Journal of Bacteriology, March 2002, p . 1449-1454, Vol . 184, No . 5

Directed Mutagenesis and Plasmid-Based Complementation in the Methanogenic Archaeon Methanosarcina acetivorans C2A Demonstrated by Genetic Analysis of Proline Biosynthesis

Jun Kai Zhang, Andrea K . White, H . Craig Kuettner, Paolo Boccazzi, and William W . Metcalf^*

Department of Microbiology, University of Illinois, Urbana, Illinois 61801

Received 7 September 2001/ Accepted 28 November 2001

Directed mutagenesis of Methanosarcina species is clearly possible . Transformation and integration of foreign DNA into the chromosome via homologous recombination have been demonstrated for Methanosarcina mazei S-6 (6) . In that study, the first to demonstrate the transformation of any Methanosarcina species, a selectable marker inserted into the grpE-dnaK intergenic region was recombined onto the M . mazei chromosome after chemical transformation . The process was, however, inefficient, requiring 50 µg of DNA to produce transformed cultures . More recently, a very efficient liposome-mediated transformation method for Methanosarcina species was developed . This method allows isolation of up to 10⁸ transformants per µg of DNA in Methanosarcina acetivorans C2A . The availability of this high-frequency transformation method led us to attempt directed mutagenesis in this species . To examine the factors involved in this type of experiment and to verify that the process of homologous recombination occurs in a reliable and predictable manner, we decided to mutagenize genes with known functions and predictable phenotypes . For this purpose, we chose to examine genes involved in the biosynthesis of proline .

Cloning the proline biosynthetic genes of M . acetivorans. Previous studies demonstrated the feasibility of cloning proline biosynthetic genes from methanoarchaea by complementation of Escherichia coli mutants (10) . Therefore, we used the same method to clone the proC gene from each of the three Methanosarcina species frequently used in our laboratory . Chromosomal DNAs, isolated as previously described (3) from M . acetivorans C2A, Methanosarcina thermophila TM1, and Methanosarcina barkeri Fusaro, were used to construct genomic libraries in the single-copy, dual-cos vector pWM357 (Table 1) . The libraries contain ca . 330,000, 14,000, and 50,000 independent clones, respectively, and were prepared essentially as described previously (2) . Numerous cosmids carrying the pro locus from each organism were subsequently obtained as plasmids that allow growth of the E . coli proC mutant XS72 on glucose-MOPS (morpholinepropanesulfonic acid) agar (19) without added proline (Pro⁺ phenotype) .

 
Two cosmids from each organism were moved into an isogenic set of Salmonella enterica serovar Typhimurium proA, proB, and proC mutants . Surprisingly, all of the cosmids complemented not only the proC mutation but also the proA and proB mutations, suggesting that each carries functional homologs of all three proline biosynthetic genes or a gene(s) that allows synthesis of proline via an alternate route . To address this issue, we carried out a series of subcloning and complementation experiments using one of the cosmids (pJK201) isolated from M . acetivorans . Random subclones of pJK201 were generated as described in Table 1 . Pro⁺ subclones were then isolated based on their ability to complement the proA mutant Salmonella serovar Typhimurium MST951(pAW3 and pAW4), the proB mutant Salmonella serovar Typhimurium MST952(pAW1 and pAW2), or the proC mutant E . coli BW10244(pCK1 and pCK2) . In all cases, the subclones isolated by complementation of a particular pro mutation failed to complement the other two pro mutations, strongly supporting the conclusion that proline biosynthesis in M . acetivorans occurs via the same three-step pathway used by Salmonella (5) . The fact that similar clones were isolated from M . barkeri and M . thermophila suggests that the same is true in these species as well .

DNA sequence analysis of the M . acetivorans proABC locus. The DNA sequences of the inserts in each of the pJK201 subclones were determined by automated dye terminator sequencing at the W . M . Keck Center for Comparative and Functional Genomics, University of Illinois . Upon analysis, these sequences could be compiled into a single continuous DNA fragment of 5,139 bp containing three large open reading frames, which, based on sequence analysis, were designated proA, proB, and proC (Fig . 1) . The predicted protein products of the proA, proB, and proC genes show very significant homology to known ProA (glutamate semialdehyde dehydrogenase), ProB (glutamate kinase), and ProC (1-pyrroline-5-carboxylate reductase) proteins, with between 30 and 50% identities to the top 20 BLAST matches for all three proteins (see below) . The results of these homology searches are in complete agreement with the complementation data presented above . Accordingly, the only complete gene found on the proA-complementing plasmids is proA, the only complete gene found on the proB-complementing plasmids is proB, and the only complete gene on the proC-complementing plasmids is proC (Fig . 1) . Thus, in combination, these data provide strong support for the function of the genes in the M . acetivorans proABC locus, i.e., the biosynthesis of proline via the standard pathway found in most organisms .

 
Although the findings regarding the function of M . acetivorans genes in the standard proline biosynthesis pathway are not surprising per se, they are at odds with the situation observed for other archaea . Recent data suggest that proline is synthesized from ornithine in several methanoarchaea (9) . Moreover, examination of the 10 complete archaeal genome sequences presently available failed to identify complete sets of genes required for the standard biochemical pathway (searches were performed in September 2001) . Thus, recognizable ProA homologs were not observed (based on a BLAST E value of <0.04 when M . acetivorans ProA is used as the query sequence) in any of the completed archaeal genome sequences, which include those of Aeropyrum pernix K1, Archaeoglobus fulgidus, Halobacterium sp. strain NRC-1, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Pyrococcus abyssi, Pyrococcus horikoshii, Sulfolobus acidocaldarius, Thermoplasma acidophilum, and Thermoplasma volcanium . In contrast, sequence comparisons of M . acetivorans ProA with dozens of other ProA proteins produced highly significant scores (the top 50 matches produced E values of <4 x 10^-44); these proteins included many with biochemically and genetically demonstrated function . Using M . acetivorans ProB as the query sequence, a possible ProB homolog was identified in Methanobacterium thermoautotrophicum (E value of 3 x 10^-4) but not in the remaining archaeal genomes (based on E values of <0.01) . When compared with the top 50 known and putative ProB proteins, M . acetivorans ProB produced scores of <7 x 10^-20 . In contrast, easily recognizable ProC proteins were found in 5 of the 10 available archaeal genomes . Thus, it appears that recognizable ProC homologs are more common, but not universal, among archaea . As described above, a proC gene was previously identified in Methanobrevibacterium smithii by genetic complementation (10), but this gene is not among the closest homologs to M . acetivorans proC . In sum, it is clear that the evolution of proline biosynthesis in Methanosarcina species is dramatically different than in the other members of the domain Archaea for which data are available; they are the only known archaea to possess the standard pathway for proline biosynthesis .

Directed mutagenesis of M . acetivorans proabc by homologous recombination-mediated allele replacement. A variety of pro mutations were constructed on plasmids for subsequent recombination onto the M . acetivorans chromosome as described in Table 1 . These gene disruption mutations consist of those with insertions of either the pac gene (in the form of the pac-ori-aph cassette or Tn5-407) or the ileS12 gene (in the form of Tn5-ileS12), which encode resistance to puromycin or pseudomonic acid, respectively, in Methanosarcina species (3, 16) . Both transposons and the pac-ori-aph cassette also encode resistance to kanamycin in E . coli to simplify the plasmid constructions . The sites and orientations of each insertion mutation are shown in Fig . 1 .

Each of the plasmid-borne pro alleles was transferred onto the M . acetivorans chromosome by simple homologous recombination after liposome-mediated transformation (3, 16) with linear DNA . For these experiments, the plasmids were linearized with restriction enzymes that cut only the plasmid backbone, as close to the insert as possible . Recombinants carrying Tn5-407 or pac-ori-aph cassette insertions were selected as puromycin-resistant (Pur^r) clones; recombinants carrying Tn5-ileS12 insertions were selected as pseudomonic acid-resistant (PA^r) clones . In both cases, selection for recombinants was carried out in medium containing 50 mM proline to allow growth of the expected auxotrophic mutants . (There is no available evidence to suggest that proline is transported by M . acetivorans. We used this relatively high proline concentration in the hope of overcoming potential transport defects.) In these experiments, transformation with ca . 2 µg of linearized plasmid DNA yielded from a few hundred to a few thousand antibiotic-resistant colonies . After purification of the recombinant clones by streaking for single colonies on solid-medium plates, 80 to 100% of these transformants (20 were tested for each mutation) required proline for growth (Pro^- phenotype) . All 11 of the pro mutations yielded Pro^- transformants .

Verification of the chromosomal structure of the M . acetivorans Pro^-. mutants. Because allele replacement has not previously been employed in M . acetivorans, we examined the physical structure of each mutant chromosome in detail by using DNA hybridization . DNA hybridizations were performed essentially as described previously (17); however, a digoxigenin-labeled probe (pJK68) and chemiluminescence detection (DIG High Prime and CDP-Star, respectively; Roche Molecular Biochemicals, Indianapolis, Ind.) were used for visualization of hybridizing bands in place of ³¹P-labeled probes . For all 11 M . acetivorans Pro^- mutants, there was complete agreement between the sizes of the hybridizing bands predicted from the known DNA sequences and those obtained experimentally (data not shown) . Thus, the mutant strains that we constructed were indistinguishable from those that would result from simple homologous recombination, and this finding provides strong support for the occurrence of this genetic process in M . acetivorans C2A .

Plasmid-based complementation of M . acetivorans proABC mutants. A series of Methanosarcina-E . coli shuttle vectors have been reported (3, 16); however, these have never been used for complementation studies because Methanosarcina mutants were not previously available . To examine whether plasmid-borne alleles could complement genomic mutations, each of the PA^r Pro^- mutants was transformed with a puromycin resistance plasmid, pJK72, carrying the entire proABC locus . The shuttle vector pWM321 (with no insert) was used as a negative control . In a similar way, each of the Pur^r Pro^- mutants was transformed with the pseudomonic acid resistance proABC plasmid, pPB36, by using the shuttle vector pPB35 as a control (Table 2) . All of the strains transformed by the proABC plasmids regained the Pro⁺ phenotype, indicating that each chromosomal mutation was complemented by the plasmid-borne, wild-type allele . Strains transformed by the vector controls remained Pro^- . Because these mutant strains are recombination proficient, it is possible that restoration of the Pro⁺ phenotype occurred by marker rescue (i.e., recombinational repair) rather than by true complementation . However, we do not believe this was the case . Selection for both antibiotics was maintained throughout the experiment . Therefore, the antibiotic resistance marker on the chromosome was not removed by recombination unless a reciprocal recombination event exchanged the markers on the plasmid and the chromosome . In each case, we were able to recover the intact original plasmid from the complemented strain, indicating that no such event had occurred (data not shown) .

 
Although all the mutations tested resulted in proline auxotrophy, it is not clear from mutant analysis alone whether proA and proB have a role in proline biosynthesis . Given the potential operon structure of the proABC locus, it was formally possible (although perhaps unlikely) that only proC was required for proline biosynthesis and that the effects of the proA and proB mutations were via polarity on proC . Accordingly, we transformed the proA::Tn5-ileS12 mutants with puromycin resistance plasmids carrying only proA (to check for polarity on both proB and proC) or proAB (to check for polarity on proC) . In a similar way, the proB::Tn5-ileS12 mutants were transformed with a puromycin resistance plasmid carrying only proAB to check for polarity on proC (Table 2) . In all cases, the plasmids were able to complement the transposon insertion mutations, indicating that these Tn5-ileS12 mutations were not polar on the downstream genes and that proA and proB are required for proline biosynthesis in M . acetivorans . Similar experiments involving complementation of Tn5-407 insertions by pseudomonic resistance plasmids yielded identical results (Table 2) .

As discussed above, the ability to complement chromosomal mutations by plasmid-borne alleles was critical in defining the functions of proA and proB . To our knowledge, this is the first example of plasmid complementation in a methanogenic archaeon . The ability to use plasmids in this way will greatly expand our ability to perform genetic analysis in Methanosarcina species . Most importantly, plasmid complementation will allow verification that a particular phenotype is caused by a mutation in a specific gene (namely, the one carried on the complementing plasmid) . However, innumerable genetic techniques require the use of replicating plasmids . Among these are the identification of essential genes and regulatory proteins and the introduction of foreign genes, or genes carrying defined mutations, into strain backgrounds with mutated alleles on the chromosome .

The use of plasmids for complementation of mutations created by the insertion of selectable markers requires, of course, multiple selectable markers . Currently, only two such markers, the ileS12 gene for resistance to pseudomonic acid (3) and the pac cassette for resistance to puromycin (16), are available for use in Methanosarcina . Our demonstration that plasmids carrying proline genes can complement proline auxotrophic mutations suggests the possibility of developing a third selectable marker from these genes . This would require a suitable proline auxotroph as a host strain, preferably one that did not already carry an antibiotic insertion in the chromosome . Experiments designed to develop such a system are presently under way in our laboratory .

These new techniques for directed mutagenesis and plasmid complementation, along with the recently presented method for random transposon-based mutagenesis (24), now complete a set of basic tools for genetic analysis of Methanosarcina species . These tools, in combination with the metabolic diversity of members of this genus (relative to other methanoarchaea), make Methanosarcina species very attractive model organisms for the study of methanogenesis and of archaeal biology in general .

Nucleotide sequence accession number. The sequence for the M . acetivorans C2A proABC locus has been deposited in GenBank under accession number AF305580 .

This work was supported by grant MCB-987459 from the National Science Foundation and by a Searle Scholars Award from the Chicago Community Trust to W.W.M .

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