Methodology Report | Open Access
An Improved Method to Knock Out the asd Gene of Salmonella enterica Serovar Pullorum
An asd-deleted (asd) mutant of Salmonella enterica serovar Pullorum (SP) was constructed using an improved method of gene knockout by combining the -suicide plasmid system with the Red Disruption system. The asd gene was efficiently knocked out by the recombinant suicide vector, which replaced the asd gene with the gene. Based on the balanced lethal host-vector system, the phenotype of the asd mutant was further defined. The improved method was simpler and more effective than previously reported conventional methods.
Salmonella enterica serovar Pullorum (SP) is highly adapted to species of fowl, although SP infections in primates have been reported . As a fowl-specific pathogen, SP has a considerable worldwide economic impact, especially in developing countries. The growing problems of antibiotic resistance and the lengthy persistence of the bacteria in chickens after infection  necessitate the development of novel and efficient measures to control this pathogen.
A balanced lethal host-vector system, based on the mutant of Salmonella typhimurium, has previously been used in a vaccine . However, because of the high specificity of SP for fowl, SP is a better live vaccine vector for mucosal immunization of fowl than other Salmonella spp. The balanced lethal host-vector system can be used to produce a live vaccine, can be used as a vaccine vector [4–7], and is also a tool with which to study the genetics and pathogenesis of SP infection. This requires construction of an SP mutant and the development of an SP balanced lethal host-vector system.
Previously, multiple attempts have failed to produce a mutant when either the -suicide plasmid system or the Red Disruption system was used to knock-out the asd gene  of SP. However, we describe here the successful ablation of the asd gene of SP using a combination of these two systems above. The basic strategy was to replace the chromosomal asd sequence with a selectable antibiotic resistance gene (Cm) using a suicide vector based on the -suicide plasmid system, and E. coli as a donor strain. After selection with the appropriate antibiotic, the Cm resistance gene can be eliminated using the helper plasmid pCP20.
2. Materials and Methods
2.1. Bacterial Strains and Plasmids
The bacterial strains and plasmids used in this study are given in Table 1. Bacteria were grown in rich liquid or solid (12 g/L agar) Luria broth (LB) medium. The media were supplemented with ampicillin (Amp, 100 g/mL), kanamycin (Km, 50 g/mL), chloramphenicol (Cm, 30 g/mL), streptomycin (Sm, 25 g/mL), or nalidixic acid (Nal, 30 g/mL) as required. NA (solid LB medium without NaCl) and NB (liquid LB medium without NaCl) with 10% sucrose were used during the gene allelic exchange experiments to select plasmids that had been excised from the chromosome.
2.2. Construction of Suicide Plasmid pGMB151-asdp1234 (Cm)
The PCR product asdp12, using primers asdp1/asdp2, and the PCR product asdp34, using primers asdp3/asdp4, were amplified from genomic DNA of SP S06004. The PCR product , which spans the cassettes and includes the flanking FRT sites of the primers, was amplified from the pKD3 plasmid. These fragments were purified and cloned into the pMD18 vector and the resulting plasmids were named pMD-asdp12, pMD-asdp34 and pMD-, respectively. The asdp12 and asdp34 fragments acted as two arms for homologous recombination. Subsequently, asdp12 and asdp34 were ligated via an XhoI site to produce asdp1234. A fragment of the gene was then cloned into the XhoI site of asdp1234 to produce pMD-asdp1234 (Cm) (Figure 1). pMD-asdp1234 (Cm) and the pGMB151 suicide plasmid were digested with BamHI and religated to produce pGMB151-asdp1234 (Cm), which was then transferred to E. coli Spy372 . pGMB151-asdp1234 (Cm) was then further transferred to E. coli , and was termed the donor strain E. coli (pGMB151-/Cm). The sequences of all primers used are given in Table 2.
2.3. Generation of the SP (Cm) Mutant by Conventional Allelic Exchange
The donor strain, E. coli (pGMB151-/Cm), and the recipient strain, SP S06004, were grown in LB at 37°C with shaking to OD 0.6-0.7 at 600 nm. The recipient strain was heat-treated at 50°C for 30 minutes immediately before conjugative mating to temporarily inactivate the host-restriction systems. Samples of the donor strain and the heat-treated recipient strain were mixed in 10 mM MgSO4 solution, the mixture was immobilized on a 0.45 m membrane filter, and the filter was incubated on LB agar at 28°C for 18 hours. The transconjugants were recovered in LB for 1 hour at 37°C, and spread onto LB agar containing Nal, Amp, and Cm . The Colonies were also streaked onto NA with Nal, Amp, and Cm and NA with Nal, Amp, Cm and sucrose to select bacteria which are sensitive to sucrose . Single bacterial colonies, of single-crossover plasmid insertions (S06004::pGMB151-/Cm), which are sensitive to sucrose, were subcultured 8–10 times on NB containing 10 mM MgSO4, 10% sucrose , 1% diaminopimelic acid (DAP) and Cm. The mutants, ie the SP (Cm) mutant, without Amp resistance were screened on LB plates that contained DAP and Cm [13, 14] (Figure 2). At the same time, The presence of the allele in the SP (Cm) mutant was confirmed by asdp5/asdp6 primers which PCR product,1360 bp, was smaller than 1796 bp amplified from wt SP S06004 (Figures 3 and 5(b)).
2.4. Antibiotic Resistance, Growth and Biochemical Characteristics of the SP (Cm) Mutant
During the selection process, the SP (Cm) mutant was cultured in medium supplemented with several antibiotics, including Nal, Amp, Cm, and/or Km. When the asd gene was replaced by the gene, the mutant became resistant to Cm and depended on exogenous DAP for growth. The SP (Cm) mutant was cultured in medium containing DAP, and also in medium without DAP, as a control, to determine if its growth was dependent on the presence of DAP. The basic biochemical characteristics of the SP (Cm) mutant were evaluated using IMViC tests.
2.5. PCR Verification of the SP (Cm) Mutant
In addition to the primers asdp1/asdp2, asdp3/asdp4 and , primers asdp5/asdp6 (asdp5 is in the asdp12 sequence and asdp6 is in the asdp34 sequence), and primers asdp5 and asdp6 (Table 2) were used to further characterize the SP (Cm) mutant. The PCR products obtained were compared with those of wt SP S06004. At the same time, the genus Salmonella was identified by PCR amplification of the hto gene [12, 15] with the primers htoF/htoR (Table 2). The rfbS gene, which specifically identifies SP [16, 17], was amplified with the primers rfbSF/rfbSR (Table 2).
2.6. Elimination of the Gene from of the SP (Cm) Mutant
Plasmid pCP20 is an and plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis . The SP (Cm) mutants were transformed with plasmid pCP20, and transformants resistant to ampicillin were selected at 30°C.
Subsequently, a few colonies were purified once, nonselectively, at 43°C, and were then tested for loss of all antibiotic resistance. The majority lost the Cm resistance gene and the FLP helper plasmid simultaneously, and comprised the SP mutant population.
2.7. Construction of the SP Balanced Lethal Host-Vector System
The SP mutant can be complemented with a foreign asd gene from the plasmid that forms the SP balanced lethal host-vector system. The SP mutant, with the plasmid containing the foreign asd gene, can grow without DAP. The pYA3334 plasmid [5, 19], which contains the asd gene, was transformed into the SP mutant to verify its growth without DAP. The pYA3334-dsRED plasmid, which contains the asd gene and the dsRED gene, was transformed into the SP mutant to express the red fluorescent protein. This was used to further demonstrate stability of the plasmid in different passages of the SP mutant (pYA3334-dsRED), using flow cytometry (FACS) analysis.
3.1. Antibiotic Resistance of the SP Mutant
During the selection process, the antibiotic resistance of the SP mutant, the SP (Cm) mutant, and other bacterial strains was determined (Table 3). As expected, the SP mutant showed the profile , which was the same as that of the wt SP S06004.
3.2. Growth and Biochemical Characteristics of the SP Mutant
DAP was an absolute requirement for growth of the SP mutant and the SP (Cm) mutant. The IMViC of the mutant was “−+−−” which was consistent with those of wt SP S06004. However, the growth velocity of the mutant and the (Cm) mutant in LB media containing DAP was slow compared with that of the wt S06004 (Figure 4).
(a) Overall of PCR identification of the SP (Cm) mutant Lanes 1, 2, 5, 6, 7, 8, 10, 11: the SP (Cm) mutant; Lanes 3, 4, 9, 12:wt, Lanes 1, 2,3: ; 4, 5, 6: asdP5/asdP; 6, 7, 8, 9: asdP1/asdP2; 10, 11, 12: asdP3/asdP4
(b) PCR with primers asdP5/asdP6 identified Wt, SP (Cm) and SP mutant, Lane 1: wt; Lanes 2, 3: First crossover of conjugants; Lanes 4, 5: Second crossover of conjugants (the SP (Cm) mutant); Lanes 6, 7: the SP mutant.
3.3. PCR Verification of the SP (Cm) and Mutants
PCR was used to identify the SP (Cm) and SP mutants. PCR amplification of the hto and the rfbS genes showed that the (Cm) mutant was SP. The PCR products were amplified using primers asdp1/asdp2, asdp3/asdp4, , asdp5/asdp6, asdp5, and asdp6 (Figure 5(a)), which demonstrated that the asd gene had been replaced by the Cm gene. PCR amplification using the primers asdp5/asdp6 showed that bacteria of the first crossover possessed two copies of an upstream fragment and a downstream fragment of the asd gene. After the second crossover, the asd gene was replaced by the Cm gene, and the gene was eliminated from the SP (Cm) mutant by plasmid pCP20 (Figures 3 and 5(b)). These results indicated that an SP mutant had been developed whose genomic DNA lacked the asd gene.
3.4. Construction of the SP Balanced Lethal Host-Vector System
When the SP mutant was transformed with plasmid pYA3334, which contains the asd gene, the recombinant SP mutant (pYA3334) could grow without DAP. This showed that the asd gene in the plasmid could functionally complement the mutant. After transformation with the plasmid pYA3334-dsRED, the SP mutant (pYA3334-dsRED) expressed red fluorescent protein (Figure 6); in contrast, there was no red fluorescence from the SP mutant (pYA3334). The FACS analysis of dsRED expression in the different passages of the SP mutant (pYA3334-dsRED) showed that pYA3334-dsRED was stable. In the 2nd and 20th passages of the SP mutant (pYA3334-dsRED), 96.3% and 95% of bacteria, respectively, showed strong red fluorescence (Figure 7).
Allelic exchange experiments  allow investigation of the functions of many unknown genes identified during the sequencing of entire genomes. A number of allele replacement methods can be used to inactivate bacterial chromosomal genes. These all require the engineering of gene disruption on a suitable plasmid. Amberg et al.  reported the successful knock-out of a gene by homologous recombination in yeast using fusion PCR technology. Kuwayama et al.  showed that genes can be directly disrupted in Saccharomyces cerevisiae by transformation with PCR fragments encoding a selectable marker and having only 35nt of flanking homologous DNA. Most bacteria, however, are not readily transformable with linear DNA, in part, because of intracellular exonucleases that degrade linear DNA. Datsenko and Wanner  developed the simple and highly efficient Red Disruption system to directly inactivate chromosomal genes in E. coli K-12 using PCR products based on the phage -Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid, such as pKD46 (or pKD20). To adapt it to more distantly related bacteria, it may be necessary to express the Red system under different control or from another low copy number vector.
Several different methods of gene knock-out have been reported in Salmonella, including the -suicide plasmid containing R6K ori, the -red system, the Red Disruption system, and a plasmid with temperature-sensitive replication. Among these methods, -suicide plasmids and the Red Disruption system have been preferred in Salmonella and E. coli, because they possess many advantages. However, the performance of the Red Disruption system in different bacteria can be variable due to intrinsic differences, such as Recombinase expression. Similarly, the major problem of the -suicide plasmid system is that its efficiency is very poor. Most bacteria subjected to homologous recombination, even under negative selection for the sacB gene [23, 24], are wild type (wt), and only a few are mutant, therefore, it is difficult to directly isolate the desired mutant. An increase in the efficiency for screening recombinants is needed. Application of the -suicide plasmid system requires two problems to be solved: (1), the requirement of antibiotic resistance in the engineered bacteria and (2), the efficiency of selection for mutants.
Previously we have made multiple attempts, to obtain mutants using the Red Disruption system, but without success (unpublished data). This is possibly because phage -Red recombinase was not expressed in S06004 from the pKD46 plasmid. It is probable that this system is not adaptable to some “recalcitrant” strains, such as SP S06004, as we showed here.
The -suicide plasmid containing R6K ori is universally used for gene ablation. The E. coli host strains SM10  and S17 for this plasmid are resistant to Km, but the recipient Salmonella strain used in this study had no special antibiotic resistance. When screening bacteria of the first crossover, it is difficult to separate donor bacteria (SM10 or S17) from recipient bacteria (SP S06004). The efficiency of screening recombinants requires improvement.
In an attempt to solve these difficulties, a new approach that combined the -suicide plasmid system with the Red Disruption system was developed. First, we used E. coli strain  instead of strain SM10 or S17, because E. coli is a mutant that depends on exogenous DAP for its growth. It was easy, therefore, to isolate donor and recipient bacteria after conjunction on an LB plate without DAP. The donor bacterium, E. coli , could not grow on medium without DAP, but the first-cross bacteria could grow. Second, the FRT-flanked resistance gene ( or ) of the pKD3 plasmid of the Red Disruption system was used to replace the gene of interest. Plasmid pCP20, which is and , shows temperature-sensitive replication and thermal induction of FLP synthesis was used to knock-out the FRT-flanked resistance gene. This improved method made the knock-out of a gene simple in comparison with the -suicide plasmid system or the Red Disruption system alone.
This improved method has been successfully applied in our lab to knock-out many bacterial genes. we anticipate that it will be widely applied for gene targeting in the future.
In this paper, we have described a new improved approach for gene targeting in Salmonella enteric serovar Pullorum to knock-out gene(s), replaceing target gene (asd gene) with Cm (or Km) gene from the Red Disruption system based on -suicide plasmid system, which is simpler in the procedures and more effective for screening recombinants than previously reported conventional methods.
The authors thank sincerely Dr. Roy Curtiss III and Dr. Cristina Marolda for their help and guide in gene knock-out and thank the cooperation of faculty members at Jiangsu Key Laboratory of Zoonosis in Yangzhou University. The authors thank sincerely the editor and reviewers for their kind comments and suggestions. This research is supported by the National Natural Science Foundation of China (30425031) and National programs for Fundamental Research and Development of China (2006CB504404).
- R. A. Ocholi, L. U. Enurah, and P. S. Odeyemi, “Fatal case of salmonellosis (Salmonella pullorum) in a chimpanzee (Pan troglodytes) in the Jos Zoo,” Journal of Wildlife Diseases, vol. 23, no. 4, pp. 669–670, 1987.
- P. Wigley, A. Berchieri Jr., K. L. Page, A. L. Smith, and P. A. Barrow, “Salmonella enterica serovar pullorum persists in splenic macrophages and in the reproductive tract during persistent, disease-free carriage in chickens,” Infection and Immunity, vol. 69, no. 12, pp. 7873–7879, 2001.
- K. L. Karem, S. Chatfield, N. Kuklin, and B. T. Rouse, “Differential induction of carrier antigen-specific immunity by Salmonella typhimurium live-vaccine strains after single mucosal or intravenous immunization of BALB/c mice,” Infection and Immunity, vol. 63, no. 12, pp. 4557–4563, 1995.
- J. E. Galán, K. Nakayama, and R. Curtiss III, “Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains,” Gene, vol. 94, no. 1, pp. 29–35, 1990.
- H. Y. Kang, J. Srinivasan, and R. Curtiss III, “Immune responses to recombinant pneumococcal PspA antigen delivered by live attenuated Salmonella enterica serovar typhimurium vaccine,” Infection and Immunity, vol. 70, no. 4, pp. 1739–1749, 2002.
- C. O. Tacket, S. M. Kelly, F. Schodel et al., “Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system,” Infection and Immunity, vol. 65, no. 8, pp. 3381–3385, 1997.
- S.-L. Yuan, P. Wang, H.-X. Tao et al., “Removal of antibiotic resistance of live vaccine strain Escherichia coli MM-3 and evaluation of the immunogenicity of the new strain,” Acta Biochimica et Biophysica Sinica, vol. 38, no. 12, pp. 844–856, 2006.
- H. Y. Kang, C. M. Dozois, S. A. Tinge, T. H. Lee, and R. Curtiss III, “Transduction-mediated transfer of unmarked deletion and point mutations through use of counterselectable suicide vectors,” Journal of Bacteriology, vol. 184, no. 1, pp. 307–312, 2002.
- X. X Huang, L. V. Phung, S. Dejsirilert et al., “Cloning and characterization of the gene encoding the z66 antigen of Salmonella enterica serovar Typhi,” FEMS Microbiology Letters, vol. 234, no. 2, pp. 239–246, 2006.
- K. A. Datsenko and B. L. Wanner, “One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 12, pp. 6640–6645, 2000.
- M. R. Sarker and G. R. Cornelis, “An improved version of suicide vector pKNG101 for gene replacement in gram-negative bacteria,” Molecular Microbiology, vol. 23, no. 2, pp. 410–411, 1997.
- P. Gay, D. Le Coq, M. Steinmetz, T. Berkelman, and C. I. Kado, “Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria,” Journal of Bacteriology, vol. 164, no. 2, pp. 918–921, 1985.
- P. Gay, D. Le Coq, M. Steinmetz, E. Ferrari, and J. A. Hoch, “Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli,” Journal of Bacteriology, vol. 153, no. 3, pp. 1424–1431, 1983.
- N. D. Cohen, E. D. McGruder, H. L. Neibergs, R. W. Behle, D. E. Wallis, and B. M. Hargis, “Detection of Salmonella enteritidis in feces from poultry using booster polymerase chain reaction and oligonucleotide primers specific for all members of the genus Salmonella,” Poultry Science, vol. 73, no. 2, pp. 354–357, 1994.
- N. D. Cohen, D. E. Wallis, H. L. Neibergs et al., “Comparison of the polymerase chain reaction using genus-specific oligonucleotide primers and microbiologic culture for the detection of Salmonella in drag-swabs from poultry houses,” Poultry Science, vol. 73, no. 8, pp. 1276–1281, 1994.
- A. R. Desai, D. H. Shah, S. Shringi et al., “An allele-specific PCR assay for the rapid and serotype-specific detection of Salmonella pullorum,” Avian Diseases, vol. 49, no. 4, pp. 558–561, 2005.
- D. H. Shah, J.-H. Park, M.-R. Cho, M.-C. Kim, and J.-S. Chae, “Allele-specific PCR method based on rfbS sequence for distinguishing Salmonella gallinarum from Salmonella pullorum: serotype-specific rfbS sequence polymorphism,” Journal of Microbiological Methods, vol. 60, no. 2, pp. 169–177, 2005.
- P. P. Cherepanov and W. Wackernagel, “Gene disruption in Escherichia coli: and cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant,” Gene, vol. 158, no. 1, pp. 9–14, 1995.
- K. Nakayama, S. M. Kelly, and R. Curtiss III, “Construction of an ASD+ expression-cloning vector: stable maintenance and high level expression of cloned genes in a Salmonella vaccine strain,” Bio/Technology, vol. 6, no. 6, pp. 693–697, 1988.
- N. Philippe, J.-P. Alcaraz, E. Coursange, J. Geiselmann, and D. Schneider, “Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria,” Plasmid, vol. 51, no. 3, pp. 246–255, 2004.
- D. C. Amberg, D. Botstein, and E. M. Beasley, “Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction,” Yeast, vol. 11, no. 13, pp. 1275–1280, 1995.
- H. Kuwayama, S. Obara, T. Morio, M. Katoh, H. Urushihara, and Y. Tanaka, “PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors,” Nucleic Acids Research, vol. 30, no. 2, p. E2, 2002.
- R. A. Edwards, L. H. Keller, and D. M. Schifferli, “Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression,” Gene, vol. 207, no. 2, pp. 149–157, 1998.
- J. L. Ried and A. Collmer, “An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in gram-negative bacteria by marker exchange-eviction mutagenesis,” Gene, vol. 57, no. 2-3, pp. 239–246, 1987.
- H. Schmidt, M. Bielaszewska, and H. Karch, “Transduction of enteric Escherichia coli isolates with a derivative of Shiga toxin 2-encoding Bacteriophage 3538 isolated from Escherichia coli O157:H7,” Applied and Environmental Microbiology, vol. 65, no. 9, pp. 3855–3861, 1999.
- F. Daigle, J. E. Graham, and R. Curtiss III, “Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences (SCOTS),” Molecular Microbiology, vol. 41, no. 5, pp. 1211–1222, 2001.
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