BACKGROUND
plasmids are self-replicating mobile genetic elements.
they are separate from the chromosome and contain a specific subset of genes from the bacterial genetic pool  <cit> .
many plasmids conjugate between different bacteria, especially related ones, leading to intra- and inter-specific dissemination of plasmid-specific genes, for instance, antibiotic resistance genes.
as a result, virtually identical plasmids are isolated repetitively in different bacterial species  <cit> .
we and others consider that inhibition of plasmid dissemination by inhibiting conjugation might be a useful strategy to enhance or complement the efficacy of antibiotics and curb the isolation of antibiotic resistant bacterial pathogens  <cit> .
with this objective in mind, we proposed to learn which genes in the recipient bacteria are needed for the production of transconjugants.
in  <dig>  curtiss et al.
<cit>  suggested already that conjugation required the active participation of both mating partners.
they demonstrated an association between energy metabolism in the f- parent and the rate of chromosome transfer in hfr × f- matings.
during the 70s, the use of different strategies facilitated the identification of recipient functions implicated in conjugation.
for example, lethal zygosis , a phenomenon initially described by clowes  <cit> , allowed the possibility of selecting for mutants in conjugation recipient ability  <cit> .
resistance to lethal zygosis was accompanied by alterations in membrane functions .
it was initially thought that the transfer of a large amount of dna from an hfr donor was responsible for recipient killing.
however, f- recipients were also killed when transfer was blocked by adding nalidixic acid to mating mixtures .
this fact suggested that extensive damage in the recipient membrane was the primary cause of lethal zygosis  <cit> .
other strategies involved the use of mutants producing bacteriophage-resistance or colicin-tolerance, since both were found to be resistant to conjugation  <cit> .
skurray et al.
<cit>  coined the term "con- mutants".
a con- mutant was defective as a conjugation recipient with either f' or hfr donor strains.
they were distinguished from recombination-deficient mutants , which were unable to inherit dna from hfr donors but not from f' donors.
the con- mutants identified by skurray  <cit>  lacked ompa, a major outer membrane protein, and resulted in  <dig>  to 1% of the parental conjugation frequency.
in addition, certain lipopolysaccharide  mutants of s. typhimurium and e. coli were also described as con- mutants  <cit> .
they differed from skurray's con- mutants both in the sugar composition of the membrane as well as in an altered permeability for certain antibiotics.
subsequently, it was demonstrated that the phosphate diester bridges in the lps backbone as well as some membrane proteins are important in forming a cell surface structure resistant to the penetration of several antibiotics  <cit> .
in summary, these experiments provided evidence for the involvement of specific recipient cell surface components  in conjugation.
unfortunately, most cell envelope mutants were obtained by treatment of recipients with random mutagens  <cit>  so the mutated sites could not be exactly determined.
more recently, other studies focused on the identification of plasmid gene products that interact with the recipient surface.
it was shown that tran and trag are involved in recipient cell recognition as well as in entry exclusion of f-like plasmids  <cit> .
in the inci <dig> plasmid r <dig>  recipient recognition is mediated by expression of seven plasmid-encoded pilv adhesins.
the pilv adhesins are thought to be located at the tips of the thin r <dig> pili in the donor cell.
the different sequences for the c-terminal segments of pilv adhesins are produced by shufflon dna rearrangement and they determine the recipient specificity in liquid matings of plasmid r <dig> through the recognition of lps on the surface of recipient cells.
for instance, pilva adhesin recognizes the glcnac  glc moiety of e. coli r <dig> type lps  <cit> .
here, we present a high-throughput screening  strategy to systematically evaluate the implication of each individual e. coli gene in bacterial conjugation.
we used an automated lux-monitored conjugation assay to screen a collection of  <dig>  random tn-insertion e. coli mutants as well as a collection of  <dig>  deletion mutants in each individual e. coli gene .
our strategy allowed us to cover > 99% of the non-essential e. coli genome and allowed us to gain a general understanding of the implication of e. coli functions in recipients of bacterial conjugation.
RESULTS
implementation of a hts lux-monitored conjugation assay to detect conjugation mutants in recipient cells
the principle of the hts conjugation assay relies on the production of visible light by psu2007::tnlux, a derivative of plasmid r <dig>  psu2007::tnlux contains a lux operon under the control of a lac promoter.
expression of lux in conjugative donor cells is repressed by the lac repressor laci, encoded on a co-resident and non-mobilizable multicopy plasmid .
upon conjugation, psu2007::tnlux, but not puc18::laciq, is transferred to recipient cells and thus light is produced.
there is a direct relationship between light emission and frequency of conjugation  <cit> .
ninety-four independent cultures of e. coli strain dh5α were tested as recipients to examine the reproducibility of the assay.
the donor strain was ucdpm <dig> .
the control assay was validated by the use of two different negative controls: the donor strain alone , and dh5α expressing the entry exclusion protein  of r <dig> .
eex inhibits transfer of a plasmid to a recipient cell harbouring the same element  <cit> , and thus it is equivalent to a conjugation recipient mutant.
the kinetics of plasmid transmission was analysed by following light production versus time .
maximum light values were obtained after  <dig> min conjugation at 37°c .
the median of the arbitrary light units  produced by the  <dig> dh5α colonies was - <dig>  with values ranging between + <dig>  and - <dig> .
when dh5α  was used as a recipient, light production was more than 500-fold lower than with dh5α , underscoring the role of the eex protein in avoiding redundant conjugation.
furthermore, light production of ucdpm <dig> was  <dig>  times lower  confirming the tight control of light production in donor cells.
the small dispersion of light values  in addition to the low quantity of light produced by the negative controls confirm that the automated conjugation assay was suitable for hts of recipient conjugation mutants.
screening of e. coli chromosomal genes involved in bacterial conjugation using keio collection
the 'keio collection' comprises a set of single-gene in-frame deletion mutants of most non-essential genes of e. coli and therefore provides a useful resource for genome-wide testing of mutational effects  <cit> .
in order to test the keio collection as recipients in conjugation, the donor strain ucdpm <dig> was conjugated to each of the  <dig>  keio collection mutants in 96-well microplates using a biomek2000® robot.
light production was measured and alu values calculated as described in methods.
a representation of alu values for the  <dig>  individual clones is shown in fig.
<dig>  the wt recipient strain , the donor strain  and the recipient expressing eex_r <dig>  were included as controls.
the median alu produced by the keio mutants was - <dig> , with values ranging between + <dig>  and - <dig>  .
their distribution is shown in fig.
<dig>  none of the  <dig>  mutants tested showed a decrease in light production comparable to eex_r <dig>  indicating that none of the mutations abolished plasmid r <dig> conjugation .
however, several mutants showed reduced light production.
the  <dig> mutants that showed alu = - <dig>   were re-assayed together with the wt strain  and the appropriate negative controls .
alu values for the mutants were now calculated considering light-wt as the average light value of the  <dig> wt conjugation mixtures.
among the re-assayed mutants only five showed alu ≤ - <dig> .
these mutants were then checked in a standard plate conjugation assay and their transfer frequencies calculated.
the rfac mutation resulted in a 5-fold reduction, the uvrd mutation resulted in a  <dig> -fold reduction and the ihfb, rimm and ybex mutations resulted in 2-fold or less reduction in conjugation frequency .
this result distinguishes between effects in conjugation proper and other effects that could limit the hts recorded frequencies, such as effects on lux expression or the bioluminescence reaction, and thus confirm that none of the keio mutants results in a dramatic drop in the conjugation frequency.
a arbitrary light units produced by the given mutant in the hts conjugation assay .
b results of a standard  <dig> h plate conjugation assay .
results are shown relative to the parental bw <dig> strain.
transfer rates are the average of at least three independent experiments.
screening for e. coli genes involved in bacterial conjugation by using tn insertion mutants
since screening the keio collection did not uncover any e. coli genes with a major role in r <dig> conjugation, random mutagenesis with mini-tn10::km  <cit>  was carried out in order to generate an additional set of e. coli mutants for hts.
in contrast to keio mutants, transposon insertion can generate mutations that inactivate more than one gene  and, more importantly, mutations that affect genes at different levels .
a mutant library was constructed by random insertion of mini-tn10::km in the e. coli chromosome.
a strategy to map transposon insertions was designed based on inverse pcr .
twenty thousand dh5α kmr mini-tn10::km insertion mutants were grown individually and conjugated with donor strain ucdpm <dig> in 96-well plates as described in methods.
alu values were calculated for each mutant.
the median alu produced by the mutants was - <dig>  .
their distribution is shown in fig.
<dig>  among the  <dig>  mutants,  <dig> showed alu ≤ - <dig> .
twenty-two mutants showed only residual growth in liquid media and were eliminated from the screening .
the remaining  <dig> mutants were re-assayed together with the appropriate controls.
alu values for these mutants were re-calculated considering light-wt as the average light value of the  <dig> wt conjugation mixtures.
only  <dig> mutants showed alu ≤ - <dig>  .
genomic dna was isolated from these strains and the transposon insertion point was identified .
the transfer rate of plasmid r <dig> to these mutants were also calculated and compared with the wt strain .
all but one of the  <dig> mutants showed a significant reduction in the transfer rate of r <dig> plasmid when used as recipients.
interestingly, in  <dig> of these  <dig> mutants the insertion was located in genes involved in lps biosynthesis.
the  <dig> insertions in lps genes were located at  <dig> different positions and interrupted one of  <dig> genes  that mapped to two regions of the e. coli chromosome .
however, since transposon insertions might be polar, expression of more than one gene within lps operons could have been affected by each insertion.
as shown in fig.
<dig>  the insertions in lps genes could be classified according to their location in  <dig> different groups:  insertions 144d <dig>  13g <dig>  121a <dig>  94h <dig>  108f <dig>  152f <dig> and 31c <dig>  located either in rfad or rfac, showed the highest impact in the r <dig> transfer .
insertion 11d <dig> in lpca gene  decreased r <dig> transfer by 7-fold .
insertions 68c <dig>  149a <dig> and 7c <dig> interrupting rfap or rfag decreases r <dig> transfer between 3- and 5-fold .
in addition, all  <dig> mutants showed a mucoid colony phenotype, as described previously for other lps mutants  <cit> , and increased susceptibility to nalidixic acid , another reported feature of bacteria defective in lps  <cit> .
the remaining  <dig> insertions targeted non lps-related genes crp, gppa, and nhaa, all with modest reductions in r <dig> transfer .
a arbitrary light units produced by the given mutant in the hts conjugation assay.
b normalized to the wild type  and expressed as a percentage.
transfer rates are the average of at least three independent experiments.
using the lux-monitored conjugation assay to evaluate the transfer rate of f plasmid to the previously identified lps mutants
the two mutant sets used in this study point out lps genes as the main non-essential recipient functions implicated in r <dig> conjugation recipient ability.
they also show that, under surface mating conditions, defective lps in recipient cells results in only minor effects when compared to conjugation of f-like plasmids in liquid media  <cit> .
to evaluate the recipient ability of our lps mutants, along with the gppa mutant , in f plasmid conjugation, a lux derivative of pox <dig> was constructed .
a donor strain harbouring the f-lux derivative  was conjugated with each of the  <dig> lps mutants both in solid and liquid matings.
the appropriate controls were included  and the resulting alu values represented in fig.
<dig>  under surface mating conditions, the  <dig> lps mutants showed similar alu values with respect to a wt recipient  indicating that recipient ability was unaffected.
under liquid mating conditions, however, the  <dig> mutants showed alu values between - <dig> and - <dig>   and therefore a significant decrease in recipient ability, to the level of the negative controls used in the experiment.
in addition, the  <dig> keio mutants which showed the lowest alu values in the hts assay with r <dig> were also tested with the donor strain harbouring an f-lux derivative in solid and liquid mating conditions.
under solid mating conditions, the tested subset of keio mutants showed alu values comparable to the wt strain bw <dig> .
in perfect agreement with the results above, only the mutant k83:b <dig>  showed an important decrement in alu values in comparison to the wild type in liquid mating conditions .
again, this value was similar to the negative controls of the experiment .
discussion
in this study we set out to determine what e. coli genes, if any, are required by the recipient bacteria in plasmid conjugation.
our screening assay used plasmid r <dig> as a test plasmid.
since it conjugates only on solid surfaces, it represents an interesting alternative to f-like plasmids, which are able to conjugate in liquid media.
previous papers reported significant differences between liquid and surface mating  <cit>  with the effects of cell envelope mutations reduced or eliminated in surface mating.
thus a fresh analysis using hts methods was warranted.
all characterized f plasmid con- mutants were lps or ompa mutants .
this fact was interpreted by frost as evidence of a receptor for either the pilus or trag/n products that mediate aggregate formation  <cit> .
alternatively, taylor proposed that con- mutants were affected in the general constitution  of the membrane  <cit> .
it must be remembered that all previous attempts to identify con- mutants used enrichment procedures, such as lethal zygosis or resistance to infection by bacteriophages, in order to select for potential mutants.
these procedures may bias the range of potential con- mutants to those with membrane alterations, leaving out genes involved in other functions .
in this respect, our work incorporated two substantial improvements over previous analyses.
first, we used an exhaustive hts conjugation assay and two independent sets of e. coli mutants that, combined, cover 99% of non-essential e. coli genes.
second, we used plasmid r <dig>  a surface mater, as a test plasmid.
surface mating is genetically simpler  and thus concentrates on the core of the mating apparatus.
due to our assay set up, some mutations might affect other processes besides conjugation per se.
for example, the ihfb mutant showed the lowest value  among the keio mutants.
however, a standard conjugation assay indicated that r <dig> plasmid transferred to an ihfb recipient at a similar rate to the wt .
since it showed decreased light production in the hts assay but a normal transfer rate in the standard conjugation assay, we assume it was affected in the process of light production.
two mutants, crp and rimm, showed a statistically significant reduction in r <dig> transfer in the standard conjugation assay , but since these genes affect a number of bacterial processes  <cit>  we consider their effect on conjugation as scarcely informative.
among the genes regulated by crp there are many membrane transport proteins  <cit> , so a crp e. coli mutant should have membrane alterations among other negative pleiotropic effects.
a mutation of rimm, encoding a 16s rrna processing protein, caused a growth delay both in liquid and solid media .
similarly to a crp mutant, a rimm mutant should be affected in numerous bacterial processes including bacterial viability, and therefore a direct implication in conjugation could not be established.
the remaining mutants showed a decrease in light production as well as a reduction of r <dig> transfer in standard conjugation assays, thus confirming a role in conjugation.
the uvrd mutant showed a transfer rate of less than 50% with respect to the wt strain.
the uvrd gene encodes dna helicase ii, which plays roles in nucleotide excision repair, mismatch repair, homologous recombination and dna replication  <cit> , including replication of rolling circle plasmids  <cit> .
uvrd turned out to be very interesting candidate for further studies since a mutation in this gene blocks the rolling-circle replication of different plasmids after nicking  <cit> .
the modest effect of uvrd mutation on r <dig> plasmid transfer could be a consequence of gene redundancy .
however, most of the con- mutants we obtained were located in genes related to the recipient cell surface structure, as in the previous reports mentioned above.
mutations in two genes encoding membrane proteins nhaa and ybex generated a slight reduction in r <dig> transfer .
the nhaa gene encodes an integral membrane na+/h+ antiporter  <cit> .
ybex is a putative integral membrane protein predicted to be involved in the transport of magnesium and cobalt ions  <cit> .
among the keio mutants, rfac showed the lowest r <dig> transfer rate in standard conjugation assays .
the rfac gene encodes a heptosyltransferase i implicated in lps inner core biosynthesis  <cit> .
mutations in rfac produce a core-defective lps in e. coli with increased permeability to a number of hydrophilic and hydrophobic agents , showing a mucoid colony phenotype, as described previously for other lps mutants  <cit> .
interestingly,  <dig> additional conjugation mutants from the mini-tn10::km insertion library were identified as presenting a mucoid phenotype as well.
furthermore, the mucoid mutants showed increased susceptibility to nalidixic acid .
indeed,  <dig> of these mutants contain insertions in lps biosynthesis genes .
the rfad and rfac genes are involved in the attachment of heptose i to 3-deoxy-d-manno-oct-2-ulosonic acid  in the first steps of the lps inner core biosynthesis  <cit> .
the lpca gene  encodes a sedoheptulose 7-phosphate isomerase used also in the first step of the lps inner core biosynthesis  <cit> .
finally, rfap and rfag encode functions involved in more distal assembly steps of the inner core or in the outer core of lps biosynthesis.
in principle, one would expect that the results from the two screenings carried out in this study will yield similar results.
however, only a mutant in one lps biosynthetic gene  was indentified among the keio collection.
alu values obtained from different keio lps biosynthetic mutants during the hts are represented in table  <dig>  although with alu values below the threshold selected for this screening ,  <dig> keio lps mutants showed negative alu values suggesting a defect in recipient ability.
in agreement with the results obtained with the random insertion strategy, mutations in rfad, rfaf, rfac and lpca genes showed the lowest alu values.
a similar trend was also observed with other mutations identified in non-lps-related genes .
the quantitative differences in alu values obtained with each strategy could be a consequence of polar effects of mini-tn10::km insertions or due to the fact that two different e. coli strains were used.
aarbitrary light units produced by the given mutant in the hts conjugation assay.
the twelfth and last mutant, 130f <dig>  presented an insertion interrupting the coding sequence of gppa, generating a similar reduction in r <dig> transfer as the lps mutants .
the gppa gene encodes a guanosine pentaphosphatase/exopolyphosphatase which is implicated in the hydrolysis of the inorganic polyphosphate chains in bacteria.
although little is known about this enzyme, the ubiquity and dynamic features of polyphosphate suggest a variety of important roles in bacteria.
its mucoid phenotype and increased susceptibility to nalidixic acid led us to think that 130f <dig> could be synthesizing an altered lps.
in summary, our genome-wide screen using two independent sets of mutants did not uncover any mutations resulting in a substantial reduction of plasmid r <dig> conjugation.
the only significant hits, the lps mutants, resulted in transfer frequencies ranging between 16- and 4-fold lower than wt , a minor effect.
nevertheless, using the lux-monitored conjugation assay with an f-lux plasmid, the same set of lps mutants showed strong conjugation inhibition  suggesting a severe defect in the recipient ability in liquid mating conditions.
however, recipient ability was restored when the conjugation experiments were carried out in surface mating conditions .
similar results were previously reported  <cit>  and suggest that lps mutants affect docking between donor and recipient in liquid medium, and not dna processing or transport.
CONCLUSIONS
in principle, we would expect to find two classes of recipient mutations affecting conjugation.
the first class of mutants would affect entry into the recipient, by lack of a suitable receptor, or entry site, or energy for the transport process.
these kinds of mutants have been found to affect phage infection and dna transformation .
the fact that we did not find mutants severely affected in these early stages suggests that conjugation does not require an active involvement of the recipient in the transport process.
this idea was proposed in our "shoot and pump" conjugation model  <cit> .
the present results reinforce our notion that the sheer push force imparted by the type four secretion system  on the pilot protein and on the ensuing dna is sufficient for the transport machinery to act as a syringe or a drilling machine, and inject the pilot protein and the trailing dna into the recipient cell.
from a certain point of view, we can say that bacterial recipient cells cannot avoid conjugation.
the second class of mutation we expected were mutations affecting the reforming of a replicative plasmid in the recipient cell.
this process involves at least recircularization of the transferred dna strand and synthesis of the lagging-strand.
recircularization of the transferred dna is probably accomplished by the transported relaxase as we discussed previously  <cit> .
lagging-strand synthesis can be effected by a number of alternative mechanisms, as shown in the analysis of replication of ssdna phages and rolling circle replicating plasmids  <cit> .
of these, only rna polymerase is an essential enzyme.
genes involved in other pathways, such as dnag, pria, prib and pric were tested as part of the keio collection and showed no effect on conjugation.
thus, either rna polymerase synthesizes the primer for lagging-strand synthesis as occurs for rolling circle plasmid replication  <cit> , or the process has to be directed by a plasmid enzyme.
a plasmid-encoded dna primase exists in some conjugation systems and, interestingly, is transported to the recipient cell as well as being essential for conjugation in some heterologous matings  <cit> .
since plasmid r <dig> contains no dna primase, it probably relies on the activity of the host rna polymerase .
other gene products possibly involved in conjugative dna processing within the recipient cells are ssb, dna polymerases, dna helicases, topoisomerases and gyrases, among others.
to the extent that they were present in the tested mutants of the keio collection, they are not required for conjugation.
what are the consequences of the fact that recipient cells contribute so little to conjugation?
first, bacterial cells that want to avoid being used as recipients have to devise ad hoc mechanisms to inhibit conjugation.
this function seems to be essential in plasmid physiology itself, and plasmids thus invented entry exclusion  <cit> .
in fact, as shown in this work, eex_r <dig> inhibits conjugation more than 500-fold, much more that the best recipient mutation found.
restriction endonucleases are an obvious alternative, which also have been shown to be powerful inhibitors of conjugation  <cit> .
second, there are interesting consequences for biotechnology and synthetic biology.
there is now a rush to engineer minimal bacterial cells, which will be used as simpler reactors in the new biotechnological industry  <cit> .
there is the potential doubt of whether these cells will be amenable to genetic manipulation, perhaps because they lose genes important for recipient ability in conjugation.
our data suggest that this will not be so, at least in the case of conjugation, and minimal cells will be as good recipients as their parental strains.

 plasmids are self-replicating mobile genetic elements. they are separate from the chromosome and contain a specific subset of genes from the bacterial genetic pool. many plasmids conjugate between different bacteria, especially related ones, leading to intra- and inter-specific dissemination of plasmid-specific genes, for instance, antibiotic resistance genes. as a result, virtually identical plasmids are isolated repetitively in different bacterial species. we and others consider that inhibition of plasmid dissemination by inhibiting conjugation might be a useful strategy to enhance or complement the efficacy of antibiotics and curb the isolation of antibiotic resistant bacterial pathogens. with this objective in mind, we proposed to learn which genes in the recipient bacteria are needed for the production of transconjugants. in curtiss et al. suggested already that conjugation required the active participation of both mating partners. they demonstrated an association between energy metabolism in the f- parent and the rate of chromosome transfer in hfr × f- matings. during the 70s, the use of different strategies facilitated the identification of recipient functions implicated in conjugation. for example, lethal zygosis, a phenomenon initially described by clowes, allowed the possibility of selecting for mutants in conjugation recipient ability. resistance to lethal zygosis was accompanied by alterations in membrane functions. it was initially thought that the transfer of a large amount of dna from an hfr donor was responsible for recipient killing. however, f- recipients were also killed when transfer was blocked by adding nalidixic acid to mating mixtures. this fact suggested that extensive damage in the recipient membrane was the primary cause of lethal zygosis. other strategies involved the use of mutants producing bacteriophage-resistance or colicin-tolerance, since both were found to be resistant to conjugation. skurray et al. coined the term "con- mutants". a con- mutant was defective as a conjugation recipient with either f' or hfr donor strains. they were distinguished from recombination-deficient mutants, which were unable to inherit dna from hfr donors but not from f' donors. the con- mutants identified by skurray lacked ompa, a major outer membrane protein, and resulted in to 1% of the parental conjugation frequency. in addition, certain lipopolysaccharide mutants of s. typhimurium and e. coli were also described as con- mutants. they differed from skurray's con- mutants both in the sugar composition of the membrane as well as in an altered permeability for certain antibiotics. subsequently, it was demonstrated that the phosphate diester bridges in the lps backbone as well as some membrane proteins are important in forming a cell surface structure resistant to the penetration of several antibiotics. in summary, these experiments provided evidence for the involvement of specific recipient cell surface components in conjugation. unfortunately, most cell envelope mutants were obtained by treatment of recipients with random mutagens so the mutated sites could not be exactly determined. more recently, other studies focused on the identification of plasmid gene products that interact with the recipient surface. it was shown that tran and trag are involved in recipient cell recognition as well as in entry exclusion of f-like plasmids. in the inci plasmid r recipient recognition is mediated by expression of seven plasmid-encoded pilv adhesins. the pilv adhesins are thought to be located at the tips of the thin r pili in the donor cell. the different sequences for the c-terminal segments of pilv adhesins are produced by shufflon dna rearrangement and they determine the recipient specificity in liquid matings of plasmid r through the recognition of lps on the surface of recipient cells. for instance, pilva adhesin recognizes the glcnac glc moiety of e. coli r type lps. here, we present a high-throughput screening strategy to systematically evaluate the implication of each individual e. coli gene in bacterial conjugation. we used an automated lux-monitored conjugation assay to screen a collection of random tn-insertion e. coli mutants as well as a collection of deletion mutants in each individual e. coli gene. our strategy allowed us to cover > 99% of the non-essential e. coli genome and allowed us to gain a general understanding of the implication of e. coli functions in recipients of bacterial conjugation.  implementation of a hts lux-monitored conjugation assay to detect conjugation mutants in recipient cells the principle of the hts conjugation assay relies on the production of visible light by psu2007::tnlux, a derivative of plasmid r psu2007::tnlux contains a lux operon under the control of a lac promoter. expression of lux in conjugative donor cells is repressed by the lac repressor laci, encoded on a co-resident and non-mobilizable multicopy plasmid. upon conjugation, psu2007::tnlux, but not puc18::laciq, is transferred to recipient cells and thus light is produced. there is a direct relationship between light emission and frequency of conjugation. ninety-four independent cultures of e. coli strain dh5α were tested as recipients to examine the reproducibility of the assay. the donor strain was ucdpm. the control assay was validated by the use of two different negative controls: the donor strain alone, and dh5α expressing the entry exclusion protein of r. eex inhibits transfer of a plasmid to a recipient cell harbouring the same element, and thus it is equivalent to a conjugation recipient mutant. the kinetics of plasmid transmission was analysed by following light production versus time. maximum light values were obtained after min conjugation at 37°c. the median of the arbitrary light units produced by the dh5α colonies was - with values ranging between + and -. when dh5α was used as a recipient, light production was more than 500-fold lower than with dh5α, underscoring the role of the eex protein in avoiding redundant conjugation. furthermore, light production of ucdpm was times lower confirming the tight control of light production in donor cells. the small dispersion of light values in addition to the low quantity of light produced by the negative controls confirm that the automated conjugation assay was suitable for hts of recipient conjugation mutants. screening of e. coli chromosomal genes involved in bacterial conjugation using keio collection the'keio collection' comprises a set of single-gene in-frame deletion mutants of most non-essential genes of e. coli and therefore provides a useful resource for genome-wide testing of mutational effects. in order to test the keio collection as recipients in conjugation, the donor strain ucdpm was conjugated to each of the keio collection mutants in 96-well microplates using a biomek2000® robot. light production was measured and alu values calculated as described in methods. a representation of alu values for the individual clones is shown in fig. the wt recipient strain, the donor strain and the recipient expressing eex_r were included as controls. the median alu produced by the keio mutants was -, with values ranging between + and -. their distribution is shown in fig. none of the mutants tested showed a decrease in light production comparable to eex_r indicating that none of the mutations abolished plasmid r conjugation. however, several mutants showed reduced light production. the mutants that showed alu = - were re-assayed together with the wt strain and the appropriate negative controls. alu values for the mutants were now calculated considering light-wt as the average light value of the wt conjugation mixtures. among the re-assayed mutants only five showed alu ≤ -. these mutants were then checked in a standard plate conjugation assay and their transfer frequencies calculated. the rfac mutation resulted in a 5-fold reduction, the uvrd mutation resulted in a -fold reduction and the ihfb, rimm and ybex mutations resulted in 2-fold or less reduction in conjugation frequency. this result distinguishes between effects in conjugation proper and other effects that could limit the hts recorded frequencies, such as effects on lux expression or the bioluminescence reaction, and thus confirm that none of the keio mutants results in a dramatic drop in the conjugation frequency. a arbitrary light units produced by the given mutant in the hts conjugation assay. b results of a standard h plate conjugation assay. results are shown relative to the parental bw strain. transfer rates are the average of at least three independent experiments. screening for e. coli genes involved in bacterial conjugation by using tn insertion mutants since screening the keio collection did not uncover any e. coli genes with a major role in r conjugation, random mutagenesis with mini-tn10::km was carried out in order to generate an additional set of e. coli mutants for hts. in contrast to keio mutants, transposon insertion can generate mutations that inactivate more than one gene and, more importantly, mutations that affect genes at different levels. a mutant library was constructed by random insertion of mini-tn10::km in the e. coli chromosome. a strategy to map transposon insertions was designed based on inverse pcr. twenty thousand dh5α kmr mini-tn10::km insertion mutants were grown individually and conjugated with donor strain ucdpm in 96-well plates as described in methods. alu values were calculated for each mutant. the median alu produced by the mutants was -. their distribution is shown in fig. among the mutants, showed alu ≤ -. twenty-two mutants showed only residual growth in liquid media and were eliminated from the screening. the remaining mutants were re-assayed together with the appropriate controls. alu values for these mutants were re-calculated considering light-wt as the average light value of the wt conjugation mixtures. only mutants showed alu ≤ -. genomic dna was isolated from these strains and the transposon insertion point was identified. the transfer rate of plasmid r to these mutants were also calculated and compared with the wt strain. all but one of the mutants showed a significant reduction in the transfer rate of r plasmid when used as recipients. interestingly, in of these mutants the insertion was located in genes involved in lps biosynthesis. the insertions in lps genes were located at different positions and interrupted one of genes that mapped to two regions of the e. coli chromosome. however, since transposon insertions might be polar, expression of more than one gene within lps operons could have been affected by each insertion. as shown in fig. the insertions in lps genes could be classified according to their location in different groups: insertions 144d 13g 121a 94h 108f 152f and 31c located either in rfad or rfac, showed the highest impact in the r transfer. insertion 11d in lpca gene decreased r transfer by 7-fold. insertions 68c 149a and 7c interrupting rfap or rfag decreases r transfer between 3- and 5-fold. in addition, all mutants showed a mucoid colony phenotype, as described previously for other lps mutants, and increased susceptibility to nalidixic acid, another reported feature of bacteria defective in lps. the remaining insertions targeted non lps-related genes crp, gppa, and nhaa, all with modest reductions in r transfer. a arbitrary light units produced by the given mutant in the hts conjugation assay. b normalized to the wild type and expressed as a percentage. transfer rates are the average of at least three independent experiments. using the lux-monitored conjugation assay to evaluate the transfer rate of f plasmid to the previously identified lps mutants the two mutant sets used in this study point out lps genes as the main non-essential recipient functions implicated in r conjugation recipient ability. they also show that, under surface mating conditions, defective lps in recipient cells results in only minor effects when compared to conjugation of f-like plasmids in liquid media. to evaluate the recipient ability of our lps mutants, along with the gppa mutant, in f plasmid conjugation, a lux derivative of pox was constructed. a donor strain harbouring the f-lux derivative was conjugated with each of the lps mutants both in solid and liquid matings. the appropriate controls were included and the resulting alu values represented in fig. under surface mating conditions, the lps mutants showed similar alu values with respect to a wt recipient indicating that recipient ability was unaffected. under liquid mating conditions, however, the mutants showed alu values between - and - and therefore a significant decrease in recipient ability, to the level of the negative controls used in the experiment. in addition, the keio mutants which showed the lowest alu values in the hts assay with r were also tested with the donor strain harbouring an f-lux derivative in solid and liquid mating conditions. under solid mating conditions, the tested subset of keio mutants showed alu values comparable to the wt strain bw. in perfect agreement with the results above, only the mutant k83:b showed an important decrement in alu values in comparison to the wild type in liquid mating conditions. again, this value was similar to the negative controls of the experiment. discussion in this study we set out to determine what e. coli genes, if any, are required by the recipient bacteria in plasmid conjugation. our screening assay used plasmid r as a test plasmid. since it conjugates only on solid surfaces, it represents an interesting alternative to f-like plasmids, which are able to conjugate in liquid media. previous papers reported significant differences between liquid and surface mating with the effects of cell envelope mutations reduced or eliminated in surface mating. thus a fresh analysis using hts methods was warranted. all characterized f plasmid con- mutants were lps or ompa mutants. this fact was interpreted by frost as evidence of a receptor for either the pilus or trag/n products that mediate aggregate formation. alternatively, taylor proposed that con- mutants were affected in the general constitution of the membrane. it must be remembered that all previous attempts to identify con- mutants used enrichment procedures, such as lethal zygosis or resistance to infection by bacteriophages, in order to select for potential mutants. these procedures may bias the range of potential con- mutants to those with membrane alterations, leaving out genes involved in other functions. in this respect, our work incorporated two substantial improvements over previous analyses. first, we used an exhaustive hts conjugation assay and two independent sets of e. coli mutants that, combined, cover 99% of non-essential e. coli genes. second, we used plasmid r a surface mater, as a test plasmid. surface mating is genetically simpler and thus concentrates on the core of the mating apparatus. due to our assay set up, some mutations might affect other processes besides conjugation per se. for example, the ihfb mutant showed the lowest value among the keio mutants. however, a standard conjugation assay indicated that r plasmid transferred to an ihfb recipient at a similar rate to the wt. since it showed decreased light production in the hts assay but a normal transfer rate in the standard conjugation assay, we assume it was affected in the process of light production. two mutants, crp and rimm, showed a statistically significant reduction in r transfer in the standard conjugation assay, but since these genes affect a number of bacterial processes we consider their effect on conjugation as scarcely informative. among the genes regulated by crp there are many membrane transport proteins, so a crp e. coli mutant should have membrane alterations among other negative pleiotropic effects. a mutation of rimm, encoding a 16s rrna processing protein, caused a growth delay both in liquid and solid media. similarly to a crp mutant, a rimm mutant should be affected in numerous bacterial processes including bacterial viability, and therefore a direct implication in conjugation could not be established. the remaining mutants showed a decrease in light production as well as a reduction of r transfer in standard conjugation assays, thus confirming a role in conjugation. the uvrd mutant showed a transfer rate of less than 50% with respect to the wt strain. the uvrd gene encodes dna helicase ii, which plays roles in nucleotide excision repair, mismatch repair, homologous recombination and dna replication, including replication of rolling circle plasmids. uvrd turned out to be very interesting candidate for further studies since a mutation in this gene blocks the rolling-circle replication of different plasmids after nicking. the modest effect of uvrd mutation on r plasmid transfer could be a consequence of gene redundancy. however, most of the con- mutants we obtained were located in genes related to the recipient cell surface structure, as in the previous reports mentioned above. mutations in two genes encoding membrane proteins nhaa and ybex generated a slight reduction in r transfer. the nhaa gene encodes an integral membrane na+/h+ antiporter. ybex is a putative integral membrane protein predicted to be involved in the transport of magnesium and cobalt ions. among the keio mutants, rfac showed the lowest r transfer rate in standard conjugation assays. the rfac gene encodes a heptosyltransferase i implicated in lps inner core biosynthesis. mutations in rfac produce a core-defective lps in e. coli with increased permeability to a number of hydrophilic and hydrophobic agents, showing a mucoid colony phenotype, as described previously for other lps mutants. interestingly, additional conjugation mutants from the mini-tn10::km insertion library were identified as presenting a mucoid phenotype as well. furthermore, the mucoid mutants showed increased susceptibility to nalidixic acid. indeed, of these mutants contain insertions in lps biosynthesis genes. the rfad and rfac genes are involved in the attachment of heptose i to 3-deoxy-d-manno-oct-2-ulosonic acid in the first steps of the lps inner core biosynthesis. the lpca gene encodes a sedoheptulose 7-phosphate isomerase used also in the first step of the lps inner core biosynthesis. finally, rfap and rfag encode functions involved in more distal assembly steps of the inner core or in the outer core of lps biosynthesis. in principle, one would expect that the results from the two screenings carried out in this study will yield similar results. however, only a mutant in one lps biosynthetic gene was indentified among the keio collection. alu values obtained from different keio lps biosynthetic mutants during the hts are represented in table although with alu values below the threshold selected for this screening, keio lps mutants showed negative alu values suggesting a defect in recipient ability. in agreement with the results obtained with the random insertion strategy, mutations in rfad, rfaf, rfac and lpca genes showed the lowest alu values. a similar trend was also observed with other mutations identified in non-lps-related genes. the quantitative differences in alu values obtained with each strategy could be a consequence of polar effects of mini-tn10::km insertions or due to the fact that two different e. coli strains were used. aarbitrary light units produced by the given mutant in the hts conjugation assay. the twelfth and last mutant, 130f presented an insertion interrupting the coding sequence of gppa, generating a similar reduction in r transfer as the lps mutants. the gppa gene encodes a guanosine pentaphosphatase/exopolyphosphatase which is implicated in the hydrolysis of the inorganic polyphosphate chains in bacteria. although little is known about this enzyme, the ubiquity and dynamic features of polyphosphate suggest a variety of important roles in bacteria. its mucoid phenotype and increased susceptibility to nalidixic acid led us to think that 130f could be synthesizing an altered lps. in summary, our genome-wide screen using two independent sets of mutants did not uncover any mutations resulting in a substantial reduction of plasmid r conjugation. the only significant hits, the lps mutants, resulted in transfer frequencies ranging between 16- and 4-fold lower than wt, a minor effect. nevertheless, using the lux-monitored conjugation assay with an f-lux plasmid, the same set of lps mutants showed strong conjugation inhibition suggesting a severe defect in the recipient ability in liquid mating conditions. however, recipient ability was restored when the conjugation experiments were carried out in surface mating conditions. similar results were previously reported and suggest that lps mutants affect docking between donor and recipient in liquid medium, and not dna processing or transport.  in principle, we would expect to find two classes of recipient mutations affecting conjugation. the first class of mutants would affect entry into the recipient, by lack of a suitable receptor, or entry site, or energy for the transport process. these kinds of mutants have been found to affect phage infection and dna transformation. the fact that we did not find mutants severely affected in these early stages suggests that conjugation does not require an active involvement of the recipient in the transport process. this idea was proposed in our "shoot and pump" conjugation model. the present results reinforce our notion that the sheer push force imparted by the type four secretion system on the pilot protein and on the ensuing dna is sufficient for the transport machinery to act as a syringe or a drilling machine, and inject the pilot protein and the trailing dna into the recipient cell. from a certain point of view, we can say that bacterial recipient cells cannot avoid conjugation. the second class of mutation we expected were mutations affecting the reforming of a replicative plasmid in the recipient cell. this process involves at least recircularization of the transferred dna strand and synthesis of the lagging-strand. recircularization of the transferred dna is probably accomplished by the transported relaxase as we discussed previously. lagging-strand synthesis can be effected by a number of alternative mechanisms, as shown in the analysis of replication of ssdna phages and rolling circle replicating plasmids. of these, only rna polymerase is an essential enzyme. genes involved in other pathways, such as dnag, pria, prib and pric were tested as part of the keio collection and showed no effect on conjugation. thus, either rna polymerase synthesizes the primer for lagging-strand synthesis as occurs for rolling circle plasmid replication, or the process has to be directed by a plasmid enzyme. a plasmid-encoded dna primase exists in some conjugation systems and, interestingly, is transported to the recipient cell as well as being essential for conjugation in some heterologous matings. since plasmid r contains no dna primase, it probably relies on the activity of the host rna polymerase. other gene products possibly involved in conjugative dna processing within the recipient cells are ssb, dna polymerases, dna helicases, topoisomerases and gyrases, among others. to the extent that they were present in the tested mutants of the keio collection, they are not required for conjugation. what are the consequences of the fact that recipient cells contribute so little to conjugation? first, bacterial cells that want to avoid being used as recipients have to devise ad hoc mechanisms to inhibit conjugation. this function seems to be essential in plasmid physiology itself, and plasmids thus invented entry exclusion. in fact, as shown in this work, eex_r inhibits conjugation more than 500-fold, much more that the best recipient mutation found. restriction endonucleases are an obvious alternative, which also have been shown to be powerful inhibitors of conjugation. second, there are interesting consequences for biotechnology and synthetic biology. there is now a rush to engineer minimal bacterial cells, which will be used as simpler reactors in the new biotechnological industry. there is the potential doubt of whether these cells will be amenable to genetic manipulation, perhaps because they lose genes important for recipient ability in conjugation. our data suggest that this will not be so, at least in the case of conjugation, and minimal cells will be as good recipients as their parental strains. 
we used an automated conjugation assay and two sets of e. coli mutant collections: the keio collection and a collection of random mini-tn10::km insertion mutants in e. coli strain dh5α. mutations in the lipopolysaccharide synthesis pathway had a modest effect on r plasmid transfer. how does the recipient cell contribute to bacterial conjugation? we conclude from our genome-wide screen that recipient bacterial cells cannot avoid being used as recipients in bacterial conjugation. results indicate that no non-essential recipient e. coli genes exist that play an essential role in conjugation. this is relevant as an indication of the problems in curbing the dissemination of antibiotic resistance and suggests that conjugation acts as a pure drilling machine, with little regard to the constitution of the recipient cell. the same mutations showed a drastic inhibition effect on f-plasmid transfer, but only in liquid matings, suggesting that previously isolated conjugation-defective mutants do in fact impair mating pair formation in liquid mating, but not conjugative dna processing or transport per se.
 how does the recipient cell contribute to bacterial conjugation? to answer this question we systematically analyzed the individual contribution of each escherichia coli gene in matings using plasmid r as a conjugative plasmid. we used an automated conjugation assay and two sets of e. coli mutant collections: the keio collection and a collection of random mini-tn10::km insertion mutants in e. coli strain dh5α. the combined use of both collections assured that we screened > 99% of the e. coli non-essential genes in our survey.  results indicate that no non-essential recipient e. coli genes exist that play an essential role in conjugation. mutations in the lipopolysaccharide synthesis pathway had a modest effect on r plasmid transfer. the same mutations showed a drastic inhibition effect on f-plasmid transfer, but only in liquid matings, suggesting that previously isolated conjugation-defective mutants do in fact impair mating pair formation in liquid mating, but not conjugative dna processing or transport per se.  we conclude from our genome-wide screen that recipient bacterial cells cannot avoid being used as recipients in bacterial conjugation. this is relevant as an indication of the problems in curbing the dissemination of antibiotic resistance and suggests that conjugation acts as a pure drilling machine, with little regard to the constitution of the recipient cell. 