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Appl Environ Microbiol. 2010 April; 76(8): 2531–2539.
Published online 2010 February 12. doi:  10.1128/AEM.02131-09
PMCID: PMC2849190

New Vector System for Random, Single-Step Integration of Multiple Copies of DNA into the Rhodococcus Genome[down-pointing small open triangle]

Abstract

We designed a new vector system for creating a random mutant library with multiple integrations of DNA fragments into the Rhodococcus genome in a single step. For this, we cotransformed two vectors into Rhodococcus by electroporation: pTip-istAB-sacB regulates the expression of the transposase (IstA) and its helper protein (IstB) under the influence of a thiostrepton-inducible promoter, and pRTSK-sacB provides the transposable-marker DNA. Both are multicopy vectors that are stable in the host cells; transposition of the transposable-marker DNA occurs only after the induction of IstA/IstB expression. With the addition of thiostrepton, all cultured cells harboring the two vectors, irrespective of the volume, can be mutated by random insertion of the transposable-marker DNA into their genome. Among the generated mutants examined, 30% showed multiple (two to five) insertion copies. The multiple integrated DNA copies were stable in the genome for more than 80 generations of serial growth without the addition of any selective antibiotics. This system can also be used for integrating various copy numbers of stably maintained protein expression cassettes in the host cell genome to modulate the expression level of biologically active recombinant proteins. We successfully applied this system to integrate multiple copies of expression cassettes for proline iminopeptidase and vitamin D3 hydroxylase into the Rhodococcus genome and verified that the clones containing double or multiple copies of the integrated cassettes produced higher levels and showed higher enzymatic activities of the target protein than clones with only a single copy of integration.

The actinobacteria or actinomycetes are a group of Gram-positive bacteria with a high G+C content. Many species of actinobacteria are well known as attractive hosts for the production of biologically active compounds since they can easily utilize cheap complex industrial media and possess excellent secretion capacities. This group includes several antibiotic producers (12, 15) and manufacturers of enzymes (5), amino acids (11), and heterologous proteins (3); hence, they are of high industrial, pharmacological, and commercial interest. Among the genera in this group are Corynebacterium, Mycobacterium, Streptomyces, Nocardia, and Rhodococcus.

While a few Rhodococcus species are pathogenic, most are benign and have been found to thrive in a broad range of environments, including soil, water, and eukaryotic cells. Rhodococcus is an experimentally advantageous system due to its relatively fast growth rate and simple developmental cycle. Rhodococcus erythropolis can grow at temperatures ranging from 4 to 35°C (41), which enables the investigation of protein production over a wide range of temperatures (24). Strains of Rhodococcus have important applications due to their ability to bioconvert cheap starting material into more valuable compounds (23) and to metabolize harmful environmental pollutants such as toluene, naphthalene, herbicides, and polychlorinated biphenyls (PCBs) (6, 17). This genetic and catabolic diversity of Rhodococcus is the result of not only its large bacterial chromosome but also the presence of large linear plasmids (37). To date, 43 species of Rhodococcus (7; reference periodically updated at http://www.bacterio.net.) have been recognized (http://www.bacterio.cict.fr/qr/rhodococcus.html). However, Rhodococcus is not yet fully characterized.

Various genetic tools have been established for the genetic manipulation of Rhodococcus. These include the development of efficient transformation techniques using electroporation (33); construction of expression vectors for protein production (24, 25); and development of shuttle vectors using cryptic, antibiotic-resistant, and temperature-sensitive plasmids (16, 18, 19, 22) derived from Rhodococcus strains as well as the generation of random mutagenesis using transposons.

Several transposon mutagenesis systems have been reported for Rhodococcus species (1, 8, 20, 21). These systems can generate a single copy of insertion into the host cell genome. To date, no efficient tool is available for the creation of random multiple gene disruptions in a single step. Previous researches on the creation of multiple integrations in a genome have been based on site-directed mutagenesis or gene disruption in sequential steps, which requires different antibiotic markers for mutant selection (9, 28, 31, 36, 39, 40).

We recently established the transposon-based vector system pTNR that can efficiently generate a random mutagenesis library by transposition in various Rhodococcus species (30). Inside the Rhodococcus cell, pTNR is unstable due to the lack of a replication origin for Rhodococcus. The expression of the transposase (IstA) and its helper protein (IstB) in pTNR is regulated under the influence of the constitutive promoter, Pnit (25). Once pTNR is electroporated into Rhodococcus cells, transposition occurs, whereby IstA and IstB are simultaneously expressed and initiate the integration of a single copy of the transposable-marker DNA into the host cell genome while the rest of the plasmid itself is lost. The transposable-marker DNA of pTNR locates between the two inverted repeats, IR1 and IR2, and encodes a replication origin for Escherichia coli and a kanamycin resistance gene, enabling easy identification of the insertion site via plasmid rescue from the genome (30).

pTNR was further modified to be used for protein expression through insertion of the protein expression cassette into the host cell genome (29). Currently, four variants of pTNR vectors are available, each with a different antibiotic resistance marker gene. Two or more variants of pTNR can be used for creating double or multiple integrations in sequential steps or even in one step if the variants are cotransposed in combinations. Nonetheless, the incidence of achieving double or multiple integrations from the cotransposed pTNR variants is very low; hence, the statistical chance of inactivating multiple genes within a definite metabolic pathway or bioprocess by such a method is extremely low. To knock out these functionally related genes, an effective method to produce huge numbers of mutations with random insertions at multiple loci is required. To that end, the present study aimed to develop an efficient genome engineering system for random integration of multiple DNA copies into the Rhodococcus genome in a single step.

MATERIALS AND METHODS

Bacterial strains, culture media, and growth conditions.

Wild-type R. erythropolis JCM3201 was obtained from Japan Collection of Microorganisms (JCM; RIKEN BioResource Center, Wako, Saitama, Japan). Rhodococcus and E. coli XL1-Blue and DH5α strains were routinely cultured in Luria-Bertani (LB) broth (1% Bacto tryptone, 0.5% Bacto yeast extract, and 1% NaCl), with Bacto agar (1.5%) added for plating. The specific antibiotics used in the culture medium to select transformants and/or mutant cells containing the transposable-marker DNA were ampicillin (100 μg/ml), kanamycin (20 μg/ml for E. coli and 200 μg/ml for Rhodococcus species), tetracycline (8 μg/ml), and chloramphenicol (34 μg/ml). Cultures and agar plates were incubated at 28°C for Rhodococcus species and at 37°C for E. coli. We used E. coli XL1-Blue and DH5α for vector maintenance and amplification. Competent Rhodococcus cells were prepared according to the procedure of Shao et al. (33).

DNA manipulations.

Plasmid DNA was isolated using a Wizard Plus SV Minipreps DNA purification system (Promega Corporation, Madison, WI). For the isolation of chromosomal DNA, ampicillin (600 μg/ml) was first added to the R. erythropolis culture 3 h before cells were collected to facilitate cell wall disruption. The collected cells were then washed with TE (Tris-EDTA) buffer, centrifuged to collect the washed cell pellet, resuspended with 500 μl of TE, and then incubated at 37°C for 1 to 2 h after the addition of lysozyme (10 mg/ml) and RNase A (50 μg/ml). The chromosomal DNA was then isolated with the use of a Maxwell 16 cell DNA purification kit (Promega Corp.) following the protocol of genomic DNA purification supplied by the manufacturer.

Oligonucleotides were obtained from Hokkaido System Science Co., Ltd. (Sapporo, Hokkaido, Japan). Restriction endonucleases, T4 DNA ligase, and alkaline phosphatase (calf intestinal phosphatase [CIP]) were purchased from New England BioLabs, Inc. (Ipswich, MA). A DNA ligation kit was obtained from Takara Bio, Inc. (Shiga, Japan). PCR was performed using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA). A Wizard SV Gel and PCR Clean-Up System (Promega Corp.) were used to purify the PCR products and isolate the DNA fragments from the gel before their subsequent cloning or sequencing.

DNA sequencing was performed with a BigDye Terminator, version 3.1, cycle sequencing kit (Applied BioSystems, Foster City, CA) according to the manufacturer's instructions, using an ABI Prism 3100 automated sequencer (Applied Biosystems). Nucleotide sequence data were then analyzed by the GENETYX-MAC software (GENETYX Corp., Tokyo, Japan).

Construction of pTip-istAB-sacB and pRTSK-sacB plasmids.

For the construction of pTip-istAB-sacB, istAB was excised from pTNR (30) after digestion with NdeI and BglII. The excised DNA was then cloned between the corresponding sites of the expression vector pTip-RT2, which possesses a thiostrepton-inducible promoter (25) to yield the istAB-expression vector pTip-istAB (10,549 bp). The sacB gene of Bacillus subtilis, which encodes levansucrase and confers sucrose sensitivity to the host cell, was amplified using plasmid pK*18mobsacB (32) along with the sense primer (5′-GGAAGATCTCGACCCACCGGCACCCGTGAGCCCCTCGCTGCGGGTGCCGGTGCGAGGAGTGAAATGAGATATTATGATATTTTC-3′; the BglII enzyme site is underlined and the TthcA site is in bold) and the antisense primer (5′-CTAGACTAGTAAAAGAAAATGCCAATAGGATATCGGCAT-3′; the SpeI site is underlined). The amplified fragment was restricted with BglII and SpeI and subcloned into the same restriction enzyme sites of pTip-istAB. The resultant vector was designated pTip-istAB-sacB (12,453 bp) (Fig. (Fig.1A1A).

FIG. 1.
Schematic map of the multiple integration two-vector system. (A) Transposase expression vector pTip-istAB-sacB. (B) The vector carrying the transposable-marker DNA, pRTSK-sacB. Thior (thiostrepton resistance), Tetr, Ampr, Chlr, PtipA, PthcA, tipAL, ColE1, ...

For the construction of pRTSK-sacB, the plasmid pTNR-KA (29) was digested with KpnI and BsrGI to excise the 2,918-bp DNA fragment that encodes the transposable-marker DNA [kanamycin resistance gene, multiple cloning site (MCS) region, and the replication origin of the cloning vector (pGEM-3zf (+) for E. coli]. The plasmid pNit-QC2 (25) was digested with KpnI and BsrGI to excise the 3,682-bp DNA fragment that encodes the genes essential for Rhodococcus replication, repA and repB, and the chloramphenicol resistance gene. Both of these excised fragments were ligated to yield the 6,600-bp vector pRTSK. The sacB gene was amplified using the plasmid pK*18mobsacB along with the relevant primers (sense, 5′-GGCGTACGAGTGAAATGAGATATTATGATATTTTC-3′; antisense, 5′-GGCGTACGAAAAGAAAATGCCAATAGGATATCGGCATTTTCTTTTGCGTTTTTATTTG-3′; the BsiWI site is underlined). The amplified sacB fragment was restricted with BsiWI and then cloned into the BsrGI-restricted site of pRTSK. The plasmid pRTSK encoding sacB was then modified to include a new MCS constructed using two sets of sense and antisense oligonucleotide primers constituting 10 unique restriction enzymes sites (sense, 5′-TTAAGGGGCCCAGATCTCATATGGCTAGCGCGGCCGCATGCATA ATATTCTCGAGA-3′; antisense primer, 5′-CTAGTCTCGAGAATATTATGCATGCGGCCGCGCTAGCCATATGAGATCTGGGCCCC-3′). The primers were individually phosphorylated by the T4 polynucleotide kinase and annealed by slow cooling down of the two primer set mixtures from 95°C to room temperature within 3 h. The annealed MCS fragment was introduced into AflII- and SpeI-restricted sites of pRTSK encoding sacB to yield the 8,466-bp vector, pRTSK-sacB (Fig. (Fig.1B1B).

Creation of random multiple insertions into the Rhodococcus genome.

Both pTip-istAB-sacB and pRTSK-sacB were cotransformed, in a single step, into the wild-type R. erythropolis by means of electroporation as previously described (30). Rhodococcal cells harboring the two vectors were selected onto LB agar plates containing both kanamycin and tetracycline, followed by incubation at 28°C. A single colony was chosen to be cultured at 28°C for 18 to 36 h in LB medium containing kanamycin and tetracycline until the culture reached an optical density at 600 nm (OD600) of 0.8 1.2 as measured by a U-1500 Spectrophotometer (Hitacchi, Ltd., Tokyo, Japan). The optimized concentration of thiostrepton (20 ng/ml) was then added, and the cells were cultured for four additional hours. Next, 100 to 200 μl of the serially diluted culture was spread onto LB agar plates containing sucrose (20%) and kanamycin (200 μg/ml) for the selection of mutant cells with various copies of transposable-marker DNA inserted in their genomes. The mutant colonies were subsequently subjected to colony PCR.

To optimize thiostrepton concentration and the incubation time required for initiating istAB gene expression by the thiostrepton-inducible promoter of the pTip-istAB vector, we conducted initial trials using several levels of thiostrepton (low levels of 5, 10, and 20 ng/ml and high levels of 1 and 10 μg/ml) along with various incubation times (2, 4, 6, 8, 16, and 24 h). We determined that thiostrepton addition at a concentration of 20 ng/ml followed by 4 to 6 h of incubation was optimal for multiple insertions.

Colony PCR, a rapid preliminary test for characterization of the mutant.

Colony PCR was carried out as a rapid preliminary test for characterization of the mutant cells with possible multiple insertion copies. For this purpose, two oligonucleotide primer sets (sense primer, 5′-CAGAGTCCCGCTCAGAAGAACTC-3′; antisense primer, 5′-GATCAAGAGACAGGATGAGGATCG-3′) were used. PCR was performed using Go Taq Green Master Mix (Promega) for 15 amplification cycles.

Southern blot hybridization.

To verify the number of copies integrated in the genome (single, double, or multiple), 48 colonies that showed a relatively intense band (by colony PCR) for the amplified kanamycin resistance gene were selected. Colonies were cultured for 24 to 36 h, followed by chromosomal DNA isolation. Five micrograms of the chromosomal DNA from each of these selected mutants was completely digested with the BamHI restriction enzyme, and the fragmented DNA was separated by electrophoresis on a 1% agarose gel. The DNA was then denatured by incubation of the gel in denaturation buffer (0.5 M NaOH, 1.5 M NaCl) for 30 min. The denatured DNA fragments were then transferred onto an Amersham Hybond-N+ nylon transfer membrane (GE Healthcare Ltd., Amersham Place, Buckinghamshire, United Kingdom) by blotting using the same denaturation buffer. Subsequently, the membranes were washed in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), prehybridized with the supplied blocking reagent at 65°C for 2 h, and hybridized for 6 h at 65°C with the denatured digoxigenin (DIG)-labeled kanamycin resistance gene probe synthesized by the same oligonucleotide primers of colony PCR against pRTSK by using a PCR DIG Probe Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany). After the membranes were washed sequentially in 2× SSC (standard saline citrate) plus 0.1% (wt/vol) SDS (sodium dodecyl sulfate) and 0.1× SSC plus 0.1% SDS, the specifically bound DNA probe on the membranes was identified using a DIG nucleic acid detection kit (Roche Diagnostics GmbH) according to the manufacturer's instructions.

Stability of the transposable DNA in the Rhodococcus genome.

Three cells that showed multiple copies of insertions were randomly selected (mutants X, Y, and Z). The growth rates of such mutants were similar to the growth rate of the wild-type Rhodococcus strain. The selected mutants were individually inoculated into 10 ml of LB broth in a 50-ml screw-cap, conical-bottom centrifuge tube (Corning Inc., Corning, NY) at 28°C without antibiotics and shaken at 140 rpm in a BR-40LF shaker (Taitec Corp., Koshigaya, Japan) until the cell density reached 1.8 to 2.4 (OD600). The cultured cells were then diluted 1/500 into fresh LB broth without antibiotics and grown at 28°C with shaking. Every 24 h, each culture was reinoculated at a dilution of 1/500 into fresh LB broth to maintain exponential growth. The cells were harvested after serial generations, and their chromosomal DNA was isolated and digested with BamHI. It was then subjected to Southern blot hybridization to verify the existence and the stability of the integrated copies of the transposable-marker DNA in the genome.

To determine the generation time, 10 ml of cell culture (OD600 of 0.2) in a 50-ml screw-cap, conical-bottom centrifuge tube was further cultured for 10 h at 28°C without antibiotics and shaken at 140 rpm. An aliquot of the culture was spread every hour onto LB plates without antibiotics, and the colonies on the plate were counted; the generation time of all mutants was approximately 2 h.

Preparation of the PIP expression cassette.

Plasmid pHN409 (25) served as a source of the proline iminopeptidase (PIP) from Thermoplasma acidophilum. PCR amplification for the PIP expression cassette was carried out using pHN409 as a template and two oligonucleotide primers, sense (5′-GGAAGATCTTACATATCGAGGCGGGCTCCCAC-3′; BglII site is underlined) and antisense (5′-CCGCTCGAGGTGTCCGTGGCGCTCATTCCAACCTC-3′; XhoI site is underlined). The amplified PIP expression cassette, which encodes the Pnit constitutive promoter (25), the pip gene, six-His-tagged sequence (at the C terminus), and the thcA transcriptional terminator (TthcA), was digested with BglII and XhoI and cloned into the corresponding sites in the MCS region of the pRTSK-sacB vector.

Preparation of the vitamin D3 hydroxylase (Vdh) expression cassette.

For preparation of the vitamin D3 hydroxylase (Vdh) expression cassette, the vdh gene of Pseudonocardia autotrophica was excised from pTipQT2-vdh-thcCD (10) after digestion with the restriction enzymes NdeI and SpeI and cloned into the corresponding restriction sites of pNit-QT2, yielding pNitQT2-vdh. Subsequently, PCR amplification for Vdh was carried out using pNitQT2-vdh as a template and the same two oligonucleotide primers used for amplification of the PIP expression cassette. The amplified Vdh expression cassette, which encodes the Pnit constitutive promoter, the vdh gene, and the TthcA transcriptional terminator, was digested with BglII and XhoI and cloned into the corresponding sites in the MCS region of the pRTSK-sacB vector.

Integration of multiple copies of PIP and Vdh expression cassettes into the R. erythropolis genome.

Both pRTSK-sacB harboring the PIP/Vdh expression cassette and pTip-istAB-sacB were cotransformed, in combinations, by electroporation into wild-type R. erythropolis. The electroporated cells were cultured onto LB plates containing kanamycin and tetracycline at 28°C. A single colony was selected and cultured in the presence of the same combination of antibiotics until an OD of 0.8 to 1.0 was obtained. Thiostrepton (20 ng/ml) was added, and the culture was incubated for an additional 4 to 6 h. The cell culture was then diluted to ~100-fold, from which 50- to 100-μl samples were spread onto LB agar plates containing kanamycin and sucrose (20%). The recovered cells were applied for colony PCR as a preliminary test for multiple copies of the expression cassette in the chromosomal DNA, followed by verification with Southern blot hybridization. Subsequently, colonies harboring single, double, or multiple PIP/Vdh expression cassettes were randomly selected for culturing into LB broth containing the proper concentration of kanamycin to be used for protein expression and biological activity assays.

Expression profile and measurement of biological activity of recombinant PIP.

To determine the expression profile of the PIP, R. erythropolis cells harboring single, double, or multiple integration copies of PIP expression cassettes were cultured in 10 ml of LB broth and harvested by centrifugation at 11,800 × g for 10 min at 4°C. Cell pellets were washed twice with buffer (50 mM Na-phosphate, 300 mM NaCl, 10% glycerol, pH 8.0), resuspended with 700 μl of sonication buffer (50 mM Na-phosphate, 300 mM NaCl, pH 8.0), and disrupted for 20 min, using a Multi Beads Shocker equipped with a cooling circulator set at 4°C (Yasui Kikai Corporation, Osaka, Japan) after the addition of lysozyme (Sigma-Aldrich Co., St. Louis, MO) at a final concentration of 2 mg/ml. The resultant lysates were centrifuged at 20,000 × g for 20 min at 4°C. The protein concentration from the different clones was determined by a Bio-Rad Bradford protein assay (4). Twenty micrograms of the protein from each clone was loaded onto a 12.5% SDS-PAGE gel, followed by staining with Coomassie brilliant blue G-250.

The peptidase activity of the recombinant PIP was determined according to the assay described by Tamura et al. (34, 35). One microgram of the crude protein was added to the assay buffer (50 mM Tris-HCl, pH 8.0) in the presence of 10 nmol of the fluorogenic substrate H-Pro-AMC (H-proline 7-amino-4-methylcoumarine; Bachem, Bubendorf, Switzerland) in a final volume of 100 μl. The tubes were incubated at 60°C for 15 min, and the reaction was terminated by the addition of 100 μl of 10% SDS and 1 ml of 0.1 M Tris-HCl (pH 9.0). The fluorescence activity of the released AMC was measured in a fluorescence spectrophotometer (F-2500; Hitachi, Ltd., Tokyo, Japan).

Expression profile and measurement of biological activity of recombinant Vdh.

The cell culture (20 ml) from different R. erythropolis clones carrying single, double, or multiple integration copies of the Vdh expression cassette, verified by Southern blotting, were harvested by centrifugation. The cell pellets were washed twice with potassium phosphate buffer ([KPB] 50 mM; pH 7.4) containing 2% glucose and resuspended in a suitable volume of the same buffer, and the concentration was adjusted to an OD600 of 5.0 in each case. This suspension was used for analyzing the biological activity. To analyze the expression profile of the Vdh protein, cell pellets from the former suspension were resuspended with 500 μl of sonication buffer and disrupted under the same conditions as those employed for the disruption of the PIP-expressing cells. The resultant lysates were centrifuged at 20,000 × g for 20 min at 4°C, and the protein concentration was determined. Fifteen micrograms from each clone was loaded onto a 12.5% SDS-PAGE gel, followed by staining with Coomassie brilliant blue G-250.

The enzymatic activity of Vdh was determined based on the bioconversion rate of the inactive vitamin D3 into the hydroxylated active form of vitamin D3 as previously described (10). Briefly, the cell pellet, twice washed with KPB, was resuspended in 1 ml of KPB containing 2% glucose, 0.5% methyl-β-cyclodextrin (MβCD; Junsei Chemical, Co., Ltd., Tokyo, Japan), 1 mM thiamine, and 0.5 mM vitamin D3 (Sigma Chemical Co., St. Louis, MO) as a substrate. The bioconversion was performed by incubating the reaction mixture at 28°C for 16 h. The mixture was then centrifuged at 20,000 × g for 20 min at 4°C, and 100 μl from the supernatant was mixed with 900 μl of methanol and recentrifuged for 10 min at the same speed and temperature. Forty microliters of the methanol solution was injected into a high-performance liquid chromatograph (HPLC) equipped with a J'sphere ODS-H80 column (75 mm by 4.6-mm internal diameter; YMC, Kyoto, Japan), and the 25-hydroxylated form of vitamin D3 was detected by UV at 265 nm.

Nucleotide sequence accession numbers.

The nucleotide sequences of pRTSK-sacB and pTip-istAB-sacB have been submitted to the DDBJ/EMBL/GenBank database under accession numbers AB545979 and AB545980, respectively.

RESULTS AND DISCUSSION

Rhodococcus species can be functionally used as an expression platform for protein expression (2, 24, 25, 26, 27), for bioconversion of cheap starting material into more important compounds (10, 23), or for metabolizing various harmful environmental pollutants (6, 14, 17). For these applications, it might be necessary to control the amount of recombinant proteins expressed in the host cells. The system developed here is a promising approach for the coexpression of heterologous proteins and the expression of different subunits of protein complexes, as well as for modulating the expression level of recombinant proteins, which is very useful in the case of toxic proteins when minuscule differences in expression level can cause cell death. This system is also valuable when a large-scale culture does not require antibiotics for retention of the plasmids in the host cells.

Strategy for the creation of multiple integrations.

Our previous transposon-based vector, pTNR, is not stable inside the host cell, and its transposition is initiated immediately upon electroporation of the vector into the Rhodococcus genome, resulting in the integration of a single copy of the insertion into a single position in the genome. In order to generate multiple insertion copies into the host cell genome, we have to stably maintain multiple copies of the vector carrying the transposable-marker DNA inside the host cell before the initiation of the transposition under the influence of a thiostrepton-inducible promoter. Since the transposase (IstA) and its helper protein (IstB) are essential for the transposition of the transposable-marker DNA, we constructed two vector systems, each containing a replication origin for Rhodococcus species (Fig. (Fig.1).1). The first vector (pRTSK-sacB) encodes the transposable-marker DNA that lies between the two inverted repeats (IR1 and IR2), and the second one (pTip-istAB-sacB) is required for the expression of the transposase and its helper protein under the influence of the thiostrepton-inducible promoter PtipA. This inducible promoter controls the expression of IstA and IstB; hence, we can initiate their expression by the addition of thiostrepton at any time desired during cell culturing. The sacB gene was incorporated into the plasmid sequence in order to eliminate the host cells that retained intact vectors upon selection onto sucrose plates. The sacB gene has been reported as a positive-selection marker for the isolation of an insertion element from Rhodococcus fascians (13) and as a counter-selectable marker for gene deletion mutagenesis in R. erythropolis SQ1 (38).

In the case of pTNR, electroporation of the vector into Rhodococcus can simultaneously create a mutagenesis, with a single copy of the insertion, only in the transposed cells, which is related to the transposition efficiency (for instance, transposed cells are ~2% of the transformants obtained by the replicating vector, pRTSK). In the present system, however, the transformant cells harboring multiple copies of the two vectors (pTip-istAB-sacB and pRTSK-sacB) constitute the starting point for the creation of random multiple integrations. Accordingly, all the cultured cells (100%) carrying the two vectors, regardless of the volume of the culture, can be theoretically mutated in a single step with various copy numbers of the transposable-marker DNA upon induction of the transposase and expression of its helper protein. The yield of the random diverse mutants by this method is much higher than any other mutant-generating system.

Creation of random multiple insertions into the Rhodococcus genome.

A simplified scheme for creation of multiple integrations into the Rhodococcus genome is shown in Fig. Fig.2.2. A single transformant colony of R. erythropolis harboring both pTip-istAB-sacB and pRTSK-sacB is selected to be cultured in LB broth for 36 to 48 h in the presence of kanamycin and tetracycline. In order to induce the expression of IstA and IstB, thiostrepton (20 ng/ml) is added, followed by reincubation of the cultured cells for a further 4 h. As soon as the IstA and IstB proteins are expressed, the transposable-marker DNA will be liberated from the pRTSK-sacB vector and integrated randomly into the host cell genome. Continuous production of IstA and IstB can subsequently lead to further random integration of additional copies of the transposable-marker DNA into the genome, resulting in the creation of random multiple insertions into rhodococcal cells. The two vectors are then eliminated from the mutant cells upon selection onto sucrose-containing plates. It should be noted that the elimination rate of the vectors from R. erythropolis is not high due to the stability of the vectors; the numbers of colonies that grow on sucrose-containing plates constitute less than 10% of the colonies that grow on the plates without sucrose (data not shown). Therefore, positive clones may first have to be isolated without sucrose selection, and the vectors can then be eliminated after amplifying the cell number.

FIG. 2.
A simplified scheme for the creation of multiple integrations into the Rhodococcus genome, using pRTSK-sacB and pTip-istAB-sacB.

It is very likely that mutated cells demonstrate different growth rates depending on the knocked out genes and that a portion of the cells will be killed if one or multiple copies of the transposable-marker DNA knocks out one or more of the genes required for cell survival.

Verification of multiple-copy integration in the rhodococcal genome.

The application of colony PCR was first used as a rapid preliminary test for characterization of the mutant cells with possible multiple insertion copies. Considering this, colonies that showed highly intense or relatively thicker bands for the amplified kanamycin gene were expected to harbor multiple insertions of the transposable-marker genes into their genomes. Southern blot hybridization was then applied to identify the number of copies integrated in the Rhodococcus genome. A total of 112 colonies, out of the 160 mutants tested, showed single integration, while 48 mutants (with a relatively high incidence of 30%) showed double and multiple integrations. Among these 48 mutants, 20 showed double, 15 showed triple, 9 showed quadruple, and 4 showed quintuple integrations of the transposable-marker DNA into their genomes.

The results demonstrated in Fig. Fig.33 are representative data for Southern blotting and amplification of marker genes by colony PCR. Southern blotting verified single, double, and multiple (three to five) integrations of the transposable-marker DNA in the Rhodococcus genome, and it was clear, in most cases, that the intensity of the marker gene amplified by colony PCR is correlated with the number of copies of the integrated DNA.

FIG. 3.
Verification of the number of copies integrated into the host cell genome. (A) A representative Southern blot analysis for 16 kanamycin-resistant mutants showing single (lanes 2, 4, 5, 8, 10, and 15), double (lanes 1, 11, and 14), and multiple (lanes ...

Stability of the integrated DNA in the host cell genome.

Southern blot hybridization was conducted for the genomes of three randomly selected mutants among the colonies that grew well on the plate. The cells with multiple copies of the integrated DNA were cultured for several generations without any selection pressure. The result indicated that the integrated copies of the transposed DNA were stable in the same position in the genome of the host cell throughout 80 generations (Fig. (Fig.4)4) of serial growth without the addition of any selective antibiotics.

FIG. 4.
Confirmation of the stability of the integrated copies of the foreign DNA in the host cell genome. Southern hybridization analysis was conducted for three randomly selected mutants with multiple insertions to verify the stability of the integrated DNA ...

Expression profile and biological activity of PIP and Vdh proteins based on the copy number of the integrated expression cassettes.

To confirm the recombinant protein expression in variable amounts based on the copy numbers of the expression cassettes integrated into the genome, we employed pip from T. acidophilum and vdh from P. autotrophica as reporter genes. We individually cloned PIP and Vdh expression cassettes into the MCS within the transposable-marker DNA encoded in the pRTS-K-sacB vector. After cotransformation of the vector harboring the cassette along with the istAB expression vector pTip-istAB-sacB into R. erythropolis, the cells were cultured for 4 h in the presence of thiostrepton. Chromosomal DNA of the resultant cells was tested by Southern blot hybridization to identify the copy number of the transposed element encoding the expression cassettes. The results indicated that 10 of the 48 clones tested (21%) for PIP cassettes were positive for double or multiple insertions, with the double insertions being predominant. In the case of the Vdh cassette, the results revealed that 14 of the 45 clones tested (31%) were positive for double and multiple insertion copies, with 4 clones showing multiple insertions and 10 clones showing double insertions. This indicated that the incidence of obtaining multiple copies of DNA insertions for certain protein expression cassettes into the host cell genome may vary from one protein to another, based on several factors such as the nature, structure, and length of the inserted gene.

Expression profile and biological activity of recombinant PIP.

Five clones showing single, double, or multiple integrations of the PIP expression cassette (Fig. (Fig.5B)5B) were randomly selected and cultured to examine protein expression level and to analyze peptidase activity.

FIG. 5.
Expression profile and biological activity of recombinant PIP. (A) The transposable-marker DNA integrated into the host cell genome. It comprises the two inverted repeats (IR1 and IR2), the origin of replication of E. coli (ori), the kanamycin resistance ...

The results revealed that the expression levels and the peptidase activities of PIP were considerably increased in clones containing double or multiple integrated copies in comparison with the clones with a single integration (Fig. 5C and D), indicating that such expression and activity levels are directly correlated with the copy numbers of the inserted cassettes. For instance, clone 5, which showed three integrations (in Fig. Fig.5C,5C, the lower band, which is more intense, is probably a combination of two similarly sized bands) exhibited peptidase activity about 3-fold higher than that of the single integration clone 4 (6.4 versus 2.2), while clones 1 and 2, which clearly showed double integrations, exhibited peptidase activity approximately 2-fold higher than that of the single integration clones 3 and 4 (Fig. (Fig.5D5D).

It is possible that the indigenous promoters within the host cell genome possibly located upstream of the integrated cassette can influence the expression level of the target proteins. Nevertheless, the integrated cassettes in this system are bounded by the two inverted repeats of the transposable-marker DNA, which could act as a terminator and prevent any influence of the indigenous promoters.

Expression profile and biological activity of recombinant Vdh.

Six clones showing single, double, or multiple integrations of the Vdh expression cassette (Fig. (Fig.6B)6B) were randomly selected and cultured to examine the protein expression level and to analyze the hydroxylase activity. Clones containing double and multiple integrated copies of the Vdh cassette exhibited higher protein expression levels than the clones with only a single integration cassette, and such expression was almost correlated with the copy numbers (Fig. (Fig.6C).6C). The biological activity of the Vdh protein was assayed by measuring the bioconversion rate of the biologically inactive vitamin D3 into the physiologically active form, 25-dihydroxyvitamin D3. The result indicated that this rate was increased from 7.1% in cells carrying a single integrated cassette (clone 3) to a level of 14.4 to 18.1% in clones harboring double and multiple integrated cassettes (Fig. (Fig.6D6D).

FIG. 6.
Expression profile and biological activity of recombinant vitamin D3 hydroxylase (Vdh). (A) The transposable-marker DNA comprises the two inverted repeats (IR1 and IR2), the origin of replication of E. coli (ori), the kanamycin resistance gene (Kanr), ...

Generally, the bioconversion rate of vitamin D3 into the 25-hydroxylated form was higher in cells containing double or multiple copies of the integrated Vdh cassettes than in the cells harboring only a single integration. Nonetheless, the bioconversion rate reached its maximal value at a certain level (17.4 to 18.14%) even in the presence of higher concentrations of the Vdh protein. This finding is in accordance with the results from our laboratory (unpublished data), which indicated that overexpression of Vdh does not accompany a higher conversion rate of vitamin D3 into the hydroxylated active form. Indeed, many factors can be implicated in the bioconversion rate of a particular substrate, such as the nature of the substrate itself (water soluble or fat soluble), substrate accessibility inside the host cell, presence or absence of other copartners, and the integration site of the expression cassette in the host cell genome.

Possible applications and advantages of the present integration system.

Our random multiple integration system is a powerful genetic tool since it has many advantages in Rhodococcus genome engineering. It can be used for the expression of proteins at different levels based on the number of stably maintained integrated cassettes without the addition of any selective markers. This is an advantage over other regular expression vectors, which are unstable in the host cells and require continuous addition of selective antibiotics to be maintained inside the cells. In addition, it is quite possible to conduct a sequential integration procedure to integrate different protein expression cassettes, each having different copy numbers, into the host cell genome. In this case, the first integration vector, pRTSK-sacB, will integrate multiple copies of an expression cassette of a certain gene along with the kanamycin resistance marker gene. The resultant cells will be further prepared to receive a second additional integration cassette of another gene in variable copy numbers with the use of pRTSA-sacB, another new vector variant in which the antibiotic resistance gene in the transposable DNA is replaced with an apramycin resistance marker. This technique can be useful for studying some biologically valuable products in which many genes, with similar or dissimilar expression levels, are required to complete the biosynthesis of certain products.

This system can also be employed for the functional characterization of the genus Rhodococcus through the development of large pools of mutants with multiple genes knocked out in a single step. Because unlimited numbers of such mutants may be encountered simultaneously, it is likely that multiple integrations can provide particular phenotypes, which enables us to study functionally related genes involved in certain biological processes and metabolic pathways in which multiple genes are believed to be implicated. Further studies using such a system are required to clarify these possible applications.

Conclusion.

The present study provides, for the first time, a random multiple integration system for the creation of a random mutant library with multiple genes knocked out in a single step. This system can be applied for the insertion of various copy numbers of stably maintained protein expression cassettes into the genome to modulate the expression levels of the target protein.

Acknowledgments

We are grateful to all members of the Proteolysis and Protein Turnover Research Group in the Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Sciences and Technology, for their technical assistance and valuable discussions.

Footnotes

[down-pointing small open triangle]Published ahead of print on 12 February 2010.

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