Structural and functional analysis of cloned DNA segments containing the replication and incompatibility regions of a miniplasmid derived from a copy number mutant of NR1.

A 1.45-megadalton segment of DNA cloned from a miniplasmid derived in vivo from a copy number mutant of the R plasmid NR1 has been shown to contain all functions essential for incompatibility and autonomous plasmid replication in Escherichia coli. Specific endonuclease cleavage sites within this DNA segment that localize functions required for replication have been mapped. A 0.45-megadalton fragment that specifies the FII incompatibility of NR1 has been identified within the replication region, and DNA fragments containing this incompatibility region, but lacking other functions required for replication, have been cloned.     

Isolation of a new broad-host-range IncQ-like plasmid, pTC-F14, from the acidophilic bacterium Acidithiobacillus caldus and analysis of the plasmid replicon

A moderately thermophilic (45 to 50 degrees C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic Acidithiobacillus caldus strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating field electrophoresis analysis of total DNA from the A. caldus cells revealed two plasmids of approximately 14 and 45 kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida and Agrobacterium tumefaciens LBA 4404, which suggested that pTC-F14 is a broad-host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open reading frames and a replicon organization like that of the broad-host-range IncQ plasmids. Three of the open reading frames encoded replication proteins which were most closely related to those of IncQ-like plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; RepC, 74%). However, the two plasmids were fully compatible and pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly, asymmetrical incompatibility was found with the less closely related IncQ plasmid R300B derivative pKE462 and the IncQ-like plasmid derivative pIE1108. Analysis of the pTC-F14 oriV region revealed five direct repeats consisting of three perfectly conserved 22-bp iterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 had a copy number of 12 to 16 copies per chromosome in both E. coli, and A. caldus. The rep gene products of pTC-F14 and pTF-FC2 were unable to functionally complement each other's oriV regions, but replication occurred when the genes for each plasmid's own RepA, RepB, and RepC proteins were provided in trans. Two smaller open reading frames were found between the repB and repA genes of pTC-F14, which encode proteins with high amino acid sequence identity (PasA, 81%; PasB, 72%) to the plasmid addiction system of pTF-FC2. This is the second time a plasmid stability system of this type has been found on an IncQ-like plasmid.     

Plasmid maintenance functions of the large virulence plasmid of Shigella flexneri.

The large virulence plasmid pMYSH6000 of Shigella flexneri contains a replicon and a plasmid maintenance stability determinant (Stb) on adjacent SalI fragments. The presence of a RepFIIA replicon on the SalI C fragment was confirmed, and the complete sequence of the adjacent SalI O fragment was determined. It shows homology to part of the transfer (tra) operon of the F plasmid. Stb stabilizes a partition-defective P1 miniplasmid in Escherichia coli. A 1.1-kb region containing a homolog of the F trbH gene was sufficient to confer stability. However, the trbH open reading frame could be interrupted without impairing stability. Deletion analysis implicated the involvement of two small open reading frames, STBORF1 and STBORF2, that fully overlap trbH in the opposite direction. These open reading frames are closely related to the vagC and vagD genes of the Salmonella dublin virulence plasmid and to open reading frame pairs in the F trbH region and in the chromosomes of Dichelobacter nodosus and Haemophilus influenzae. Stb appears to promote better-than-random distribution of plasmid copies and is a plasmid incompatibility determinant. The F homolog does not itself confer stability but exerts incompatibility against the activity of the Stb system. Stb is likely to encode either an active partition system or a postsegregational killing system. It shows little similarity to previously studied plasmid stability loci, but the genetic organization of STBORF1 and STBORF2 resembles that of postsegregational killing mechanisms.     

Cloning and nucleotide sequence of a negative regulator gene for Klebsiella aerogenes arylsulfatase synthesis and identification of the gene as folA.

A negative regulator gene for synthesis of arylsulfatase in Klebsiella aerogenes was cloned. Deletion analysis showed that the regulator gene was located within a 1.6-kb cloned segment. Transfer of the plasmid, which contains the cloned fragment, into constitutive atsR mutant strains of K. aerogenes resulted in complementation of atsR; the synthesis of arylsulfatase was repressed in the presence of inorganic sulfate or cysteine, and this repression was relieved, in each case, by the addition of tyramine. The nucleotide sequence of the 1.6-kb fragment was determined. From the amino acid sequence deduced from the DNA sequence, we found two open reading frames. One of them lacked the N-terminal region but was highly homologous to the gene which codes for diadenosine tetraphosphatase (apaH) in Escherichia coli. The other open reading frame was located counterclockwise to the apaH-like gene. This gene was highly homologous to the gene which codes for dihydrofolate reductase (folA) in E. coli. We detected 30 times more activity of dihydrofolate reductase in the K. aerogenes strains carrying the plasmid, which contains the arylsulfatase regulator gene, than in the strains without plasmid. Further deletion analysis showed that the K. aerogenes folA gene is consistent with the essential region required for the repression of arylsulfatase synthesis. Transfer of a plasmid containing the E. coli folA gene into atsR mutant cells of K. aerogenes resulted in repression of the arylsulfatase synthesis. Thus, we conclude that the folA gene codes a negative regulator for the ats operon.     

Control of cell division by sex factor F in Escherichia coli. I. The 42.84-43.6 F segment couples cell division of the host bacteria with replication of plasmid DNA

The F plasmid of Escherichia coli was used to study the genetic background of the control circuit in the bacteria that co-ordinates DNA replication and cell division of the host cells. When DNA replication of the F plasmid was blocked by growing cells carrying an amber-suppressible replication-defective F plasmid mutant under restrictive conditions, the cells continued to divide for about one generation until F plasmid was supposedly diluted to one copy per cell, and then they stopped dividing and formed non-septated filamentous cells. These observations suggest that completion of a round of replication is a necessary and sufficient condition of F DNA synthesis in the cell division of F+ bacteria; i.e. cell division of the F+ bacteria is coupled with DNA replication of the F plasmid. The observation that Giemsa-stainable materials in the filamentous cells were clustered in the center indicates that partitioning of chromosomal DNA (and presumably of F plasmid DNA) is also coupled with plasmid DNA replication. The function necessary for this coupling is carried by the 42.84-43.6 F (BamHI-PstI) segment, which is located outside the region essential for replication of the F plasmid. The nucleotide sequence demonstrates the existence of two open reading frames in this region, which encode polypeptides of 72 and 101 amino acids, respectively. These two reading frames are most likely to be transcribed as a single polycistronic message in the direction from the BamHI site at 42.84 F to the PstI site at 43.6 F. The expression of this "operon" is likely to be controlled by plasmid DNA replication.     

The sequence of the bacteriophage P1 genome region serving as hot target for IS2 insertion.

A restriction fragment of the bacteriophage P1 genome known to serve as a hot target for IS2 insertion in its host, Escherichia coli K12, was entirely sequenced. It is 1756 bp long and it contains four long open reading frames, all in the same orientation. The two middle frames overlap partially. Eight of the nine studied IS2 insertions affecting phage reproduction map within three of these reading frames. No common feature was found between the nine target sites which have served for IS2 integration. However, there are two structural elements which might possibly contribute to rendering the studied DNA segment a hot region for IS2 insertion. The first is formed by two neighbouring, 30 and 40 bp regions of homology with an internal segment of IS2. The second is the pentanucleotide 5' GGTAT3', which is carried nine times in the sequenced fragment and which is found always in at least one copy within a variable distance of less than 100 bp of each inserted IS2 element.     

Genetic Organization of the Basic Replicon of Plasmid pMTH4 of a Facultatively Methylotrophic Bacterium <Emphasis Type="Italic">Paracoccus methylutens</Emphasis> DM12

Two functional regions within the basic replicon of plasmid pMTH4 of Paracoccus methylutens DM12 have been distinguished that are responsible for the replication of the plasmid (REP) and its stabilization (STA). In the REP region, a gene encoding the putative replication initiation protein RepA has been identified, with the highest similarity to the replication protein of plasmid pALC1 (Paracoccus alcaliphilus). The potential origin of replication (oriV), consisting of five long repeated sequences (iterons) as well as putative DnaA and IHF boxes, has been localized in the promoter region of the gene repA. The STA region was found to ensure stability for heterogeneous plasmid pABW3 that is unstable itself in paracocci. The mini-STA region (850 bp) contains two short open reading frames, one of which shows similarity to the RelB protein of Escherichia coli. Our investigations suggest that the stabilizing system of pMTH4 is based on the toxin and antidote principle.     

P1 plasmid replication: replicon structure

Bacteriophage P1 lysogenizes Escherichia coli as a unit-copy plasmid. We have undertaken to define the plasmid-encoded elements implicated in P1 plasmid maintenance. We show that a 2081 base-pair fragment of the 90,000 base P1 plasmid confers the capacity for controlled plasmid replication. DNA sequence analysis reveals several open reading frames in this fragment. The largest is shown to encode a 32,000 Mr protein required for plasmid replication. The corresponding gene, repA, has been identified genetically. A set of five 19 base-pair repeats is located upstream from repA; a set of nine similar repeats is located immediately downstream from repA. Each set of repeats, when cloned into pBR322, exerts incompatibility towards a P1 replicon. The upstream set, designated incC, consists of direct repeats that are spaced about two turns of the DNA helix apart; the downstream set, designated incA, consists of nine repeats arranged three in one orientation and six in the other. Spacing between incA repeats were three or four turns of the helix apart. The organization of the plasmid maintenance regions of P1 and the unit-copy sex factor plasmid, F, is strikingly similar. Although the DNA sequences of this region in the two plasmids exhibit little homology, a 9 base-pair sequence that appears four times in the origin region of members of the Enterobacteriaceae also occurs twice as direct repeats in similar positions in P1 and F. This sequence, where it occurs in E. coli, has been postulated to be the binding site for the essential replication protein determined by dnaA. The dnaA protein appears not to be essential for the replication of either plasmid; therefore, the function of the sequence in P1 and F may be regulatory.     

Structure and function of the F plasmid genes essential for partitioning

The F plasmid in Escherichia coli has its own partition mechanism controlled by the sopA and sopB genes, and by the cis-acting sopC region. The DNA sequence of the entire partition region and its flanking regions is described here. Two large open reading frames coding for 43,700 Mr and 35,400 Mr proteins correspond to sopA and sopB, respectively. The sopB reading frame is located immediately downstream from the sopA reading frame. Twelve 43 base-pair direct repeats exist in the sopC region without any spacer regions, and one pair of seven base-pair inverted repeats exists in each of the direct repeats. Analysis of deletions in the sopC region showed that the direct repeats play an important role in plasmid partition and IncD incompatibility. IncG incompatibility is exhibited by pBR322 derivatives carrying the sopB gene alone. When compared with the partition genes parA and parB of plasmid P1, homology in amino acid sequence was found between the SopA protein of F and the ParA protein of P1, and also between SopB protein of F and ParB protein of P1. In addition, homology was found between Rep proteins of F and P1.     

Characterization of a member of the highly repeated long interspersed rat DNA family with long open reading frames

A highly repetitive long interspersed sequence from rat DNA has been isolated and partly characterized. This sequence comprises at least a 1300 base-pair and a 2400 base-pair EcoRI fragment and probably additional elements. The 2400 base-pair segment has been analyzed in detail. It appears to be part of the chromosomal DNA in rat cells. The 2400 base-pair repeat is likely to be distributed over several regions in the rat genome. The 2400 base-pair segment has been cloned, mapped for restriction sites, and part of its nucleotide sequence has been determined. The 2400 base-pair sequence is a member of a typical highly repetitive long interspersed sequence with high copy number and restriction site polymorphism. There are sequence homologies to mouse and human DNA. A striking homology has been detected to the flanking sequences of a repetitive mouse DNA sequence that has been described to be located adjacent to one of the kappa-immunoglobulin variable genes. Elements in the 2400 base-pair rat repeat are transcribed in cells from most rat organs and from several continuous rat cell lines. This RNA from rat cell lines was found polyadenylated or not polyadenylated. The nucleotide sequence of parts of the 2400 base-pair DNA segment revealed open reading frames for polypeptide sequences. Such open reading frames have been detected in two different segments of the 2400 base-pair DNA repeat. Open reading frames exist in the two complementary strands in the same DNA segment. The hypothetical polypeptide whose sequence has been determined in toto has a length of 190 amino acid residues and is enriched in hydrophobic amino acids, reminiscent of the amino acid composition in membrane proteins. Hence, it is conceivable that the 2400 base-pair repeat sequence from rat DNA, at least in part, encodes messenger RNAs that might be translated into functional proteins.     

Partitioning of plasmid R1. Structural and functional analysis of the parA locus

The stability locus, parA+, of plasmid R1 is shown to be localized within a 1500 base-pair region of DNA on the largest EcoRI restriction fragment of plasmid R1. The nucleotide sequence of the region revealed the presence of two open reading frames, one of 320 codons, and another of 60 codons. The larger open reading frame encodes a polypeptide of 36,000 Mr. Deletions covering the promoter distal end of the 36,000 Mr reading frame give rise to synthesis of large amounts of truncated protein. Construction of promoter fusions between the parA+ promoter and the lacZ gene showed that the parA+ region encodes a factor that negatively regulates the expression of the 36,000 Mr protein. The locus exerting parA+-associated incompatibility, denoted incA+, was mapped to a 60 base-pair region covering the parA+ promoter. Most likely, this region is involved both in the negative regulation of the parA+ operon and in the parA+-associated incompatibility. Two explanations are suggested to explain this possible dual function of the parA+ promoter region. The parA+ region was cloned into an unstably inherited (par-) derivative of a mini-F derivative. The low copy number plasmid mini-F devoid of its own partition genes was stabilized more than 100-fold by carrying the parA+ genes. This observation is in accordance with the proposal that the parA+ locus specifies the true partition function of plasmid R1.     

Interactions of plasmid-encoded replication initiation proteins with the origin of DNA replication in the broad host range plasmid RK2

The TrfA proteins, encoded by the broad host range plasmid RK2, are required for replication of this plasmid in a variety of Gram-negative bacteria. Two TrfA proteins, 33 and 44 kDa in molecular mass (designated TrfA-33 and TrfA-44, respectively), are expressed from the trfA gene of RK2 through the use of two alternative in-frame start codons within the same open reading frame. The two proteins have been purified from Escherichia coli to near homogeneity as a mixture of wild-type TrfA-44/33, as TrfA-33 alone and as a functional variant form of TrfA-44, designated TrfA-44(98L), which contains a leucine in place of the TrfA-33 methionine start codon. Cross-linking experiments demonstrated that TrfA-33 can multimerize in solution. By using gel mobility shift and DNase I footprinting techniques the binding properties of TrfA-33, TrfA-44(98L), and TrfA-44/33 to the origin of replication of plasmid RK2 were analyzed. All three protein preparations were able to bind very specifically to the cluster of five direct repeats (iterons) contained in the minimal origin of replication. Each protein preparation produced a ladder of TrfA/minimal oriV complexes of decreasing electrophoretic mobility. The DNase I protection pattern on the five iterons was identical for all three protein preparations and extended from the beginning of the first iteron to 5 base pairs upstream of the fifth iteron. Studies on the affinity of the proteins for DNA fragments containing one, two, or all five iterons of the origin revealed a strong preference of TrfA protein for DNA containing at least two iterons. To study the stability of TrfA.DNA complexes, association and dissociation rates of TrfA-33 and DNA fragments with one, two, or five iterons were measured. This analysis showed that unlike complexes involving two or five iterons the TrfA/one iteron complexes were highly unstable, suggesting some form of cooperativity between proteins or iterons in the formation of stable complexes and/or the requirement of specific sequences bordering the iterons at the RK2 origin of replication for the stabilization of TrfA/DNA complexes.     

Construction of new vectors for cloning of promoters

Vectors for cloning promoter-DNA fragments were derived from plasmid pBR313 (Bolivar et al., 1977). These have several unique restriction sites and carry the trpA gene from Escherichia coli as a selective marker. The selection is based on an enhancement of the growth rate of those bacteria in which the expression of trpA is directed by the cloned promoter. The expression of trpA can be determined quantitatively, independently of the copy number of the vector, and should reflect the apparent strength of the promoter, since the DNA segment located before trpA contains translational stop signals in all three reading frames.     

Genes associated with meningococcal capsule complex are also found in Neisseria gonorrhoeae.

A homolog of the meningococcal cps locus region E has been identified in Neisseria gonorrhoeae immediately upstream of the gonococcal region D locus. Region E has no detectable function in capsule biosynthesis in Neisseria meningitidis or in lipopolysaccharide biosynthesis in either organism. The open reading frame is homologous to proteins of unknown function in Escherichia coli and Haemophilus influenzae. Further analysis of the N. meningitidis cps cluster has identified a second copy of region D encoding three additional open reading frames, including homologs of DNA methyltransferases. The organization of the region D and E genes in N. gonorrhoeae and N. meningitidis in relation to the cps genes provides some insight into the evolutionary origin of encapsulation in N. meningitidis.     

The mobilization and origin of transfer regions of a Thiobacillus ferrooxidans plasmid: relatedness to plasmids RSF1010 and pSC101

The components for the mobilization function of a plasmid DNA during conjugation include a cis-acting sequence (the origin of transfer, oriT) and a transacting sequence coding for mobilization (Mob) proteins. By genetic and deletion analysis, we have located the mobilization region of pTF1, a cryptic plasmid previously isolated from a Thiobacillus ferrooxidans strain. Within a 2797 bse-pair sequenced region, several open reading frames (ORFs) were predicted; two of the ORFs are divergently transcribed and they encode proteins of calculated molecular masses, 42.6kD (ORF2) and 11.4kD (ORF6). Surprisingly, these protein sequences are substantially similar to two of the previously characterized mobilization proteins of the Escherichia coli IncQ plasmid, RSF1010. Moreover, the pTF1 ORF2 (now designated MobL) sequence is also found to be similar to a presumed mobilization protein of plasmid pSC101. Regions of sequence identity of plasmids pTF1, RSF1010 and pSC101 include their oriT sites. By alkaline agarose gel electrophoresis and DNA sequencing, we have established the location of the relaxation complex nick site within the oriT of pTF1. An identical nick site, which is adjacent to a characteristic 10 base-pair inverted repeat sequence, is also found for plasmid RSF1010. A recombinant plasmid containing a 42 base-pair synthetic piece of DNA encompassing the pTF1 inverted repeat and nick sequence was shown to be oriT-active.     

Sequence analysis of a group of low molecular-weight plasmids carrying multiple IS903 elements flanking a kanamycin resistance aph gene in Salmonella enterica serovars

A group of low molecular-weight ColE1-like plasmids carrying the aph sequence type aph(ii) from three different Salmonella serovars were sequenced. These plasmids carry two or more copies of IS903 elements, with up to 21bp sequence differences to one another, two of which flank the aph gene. This group of plasmids did not appear to carry any known mobilization genes and instead carry three open reading frames encoding hypothetical proteins of unknown function possibly organized in an operon. The plasmid replication region (RNA I/II--rom) of this plasmid group showed extensive homology to that of pKPN2 plasmid of Klebsiella pneumoniae and pCol-let plasmid of Escherichia coli. Three of the four plasmids had identical sequences, and the fourth had an extra copy of IS903 with target duplication, suggesting a recent divergence in the different Salmonella serovars from a common ancestor.     

Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE-bound iterons inhibits DNA replication of mini-F plasmid in Escherichia coli.

In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences. To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis. Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F plasmids lost the function to control the copy number. The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein. Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number.     

Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K

The three replication origins of the antibiotic resistance plasmid R6K require for their activity in Escherichia coli a DNA segment containing seven 22 base-pair direct repeats and a plasmid-encoded initiation protein (pi). The pi protein functions in the negative control of R6K replication, in addition to its requirement for the initiation of replication. Construction of a plasmid containing the pi structural gene (pir) downstream from the inducible pR promoter of bacteriophage lambda provided high levels of production of pi protein in E. coli. The pi protein was purified and shown to possess general DNA binding properties with a preference for DNA fragments containing the gamma origin of replication, the operator region of the pir gene and the R6K beta-origin region. Velocity sedimentation analysis indicates that the pi protein exists as a dimer in its native form. Agarose gel electrophoresis analysis of pi-gamma-origin complexes suggests that one pi dimer binds to each copy of the 22 base-pair direct repeats in the gamma origin region. Purified mutant pi protein obtained from a temperature-sensitive initiation mutant (pir 105-ts) exhibited temperature-sensitive binding activity to the gamma-origin region, whereas two mutant proteins exhibiting a high copy number phenotype were unaltered (pir104-cop) or slightly reduced (pir1-cop) in binding activity. The patterns of DNase I protection and enhancement were similar for the wild-type and mutant proteins examined.