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Keywords:

  • Methylobacterium extorquens AM1;
  • Methylotroph;
  • Methanol oxidation;
  • Electroporation

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Methylobacterium extorquens AM1 is a pink-pigmented facultative methylotroph which is widely used for analyzing pathways of C1 metabolism with biochemical and molecular biological techniques. To facilitate this approach, we have applied a new method to construct insertion or disruption mutants with drug resistance genes by electroporation. By using this method, mutants were obtained in four genes present in the mxa methylotrophy gene cluster for which the functions were unknown, mxaR, mxaS, mxaC and mxaD. These mutants were unable to grow on methanol except the mutant of mxaD, which showed reduced growth on methanol.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Methylobacterium extorquens AM1 is a pink-pigmented facultative methylotroph which was originally isolated as an airborne contaminant of liquid methylamine culture [1]. This microorganism can grow on C1 compounds such as methanol and methylamine using the serine cycle for carbon assimilation, but it is unable to grow on methane. It also grows on ethanol and some organic acids as a sole carbon and energy source. This organism has been well characterized physiologically and biochemically [2], and its molecular genetics is well established. A number of chromosomal regions of M. extorquens AM1 have been characterized that are involved in C1 metabolism, showing that many C1 genes are clustered [3–5]. The function of many of these genes has been determined by a combination of mutant and gene sequence analysis. However, a subset of these clustered genes have unknown functions, as they show little or no similarity to other proteins or show similarity to proteins with unknown function in protein databases.

Constructing mutants in open reading frames is an important step in elucidating the function of unknown genes. The usual approach in M. extorquens AM1 is an allelic exchange procedure, which involves inserting a drug resistance gene into the open reading frame of interest, and exchanging this with the chromosomal gene copy via homologous double-crossover recombination events [3]. This approach is cumbersome, because it involves two subsequent subcloning steps to obtain the construction for generating the allelic exchange, and the mating step requires purification of the recipient from the donor, even in the presence of a counter-selective marker. In addition, the majority of the drug-resistant strains are the result of single-crossover recombination events, and in many cases a large number of recombinants must be screened for loss of the vector drug resistance marker to obtain the desired double-crossover recombinants.

Allelic exchange mutagenesis with linear fragments is a more attractive procedure for generating insertional mutants in strains such as M. extorquens AM1, as it simplifies both the cloning and mutation processes. However, until now there has been no published procedure for transferring linear DNA fragments into M. extorquens AM1. Procedures are available for electroporation of closed circular plasmids [6, 7]. Therefore, we have adapted these procedures for the electroporation of linear DNA fragments into M. extorquens AM1 to generate chromosomal insertion mutants by allelic exchange.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Strains and plasmids used are listed in Table 1. M. extorquens AM1 rif [8] was used as the wild-type strain and for mutation.

Table 1.  Bacterial strains and plasmids used in this study
Strain or plasmidRelevant trait(s)Source or reference
E. coli strains
DH5αr-m+recA1 lacZYAφ80 dlacΔ(lacZ)M15Bethesda Research Laboratories
S17-1recA oriT integrated RP4-2-Tc::Mu-Km::Tn7[21]
JM110dam dcm supE44 hsdR17 thi leu rpsL lacY F′[traD36 proAB+ lacIq lacZΔM15][22]
M. extorquens AM1 mutant strains
R1mutated by pHT9XbHKan, mxaR mutantThis study
SH11mutated by pHT9BgHKanHThis study
SN21mutated by pHT9BgHKanN, mxaS mutantThis study
SA31mutated by pHT9BgHKanA, mxaS mutantThis study
C31mutated by pCMPN5Kan2, mxaC mutantThis study
D11mutated by pUC19A3S1Kan, mxaD mutantThis study
Plasmids
pRK310Tcr IncP1[12]
pUC18/19AprlacZ[22]
pUC4KApr Knr from Tn903Pharmacia
pHT98.6-kb HindIII subclone containing mxaFJGIR in pRK310This study
pDN94.4-kb HindIII-XhoI subclone containing mxaACKLD in pVK100[18]
pHT9Bf3-kb BamHI suclone containing mxaRSA in pRK310This study
pCM590.8-kb subclone containing mxaC in pRK310[18]
pRK310A3S11.9-kb subclone containing mxaD in pRK310[18]
pRK310A3S2as above but in opposite orientation[18]
pHT9XbH193-kb XbaI-HindIII subclone in pUC19This study
pHT9XbHKanKanr inserted in BglII site in pHT9XbH19This study
pHT9BgH181.4-kb BglII-HindIII subclone in pUC18This study
pHT9BgHKanHKanr inserted in HincII site in pHT9BgH18This study
pHT9BgHKanNKanr inserted in NruI site in pHT9BgH18This study
pHT9BgHKanAKanr inserted in AscI site in pHT9BgH18This study
pCMPN52.1-kb PstI-NruI subclone containing mxaC in pUC19[18]
pCMPN5Kan2Kanr inserted in BglII site in pCMPN5This study
pUC19A3S11.9-kb fragment containing mxaD in pUC19[18]
pUC19A3S1KanKanr inserted in BsaBI site in pUC19A3S1This study

2.1Growth conditions

The growth medium originally described by Attwood and Harder [9] was used for M. extorquens AM1. Methanol, methylamine hydrochloride or sodium succinate were used as carbon sources, at 0.5% (v/v), 0.2% (w/v) and 0.46% (w/v), respectively. Escherichia coli was grown on LB medium [10]. Antibiotics used were: kanamycin, 25 μg ml−1; tetracycline, 10 μg ml−1; ampicillin, 50 μg ml−1 for E. coli, 300 μg ml−1 for M. extorquens AM1.

2.2Electroporation

Electro-competent cells were prepared as follows. M. extorquens AM1 was grown to exponential phase on succinate medium. The culture was chilled for 15 min on ice and centrifuged at 2000 rpm at 4°C for 10 min. Cells were washed with ice-cold sterile distilled water, then washed with ice-cold sterile 10% (v/v) glycerol. Cells were suspended in 10% glycerol, dispensed in 100-μl aliquots and kept at −70°C. Electro-competent cells (50 μl) were mixed with DNA solution (2 μl) and transferred into a cuvette chilled on ice. Electroporation was carried out using a Gene Pulser (Bio Rad) with the following parameters: 2.5 kV, 400 Ω, 25 μF for 2-mm gap cuvette, or 2 kV, 200 Ω, 25 μF for 1-mm gap cuvettes. After the cells had been pulsed, ice-cold sterile Nutrient Broth (Difco) was added to the cuvette immediately, the cell suspension was transferred into a test tube, and it was then shaken at 30°C for 1–24 h. An aliquot was spread on an agar medium with succinate and appropriate antibiotics. Colonies usually appeared after 3 days.

2.3DNA manipulations

Plasmid DNA from E. coli strains was prepared as described by Sambrook et al. [10]. Restriction enzyme digestion, DNA ligation and other DNA modifications were performed according to vendors' recommendations.

2.4DNA hybridization

Chromosomal DNA was isolated by the method of Marmur [11] with slight modifications. Chromosomal DNA (5 μg) digested with restriction enzyme(s) was separated by agarose gel electrophoresis, blotted onto a nylon membrane and hybridized with an appropriate probe. Labeling of the probe and detection of hybridized DNA were done with DIG labeling and detection kit (Boehringer Mannheim). Probes used were pUC18 and the 1.2-kb EcoRI fragment from pUC4K.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Electroporation of pRK310 into M. extorquens AM1

pRK310, a broad-host-range vector [12] known to be maintained in M. extorquens AM1 [3], was used to select optimal electroporation conditions. Efficiencies of 105–106 cells per μg of plasmid DNA were obtained using different conditions, which is a significantly higher electroporation efficiency than reported previously for Methylobacterium strains [6, 7]. Optimal conditions were 2.5 kV, 400 Ω, 25 μF for 2-mm gap cuvette, or 2 kV, 200 Ω, 25 μF for 1-mm gap cuvettes, and time constants were 8.8–9.4 ms or 4.3–4.8 ms, respectively. Nutrient broth was chosen for the post-incubation medium, as it gave a higher recovery rate than minimum medium with succinate. Post-incubation for 1–24 h showed no significant difference for numbers of tetracycline-resistant colonies.

3.2Mutation of mxaF by electroporation-mediated allelic exchange

The kanamycin resistance gene from pUC4K was inserted into the SalI site in mxaF, the gene of the large subunit of methanol dehydrogenase (MDH) [8]. The gene fragment containing mxaF::Kan was subcloned into pAYC61, a suicide vector [13]. When this plasmid was transferred to M. extorquens AM1 by conjugation from E. coli S17-1, kanamycin-resistant colonies appeared at high frequency and those that were tetracycline-sensitive were defective in growth on methanol as expected (data not shown). When the intact plasmid was introduced into M. extorquens AM1 by electroporation using the conditions described above, kanamycin-resistant colonies appeared. Efficiencies of 102–103 cells per μg of plasmid DNA were obtained. Each colony was patched onto agar medium containing kanamycin, tetracycline or ampicillin. Double-crossover mutants were identified as kanamycin-resistant and tetracycline-sensitive. All tetracycline-resistant colonies were ampicillin-resistant, suggesting that either marker could be used for screening, although it was reported that ampicillin resistance gene from pBR322 was not expressed in M. extorquens AM1 [14].

3.3Mutation of other mxa genes

M. extorquens AM1 has a gene cluster involving methylotrophy functions, mxaFJGIR(S)ACKLD[15]. These genes are all transcribed in the same direction and may be a single transcriptional unit. Mutants have been isolated in mxaFJGIACKL, and these are all required for methanol oxidation. As noted above, mxaF encodes the large subunit of MDH, mxaI encodes the MDH small subunit, and mxaG encodes the MDH electron acceptor, cytochrome cL. The exact function of mxaJ is unknown, but it may be an assembly protein [16]. mxaAKL genes are involved in insertion of calcium ions into MDH [17]. No mutants are currently available in M. extorquens AM1 for mxaR, mxaS, or mxaD, and the chemical mutants available in mxaC cannot be rescued by a plasmid with only mxaC[18]. Therefore, insertion mutants were generated in these four genes using the electroporation procedure described for mxaF, except that pUC18- or pUC19-based plasmids were used instead of pAYC61 and the plasmids were linearized (Table 1, Fig. 1). The direction of the inserted drug resistance gene was determined by restriction enzyme digestion, and in each case a plasmid was chosen that had the kanamycin resistance gene in the same orientation as that of transcription of the gene of interest. In past work with insertion mutations in M. extorquens AM1, this orientation avoids polarity on neighboring genes [3–5]. Each plasmid was linearized with an appropriate restriction enzyme, precipitated by ethanol, dissolved in deionized water, and then used for electroporation. Recombinants were selected on agar plates containing kanamycin and screened for ampicillin resistance. Efficiencies were around 102–103 cells per μg of DNA. Most of the colonies obtained on kanamycin were ampicillin-sensitive. Double-crossover mutations were confirmed by DNA hybridization with digoxigenin-labelled pUC18 or the kanamycin resistance gene from pUC4K as a probe.

image

Figure 1. Physical map of the BamHI-XhoI DNA fragment. The open boxes represent open reading frames of mxaRSACKLD. Arrowheads indicate the restriction enzyme sites used for mutation. Arrows represent the cloned genes used for complementation analysis, whose directions indicate those of the pRK310 lacZ promoter in the plasmids listed above the genes. Restriction enzyme sites: B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; P, PstI; S, SalI; X, XhoI.

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The mxaC insertion mutant, C31, did not grow on methanol (Table 2). Strain C31 can be complemented by either pDN9 or pCM59, both of which have mxaC. The insertion mutant of mxaD (D11) grew less well than the wild-type did on methanol agar plates, while it grew as well as the wild-type on succinate or methylamine plates. The reduced growth on methanol was recovered by pRK310A3S1 but not pRK310A3S2, which contain mxaD in opposite orientations with respect to the lac promoter in pRK310 with pRK310A3S1 in the correct orientation (Fig. 1) [18]. These results suggest that mxaD is involved in methanol utilization but is not essential to growth on methanol.

Table 2.  Complementation of mutants with plasmids
StrainPlasmidGenesGrowth on:
   methanolsuccinate
Wild-typenone++++
C31none++
 pDN9mxaACKLD++++
 pCM59mxaC++++
D11none+++
 pDN9mxaACKLD++++
 pRK310A3S1mxaD++++
 pRK310A3S2mxaD+++
R1none+
 pHT9mxaFJGIR+++
 pHT9BfmxaRSA++++
SH11none++++
SN21none++
 pHT9mxaFJGIR++++
 pHT9BfmxaRSA++++
SA31none++
 pHT9mxaFJGIR++++
 pHT9BfmxaRSA++++

Between mxaFJGI and mxaACKLD, two probable open reading frames, mxaRS, are present that show identity to previously known open reading frames required for methanol oxidation in Paracoccus denitrificans[15]. However, in M. extorquens AM1 mxaS has atypical codon usage at the start of the putative open reading frame and this portion of the putative mxaS does not show identity to the P. denitrificans mxaS. Therefore, it is not known whether mxaS encodes a gene product in M. extorquens AM1 [15]. Three constructions were used to analyze the mxaS region by mutation (Fig. 1). One has the kanamycin resistance gene inserted in a HincII site existing in the probable intergenic region between mxaR and mxaS. The mutant obtained in this case (SH11) was able to grow on methanol as well as wild-type, suggesting that it interrupted a non-coding region and that the inserted gene had little or no polar effect on neighboring genes. The insertion mutant obtained at the same position but in the opposite orientation also grew on methanol but less well than the wild-type, suggesting a mild polar effect. A second mutant (SN21) contains a deletion of a 202-bp NruI fragment, which removes about half of the probable intergenic region and deletes the first 23 amino acids of MxaS (Fig. 2). This mxaS deletion is within the region that does not show similarity to mxaS of P. denitrificans (Fig. 2). Mutant SN21 did not grow on methanol (Table 2). A third mutant (SA31) was obtained by inserting the kanamycin resistance gene in the AscI site present in the middle of the portion of mxaS that shows identity to mxaS of P. denitrificans. Strain SA31 showed no growth on methanol either. A plasmid containing the mxaRSA region (pHT9Bf) complemented the growth of the latter two mutants on methanol. These results suggest that mxaS is translated and its product is involved in methanol oxidation, even though it contains atypical codon usage at the start of the open reading frame.

image

Figure 2. Nucleotide sequence of the intergenic region between mxaR and mxaS. Amino acid sequences of the C-terminal part of MxaR and N-terminal part of the putative MxaS are shown. Restriction enzyme sites used for mutation are underlined. Accession number in the EMBL database is Y07864. Numbers at the right edge of the sequence are the same as those in [13], which start at the BamHI site in Fig. 1.

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The mxaR gene is also a gene of unknown function. Sequence analysis has shown that the mxaR gene product is similar to MoxR of P. denitrificans and has predicted MgATP binding motifs A and B [15]. An mxaR insertion mutant (R1) was obtained and it did not grow on methanol. The mutant grew on succinate less well than wild-type (Table 2). Growth on methanol was restored by pHT9Bf, which contains mxaRSA. Although the function of the mxaR gene product is still unknown, apparent homologs of this gene have been found in the genome sequences of Bacillus subtilis[19] and Archeoglobus fulgidus[20], and so it probably represents a new gene family of widespread distribution.

In this article, we have shown that gene disruption by homologous recombination in M. extorquens AM1 is possible by electroporation of linear fragments. This method facilitates the generation of mutants in genes that have been cloned and sequenced.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

H.T. was supported by Postdoctoral Fellowships for Research Abroad from the Japan Society of the Promotion of Science. This work was funded in part by a grant to M.E.L. from the NIH (GM36294).

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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