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PubMed2004

Novel cassette-based shuttle vector system for gram-positive bacteria.

Our understanding of staphylococcal pathogenesis depends on reliable genetic tools for gene expression analysis and tracing of bacteria. Here, we have developed and evaluated a series of novel versatile Escherichia coli-staphylococcal shuttle vectors based on PCR-generated interchangeable cassettes. Advantages of our module system include the use of (i) staphylococcal low-copy-number, high-copy-number, thermosensitive and theta replicons and selectable markers (choice of erythromycin, tetracycline, chloramphenicol, kanamycin, or spectinomycin); (ii) an E. coli replicon and selectable marker (ampicillin); and (iii) a staphylococcal phage fragment that allows high-frequency transduction and an SaPI fragment that allows site-specific integration into the Staphylococcus aureus chromosome. The staphylococcal cadmium-inducible P(cad)-cadC and constitutive P(blaZ) promoters were designed and analyzed in transcriptional fusions to the staphylococcal beta-lactamase blaZ, the Vibrio fischeri luxAB, and the Aequorea victoria green fluorescent protein reporter genes. The modular design of the vector system provides great flexibility and variety. Questions about gene dosage, complementation, and cis-trans effects can now be conveniently addressed, so that this system constitutes an effective tool for studying gene regulation of staphylococci in various ecosystems.

Artificial Gene Fusion↗
PubMed2006

Sequence analysis of the 144-kilobase accessory plasmid pSmeSM11a, isolated from a dominant Sinorhizobium meliloti strain identified during a long-term field release experiment.

The genome of Sinorhizobium meliloti type strain Rm1021 consists of three replicons: the chromosome and two megaplasmids, pSymA and pSymB. Additionally, many indigenous S. meliloti strains possess one or more smaller plasmids, which represent the accessory genome of this species. Here we describe the complete nucleotide sequence of an accessory plasmid, designated pSmeSM11a, that was isolated from a dominant indigenous S. meliloti subpopulation in the context of a long-term field release experiment with genetically modified S. meliloti strains. Sequence analysis of plasmid pSmeSM11a revealed that it is 144,170 bp long and has a mean G+C content of 59.5 mol%. Annotation of the sequence resulted in a total of 160 coding sequences. Functional predictions could be made for 43% of the genes, whereas 57% of the genes encode hypothetical or unknown gene products. Two plasmid replication modules, one belonging to the repABC replicon family and the other belonging to the plasmid type A replicator region family, were identified. Plasmid pSmeSM11a contains a mobilization (mob) module composed of the type IV secretion system-related genes traG and traA and a putative mobC gene. A large continuous region that is about 42 kb long is very similar to a corresponding region located on S. meliloti Rm1021 megaplasmid pSymA. Single-base-pair deletions in the homologous regions are responsible for frameshifts that result in nonparalogous coding sequences. Plasmid pSmeSM11a carries additional copies of the nodulation genes nodP and nodQ that are responsible for Nod factor sulfation. Furthermore, a tauD gene encoding a putative taurine dioxygenase was identified on pSmeSM11a. An acdS gene located on pSmeSM11a is the first example of such a gene in S. meliloti. The deduced acdS gene product is able to deaminate 1-aminocyclopropane-1-carboxylate and is proposed to be involved in reducing the phytohormone ethylene, thus influencing nodulation events. The presence of numerous insertion sequences suggests that these elements mediated acquisition of accessory plasmid modules.

Agrobacterium tumefaciens↗
PubMed2006

A Type Ib ParB protein involved in plasmid partitioning in a gram-positive bacterium.

Our current understanding of segregation of prokaryotic plasmids has been derived mainly from the study of the gram-negative bacterial plasmids. We previously reported a replicon of the cryptic plasmid from a gram-positive bacterium, Leifsonia xyli subsp. cynodontis. The replicon contains a putative plasmid partition cassette including a Walker-type ATPase followed by open reading frame 4 without sequence homologue. Here we reported that the orf4 gene was essential for maintaining the plasmid stability in L. xyli subsp. cynodontis. Furthermore, the purified orf4 protein specifically and cooperatively bound to direct repeat sequences located upstream of the parA gene in vitro, indicating that orf4 is a parB gene and that the direct repeat DNA sequences constitute a partition site, parS. The location of parS and the features of ParA and ParB proteins suggest that this plasmid partition cassette belongs to type Ib, representing the first type Ib cassette identified from a gram-positive bacterial plasmid.

Bacterial Proteins↗
PubMed2006

Localization of the naturally occurring plasmid ColE1 at the cell pole.

The naturally occurring plasmid ColE1 was found to localize as a cluster in one or both of the cell poles of Escherichia coli. In addition to the polar localization of ColE1 in most cells, movement of the plasmid to the midcell position was observed in time-lapse studies. ColE1 could be displaced from its polar location by the p15A replicon, pBAD33, but not by plasmid RK2. The displacement of ColE1 by pBAD33 resulted in an almost random positioning of ColE1 foci in the cell and also in a loss of segregational stability, as evidenced by the large number of cells carrying pBAD33 with no visible ColE1 focus and as confirmed by ColE1 stability studies. The addition of the active partitioning systems of the F plasmid (sopABC) or RK2 (O(B1) incC korB) resulted in movement of the ColE1 replicon from the cell pole to within the nucleoid region. This repositioning did not result in destabilization but did result in an increase in the number of plasmid foci, most likely due to partial declustering. These results are consistent with the importance of par regions to the localization of plasmids to specific regions of the cell and demonstrate both localization and dynamic movement for a naturally occurring plasmid that does not encode a replication initiation protein or a partitioning system that is required for plasmid stability.

Bacterial Proteins↗
PubMed1980

Integration of specialized transducing bacteriophage lambda cI857 St68 h80 dgnd his by an unusual pathway promotes formation of deletions and generates a new translocatable element.

Molecular and genetic studies have revealed that several illegitimate recombinational events are associated with integration of the specialized transducing bacteriophage lambda cI57 St68 h80 dgnd his into either the Escherichia coli chromosome or into a plasmid. Most Gnd+ His+ transductants did not carry the prophage at att phi-80, and 10% were not immune to lambda, i.e., "nonlysogenic." Integration of the phage was independent of the phage Int and Red gene products and of the host's general recombination (Rec) system. In further studies, bacterial strains were selected which carried the phage integrated into an R-factor, pSC50. Restriction endonuclease analysis of plasmid deoxyribonucleic acid (DNA) purified from these strains showed that formation of the hybrid plasmids resulted from recombination between a single region of pSC50 and one of several sites within the lambda-phi 80 portion of the phage. Furthermore the his-gnd region of the phage, present in the chromosome of one nonlysogenic transductant, was shown to be able to translocate to pSC50. Concomitant deletion of phage DNA sequences or pSC50 DNA was frequently observed in conjunction with these integration or translocation events. In supplemental studies, a 22- to 24-megadalton segment of the his-gnd region of the chromosome of a prototrophic recA E. coli strain was shown to translocate to pSC50. One terminus of this translocatable segment was near gnd and was the same as a terminus of the his-gnd segment of the phage which translocated from the chromosome of the nonlysogenic transductant. These data suggest that integration of lambda cI857 St 68 h80 dgnd his may be directed by a recombinationally active sequence on another replicon and that the resulting cointegrate structure is subject to the formation of deletions which extend from the recombinationally active sequence. Translocation of the his-gnd portion of the phage probably requires prior replicon fusion, whereas the his-gnd region of the normal E. coli chromosome may comprise a discrete, transposable element.

Bacteriophage lambda↗
PubMed1981

Replication control and switch-off function as observed with a mini-F factor plasmid.

Mini-F is a fragment of the F plasmid, consisting of 9,000 base pairs, which carries all of the genes and sites required for replicon maintenance and control. Its copy number is one to two per chromosome. This plasmid is joined to ColE1, whose copy number is 16 to 20. Under normal circumstances the composite plasmid replication exhibited ColE1 characteristics, maintaining a high copy number. However, when ColE1 replication was inhibited by deoxyribonucleic acid polymerase I inactivation, its replication exhibited mini-F characteristics, maintaining a low copy number. These observations are in complete agreement with those of Timmis et al. (Proc. Natl. Acad. Sci. U.S.A. 71:4556-4560, 1974), who examined the behavior of a recombinant plasmid formed between pSC101 and ColE1. The transition from high to low copy number allowed us to examine the control system acting in cells carrying plasmids exhibiting intermediate copy numbers. The initiation of the mini-F replication system as represented by deoxyribonucleic acid synthesis of the composite plasmid was completely blocked when there were multiple copies of mini-F in a cell. It was not restored until the copy number was lowered to one to two, after which replication was first detected. ppF, a mini-F replicon packaged in a phage lambda head behaved similarly: its replication was completely shut off when the resident mini-F genome copy number was high and was inhibited partially when the resident mini-F genome copy number was low. These experiments clearly demonstrate that there is a switch-off mechanism acting on deoxyribonucleic acid synthesis (initiation) in a cell carrying mini-F, and its intensity is related to the plasmid copy number. This result supports the "inhibitor dilution model" proposed by Pritchard et al. (Symp. Soc. Gen. Microbiol. 19:263-297, 1969). The nature of the hypothetical inhibitor is discussed.

DNA Replication↗
PubMed1982

Interference of plasmid pCM194 with lysogeny of bacteriophage SP02 in Bacillus subtilis.

Three observations indicated that the 2-megadalton chloramphenicol resistance plasmid pCM194 interferes with SP02 lysogeny of Bacillus subtilis. SP02 plaques formed on B. subtilis(pCM194) appeared almost clear, whereas plaques produced on plasmid-free or pUB110-containing cells contained large turbid centers. The number of phages spontaneously liberated by B. subtilis(SP02) was increased 10-fold or more when pCM194 was also present in the lysogens. Lastly, growth of B. subtilis(SP02, pCM194) for approximately 20 to 25 generations resulted in essentially complete loss of the prophage. This interference was not observed with pUB110 or pE194, and the pCM194 interference was not directed against B. subtilis temperate phage phi 105, which is unrelated to SP02. Lytic replication of SP02 appeared to be unaffected by pCM194. pCM194 interference with SP02 lysogeny was demonstrable in recombination-proficient strains and a recE mutant of B. subtilis. SP02 prophage which were noninducible due to the phage ind mutation were resistant to pCM194 interference. pCM194 interference was lost when the entire pCM194 molecule was joined at its unique HpaII site or at one of the two MboI sites to pUB110 or pUB110 derivatives. pBR322 joined to pCM194 at the same MboI site or at the HindIII site produced chimeras that retained the ability to interfere with SP02 lysogeny. A three-part plasmid constructed by joining pBR322 to pCM194 (at HindIII sites) and to pE194 (at PstI sites) was compatible with the SP02 prophage and showed a temperature-sensitive replication phenotype characteristic of the pE194 replicon. One explanation for the interference involves competition for a host component between an SP02 genome attempting to establish lysogeny and plasmids whose replication is directed by the pCM194 replicon.

Bacillus subtilis↗
PubMed1982

Multiple mechanisms for expression of incompatibility by broad-host-range plasmid RK2.

By cloning fragments of plasmid DNA, we have shown that RK2 expresses incompatibility by more than one mechanism. One previously identified (R. J. Meyer, Mol. Gen, Genet. 177:155--161, 1979; Thomas et al., Mol. Gen. Genet. 181:1--7, 1981) determinant for incompatibility is linked to the origin of plasmid DNA replication. When cloned into a plasmid vector, this determinant prevents the stable inheritance of a coresident RK2. However, susceptibility to this mechanism of incompatibility requires an active RK2 replicon and is abolished if another replicator is provided. We have also cloned a second incompatibility determinant, encoded within the 54.1- to 56.4-kilobase region of RK2 DNA, which we call IncP-1(II). An RK2 derivative remains sensitive to IncP-1(II), even when it is not replicating by means of the RK2 replicon. The 54.1- to 56.4-kilobase DNA does not confer susceptibility to the IncP-1(II) mechanism, nor does it encode a detectable system for efficient plasmid partitioning. The incompatibility may be related to the expression of genes mapping in the 54.1- to 56.4-kilobase region, which are required for plasmid maintenance and suppression of plasmid-encoded killing functions.

Cloning, Molecular↗
PubMed1983

Isolation and characterization of P1 minireplicons, lambda-P1:5R and lambda-P1:5L.

We have isolated two new classes of P1 miniplasmids, called lambda-P1:5R and lambda-P1:5L, by the in vivo extension of a cloned P1 fragment, EcoRI-5, which by itself is not capable of plasmid replication. The lambda-P1:5R plasmids contain EcoRI-5 plus a variable portion of the adjacent P1 EcoRI fragment 8. They have a copy number like that of P1 (about 1 per host chromosome), are faithfully segregated at cell division, and are subject to incompatibility exerted by either a single copy of P1 or a single copy of EcoRI-5. In contrast, the lambda-P1:5L plasmids contain EcoRI-5 and a portion of adjacent P1 DNA that includes at least P1 EcoRI fragments 15, 18, and 23 and a part of fragment 17. These plasmids have a copy number of about 15 per cell chromosome. Despite this they are segregated to daughter cells somewhat less faithfully than are lambda-P1:5R plasmids. They are sensitive to incompatibility exerted by a single copy of P1, but not to incompatibility exerted by a single copy of EcoRI-5. lambda-P1:5L plasmids are, however, sensitive to incompatibility exerted by multiple copies of EcoRI-5. These results show that the relative copy numbers of exerting and responding elements are important for the incompatibility phenotype and strongly suggest that lambda-P1:5L plasmids lack a repressor of replication that can be supplied in trans from P1 but not from EcoRI fragment 5. We suggest that P1 normally uses the 5R replicon and that the 5L replicon may be a backup system that ensures plasmid maintenance should the primary replication event fail to initiate.

Bacteriophage lambda↗
PubMed1985

Incompatibility mutants of IncFII plasmid NR1 and their effect on replication control.

DNA from the replication control region of plasmid NR1 or of the Inc- copy mutant pRR12 was cloned into a pBR322 vector plasmid. These pBR322 derivatives were mutagenized in vitro with hydroxylamine and transformed into Escherichia coli cells that harbored either NR1 or pRR12. After selection for the newly introduced pBR322 derivatives only, those cells which retained the unselected resident NR1 or pRR12 plasmids were examined further. By this process, 134 plasmids with Inc- mutations in the cloned NR1 or pRR12 DNA were obtained. These mutants fell into 11 classes. Two of the classes had plasmids with deletions or insertions in the NR1 DNA and were not examined further. Plasmids with apparent point mutations were classified by examining (i) their ability to reconstitute a functional NR1-derived replicon (Rep+ or Rep-), (ii) the copy numbers of the Rep+ reconstituted replicons, (iii) the cross-reactivity of incompatability among the various mutant classes and parental plasmids, and (iv) the trans effects of the mutants on the copy number and stable inheritance of a coresident plasmid.

Cloning, Molecular↗
PubMed1985

Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria.

The replication and maintenance properties of the broad-host-range plasmid RK2 and its derivatives were examined in nine gram-negative bacterial species. Two regions of RK2, the origin of replication (oriV) and a segment that encodes for a replication protein (trfA delta kilD, designated trfA*), are sufficient for replication in all nine species tested. However, stable maintenance of this minimal replicon (less than 0.3% loss per generation under nonselection conditions) is observed only in Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, and Azotobacter vinelandii. Maintenance of this minimal replicon is unstable in Rhizobium meliloti, Agrobacterium tumefaciens, Caulobacter crescentus, Acinetobacter calcoaceticus, and Rhodopseudomonas sphaeroides. A maintenance function has been localized to a 3.1-kilobase (kb) region of RK2 encoding three previously described functions: korA (trfB korB1 korD), incP1-(II), and korB. The 3.1-kb maintenance region can increase or decrease the stability of maintenance of RK2 derivatives dependent on the host species and the presence or absence of the RK2 origin of conjugal transfer (oriT). In the case of A. calcoaceticus, stable maintenance requires an RK2 segment that includes the promoter and the kilD (kilB1) functions of the trfA operon in addition to the 3.1-kb maintenance region. The broad-host-range maintenance requirements of plasmid RK2, therefore, are encoded by multiple functions, and the requirement for one or more of these functions varies among gram-negative bacterial species.

Base Sequence↗
PubMed1989

Replication and segregational stability of Bacillus plasmid pBAA1.

A cryptic plasmid, pBAA1, was identified in an industrial Bacillus strain. The plasmid is 6.8 kilobases in size and is present in cells at a copy number of approximately 5 per chromosome equivalent. The plasmid has been maintained under industrial fermentation conditions without apparent selective pressure and so is assumed to be partition proficient. The minimal replicon was localized to a 1.4-kilobase fragment which also contains the functions required for copy number control. The very low level of segregational instability of the minimal replicon suggests that it also contains functions involved in plasmid maintenance. Comparison with other plasmids indicates that pBAA1 belongs to the group of small gram-positive plasmids which replicate by a rolling cycle-type mechanism. A sequence was identified which is required for the efficient conversion of the single plus strand to the double-stranded form during plasmid replication. Deletion of this sequence resulted in a low level of segregational plasmid instability.

Amino Acid Sequence↗
PubMed1992

High-molecular-weight linear multimer formation by single-stranded DNA plasmids in Escherichia coli.

We inserted foreign DNA segments into plasmids which replicate by a rolling-circle mechanism in Escherichia coli and observed the appearance of high-molecular-weight plasmid multimers (HMW). This phenomenon, which occurs more frequently with GC-rich segments, depends on the mode of replication of the plasmid and on host homologous recombination functions. We found that (i) HMW are formed upon insertion of a foreign DNA segment into a single-stranded DNA plasmid, whereas the same DNA insert has no such effect on a theta replicon, and (ii) HMW are not present in a recA mutant strain but are found in a lexA (Ind-) mutant. Enzymatic studies allowed us to define the HMW structure as linear double-stranded tandem head-to-tail plasmid repeats. Use of heteroplasmid strains showed that HMW production by one plasmid does not affect another resident plasmid, indicating that no host functions are phenotypically inactivated. This distinguishes our system from the HMW observed with various replicons in the absence of RecBCD enzyme activity. We propose that the role of the foreign insert is to protect the DNA from RecBCD exonuclease attack.

DNA Mutational Analysis↗
PubMed1993

Identification, characterization, and nucleotide sequence of a region of Enterococcus faecalis pheromone-responsive plasmid pAD1 capable of autonomous replication.

A 5-kbp region of pAD1, previously shown to be capable of supporting replication, copy control, and stable inheritance of the plasmid, was cloned into a replicon probe vector and subjected to transposon insertional mutagenesis. Transposon inserts identifying essential replication, copy control, and stability functions were isolated. Deletion of stability functions not essential for replication resulted in delimitation of a basic replicon. The complete DNA sequence of this approximately 3-kbp region and the precise positions of several transposon inserts were determined, and the phenotypic effects of the transposon inserts were correlated with the physical locations of individual determinants. The following three genes, apparently involved in plasmid maintenance, were identified; repA, which encodes a protein required for replication; repB, which encodes a protein involved in copy control; and repC, which may be involved in stable inheritance. In addition, two clusters of repeats composed of a consensus sequence, TAGTARRR, were identified, one located between the divergently transcribed repA and repB genes and another located downstream of repC. The region between repA and repB contained 25 repeats divided into two subregions of 13 and 12 repeats separated by 78 bp. The region located downstream of repC contained only three repeats but may be essential for plasmid replication, since deletion of this determinant resulted in loss of ability to replicate in Enterococcus faecalis. We hypothesize that the repeat units represent protein-binding sites required for assembly of the replisome and control of plasmid copy number. Another region of unrelated repeat units that may also be involved in replication is located within the repA gene. Possible mechanisms of action of these determinants are discussed.

Amino Acid Sequence↗
PubMed1996

Transformation of Coxiella burnetii to ampicillin resistance.

A 5.8-kb chromosomal fragment isolated from Coxiella burnetii initiates plasmid replication in Escherichia coli and was characterized as an autonomous replication sequence, ars (M. Suhan, S.-Y. Chen, H.A. Thompson, T.A. Hoover, A. Hill, and J.C. Williams, J. Bacteriol. 176:5233-5243, 1994). In the present study, an ars replicon was used to transform C. burnetii to ampicillin resistance. Plasmid pSKO(+)1000 contained the C. burnetii ars sequence cloned into a ColE1-type replicon encoding beta-lactamase. pSKO(+)1000 was introduced into C. burnetii by electroporation. Ampicillin-resistant cells were selected, and survivors were examined for the transformed genotype by Southern hybridization. Transformants stably maintained the pSKO(+)1000 bla DNA sequence in the chromosome as a result of homologous recombination. The recombination event resulted in the duplication of the 5.8-kb ars sequence in the C. burnetii chromosome. The bla gene was also located in an episome. However, an ampicillin resistance plasmid lacking the C. burnetii ars sequence did not stably transform C. burnetii. A biological assay analyzing beta-lactamase activity of C. burnetii transformants during acid activation in vitro provided evidence for expression of the bla (beta-lactamase) gene.

Ampicillin↗
PubMed1997

Genome structure and phylogeny in the genus Brucella.

PacI and SpeI restriction maps were obtained for the two chromosomes of each of the six species of the genus Brucella: B. melitensis, B. abortus, B. suis, B. canis, B. ovis, and B. neotomae. Three complementary techniques were used: hybridization with the two replicons as probes, cross-hybridization of restriction fragments, and a new mapping method. For each type strain, a unique I-SceI site was introduced in each of the two replicons, and the location of SpeI sites was determined by linearization at the unique site, partial digestion, and end labeling of the fragments. The restriction and genetic maps of the six species were highly conserved. However, numerous small insertions or deletions, ranging from 1 to 34 kb, were observed by comparison with the map of the reference strain of the genus, B. melitensis 16M. A 21-kb Spel fragment specific to B. ovis was found in the small chromosome of this species. A 640-kb inversion was demonstrated in the B. abortus small chromosome. All of these data allowed the construction of a phylogenetic tree, which reflects the traditional phenetic classification of the genus.

Brucella↗
PubMed1997

Role of the RepA1 protein in RepFIC plasmid replication.

Using a sensitive primer extension technique, we have carried out studies to localize the start site of replication of the replicon RepFIC. In the course of these studies, we have found evidence that supports the hypothesis that transcription is an integral component of the initiation of replication. On the basis of our findings, we suggest that the transcript is processed to act as a primer, and therefore we propose that the transcript has a dual role as primer of replication and mRNA for the RepA1 protein. We present a model, based on our evidence, for the initiation of replication of the replicon RepFIC. This model provides as well an alternative explanation for what has been called the cis action of RepA1, and we show that RepA1 may act in trans as well as in cis.

Bacterial Proteins↗