A mariner-Based Transposition System for Listeria monocytogenes

In this study, we developed a new mariner-based transpositionsystem for Listeria monocytogenes. The mariner-based systemhas a high rate of transposition and a low rate of plasmid retention,and transposition is very random, making it an ideal tool forhigh-throughput transposon mutagenesis in L. monocytogenes.

INTRODUCTION

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INTRODUCTION

Listeria monocytogenes is a saprophytic gram-positive bacterialrod that is ubiquitous in nature and is an opportunistic food-bornepathogen of humans and a variety of other vertebrates (24).During infection, L. monocytogenes multiplies intracellularlyin the cytosols of host cells (19, 23). Intracellular survivalof L. monocytogenes relies largely on its ability to subverthost functions by escaping phagocytic vacuoles and spreadingfrom cell to cell without exiting the intracellular milieu.In the last two decades, multiple studies have aimed at identifyingvirulence factors and deciphering the mechanisms by which L.monocytogenes survives in a wide range of environments (9, 13).

One of the most valuable genetic tools used to study bacteriais the transposon. Transposons can be used to perform high-throughputmutagenesis of an entire chromosome, generating banks of mutantsthat can be screened for identification of factors related tospecific bacterial functions. The transposon delivery systemsthat are currently available for use with L. monocytogenes arenot ideal for these types of studies (4, 7, 14). For example,the most commonly used transposon delivery system, Tn917-LTV3,is more than 22 kb in size and has a low efficiency of transpositionand a high rate of vector retention (4). In this study, we aimedat designing a transposon delivery system that is more suitableto high-throughput mutagenesis.

In recent years, Himar1 mariner has been used as the transposonof choice in performing high-throughput mutagenesis in manydifferent bacterial species, including low-GC-content gram-positivespecies (1, 2, 18). Himar1 was originally isolated from thehorn fly, Haematobia irritans, and is a member of the Tc1/marinersuperfamily of transposable elements (21). The Tc1/marinersare the most-widespread transposons in nature. These elementsrequire no factors for transposition other than their self-encodedtransposases (16), a feature that makes them ideal candidatesfor development into generalized genetic tools. Moreover, themariner requirement for insertion is the dinucleotide TA, whichmakes it perfect for transposition into low-GC-content organismssuch as L. monocytogenes (39% GC). We reasoned that a mariner-basedtransposition system would be an excellent tool for the entirecommunity of scientists working with L. monocytogenes.

Construction of mariner-based transposon delivery vectors.

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Construction of mariner-based...

The plasmids and primers used for construction of a marinerdelivery vector for L. monocytogenes are listed in Tables 1and 2, respectively. pPL2 (17) and pDG780 (12) were digestedwith SacI and XhoI, and the gram-positive kanamycin (kan) resistancecassette from pDG780 was ligated into pPL2 to create pMC14.The gram-positive chloramphenicol acetyltransferase gene (cat)from pPL2 was amplified by PCR with primer pair Marq155/156and ligated into pCR2.1-Topo (Invitrogen) to create pMC1. pMC1was digested with BamHI and ligated into the BglII site of pMMOrf(15) between the 5' and 3' Himar1-inverted terminal repeats(ITR), creating pMC3. The Himar1 transposase gene (tpase) (16)was amplified by PCR from pNF1100 (provided by Nancy Freitag)with primer pair Marq188/234. The promoter regions of the Bacillussubtilis CU1065 (provided by John Helmann) mrgA and katA geneswere amplified by PCR, using primer pairs Marq247/248 and Marq249/250,respectively. P_mrgA, P_katA, and tpase PCR fragments were digestedwith BamHI, and each promoter was individually ligated to tpase.The P_mrgA-tpase and P_katA-tpase ligation products were amplifiedby PCR with primer pairs Marq247/188 and Marq249/188, respectively.The P_mrgA-tpase and P_katA-tpase PCR products and pMC14 weredigested with KpnI and SphI. The pMC14 fragment comprising P4oriT,p15Aori, gram-negative cat, and gram-positive kan genes wasligated with P_mrgA-tpase and P_katA-tpase, respectively. TheITR-cat-ITR fragment from pMC3 was ligated at the KpnI and XhoIsites, and the gram-positive cat gene was later replaced byermC. This replacement was done by amplifying ermC from pPL3e(10) with primer pair Marq205/206, digesting the PCR productsand vector with NdeI, and replacing the cat gene between theITR with ermC. Last, the temperature-sensitive origin of replicationpE194ts ori in pKSV7 (22) was amplified by PCR, using primerpair Marq194/195, digested with KpnI, and ligated into eachvector, creating pMC38 (P_mgrA) and pMC39 (P_katA) (Fig. 1). Thevectors are 8,172 bp (pMC38) and 8,297 bp (pMC39), and the transposonitself is 1,395 bp.

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TABLE 1. Bacterial strains and vectors used in this study

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TABLE 2. Oligonucleotide primers used in this study

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FIG. 1. Physical map of the mariner-based transposon delivery vectors pMC38 and pMC39. The vectors comprise the Escherichia coli p15A low-copy-number replication origin (5), the RP4 ori for conjugative transfer (20), the pE194ts gram-positive temperature-sensitive replication origin (11, 25), a gram-negative chloramphenicol resistance gene (cat) (5), a gram-positive noninducible erythromycin resistance gene (ermC) (25) flanked by the 29-bp ITR of the Himar1 mariner (15), a gram-positive kanamycin resistance gene (kan) (12) as a screening marker for loss of the plasmid, and the Himar1 mariner transposase gene (tpase) (16). The indicated restriction sites represent those used for cloning, but they are not necessarily unique. Gm^–, gram negative.

The Himar1 transposase and the ITR sequences are the only twofactors required for transposition; thus, successful expressionof the transposase in L. monocytogenes is the key element. Inour initial constructs, the L. monocytogenes p60 (pMC25) andactA (pMC30) promoters were used to direct transcription ofthe transposase gene. This approach failed, as the entire plasmidinvariably integrated into the chromosome. However, when weused B. subtilis to evaluate the efficiency of transpositionwith these same vectors, we obtained a very low level of plasmidretention (data not shown). We reasoned that the Listeria speciespromoters were responsible for mediating plasmid integrationinto the chromosome of L. monocytogenes and sought to use twoB. subtilis ${sigma}$ ^A-dependent promoters (P_mrgA and P_katA) with nosequence similarity to the Listeria genome (6). This approachwas very successful.

Evaluation of the mariner-based transposon delivery vector.

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Evaluation of the mariner-based...

pMC38 and pMC39 were preferentially transferred into L. monocytogenesstrain 10403S by electroporation, as the efficacy of transferby conjugation was very low. Transformants were selected at30°C on brain heart infusion (BHI) plates supplemented witherythromycin at 5 µg/ml. Individual colonies were grownovernight in BHI with erythromycin and kanamycin (10 µg/ml)at 30°C with shaking. The cultures were diluted 1/200 inbroth with erythromycin, grown for 1 h at 30°C with shaking,and then shifted to 40°C for about 6 h until the opticaldensity at 600 nm was between 0.3 and 0.5. Aliquots of the culturewere plated on BHI agar supplemented with erythromycin and incubatedat 40°C. Individual colonies were picked and plated in parallelon BHI agar supplemented with either erythromycin or kanamycinto evaluate the rate of plasmid retention. The rate of plasmidretention was calculated by dividing the number of kanamycin-resistantcolonies (due to plasmid retention) by the number of erythromycin-resistantcolonies (total number of mutants with the plasmid or with thetransposon only). The same procedures were used to evaluatethe Tn917-based transposition system, Tn917-LTV3 (4), exceptthat 1 µg/ml erythromycin plus 25 µg/ml lincomycinwere used for selection of transposon mutants and 12.5 µg/mltetracycline was used to evaluate plasmid retention.

In general, we obtained 10-fold-more mutants with the mariner-basedvectors than with the Tn917-based vector (Table 3). Plasmidretention was less than 2.5% for the mariner-based vectors,whereas it was more than 50% with the Tn917-based vector. Therefore,by comparing the numbers of transposon insertion mutants (coloniesthat have lost the plasmid) generated by the two systems, weestimated that the efficiency of transposition of the mariner-basedvectors was more than 20-fold higher than that of the Tn917-basedvector.

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TABLE 3. Comparison of transposon delivery vectors

To evaluate the randomness of transposition, we arbitrarilypicked 100 erythromycin-resistant colonies from one library,performed Southern blot analysis, and identified the sites oftransposon insertion. For Southern blot analysis, a 400-bp fragmentwithin the ermC gene was amplified from pMC38 by PCR, usingprimer pair Marq206/254. The probe was labeled with alkalinephosphatase, using the AlkaPhos Direct labeling system fromAmersham Biosciences. Of the 100 mutants, 84 had a single transposoninsertion, whereas 16 had two-to-three insertions (data notshown). To identify the sites of transposon insertion, we initiallyperformed arbitrary PCR to amplify the DNA sequences flankingthe transposon (8). DNA was amplified from either end of thetransposon with a series of two rounds of PCR with Taq polymerasein the first round and Expand High Fidelity polymerase (Roche)in the second round. In each round, a transposon-specific primerand an arbitrary primer were used. The arbitrary primers Marq207and Marq208 were previously identified as ARB1B and ARB2 byGarsin et al. (8). The approximate locations of transposon-specificprimers are illustrated in Fig. 1. In the first round, DNA fragmentsfrom the left and right ends of the transposon were amplifiedwith primer pairs Marq207/255 and Marq207/269, respectively.For the second round, 5 µl of a 1/25 dilution from thefirst round of PCR was used in a 20-µl reaction. DNA fragmentsfrom the left and right ends of the transposon were amplifiedwith primer pairs Marq208/256 and Marq208/270, respectively.The PCR products were sequenced, using primers Marq257 and Marq271for the left and right ends of the transposon, respectively.The Biotechnology Research Center of Cornell University performedthe sequencing with an Applied Biosystems automated 3730 DNAanalyzer. The ListiList website (http://genolist.pasteur.fr/ListiList/)was used for sequence analysis.

Readable sequencing results were obtained from 77 transposoninsertion mutants, including one pair of siblings and a seriesof three genes that were hit twice. For the 77 mutants, 36 insertionswere in positive-strand open reading frames, 29 in negative-strandopen reading frames, and 12 in intergenic regions (Fig. 2).The mutants distributed evenly along the L. monocytogenes genome,and there was no bias in terms of the transposon orientation.We further aligned all the insertion sites, and no sequencespecificity beyond the known requirement of the dinucleotideTA was found (data not shown).

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FIG. 2. Locations of 77 sequenced Himar1 insertions in the chromosome of L. monocytogenes. The bars located outside the circle indicate transposons that were oriented in the same direction as the positive strand, whereas the bars located inside the circle indicate transposons that were oriented in the opposite direction. Each quarter of the chromosome is identified with the approximate base pair, except for bp 1, which is identified by an arrow.

Taken together, these results show that the newly designed marinerdelivery vectors are powerful genetic tools to study L. monocytogenes.The mariner-based system outachieves the Tn917-based systemin the following aspects: transposition efficiency, randomness,and a low rate of plasmid retention. The temperature-sensitivereplicon (pE194ts ori) and the noninducible erythromycin resistancegene (ermC) are common to many gram-positive bacteria. We believethat this mariner-based system will be a great tool for theentire community of scientists working with L. monocytogenesand other low-GC gram-positive bacteria.

ACKNOWLEDGMENTS

We thank Nancy Freitag for providing the plasmid pNF1100, JohnHelmann for providing the Bacillus subtilis CU1065 strain andthe plasmid pDG780, Darren Higgins for providing the plasmidpPL3e, David Lampe for providing the plasmid pMMORF, and DanielPortnoy for providing the plasmid pPL2. We are grateful to JoePeters for helpful suggestions and discussions and to EmilySlepkov for careful reading of the manuscript.

This work was in part supported by U.S. Public Health Servicegrant AI52154 to H.M. from NIAID.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Immunology, VMC C5-169, Cornell University, Ithaca, NY 14853-6401. Phone: (607) 253-3273. Fax: (607) 253-3384. E-mail: hm72{at}cornell.edu

Published ahead of print on 16 February 2007.

Present address: Department of Biological Sciences, ClemsonUniversity, Clemson, SC 29634.

REFERENCES

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