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Shunpeng Li, Department of Microbiology, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, Nanjing, 210095, China. E-mail: email@example.com
Aims: To investigate the key parameters controlling the exogenous methyl parathion hydrolase (MPH) gene mpd-targeting frequency at the ribosomal RNA operon (rrn) site of Sphingomonas species which has a wide range of biotechnological applications.
Methods and Results: Targeting vectors with different homology lengths and recipient target DNA with different homology identities were used to investigate the parameters controlling the targeting frequency at the Sphingomonas species rrn site. Targeting frequency decreased with the reduction of homology length, and the minimal size for normal homologous recombination was >100 bp. Homologous recombination could succeed even if there were 3–4% mismatches; however, targeting frequency decreased with increasing sequence divergence. The Red recombination system could increase the targeting frequency to some extent. Targeting of the mpd gene to the rrn site did not affect cell viability and resulted in an increase of MPH-specific activity in recombinants.
Conclusions: Targeting frequency was affected by homology length, identity and the Red recombination system. The rrn site is a good target site for the expression of exogenous genes.
Significance and Impact of the Study: This work is useful as a foundation for a better understanding of recombination events involving homologous sequences and for the improved manipulation of Sphingomonas genes in biotechnological applications.
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The genus Sphingomonas is a group of widely distributed, Gram-negative, rod-shaped, chemoheterotrophic, strictly aerobic bacteria that typically produce yellow-pigmented colonies (Fredrickson et al. 1995; Balkwill et al. 1997). They possess ubiquinone-10 as the major respiratory quinone, and contain glycosphingolipids instead of lipopolysaccharide in their cell envelopes. It is well known that Sphingomonas species have versatile metabolic abilities, produce extracellular biopolymers (Fredrickson et al. 1995; Hashimoto and Murata 1998; Marja et al. 2002), and have been utilized for a wide range of biotechnological applications, from bioremediation of environmental contaminants to production of extracellular polymers, such as sphingans which are extensively used in the food industry among others (Sutherland 1999). Because of their intrinsic potential in biotechnological applications, there has been great interest in the genetic engineering of Sphingomonas species to obtain strains with improved properties. However, the majority of the genetic studies on Sphingomonas species have focussed on the construction of mutants generated with Tn transposons (Pollock et al. 1998; Story et al. 2001).
Ribosomal RNA (rRNA) genes of bacteria have a high degree of sequence conservation, and exist mostly in multiple copies, such that inactivation of some of the copies is not lethal (Condon et al. 1993; Asai et al. 1999). Manipulations of these genes in Sphingomonas species have been made possible by the efficient methods of gene targeting. In a previous study, the 16S rRNA gene of a carbofuran-degrading Sphingomonas species was successfully used as the homologous recombination target site for integration of a methyl parathion hydrolase (MPH) gene mpd to construct a multifunctional pesticide-degrading micro-organism (Jiang et al. 2005). It was known that the frequency of homologous recombination between an introduced vector and chromosomal DNA sequences was influenced by many factors. So far, several studies have investigated the parameters for efficient homologous recombination in Escherichia coli (Shen and Huang 1986), yeast (Jinks-Robertson et al. 1993; Predrag et al. 2000), the protozoan parasite Leishmania (Barbara and Carole 1997) and others. However, the parameters that may influence the frequency of gene targeting and the minimal efficient processing segment (MEPS) required for homologous recombination in Sphingomonas species have not been extensively studied. Given the importance of gene-targeting technology in the genetic manipulation of Sphingomonas species, we used the novel MPH gene mpd (responsible for hydrolyzing methyl parathion to yellow p-nitrophenol which can been visualized on an agar plate) as the reporter gene to study critical parameters (homology length and homology identity) controlling efficient homologous recombination at the Sphingomonas species rrn site. To further investigate whether an rRNA gene would have advantages when used as a homologous recombination target site for expression of an exogenous gene, cell viability of recombinants and expression of MPH in recombinants were also studied.
Materials and methods
Bacterial strains, plasmids and culture conditions
The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli and Pseudomonas putida were cultured in Luria–Bertani (LB) medium at 37°C and 30°C, respectively. Sphingomonas species were cultured in 1/3 strength LB medium (3·3 g of tryptone, 1·7 g yeast extract, 3·3 g NaCl per litre and pH 7·0) at 30°C. An amount of 20·0 g of agar per litre was added for the preparation of solid media. Antibiotics were added when required at the following concentrations: streptomycin (Str), 100 μg ml−1; ampicillin (Amp), 50 μg ml−1 and kanamycin (Km), 25 μg ml−1.
Ampr, 1·3, 0·85, 0·28 and 0·1 kb 16S rRNA gene fragment flanking mpd, cloned at the BamHI/XhoI sites of pWM91, respectively
pWSMK-1, -2, -3, -U series
Ampr, Kmr, 3·8-kb Km-sacB cassette inserted into the BamHI site of pWSM-1, -2, -3, and -U, respectively
Ampr, Kmr, 3·1-kb Red (γ-β-exo) cassette inserted into the NotI and SpeI site of pWSMK-U
Chemicals, enzymes and DNA manipulations
Methyl parathion (50% and 98% purity) was purchased from Zhenjiang pesticide factory (Zhenjiang, China). Restriction enzymes, T4 DNA ligase and calf intestinal phosphatase were purchased from TaKaRa Biotechnology Co. Ltd (Dalian, China). Specific primers used for PCR amplification were synthesized by Bioasia Co. Ltd (Shanghai, China). All DNA manipulations were performed according to standard procedures (Sambrook et al. 1989).
Construction of homologous recombination vectors
A 16S rRNA gene fragment from Sphingomonas sp. CDS-1 of approx. 1·5 kb was obtained by PCR amplification using the F27 (5′-agagtttgatcctggctcag-3′; E. coli bases 8–27)/R1492 (5′-taccttgttacgactt-3′; E. coli bases 1507–1492) primer pair, and bi-directionally sequenced by TaKaRa Biotechnology Co. Ltd. The 1·3-kb functional mpd gene fragment was amplified with the primer pair F1 (5′-atgctagctccgtccaatctcc-3′)/R1 (5′-cagctagctatcacttggggttg-3′) with pDT3 as the template (Cui et al. 2001; Liu et al. 2003). An NheI site (underlined) as well as an additional stop codon (TGA) was introduced with primers. The amplified mpd gene products were digested with NheI, inserted into the unique NheI cloning site of the 16S rRNA gene, and transformed into competent E. coli cells. Transformants were selected if they formed yellow transparent halos of p-nitrophenol around the colonies on agar plates containing 100 μg ml−1 methyl parathion. The16S rRNA gene together with the mpd gene were digested with XhoI and BamHI, and inserted into the XhoI and BamHI sites of pWM91 (Fig. 1a) to generate pWSM-1. Recombinant plasmids with conditional replication oriRR6Kγ were transformed into E. coli DH5αλpir expressing the R6Kπ replicase for propagation. A 3·8-kb BamHI-restricted fragment containing sacB-Km cassette from pIB279 (Blomfield et al. 1991) was inserted into the BamHI site of pWSM-1 to produce pWSMK-1.
To investigate the effect of homology length on recombination frequency, 16S rRNA gene fragments of different lengths together with the mpd gene were amplified by PCR using the H1 (5′-tgctcgagaagtccaaagatt-3′)/H2 (5′-ttggatccacgagctgacgac-3′), H3 (5′-gtctcgagaattggacaatggg-3′)/H4 (5′-taggatccccacccacctctcc-3′), or UR-5 (5′-aatctcgagtttgatcctggctcagaacgaacgctggcggcatgcctaacacatgcgatcggctagctccgtccaatctccg-3′)/UR-3 (5′-taaggatccatggtgtgacgggcggtgtgtacaaggcctgggaacgtattcaccgctagctatcacttggggttgacgaccg-3′) primer pairs (XhoI and BamHI underlined), and inserted into the XhoI and BamHI sites of pWM91 to generate pWSM-2, -3 and -U, respectively. Vectors pWSMK-2, -3 and -U with different homology lengths were constructed as described for pWSMK-1 (Fig. 1b). These vectors were transformed into E. coli SM10λpir (expressing RP4-conjugative functions) for efficient two-parental conjugal transfers.
Two-parental mating and selection of recombinants
Donor strains E. coli SM10λpir harbouring mobilizable homologous recombination vectors were mixed with the Strr recipient Sphingomonas species cells at a ratio of 1 : 2 and then filtered through a 0·45 μm cellulose nitrate membrane filter. The filter was placed on the surface of a 1/3 LB agar plate and further incubated for 24 h. The cells on the filter surface were then suspended in 3 ml of sterile H2O, and 0·1–0·5 ml of the suspension was spread onto 1/3 LB agar plates containing Str, Km and Amp antibiotics that counter-select the donor strain and select exconjugants. We confirmed the ability of exconjugants to form yellow transparent halos on methyl parathion plates and selected these as single-crossover homologous recombinants. Single-crossover recombination frequency was calculated as the number of exconjugants per number of recipients (Furlaneto et al. 2000). To obtain double-crossover recombinants, the single-crossover recombinants were cultured overnight in 1/3 LB medium without antibiotics and subsequently spread onto 1/3 LB agar plates containing 5–10% sucrose. The sucrose resistant (SucR) transformants that were able to hydrolyse methyl parathion were selected as positive double-crossover homologous recombinants (Blomfield et al. 1991).
Random insertion of mpd gene into the chromosome of Sphingomonas sp. CDS-1
The mpd gene fragment was amplified with the primer pair pNot1 (5′-tagcggccgctccgtccaatctcc-3′)/pNot2 (5′-tagcggccgctatcacttggggttg-3′) (NotI instead NheI site was introduced). The amplified mpd gene products were digested with NotI and inserted into the unique NotI cloning site of pUT-Km (Victor et al. 1990). The constructed mini-Tn5-derived transposon carrying a Km resistance marker and the mpd gene permit the random insertion of the mpd gene into the chromosome of Gram-negative bacteria. Transposon insertion with mini-Tn5-derived transposon was performed by triparental mating. Sphingomonas sp. CDS-1, E. coliλpir harbouring the constructed transposon, and E. coli HB101 with the plasmid RK600 were used as recipient, donor and helper strain, respectively. Exconjugants resistant to Str and Km antibiotics were selected as the randomly inserted recombinants.
Activity assay of recombinant methyl parathion hydrolase
The gene targeting at the Sphingomonas species rrn site was carried out as previously described (Jiang et al. 2005), and confirmed by PCR with the P1 (5′-tttccttgtccttgtggg-3′)/P2 (5′-cctgcctccttgcggtta-3′) primer pair (the location of P1/P2 is indicated in Fig. 1b). In order to determine whether the targeting frequency at the Sphingomonas sp. CDS-1 rRNA gene site could be influenced by the length of homology, multiple vectors with varying lengths of homology were constructed: pWSMK-1, -2, -3 and -U, which contain 1271 bp (left and right flank is 493 and 778 bp), 855 bp (317 and 538 bp), 284 bp (154 and 130 bp) and 99 bp (50 and 49 bp) (Fig. 1b). The relative targeting frequencies with respect to different homology lengths were investigated and are shown in Table 2. The highest targeting frequency was obtained when homologous sequences were 493 and 778 bp on each flank. A decrease in the length of homology on both flanks greatly reduced the targeting frequency. With homologous sequences of 154 and 130 bp on each flank, the targeting frequency was diminished by 76·3-fold when compared with homologous sequences of 317 and 538 bp. Vector pWSMK-U, whose homologous sequences were the universally conserved 99 bp regions of the 16S rRNA gene (based on an alignment of 500 bacterial 16S rRNA gene sequences, Van de Peer et al. 1996), did not yield any exconjugants, so we deduced that the MEPS required for normal homologous recombination at the 16S rRNA gene site of Sphingomonas species might be longer than 100 bp.
Table 2. Effect of homologous sequence length on targeting frequency at Sphingomonas sp. CDS-1 rrn site
Homology length (bp)
Single-crossover homologous recombination frequency (exconjugants/recipients)*
*The data are represented as the mean ± standard deviation for triplicate experiments.
†No homologous recombination event was detected.
5·0 × 10−7 ± 2·10 × 10−7
5·8 × 10−8 ± 2·96 × 10−8
7·6 × 10−10 ± 2·52 × 10−10
However, the targeting frequency of the vector pWSMK-U was increased by using the bacteriophage λ Red recombination system. Approximately 3·1-kb λ Red (γ-β-exo) recombinase gene together with its arabinose inducible promoter from pKD46 (Kirill and Wanner 2000) were inserted into the NotI and SpeI sites of pWSMK-U to generate pWSMKR-U. pWSMKR-U was transformed into recipient Sphingomonas sp. CDS-1 in the presence of 1 mmol l−1l-arabinose by two-parental conjugation. Utilization of the Red system increased the targeting frequency of pWSMKR-U to 5·1 × 10−10 ± 0·59 × 10−10 when compared with that of the pWSMK-U vector (no exconjugants were found). The results indicated that direct homologous recombination by the Red system with short homologous sequences (<100 bp) was successful at the Sphingomonas species rrn site.
Effect of sequence identity on recombination
The 16S rRNA gene has a high degree of sequence conservation at the genus level. This conserved gene may be able to serve as a universal target site as homologous recombination is not restricted to sequences of perfect homology. To determine the degree of homology necessary for efficient gene targeting at the Sphingomonas species rrn site, we used the 1271 bp 16S rRNA gene of CDS-1 as donor DNA and the 16S rRNA genes of other Sphingomonas species as the recipient target DNAs. The donor DNA shared 91–97% identity with recipient target DNA sequences (Table 3). We found that recombination events could occur when the identity between donor and recipient DNA was over 96%. However, there was a significant decrease in the targeting frequency with increasing sequence divergence. In instances where the genomic target DNA contained 3–4% mismatches with the donor DNA, integration frequency was reduced at least 3·3-fold to 35·7-fold relative to recombination between identical sequences (Tables 2 and 3).
Table 3. Effect of homologous sequence identity on targeting frequency
Target Sequence identity with donor sequence (%)*
Single-crossover homologous recombination frequency (exconjugants/recipients)†
*The identity of target 16S rRNA gene sequences with donor sequence was calculated using BioEdit ClustalW (Tom Hall Isis Pharmaceuticals) at the level of the nucleotide sequence.
†The data are represented as the mean ± standard deviation for triplicate experiments.
‡No homologous recombination event was detected.
1·5 × 10−7 ± 0·82
1·4 × 10−8 ± 0·49
Our attempts to target the 16S rRNA genes of DB-1, DSP-2 and Y57 strains, whose target sequences share 91–93% identity with donor DNA, were unsuccessful. The data show that a relatively high degree of homology between donor and target sequences is necessary for efficient gene targeting.
Cell viability of recombinants
The growth curves of recombinants in MSM medium supplemented with appropriate pesticides were similar to that of their wild-type counterparts (Fig. 2). It was reported that rrn disruption in Bacillus and E. coli did not cause any significant decrease in either growth rate or rRNA synthesis (Asai et al. 1999). As there is a feedback control of rRNA expression, when rrn gene dosage is decreased, there is a compensatory increase in the expression of the remaining intact copies (Condon et al. 1993). Our results also show that inactivation of one of the ribosomal operons in Sphingomonas species did not affect cell viability.
Expression of methyl parathion hydrolase in recombinants
Although rRNA operons in a chromosome will recombine with each other at an extremely low level, our double-crossover recombinants did not lose the mpd gene after >150 generations in culture without selective pressure, indicating that these recombinants were stable. The specific activities of MPH of Sphingomonas double-crossover recombinants were higher than that of the Ps. putida strain DLL-1 from which the mpd gene was cloned (Table 4). The average specific MPH activities of three randomly selected recombinants constructed by mini-transposon system were lower than that of the homologous recombinants whose mpd gene integrated into the rrn site (Table 4).
Table 4. Specific activities of MPH in cell lysate at different times
Late lag phase
Specific activity (mU μg−1 protein)†
Specific activity (mU μg−1 protein)
Specific activity (mU μg−1 protein)
*One unit (U) of MPH activity was defined as the amount of enzyme required to hydrolyse 1 μmol methyl parathion in 1 min at 35°C. MPH activity was determined at 35°C by measuring the increase in absorbance at 399·5 nm resulting from the p-nitrophenol released by the hydrolysis of methyl parathion. Enzyme samples (20 μl) were added to an assay mixture containing 974 μl PBS buffer (0·2 mol l−1, pH 8·0) and 6 μl methyl parathion (10 mg ml−1). Samples boiled for 5 min were used as controls.
†The data are represented as mean ± standard deviation for triplicate experiments.
‡No MPH activity was detected.
68·7 ± 4·69
300·6 ± 13·82
4·4 ± 0·11
293·9 ± 3·13
1254·7 ± 4·45
4·3 ± 0·03
257·5 ± 6·68
654·1 ± 18·47
2·5 ± 0·11
78·2 ± 5·07
330·2 ± 12·60
4·2 ± 0·17
275·7 ± 11·44
1146·8 ± 6·33
4·2 ± 0·17
188·2 ± 5·02
468·6 ± 22·84
2·5 ± 0·07
94·4 ± 6·85
424·7 ± 10·71
4·5 ± 0·22
351·6 ± 8·07
1684·3 ± 14·28
4·8 ± 0·07
284·5 ± 6·11
762·5 ± 8·56
2·7 ± 0·06
74·1 ± 8·79
284·6 ± 17·01
3·9 ± 0·22
278·5 ± 9·42
1027·6 ± 15·53
3·7 ± 0·10
213·0 ± 10·12
423·8 ± 6·17
2·0 ± 0·09
308·8 ± 3·31
1241·4 ± 6·94
4·0 ± 0·05
704·4 ± 8·14
2810·5 ± 27·07
4·0 ± 0·07
747·3 ± 11·34
1773·0 ± 9·17
2·4 ± 0·03
65·3 ± 2·51
287·7 ± 5·28
251·9 ± 4·26
The most critical parameters for efficient homologous recombination in many systems tested so far were the length of homologous sequences and the degree of homology between the donor and genomic target DNAs. Shen and Huang (1986) reported that the frequencies of recombination were linearly dependent on the substrate length when reciprocal recombination in E. coli occurred between homologous regions present on a plasmid and the phage lambda genome. Several results also indicated that different types of recombination events required different minimal amounts of homology (Sugawara and Haber 1992; Jinks-Robertson et al. 1993; Hua et al. 1997). DNA sequence divergence affected the efficiency of homologous recombination in a wide variety of systems, ranging from bacteria to yeast and mammals (Barbara and Carole 1997; Predrag et al. 2000). Several studies showed an exponential decrease in the frequency of recombination with increasing sequence divergence (Datta et al. 1997; Vulic et al. 1997).
In our study, we investigated the effects of length and identity of homology on the targeting frequency in Sphingomonas species. Our results are in general agreement with the data obtained in other systems, but they also indicate that the influence of homology length and identity at the rrn site could be more complex. We deduced that the requirement for a longer MEPS in the Sphingomonas species rrn locus to saturate the recombination machinery than that required in other bacteria might be necessary to disfavour exchange between short repetitive DNA sequences, which could lead to genome instability. Although the direct homologous recombination frequencies obtained using the Red system with 35–60 bp homologous sequences in E. coli, Candida albicans and Saccharomyces cerevisiae were relatively high, the targeting frequency that we obtained with this system, using 99 bp homologous sequences in our study, was relatively low. It was puzzling and requires further study. Although the frequency of targeting would decrease with decreasing DNA homology, we explored the possibility that a vector with conserved rRNA genes that serve as homologous sequences may be able to act as a universal targeting vehicle to some extent. The failure to integrate the mpd gene into low identity target sequences (91–93%) might be due to a mismatch repair system in the Sphingomonas species, which suppresses recombination between divergent DNA sequences (Datta et al. 1996).
An increase in the specific activity of MPH in homologous recombinants might be due to the strong activity of rrn promoter. The housekeeping rrn operon encodes ribosome RNA and some transfer RNA, and is transcribed constantly because rRNAs and tRNAs are needed in every phase of cell growth. This would result in robust transcription of the mpd gene integrated into this locus (Ana et al. 2000). Thus, rrn site is a good integration target for improving the transcription of exogenous genes.
In summary, the data presented here are useful for obtaining a better understanding of recombination events involving homologous sequences and also for the improved manipulation of Sphingomonas species genes for biotechnological applications.
We thank Dr Zhu Jun from Harvard Medical School, Prof. Ian Blomfield from University of Kent and E. coli Genetic Stock Center, Yale University for providing bacterial strains and plasmid vectors. We are also grateful to Dr Gu Xiangyang from Nanjing Agricultural University for revision of this paper. This work was supported by grants from National Natural Science Foundation of China (30600016 and 40471073) and National Programs for High Technology Research and Development of China (2004AA246070).