Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK1
Author for correspondence: John A. C. Archer. Tel: +44 1223 333 999. Fax: +44 1223 333 992. e-mail: jaca1{at}mole.bio.cam.ac.uk
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ABSTRACT |
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Keywords: transposon, kanamycin, Gram-positive, Mycobacterium, Nocardia
Abbreviations: iPCR, inverse PCR
a Present address: Paradigm Therapeutics, Department of Physiology, Downing Street, Cambridge, CB2 3EG, UK.
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INTRODUCTION |
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Originally primarily of veterinary significance, Rhodococcus has become clinically important in the immuno-compromised, particularly in patients with AIDS (Mosser & Hondalus, 1996 ). Rhodococci and members of related genera such as the nocardiae have been shown to cause a number of complaints, including brain abscesses, sub-acute endocarditis and pulmonary disease (Berkowitz, 1994
).
Rhodococcus appears specialized to recycle complex plant-derived compounds in the soil, including a wide range of aromatic hydrocarbons. As a result, Rhodococcus isolates have been shown to catalyse a wide range of biotransformations of industrial relevance, such as xenobiotic degradation and surfactant synthesis (Goodfellow & Alderson, 1977 ; Larkin et al., 1998
; Warhurst & Fewson, 1994
), and the manufacture of other commercially important products (Allen et al., 1997
; Larkin et al., 1998
).
Currently, Rhodococcus genetics lacks a transposon system which functions efficiently enough to permit the generation of comprehensive random insertion libraries. Such systems have been widely used in the characterization of genes in Escherichia coli and many other genera (Kleckner, 1981 ). The development of these systems for many Gram-positive bacteria (in particular the recalcitrant actinomycetes) has, until recently, been hampered by a general lack of genetic tools. In a strain of Rhodococcus fascians, tagged mutagenesis has been achieved by the random illegitimate insertion into the chromosome of suicide vectors delivered by electroporation (Crespi et al., 1994
). However, high frequencies of illegitimate recombination have not been observed upon introduction of various non-replicating vectors into other strains of Rhodococcus; consequently this technique is not generally applicable (Powell & Archer, 1998
; Shao & Behki, 1995
; Yao et al., 1994a
).
In vivo transposition systems based on suicide delivery suffer from the combined effects of low transformation efficiency in wild-type strains and low transposition frequency, precluding the generation of insertion libraries (Guilhot et al., 1994 ; Pelicic et al., 1997
). Replicative delivery systems avoid this problem but require both a functioning replicon and a means of selecting for transposition events. Selection can be achieved with a counter-selectable marker such as sacB, a temperature-sensitive replicon, or by insertional activation of a promoterless antibiotic resistance gene carried on the transposable element. In Mycobacterium tuberculosis, it has proven necessary to use both sacB counter-selection and temperature-sensitive plasmid replicons simultaneously to ensure clean selection (Pelicic et al., 1997
). The use of replicative vectors runs a risk of generating a large number of sibling colonies due to the sustained period of growth prior to selection. Although a number of Rhodococcus IS elements have been isolated, none have been engineered into a usable mutagensis system (Denome & Young, 1995
; Jager et al., 1995
; Lessard et al., 1999
; Nagy et al., 1997
; Yao et al., 1994a
).
In vitro mutagenesis of DNA using the Tn552 system followed by integration back into the host chromosome has proven effective in Mycobacterium smegmatis. This approach depends on the frequency of homologous recombination, which has been shown to be low in strains of Rhodococcus (Barnes et al., 1997 ; Powell & Archer, 1998
) and its utility in this genus has yet to be demonstrated.
The recent development of the Tn5-based Transposome system eliminates a number of these problems. Tn5 is one of the simplest and best characterized transposons. In vitro transposition requires only Tn5 transposase, transposon DNA flanked by Tn5 inverted repeats, and target DNA. The transposome system consists of Tn5 transposase bound to transposon DNA in vitro. This bound complex is prevented from completion of the transposition reaction by the absence of magnesium ions. Following electroporation of the proteinDNA complex into a bacterial cell, the reaction proceeds due to the presence of magnesium ions, resulting in insertion of the transposon DNA at random into the genome. For a poorly characterized host like Rhodococcus this approach has a number of advantages. Firstly, the expression of transposase in the target strain is not required. Secondly, the transposition frequency once the complex has been transformed into the cell is very high. There is therefore no requirement for replicative delivery and the concomitant problems of selecting for transposition events. Transposome insertion frequencies are limited only by the rate of DNA transformation. The lack of requirement for host-encoded factors means that the system has the potential to operate in a very wide range of organisms (Goryshin et al., 2000 ).
The use of this system in the genus Rhodococcus or the related genera Corynebacterium, Nocardia or Mycobacterium has not previously been investigated. In this paper we describe the use of the EZ::TN(KAN-2)Tnp Transposome to create random transposon libraries in Rhodococcus rhodochrous CW25 and in a wild-type Rhodococcus soil isolate. The transposable element in this case consists of the Tn903 kanamycin resistance gene flanked by two 19 bp Tn5 inverted repeats. During the course of this work the use of the kanamycin-resistance transposome to create a transposon library in M. smegmatis was reported (Derbyshire et al., 2000 ).
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METHODS |
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Transformation of Rhodococcus spp.
Ten millilitres of a stationary-phase culture was used to inoculate a 500 ml flask containing 200 ml LB supplemented with 2% (w/v) glycine for R. rhodochrous CW25 and 1% glycine plus 0·5% (w/v) glucose for Rhodococcus sp. RC89. The flask was incubated at 30 °C with shaking until the culture reached late exponential phase or an OD550 of no more than 0·9 units using a Beckman DU 650 spectrophotometer. Cells were then incubated on ice for 20 min and pelleted by centrifugation (Sorvall RC-5B) at 4000 r.p.m. for 20 min at 4 °C. The cell pellet was washed twice in 100 ml sterile 10% (v/v) glycerol. Washes were carried out at 4 °C and cells were stored on ice at all other times. Aliquots (3 µl) of cells were diluted in 1 ml LB and the OD550 was measured. The concentration of cells for electroporation was adjusted to give an OD550 of 0·4 in the diluted sample.
Mutagenesis.
Electrocompetent Rhodococcus cells (40 µl) were placed on ice in a 0·1 cm electrode gap Bio-Rad electroporation cuvette and mixed with 20 ng (1 µl) EZ::TN(KAN-2)Tnp Transposome (Epicentre Technologies). Electroporation was carried out at 1·6 kV, 25 µF, 200 . Cells were resuspended in 1 ml SMME (0·7 M sucrose, 50 mM maleic acid, 10 mM MgCl2.6H2O and 10 mM CaCl2, pH 6·5) (Haynes & Britz, 1990
) and immediately plated in 100 µl aliquots onto LA plates containing 1% glucose. Plates were incubated for 12 h at 30 °C, then overlaid with soft agar containing sufficient kanamycin to give a final concentration of antibiotic in the plate (taking the volume of LA into account) of 400 µg ml-1 for R. rhodochrous CW25 and 100 µg ml-1 for Rhodococcus sp. RC89. The soft agar was allowed to set and plates incubated at 30 °C. Transposants appeared within 5 d.
DNA extraction and manipulation.
DNA extractions were based on the salting out method of Pospiech & Neumann (1995) . Rhodococcus cultures were grown in 10 ml LB containing the appropriate concentration of kanamycin and pelleted (Sorvall RC-5B) at 4500 r.p.m. for 12 min. Samples were resuspended in 5 ml SET buffer (75 mM NaCl, 25 mM EDTA pH 8, 20 mM Tris pH 7·5). Lysozyme (5 mg ml-1) was added and samples were incubated at 37 °C for 1 h with shaking. Proteinase K (1 mg ml-1 final concentration) plus 2% (w/v) SDS (final concentration) were then added and samples were incubated at 60 °C for 2 h. NaCl (5 M; 2 ml) was added; the samples were mixed and allowed to cool to less than 37 °C. Chloroform (5 ml) was then added and the samples were incubated at 4 °C for 30 min with shaking. Cell debris was pelleted by centrifugation at 4500 r.p.m. at 4 °C and supernatants transferred to a 15 ml Corex tube. DNA was precipitated with 0·6 vol. 2-propanol at -20 °C for 1 h and recovered by centrifugation at 8000 r.p.m. for 15 min. DNA pellets were left to dry and were then resuspended in 100 µl warm sterile distilled water.
Restriction enzymes and T4 DNA ligase were from New England Biolabs and were used according to the manufacturers instructions. Ligations were carried out at room temperature for 2 h.
Southern hybridization.
Probes were labelled by the incorporation of digoxigenin-11-dUTP by PCR using the Roche PCR DIG Probe Synthesis Kit, used according to the manufacturers instructions. All hybridizations were carried out using the DIG system for filter hybridization (Roche) according to the manufacturers instructions. DNA was transferred onto positively charged nylon membranes (Boehringer Mannheim) and fixed by UV cross-linking. Hybridization and post-hybridization washes were carried out at 68 °C in a Hybaid shakenstack hybridization oven.
Inverse PCR (iPCR).
To sequence tagged genome insertions, total DNA was extracted and 12 µg samples were digested with the appropriate restriction enzymes (MluI, NcoI and PstI were used). Digested DNA was cleaned up by extraction with phenol/chloroform (1:1) and precipitated with 2-propanol. Digested samples for each transposant were pooled in 17 µl sterile distilled water and ligation carried out at room temperature for 2 h. Ligations were heat-killed and then drop-dialysed for 1 h on 0·025 µm Millipore filters before proceeding to iPCR. After drop dialysis, iPCR was carried out as follows using GibcoBRL reagents: 1xPCR buffer, 1·5 mM MgCl2, 200 µM of each dNTP (Promega), 0·2 µM of forward primer (KAN-2 FP-1, 5'-ACCTACAACAAAGCTCTCATCAACC-3') and of reverse primer (KAN-2 RP-1, 5'-GCAATGTAACATCAGAGATTTTGAG-3'), 2·5 U Taq DNA polymerase and 1 µl template DNA (from ligation). Sterile distilled water was added to a final volume of 50 µl. PCR reaction conditions were as follows: initial denaturation at 96 °C for 5 min; followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 1 min and extension at 72 °C for 1 min; with a final extension at 72 °C for 5 min. Following iPCR, samples were run on 0·85% agarose gels, products were cut out and DNA extracted using the Concert rapid gel extraction system (GibcoBRL). Finally, salt was removed by drop dialysis of samples prior to sequencing.
DNA sequencing and analysis.
Sequencing reactions were carried out using ABI PRISM BigDye sequencing kits on an ABI PRISM 377 DNA sequencer. KAN-2 FP-1 and KAN-2 RP-1 primers used for iPCR were also used for sequencing. Sequences were assembled using Staden software programs PREGAP and GAP4 (Bonfield et al., 1995 ) on a Silicon Graphics Unix computer. DNA and protein similarity searches were carried out with BLAST 2.0 using the WWW interface at the NCBI (National Center for Biotechnology Information) (Altschul et al., 1997
). The Prosite (Bairoch et al., 1997
) and Pfam (Sonnhammer et al., 1998
) profile databases were searched using the server of the Bioinformatics group at the ISREC (Swiss Institute for Experimental Cancer Research) and the ExPASy WWW molecular biology server of the Geneva University Hospital and the University of Geneva. Other analyses were carried out using the GCG (Genetics Computer Group, Madison, Wisconsin) package, version 9.1.
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RESULTS |
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The generation of significant numbers of insertion mutants using the transposome system is critically dependent on the electroporation efficiency of the strain, particularly with heterologously derived DNA, as is the case for both pCY104 and the transposome system. Electroporation efficiencies are most easily measured using a replicative vector. Highly electrocompetent E. coli will generate 109 c.f.u. µg-1 of replicative, homologously derived plasmid DNA and 105 c.f.u. (20 ng)-1 of transposome preparation. Such high efficiencies with replicative plasmid DNA are not readily achievable using wild-type strains of Rhodococcus and it would therefore be expected that the number of transposome insertions generated would also be lower.
It has been shown that recovery after electroporation on solid media prior to selection results in a higher frequency of transformants than recovery in liquid media (Powell, 1998 ). Although selection can be carried out by resuspension of the cells from non-selective solid media and subsequent replating on selective media, this generates a significant number of sibling colonies, thus obscuring the true number of independent transformants. To overcome this problem, the use of soft agar overlays was investigated. Kanamycin levels ranged from 50 to 450 µg ml-1. Using R. rhodochrous CW25 it was found that at levels below 400 µg ml-1 a significant number of spontaneous resistant mutants arose, whereas at levels above 500 µg ml-1 the number of transformants was reduced. A soft agar overlay supplemented with kanamycin to give a final concentration of 400 µg ml-1 in each plate was therefore used to select transformants for the remainder of the work.
To determine the optimum recovery time after electroporation, 100 µl of each sample was plated onto LAG (Luria agar with 1% glucose) and incubated at 30 °C for 0, 6 and 12 h prior to addition of selective overlay. Selection immediately after plating cells on LA with glucose greatly reduced efficiency, 5x103 c.f.u. (0·5 µg DNA)-1, as compared with those allowed to recover for longer periods post-electroporation. The longer recovery times of 6 and 12 h gave rise to 2x104 and 7x104 c.f.u. (0·5 µg DNA)-1, respectively. After 12 h of recovery, the number of transformants was increased 14-fold compared with immediate plating onto selective media. If cells were left to recover for longer periods (more than 12 h), the amount of background growth no longer allowed for selection of transformants.
Growth on glycine and/or isoniazid has been shown to enhance the electroporation efficiencies of strains of Corynebacterium glutamicum (Haynes & Britz, 1990 ), Mycobacterium bovis BCG (Cho et al., 1998
) and Rhodococcus equi (Sekizaki et al., 1998
), most likely by affecting growth of the cell wall. R. rhodochrous CW25 grown in the presence of glycine (2%), or glycine (2%) plus isoniazid (100 µg ml-1) gave rise to a 10-fold increase [7x105 c.f.u. (0·5 µg DNA)-1] in electroporation efficiency (using the replicative vector pCY104) as compared to cells grown in unsupplemented media. These high-efficiency cells were stored at -70 °C and subsequently used for electroporation with transposome.
Electroporation with transposome
A preliminary experiment was carried out in which R. rhodochrous CW25 (grown without media supplementation) was electroporated with 20 ng of the transposome preparation and spread onto non-selective LA plates. Following overnight incubation, cells were resuspended and plated onto LA supplemented with 400 µg kanamycin ml-1. A number of transposants were isolated and chromosomal insertions confirmed by Southern blotting, but electroporation/transposition frequencies could not be determined as the nature of the recovery gave rise to large numbers of siblings (data not shown). To confirm these results, the experiment was repeated using the glycine-grown electrocompetent cells described above. Transposants were selected using the kanamycin overlay technique to prevent isolation of sibling colonies. One thousand five hundred kanamycin-resistant colonies were obtained from 20 ng transposome complex.
Confirmation of insertion by Southern blotting
To confirm insertion of the transposome, chromosomal DNA was prepared from 23 transposant colonies chosen at random. The genomic DNA was digested to completion with PstI and Southern blot analysis was performed using the Tn903 kanamycin resistance gene as a probe. The Southern blot shown in Fig. 1 demonstrates that all insertions were unique and random insertion of the transposon occurred, in all cases at different places on the chromosome with the exception of the colonies in lanes 22 and 23, which were siblings from the preliminary experiment described earlier in which the overlay technique was not used. These results were confirmed by an additional blot in which samples were digested with EcoRI (data not shown).
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Rhodococcus sp. RC89 was transformed by electroporation with pCY104 and allowed to recover on LA with 1% glucose for 12 h at 30 °C. Soft agar overlays were added to give a range of final kanamycin concentrations from 100 to 400 µg ml-1. A concentration of 100 µg kanamycin ml-1 was found to be optimal. The pCY104 transformation efficiency of Rhodococcus sp. RC89 was 3x104 c.f.u. (0·5 µg DNA)-1.
Electroporation with 20 ng (1 µl) transposome gave rise to 500 Rhodococcus sp. RC89 transposants. Four hundred transposants were patched onto minimal media with succinate and 24 (6·5%) failed to grow, indicating efficient library coverage. Around 200 mutants were screened for their inability to utilize toluene and benzene as sole sources of carbon and energy. Two mutants incapable of utilizing these compounds and five further mutants that grew slowly on them were isolated. The insertion site of one of the mutants was sequenced and transposition confirmed by the presence of 9 bp repeats. The disrupted gene was 57% identical to a diaminobutyric acid aminotransferase from Bacillus halodurans (NCBI accession no. BAB04638). As there is no evidence to suggest that this gene product is involved in the degradation of monoaromatic hydrocarbons, it is likely that the associated phenotype is due to a polar effect. A number of Rhodococcus sp. RC89 aromatic hydrocarbon degradation mutants are currently under investigation.
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DISCUSSION |
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Because of polar effects, it is difficult to calculate exactly the number of insertions required for a high confidence library. However, given a plausible genome complement of 4000 genes arranged in 2500 transcriptional units, this number should lie between 3000 and 10000 insertions (Blattner et al., 1997 ). This number is readily achievable by simple scale-up with the system described in this paper for both R. rhodochrous CW25 and the wild-type strain Rhodococcus sp. RC89. Library construction would be facilitated by optimization of the electroporation efficiency for each strain. We have found that pCY104 has a particularly broad host range amongst the rhodococci and is therefore an ideal test vector. This is the first report of transposon mutagenesis on this scale in Rhodococcus.
The advantages of this system over the standard in vivo transposase expression approach are the high efficiency, the lack of requirement for complex replicative vectors or native inducible promoters to drive transposase expression, and the generation of unique insertions without risk of isolating sibling colonies (using the overlay technique described here). Transposome electroporation is likely to become the method of choice for generating transposon insertion libraries in Rhodococcus.
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ACKNOWLEDGEMENTS |
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Received 1 May 2001;
revised 23 May 2001;
accepted 29 May 2001.