Departments of 1Microbiology, Box 357242, and 2Chemical Engineering, Box 351750, University of Washington, Seattle, WA 98195-1750, USA
Author for correspondence: Mary E. Lidstrom. Tel: +1 206 616 5282. Fax: +1 206 616 5721. e-mail: lidstrom{at}u.washington.edu
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ABSTRACT |
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Keywords: plasmids, cloning vectors, expression vectors, promoter-probe vectors, Methylobacterium extorquens AM1
Abbreviations: Ap, ampicillin; ßGal, ß-galactosidase; bhr, broad-host-range; Km, kanamycin; LB, LuriaBertani (medium); oligo, oligodeoxyribonucleotide; ori, origin(s) of DNA replication; P, promoter; Rif, rifamycin; Sm, streptomycin; t, terminator of transcription; Tc, tetracycline; wt, wild-type
The GenBank accession numbers for the sequences reported in this paper are AF327711, AF327712, AF327713, AF327714, AF327715, AF327716, AF327717, AF327718, AF327719 and AF327720.
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INTRODUCTION |
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The majority of bhr vectors are based upon IncP or IncQ replicons. Most IncP plasmids are derived from RK2, a 60·1 kb self-transmissible plasmid (Pansegrau et al., 1994 ). RK2 has an estimated copy number in E. coli of five to seven per chromosome (Figurski et al., 1979
), and plasmids containing the IncP origin of transfer, oriT, can be mobilized by IncP transfer proteins provided in trans (Figurski & Helinski, 1979
). Currently available vector tools based on IncP replicons are low- to medium-copy in E. coli, and few are fully sequenced or have a large number of available restriction sites.
Methylobacterium extorquens AM1 is an example of an organism for which improved genetic tools are needed. The -proteobacterium M. extorquens AM1 is the best-studied representative of the methylotrophic bacteria, which are those capable of using single-carbon (C1) substrates as their sole source of carbon and energy (Lidstrom, 1991
). Methylotrophs play a critical role in the biogeochemical cycling of C1 compounds (Hanson & Hanson, 1996
) and have the potential to convert alternate feedstocks, such as methanol or methane, into fine chemicals and/or biopolymers. M. extorquens AM1 is a facultative methylotroph capable of growth on C1 substrates, such as methanol and methylamine, as well as on a limited number of multi-carbon compounds, such as succinate and pyruvate. The ability to grow on nonmethylotrophic compounds and the ease of DNA transfer by either conjugation (Windass et al., 1980
) or electroporation (Toyama et al., 1998
) have made M. extorquens AM1 a model organism for the genetic dissection of the methylotrophic metabolism. In addition, the availability of genome-level sequence data for this strain (http://faculty.washington.edu/lidstrom/genome/genome/genome.html) is providing a rich database of information on metabolic potential. Thus far, only large (1923 kb) IncP vectors with limited cloning sites available have been used with success in M. extorquens AM1. These include the cloning vectors pRK310 (Ditta et al., 1985
) and pVK100 (Knauf & Nester, 1982
), and the promoter-probe vectors pHX200 (Xu et al., 1993
) and pGD500 (Ditta et al., 1985
). No IncQ vectors or smaller IncP vectors have been found to be maintained in this strain (Lidstrom, 1992
). Here we present the isolation of a spontaneous mutant of a small IncP plasmid that functions efficiently in M. extorquens AM1, the development of improved bhr cloning vectors based upon this plasmid, and their modification into host-specific promoter-probe and expression tools.
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METHODS |
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Construction of the minimal transferable and selectable replicon for use in M. extorquens AM1.
The 2·0 kb NdeINotI region of pDN19X containing the traJ' allele that allowed efficient replication and/or transfer in M. extorquens AM1 was transferred into the corresponding region of pTJS75 (Schmidhauser & Helinski, 1985 ), a parent vector of pDN19 (Nunn et al., 1990
), to create pCM48. The plasmid pCM48 was then cut with MunI and BlnI, and the DNA ends were blunted and self-ligated to produce pCM50. pCM50 was then digested with HindIII and XmnI, and the DNA ends were blunted and self-ligated to produce the 5·3 kb minimal transferable and selectable replicon pCM51. Two additional plasmids were created to confirm that the traJ' allele present in pDN19X is sufficient to maintain and/or transfer the plasmid into M. extorquens AM1. The 2·0 kb NdeINotI region surrounding the substitution in pDN19X was swapped into the corresponding region of pDN19 to create the plasmid pCM46. Similarly, the 2·0 kb NdeINotI region from pDN19 encoding the full-length TraJ was cloned into pDN19X to create the plasmid pCM47.
To compare the reduced gene complement of the minimal selectable and transferable replicon pCM51 to the cloning vector previously used with M. extorquens AM1, pRK310 (Ditta et al., 1985 ), we have pieced together the complete sequence of this large IncP vector by determining the sequence over the junctions created during the partial digestion steps in its cloning history (Fig. 1
).
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Construction of low-background promoter-probe vectors.
Two low-background bhr promoter-probe vectors with different reporters were created using the cloning vectors pCM62 and pCM66 as their vector backbone. The 1·0 kb HindIIINcoI fragment from pCM75 containing xylE was inserted into pCM62 cut with AflIII and HindIII to create pCM76. Similarly, the 3·2 kb EcoRI fragment of pLacZ2.1+ (R. Meima & M. E. Lidstrom, unpublished) containing lacZ was blunted and cloned into the BamHI site of pCM66 which had been similarly blunted to create pCM66LacZ. pCM76 and pCM66LacZ were each found to have a high background reporter gene activity in M. extorquens AM1 (data not shown). To reduce this background activity, an E. coli terminator, trrnB, was introduced upstream of the multiple cloning sites on the two vectors. The 0·5 kb XbaIXmnI fragment of pMTL20T1T2 (Cordes et al., 1996 ) containing trrnB was excised, blunted and ligated into the blunted EcoRI site of pCM76 to create pCM130 (Fig. 3
). The 1·0 kb EcoRIRsrII fragment of pCM130 containing trrnB was then transferred into pCM66LacZ, cut with EcoRI and RsrII, to create pCM132 (Fig. 3
). In order to test the ability of these vectors to detect promoter activity, the PmxaF of M. extorquens AM1 was introduced into both plasmids. The 0·4 kb EcoRIBamHI fragment from pCM27 was cloned into pCM130 cut with EcoRI and BamHI to create pCM131. Similarly, the 0·4 kb EcoRI fragment from pCM27 was inserted into the EcoRI site of pCM132 to produce pCM133.
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In addition to pCM80 and pCM160, an additional expression vector, pCM110, was created that would provide minimal expression in E. coli to allow toxic genes to be introduced into M. extorquens AM1 (Fig. 4). pCM110 was constructed by inserting the 0·4 kb HindIIINsiI fragment of pCM27 containing PmxaF into pCM60 (C. J. Marx & M. E. Lidstrom, unpublished results) which had been cut with HindIII and NsiI. To compare expression from the PmxaF in pCM80 to that in pCM110, the 1·0 kb EcoRI fragment from pCM20 containing xylE was inserted into the EcoRI site in pCM110 to create pCM111.
Reporter gene assays and SDS-PAGE analysis of vector expression.
Cell extracts for enzyme assays and SDS-PAGE gels were prepared from mid-exponential cultures of M. extorquens AM1 harvested at an OD600 of 0·70·8, as determined using 1·0 cm cuvettes in a Beckmann DU 640B spectrophotometer. XylE and ß-galactosidase (ßGal) assays are reported as the mean and standard deviation of three replicates and were performed as described previously (Zukowski et al., 1983 ; Kataeva & Golovleva, 1990
; Miller, 1972
). XylE activities in E. coli JM109 were assayed in extracts prepared from cultures grown to an OD600 of 0·61·0 in LB. The total protein was estimated either by direct spectrophotometric methods (Kalb & Bernlohr, 1977
; Whitaker & Granum, 1980
) for enzyme assays, or by the Bradford method using the Protein Assay Kit (Bio-Rad), using BSA as a standard, for SDS-PAGE analysis on 15% gels. The relative intensities of selected bands on Coomassie-blue-stained SDS-PAGE gels were determined using Kodak 1D Image Analysis Software (Eastman Kodak).
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RESULTS AND DISCUSSION |
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Initially, it appeared that pDN19 was transferred from E. coli to M. extorquens AM1 with an efficiency 1000 times less than that observed for pRK310. Following transfer of pDN19 from M. extorquens AM1 back into E. coli, however, the plasmid could be reintroduced into M. extorquens AM1 at the same high transfer efficiency as pRK310 (data not shown). This result suggested that the original pDN19 plasmid may have acquired a mutation that increased its transfer efficiency or the ability of M. extorquens AM1 to maintain this plasmid. No difference was observed in the maintenance of this plasmid derivative in E. coli (data not shown). We designated this pDN19 derivative as pDN19X.
Sequencing of pDN19 and the identification of the spontaneous mutation present in pDN19X
The exact sequence of the RK2-derived vector pDN19 (Nunn et al., 1990 ) was not known. Therefore, single-strand sequence was obtained for pDN19. A map of pDN19 and the features present on this plasmid are presented in Fig. 1
. Seven single-nucleotide differences were observed relative to the reported sequence of RK2. These included single-nucleotide deletions and insertions, and nucleotide replacements. The only changes present in coding regions disrupt upf-16.5, a putative ORF of unknown function (Pansegrau et al., 1994
). The single-strand sequence of pDN19X revealed a single nucleotide difference relative to the parent plasmid, pDN19, which was located within traJ. This C to A transversion results in an early stop codon in traJ, whose gene product recognizes the origin of transfer, oriT, initiating the DNA-processing events required for conjugal transfer (Ziegelin et al., 1989
). This early stop codon creates a TraJ' polypeptide that is missing the final 85 of 123 amino acids.
To determine whether the traJ' allele of pDN19X is sufficient to increase the maintenance and/or transfer of this plasmid in M. extorquens AM1, a 2·0 kb region encompassing traJ was swapped between pDN19X and the corresponding region of pDN19 to create pCM46. Similarly, the region from pDN19 encoding the full-length TraJ was cloned into pDN19X to create pCM47. The plasmid pCM46 bearing the mutation was maintained and/or transferred as had been observed for pDN19X, whereas pCM47 lacking the mutation lost this capacity. This confirmed that the ability of pDN19X to be efficiently maintained and/or transferred in M. extorquens AM1 was due to the region containing the traJ' allele, and not due to a distal region of the plasmid. The plasmid pRK310 is transferred and maintained in M. extorquens efficiently, and it has the same traJ allele as pDN19. In addition, all necessary tra functions including traJ are provided by the helper plasmid (pRK2013 or pRK2073) during triparental matings. Therefore, it is not clear why an alteration in TraJ would be required for efficient maintenance and/or transfer of pDN19 into M. extorquens AM1. It is possible that the effect may be due to altered expression of downstream genes, such as the essential initiator gene trfA. TrfA has been shown to affect copy number and host range (Haugan et al., 1995 ). In addition, our sequencing has shown that pDN19 lacks the native trfA promoter and the only known upstream promoter is that for traJ. It was not possible to compare expression of trfA or copy number between pDN19 and pDN19X, because pDN19 is apparently not maintained in M. extorquens AM1.
Reduction of pDN19X to the minimal transferable and selectable replicon pCM51
The nucleotide sequence of pDN19X was utilized to guide the removal of potentially nonessential plasmid regions in order to create a minimal transferable and selectable bhr replicon that could be utilized as the backbone for further vector development. Three regions of the 7·8 kb pDN19X plasmid were sequentially excised to create the 5·3 kb minimal transferable and selectable replicon pCM51 (Fig. 1). The plasmid pCM51 consists solely of the IncP oriV and oriT, traJ', trfA and tetA and tetR. It behaved identically to pDN19X with regard to transfer and maintenance (data not shown), indicating that the regions of the parent plasmid that had been removed did not affect plasmid transfer or maintenance in M. extorquens AM1. Furthermore, pCM51 possesses a considerably reduced gene complement relative to the 19·1 kb IncP cloning vector previously used with M. extorquens AM1, pRK310 (Ditta et al., 1985
) (Fig. 1
). Achieving a small, functional replicon was critical in the development of improved bhr cloning vectors, and the subsequent elaboration of these vectors into more sophisticated genetic tools.
Development of facile bhr cloning vectors pCM62 and pCM66
The first goal of this study was to develop improved bhr cloning vectors that could be maintained in M. extorquens AM1 and other bacterial species, based on the minimal transferable and selectable replicon pCM51. This was accomplished by fusing a portion of pUC19 containing its multiple cloning site and the ColE1 ori to the region of pCM50 present in the small IncP replicon pCM51, creating pCM62 (Fig. 2). A kanamycin-resistant derivative, pCM66, was constructed by inserting the kanamycin resistance cassette from pUC4K for use in bacteria such as methanotrophs in which tetracycline is a poor marker (Fig. 2
). Both of these plasmids were maintained in and transferred into M. extorquens AM1 with efficiency equal to pCM51 (data not shown). Routine alkaline lysis plasmid minipreps of pCM62 and pCM66 from E. coli cloning strains, however, indicated that the presence of the ColE1 ori raised their copy number in E. coli to that typical of pUC19 and related plasmids (data not shown).
The bhr cloning vectors pCM62 and pCM66 have a number of features that simplify their routine use: (1) high copy number in E. coli; (2) small size (7·2 and 8·0 kb, respectively); (3) complete sequences; (4) variety of unique restriction sites; (5) bluewhite screening via lacZ; (6) conjugative mobilization between bacterial species; and (7) readily adaptable into species-specific promoter-probe and expression vectors.
A number of proteobacterial species other than E. coli and M. extorquens AM1 have been found to maintain pCM62 and/or pCM66. These include the -proteobacteria Agrobacterium tumefaciens (L. Chen & E. Nester, personal communication), Methylobacterium strains CM4 and DM4 (S. Vuillemaier & T. Leisinger, personal communication), and Rhodobacter sphaeroides (J. Hickman & T. Donohue, personal communication), the ß-proteobacterium Ralstonia eutropha (O. Lenz & B. Friedrich, personal communication) and the
-proteobacteria Methylococcus capsulatus Bath (S. Stolyar & M. E. Lidstrom, unpublished) and Pseudomonas aeruginosa (T. Motley & S. Lory, personal communication). These reports suggest that the improved bhr cloning vectors and the promoter-probe vectors described below will be generally applicable to a wide variety of Gram-negative bacteria.
Conversion of bhr cloning vectors into low-background promoter-probe vectors pCM130 and pCM132 and their demonstration in M. extorquens AM1
The second goal of this work was to construct facile promoter-probe vectors for use in M. extorquens AM1, as well as other bacterial species. Two large IncP promoter-probe vectors have been used in M. extorquens AM1, pHX200 bearing xylE as a reporter (Xu et al., 1993 ) and the lacZ-containing pGD500 (Ditta et al., 1985
). These plasmids are each greater than 20 kb in size, are not fully sequenced, and have limited cloning sites available upstream of their reporter genes. In addition, pGD500 has a high background reporter activity in M. extorquens AM1 (Morris & Lidstrom, 1992
). In order to facilitate the identification and dissection of promoter regions in M. extorquens AM1, we created two promoter-probe vectors, pCM130 and pCM132, that are based upon the cloning vectors pCM62 and pCM66, respectively (Fig. 3
). These promoter-probe vectors were created from their respective cloning vectors in two cloning steps. The reporter genes xylE and lacZ were first introduced into one side of the polylinker. This was followed by the introduction of a transcriptional terminator into the opposite side of the polylinker, in order to reduce background activity. The resulting bhr promoter-probe vectors have low background reporter gene activity in M. extorquens AM1 (Table 2
) as compared to pHX200, facilitating their use in analysing weak promoters as well as strong promoters.
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Conversion of bhr cloning vectors into expression vectors pCM80 and pCM160 specifically designed for use in M. extorquens AM1
The final goal of this work was to create expression tools appropriate for use in M. extorquens AM1 based on these improved bhr cloning vectors. The three-step approach we outline below can be readily adapted to generate expression tools specific for any bacterial species found to maintain these plasmids. Firstly, amplify by PCR a known promoter region and insert this fragment into the Plac-proximal side of the pCM62 or pCM66 polylinker, taking advantage of sites introduced on the primers, if necessary. Secondly, fill in the upstream site used for the insertion of the promoter fragment to preserve it for eventual use as a unique site in the polylinker. Thirdly, design and insert a linker with ends compatible to the downstream site used for insertion that can reconstitute the original polylinker within lacZ. This strategy allows the development of an expression vector that retains all of the advantages outlined for these cloning vectors.
For the conversion of pCM62 into an expression vector specifically designed for use in M. extorquens AM1, we chose to utilize PmxaF (Morris & Lidstrom, 1992 ). In the promoter-probe vectors described above, this promoter provides high-level expression during growth on both methylotrophic and nonmethylotrophic substrates. The expression vectors pCM80 and the kanamycin-resistant derivative, pCM160 (Fig. 4
), were constructed using the strategy outlined above.
Demonstrations of the utility of the M. extorquens AM1 expression vector pCM80
As a first test of the ability of pCM80 to express cloned genes in M. extorquens AM1, we used the gene encoding GFP from pGFPuv (Clontech). gfp was cloned into the cloning vector pCM62 (containing the Plac promoter) and into pCM80 to produce pCM87 and pCM88, respectively. In vivo GFP activity of M. extorquens AM1 bearing pCM87, pCM88, or the empty vector pCM80 was assessed on plates by observing the fluorescence of colonies during a brief exposure to UV light from a UV transilluminator (TFX-35M, Life Technologies). M. extorquens AM1 bearing pCM88 exhibited substantial fluorescence upon UV exposure, whereas colonies with pCM87 were indistinguishable from those carrying the empty vector pCM80. These results suggested that substantial expression of a heterologous gene could be achieved in M. extorquens AM1 using pCM80, while the Plac promoter of pCM62 did not appear to direct significant expression.
To quantify gene expression in M. extorquens AM1 using pCM80, two constructs, pCM63 and pCM81, were made that contain xylE inserted into the polylinker of either pCM62 or pCM80, respectively. Only low-level XylE expression in M. extorquens AM1 was observed from the Plac of pCM62 (Table 2). Similar results using plasmids bearing the Plac derivatives Ptac and Pspac further suggest that Plac-derived expression vectors will not be useful for high-level expression in M. extorquens AM1 (C. J. Marx & M. E. Lidstrom, unpublished results). In contrast, a high level of XylE activity was detected in extracts prepared from M. extorquens AM1 bearing pCM81, which expresses XylE from the PmxaF of pCM80 (Table 2
). Similar results were found for a pRK310-derived expression vector that bears PmxaF (C. J. Marx & M. E. Lidstrom, unpublished results). The modest induction observed using pCM81 during growth on methanol is similar to that observed from cells bearing the plasmid pCM131; however, the magnitude of XylE activity is roughly twofold higher. pCM131 differs from pCM81 by the presence of the trrnB upstream of PmxaF in pCM131 and the orientation of PmxaF::xylE relative to the remainder of the plasmid (Figs 3
and 4
).
One concern about the use of pCM80 is its high copy number in E. coli and the presence of Plac upstream of PmxaF. This could lead to significant expression of cloned genes in E. coli, preventing the use of this vector for the expression of genes in M. extorquens AM1 that are toxic in E. coli. Accordingly, XylE activity was measured in cell extracts prepared from E. coli strain JM109 bearing various plasmids that had been grown in LB and harvested at an OD600 of 0·61·0 (Table 2). Significant expression was observed from both pCM63 and pCM81 in JM109. An additional expression vector, pCM110, was constructed from an IncP plasmid that lacks both the ColE1 ori and Plac. A construct containing xylE cloned into pCM110 (pCM111) was created to compare its XylE expression level to that of pCM81 in both organisms. pCM111 provided a high level of expression in M. extorquens AM1, 1.52-fold higher than that achieved with pCM81; however, extracts from E. coli JM109 bearing pCM111 had a basal level of XylE activity, nearly at the background. These results indicate that the PmxaF is expressed at very low levels in E. coli. Therefore, while facile expression vectors such as pCM80 are useful for the expression of most cloned genes, an alternative expression vector, such as pCM110, may be required to express genes that are toxic in E. coli.
To determine the percentage of the total cell protein that could be obtained using pCM80, SDS-PAGE was used to estimate the relative content of the XylE and GFP polypeptides expressed from pCM81 and pCM88, respectively (Fig. 5). Protein bands could be identified specifically in the extracts from cells containing pCM81 and pCM88 that corresponded to the 35·1 and 26·8 kDa molecular masses predicted for XylE and GFP, respectively. Image analysis of the Coomassie-blue-stained SDS-PAGE gel indicated that pCM80 can express heterologous proteins at 59% of the total cell protein, with a modest increase in protein levels during growth on methanol relative to succinate. This level of induction corroborates the XylE activity data obtained from M. extorquens AM1 containing pCM81.
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Conclusions
We report the development of improved bhr cloning vectors and low-background promoter-probe vectors, and demonstrate a simple strategy to convert these plasmids into species-specific expression vectors. This suite of tools possesses a number of advantages over previously described bhr genetic tools in terms of their ease of use and their ability to be adapted for multiple purposes. These genetic tools will greatly facilitate the genetic dissection of the metabolism of M. extorquens AM1. The observation that these vectors can be maintained in a wide variety of bacterial species suggests that these vectors, and future elaborations upon them, will help fulfil the need for more sophisticated bhr genetic tools in a variety of bacteria. Such tools are critical for the facile genetic analysis of the wide spectrum of bacterial species for which genome-level sequence data are now available.
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ACKNOWLEDGEMENTS |
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Received 9 January 2001;
revised 10 April 2001;
accepted 12 April 2001.