Analogs of the dnaB gene of Escherichia coli K-12 associated with conjugative R plasmids.

The dnaB266(Am) mutation in Escherichia coli K-12 is an amber mutation such that strains carrying this mutation are not viable in a sup+ strain. With five different R plasmids, it has been possible to construct viable R+ derivatives of this amber mutant and show that the plasmids themselves do not carry amber suppressors. This is interpreted as evidence for the presence of dnaB analog genes associated with these plasmids. Plasmid-positive strains carrying these genes often showed some degree of cryosensitivity of DNA synthesis and colony-forming ability. These observations indicate that the presence of dnaB analog genes in association with R plasmids must be relevant to the plasmid state or to some aspect of conjugative ability.     

dnaB125, a dnaB nonsense mutation

A temperature-sensitive dnaB mutation, dnaB125, was shown to be a suppressed amber mutation. The effects of inserting different amino acids at the mutated site via amber suppressors were examined for both Escherichia coli and bacteriophage gamma growth. In addition, the dnaB125 amber allele was shown to be different from the previously described dnaB amber allele, dnaB266. The extent of residual deoxyribonucleic acid synthesis observed in a supF(Ts) dnaB125 strain at high temperature revealed that the dnaB protein was present in excess and that deoxyribonucleic acid synthesis could continue for several generation equivalents without further production of dnaB protein.     

Effect of temperature on formation of lysozyme in E. coli CRT 266 (dnaB ts) after infection with bacteriophage T3]

Lysozyme formation induced by bacteriophage T3 was studied in the ts-mutant E. coli CRT 266 (dnaBts) and in the wild-type E. coli CR 34--45 (dnaB+) at different temperatures. It was found that lysozyme was formed in E. coli CRT 266, however, no lysozyme synthesis took place at 41.5 degrees C. These results indicate that the expression of the lysozyme gene is disturbed in the ts-mutant at 41.5 degrees C.     

Mechanism of dnaB protein action. I. Crystallization and properties of dnaB protein, an essential replication protein in Escherichia coli

Purification and crystallization of dnaB protein from Escherichia coli was performed on a large scale by a simple procedure. From 1.5 kg of cells, 520 mg of dnaB protein were obtained in a 58% yield with a purity greater than 99%. The E. coli cells harbor a high copy-number plasmid carrying the dnaB gene and overproduce the enzyme over 200-fold. The subunit molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 50,000. Based on a native Mr = 290,000 and cross-linking studies that yielded six bands, dnaB protein is judged to be a hexamer, confirming the results of Reha-Krantz, L. J., and Hurwitz, J. (1978) J. Biol. Chem. 253, 4043-4050.     

Effects of temperature-sensitive variants of the Bacillus subtilis dnaB gene on the replication of a low-copy-number plasmid.

The dnaB gene of Bacillus subtilis is involved in the initiation of DNA replication and also in the binding of the chromosomal origin to the bacterial membrane. We studied the effect of temperature-sensitive dnaB mutants (dnaB1 and dnaB19) on the replication and on the DNA-membrane binding of the plasmid pKW1, which was derived from the low-copy-number plasmid pBS2. In the dnaB19 mutant, pKW1 was not able to replicate at the restrictive temperature. In the dnaB1 mutant, however, the dimeric form of pKW1 DNA was preferentially produced as the restrictive temperature, but the replication of the monomeric form was totally blocked. We also examined the effects of the dnaB(Ts) gene on the DNA-membrane binding of both the double-stranded and single-stranded DNA from pKW1. The single-stranded DNA from pKW1 was prepared from the DNA of the phage M13 mp19, which contained the origin of replication of pKW1. In the dnaB1 mutant, pKW1 DNA in both the double-stranded and single-stranded form was released from the membrane at the restrictive temperature. On the other hand, in the dnaB19 mutant, only double-stranded DNA, and not single-stranded DNA, was released from the membrane at the restrictive temperature. These results suggest that the product of the dnaB gene has at least two domains which influence the replication of DNA and the binding of DNA to the cell membrane in separate ways.     

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.     

Suppression of dnaC alleles by the dnaB analog (ban protein) of bacteriophage P1.

The dnaB analog protein produced by the ban gene of bacteriophage P1 was shown to suppress several Escherichia coli dnaC alleles. Suppression of dnaC7 temperature sensitivity in P1 lysogens of a dnaC7 mutant was complete at all temperatures. For the dnaC2 and dnaC28 alleles, suppression was observed only at intermediate temperatures. Though these intermediate temperatures were sufficient to completely restrict the mutants, at higher temperatures the suppression was not observed. No suppression of the dnaC1 allele was detected. These results have implications concerning the requirement for the dnaB-dnaC complex at the various stages of deoxyribonucleic acid replication.     

The dnaB gene product of Escherichia coli. I. Purification, homogeneity, and physical properties.

The dnaB gene product was purified to homogeneity and its physical properties were characterized. Purification was aided by the use of the Escherichia coli strain. MV12/28, which overproduced the dnaB gene product 10-fold (Wickner, S. H., Wickner, R. B., and Raetz, C. R. H. (1976) Biochem. Biophys. Res. Commun. 70, 389-396) and by taking advantage of the enzyme's high affinity for both DEAE-cellulose and phosphocellulose. The most highly purified fractions gave a single stained band on native, polyacrylamide gels and dnaB enzymatic activity was coincident with this band. On denaturing sodium dodecyl sulfate-polyacrylamide gels, a single band was observed corresponding to a molecular weight of 48,000 +/- 2,000. The native molecular weight of 290,000 +/- 12,000 was calculated from determinations of the sedimentation coefficient, which was 11.3 S, and the Stokes radius, which was 60 A. Cross-linking the protein with dimethyl suberimidate yielded six bands. We conclude that the enzyme consists of six identical subunits. The apparent pI was 4.9 and the amino acid composition was typical except for the absence of cysteine.     

Analysis of the dnaB function of Escherichia coli K12 and the dnaB-like function of P1 prophage

The dnaB function of Escherichia coli K12 was studied with a series of isogenic strains differing from each other only by a mutation in the dnaB gene. The strains showed different phenotypes depending on the particular dnaB mutation they carry. A clear example is provided by a strain carrying dnaB266 mutation which turned out to be an amber mutation. When the mutation was suppressed by different suppressors, the strains showed different phenotypes. Thus, dnaB proteins which differ from each other by only one amino acid at the mutation site give different phenotypes. Mutation dnaB266 is lethal to the host when not suppressed. Hence the dnaB protein is essential for bacterial growth.Three P1 mutants, P1mcb-4, P1mcb-5 and P1mcb-8, were isolated which converted the temperature-sensitive bacterial growth of dnaB266-supE to resistant growth. Lysogenization with P1mcb allowed growth of dnaB266su⁻ strain which was absolutely defective in the bacterial dnaB function, indicating that the dnaB-like function of P1 prophage can substitute for the bacterial dnaB function. However, lysogenization by P1mcb did not support the growth of λ and λπ phages on dnaB 266su⁻. While P1mcb-4 and P1mcb-5 prophages altered the phenotypes of other dnaB strains to permit the growth of bacterial and λ phage at 32 °C and 42 °C, P1mcb-8 prophage supports the growth of λ phages and bacteria at 42 °C but not λ phage growth on groP-bacteria at 32 °C. The alteration of phenotypes of the P1mcb lysogens varied depending on the dnaB mutations they carried. Mutual interaction between the bacterial dnaB protein and the phage dnaB-like protein which results in different phenotypes of lysogens is suggested.     

The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase isoenzymes.

In this paper we describe the cloning and sequence analysis of the tyrB and aspC genes from Escherichia coli K12, which encode the aromatic aminotransferase and aspartate aminotransferase respectively. The tyrB gene was isolated from a cosmid carrying the nearby dnaB gene, identified by its ability to complement a dnaB lesion. Deletion and linker insertion analysis located the tyrB gene to a 1.7-kilobase NruI-HindIII-digest fragment. Sequence analysis revealed a gene encoding a 43 000 Da polypeptide. The gene starts with a GTG codon and is closely followed by a structure resembling a rho independent terminator. The aspC gene was cloned by screening gene banks, prepared from a prototrophic E. coli K12 strain, for plasmids able to complement the aspC tyrB lesions in the aminotransferase-deficient strain HW225. Sub-cloning and deletion analysis located the aspC gene on a 1.8-kilobase HincII-StuI-digest fragment. Sequence analysis revealed the presence of a gene encoding a 43 000 Da protein, the sequence of which is identical with that previously obtained for the aspartate aminotransferase from E. coli B. Considerable overproduction of the two enzymes was demonstrated. We compared the deduced protein sequences with those of the pig mitochondrial and cytoplasmic aspartate aminotransferases. From the extensive homology observed we are able to propose that the two E. coli enzymes possess subunit structures, subunit interactions and coenzyme-binding and substrate-binding sites that are very similar both to each other and to those of the mammalian enzymes and therefore must also have very similar catalytic mechanisms. Comparison of the aspC and tyrB gene sequences reveals that they appear to have diverged as much as is possible within the constraints of functionality and codon usage.     

Regulation of dnaB function in DNA replication in Escherichia coli by dnaC and lambda P gene products

The dnaB protein of Escherichia coli, a multifunctional DNA-dependent ribonucleotide triphosphatase and dATPase, cross-links to ATP on ultraviolet irradiation under conditions that support rNTPase and dATPase activities of dnaB protein. The covalent cross-linking to ATP is specifically inhibited by ribonucleotides and dATP. Tryptic peptide mapping demonstrates that ATP cross-links to only the 33-kDa tryptic fragment (Fragment II) of dnaB protein. The presence of single-stranded DNA alters the covalent labeling of dnaB protein by ATP, suggesting a possible role of DNA on the mode of nucleotide binding by dnaB protein. Present studies demonstrate that the dnaC gene product binds ribonucleotides independent of dnaB protein. On dnaB-dnaC protein complex formation, covalent incorporation of ATP to dnaB protein decreases approximately 70% with a concomitant increase of ATP incorporation to dnaC protein by approximately 3-fold. The mechanism of this phenomenon has been analyzed in detail by titrating dnaB protein with increasing amounts of dnaC protein. The binding of dnaC protein to dnaB protein appears to be a noncooperative process. The lambda P protein, which interacts with dnaB protein in the bacteriophage lambda DNA replication, does not bind ATP in the presence or absence of dnaB protein. However, lambda P protein enhances the covalent incorporation of ATP to dnaB protein approximately 4-fold, suggesting a direct physical interaction between lambda P and dnaB proteins with a probable change in the modes of nucleotide binding to dnaB protein. The lambda P protein likely forms a lambda P-dnaB-ATP dead-end ternary complex. The implications of these results in the E. coli and bacteriophage lambda chromosomal DNA replication are discussed.     

Analysis of an Escherichia coli dnaB temperature-sensitive insertion mutation and its cold-sensitive extragenic suppressor.

An Escherichia coli mutant, ts121, was isolated following random insertional mutagenesis using phage lambda Mu transposition. The mutant phenotype includes inability to form colonies at temperatures above 38 degrees C and inability to propagate phage lambda at all temperatures. A lambda i434 cI- (ts121)+ transducing phage was isolated on the basis of its ability to form plaques on ts121 mutant bacteria. Using this transducing phage, it was shown through complementation and protein analyses, that the ts121 mutation is located in the dnaB gene. The exact insertion event was identified by polymerase chain reaction amplification of the DNA sequences containing the insertion junction. The mutational insertion event in ts121 was mapped precisely between base pairs 1514 and 1515 of the dnaB gene. This result predicts that the mutant dnaB protein has lost its six terminal amino acids. The reading frame shifts into Mu-specific DNA sequences resulting in an additional 20 amino acid residues. The E. coli wild type dnaB protein participates in host replication and interacts with lambda P protein to initiate phage lambda DNA replication. Our results demonstrate that the extreme carboxyl end of the dnaB protein is required for productive interaction with the lambda P replication protein at all temperatures, and is important for dnaB function at temperatures above 38 degrees C. Cold-sensitive extragenic suppressors of the ts121 mutation were isolated on the basis of their ability to restore colony formation at 42 degrees C. One of these extragenic suppressors was mapped at 54 min on the E. coli genetic map and localized to the suhB gene, whose product may affect the expression of a number of genes at the translational level.     

Origin of Haemophilus influenzae R factors.

The Haemophilus influenzae R plasmids specifying resistance against one, two, or three antibiotics which have emerged in different parts of the world were shown to have closely related but not identical plasmid cores. The gene for ampicillin resistance in the H. influenzae plasmid pKRE5367 is part of a transposon similar to Tn3, which was transposed from pKRE5367 onto RSF1010 in Escherichia coli. An indigenous H. influenzae plasmid (pW266) was isolated. Its properties correspond to those of the H. influenzae R plasmids, except for the presence of a drug resistance transposon. The in vitro-generated H. influenzae R plasmids carrying an ampicillin resistance transposon, a tetracycline resistance transposon, and a transposon for combined tetracycline-chloramphenicol resistance resembled the natural isolates. The findings support the hypothesis that the R plasmids of H. influenzae are of multiclonal evolutionary origin.     

Defective replication activity of a dominant-lethal dnaB gene product from Escherichia coli.

dnaB protein of Escherichia coli is an essential replication protein. A missense mutant has been obtained which results in replacement of an arginine residue with cysteine at position 231 of the protein (P. Shrimankar, L. Shortle, and R. Maurer, unpublished data). This mutant displays a dominant-lethal phenotype in strains that are heterodiploid for dnaB. Biochemical analysis of the altered form of dnaB protein revealed that it was inactive in replication in several purified enzyme systems which involve specific and nonspecific primer formation on single-stranded DNAs, and in replication of plasmids containing the E. coli chromosomal origin. Inactivity in replication appeared to be due to its inability to bind to single-stranded DNA. The altered dnaB protein was inhibitory to the activity of wild type dnaB protein in replication by sequestering dnaC protein which is also required for replication. By contrast, it was not inhibitory to dnaB protein in priming of single-stranded DNA by primase in the absence of single-stranded DNA binding protein. Sequestering of dnaC protein into inactive complexes may relate to the dominant-lethal phenotype of this dnaB mutant.     

Escherichia coli deoxyribonucleic acid synthesis mutants: their effect upon bacteriophage P2 and satellite bacteriophage P4 deoxyribonucleic acid synthesis.

Escherichia coli C strains containing different deoxyribonucleic acid (DNA) synthesis mutations have been tested for their support of the DNA synthesis of bacteriophage P2 and its satellite phage P4. Bacteriophage P2 requires functional dnaB, dnaE, and dnaG E. coli gene products for DNA synthesis, whereas it does not require the products of the dnaA, dnaC, or dnaH genes. In contrast, the satellite virus P4 requires functional dnaE and dnaH gene products for DNA synthesis and does not need the products of the dnaA, dnaB, dnaC, and dnaG genes. Thus the P2 and P4 genomes are replicated differently, even though they are packaged in heads made of the same protein.     

Restoration of F' superinfection inhibition in a DnaB mutant of Escherichia coli upon construction of heterozygous DnaB merodiploids or P1 lysogens carrying a dnaB analogue.

F-prime derivatives of the Escherichia coli strain CR34 bearing the thermosensitivity mutation dnaB43 display low levels of plasmid-determined superinfection inhibition in conjugational crosses at 30 C. Salt-mediated phenotypic suppression of this temperature sensitivity fails to restore normal levels of inhibition, indicating its alteration is not a secondary effect of dnaB43 a-tion on growth or deoxyribonucleic acid syntheiss. Superinfection inhibition is fully restored in mutant cells made merodiploid for the dnaB region by introduction of the F' dnaB-+ plasmid F134-1. dnaB43-bearing strains lysogenized with P1 phage contribution dnaB-analogue protein show eight to nine times more superinfection inhibition than do the same cells carrying P1 prophage repressed dnaB-analogue protein production. Taken together, this evidence suggests a direct causal relationship between dnaB43 and the altered superinfection inhibition phenotype.     

In vivo mutagenesis of bacteriophage Mu transposase.

We devised a method for isolating mutations in the bacteriophage Mu A gene which encodes the phage transposase. Nine new conditional defective A mutations were isolated. These, as well as eight previously isolated mutations, were mapped with a set of defined deletions which divided the gene into 13 100- to 200-base-pair segments. Phages carrying these mutations were analyzed for their ability to lysogenize and to transpose in nonpermissive hosts. One Aam mutation, Aam7110, known to retain the capacity to support lysogenization of a sup0 host (M. M. Howe, K. J. O'Day, and D. W. Shultz, Virology 93:303-319, 1979) and to map 91 base pairs from the 3' end of the gene (R. M. Harshey and S. D. Cuneo, J. Genet. 65:159-174, 1987) was shown to be able to complement other A mutations for lysogenization, although it was incapable of catalyzing either the replication of Mu DNA or the massive conservative integration required for phage growth. Four Ats mutations which map at different positions in the gene were able to catalyze lysogenization but not phage growth at the nonpermissive temperature. Phages carrying mutations located at different positions in the Mu B gene (which encodes a product necessary for efficient integration and lytic replication) were all able to lysogenize at the same frequency. These results suggest that the ability of Mu to lysogenize is not strictly correlated with its ability to perform massive conservative and replicative transposition.