Leptospira and E. coli caught in the act

I found this web photo of E. coli mating with the spirochete Leptospira biflexa in a process called conjugation (image source, Mathieu Picardeau, Pasteur Institute).  The donor E. coli cell is transferring a copy of a plasmid bearing antibiotic resistance genes to the recipient spirochete.  The DNA is most likely pushed through a pore that forms between the mating pair where the outer membranes come together.

There are many types of plasmids, but only self-transmissible plasmids are capable of transferring copies of themselves to other bacteria by conjugation. These plasmids carry a set of at least 20 genes collectively called tra (transfer), which encode all of the proteins necessary to carry out conjugation.  The conjugational proteins assemble into several structures, including the sex pili, which bring the mating pair together, the relaxosome, which processes the DNA for transfer, and the poorly characterized pore through which the DNA traverses.  The plasmids can also harbor additional genes that have no role in conjugation, including genes encoding resistance to antibiotics.  RP4 is one example of a self-transmissible plasmid that can transfer itself to a wide range of bacteria species.  Self-transmissible plasmids have been found in many different bacteria, yet none have been discovered in spirochetes.

Transformation is the microbiologist's favorite genetic tool for delivering DNA of their choosing into bacteria. Unfortunately for those interested in leptospirosis, transformation of disease-causing species of Leptospira such as L. interrogans is difficult.  Conjugation employing a laboratory strain of E. coli as a donor provides scientists another route for delivering DNA into Leptospira.  For example, the plasmid illustrated below (Figure 1 from Picardeau, 2008) has been used to ferry the Himar1 transposon into Leptospira for random insertional mutagenesis.  Many readers may be most familiar with the F conjugational plasmid of E. coli, but the conjugational machinery found on the RP4 self-transmissible plasmid is used here since it is able to deliver DNA to a wide range of bacteria species.

Figure 1 from Picardeau, 2008.  The Himar1 transposon consists of the arrowheads and everything in between, including the kanamycin-resistance gene (Km^R).  The RP4 oriT element and the genes encoding the C9 tranposase and spectinomycin resistance (Spc^R) lie outside of the transposon.


The critical element of the plasmid is the RP4 oriT sequence where relaxase, a component of the relaxosome, nicks the DNA to initiate the transfer process.  The tra genes were removed to permit easy manipulation of the plasmid.  To perform conjugation, the plasmid was transformed into a special E. coli strain that encodes the RP4 tra genes on its chromosome.  The E. coli cells were then mixed with Leptospira and concentrated onto a filter to facilitate mating.  After allowing them to mate for 20 hours, the mating mixture was plated onto Leptospira medium agar plates containing the antibiotic kanamycin to recover Leptospira mutants with the transposon on one of its two chromosomes.  The plasmid itself is unable to replicate in Leptospira, so the transposon must hop onto a chromosome following plasmid transfer to enable growth of kanamycin-resistant Leptospira into colonies.  The donor E. coli bacteria had been genetically modified to require the nutrient diaminopimelate (DAP) to counterselect the donor on the agar plates, which were lacking DAP.

It is not feasible to screen L. interrogans insertion mutants for a desired phenotype (trait) following a single mating experiment since only a few hundred kanamycin-resistant colonies can be recovered.  Tens of thousands of mutants would be necessary to ensure coverage of (almost) all L. interrogans genes.

One application of this genetic tool is to perform multiple mating experiments to generate a library of mutants with insertions of Himar1 in different L. interrogans genes.  The sequence of the insertion site of the transposon in the chromosome can be obtained easily with today's sequencing technology.  Further experiments can be performed to examine any mutants with insertions in genes that hold the investigator's interest.  Several labs have teamed up to embark on a similar approach by delivering the Himar1 transposon into L. interrogans by transformation (see the Murray 2009 paper), which does not yield as many colonies as conjugation.

References

Picardeau, M. (2008).  Conjugative transfer between Escherichia coli and Leptospira spp. as a new genetic tool.  Applied and Environmental Microbiology 74(1):319-322.  DOI: 10.1128/AEM.02172-07

Murray G.L., Morel, V., Cerqueira G.M., Croda, J., Srikram, A.,  Henry, R., Ko, A.I., Dellagostin, O.A., Bulach, D.M., Sermswan, R.W., Adler, B., and Picardeau, M. (2009).  Genome-wide transposon mutagenesis in pathogenic Leptospira species.  Infection and Immunity 77(2):810-816.  DOI: 10.1128/IAI.01293-08