Plasmid pSC101 replication mutants generated by insertion of the transposon Tn1000.

A derivative of pSC101, pLC709, was constructed by ligation of the HincII-A fragment of pSC101 to the mini-colEI plasmid pVH51 and to a DNA fragment encoding resistance to the antibiotics streptomycin and spectinomycin. Insertions of the transposon Tn1000 (gamma-delta) into the pSC101 replication region of pLC709 were isolated following cotransfer of the plasmid with the sex factor F. The sites of insertion of the transposon were determined by restriction enzyme analysis and the replication and incompatibility properties of the insertion plasmids and DNA fragments cloned from them were analysed. The insertion mutations defined a locus, inc, of approximately 200 base-pairs that is responsible for pSC101-specific incompatibility. Two mutations adjacent to this region inactivate pSC101 replication but can be complemented in trans by a wild-type pSC101 plasmid, and thus define a trans-acting replication function, rep. The inc locus is within a larger region of some 450 base-pairs that is essential for pSC101 replication and that includes the origin of replication. This 450 base-pair segment can replicate in the presence of a helper plasmid that supplies the rep function in trans.

An essential replication gene, repA, of plasmid pSC101 is autoregulated.

Measurements of the rate of replication of a mutant pSC101 plasmid, cloned into a ColE1 vector, showed that insertions of the transposon Tn1000 into the repA gene of pSC101 abolished replication activity, but could be complemented in trans, albeit at a low level. The promoter of the repA gene was mapped by the construction of repA-lacZ gene fusions, and one of the fusions was used to demonstrate that repA protein, provided in trans, could repress expression of beta-galactosidase activity. This repression was primarily due to reduction of transcription of the repA-lacZ fusion. The sequence analysis of mutants of the repA-lacZ fusion gene which were no longer sensitive to the presence of repA protein showed that the site of action of repA was a 22 base-pair sequence, present as an inverted repeat, overlapping the repA promoter. The repA gene is thus autoregulated.

A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors.

We have constructed a plasmid cloning vector, pGB2, which is derived from the Escherichia coli plasmid pSC101. The plasmid, which specifies resistance to spectinomycin and streptomycin, contains unique restriction sites for the enzymes HindIII, PstI, SalI, BamHI, SmaI and EcoRI. pGB2 shows no sequence homology, as detected by DNA-DNA hybridization, to several widely used vectors such as pBR322, pUC8 and phage lambda L47.1. Amongst other applications, DNA fragments can be cloned into the plasmid and then radioactive plasmid DNA can be used as a probe to screen recombinant DNA libraries.

Replication functions encoded by the plasmid pSC101.

We describe the mapping of several genetic loci involved in the replication of the pSC101 plasmid. These include the origin of replication and a short segment of DNA that encodes a pSC101 incompatibility function. This short segment lies within the origin region. Flanking the incompatibility segment are two loci, repA and repB, which are required for replication. The product of the repA locus is shown to be trans-acting.

Control of cyclic chromosome replication in Escherichia coli.

The biochemical basis for cyclic initiation of bacterial chromosome replication is reviewed to define the processes involved and to focus on the putative oscillator mechanism which generates the replication clock. The properties required for a functional oscillator are defined, and their implications are discussed. We show that positive control models, but not negative ones, can explain cyclic initiation. In particular, the widely accepted idea that DnaA protein controls the timing of initiation is examined in detail. Our analysis indicates that DnaA protein is not involved in the oscillator mechanism. We conclude that the generations of a single leading to cyclic initiation is separate from the initiation process itself and propose a heuristic model to focus attention on possible oscillator mechanisms.

Tn916 target DNA sequences bind the C-terminal domain of integrase protein with different affinities that correlate with transposon insertion frequency.

The conjugative transposon Tn916 inserts with widely different frequencies into a variety of target sites with related nucleotide sequences. The binding of chimeric proteins, consisting of maltose-binding protein fused to Tn916 integrase, to three different target sequences for Tn916 was examined by DNase I protection experiments. The C-terminal DNA binding domain of the Tn916 integrase protein was shown to protect approximately 40 bp, spanning target sites in the orfA and cat genes of the plasmid pIP501 and in the cylA gene of the plasmid pAD1. Competition binding assays showed that the affinities of the three target sites for Tn916 integrase varied over a greater than 3- but less than 10-fold range and that the cat target site bound integrase at a lower affinity than did the other two target sites. A PCR-based assay for transposition in Escherichia coli was developed to assess the frequency with which a defective minitransposon inserted into each target site. In these experiments, integrase provided in trans from a plasmid was the sole transposon-encoded protein present. This assay detected transposition into the orfA and cylA target sites but not into the cat target site. Therefore, the frequency of transposon insertion into a particular target site correlated with the affinity of the target for the integrase protein. Sequences within the target fragments similar to known Tn916 insertion sites were not protected by integrase protein. Analysis ot he electrophoretic behavior of circularly permuted sets of DNA fragments showed that all three target sites contained structural features consistent with the presence of a static bend, suggesting that these structural features in addition to the primary nucleotide sequence are necessary for integrase binding and, thus, target site activity.

Conjugative transposition: Tn916 integrase contains two independent DNA binding domains that recognize different DNA sequences.

Transposition of the conjugative transposon Tn916 requires the activity of a protein, called Int, which is related to members of the integrase family of site-specific recombinases. This family includes phage lambda integrase as well as the Cre, FLP and XerC/XerD recombinases. Different proteins, consisting of fragments of Tn916 Int protein fused to the C-terminal end of maltose binding protein (MBP) were purified from Escherichia coli. DNase I protection experiments showed that MBP-INT proteins containing the C-terminal end of Int bound to the ends of the transposon and adjacent plasmid DNA. MBP-INT proteins containing the N-terminal end of Int bound to sequences within the transposon close to each end. Competition binding experiments showed that the sites recognized by the C- and N-terminal regions of Int did not compete with each other for binding to MBP-INT. We suggest that Tn916 and related conjugative transposons are unique among members of the integrase family of site-specific recombination systems because the presence of two DNA binding domains in the Int protein might allow Int to bridge recombining sites, and this bridging seems to be the sole mechanism ensuring that only correctly aligned molecules undergo recombination.

Xis protein of the conjugative transposon Tn916 plays dual opposing roles in transposon excision.

The binding of Tn916 Xis protein to its specific sites at the left and right ends of the transposon was compared using gel mobility shift assays. Xis formed two complexes with different electrophoretic mobilities with both right and left transposon ends. Complex II, with a reduced mobility, formed at higher concentrations of Xis and appeared at an eightfold lower Xis concentration with a DNA fragment from the left end of the transposon rather than with a DNA fragment from the right end of the transposon, indicating that Xis has a higher affinity for the left end of the transposon. Methylation interference was used to identify two G residues that were essential for binding of Xis to the right end of Tn916. Mutations in these residues reduced binding of Xis. In an in vivo assay, these mutations increased the frequency of excision of a minitransposon from a plasmid, indicating that binding of Xis at the right end of Tn916 inhibits transposon excision. A similar mutation in the specific binding site for Xis at the left end of the transposon did not reduce the affinity of Xis for the site but did perturb binding sufficiently to alter the pattern of protection by Xis from nuclease cleavage. This mutation reduced the level of transposon excision, indicating that binding of Xis to the left end of Tn916 is required for transposon excision. Thus, Xis is required for transposon excision and, at elevated concentrations, can also regulate this process.

Specific binding of integrase to the origin of transfer (oriT) of the conjugative transposon Tn916.

Purified integrase protein (Int) of the conjugative transposon Tn916 was shown, using nuclease protection experiments, to bind specifically to a site within the origin of conjugal transfer of the transposon, oriT. A sequence similar to the ends of the transposon that are bound by the C-terminal DNA-binding domain of Int was present in the protected region. However, Int binding to oriT required both the N- and C-terminal DNA-binding domains of Int, and the pattern of nuclease protection differed from that observed when Int binds to the transposon ends and flanking DNA. Binding of Int to oriT may be part of a mechanism to prevent premature conjugal transfer of Tn916 prior to excision from the donor DNA.

Initiation of chromosome replication in Escherichia coli after induction of dnaA gene expression from a lac promoter.

Escherichia coli HB282 carries a dnaA46(Ts) allele on the chromosome, a wild-type dnaA allele under the control of the lacUV5 promoter on the multicopy plasmid pBC32, and an overproducing lac repressor allele on an F' factor. When the plasmid dnaA gene is repressed, the strain is thermosensitive. After a temporary deficiency in active dnaA protein at nonpermissive temperature, the addition of isopropyl-beta-D-thiogalactopyranoside to the culture was found to produce a burst of initiations within 5 to 10 min at 30% of the origins in 90% of the cells. Initiations then continued at a rate slightly faster than the mass-doubling time such that after 2 h the origin-to-mass ratio of the control culture was restored.

Determination of deoxyribonucleic acid replication time in exponentially growing Escherichia coli B/r.

The time necessary to replicate the chromosome (C period) was measured in Escherichia coli B/r (ATCC 12407) and a low-thymine-requiring derivative of that strain. In the Thy- strain, C was measured as a function of growth rate and exogenous thymine concentration either from step-up or chloramphenicol experiments. In the Thy+ parental strain, C was measured only as a function of the growth rate and only by the chloramphenicol method. The C period was found to decrease with growth rate and, in the Thy- strain, the C period also decreased with increasing thymine concentration. It approached a value of approximately 37 min at high growth rates.

Deoxyribonucleic acid synthesis after inhibition of initiation of rounds of replication in Escherichia coli B/r.

The theory describing the effect of inhibition of initiation of rounds of deoxyribonucleic acid (DNA) replication on the accumulation of DNA is derived, and an analysis is presented which allows the determination of the time C taken to replicate the bacterial chromosome from the kinetic changes in the accumulation of DNA. This analysis is applied to experiments in which inhibition of initiation was achieved by inhibiting protein or protein and ribonucleic acid synthesis with chloramphenicol or rifampin. The results for both antibiotics are identical and indicate that there is a delay of 6 to 11 min in the effect of the antibiotics on initiation of rounds of replication. If this delay is taken into account, then the value of the C period estimated from such experiments agrees with values obtained by other methods, whereas by conventional data evaluation of such experiments the C period would be overestimated. In the low thymine-requiring derivative of Escherichia coli B/r ATCC 12407 used here, the C period was found to be between 38 and 41 min for cultures growing with a mass doubling time of 29 min in glucose-amino acids medium, supplemented with 20 micrograms of thymine/ml.

Coupling sequences flanking Tn916 do not determine the affinity of binding of integrase to the transposon ends and adjacent bacterial DNA.

Coupling sequences are the 6 bp flanking the conjugative transposon Tn916 and are thought to play a role in determining the frequency of conjugative transposition. The affinity of binding of a chimeric protein, which consisted of maltose binding protein fused to the carboxy-terminal DNA binding domain of Tn916 integrase (Int), to different double-stranded oligonucleotide substrates containing coupling sequences associated with high- and low-frequency conjugative transposition was measured using a competition binding assay. The relative affinity of the chimeric protein was unaffected by the nature of the coupling sequences tested. The same results were obtained when the coupling sequences were placed in a different surrounding sequence context. It therefore appears that the effects of different coupling sequences on the frequency of conjugative transposition are not due simply to differences in Int binding.

Interactions of the integrase protein of the conjugative transposon Tn916 with its specific DNA binding sites.

The binding of two chimeric proteins, consisting of the N-terminal or C-terminal DNA binding domain of Tn916 Int fused to maltose binding protein, to specific oligonucleotide substrates was analyzed by gel mobility shift assay. The chimeric protein with the N-terminal domain formed two complexes of different electrophoretic mobilities. The faster-moving complex, whose formation displayed no cooperativity, contained two protein monomers bound to a single DNA molecule. The slower-moving complex, whose formation involved cooperative binding (Hill coefficient > 1.0), contained four protein monomers bound to a single DNA molecule. Methylation interference experiments coupled with the analysis of protein binding to mutant oligonucleotide substrates showed that formation of the faster-moving complex containing two protein monomers required the presence of two 11-bp direct repeats (called DR2) in direct orientation. Formation of the slower-moving complex required only a single DR2 repeat. Binding of the N-terminal domains in vivo could serve to position two Int monomers on the DNA near each end of the transposon and assist in bringing together the ends of the transposon so that excision can occur. The chimeric protein with the C-terminal domain of Int also formed two complexes of different electrophoretic mobilities. The major, slower-moving complex, whose formation involved cooperative binding, contained two protein molecules bound to one DNA molecule. This finding suggested that while the C-terminal domain of Int can bind DNA as a monomer, a cooperative interaction between two monomers of the C-terminal domain may help to bring the ends of the transposon together during excision.

Conjugative transposition.

Conjugative transposons are important determinants of antibiotic resistance, especially in gram-positive bacteria. They are remarkably promiscuous and can conjugate between bacteria belonging to different species and genera. Transposon-promoted conjugation may be similar to F plasmid-promoted conjugation, as it appears that only one strand of the transposon DNA is transferred from donor to recipient. The recent determination of the entire nucleotide sequence of Tn916 allowed us to make specific predictions about the possible function of different open reading frames and the position of a (hypothetical) origin of transfer. The mechanism of recombination during conjugative transposition differs from that of other transposons, as shown by the absence of a duplication of the target sequence upon integration. The current model for recombination postulates that staggered double-stranded cleavages occur at each end of the transposon. One DNA strand is cut six bases from the end of the transposon, and the other strand is cut immediately adjacent to the end. The ends of the excised transposon are then ligated to form a circular intermediate with a six-base heteroduplex. Staggered cleavages of the circular intermediate and the target DNA allow the transposon to insert into the target, where it is flanked by heteroduplex regions that are resolved by replication. All hosts examined contain preferential target sites: these are not specific sequences but apparently consist of bent DNA. The site-specific recombinases encoded by conjugative transposons belong to the integrase family. Like phage lambda integrase, the integrase of Tn916 has two DNA-binding domains that recognize different sequences, one within the ends of the element and one that includes target DNA. The affinity of Tn916 integrase for target sites correlates with the frequency of integration into a particular site. The similarity between conjugative transposons and phage lambda is striking and suggests that both use the same mechanism of recombination. In lambda, however, recombining sites must be homologous. Homology may be necessary because of branch migration, which is thought to occur during recombination. In conjugative transposition, the recombining sites are nearly always different, and therefore branch migration probably does not occur. This review presents a speculative model for the alignment of the ends of Tn916 during excision that was adapted from one recently proposed for lambda.

Cloning and sequence analysis of the rpsL and rpsG genes of Mycobacterium smegmatis and characterization of mutations causing resistance to streptomycin.

The Mycobacterium smegmatis rpsL and rpsG genes, encoding the ribosomal proteins S12 and S7, were cloned, and their DNA sequence was determined. The third nucleotide of the S12 termination codon overlapped the first nucleotide of the S7 translation initiation codon. A collection of 28 spontaneous streptomycin-resistant mutants of M. smegmatis were isolated. All had single-base-pair substitutions in the rpsL gene which were changed to a streptomycin-sensitive phenotype by complementation with a low-copy-number plasmid carrying the wild-type M. smegmatis rpsL gene. A total of eight different mutations were found in two specific regions of the rpsL gene. Fifty-seven percent (16 of 28) altered the Lys codon at position 43. Forty-six percent of the mutations (13 of 28) were due to a transition changing an AAG Lys codon to an AGG Arg codon, with eight changes at codon 43 and five at codon 88.

Synthesis and function of ribonucleic acid polymerase and ribosomes in Escherichia coli B/r after a nutritional shift-up.

The syntheses of stable ribosomal ribonucleic acid (RNA) and transfer RNA in bacteria depend on the concentration and activity of RNA polymerase and on the fraction of active RNA polymerase synthesizing stable RNA. These parameters were measured in Escherichia coli B/r after a nutritional shift-up from succinate-minimal to glucose-amino acids medium and were found to change in complex patterns during a 1- to 2-h period after the shift-up before reaching a final steady-state level characteristic for the postshift growth medium. The combined effect of these changes was an immediate, one-step increase in the exponential rate of stable RNA synthesis and thus of ribosome synthesis. This suggests that the distribution of transcribing RNA polymerase over ribosomal and nonribosomal genes and the polymerase activity are continuously adjusted during postshift growth to some growth-limiting reaction whose rate increases exponentially. It is proposed that this reaction is the production of amino-acylated transfer RNA and that is exponentially increasing rate results in part from a gradually increasing concentration of aminoacyl transfer RNA synthetases after a shift-up. This idea was tested and is supported by a computer simulation of a nutritional shift-up.