An Improved Method to Knock Out the asd Gene of Salmonella enterica Serovar Pullorum

An asd-deleted (Δasd) mutant of Salmonella enterica serovar Pullorum (SP) was constructed using an improved method of gene knockout by combining the π-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 CmR gene. Based on the balanced lethal host-vector system, the phenotype of the Δasd mutant was further defined. The improved method was simpler and more effective than previously reported conventional methods.


Introduction
Salmonella enterica serovar Pullorum (SP) is highly adapted to species of fowl, although SP infections in primates have been reported [1]. 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 [2] necessitate the development of novel and efficient measures to control this pathogen.
A balanced lethal host-vector system, based on the Δasd mutant of Salmonella typhimurium, has previously been used in a vaccine [3]. 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][5][6][7], and is also a tool with which to study the genetics and pathogenesis of SP infection. This requires construction of an SP Δasd mutant and the development of an SP balanced lethal host-vector system.
Previously, multiple attempts have failed to produce a mutant when either the π-suicide plasmid system or the Red Disruption system was used to knock-out the asd gene [8] 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 π-suicide plasmid system, and E. coli χ7213 as a donor strain. After selection with the appropriate antibiotic, the Cm resistance gene can be eliminated using the helper plasmid pCP20.

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 μg/mL), kanamycin (Km, 50 μg/mL), chloramphenicol (Cm, 30 μg/mL), streptomycin (Sm, 25 μg/mL), or nalidixic acid (Nal, 30 μg/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.

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 Cm R , which spans the Cm R cassettes and includes the flanking FRT sites of the Cm F /Cm R 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-Cm R , 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 Cm R 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 [9]. pGMB151-asdp1234 (Cm) was then further transferred to E. coli χ7213, and was termed the donor strain E. coli χ7213 (pGMB151-Δasd/Cm). The sequences of all primers used are given in Table 2.

Generation of the SP Δasd (Cm) Mutant by Conventional
Allelic Exchange. The donor strain, E. coli χ7213 (pGMB151-Δasd/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 MgSO 4 solution, the mixture was immobilized on a 0.45 μm 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 [11]. 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 [11]. Single bacterial colonies, of single-crossover plasmid insertions (S06004::pGMB151-Δasd/Cm), which are sensitive to sucrose, were subcultured 8-10 times on NB containing 10 mM MgSO 4 , 10% sucrose Table 3: Antibiotics resistance of bacteria during the selection of Δasd mutant. [12], 1% diaminopimelic acid (DAP) and Cm. The mutants, ie the SP Δasd (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 Δasd allele in the SP Δasd (Cm) mutant was confirmed by asdp5/asdp6 primers which PCR product,1360 bp, was smaller than 1796 bp amplified from wt SPS06004 (Figures 3 and 5(b)).

Antibiotic Resistance, Growth and Biochemical
Characteristics of the SP Δasd (Cm) Mutant. During the selection process, the SP Δasd (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 Cm R gene, the mutant became resistant to Cm and depended on exogenous DAP for growth. The SP Δasd (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 Δasd (Cm) mutant were evaluated using IMViC tests.

PCR Verification of the SP Δasd (Cm) Mutant.
In addition to the primers asdp1/asdp2, asdp3/asdp4 and Cm F /Cm R , primers asdp5/asdp6 (asdp5 is in the asdp12 sequence and asdp6 is in the asdp34 sequence), and primers asdp5/Cm F and asdp6/Cm R ( Table 2) were used to further characterize the SP Δasd (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).

Elimination of the Cm R Gene from of the SPΔasd (Cm)
Mutant. Plasmid pCP20 is an Amp R and Cm R plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis [18]. The SP Δasd (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 Δasd mutant population.

Construction of the SP Balanced Lethal Host-Vector
System. The SP Δasd mutant can be complemented with a foreign asd gene from the plasmid that forms the SP balanced lethal host-vector system. The SP Δasd 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 Δasd mutant to verify its growth without DAP. The pYA3334-dsRED plasmid, which contains theasd gene and the dsRED gene, was transformed into the SP Δasd mutant to express the red fluorescent protein. This was used to further demonstrate stability of the plasmid in different passages of the SP Δasd mutant (pYA3334-dsRED), using flow cytometry (FACS) analysis.

Antibiotic Resistance of the SP Δasd
Mutant. During the selection process, the antibiotic resistance of the SP Δasd mutant, the SP Δasd (Cm) mutant, and other bacterial strains was determined ( Table 3). As expected, the SP Δasd mutant showed the profile Nal R Amp S Cm S Sm S , which was the same as that of the wt SP S06004. containing DAP was slow compared with that of the wt S06004 ( Figure 4).

PCR Verification of the SP Δasd (Cm) and Δasd
Mutants. PCR was used to identify the SP Δasd (Cm) and SP Δasd mutants. PCR amplification of the hto and the rfbS genes showed that the Δasd (Cm) mutant was SP. The PCR products were amplified using primers asdp1/asdp2, asdp3/asdp4, Cm F /Cm R , asdp5/asdp6, asdp5/Cm F , and asdp6/Cm R (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 Cm R gene was eliminated from the SP Δasd (Cm) mutant by plasmid pCP20 (Figures 3 and 5(b)). These results indicated that an SP Δasd mutant had been developed whose genomic DNA lacked the asd gene.

Construction of the SP Balanced Lethal Host-Vector
System. When the SP Δasd mutant was transformed with plasmid pYA3334, which contains the asd gene, the recombinant SP Δasd mutant (pYA3334) could grow without DAP. This showed that the asd gene in the plasmid could functionally complement the Δasd mutant. After transformation with the plasmid pYA3334-dsRED, the SP Δasd mutant (pYA3334-dsRED) expressed red fluorescent protein ( Figure 6); in contrast, there was no red fluorescence from the SP Δasd mutant (pYA3334). The FACS analysis of dsRED expression in the different passages of the SP Δasd mutant (pYA3334-dsRED) showed that pYA3334-dsRED was stable. In the 2nd and 20th passages of the SP Δasd mutant (pYA3334-dsRED), 96.3% and 95% of bacteria, respectively, showed strong red fluorescence (Figure 7).

Discussion
Allelic exchange experiments [20] 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. [21] reported the successful knock-out of a gene by homologous recombination in yeast using fusion PCR technology. Kuwayama et al. [22] showed that genes can be directly part, because of intracellular exonucleases that degrade linear DNA. Datsenko and Wanner [10] 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 λ-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 π-suicide plasmid containing R6K ori, the λ-red system, the Red Disruption system, and a plasmid with temperature-sensitive replication. Among these methods, π-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 π-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 πsuicide 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 λ-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 π-suicide plasmid containing R6K ori is universally used for gene ablation. The E. coli host strains SM10 [25] 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 π-suicide plasmid system with the Red Disruption system was developed. First, we used E. coli strain χ7213 [26] instead of strain SM10 or S17, because E. coli χ7213 is a Δasd 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 χ7213, could not grow on medium without DAP, but the first-cross bacteria could grow. Second, the FRT-flanked resistance gene (Cm R or Km R ) of the pKD3 plasmid of the Red Disruption system was used to replace the gene of interest. Plasmid pCP20, which is Amp R and Cm R , shows temperature-sensitive replication and thermal induction of FLP synthesis was used to knockout the FRT-flanked resistance gene. This improved method made the knock-out of a gene simple in comparison with the π-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.

Conclusions
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 π-suicide plasmid system, which is simpler in the procedures and more effective for screening recombinants than previously reported conventional methods.