Abstract: A simpler method for the efficient and precise deletion of genes in Salmonella sp. was developed. To demonstrate this approach, the prgH gene of Salmonella typhimurium SL7207 was deleted by homologous recombination with a temperature-sensitive plasmid containing a cassette that two DNA fragments as homologous arms flanking chloramphenicol resistance gene (Cm^R) which replaced the prgH gene. During screening mutant at 44°C, the temperature-sensitive plasmid was lost easily only the mutant which prgH gene was replaced by Cm^R gene could grow in the LB media with chloramphenicol. The results showed that the method was simpler, more effective to delete target gene in genomic DNA of Salmonell sp. than those conventional methods.

INTRODUCTION

Allelic exchange experiments allow investigation of the functions of many unknown genes identified during the sequencing of entire genomes (Zhou et al., 2010).

Deleting directed gene is a central technique used to demonstrate gene function in many studies (Zhou et al., 2010). Several main different methods of gene deletions have been reported in Salmonella sp. including use of the R6K-suicide plasmid (Miller and Mekalanos, 1988; Philippe et al., 2004), the λ-Red disruption system (Kuwayama et al., 2002; Datsenko and Wanner, 2000; Doublet et al., 2008), the suicide plasmid combined with the Red system (Geng et al., 2009) or the plasmid with temperature-sensitive with SacB gene (pKO3) (Link et al., 1997; Hamann et al., 2005; Hamilton et al., 1989).

Although, these methods were reported many times few of them were applied successfully when we attempted to delete prgH (Kubori et al., 1998; Kimbrough and Miller, 2000) gene of Salmonella sp. In this study, researchers reported a simpler and precise method to knock out prgH gene of Salmonella sp.

MATERIALS AND METHODS

Bacteria and plasmids: The strains and plasmids used in this study were shown in Table 1. Strains were grown in rich liquid or solid (12 g L^-1 agar) Luria Broth (LB) medium. The media were supplemented with ampicillin (Amp, 100 μg mL^-1), chloramphenicol (Cm, 50 μg mL^-1),streptomycin (Sm, 25 μg mL^-1).

Table 1:	Strains and plasmids used in this study

Construction of plasmid pKD46-prgH-u+d (Cm^R) for knocking out of prgH gene: The plasmid pKD46-prgH-u+d (Cm^R) was constructed following the process shown in Fig. 1. All of the primers used in this study were shown in Table 2.

Screening the ΔprgH/Cm^R mutant of S. typhimurium SL7207: Plasmid pKD46-prgH-u+d (Cm^R) was transformed to S. typhimurium SL7207 by electroporation instrument and the recombinants (SL7207::pKD46-prgH-u+d (Cm^R)) with Amp and Cm resistance were cultured in 28°C, one colony was cultured in the LB media with Cm at 28°C and subcultured (100 μL of the culture was used to inoculate 4 mL of fresh media) for 12 h at 44°C and repeat 8-10 times (Fig. 2). At last, serial dilutions of the culture were prepared and plated 250 μL of the 10^-4, 10^-5 and 10^-6 dilutions onto LB (Cm) agar plates and incubated for ~16 h at 44°C. Each colony screened should be patched on an LB (Cm) agar plate and an LB (Amp) plate. Only those colonies that grew on LB (Cm) plate and did not grow in LB (Amp) plate would be correct. On average 50-100 positive colonies were screened.

The colonies of the ΔprgH/Cm^R mutant that could only grow on the LB (Cm) plates were furtheridentified by PCR and its characteristics related to prgH gene was verified. The SL7207 ΔprgH mutant was received from the ΔprgH/Cm^R mutant by pCP20 plasmid (Fig. 2) (Datsenko and Wanner, 2000; Doublet et al., 2008).

Verification of the SL7207 ΔprgH/Cm^R mutant PCR identification of SL7207ΔprgH/Cm^R mutant: Primers asdp5/asdp6 (asdp5 is in the asdp12 sequence and asdp6 is in the asdp34 sequence) were used to further identify the ΔprgH/Cm^R mutant. The stn gene (Geng et al., 2009) was amplified with the primer set stnF/stnR (Table 2) which specifically identified Salmonella sp.

Fig. 1:	The construction of plasmid pKD46-prgH-μ+d (CmR)

Table 2:	The primer sequences of PCR amplification

Fig. 2:	The screening strategy for the ΔprgH mutant

Detecting of mRNA of prgH gene by Reverse Transcription-PCR (RT-PCR): The SL7207ΔprgH/Cm^R mutant and parent SL7207 were cultured and their mRNA were extracted by mRNA extraction Kit of RNAiso Plus respectively, prgH gene was detected of by RT-PCR to determine whether prgH gene was expression the primers were shown in Table 2.

Biochemical characteristics: The basic biochemical characteristics of the ΔprgH/Cm^R mutant were evaluated by biochemical tube test.

RESULTS AND DISCUSSION

PCR identification of SL7207ΔprgH/Cm^R mutant: The PCR products of primer set asdp5/asdp6 showed that bacteria with Plasmid pKD46-prgH-u+d (Cm^R) possessed two copies of an upstream fragment and a downstream fragment of the prgH gene, the SL7207 had only one copy. After mutant being screened, the prgH gene was replaced by the Cm^R gene, the amplicons were 1,697 bp (parent) and 1,697bp and 1,841 bp (the recombinant with Plasmid pKD46-prgH-u+d (Cm^R), 1,841 bp (ΔprgH/Cm^R mutant) (Fig. 3). These results indicated that an ΔprgH/Cm^R mutant had been constructed whose genomic DNA lacked the prgH gene. The stn gene was amplified with the primer set stnF/stnR, the amplicon size was 260 bp (Fig. 4), the result showed that the mutant was Salmonella sp.

Detecting of mRNA of prgH gene by reverse transcription-PCR (RT-PCR): The result of RT-PCR showed that SL7207ΔprgH/Cm^R mutant did not express mRNA of prgH gene as a control, the parent SL7207 could express mRNA of prgH gene (Fig. 5).

Fig. 3:	PCR identification by prgHp5/p6 of SL7207 ΔprgH/CmR M DNA marker DL20001; 2: SL7207; 3-4: the recombinants; 5: SL7207 ΔprgH/CmR

Fig. 4:	PCR identification based on stn gene of Salmonella sp. M DNA Marker DL2000; 1: SL7207 ΔprgH/CmR; 2: SL7207

Table 3:	Biochemical identification of the SL7207 (ΔprgH/CmR) and parent strain SL7207

This illustratedb further that the SL7207ΔprgH/Cm^R mutant did not have the prgH gene which had been knocked out from genomic DNA of the parent SL7207.

Biochemical characteristics: The results of biochemical tube test of the mutant were consistent with those of parent SL7207 (Table 3).

Table 4:	The difference of different methods of gene deletions in Salmonella sp.

Fig. 5:	RT-PCR identification by prgH-(RT) primers M:DNA Marker DL2000; 1: SL7207; 2: SL7207; 3: SL7207 (ΔprgH/CmR)

The SL7207 (ΔprgH/Cm^R) mutant was constructed successfully during 3 weeks using this method based on temperature-sensitive plasmid with RepA101 (ts). Because target gene was replaced by Cm^R gene in the temperature-sensitive plasmid. At 44°C, this plasmid was lost easily from the recombinants at the same time, the parent SL7207 had no Cm restance, SL7207 without pKD46-prgH-u+d (Cm^R) was dead in the LB (Cm) media. Only when target gene (prgH) was replaced by Cm^R gene via allele replacement and the recombinants [SL7207::pKD46-prgH-u+d (Cm)] lost plasmid, the mutant with Cm^R gene and without prgH gene could grow in the Cm and 44° and could not grow on LB ( Amp) plates. And so, by subculturing 8-10 times, the mutant could be retrieved. Comparing with the Red system (Table 4), the method need not what pKD46 plasmid was transformated into target bacteria and induced by arabinose to express phage λRed recombinase which was unsure in the try (Datsenko and Wanner, 2000). Although, λ-Red Disruption system looked simple and was applied in E. coli and other G-bacteria, researchers tried many times with Red system in Salmonella sp. but at last failed. As a control, researchers cloned the cassette into the R6K-suicide plasmid with SacB gene (Geng et al., 2009) and the temperature-sensitive plasmid. In theory, the recombinant bacteria with the R6K-suicide plasmid was very sensitive to sucrose because levansucrase was expressed by the SacB gene which was lethal to bacteria in the presence of 10% sucrose and utilized as a positive and counter-selectable markers in the template plasmid (Gay et al., 1983) but during screening mutant, it is difficult to receive a clony with suicide plasmid which is sensitive to sucrose in spite that we success in knocking out this gene.

CONCLUSION

In this improved method, some uncertain factors were avoid by temperature-sensitive plasmid replacing R6K-suicide plasmid such as phageλRed recombinase, levansucrase only temperature was used to make plasmid lose and speed process of screening mutant under the Cm antibiotic which was positive and counter-selectable markers in the mutant. In brief, this method was simpler than the others to generate precise gene deletions in Salmonella sp.

ACKNOWLEDGEMENTS

The researchers thank sincerely Dr. Cristina Marolda and CGSC (Coli Genetic Stock Center) in USA 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. This research was supported by key projects in the National Science and Technology Pillar Program (2009BADB9B01), National Programs for Fundamental Research and Development of China (2006CB504404), National Nature Science Foundation of China (No. 30871860, 30425031) and the Government of Jiangsu Province (Nos. BK2008011 and BK2010039).