Construction of prcK and prcR Mutant Strains of Lactobacillus paracasei HD 1 . 7 and the Impact on the Production of Paracin 1 . 7

Gene knockouts of prcK, prcR and both together were constructed in L. paracasei HD1.7. The antimicrobial activities of the prcK, prcR and prcKprcR mutant strains against B. subtilis were 23.6%, 21.9% and 36.6% lower than that of the parental strain, respectively, indicating that these genes affect production of bacteriocin antimicrobial peptides. qRT-PCR assays showed that the relative transcription levels of prcK and prcR mRNA in the DK and DR strains were 0.36 and 0.33 times of that in parental bacteria, respectively. Our data suggest that prcK and prcR are quorum sensing related genes that influence production of the bacteriocin Paracin 1.7. This research provides the basis for exploring the functions of these genes in the production of Paracin 1.7 and more generally for the exploration of the biological preservatives instead of chemical preservatives.


Introduction
Lactobacillus paracasei HD1.7 (CCTCCM 205015) was isolated from Chinese sauerkraut juice in 2003.In previous studies, the fermentation broth of L. paracasei HD1.7 contains a type of peptide, Paracin 1.7, a bacteriocin, that could inhibit the growth of several Gram-positive bacteria (G + ), Gram-negative bacteria (G -) and yeast. 1 The bacterial production process of Paracin 1.7 had characteristics of quorum sensing.Nakayama identified a series of genes in L. paracasei E93490 that were assigned as putative quorum sensing components, and predicted that the signaling molecule of L. paracasei E93490 might have antibacterial activity. 2The antibacterial activity may be similar to Paracin 1.7 produced by L. paracasei HD1. 7.
4][5] The RR, a DNA binding protein, activates related genes transcription; the phosphorylated RR can bind to the target promoter, directly or indirectly regulating expression of genes. 6Quorum sensing in L. paracasei is not well understood.Putative histidine protein kinase (prcK) and response regulator (prcR) genes have been identified in L. paracasei E93490 by PCR, but functional studies were not conducted. 2herefore, in this work, we investigated the functions of the prcK and prcR genes in quorum sensing and in the potentially related process of the production of antimicrobial peptides.
][9][10] Insertional inactivation has been the main method applied to G + bacteria. 11In this method, the flanking sequences of the exogenous DNA imported into host cells and of the target gene in the chromosome of host cells are homologous.The marker gene in the exogenous DNA fragment is therefore inserted into the target gene via homologous recombination, leading to the inactivation of the target gene by the replacement of its DNA.
In this study, we constructed suicide plasmids to create insertional inactivationbased gene knockouts.DNA was incorporated into the chromosome of L. paracasei HD1.7 to produce knockouts of prcK, prcR and prcKprcR, with the tetracycline resistance gene used as a marker (replacement DNA) in each case.Growth of colonies on plates containing tetracycline indicated that homologous recombination between the suicide plasmids and the host cell had occurred and that knockout mutant strains were produced.Antimicrobial tests were used to show the effects of deletion of prcK and prcR on the production of Paracin 1.7, and qRT-PCR was performed to determine whether the expression of prcK and prcR mRNA was affected.The data provided the basis for further exploration of the functions of these genes in the production of Paracin 1.7.

Bacterial strains, plasmids, growth medium and culture conditions
Bacterial strains and plasmids used in this work are listed in Table 1.L. paracasei HD1.7 strains were propagated in De Man-Rogosa-Sharpe (MRS) broth (Top Biotech Co., Qingdao, China) or agar at 30°C for 24 h.Where appropriate, tetracycline was added to the culture medium at 50 mg/mL.E. coli DH5α was grown in Luria-Bertani (LB) broth or agar at 37°C with vigorous agitation.E. coli DH5α transformant cells harbouring recombinant plasmids were selected onto LB agar plates supplemented with 100 mg/mL (final concentration) of ampicillin, 16 mL of X-Gal and 4 mL of IPTG per plate.B.subtilis ATCC 11774 was grown in Beef extract peptone (BP) broth or agar at 37°C, and this strain was used as an indicator strain when detect the antimicrobial activity.

DNA manipulation and transformation procedures
Genomic DNA isolation from L. paracasei HD1.7 was performed with TIAN-GEN genomic tips (Beijing, China).Plasmid isolation from E. coli DH5α transformants was done using TIANGEN plasmid Kit (Beijing, China).For amplication of DNA fragment was used procedure of PCR.The GE system 12 was used for the reaction system a PCR procedure.For screening purposes, DNA extractions from L. paracasei HD1.7 and E. coli DH5α colonies to be used as the template for PCR were carried out.PCR products were separated by 1% agar gel electrophoresis and were recovered using the Gel Extraction Kit (Tiangen Biotech CO., Beijing, China).The primers synthesis used in PCR reaction (Table 2) and PCR products sequencing were performed by Invitrogen Corporation.
Electroporation of L. paracasei HD1.7 was also carried out according to the method of Ge. 13 Transformation of E. coli DH5α competent cells were performed according to the Hannahan method. 14lasmids and restriction digestion products

Construction of prcK, prcR and prcKprcR knockout mutant of L. paracasei HD1.7, respectively
Restriction enzymes, T4 DNA ligase and DNA-modifying enzymes were used as recommended by the manufacturer (Tiangen, China).To delete the prcK, prcR and prcKprcR from the chromosome of L. paracasei HD1.7 by homologous recombination, plasmid pYTKLKRT, pYTRLRRT and pYTKRT were constructed (Figure 1).Three recombinant plasmids were transformed by electroporation into L. paracasei HD1.7 cells.The prcK and prcR mutant strains were selected by plating out appropriate dilutions on MRS agar containing 50 μg/mL (final concentration) of tetracycline.Restriction enzyme analysis and PCR identification were used to investigate whether the suicide plasmids met the experimental design.

Bacteriocin production of the original and the mutant strains
To evaluate bacteriocin production, the cultures of the parental and the mutant strains were inoculated into MRS broth and incubated at 37°C for 24 h, respectively.The supernatants from cultures were collected for determination of bacteriocin activity using the agar-well diffusion method described by Nwuche. 15To eliminate the antimicrobial effect of lactic acid, the pH of the supernatants were adjusted to 5.5 with 1 M NaOH.Titers were defined as the reciprocal of the highest dilution that inhibited the growth of the indicator strain.The results of the bacteriocin activity assays are presented in arbitrary units per milliliter (AU/mL).

RNA isolation, cDNA synthesis and qRT-PCR
The cultures of the parental and the mutant strains were inoculated into MRS broth and incubated at 37°C for 12 h, respectively.Both these cultures were harvested to extract and purify their RNA.Isolation total RNA was carried out with RNAprep Pure Cell/Bacteria Kit (Tiangen, China) in accordance with the manufacturer's recommendations.RNA concentration was measured at 260 nm and RNA purity was determined by measuring the absorbance ration at 260 nm/280 nm with A560 spectrophotometer (AOE INSTRUN-MENTS, Shanghai, China).
Reverse transcription was completed using BioRT cDNA First Strand Synthesis Kit (Bioer Technology, China) as instructed.Controls without reverse transcriptase were included in the qRT-PCR runs in order to confirm absence of contaminating DNA.
qRT-PCR amplications were performed with at least 3 replicates using RealMasterMix SYBR Green reagents (Tiangen, China) in a 7500 Real-Time PCR System (Applied Biosystems, Inc., USA).The housekeeping gene ldh was used as internal control L. paracasei HD1.7.The suitability of ldh was verified by isolation of both genomic DNA and RNA during the experiments.Transcriptional levels of prcK and prcR were normalized to the transcriptional level of the ldh gene.

Antimicrobial activity of knockout mutant strains
Three single colonies growing well on the MRS resistant plates with tetracycline were selected as prcK knockout mutant strain (DK), prcR knockout mutant strain (DR), and prcKprcR knockout strain (DKR).
The antimicrobial activity of DK, DR and DKR were shown in Figure 2. The results showed that the inhibition degrees of DK, DR and DKR were 23.61% (1538.29±46.27AU/mL), 21.93% (1572.26±39.04AU/mL), and 36.61% (1276.53±21.26AU/mL) lower than that of original strain (2013.80±26.54AU/ml), respectively, this indicated that the outputs of bacteriocin produced by three mutant strains were less than that of original strain, and the output of DKR was obviously less than those of DK and DR.
However, DK and DR still had inhibition ability to the growth of B. subtilis.That was probably due to L. paracasei HD1.7 having several quorum sensing systems.Other systems would not be impacted if one system was disrupted.Therefore, the mutants still could produce some antimicrobial peptides.Furthermore, the inhibition degree of DKR was 36.6% lower than that of the wild-type strain, obviously a greater effect than in DK or DR.Knocking out one gene would negatively influence the regulation of the quorum sensing system and lead to a decrease in production of antimicrobial peptides.Knocking out two genes in DKR apparently further increased the negative influence on the regulation of quorum sensing system, leading to a lower production of antimicrobial peptides in DKR than in DK or DR.

PCR analysis of prcK and prcR knockout mutant strains
Amplification of both Tet R and (prcK + Tet R ) by PCR was used to identify whether homologous recombination between suicide plasmids and genome of host cells had accomplished, which was based on the theoretical design.For PCR analysis of DK, genomic DNA of DK and the primers Tet-up and Tet-down were used as template and the primers, respectively.The original strain L. paracasei HD1.7 was used in the negative control experiment.It was the same with DR and DKR.The results of agarose gel electrophoresis were shown in Figure 3.There was a 1400 bp DNA fragment (Tet R ) that was amplified in DK, DR, and DKR, but no similar fragment in the negative control experiment, which demonstrated that Tet R was inserted into the genomes of DK, DR, and DKR.
For PCR identification of DK, genomic DNA of DK and the primers prcKL-up and prcKR-down were used as template and the primers, respectively.The original strain L. paracasei HD1.7 was used in the negative control experiment.DR (primers prcRL-up and prcRR-down) and DKR (primers prcKL-up and prcRR-down) were also identified as described above.
The results of agarose gel electrophoresis were shown in Figure 4.The results of Figure 4A showed that there was a 4140 bp DNA fragment (prcK + Tet R ) that were amplified in DK and a 2740 bp fragment (only prcK) in the negative control experiment.The results of Figure 4B showed that there was a 4820 bp DNA fragment (prcR + Tet R ) that was amplified in DR and a 3420 bp fragment (only prcR) in the negative control experiment.The results of Figure 4C showed that there was a 4130 bp DNA fragment (prcKL + prcRR + Tet R ) that was amplified in DKR and a 3500 bp fragment (only prcK+prcR) in the negative control experiment.It was demonstrated that Tet R was successfully inserted into prcK, prcR and prcKprcR, respectively.Therefore, double cross-over occurred in DK, DR and DKR.The prcK, prcR and prcKprcR knockout mutant strains were constructed successfully.

qRT-PCR analysis of prcK and prcR knockout mutant strains
Relative transcriptional expression of prcK and prcR in the parental and the mutant L. paracasei HD1.7 are present in Figure 5.There were significant reductions (p < 0.01) in prcK and prcR transcriptional levels in the parental and the mutant L. paracasei HD1.7.The level of prcK mRNA in DK was 0.36:1 compared with the parental strain.For DR, the corresponding ratio was 0.33:1.These results indicate that the bacteriocin produced by the mutant L. paracasei HD1.7 was indeed mediated by the action of prcK and prcR genes.As a consequence, the results of qRT-PCR analysis are consistent with the analysis of PCR and antimicrobial activity.

Discussion and Conclusions
Quorum sensing of Gram-positive bacteria is often regulated by three-component regulatory system composed of autoinducing peptide, sensor kinase and response regulator. 16hen the extracellular AIPs concentration reaches the threshold, specific receptor enzyme proteins can be activated and bind to the histidine protein kinase (HPK) receptors on the cell membrane.The HPK autophosphorylates its own histidine residue and then transfers the phosphate group to the asparitic acid in a response regulator (RR).It has been confirmed that the process of producing bacteriocin from a variety of lactic acid bacteria is regulated by the QS system, such as L. sanfranciscensis, 17 L. acidophilus 18,19 and L. plantarum. 20,21 series of genes mediated quorum sensing and production of Paracin1.7 by L. paracasei HD1.7 has been reported before.Previously, We have demonstrated that the process of producing bacteriocin of L. paracasei HD1.7 is regulated by quorum sensing via the cell density test. 13Here, we showed that the inhibition ability of L. paracasei HD1.7 decreased after knocking out the prcK and prcR genes.This finding suggests that prcK and prcR are quorum sensing related genes and influence the production of antimicrobial peptides as plnB has been observed for L. paraplantarum L-XM1. 22ifferent studies have shown the role of prcK and prcR in the quorum sensing system.In the study of Nakayama, 2 the fragment of L. paracasei E93490 amplified by degenerate primers was located in the prcK gene prior to prcR.Their predictive products PrcK and PrcR are similar to cognate HPK and RR, respectively, and the highest sequence similarity was L. sake SppK and L. plantarum PlnB, both of which formed a 3CRS that regulates the formation of bacteriocin.PrcK is expected to have six transmembrane alpha-helices at its N-terminal moiety, which can be used as a sensor domain.Similarly, the effect of prcK and prcR of L. paracasei HD1.7 on regulation of bacteriocin production needs further investigation.
In conclusion, the present study shows that the prcK and prcR genes of L. paracasei HD1.7 were down-regulated in response to the inhibition ability of DK and DR.These findings suggested that prcK and prcR are quorum sensing related genes and influence the production of antimicrobial peptides.This provides the basis for further exploration of the productions of natural preservatives.

Figure 3 .
Figure 3. PCR screening results using different templates with Tet-up and Tet-down primers.M: DNA Marker DL 2000; Lane 1, 3, 5: the PCR product using △K, △R and △KR gDNA as template, respectively; Lane 2, 4, 6: the PCR product using the original strain gDNA as template.

Figure 4 .
Figure 4. PCR screening for gene knockouts using various primer pairs.(A) The PCR identification result with prcKL-up and prcKR-down primers.(B) The PCR identification result with prcRL-up and prcRR-down primers.(C) The PCR identification result with prcKL-up and prcRR-down primers.M: DNA Marker DL 15000 + 2000; Lane 1, 3, 5: the PCR product using the original strain gDNA as template; Lane 2, 4, 6: the PCR product using △K, △R and △KR gDNA as template, respectively.

Figure 5 .
Figure 5. Relative transcriptional expression of prcK and prcR in the parental (■) and the mutant (□) L. paracasei HD1.7.Asterisk indicates a statistically significant difference (p < 0.01) with respect to the control group.