Role of sog polypeptides specified by plasmid ColIb-P9 and their transfer between conjugating bacteria.

The sog gene of the conjugative plasmid ColIb-P9 specifies two sequence-related polypeptides with the N-terminal third of the larger product having DNA primase activity. To resolve the function of the C-terminal portion of the polypeptides, we constructed a ColIb mutant containing a Tn5 insertion in the 3' region of sog. The mutation truncated sog gene products without inactivating DNA primase and rendered the plasmid defective in conjugation. Tests for the presence of conjugative pili, for complementation by a sog+ recombinant, and for mobilization of small origin of transfer (oriT) recombinant plasmids indicated that the mutant ColIb allows conjugative aggregation of cells but it is defective in DNA transfer at some stage subsequent to its initiation at oriT. Physical evidence is given that normal sog polypeptides are among a group of proteins transferred selectively from the donor to the recipient cell by a conjugation-specific process. No transfer of the mutant sog proteins was detected. It is proposed that the C-terminal region of sog polypeptides facilitates transfer of single-stranded ColIb DNA between conjugating cells following initiation of transfer at the oriT site, and that in this role the proteins are transmitted to the recipient cell.     

Role and specificity of plasmid RP4-encoded DNA primase in bacterial conjugation.

The role of the DNA primase of IncP plasmids was examined with a derivative of RP4 containing Tn7 in the primase gene (pri). The mutant was defective in mediating bacterial conjugation, with the deficiency varying according to the bacterial strains used as donors and recipients. Complementation tests involving recombinant plasmids carrying cloned fragments of RP4 indicated that the primase acts to promote some event in the recipient cell after DNA transfer and that this requirement can be satisfied by plasmid primase made in the donor cell. It is proposed that the enzyme or its products or both are transmitted to the recipient cell during conjugation, and the role of the enzyme in the conjugative processing of RP4 is discussed. Specificity of plasmid primases was assessed with derivatives of RP4 and the IncI1 plasmid ColIb-P9, which is known to encode a DNA primase active in conjugation. When supplied in the donor cell, neither of the primases encoded by these plasmids substituted effectively in the nonhomologous conjugation system. Since ColIb primase provided in the recipient cell acted weakly on transferred RP4 DNA, it is suggested that the specificity of these enzymes reflects their inability to be transmitted via the conjugation apparatus of the nonhomologous plasmid.     

Plasmid RP4 specifies a deoxyribonucleic acid primase involved in its conjugal transfer and maintenance.

We surveyed plasmids representative of most incompatibility groups for their conferred deoxyribonucleic acid (DNA) primase activity. RP4 (IncP) was one of the few with such activity although, unlike the derepressed IncIalpha plasmids (which also specify a primase), it did not suppress the dnaG mutation. Using deletion and Tn7 derivatives of RP4, we located the presumed primase structural gene (pri) in the 37- to 42-kilobase region. Tn7 insertions in the adjacent Tra1 region also reduced or caused overproduction of primase. We purified the RP4 primase to a single polypeptide of molecular weight 118,000. It is an anisometric molecule and functions as a monomer, initiating complementary strand synthesis on phi X174 DNA in Escherichia coli dnaG cell extracts in the presence of ribonucleotide triphosphates and rifampin. It is immunologically unrelated to either the E. coli dnaG or the IncIalpha plasmid-specified DNA primases. RP4 pri mutants conjugated with a lower efficiency into some bacterial species, including Salmonella typhimurium. Back-transfer experiments showed that this effect was recipient specific. There was also a comparable reduction in mobilization efficiency of R300B by RP4 pri into such recipients. Loss of RP4 primase led to detectable plasmid instability. The RP4-specified primase therefore seems to serve two functions: the single DNA strand transferred during conjugation is primed by it in the recipient cell, and it appears to be necessary for the efficient priming of discontinuous plasmid DNA replication despite the presence of the chromosomal priming system.     

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.     

DNA-independent transport of plasmid primase protein between bacteria by the I1 conjugation system

The ColIb-P9 (IncI1)-encoded conjugation system supports transfer of the plasmid T-strand plus hundreds of molecules of the Sog polypeptides determined by the plasmid primase gene. Here, we report that Sog primase is abundantly donated to the recipient cell from cells carrying a non-transferable ColIb plasmid deleted of the nic site essential for DNA export. Such DNA-independent secretion of Sog primase is typical of authentic conjugation, both in being blocked when the recipient cell specifies the entry exclusion function of ColIb and in requiring the thin I1 pilus encoded by the ColIb pil system under the mating conditions used. It is proposed that Sog polypeptides form a complex with the ColIb T-strand during conjugation and aid DNA transport through processive secretion of the proteins into the recipient cell. Functional and genetic relationships between the ColIb conjugation system and other type IV secretion pathways are discussed.     

Temperature-Sensitive Mutants for the Replication of Plasmids in Escherichia coli: Requirement for Deoxyribonucleic Acid Polymerase I in the Replication of the Plasmid ColE1

An Escherichia coli mutant (polA1), defective in deoxyribonucleic acid (DNA) polymerase I, (EC 2.7.7.7) is unable to maintain colicinogenic factor E1 (ColE1), whereas several sex factor plasmids are maintained normally in this strain. polA1 mutant strains containing these sex factor plasmids do not exhibit a readily detectable plasmid-induced polymerase activity. A series of E. coli mutants that are temperature sensitive for ColE1 maintenance, but able to maintain other plasmids, were isolated and shown to fall into two phenotypic groups. Mutants in one group are defective specifically in ColE1 maintenance at 43 C, but exhibit normal DNA polymerase I activity. Mutations in the second group map in the polA gene of E. coli, and bacteria carrying these mutations are sensitive to methylmethanesulfonate (MMS). Revertants that were selected either for MMS resistance or the ability to maintain ColE1 were normal for both properties. The DNA polymerase I enzyme of two of these mutants shows a pronounced temperature sensitivity when compared to the wild-type enzyme. An examination of the role of DNA polymerase I in ColE1 maintenance indicates that it is essential for normal replication of the plasmid. In addition, the presence of a functional DNA polymerase I in both the donor and recipient cell is required for the ColV-promoted conjugal transfer of ColE1 and establishment of the plasmid in the recipient cell.     

Extrachromosomal DNA isolated from tomato big bud and Candidatus Phytoplasma australiense phytoplasma strains

The nucleotide sequences of two extrachromosomal elements from tomato big bud (TBB) and one extrachromosomal element from Candidatus Phytoplasma australiense (Ca. P. australiense) phytoplasmas were determined. Both TBB plasmids (3319 and 4092 bp) contained an open reading frame ( approximately 570 bp) with homology to the rolling circle replication initiator protein (Rep). This gene was shorter than the rep genes identified from other phytoplasma plasmids, geminiviruses and bacterial plasmids. Both TBB extrachromosomal DNAs (eDNAs) encoded a putative DNA primase (dnaG) gene, a chromosomal gene required for DNA replication and which contains the conserved topoisomerase/primase domain. We speculate that the replication mechanism for the TBB phytoplasma eDNA involves the dnaG gene instead of the rep gene. The Ca. P. australiense eDNA (3773 bp) was shown to be circular and contained four open reading frames. The rep gene was encoded on ORF 1 and had homology to both plasmid (pLS1) and geminivirus-like domains.     

Replication of the Bacteriocinogenic Plasmid Clo DF13 in Thermosensitive Escherichia coli Mutants Defective in Initiation or Elongation of Deoxyribonucleic Acid Replication

The replication of the bacteriocinogenic plasmid Clo DF13 has been studied in the seven temperature-sensitive Escherichia coli mutants defective in deoxyribonucleic acid (DNA) replication (dnaA-dnaG). Experiments with dna initiation mutants revealed that the replication of the Clo DF13 plasmid depends to a great extent on the host-determined dnaC (dnaD) gene product, but depends slightly on the dnaA gene product. The synthesis of Clo DF13 plasmid DNA also requires the dnaF and dnaG gene products, which are involved in the elongation of chromosomal DNA replication. In contrast, the Clo DF13 plasmid is able to replicate in the dnaB and dnaE elongation mutants at the restrictive temperature. When de novo protein synthesis is inhibited by chloramphenicol in wild-type cells, the Clo DF13 plasmid continues to replicate for at least 12 h, long after chromosomal DNA synthesis has ceased, resulting in an accumulation of Clo DF13 DNA molecules of about 500 copies per cell. After 3 h of chloramphenicol treatment, the Clo DF13 plasmid replicates at a rate approximately five times the rate in the absence of chloramphenicol. Inhibition of protein synthesis by chloramphenicol does not influence the level of Clo DF13 DNA synthesis at the restrictive temperature in the dna mutants, except for the dnaA mutant. Chloramphenicol abolishes the inhibition of Clo DF13 DNA synthesis in the dnaA mutant at the nonpermissive temperature. Under these conditions, Clo DF13 DNA synthesis was slightly stimulated in the first 30 min after the temperature shift, and continued for more than 3 h at an almost uninhibited level.     

The ssb gene of plasmid ColIb-P9.

The IncI1 plasmid ColIb-P9 was found to carry a single-stranded DNA-binding (SSB) protein gene (ssb) that maps about 11 kilobase pairs from the origin of transfer in the region transferred early during bacterial conjugation. The cloned gene was able to suppress the UV and temperature sensitivity of an ssb-1 strain of Escherichia coli K-12. The nucleotide sequence of the ColIb ssb gene was determined, giving a predicted molecular weight of 19,110 for the SSB protein. Sequence data show that ColIb ssb is very similar to the ssb gene on plasmid F, which is also known to map in the leader region. High-level expression of ssb on ColIb required derepression of the transfer (tra) genes and the activity of the positive regulatory system controlling these genes, suggesting that the SSB protein contributes to the conjugative processing of DNA. A mutant of ColIbdrd-1 carrying a Tn903-derived insertion in ssb was constructed, but it was unaffected in the ability to generate plasmid transconjugants and it was maintained apparently stably in donor cells both following mating and during vegetative growth. Hence, no biological role of ColIb SSB protein was detected. However, unlike the parental plasmid, such ColIb ssb mutants conferred a marked Psi+ (plasmid-mediated SOS inhibition) phenotype on recA441 and recA730 strains, implying a functional relationship between SSB and Psi proteins.     

Primase, the dnaG protein of Escherichia coli. An enzyme which starts DNA chains.

Conversion of the viral DNA of phage G4 to the duplex form provided an opportunity to isolate and determine the function of the dnaG protein, the product of a gene known to be essential for replication of the Escherichia coli chromosome. This stage of G4 DNA replication requires action of three proteins: the E. coli DNA-binding protein, the dnaG protein, and the DNA polymerase III holoenzyme. The dnaG protein has been purified approximately 25,000-fold to near-homogeneity. The native protein contains a single polypeptide of 60,000 daltons. It has been assayed for its activity on G4 DNA in three ways: (a) RNA synthesis, (b) complementation for replication of an extract of a temperature-sensitive dnaG mutant, and (c) priming of DNA replication by DNA polymerase III holoenzyme. The dnaG protein is highly specific for G4 DNA and synthesizes a unique 29-residue RNA primer to be described in the suceeding paper. Other single-stranded and duplex DNA templates are inactive. RNA primer synthesis by the dnaG protein has an apparent Km for ribonucleoside triphosphates near 10 micrometer, and a narrow optimum for Mg2+. The sharp specificity of the dnaG protein in choice of template and the utilization of either deoxyribonucleotides or ribonucleotides to produce a hybrid piece only a few residues long (as described in a succeeding paper) suggests that the dnaG protein previously named RNA polymerase by renamed primase.     

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.     

Transfer of tra proteins into the recipient cell during bacterial conjugation mediated by plasmid ColIb-P9.

Selective transfer of the two products of the ColIb primase gene, sog, from donor to recipient cell during conjugation was demonstrated by two independent methods. The transfer of these tra proteins was unidirectional and dependent on DNA transfer. The Sog polypeptides were localized to the cytoplasm of the donor cell, but they appeared to interact with other tra gene products located in the inner membrane. After cell mating, the transferred polypeptides were found to be in the cytoplasm of the recipient cell, and it is estimated that as many as 500 Sog polypeptides were transferred per round of conjugation. It is proposed that these proteins are transferred as a result of an interaction with the single-stranded DNA and that the transferred strand may be coated with Sog polypeptides.     

Partial suppression of the phenotype of Escherichia coli K-12 dnaG mutants by some I-like conjugative plasmids.

Three I-like conjugative plasmids, ColIdrd1, R144drd3, and R64drd11, which are derepressed for functions involved in conjugation, were found to suppress at least partially the phenotype of temperature-sensitive dnaG mutants of Escherichia coli K-12, as judged from the kinetics of deoxyribonucleic acid synthesis at elevated temperature in newly formed and established plasmid-containing strains. In contrast, the corresponding wild-type plasmids and three F-like derepressed conjugative plasmids, F101, R100drd1, and R1drd16, all failed to suppress. Suppression is presumably caused by a different plasmid-determined function from that which promotes survival of ultraviolet-irradiated bacteria, because both the wild-type I-like plasmids and their drd mutants protected irradiated bacteria. One possible interpretation of these results is that the product of a gene carried by certain I-like plasmids can substitute for the bacterial dnaG gene product during ongoing deoxyribonucleic acid replication.     

Conserved regions at the DNA primase locus of IncP alpha and IncP beta plasmids

Genes specifying DNA primases (pri) are common in all IncP plasmids examined so far. These plasmids suppress the thermosensitive character of the Escherichia coli dnaG3 mutation. The mechanism of suppression appears to be identical to that known for RP4 and IncI alpha plasmids. The DNA primases of both these plasmid types can substitute for the dnaG protein in chromosomal DNA replication. The pri genes of the alpha and beta subgroup of IncP plasmids are related to each other as judged from Southern hybridization and immunological data. Extensive DNA and protein sequence homology has been detected although the gene products of the alpha and beta subgroups exhibit substantial differences in size. The arrangement of overlapping genes at the pri locus of IncP alpha plasmids also appears to be present in the IncP beta group.     

Plasmid ColE1 DNA replication in Escherichia coli strains temperature-sensitive for DNA replication

Mutants of the dnaA, dnaC, dnaD, polC, dnaF and dnaG gene loci were tested for their capacity for colicinogenic plasmid E1 (ColE1) replication at a non-permissive temperature. It was found that ColE1 replication was independent of the dnaA gene function and dependent on dnaC, D, F and G. ColE1 replication in the polC mutant E486 continued for several hours but at a greatly reduced rate. No effect was found of the dnaG mutation on thymine-deprivation-induced "priming" of ColE1 replication at the non-permissive temperature. The mutants also were tested for aberrant replication intermediates of plasmid DNA as well as a temperature sensitive supercoiled DNA-protein relaxation complex. RNA-containing supercoils were found to accumulate in a poIC mutant also blocked for protein synthesis.     

alpha-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected Heliothis zea Larvae and Spodoptera frugiperda Cells

We performed three types of experiments to test the hypothesis that abortive infection of T5 bacteriophage in Escherichia coli (ColIb+) is due to internally released colicin. (i) We measured the sensitivity of cells to colicin under a variety of conditions and then looked at the plating efficiency of T5 in ColIb+ cells under these same conditions. Cells grown at 42 degrees C or with hexanol had a reduced sensitivity to externally added colicin and an increased efficiency for T5 when the ColIb plasmid was present in the infected cells. Phage growth was far from normal, however. (ii) We measured the colicin sensitivity of a mutant bacterium that grew T5 normally even in the presence of the ColIb plasmid and measured the plating efficiency of T5 on another mutant that was colicin tolerant. Here again, the correlation between colicin activity and inhibition of phage replication was not complete. (iii) We looked for colicin-negative plasmid mutants and tested the ability of cells containing these plasmids to support the growth of T5. These experiments used Tn5, a kanamycin resistance transposon, as the mutagen. All possible combinations of colicin production and phage inhibition were found, including mutants that produced no colicin but still inhibited phage production.