strains with integrative suppression near the terminus terC were viable only in minimal medium. The rich medium sensitivity of these strains was correlated with a loss of regulation of initiation. The other constraint was a requirement for a specific orientation in certain regions of the chromosome. The two branches defined by normally initiated replication, between oriC and terC, were also symmetrical with respect to these plasmid orientation constraints. In studying the possible reasons for a plasmid orientation constraint, we found that, of the two forks initiated in bidirectional replication from the integrated plasmid, one was capable of moving across the terC region with a higher movability than the other.     

Replication fork arrest at relocated replication terminators on the Bacillus subtilis chromosome.

The replication terminus region of the Bacillus subtilis chromosome, comprising TerI and TerII plus the rtp gene (referred to as the terC region) was relocated to serC (257 degrees) and cym (10 degrees) on the anticlockwise- and clockwise-replicating segments of the chromosome, respectively. In both cases, it was found that only the orientation of the terC region that placed TerI in opposition to the approaching replication fork was functional in fork arrest. When TerII was opposed to the approaching fork, it was nonfunctional. These findings confirm and extend earlier work which involved relocations to only the clockwise-replicating segment, at metD (100 degrees) and pyr (139 degrees). In the present work, it was further shown that in the strain in which TerII was opposed to an approaching fork at metD, overproduction of the replication terminator protein (RTP) enabled TerII to function as an arrest site. Thus, chromosomal TerII is nonfunctional in arrest in vivo because of a limiting level of RTP. Marker frequency analysis showed that TerI at both cym and metD caused only transient arrest of a replication fork. Arrest appeared to be more severe in the latter situation and caused the two forks to meet at approximately 145 degrees (just outside or on the edge of the replication fork trap). The minimum pause time erected by TerI at metD was calculated to be approximately 40% of the time taken to complete a round of replication. This significant pause at metD caused the cells to become elongated, indicating that cell division was delayed. Further work is needed to establish the immediate cause of the delay in division.     

Impediment to replication fork movement in the terminus region of the Bacillus subtilis chromosome

The terminus regions of the chromosomes of three strains of Bacillus subtilis 168 were radioactively labelled by supplying [3H]thymine towards the end of a round of replication. These strains lacked or contained the prophage SP beta c2. Following restriction endonuclease digestion of the purified DNA and fluorography, an SP beta c2-related perturbation of the terminus-labelling profile was observed, which was completely consistent with the previously suggested existence of an impediment to replication fork movement (terC) within a BamHI 24.8 X 10(3) base fragment (Weiss & Wake, 1983). The present data suggest that terC is located within the 11.4 X 10(3) base BamHI + SalI double-digest portion of this BamHI fragment.     

Cloning and localization of the Bacillus subtilis chromosome replication terminus, terC

A 10.9-kb segment of the Bacillus subtilis 168 chromosome has been cloned in an Escherichia coli plasmid and shown to contain terC (the replication terminus of the chromosome). The terC-containing portion of this plasmid has been subcloned within each of two overlapping fragments of DNA, 1.75 and 1.95 kb, again in E. coli plasmids. These have afforded a more precise definition of the location of terC in the B. subtilis chromosome and provided material for a detailed analysis of the structure and functioning of this site.     

Nonrandom cosmid cloning and prophage SP beta homology near the replication terminus of the Bacillus subtilis chromosome.

From a library of Bacillus subtilis DNA cloned with the Escherichia coli cosmid vector pHC79, 85 recombinant cosmids containing DNA from near the replication terminus, terC, were identified. The DNA inserts of these cosmids were confined to three regions of a 350-kilobase segment of the chromosome extending from the left end of the SP beta prophage to approximately 75 kilobases on the right of terC. All B. subtilis genes known to reside in this segment, as well as the portion of the SP beta prophage that is expressed early in the lytic cycle of the phage, appeared to be absent from the library. A region of SP beta homology distinct from the prophage and just to the left of terC was identified.     

Unidirectional theta replication of the structurally stable Enterococcus faecalis plasmid pAM beta 1.

Numerous bacterial replicons remain poorly characterized due to difficulties in localization of the replication origin. We have circumvented this problem in the characterization and fine mapping of the origin of plasmid pAM beta 1 by exploiting the Bacillus subtilis termination signal, terC. In terC-containing derivatives, theta-form molecules with two invariant endpoints accumulate. The endpoints, which correspond to plasmid origin and terC, were mapped with single-nucleotide precision. Analysis of the replication intermediates of wild-type molecules by two-dimensional gel electrophoresis confirmed the location of the plasmid origin. Our results demonstrate that pAM beta 1 replication proceeds unidirectionally by a theta mechanism. This work confirms the use of termination signals to localize origins, suggests that termination in B. subtilis occurs by a mechanism similar to that of Escherichia coli and establishes that in addition to rolling circle replicating plasmids, Gram positive bacteria harbour plasmids which replicate by a theta mechanism.     

Relocation of the replication terminus, terC, of Bacillus subtilis to a new chromosomal site

The terC-deletion strain of Bacillus subtilis 168, SU153 [Iismaa and Wake, J. Mol. Biol. 195 (1987) 299-310] was used for the re-insertion of a 1.75-kb segment of DNA containing terC at a site approx. 25 kb from its original position. The relocated terC in the new strain, SU160, was oriented normally with respect to the approaching clockwise replication fork, and was positioned such that this fork was the first to reach it. The relocated terC was effective in causing arrest of the clockwise fork, as evidenced by the appearance of a unique DNA species with a characteristic mobility in agarose gel electrophoresis and with a predicted single-strand composition. Thus, the previously cloned 1.75-kb terC-containing segment [Smith et al., Gene 38 (1985) 9-17] has not been altered with respect to TerC function and contains sufficient sequence for this function. The findings reported here provide the opportunity for establishing the minimal and essential sequence features of terC, and for examining its possible polarity of action in causing fork arrest.     

Requirement of IS911 replication before integration defines a new bacterial transposition pathway

Movement of transposable elements is often accompanied by replication to ensure their proliferation. Replication is associated with both major classes of transposition mechanisms: cut-and-paste and cointegrate formation (paste-and-copy). Cut-and-paste transposition is often activated by replication of the transposon, while in cointegrate formation replication completes integration. We describe a novel transposition mechanism used by insertion sequence IS911, which we call copy-and-paste. IS911 transposes using a circular intermediate (circle), which then integrates into a target. We demonstrate that this is derived from a branched intermediate (figure-eight) in which both ends are joined by a single-strand bridge after a first-strand transfer. In vivo labelling experiments show that the process of circle formation is replicative. The results indicate that the replication pathway not only produces circles from figure-eight but also regenerates the transposon donor plasmid. To confirm the replicative mechanism, we have also used the Escherichia coli terminators (terC) which, when bound by the Tus protein, inhibit replication forks in a polarised manner. Finally, we demonstrate that the primase DnaG is essential, implicating a host-specific replication pathway.     

Sequence features of the replication terminus of the Bacillus subtilis chromosome.

The sequence of 1267 nucleotides spanning the replication terminus, terC, of the Bacillus subtilis 168 chromosome has been determined. The site of arrest of the clockwise fork, which defines terC, has been localized to a 30-nucleotide portion (approximately) within this sequence. The arrest site occurs in an A + T-rich region between two open reading frames and very close to one of two imperfect inverted repeats (47-48 nucleotides each) which are separated by 59 nucleotides. The closeness of approach of the arrested clockwise fork to the first imperfect inverted repeat encountered in this region raises the possibility of a role for the inverted repeats in the mechanism of fork arrest.     

The normal replication terminus of the Bacillus subtilis chromosome, terC, is dispensable for vegetative growth and sporulation

The Bacillus subtilis strains CU1693, CU1694 and CU1695 were shown by hybridization analysis to carry large deletions of the terminus region that originated within discrete fragments of the SP beta prophage genome. The absence of terC in CU1693 was demonstrated definitively by the identification of a novel junction fragment comprising SP beta DNA and DNA that lies on the other side of terC in the parent strain. This represented the deletion of approximately 230 kb of CU1693 DNA, with the removal of approximately 150 kb to the left of terC and approximately 80 kb to the right of terC. The lack of hybridization of CU1694 and CU1695 DNA to cloned DNA carrying the terC sequence and to cloned DNAs flanking terC suggested that terC is absent from the chromosome of each of these strains also, and that the deletions in CU1694 and CU1695 extend beyond the segment of the terminus region that has been mapped and cloned. The normal growth rate and morphology of CU1693, CU1694 and CU1695 relative to the parent strain when grown in complex medium indicated dispensability of terC for vegetative growth and division. B. subtilis SU153 was constructed using a specific deletion-insertion vector that was designed to effect the deletion of 11.2kb of DNA spanning terC, with the removal of approximately 9.7kb to the left of terC and approximately 1.kb to the right of terC. This manipulation did not introduce any readily detectable auxotrophic requirement. Physiological characterization of SU153 confirmed the dispensability of terC for vegetative growth and cell division, and also established the lack of requirement of terC for the specialized cell division that is associated with formation of the bacterial endospore.     

A checkpoint involving RTP, the replication terminator protein, arrests replication downstream of the origin during the Stringent Response in Bacillus subtilis

Regulation of DNA replication in Bacillus subtilis involves a post-initiation mechanism which is subject to control by the Stringent System, an essential regulatory network, mediated by the alarmone, ppGpp. In detailed studies using DNA-DNA hybridization procedures, we have now shown that, following the induction of the Stringent Response, replication is blocked downstream of the origin, on the left, close to the hut marker (-175 kb) and on the right, beyond the soft10 marker (+199 kb). In addition, we provide evidence that inhibition of replication under these conditions requires the replication terminator protein (RTP). In a mutant lacking RTP, a protein normally involved in termination of chromosomal replication through recognition of specific terminator sequences, replication continues past the sites normally blocked by the Stringent Response. These data strengthen the argument that this second level of control of DNA replication occurs at specific sites, the Stringent Terminus (STer) sites, either side of oriC Such sites are presumably related to the sequence involved in RTP recognition at the terminus, terC. We propose that the binding of RTP must be modulated, perhaps through the action of ppGpp, to recognize post-initiation control sequences during the Stringent Response, in order to block replisome movement. This, therefore, acts as a checkpoint in chromosome elongation.     

DNA replication termination in Escherichia coli parB (a dnaG allele), parA, and gyrB mutants affected in DNA distribution.

We investigated the Escherichia coli mutants carrying the parB, parA, and gyrB mutations, all of which display faulty chromosome partitioning at the nonpermissive temperature, to see whether their phenotype reflected a defect in the termination of DNA replication. In the parB strain DNA synthesis slowed down at 42 degrees C and the SOS response was induced, whereas in the parA strain DNA synthesis continued normally for 120 min and there was no SOS induction. To see whether replication forks accumulated in the vicinity of terC at the nonpermissive temperature, the mutants were incubated for 60 min at 42 degrees C and then returned to low temperature and pulse-labeled with [3H]thymidine. In all cases the restriction pattern of the labeled DNA was incompatible with that of the terC region, suggesting that replication termination was normal. In the parA mutant no DNA sequences were preferentially labeled, whereas in the parB and gyrB strains there was specific labeling of sequences whose restriction pattern resembled that of oriC. In the case of parB this was confirmed by DNA-DNA hybridization with appropriate probes. This test further revealed that the parB mutant over initiates at oriC after the return to the permissive temperature. Like dna(Ts) strains, the parB mutant formed filaments at 42 degrees C in the absence of SOS-associated division inhibition, accompanied by the appearance of anucleate cells of nearly normal size (28% of the population after 3 h), as revealed by autoradiography. The DNA in the filaments was either centrally located or distributed throughout. The parB mutation lies at 67 min, and the ParB- phenotype is corrected by a cloned dnaG gene or by a plasmid primase, strongly suggesting that parB is an allele of dnaG, the structural gene of the E. coli primase. It is thus likely that the parB mutant possesses an altered primase which does not affect replication termination but causes a partial defect in replication initiation and elongation and in chromosome distribution.