|Home | About | Journals | Submit | Contact Us | Français|
Methanococcus maripaludis is an anaerobic, methane-producing archaeon that utilizes H2 or formate for the reduction of CO2 to methane. Tryptophan auxotrophs were constructed by in vitro insertions of the Tn5 transposon into the tryptophan operon, followed by transformation into M. maripaludis. This method could serve for rapid insertions into large cloned DNA regions.
Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon. It is a mesophile that utilizes H2 or formate for the reduction of CO2 to methane as a source of energy. Although M. maripaludis is an autotroph that can grow with CO2 as its sole source of carbon, it also can assimilate acetate and amino acids [1–4]. Many factors make M. maripaludis a useful model organism in which to study the function of genes in vivo. First, genetics tools have been developed, including selectable resistance markers, efficient transformation systems, gene deletion methods involving substitutions with resistance markers or marker-less in-frame deletions, and expression vectors [5–8]. Second, M. maripaludis grows quickly compared to many other methanogens and is easily cultured on plates . Third, the sequence of the M. maripaludis strain S2 genome is relatively small, containing only 1,722 ORFs .
A variety of methods, including chemical mutagenesis and random insertions with a suicide vector, have been previously explored for random mutagenesis of methanococci, but none have proven to be ideal [11–13]. This study presents random insertions in the tryptophan operon, by in vitro Tn5 transposition followed by transformation into M. maripaludis.
The tryptophan operon of M. maripaludis is an excellent region of the genome for developing a random in vitro transposon insertion method. First, aromatic amino acid auxotrophs have been constructed and tryptophan is easily incorporated [7,14]. Second, all the genes for the biosynthesis of tryptophan from chorismate are located in one operon. This type of arrangement is unusual in methanococci, where it is rare for biosynthetic genes involved in a single pathway to be clustered in the chromosome [10,15]. Third, all the insertions would result in the same tryptophan auxotroph phenotype.
The bacteria and plasmids used in this work are listed in Table 1. E. coli was grown in Luria-Bertani medium with ampicillin (100 µg/ml) when needed. M. maripaludis was grown at 37 °C in the mineral medium McN, McNA (McN plus 10 mM sodium acetate), or McC (McNA plus 0.2 % [wt/vol] Casamino acids and 0.2 % [wt/vol] yeast extract) as described previously . M. maripaludis was grown with 276 kPa H2/CO2 gas (80:20 [vol/vol]) in broth and with 137 kPa H2/CO2 gas (80:20 [vol/vol]) in agar plates. Puromycin (2.5 µg/ml) or aromatic amino acids (1 mM each) were added when needed.
Most of the tryptophan operon (Fig. 1) from M. maripaludis strain JJ  was cloned into XbaI – KpnI sites of pUC18 vector, thus obtaining the plasmid pUC18-trp-JJ. For the cloning, the tryptophan operon was amplified by PCR using the primers trp-for3 and trp-rev3 (Table 1), which were identical to sequences of the trpC and trpA genes from the closely related sequenced strain S2 [10,16]. Due to the length of the expected PCR fragment (5,952 bp), the PCR reaction included Herculase Enhanced DNA Polymerase (Stratagene, La Jolla, CA) and 2 % (v/v) DMSO. The conditions for the PCR incubation were 2 min at 92 °C for initial denaturation; 30 cycles of 12 sec. at 92 °C, 30 sec. at 60 °C and 6 min at 68 °C for denaturation, annealing and extension, respectively, and 5 min at 72 °C for final extension. Sequencing of the pUC18-trp-JJ was performed at the DNA sequencing core (The University of Michigan) using universal M13 primers and trp-for1 and trp-rev1 primers (Table 1).
The pKJ331 plasmid was constructed at Ken Jarrell’s laboratory (Queen’s University, Canada) by cutting out the puromycin resistant cassette from pPAC60  with PstI and KpnI and cloning it into the same sites of pMOD vector (Epicentre Biotechnologies, Madison, WI). The kanamycin resistance gene was PCR amplified from genomic DNA of Silicibacter pomeroyi DSS-3 mutant 41-H6 , which contains a Tn5™<KAN-2> transposon insertion, using the primers Kan2-for-Hind and Kan2-rev-Pst (Table 1). Following restriction digestion with HindIII and PstI, the PCR fragment was cloned into the HindIII-PstI sites of pKJ331, yielding pKJ331-kan.
The Tn5™<KAN-2-pac> was isolated by digesting the pKJ331-kan plasmid with PvuII and ScaI and purifying the 2,399 bp band following agarose gel electrophoresis.
The locations of the transposons (fig. 1) were mapped by PCR amplification using a primer located by the end of the transposon (KAN-2-PAC-out1 or KAN-2 RP-1-out2), the primers trp-for1 or trp-for2 (Table 1), genomic DNA of the ten tryptophan auxotrophs, and Taq polymerase (New England Biolab, Ipswich, MA).
The tryptophan operon was selected for developing the in vitro transposon insertion in M. maripaludis. Most of the trp operon from M. maripaludis strain JJ  was cloned in the vector pUC18 obtaining the plasmid pUC18-trp-JJ. Partial sequencing of the pUC18-trp-JJ from both ends of the cloned DNA included 1,665 bp from the trpC end and 2,109 bp from the trpA end (Fig. 1) confirmed that the JJ genes possessed 94.4 % and 92.7 % identity, respectively, to the M. maripaludis S2 tryptophan operon .
A Tn5™<KAN-2-pac> transposon containing puromycin and kanamycin selection markers for M. maripaludis and E. coli, respectively, was constructed. The plasmid pKJ331 contained the puromycin resistant gene under the regulations of S-layer promoter and mcr terminator from Methanococcus voltae between the mosaic elements of Tn5 transposon. Additionally, the kanamycin resistance gene was cloned in pKJ331, yielding pKJ331-kan in order to have a selection for E. coli.
The transposition reaction was performed using the plasmid pUC18-trp-JJ as target DNA, the isolated Tn5™<KAN-2-pac> transposon, and the Tn5 transposase (Epicentre Biotechnologies). In addition, the Tn5™<KAN-2> transposon, provided with the transposase kit, was used as a control. The resulting plasmids were transformed into E. coli. The transposition efficiency was calculated from the number of colonies on ampicillin plus kanamycin plates divided by the number of colonies in ampicillin alone plates and was 0.02–0.03 and 0.12–0.35 with Tn5™<KAN-2-pac> transposon and the Epicentre prepared Tn5™<KAN-2> transposon, respectively. The mixture of pUC18-trp-JJ with the Tn5™<KAN-2-pac> transposon were then enriched following growth of the E. coli transformants in LB liquid medium with kanamycin and ampicillin. The plasmids were then isolated by miniprep (GenScript Corp., Piscataway, NJ).
The mixture of plasmids was used for transformation in both strains of M. maripaludis . Following transformation, the cells were plated in complex medium McC medium  with puromycin (Table 2). The efficiency of transformation was higher using the M. maripaludis JJ strain. Increasing the amount of DNA from 0.5 µg to 1 µg only increased the transformation efficiency by 27 %. Isolation of M. maripaludis aromatic amino acid auxotrophs was achieved by replica plating in defined media (Table 3). The number of aromatic amino acid auxotrophs was low probably due to single cross over insertions of the entire transformed plasmid. Ten isolates from M. maripaludis JJ were subjected to further analysis. All ten isolates were tryptophan auxotrophs. The location of the transposons (fig. 1) were mapped to six locations within the trpD, trpE or trpG .
Originally, the pKJ331 plasmid was constructed to isolate the Tn5™<pac> transposon, containing the puromycin cassette only, in order to perform in vitro transposition with genomic DNA of Methanococcus spp., followed by direct transformation into methanogens. However, that approach failed to produce mutants (Ken Jarrell, personal communication). In addition, repeating the last approach in this study with the Tn5™<KAN-2-pac> transposon also failed to obtain results in M. maripaludis (unpublished data). The in vitro transposition method required stopping the reaction by adding SDS solution (0.1 % SDS final concentration) and incubation at 70 °C for 10 minutes (EZ-Tn5™ <KAN-2> insertion kit manual, Epicentre Biotechnologies, Madison, WI). Avoiding the stop treatment following the transposition reaction using Tn5™<KAN-2> transposon and the control DNA target (supplied by the Epicentre Biotechnologies transposon kit) decreased the efficiency of transposition by 95%, compared to the recommended protocol. M. maripaludis appears to be very sensitive to SDS assuming this is the reason for the unsuccessful transformation of M. maripaludis directly with the transposition mixture.
A similar method, using in vitro transposition following by enrichment of transposon insertions in E. coli, was used for demonstrate the function of the proline biosynthetic genes in Methanosarcina acetivorans . This newly developed method for M. maripaludis demonstrated the function of the genes trpD, trpE or trpG in the biosynthesis of tryptophan. Even though the function of the proteins encoded by trpD, trpE or trpG was previously predicted by homology, only experimental results confirmed that prediction in vivo. For example, a knockout of the M. maripaludis ORF Mmp0006 did not produce an aromatic amino acids auxotroph , even though the function of the Mmp0006 was expected to be dehydroquinate synthase II in M. maripaludis based on the homology to the previously biochemical characterized ORF MJ1249 from Methanocaldococcus jannaschii .
In conclusion, this method could be used for rapid knockout genes in a large genomic cloned region. For example the fosmid library built for the genome sequence of M. maripaludis  could serve as target for in vitro transposition, in a mixture or as isolated fosmid, followed by transformation in M. maripaludis. Even the small genome of M. maripaludis contains more than 50% ORFs with unknown function. This method could speed up the discovery of new functions in this organism.
This work was supported by a grant from NIH to WBW. The authors would like to thank Sonia Bardy and Ken F. Jarrell, Queen’s University, Canada, for constructing and providing the plasmid pKJ331, to Erinn C. Howard, Department of Microbiology, University of Georgia, for providing genomic DNA of Silicibacter pomeroyi DSS-3 mutant 41-H6 and to Meghan M. Drake, Biological and Environmental Sciences Directorate, Oak Ridge National Laboratory, for English corrections of this manuscript.