Two types of cold sensitivity associated with the A184-->V change in the DnaA protein

Multicopy dnaA(Ts) strains carrying the dnaA5 or dnaA46 allele are high-temperature resistant but are cold sensitive for colony formation. The DnaA5 and DnaA46 proteins both have an A184-->V change in the ATP binding motif of the protein, but they also have one additional mutation. The mutations were separated, and it was found that a plasmid carrying exclusively the A184-->V mutation conferred a phenotype virtually identical to that of the dnaA5 plasmid. Strains carrying plasmids with either of the additional mutations behaved like a strain carrying the dnaA+ plasmid. In temperature downshifts from 42 degrees C to 30 degrees C, chromosome replication was stimulated in the multicopy dnaA46 strain. The DNA per mass ratio increased threefold, and exponential growth was maintained for more than four mass doublings. Strains carrying plasmids with the dnaA(A184-->V) or the dnaA5 gene behaved differently. The temperature downshift resulted in run out of DNA synthesis and the strains eventually ceased growth. The arrest of DNA synthesis was not due to the inability to initiate chromosome replication because marker frequency analysis showed high initiation activity after temperature downshift. However, the marker frequencies indicated that most, if not all, of the newly initiated replication forks were stalled soon after the onset of chromosome replication. Thus, it appears that the multicopy dnaA(A184-->V) strains are cold sensitive because of an inability to elongate replication at low temperature. The multicopy dnaA46 strains, on the contrary, exhibit productive initiation and normal fork movement. In this case, the cold-sensitive phenotype may be due to DNA overproduction.     

Formation of Escherichia coli Hfr strains by integrative suppression with the P group plasmid RP1.

Hfr strains of Escherichia coli were obtained by integrative suppression of a dnaA(Ts) mutation by the Inc P-1 plasmid RP1 without prior creation of an unnatural homology between the plasmid and the E. coli chromosome. Unmodified RP1 mobilized the polarized transfer of the chromosome in a counterclock-wise direction from a distinct origin between 81 min (pyrE) and 82 min (dnaA) with pyrE as a leading marker. Inheritance of RP1-Hfr chromosomal and antibiotic resistance genes was due to recombination with the recipient chromosome, as shown by the need for a functional recA system. The acquisition of temperature resistance and donor ability was accompanied by the disappearance of free plasmid when the selection pressure for integration was maintained (growth at 41 degrees C); the loss of temperature resistance and donor ability was accompanied by the reappearance of autonomous RP1 when the selection pressure was removed (growth at 30 degrees C).     

Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae.

Genes CDC24 and CDC42 are required for the establishment of cell polarity and for bud formation in Saccharomyces cerevisiae. Temperature-sensitive (Ts-) mutations in either of these genes cause arrest as large, unbudded cells in which the nuclear cycle continues. MSB1 was identified previously as a multicopy suppressor of Ts- cdc24 and cdc42 mutations. We have now sequenced MSB1 and constructed a deletion of this gene. The predicted amino acid sequence does not closely resemble any other in the available data bases, and the deletion does not produce any readily detectable phenotype. However, we have used a colony-sectoring assay to identify additional genes that appear to interact with MSB1 and play a role in bud emergence. Starting with a strain deleted for the chromosomal copy of MSB1 but containing MSB1 on a high-copy-number plasmid, mutants were identified in which MSB1 had become essential for viability. The new mutations defined two genes, BEM1 and BEM2; both the bem1 and bem2 mutations are temperature sensitive and are only partially suppressed by MSB1. In bem1 cells, a single copy of MSB1 is necessary and sufficient for viability at 23 or 30 degrees C, but even multiple copies of MSB1 do not fully suppress the growth defect at 37 degrees C. In bem2 cells, a single copy of MSB1 is necessary and sufficient for viability at 23 degrees C, multiple copies are necessary for viability at 30 degrees C, and even multiple copies of MSB1 do not suppress the growth defect at 37 degrees C. In a wild-type background (i.e., a single chromosomal copy of MSB1), both bem1 and bem2 mutations cause cells to become large and multinucleate even during growth at 23 degrees C, suggesting that these genes are involved in bud emergence. This suggestion is supported for BEM1 by other evidence obtained in a parallel study (J. Chant, K. Corrado, J. Pringle, and I. Herskowitz, submitted for publication). BEM1 maps centromere distal to TYR1 on chromosome II, and BEM2 maps between SPT15 and STP2 on chromosome V.     

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.     

Overinitiation of chromosome and plasmid replication in a dna Acos mutant of Escherichia coli K12. Evidence for dnaA-dnaB interactions

The dnaAcos mutations are phenotypic suppressors of dnaAts46 that are co-transduced with dnaA, render the cell cold sensitive, and cause an excess of chromosome replication relative to cell mass when the cells are shifted from 42 degrees C to 32 degrees C. We have used pulse labelling and DNA-DNA hybridization to follow the effect of a temperature shift on the replication of the chromosome and of the plasmids pSC101, RTF-Tc, and lambda dv in such strains. After a shift of a dnaAcos strain from 42 degrees C to 32 degrees C (non-permissive temperature), initiation of the chromosome and replication of the plasmid pSC101 are stimulated, while the dnaA-independent plasmid RTF-Tc is not affected. The presence of pSC101 does not affect the level of overinitiation of the chromosome. The presence of lambda dv suppresses the cold sensitivity of dnaAcos mutants and allows the cells to grow at both 32 degrees C and 42 degrees C. The presence of lambda dv suppresses the overinitiation of chromosome and of pSC101 replication at 32 degrees C. Previous reports had shown that these suppressions involve an interaction between the dnaA product and the lambda P protein, which is also known to interact with dnaB. We show here that the mutant prophage P1 bac-crr, which produces high levels of a dnaB analogue, suppresses the dnaAcos phenotype, while wild type P1 does not. These results suggest that initiation involves interactions between the dnaA and dnaB products.     

Effect of dna mutations on the replication of plasmid pSC101 in Escherichia coli K-12.

Escherichia coli strains with mutations in genes dnaB, dnaC, and dnaG were tested for their capacity to replicate pSC101 deoxyribonucleic acid (DNA) at a nonpermissive temperature. Only a small amount of radioactive thymine was incorporated into pSC101 DNA in the dna mutants at 42 degrees C, whereas active incorporation into plasmid DNA took place in wild-type strains under the same conditions. The effects of the dnaB and dnaC mutations were greater on plasmid DNA synthesis than on host chromosomal DNA synthesis, suggesting that these gene products are directly involved in the process of pSC101 DNA replication. In dnaG mutants, both plasmid and chromosomal DNA synthesis were blocked soon after the shift to high temperature; although the extent of inhibition of the plasmid DNA synthesis was greater during the early period of temperature shift to 42 degrees C as compared with that of the host DNA synthesis, during the later period it was less. It was found that the number of copies of pSC101 per chromosome in dnaA and dnaC strains, grown at 30 degrees C, was considerably lower than that in wildtype strains, suggesting that the replication of pSC101 in these mutant strains was partially suppressed even under the permissive conditions. No correlation was found between the number of plasmid copies and the tetracycline resistance level of the host bacterium.     

Chromosome-plasmid interaction in Escherichia coli K-12 carrying a thermosensitive plasmid, Rts1, in autonomous and in integrated states.

An Hfr strain of Escherichia coli K-12 was obtained by integrative suppression with a thermosensitive plasmid, Rts1. The R plasmid was integrated into the chromosome between rif and thr, and transfer of the chromosome occurred counterclockwise. The thermosensitivity of host cell growth due to the dnaA mutation was markedly but not completely reduced in this integratively suppressed Hfr strain. When the dnaA mutation was removed by transducing the dnaA+ genome to this Hfr, the thermosensitivity of cell growth due to existence of Rts1 was suppressed in contrast to strains carrying it autonomously. Thermosensitivity of cell growth appeared again when the plasmid was detached from the chromosome to exist autonomously. Contrary to the effect on cell growth, the transfer of the chromosome and the plasmid itself and the ability to "restrict" T-even phages were still thermosensitive in all of these strains carrying Rts1, irrespective of its state of existence. The detached plasmid as well as the original Rts1 were segregated upon growth at 42 C. These data are discussed in relation to chromosome-plasmid interaction. One of the most important conculusions is that some plasmid genes, related to their replication, are phenotypically suppressed by the chromosome when it is integrated.     

Construction, transfer and properties of a novel temperature-sensitive integrable plasmid for genomic analysis of Staphyiococcus aureus

As an alternative approach to genetic transfer and analysis, a novel integrable plasmid system was developed that should prove useful for mapping and cloning various genes in Staphylococcus aureus and other Gram-positive bacteria. The use of a restriction-deficient recipient strain and an improved protocol for protoplast plasmid transformation facilitated direct cloning of a recombinant plasmid (pPQ126) in S. aureus NCTC 8325-4. Plasmid pPQ126 (13.6 kb) is a novel, temperature-sensitive integrable plasmid containing genes encoding resistance to erythromycin and chloramphenicol (from plasmid pTV1ts), and resistance to gentamicin (from transposon Tn4001). When introduced into an appropriate recipient strain at the permissive temperature (30 degrees C), pPQ126 replicates autonomously. Integration of pPQ126 is directed into homologous chromosomal target sequences (chromosomal insertions of Tn551 or Tn4001) by growing a population of cells containing autonomous pPQ126 in the presence of gentamicin, erythromycin, and chloramphenicol at 39 degrees C (nonpermissive temperature). Elevated temperature both selects for and maintains pPQ126 as an integrated replicon. Integration of pPQ126 occurs at significantly reduced frequency in a recombination-deficient host, and does not occur in the absence of host chromosomal homology. Integrated pPQ126 excises from the chromosome under permissive conditions (30 degrees C), and excision results in derivatives of pPQ126 that harbour DNA of chromosomal origin.     

Mutations affecting replication and copy number control in plasmid mini-F both reside in the gene for the 29-kDa protein

We have isolated and characterized cop, copts, and repam mutants of plasmid mini-F after in vitro mutagenesis with hydroxylamine. cop mutants exhibit a copy number of about 10 per cell. The copts mutants are cold-sensitive and have, at 25 degrees C, a copy number of about 30-40 copies per cell, which drops to 4 copies at 42 degrees C. The cop and repam mutations affect the 29-kDa E protein. The Copts phenotype results from the simultaneous occurrence of two mutations, a cop mutation in the E protein and a temperature-dependent mutation (termed ecp) enhancing the Cop phenotype at low temperature. The latter new type of mutation is located within the DNA region 44.1-44.85F. Complementation experiments with plasmid cointegrates show that the wild-type gene is dominant over the cop allele. The nucleotide sequences of the cop and the repam mutations have been determined.     

Incompatibility properties of the IncFIII/FIV haemolytic plasmid pSU316 when integrated in the Escherichia coli chromosome

The haemolytic plasmid pSU316 is incompatible with members of the IncFIII and IncFIV incompatibility groups. Plasmid pSU307 (pSU316 hlyC::Tn5) was inserted by integrative suppression into the chromosome of JW112, a temperature-sensitive dnaA mutant of Escherichia coli. The incompatibility properties of this strain (SU51) were studied and it was found that: (1) plasmid pSU306 (pSU316 hlyA::Tn802) was rapidly lost from strain SU51 both at 30 degrees C and 42 degrees C; (2) the IncFIII plasmid pSU397 (ColB-K98::Tn802) was lost from strain SU51 and at 42 degrees C but not at 30 degrees C; and (3) the IncFIV plasmid R124 was stably maintained in strain SU51 at both temperatures. Revertants of pSU307 to the autonomous state could be obtained from SU51. These revertants exerted incompatibility towards the prototype plasmids pSU306, pSU397 and R124 in the same way as pSU307 itself. Thus, strain SU51 provided a suitable method for distinguishing the three different incompatibility determinants of plasmid pSU316.     

Integrative and free Spiroplasma citri oriC plasmids: expression of the Spiroplasma phoeniceum spiralin in Spiroplasma citri.

The replication region (oriC) of the Spiroplasma citri chromosome has been recently sequenced, and a 2-kbp DNA fragment was characterized as an autonomously replicating sequence (F. Ye, J. Renaudin, J. M. Bové, and F. Laigret, Curr. Microbiol. 29:23-29, 1994). In the present studies, we have combined this DNA fragment, containing the dnaA gene and the flanking dnaA boxes, with a ColE1-derived Escherichia coli replicon and the Tet M determinant, which confers resistance to tetracycline. The recombinant plasmid, named pBOT1, was introduced into S. citri cells, in which it replicated. Plasmid pBOT1 was shuttled from E. coli to S. citri and back to E. coli. In S. citri, replication of pBOT1 did not require the presence of a functional dnaA gene on the plasmid. However, the dnaA box region downstream of the dnaA gene was essential. Upon passaging of the S. citri transformants, the plasmid integrated into the spiroplasmal host chromosome by recombination at the replication origin. The integration process led to duplication of the oriC sequences. In contrast to the integrative pBOT1, plasmid pOT1, which does not contain the E. coli replicon, was stably maintained as a free extrachromosomal element. Plasmid pOT1 was used as a vector to introduce into S. citri the G fragment of the cytadhesin P1 gene of Mycoplasma pneumoniae and the spiralin gene of Spiroplasma phoeniceum. The recombinant plasmids, pOTPG with the G fragment and pOTPS with the spiralin gene, were stably maintained in spiroplasmal transformants. Expression of the heterologous S. phoeniceum spiralin in S. citri was demonstrated by Western immunoblotting.     

Mutations in Escherichia coli dnaA which suppress a dnaX(Ts) polymerization mutation and are dominant when located in the chromosomal allele and recessive on plasmids.

Extragenic suppressor mutations which had the ability to suppress a dnaX2016(Ts) DNA polymerization defect and which concomitantly caused cold sensitivity have been characterized within the dnaA initiation gene. When these alleles (designated Cs, Sx) were moved into dnaX+ strains, the new mutants became cold sensitive and phenotypically were initiation defective at 20 degrees C (J.R. Walker, J.A. Ramsey, and W.G. Haldenwang, Proc. Natl. Acad. Sci. USA 79:3340-3344, 1982). Detailed localization by marker rescue and DNA sequencing are reported here. One mutation changed codon 213 from Ala to Asp, the second changed Arg-432 to Leu, and the third changed codon 435 from Thr to Lys. It is striking that two of the three spontaneous mutations occurred in codons 432 and 435; these codons are within a very highly conserved, 12-residue region (K. Skarstad and E. Boye, Biochim. Biophys. Acta 1217:111-130, 1994; W. Messer and C. Weigel, submitted for publication) which must be critical for one of the DnaA activities. The dominance of wild-type and mutant alleles in both initiation and suppression activities was studied. First, in initiation function, the wild-type allele was dominant over the Cs, Sx alleles, and this dominance was independent of location. That is, the dnaA+ allele restored growth to dnaA (Cs, Sx) strains at 20 degrees C independently of which allele was present on the plasmid. The dnaA (Cs, Sx) alleles provided initiator function at 39 degrees C and were dominant in a dnaA(Ts) host at that temperature. On the other hand, suppression was dominant when the suppressor allele was chromosomal but recessive when it was plasmid borne. Furthermore, suppression was not observed when the suppressor allele was present on a plasmid and the chromosomal dnaA was a null allele. These data suggest that the suppressor allele must be integrated into the chromosome, perhaps at the normal dnaA location. Suppression by dnaA (Cs, Sx) did not require initiation at oriC; it was observed in strains deleted of oriC and which initiated at an integrated plasmid origin.     

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.     

Identification and characterization of a novel allele of Escherichia coli dnaB helicase that compromises the stability of plasmid P1

Bacteriophage P1 lysogenizes Escherichia coli cells as a plasmid with approximately the same copy number as the copy number of the host chromosome. Faithful inheritance of the plasmids relies upon proper DNA replication, as well as a partition system that actively segregates plasmids to new daughter cells. We genetically screened for E. coli chromosomal mutations that influenced P1 stability and identified a novel temperature-sensitive allele of the dnaB helicase gene (dnaB277) that replaces serine 277 with a leucine residue (DnaB S277L). This allele conferred a severe temperature-sensitive phenotype to the host; dnaB277 cells were not viable at temperatures above 34 degrees C. Shifting dnaB277 cells to 42 degrees C resulted in an immediate reduction in the rate of DNA synthesis and extensive cell filamentation. The dnaB277 allele destabilized P1 plasmids but had no significant influence on the stability of the F low-copy-number plasmid. This observation suggests that there is a specific requirement for DnaB in P1 plasmid maintenance in addition to the general requirement for DnaB as the replicative helicase during elongation.     

A missense mutation in the rpoC gene affects chromosomal replication control in Escherichia coli.

An RNA polymerase mutant with a single-base-pair change in the rpoC gene affects chromosome initiation control. The mutation, which is recessive, is a G to A transition leading to the substitution of aspartate for glycine at amino acid residue 1033 in the RNA polymerase beta' subunit. The chromosome copy number is increased twofold in the mutant at semipermissive growth temperatures (39 degrees C). In a delta oriC strain, in which chromosome initiation is governed by an F replicon, chromosome copy number is not affected. Plasmid pBR322 copy number is also increased in the mutant at 39 degrees C. The mutation causes a more than fivefold increased expression of the dnaA gene at 39 degrees C. It is conceivable that it is this high DnaA concentration which causes the high chromosome copy number and that the mutant RNA polymerase beta' subunit exerts its effect by altering the expression of the dnaA gene. However, other factors must be affected as well to explain why the RNA polymerase mutant can grow in a balanced fashion with a high chromosome concentration. This is in contrast to wild-type cells, which exhibit higher origin concentrations when DnaA protein is overproduced, but in which the overall DNA concentration is only moderately affected.     

A single amino acid difference between Rep proteins of P1 and P7 affects plasmid copy number

P1 and P7 are closely related plasmid prophages which are members of the same incompatibility group. We report the complete DNA sequence of the replication region of P7 and compare it to that of P1. The sequence predicts a single amino acid difference between the RepA proteins of these two plasmids, no differences in methylation sites or regions where dnaA protein is expected to bind, and no difference in the spacing of the major features of the two replicons. A P1 replicon with a mutation in repA, the gene that encodes an essential replication protein, is complemented for replication by providing either the P1 RepA protein (RepA1) or the P7 RepA protein (RepA7) in trans. Furthermore, when either of these proteins is supplied in trans, the plasmid copy number of P1 cop mutants drops to that of P1 cop+. However, when RepA7 is supplied, the copy number of P1 cop and P1 cop+ is higher than that when RepA1 is supplied. This indicates that the single amino acid difference between the two versions of the RepA protein plays an important role in determining the plasmid copy number.     

Characterization of a 56-kb plasmid of Erwinia amylovora Ea322: its noninvolvement in pathogenicity

A plasmid from Erwinia amylovora strain Ea322, pCPP60, was studied for its involvement in the phytopathogenicity of this strain. Eviction through incompatibility and curing with acridine orange did not affect the pathogenic capability of Ea322. The plasmid was characterized as self-transmissible with a narrow host range. Hybridization of its origin of replication with plasmids of different incompatibility groups revealed affiliation with IncF. The exact subgroup was not determined, although it does not belong to IncFI, IncFII, IncFIV, or IncFV. A sequence of 800 bp, required for conjugation in cis, was cloned in pUC9. A "miniplasmid" containing the origin of replication in a 1.2-kb sequence was constructed. Its high copy number was in contrast with the stringently controlled copy number of the native plasmid of one to three copies per chromosome equivalent.     

Replication properties of mini-Rts1 derivatives deleted for DnaA boxes in the replication origin

Mini-Rts1 was found to be unable to replicate in a dnaA-null mutant. However, a mini-Rts1 derivative lacking entire tandem DnaA boxes in the replication origin retained the replication ability in a dnaA+ host although its copy number was about half that of the mini-Rts1 having complete DnaA boxes. Mini-Rts1cop1 that contains a high copy number mutation in repA was found to replicate more efficiently than mini-Rts1 of wild repA when DnaA boxes were deleted. In addition, the copy number of mini-Rts1cop1 without DnaA boxes increased 1.5-fold upon removal of incI iterons, whereas that of mini-Rts1 without DnaA boxes did not increase after the iterons were deleted. These indicate that the RepAcop1 protein can initiate the replication of mini-Rts1 efficiently even when DnaA boxes are absent from the origin of replication.     

Replication and incompatibility properties of plasmid pE194 in Bacillus subtilis.

pE194, a 3.5-kilobase multicopy plasmid, confers resistance to the macrolide-lincosamide-streptogramin B antibiotics in Bacillus subtilis. By molecular cloning and deletion analysis we have identified a replication segment on the physical map of this plasmid, which consists of about 900 to 1,000 base pairs. This segment contains the replication origin. It also specifies a trans-acting function (rep) required for the stable replication of pE194 and a negatively acting copy control function which is the product of the cop gene. The target sites for the rep and cop gene products are also within this region. Two incompatibility determinants have been mapped on the pE194 genome and their properties are described. One (incA) resides within the replication region and may be identical to cop. incB, not located in the replication region, expresses incompatibility toward a copy control mutant (cop-6) but not toward the wild-type replicon.