BACKGROUND
the stabilization functions carried by low-copy-number plasmids from a wide range of bacteria ensure their stable inheritance during cell division  <cit> .
putative stabilization modules  are also encoded on bacterial chromosomes .
such modules could be used to construct vectors for biotechnological applications.
the properties of the stabilization modules may vary depending on the host they are expressed in, so a thorough analysis is required before use.
several test vectors are available for studying stabilization functions in bacteria.
most of them rely on narrow-host-range replicons and can be used only in certain e. coli strains or other narrowly defined hosts  <cit> .
pala <dig>  <cit>  and pog <dig>  <cit>  are based on dual pmb <dig> and p <dig> or p <dig> replicons, respectively.
the high-copy-number pmb <dig> replicon requires poli for replication, so the plasmid is stable in pola+ strains but when transformed into a pola mutant, it depends on the phage vegetative replication system and consequently becomes highly unstable as a single-copy molecule unless a stabilization cassette is included.
the standard method for testing putative stabilization functions relies on a classical segregation test, in which a strain with the plasmid is cultured for a certain number of generations without selection and then the number of cells still carrying the plasmid is estimated by the time-consuming replica plating of colonies or serial dilutions .
introduction of the reporter gene lacz in pou <dig>  <cit>  simplifies the screening for plasmid loss, but this very useful test vector can only be applied for e. coli and its closely related species since it relies on the narrow-host-range r <dig> replicon of the incfii incompatibility group  <cit> .
this paper presents a set of highly unstable broad-host-range plasmids based on the rk <dig> replicon of incp- <dig> group  <cit>  designed to test stabilization functions in diverse bacterial species.
pabb <dig> and its derivatives are single-copy vectors specifying chloramphenicol resistance .
the multiple cloning site  is flanked by laco operators serving as binding sites for laci repressor to build a roadblock for polymerizing parb-type proteins encoded by the tested partitioning cassettes of type ia  <cit> .
the unstable vectors contain the xyle  or lacz  reporter gene enabling easy and quick detection of bacterial colonies retaining the plasmid with the potential stabilization cassette.
the plasmid segregation process can also be monitored in liquid cultures by a quantitative xyle activity assay.
variants of the unstable vector mobilizable by the rk <dig>  conjugative system integrated into the e. coli chromosome or by the ra <dig>  one integrated into the pseudomonas putida chromosome are also available.
RESULTS
construction of a highly unstable broad-host-range plasmid
the main aim of this project was to engineer an unstable cloning vector suitable for easy monitoring of segregation functions in a wide range of bacteria.
we chose prk <dig>  <cit> , a derivative of the rk <dig> replicon from the incp-1α incompatibility group, to construct a highly unstable broad-host-range  test vector.
the prk <dig> cloning vector contains four following rk <dig> fragments: i/ a region encoding ssb , the replication initiator protein trfa, upf <dig>  of unknown function  <cit> , and trba, a regulatory protein of rk <dig> conjugative transfer operons  <cit> ; ii/ orivrk <dig> with eight iterons constituting trfa binding sites  <cit> ; iii/ part of the central control operon kora-incc encoding kora, the primary repressor of trfap  <cit> , since strong trfap is unclonable when unregulated  <cit> , and iv/ the traj trak intergenic region with oritrk <dig> to facilitate mobilization by the rk <dig> conjugation system  <cit> .
additionally, the vector carries a tetracycline resistance cassette  and a lacp-lacz fragment with mcs for α-complementation and easy identification of cloned inserts.
prk <dig> had previously been reported as slightly unstable  <cit> , but our plasmid retention tests showed its almost 100 % stability, since after  <dig> generations of growth of e. coli dh5α  under non-selective conditions in l broth without antibiotics almost 100 % of cells retained the plasmid .
the strategy to obtain a truly unstable derivative of the rk <dig> minireplicon was to limit the expression of trfa, first by introducing a promoter-down mutation in trfap and, if required, by adding korb, a second repressor acting cooperatively with kora on trfap  <cit> , to the system.
it has previously been shown that the t → c mutation in the - <dig> sequence of trfap  decreases the promoter activity at least 10-fold  <cit> .
site-directed pcr mutagenesis was used to introduce trfap- <dig> mutation together with an aatii restriction site into prk <dig>  plasmid dna sequencing confirmed the introduction of the desired mutation into the - <dig> sequence of trfap, but also an unexpected deletion of 1974 bp encompassing laczα with mcs and the traj-trak region with oritrk <dig>  the obtained 8716-bp derivative pakb <dig>   was still very stably maintained in e. coli dh5α cells demonstrating 100 % retention after  <dig> generations of growth under non-selective conditions .fig.
<dig> milestones in construction of unstable bhr vector.
circular maps of intermediate  and final vectors  are drawn to scale.
only intact orfs are indicated.
unique or double restriction sites important for cloning are shown, those described in the text are in bold.
t <dig> marks the divergent transcription terminator sequence tpro/tlyz of p <dig> prophage  <cit>
to facilitate insertion of potentially large dna fragments bearing stability cassettes it was required to downsize the cloning vector.
hence, pakb <dig>  was modified by ncoi digestion and self-ligation to delete a 527-bp fragment of the upf <dig>  gene of unknown function  <cit> , the last orf in the ssb-trfa-upf <dig>  operon present only in one subgroup of incp- <dig> plasmids .
although an upf <dig>  function in copy number control  has not been reported yet, the new derivative, pabb <dig>  was slightly less stable in e. coli dh5α cells in comparison to pakb <dig>  and after  <dig> generations of growth without selection 20 % of cells lost the pabb <dig> plasmid .
in the next step the tetracycline resistance operon  of pabb <dig> was replaced with a shorter cat gene  conferring the cmr phenotype.
the resulting plasmid pabb <dig>   demonstrated stability in e. coli dh5α comparable to that of pabb <dig> .fig.
<dig> standard stability tests of constructed vectors.
e. coli dh5α transformants were grown overnight with antibiotic  and for  <dig> generations without antibiotic.
every  <dig> generations appropriate dilution was plated on l agar to obtain approximately  <dig> colonies.
the colonies were replica plated to test for chloramphenicol resistance.
plasmid retention was expressed as percentage of cmr colonies.
the results shown are average from three experiments with standard deviation.
a retention of constructed vectors.
b plasmid copy number estimated by realtime qpcr.
plasmid copy relative to the chromosome was assayed in three independent biological samples with three technical replicates each.
average results for plasmids are presented with sd as follows:  <dig> ;  <dig> ;  <dig> ;  <dig> ;  <dig> ;  <dig> ;  <dig> ;  <dig> , respectively.
c stabilizing properties of active partitioning operon from ra <dig> in pabb <dig> and pabb <dig> vectors.
d effect of iptg-induced expression of p. aeruginosa para-parb operon on pabb <dig> plasmid retention.
dh5α cultures were grown in l broth with various concentrations of iptg
the initial manipulations did not sufficiently decrease the stability of the rk <dig> minireplicon, so it was decided to proceed with the addition of korb encoding a co-repressor of trfap, to the system.
it was also important to inactivate incc since incc and korb constitute the active partitioning system of rk <dig>  <cit> .
two restriction sites, apai and xhoi were introduced into pabb <dig> to facilitate substitution of incc <dig> orf with korbrk <dig> and to give pabb <dig>  before korb cloning, a smai-ncoi fragment encoding klcapra3-xyle-tpro/lyzp <dig> was inserted into pabb <dig> between the trfa and cat genes to give pabb <dig> .
klcapra <dig> is a strong promoter and xyle encodes catechol  <dig> - dioxygenase, whose activity is easy to be monitored following bacteria growth on plates  <cit>  or in liquid cultures .
subsequently, the korbrk <dig> was inserted into pabb <dig> between the xhoi and apai sites.
the obtained pabb <dig> plasmid  demonstrated a high loss rate in e. coli dh5α strain .
pabb <dig>  comprising a modified rk <dig> replication system, the kora-korb operon, the klcap-xyle-tpro/lyzp <dig> reporter cassette and the cm resistance marker, was used in the next step to prepare the final version of the unstable bhr vector for cloning and testing stabilization cassettes in a wide range of bacterial species.
the mcs introduced between the unique ncoi and bsshii  sites in pabb <dig> to give pabb <dig>  contained bglii, ecori, snabi, xhoi, ecl <dig> and saci restriction sites and was surrounded by laco operator sequences.
the binding of laci repressor to the laco operators was expected to act as a roadblock  <cit>  for potentially polymerizing parb partitioning proteins encoded by type ia partition cassettes  <cit>  that might be analyzed using this vector.
parb spreading that follows its binding to pars  may lead to transcriptional silencing of nearby genes and therefore affect results of segregation studies .
finally, the laciq allele  <cit>  was inserted into pabb <dig> to obtain the final construct, the pabb <dig> vector .
the laciq mutation refers to a change in the − <dig> motif of lacip causing overexpression of laci   <cit>  and is often used in recombinant strains or vectors to provide tighter control of laczp  expression in the absence of the iptg inducer.
the high instability of pabb <dig> was confirmed by the standard stability test: only approximately 2 % of cells retained the plasmid after  <dig> generations of growth without selection .
plasmid copy number
the copy number of chosen plasmid constructs described above was determined in e. coli cells by qpcr  <cit> .
the prk <dig> derivatives pakb <dig> , pabb <dig> and pabb <dig> were present in  <dig> to  <dig>  copies per chromosome, similarly to prk <dig> itself .
the number of pabb <dig> and pabb <dig> copies was  <dig>  -  <dig>  per e. coli chromosome.
this lower copy number is due to the tight regulation of trfap- <dig> and underlies the instability of these test vectors.
the copy number of pmpb <dig>  arabadp-trfa, based on pbad <dig>  <cit> , was established at  <dig> copies per chromosome in correlation with a published data  <cit> .
for comparison also a single-copy-number plasmid ra <dig> of incu group  <cit>  was used and demonstrated 1– <dig> copies per chromosome .
plasmidic and chromosomal partitioning cassettes stabilize test plasmids in e. coli
the type ia active-partitioning cassette kora-incc-korb-orf11-pars from ra <dig> plasmid  <cit>  was chosen to check the applicability of the constructed vectors in a stabilization assay in bacteria.
the cassette was cloned into pabb <dig> and pabb <dig> vectors to obtain pakb <dig>  and pakb <dig> , respectively, and both plasmids were tested in the standard stabilization assay in e. coli dh5α strain.
the ra <dig> partitioning cassette did not drastically improve the pabb <dig> plasmid segregation rate: approximately 10 % of cells retained the pakb <dig>  plasmid after  <dig> generations of growth without selection .
remarkably, the same ra <dig> fragment cloned into pabb <dig> exhibited the expected stabilization function and pakb <dig>  was retained in approximately  <dig> and 60 % of e. coli dh5α cells after  <dig> and  <dig> generations of growth without selection, respectively .
the only difference between pabb <dig> and pabb <dig> is the presence of the laciq allele in the latter .
since it has been shown previously that korbra <dig>  spreads on dna after binding to pars and silences nearby genes  <cit> , the different stability of pakb <dig>  and pakb <dig>  convincingly demonstrates that overproduction of laci and its binding to laco sequences flanking the cloned stabilization cassette blocks effectively the korb spreading.
the usefulness of the constructed vectors was also checked with a synthetic chromosomal partition cassette laciq-tacp-para-parb-pars from p. aeruginosa  <cit>  cloned into pabb <dig> to give pabb <dig>
the pabb <dig> plasmid was present in more than 50 % of e. coli cells after  <dig> generations of growth without selection, in comparison to only 10 % of cells retaining empty pabb <dig> .
after  <dig> generations pabb <dig> was still present in 14 % of cells, whereas the retention of pabb <dig> dropped below 1 %.
the stabilization effect of the ra <dig> partitioning cassette cloned in pakb <dig>  was stronger than that demonstrated by the para-parb-parsp.a cassette present in pabb <dig> .
these differences in the stabilization potential could reflect the individual properties of each cassette, but the rather modest effect of the synthetic para-parb-pars cassette of p. aeruginosa could also be due to the low amount of partitioning proteins produced since the para-parb operon in pabb <dig> is expressed at a low basal level from the strongly repressed tacp.
to check which explanation was correct, different concentrations of iptg were used to boost the production of the partition proteins para and parb.
in support of the latter possibility iptg at  <dig> - <dig>  mm improved the stability of pabb <dig> .
a further increase in iptg concentration  did not improve the plasmid stability  probably due to the antagonistic effect of iptg on the action of laci as the roadblock to parb, known to spread on dna starting from pars  <cit> .
catechol  <dig> -dioxygenase activity assay as an estimate of plasmid stability
the results presented in fig.
<dig> come from a standard stabilization assay with the use of replica plating to estimate the proportion of colonies retaining the plasmid tested .
the xyle reporter cassette present in pabb <dig> and pabb <dig> allows the plasmid segregation to be assayed using a simple plate test to visualize colonies that express the xyle reporter gene and hence must have retained the plasmid.
the cultures of transformants were grown without selection for a certain number of generations, diluted and plated onto l agar without antibiotics to get  <dig> to  <dig> colonies.
the colonies were sprayed with 10 mm catechol and those derived from cells that had lost the test plasmid with xyle remained opalescent, those in which the test plasmid was stably maintained turned yellow quickly , whereas colonies of strains carrying an unstable plasmid with the xyle gene, were in various shades of yellow.fig.
<dig> detection of plasmid retention on plates, in liquid cultures and by high-throughput quantification.
a transformants of e. coli dh5α with stable  and unstable  plasmids were grown for  <dig> generations without selection and iptg and then 100 μl of 106-fold dilutions was plated on l agar, sprayed with 10 mm catechol and photographed.
b overnight cultures of e. coli dh5α transformants bearing indicated plasmids were grown for  <dig> generations in l broth and  <dig>  mm iptg where marked.
catechol was added to 1 mm to the cultures and after 5 min of incubation tubes were photographed.
c comparison of segregation data obtained in the standard replica plating test and visualization test  with the use of catechol as in .
the results shown are representative of three independent experiments.
d/f high-throughput quantification of plasmid retention in single-cell subcultures.
overnight cultures of dh5α and dh5α grown in l broth with antibiotic  were diluted to  <dig> cells ml− <dig> in l broth and aliquoted into 100-well plates .
after growth in bioscreen  the subcultures were diluted 100-fold into a new plate and 1/ <dig> vol of 10 mm catechol was added to each well and photographed .
similar tests  were carried out after  <dig> generations of growth without selection .
the photographs were taken after 10 min of incubation with catechol.
the plasmid retention in liquid cultures  corresponds to the percentage of single-cell subcultures turning yellow.
e/g the colour development quantified by od <dig> after addition of catechol to the single-cell subcultures in the wells from  and , respectively.
dh5α strain was used as a negative control.
plasmid segregation during growth without selection in the wells for  <dig> generations  and for total  <dig> generations  was reflected by variable level of xyle activity .
od405/od <dig> ratio for each culture was plotted using boxplotr .
boxes indicate the 25th and 75th percentiles and center lines show the medians.
whiskers mark minimum and maximum values in accordance with spear criteria, and non-overlapping notches indicate that population medians are different with 95 % confidence as determined by r software
a quick semi-quantitative test for plasmid stability can also be performed for liquid cultures directly.
addition of catechol to overnight cultures  to 1 mm final concentration clearly distinguishes those in which xyle is produced by the majority of cells  from the ones where the plasmid is hardly retained  .
care must be taken to determine the initial rate of reaction, i.e., to measure od <dig> within a few minutes  after substrate addition  <cit> .
comparison of the standard plasmid stability assay with plate catechol  <dig> -dioxygenase determination
e. coli dh5α transformants bearing appropriate test plasmids were cultivated in l broth without antibiotics as earlier and tested for plasmid retention after approximately  <dig>   <dig> and  <dig> generations using, in parallel, the standard stabilization assay and the xyle plate test described above.
as shown in fig.
3c the results of plasmid retention estimation are quite similar for the two assays, justifying the use of the quicker xyle test.
high-throughput analysis of plasmid stabilization functions
plasmid retention was quantified in liquid cultures of dh5α and dh5α using a high-throughput procedure.
in this experiment the production of para and parb in dh5α was not induced by iptg to have two plasmids  stabilized to different extent by the various partition cassettes .
dh5α and dh5α strains were used as controls.
cultures of the transformants were grown overnight under selection  and then diluted to  <dig> cells ml− <dig> in l broth and aliquoted into 100-well plates .
the plates were incubated at 37 °c with shaking for ca.
<dig> generations.
in parallel the overnight start cultures were diluted 105-fold and grown in tubes for  <dig> generations without selection and then diluted, aliquoted and incubated as above .
overnight subcultures derived from single cells and grown in the 100-well plates were diluted 100-fold, od <dig> values were measured and after addition of 10 mm catechol to each well  od <dig> was read after 10 min.
the results obtained after  <dig> generations for dh5α showed an almost complete lack of the plasmid indicating its high instability .
in the case of dh5α and dh5α xyle activity was clearly detectable in  <dig> and 90 % of wells, respectively, after  <dig> generations of growth without antibiotic .
after  <dig> generations of growth without antibiotic the corresponding values were  <dig> and 82 % for dh5α and dh5α, respectively .
these results reflected well the differences in stability of the two plasmids observed in the standard and colony visualization assays when dh5α was grown without iptg .
to normalize obtained data the od405/od <dig> ratio was calculated.
it was around 2– <dig> for the control dh5α strain and varied between  <dig> and  <dig> for the dh5α and dh5α strains .
when the ratios were plotted and analyzed, the medians reflecting plasmid retention rates in the subcultures were similar for the two plasmids following growth without selection for  <dig> generations, and substantially higher for dh5α compared with dh5α after  <dig> generations of growth without selection, as observed before.
the high-throughput approach is obviously more reliable than the standard and colony visualization method since human error is minimized and such a quantitative procedure may help to demonstrate even small, but statistically significant differences in stabilities between various plasmids in a given host, the same plasmid in various hosts, or between variants of the same plasmid.
modifications of the test vectors to expand their applicability
the test plasmid pabb <dig> based on the rk <dig> minireplicon can propagate in a variety of species.
since many bacterial species are not easily transformable, two different orit regions amplified from bhr conjugative plasmids, rk <dig> and ra <dig>  were inserted additionally to pabb <dig> to give pesb <dig> and pesb <dig>  respectively.
such vectors are mobilizable during conjugation of the recipient with two different bacterial species, e. coli s17- <dig> with integrated rk <dig> plasmid  <cit>  or p. putida kt <dig> with integrated conjugation module of ra <dig> , which may significantly extend the range of recipient strains.
the suitability of pesb <dig> vector for investigating stability mechanisms other than active partition was in the meantime confirmed by d. bartosik’s group studying plasmidic toxin-antidote  systems.
the use of pesb <dig> vector with cloned hipab system  of pkon <dig> from paracoccus kondratievae  <cit>  allowed analysis of its stabilization functions in various species of alphaproteobacteria e.g., p. pantotrophus and ochrobactrum sp.
.
quick identification of colonies carrying the constructed vectors with the use of the color reaction enabled by xyle cassette could not be applied to some analyzed species e.g., p. aeruginosa, since when pao <dig> was transformed with xyle plasmids it formed yellow colonies due to the intrinsic substrates for catechol  <dig> -dioxygenase.
an alternative test vector, pesb <dig> , was constructed with the klcap-lacz cassette enabling monitoring of plasmid presence by formation of blue colonies in the presence of x-gal.
the partition operon kora-incc-korb-orf11-pars of ra <dig> was inserted between the ecori and xhoi sites in pesb <dig> to give pesb <dig>  p. aeruginosa pao <dig> was transformed with pesb <dig> or pesb <dig>  and transformants were used to estimate plasmid retention .
pesb <dig> or pesb <dig> were also used to transform e. coli s17- <dig> and transformants were applied as donors in conjugation with a rifr derivative of a. tumefaciens lba1010r.
the transconjugants were grown under selective conditions, then diluted appropriately and plated on l agar with x-gal.
the retention rates of both plasmids assessed by the number of blue colonies are shown in fig.
4b.
pesb <dig> was less unstable in the both strains tested in comparison with the original pabb <dig> in e. coli dh5α  probably due to variations in the functioning of the copy-number control circuit of the rk <dig> minireplicon.
the presence of the ra <dig> partition cassette still significantly increased the pesb <dig> retention in p. aeruginosa pao <dig> strain but had a much lower impact on plasmid stability in a. tumefaciens lba1010r.
the reasons for the observed differences in empty vector stability and the stabilization effects of a given cassette in various bacterial species await elucidation.fig.
<dig> functionality of test vectors in various bacterial species.
a
p. aeruginosa pao <dig> was transformed with pesb <dig> and pesb <dig> .
three transformants were grown for  <dig> generations without selection.
the retention of plasmid was estimated by percentage of blue colonies on l agar plates with x-gal.
the results shown are average from three experiments with standard deviation.
b
a. tumefaciens lba1010r rifr was conjugated with e. coli s17- <dig> and s17- <dig> donor strains.
obtained transconjugants were selected on l agar supplemented with chloramphenicol and rifampicin.
three independent transconjugants were grown for  <dig> generations without selection.
the retention of plasmid was estimated by percentage of blue colonies on l agar plates with x-gal.
the results shown are average from three experiments with standard deviation
CONCLUSIONS
we have manipulated the broad-host-range rk <dig> minireplicon prk <dig> to obtain a highly unstable pabb <dig> vector.
to facilitate easy monitoring of plasmid retention, a xyle or lacz reporter system was inserted.
a multiple cloning site surrounded by roadblocks for protein spreading enables analysis of various partition cassettes.
to broaden applications of the vector to a variety of hosts we added orit regions from rk <dig> or ra <dig> bhr plasmids so they could be mobilized by the widely used s17- <dig>  and p. putida kt <dig> with the tra module of ra <dig> constructed in this work, respectively.
the constructed vectors were demonstrated to be useful for cloning chromosomal and plasmid partition operons that produce type ia parb-like proteins able to spread on dna.
retention of the vectors varied depending on the host so they need to be tested in a given strain prior to application.
analysis of the stabilization properties of the cloned partitioning operons in three different hosts  confirmed their variability and at the same time necessity to conduct such experiments.
estimation of plasmid stability using catechol  <dig> -dioxygenase assay in liquid cultures facilitates large scale or high-throughput experiments in bacteria, since hundreds or even thousands of variants can be monitored easily.
it can be used to screen for new stabilization cassettes in meta-genomic approaches, to study stability functions in diverse bacteria and to screen mutant proteins affecting plasmid stability as well as inhibitors of the systems.
the test plasmids described here with an easy detection/monitoring system should be useful for studies of various stabilization functions in a wide range of strains in which the rk <dig> replicon can propagate.

 the stabilization functions carried by low-copy-number plasmids from a wide range of bacteria ensure their stable inheritance during cell division. putative stabilization modules are also encoded on bacterial chromosomes. such modules could be used to construct vectors for biotechnological applications. the properties of the stabilization modules may vary depending on the host they are expressed in, so a thorough analysis is required before use. several test vectors are available for studying stabilization functions in bacteria. most of them rely on narrow-host-range replicons and can be used only in certain e. coli strains or other narrowly defined hosts. pala and pog are based on dual pmb and p or p replicons, respectively. the high-copy-number pmb replicon requires poli for replication, so the plasmid is stable in pola+ strains but when transformed into a pola mutant, it depends on the phage vegetative replication system and consequently becomes highly unstable as a single-copy molecule unless a stabilization cassette is included. the standard method for testing putative stabilization functions relies on a classical segregation test, in which a strain with the plasmid is cultured for a certain number of generations without selection and then the number of cells still carrying the plasmid is estimated by the time-consuming replica plating of colonies or serial dilutions. introduction of the reporter gene lacz in pou simplifies the screening for plasmid loss, but this very useful test vector can only be applied for e. coli and its closely related species since it relies on the narrow-host-range r replicon of the incfii incompatibility group. this paper presents a set of highly unstable broad-host-range plasmids based on the rk replicon of incp- group designed to test stabilization functions in diverse bacterial species. pabb and its derivatives are single-copy vectors specifying chloramphenicol resistance. the multiple cloning site is flanked by laco operators serving as binding sites for laci repressor to build a roadblock for polymerizing parb-type proteins encoded by the tested partitioning cassettes of type ia. the unstable vectors contain the xyle or lacz reporter gene enabling easy and quick detection of bacterial colonies retaining the plasmid with the potential stabilization cassette. the plasmid segregation process can also be monitored in liquid cultures by a quantitative xyle activity assay. variants of the unstable vector mobilizable by the rk conjugative system integrated into the e. coli chromosome or by the ra one integrated into the pseudomonas putida chromosome are also available.  construction of a highly unstable broad-host-range plasmid the main aim of this project was to engineer an unstable cloning vector suitable for easy monitoring of segregation functions in a wide range of bacteria. we chose prk, a derivative of the rk replicon from the incp-1α incompatibility group, to construct a highly unstable broad-host-range test vector. the prk cloning vector contains four following rk fragments: i/ a region encoding ssb, the replication initiator protein trfa, upf of unknown function, and trba, a regulatory protein of rk conjugative transfer operons; ii/ orivrk with eight iterons constituting trfa binding sites; iii/ part of the central control operon kora-incc encoding kora, the primary repressor of trfap, since strong trfap is unclonable when unregulated, and iv/ the traj trak intergenic region with oritrk to facilitate mobilization by the rk conjugation system. additionally, the vector carries a tetracycline resistance cassette and a lacp-lacz fragment with mcs for α-complementation and easy identification of cloned inserts. prk had previously been reported as slightly unstable, but our plasmid retention tests showed its almost 100 % stability, since after generations of growth of e. coli dh5α under non-selective conditions in l broth without antibiotics almost 100 % of cells retained the plasmid. the strategy to obtain a truly unstable derivative of the rk minireplicon was to limit the expression of trfa, first by introducing a promoter-down mutation in trfap and, if required, by adding korb, a second repressor acting cooperatively with kora on trfap, to the system. it has previously been shown that the t → c mutation in the - sequence of trfap decreases the promoter activity at least 10-fold. site-directed pcr mutagenesis was used to introduce trfap- mutation together with an aatii restriction site into prk plasmid dna sequencing confirmed the introduction of the desired mutation into the - sequence of trfap, but also an unexpected deletion of 1974 bp encompassing laczα with mcs and the traj-trak region with oritrk the obtained 8716-bp derivative pakb was still very stably maintained in e. coli dh5α cells demonstrating 100 % retention after generations of growth under non-selective conditions.fig. milestones in construction of unstable bhr vector. circular maps of intermediate and final vectors are drawn to scale. only intact orfs are indicated. unique or double restriction sites important for cloning are shown, those described in the text are in bold. t marks the divergent transcription terminator sequence tpro/tlyz of p prophage to facilitate insertion of potentially large dna fragments bearing stability cassettes it was required to downsize the cloning vector. hence, pakb was modified by ncoi digestion and self-ligation to delete a 527-bp fragment of the upf gene of unknown function, the last orf in the ssb-trfa-upf operon present only in one subgroup of incp- plasmids. although an upf function in copy number control has not been reported yet, the new derivative, pabb was slightly less stable in e. coli dh5α cells in comparison to pakb and after generations of growth without selection 20 % of cells lost the pabb plasmid. in the next step the tetracycline resistance operon of pabb was replaced with a shorter cat gene conferring the cmr phenotype. the resulting plasmid pabb demonstrated stability in e. coli dh5α comparable to that of pabb.fig. standard stability tests of constructed vectors. e. coli dh5α transformants were grown overnight with antibiotic and for generations without antibiotic. every generations appropriate dilution was plated on l agar to obtain approximately colonies. the colonies were replica plated to test for chloramphenicol resistance. plasmid retention was expressed as percentage of cmr colonies. the results shown are average from three experiments with standard deviation. a retention of constructed vectors. b plasmid copy number estimated by realtime qpcr. plasmid copy relative to the chromosome was assayed in three independent biological samples with three technical replicates each. average results for plasmids are presented with sd as follows:;;;;;;;, respectively. c stabilizing properties of active partitioning operon from ra in pabb and pabb vectors. d effect of iptg-induced expression of p. aeruginosa para-parb operon on pabb plasmid retention. dh5α cultures were grown in l broth with various concentrations of iptg the initial manipulations did not sufficiently decrease the stability of the rk minireplicon, so it was decided to proceed with the addition of korb encoding a co-repressor of trfap, to the system. it was also important to inactivate incc since incc and korb constitute the active partitioning system of rk. two restriction sites, apai and xhoi were introduced into pabb to facilitate substitution of incc orf with korbrk and to give pabb before korb cloning, a smai-ncoi fragment encoding klcapra3-xyle-tpro/lyzp was inserted into pabb between the trfa and cat genes to give pabb. klcapra is a strong promoter and xyle encodes catechol - dioxygenase, whose activity is easy to be monitored following bacteria growth on plates or in liquid cultures. subsequently, the korbrk was inserted into pabb between the xhoi and apai sites. the obtained pabb plasmid demonstrated a high loss rate in e. coli dh5α strain. pabb comprising a modified rk replication system, the kora-korb operon, the klcap-xyle-tpro/lyzp reporter cassette and the cm resistance marker, was used in the next step to prepare the final version of the unstable bhr vector for cloning and testing stabilization cassettes in a wide range of bacterial species. the mcs introduced between the unique ncoi and bsshii sites in pabb to give pabb contained bglii, ecori, snabi, xhoi, ecl and saci restriction sites and was surrounded by laco operator sequences. the binding of laci repressor to the laco operators was expected to act as a roadblock for potentially polymerizing parb partitioning proteins encoded by type ia partition cassettes that might be analyzed using this vector. parb spreading that follows its binding to pars may lead to transcriptional silencing of nearby genes and therefore affect results of segregation studies. finally, the laciq allele was inserted into pabb to obtain the final construct, the pabb vector. the laciq mutation refers to a change in the − motif of lacip causing overexpression of laci and is often used in recombinant strains or vectors to provide tighter control of laczp expression in the absence of the iptg inducer. the high instability of pabb was confirmed by the standard stability test: only approximately 2 % of cells retained the plasmid after generations of growth without selection. plasmid copy number the copy number of chosen plasmid constructs described above was determined in e. coli cells by qpcr. the prk derivatives pakb, pabb and pabb were present in to copies per chromosome, similarly to prk itself. the number of pabb and pabb copies was - per e. coli chromosome. this lower copy number is due to the tight regulation of trfap- and underlies the instability of these test vectors. the copy number of pmpb arabadp-trfa, based on pbad, was established at copies per chromosome in correlation with a published data. for comparison also a single-copy-number plasmid ra of incu group was used and demonstrated 1– copies per chromosome. plasmidic and chromosomal partitioning cassettes stabilize test plasmids in e. coli the type ia active-partitioning cassette kora-incc-korb-orf11-pars from ra plasmid was chosen to check the applicability of the constructed vectors in a stabilization assay in bacteria. the cassette was cloned into pabb and pabb vectors to obtain pakb and pakb, respectively, and both plasmids were tested in the standard stabilization assay in e. coli dh5α strain. the ra partitioning cassette did not drastically improve the pabb plasmid segregation rate: approximately 10 % of cells retained the pakb plasmid after generations of growth without selection. remarkably, the same ra fragment cloned into pabb exhibited the expected stabilization function and pakb was retained in approximately and 60 % of e. coli dh5α cells after and generations of growth without selection, respectively. the only difference between pabb and pabb is the presence of the laciq allele in the latter. since it has been shown previously that korbra spreads on dna after binding to pars and silences nearby genes, the different stability of pakb and pakb convincingly demonstrates that overproduction of laci and its binding to laco sequences flanking the cloned stabilization cassette blocks effectively the korb spreading. the usefulness of the constructed vectors was also checked with a synthetic chromosomal partition cassette laciq-tacp-para-parb-pars from p. aeruginosa cloned into pabb to give pabb the pabb plasmid was present in more than 50 % of e. coli cells after generations of growth without selection, in comparison to only 10 % of cells retaining empty pabb. after generations pabb was still present in 14 % of cells, whereas the retention of pabb dropped below 1 %. the stabilization effect of the ra partitioning cassette cloned in pakb was stronger than that demonstrated by the para-parb-parsp.a cassette present in pabb. these differences in the stabilization potential could reflect the individual properties of each cassette, but the rather modest effect of the synthetic para-parb-pars cassette of p. aeruginosa could also be due to the low amount of partitioning proteins produced since the para-parb operon in pabb is expressed at a low basal level from the strongly repressed tacp. to check which explanation was correct, different concentrations of iptg were used to boost the production of the partition proteins para and parb. in support of the latter possibility iptg at -  mm improved the stability of pabb. a further increase in iptg concentration did not improve the plasmid stability probably due to the antagonistic effect of iptg on the action of laci as the roadblock to parb, known to spread on dna starting from pars. catechol -dioxygenase activity assay as an estimate of plasmid stability the results presented in fig.  come from a standard stabilization assay with the use of replica plating to estimate the proportion of colonies retaining the plasmid tested. the xyle reporter cassette present in pabb and pabb allows the plasmid segregation to be assayed using a simple plate test to visualize colonies that express the xyle reporter gene and hence must have retained the plasmid. the cultures of transformants were grown without selection for a certain number of generations, diluted and plated onto l agar without antibiotics to get to colonies. the colonies were sprayed with 10 mm catechol and those derived from cells that had lost the test plasmid with xyle remained opalescent, those in which the test plasmid was stably maintained turned yellow quickly, whereas colonies of strains carrying an unstable plasmid with the xyle gene, were in various shades of yellow.fig. detection of plasmid retention on plates, in liquid cultures and by high-throughput quantification. a transformants of e. coli dh5α with stable and unstable plasmids were grown for generations without selection and iptg and then 100 μl of 106-fold dilutions was plated on l agar, sprayed with 10 mm catechol and photographed. b overnight cultures of e. coli dh5α transformants bearing indicated plasmids were grown for generations in l broth and  mm iptg where marked. catechol was added to 1 mm to the cultures and after 5 min of incubation tubes were photographed. c comparison of segregation data obtained in the standard replica plating test and visualization test with the use of catechol as in. the results shown are representative of three independent experiments. d/f high-throughput quantification of plasmid retention in single-cell subcultures. overnight cultures of dh5α and dh5α grown in l broth with antibiotic were diluted to cells ml− in l broth and aliquoted into 100-well plates. after growth in bioscreen the subcultures were diluted 100-fold into a new plate and 1/ vol of 10 mm catechol was added to each well and photographed. similar tests were carried out after generations of growth without selection. the photographs were taken after 10 min of incubation with catechol. the plasmid retention in liquid cultures corresponds to the percentage of single-cell subcultures turning yellow. e/g the colour development quantified by od after addition of catechol to the single-cell subcultures in the wells from and, respectively. dh5α strain was used as a negative control. plasmid segregation during growth without selection in the wells for generations and for total generations was reflected by variable level of xyle activity. od405/od ratio for each culture was plotted using boxplotr. boxes indicate the 25th and 75th percentiles and center lines show the medians. whiskers mark minimum and maximum values in accordance with spear criteria, and non-overlapping notches indicate that population medians are different with 95 % confidence as determined by r software a quick semi-quantitative test for plasmid stability can also be performed for liquid cultures directly. addition of catechol to overnight cultures to 1 mm final concentration clearly distinguishes those in which xyle is produced by the majority of cells from the ones where the plasmid is hardly retained. care must be taken to determine the initial rate of reaction, i.e., to measure od within a few minutes after substrate addition. comparison of the standard plasmid stability assay with plate catechol -dioxygenase determination e. coli dh5α transformants bearing appropriate test plasmids were cultivated in l broth without antibiotics as earlier and tested for plasmid retention after approximately and generations using, in parallel, the standard stabilization assay and the xyle plate test described above. as shown in fig. 3c the results of plasmid retention estimation are quite similar for the two assays, justifying the use of the quicker xyle test. high-throughput analysis of plasmid stabilization functions plasmid retention was quantified in liquid cultures of dh5α and dh5α using a high-throughput procedure. in this experiment the production of para and parb in dh5α was not induced by iptg to have two plasmids stabilized to different extent by the various partition cassettes. dh5α and dh5α strains were used as controls. cultures of the transformants were grown overnight under selection and then diluted to cells ml− in l broth and aliquoted into 100-well plates. the plates were incubated at 37 °c with shaking for ca. generations. in parallel the overnight start cultures were diluted 105-fold and grown in tubes for generations without selection and then diluted, aliquoted and incubated as above. overnight subcultures derived from single cells and grown in the 100-well plates were diluted 100-fold, od values were measured and after addition of 10 mm catechol to each well od was read after 10 min. the results obtained after generations for dh5α showed an almost complete lack of the plasmid indicating its high instability. in the case of dh5α and dh5α xyle activity was clearly detectable in and 90 % of wells, respectively, after generations of growth without antibiotic. after generations of growth without antibiotic the corresponding values were and 82 % for dh5α and dh5α, respectively. these results reflected well the differences in stability of the two plasmids observed in the standard and colony visualization assays when dh5α was grown without iptg. to normalize obtained data the od405/od ratio was calculated. it was around 2– for the control dh5α strain and varied between and for the dh5α and dh5α strains. when the ratios were plotted and analyzed, the medians reflecting plasmid retention rates in the subcultures were similar for the two plasmids following growth without selection for generations, and substantially higher for dh5α compared with dh5α after generations of growth without selection, as observed before. the high-throughput approach is obviously more reliable than the standard and colony visualization method since human error is minimized and such a quantitative procedure may help to demonstrate even small, but statistically significant differences in stabilities between various plasmids in a given host, the same plasmid in various hosts, or between variants of the same plasmid. modifications of the test vectors to expand their applicability the test plasmid pabb based on the rk minireplicon can propagate in a variety of species. since many bacterial species are not easily transformable, two different orit regions amplified from bhr conjugative plasmids, rk and ra were inserted additionally to pabb to give pesb and pesb respectively. such vectors are mobilizable during conjugation of the recipient with two different bacterial species, e. coli s17- with integrated rk plasmid or p. putida kt with integrated conjugation module of ra, which may significantly extend the range of recipient strains. the suitability of pesb vector for investigating stability mechanisms other than active partition was in the meantime confirmed by d. bartosik’s group studying plasmidic toxin-antidote systems. the use of pesb vector with cloned hipab system of pkon from paracoccus kondratievae allowed analysis of its stabilization functions in various species of alphaproteobacteria e.g., p. pantotrophus and ochrobactrum sp.. quick identification of colonies carrying the constructed vectors with the use of the color reaction enabled by xyle cassette could not be applied to some analyzed species e.g., p. aeruginosa, since when pao was transformed with xyle plasmids it formed yellow colonies due to the intrinsic substrates for catechol -dioxygenase. an alternative test vector, pesb, was constructed with the klcap-lacz cassette enabling monitoring of plasmid presence by formation of blue colonies in the presence of x-gal. the partition operon kora-incc-korb-orf11-pars of ra was inserted between the ecori and xhoi sites in pesb to give pesb p. aeruginosa pao was transformed with pesb or pesb and transformants were used to estimate plasmid retention. pesb or pesb were also used to transform e. coli s17- and transformants were applied as donors in conjugation with a rifr derivative of a. tumefaciens lba1010r. the transconjugants were grown under selective conditions, then diluted appropriately and plated on l agar with x-gal. the retention rates of both plasmids assessed by the number of blue colonies are shown in fig. 4b. pesb was less unstable in the both strains tested in comparison with the original pabb in e. coli dh5α probably due to variations in the functioning of the copy-number control circuit of the rk minireplicon. the presence of the ra partition cassette still significantly increased the pesb retention in p. aeruginosa pao strain but had a much lower impact on plasmid stability in a. tumefaciens lba1010r. the reasons for the observed differences in empty vector stability and the stabilization effects of a given cassette in various bacterial species await elucidation.fig. functionality of test vectors in various bacterial species. a p. aeruginosa pao was transformed with pesb and pesb. three transformants were grown for generations without selection. the retention of plasmid was estimated by percentage of blue colonies on l agar plates with x-gal. the results shown are average from three experiments with standard deviation. b a. tumefaciens lba1010r rifr was conjugated with e. coli s17- and s17- donor strains. obtained transconjugants were selected on l agar supplemented with chloramphenicol and rifampicin. three independent transconjugants were grown for generations without selection. the retention of plasmid was estimated by percentage of blue colonies on l agar plates with x-gal. the results shown are average from three experiments with standard deviation  we have manipulated the broad-host-range rk minireplicon prk to obtain a highly unstable pabb vector. to facilitate easy monitoring of plasmid retention, a xyle or lacz reporter system was inserted. a multiple cloning site surrounded by roadblocks for protein spreading enables analysis of various partition cassettes. to broaden applications of the vector to a variety of hosts we added orit regions from rk or ra bhr plasmids so they could be mobilized by the widely used s17- and p. putida kt with the tra module of ra constructed in this work, respectively. the constructed vectors were demonstrated to be useful for cloning chromosomal and plasmid partition operons that produce type ia parb-like proteins able to spread on dna. retention of the vectors varied depending on the host so they need to be tested in a given strain prior to application. analysis of the stabilization properties of the cloned partitioning operons in three different hosts confirmed their variability and at the same time necessity to conduct such experiments. estimation of plasmid stability using catechol -dioxygenase assay in liquid cultures facilitates large scale or high-throughput experiments in bacteria, since hundreds or even thousands of variants can be monitored easily. it can be used to screen for new stabilization cassettes in meta-genomic approaches, to study stability functions in diverse bacteria and to screen mutant proteins affecting plasmid stability as well as inhibitors of the systems. the test plasmids described here with an easy detection/monitoring system should be useful for studies of various stabilization functions in a wide range of strains in which the rk replicon can propagate. 
the efficiency of stabilization may vary depending on the plasmid host so a thorough analysis of stabilization functions is required before use. the vectors are suitable for testing of various stabilization functions, including plasmid and chromosomal partitioning cassettes encoding parb homologues capable of spreading on dna. the range of bhr vectors with different reporter cassettes and alternative mobilization systems expands their application in diverse bacterial species. low-copy-number vectors of potential wide application in biotechnology need to encode stabilization modules ensuring their stable inheritance. the xyle or lacz reporter systems facilitate easy monitoring of plasmid segregation.
 low-copy-number vectors of potential wide application in biotechnology need to encode stabilization modules ensuring their stable inheritance. the efficiency of stabilization may vary depending on the plasmid host so a thorough analysis of stabilization functions is required before use.  to facilitate such analysis highly unstable, mobilizable, broad-host-range vectors based on rk replicon were constructed. the vectors are suitable for testing of various stabilization functions, including plasmid and chromosomal partitioning cassettes encoding parb homologues capable of spreading on dna. the xyle or lacz reporter systems facilitate easy monitoring of plasmid segregation.  the range of bhr vectors with different reporter cassettes and alternative mobilization systems expands their application in diverse bacterial species. keywords broad-host-rangecloning vectorrk2laczxyle reporterstability functionsthis work was supported by the national science centre in poland and in part by grant pbz-mnisw-04/1/ issue-copyright-statement© the author 2016 