Effect of LuxS/AI-2-mediated quorum sensing system on bacteriocin production of Lactobacillus plantarum NMD-17

Lactobacillus plantarum NMD-17 separated from koumiss could produce a bacteriocin named plantaricin MX against Gram-positive bacteria and Gram-negative bacteria. The bacteriocin synthesis of L. plantarum NMD-17 was remarkably induced in co-cultivation with Lactobacillus reuteri NMD-86 as the increase of cell numbers and AI-2 activity, and the expressions of luxS encoding signal AI-2 synthetase, plnB encoding histidine protein kinase, plnD encoding response regulator, and plnE and plnF encoding structural genes of bacteriocin were significantly upregulated in co-cultivation, showing that the bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation may be regulated by LuxS/AI-2-mediated quorum sensing system. In order to further demonstrate the role of LuxS/AI-2-mediated quorum sensing system in the bacteriocin synthesis of L. plantarum NMD-17, plasmids pUC18 and pMD18-T simple were used as the skeleton to construct the suicide plasmids pUC18-UF-tet-DF and pMD18-T simple-plnB-tet-plnD for luxS and plnB-plnD gene deletion, respectively. luxS and plnB-plnD gene knockout mutants were successfully obtained by homologous recombination. luxS gene knockout mutant lost its AI-2 synthesis ability, suggesting that LuxS protein encoded by luxS gene is key enzyme for AI-2 synthesis. plnB-plnD gene knockout mutant lost the ability to synthesize bacteriocin against Salmonella typhimurium ATCC14028, indicating that plnB-plnD gene was a necessary gene for bacteriocin synthesis of L. plantarum NMD-17. Bacteriocin synthesis, cell numbers, and AI-2 activity of luxS or plnB-plnD gene knockout mutants in co-cultivation with L. reuteri NMD-86 were obviously lower than those of wild-type strain in co-cultivation at 6–9 h (P < 0.01). The results showed that LuxS/AI-2-mediated quorum sensing system played an important role in the bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation.


Introduction
Bacteriocins are antimicrobial peptides secreted by bacteria that inhibit the growth of closely related microorganisms (Cavicchioli et al. 2019).In recent years, bacteriocins produced by lactic acid bacteria have received special attention due to their potential use as food preservatives (De Giani et al. 2019;Qiao et al. 2020;Dai et al. 2022;Kumari et al. 2022).However, commercialized bacteriocins are limited in number, e.g., nisin and pediocin PA-1 (Turcotte et al. 2004;Anastasiadou et al. 2008).Many bacteriocins are not used as biopreservatives in food due to its narrow inhibitory spectrum, narrow pH range, heat instability, or relatively low yield.
L. plantarum NMD-17 was isolated from "koumiss," a traditional fermented dairy product from Inner Mongolia in China.The bacteriocin named plantaricin MX produced by L. plantarum NMD-17 had the characteristics of broad spectrum, wider pH tolerance, heat stability, and generational stability.However, the amount of bacteriocin synthesis needs to be further increased.Our studies demonstrated that the bacteriocin synthesis of L. plantarum NMD-17 was remarkably enhanced in co-cultivation with L. reuteri NMD-86, accompanied by an increase in cell numbers of L. plantarum NMD-17 and AI-2 activity, which was associated with quorum sensing (QS) (Man and Xiang 2021).
1 3 QS is a bacterial intercommunication system that controls the expression of multiple genes in response to population density (Mukherjee and Bassler 2019;Zhu et al. 2020).QS has been identified in both Gram-negative and Gram-positive species of bacteria, and is used to regulate diverse functions, such as biofilm formation (Rajkumari et al. 2018), antibiotic susceptibility (Kaur et al. 2020), flagellar motility (Yang and Defoirdt 2015), and sporulation (Lazazzera 2000).A number of chemically distinct families of QS molecules have been identified.The most intensively investigated family is peptide autoinducers in Gram-positive bacteria.A second type of QS system is found in a wide variety of bacteria, including both Gram-negative and Gram-positive species, termed the LuxS or autoinducer 2 (AI-2)-mediated quorum sensing system, and the structure of AI-2 was reported to be a furanosyl-borate diester (Join et al. 2007;Xiong et al. 2020).AI-2 is formed chemically from 4,5-dihydroxy-2,3-pentanedione that is generated by the action of LuxS AI synthase on S-ribosylhomocysteine.The AI-2-mediated quorum sensing system may operate as a universal system for many bacteria possessing a characteristic luxS gene that encodes LuxS AI synthase.The luxS gene is highly conserved among many species of Gram-positive bacteria and Gram-negative bacteria (Asanuma et al. 2004).The previous study of QS system mainly focused on pathogenic bacteria, e.g., Pseudomonas fluorescens (Li et al. 2018a, b), Pseudomonas aeruginosa (Ahmed and Salih 2019), Staphylococcus epidermidis (Xue et al. 2015), Streptococcus mutans (Merritt et al. 2003), and Streptococcus suis (Han and Lu 2009).But the effects of LuxS/AI-2-mediated quorum sensing system on bacteriocin synthesis of lactic acid bacteria (LAB) strains in co-culturation are less well known; one logical possibility is that it functions to allow bacteria to optimize gene expression in response to the total cell density of all luxS-containing species occupying the same niche.
The aim of our study was to construct the suicide plasmids for luxS and plnB-plnD gene knockout, successfully acquire luxS and plnB-plnD gene knockout mutants by electrotransformation, and analyze the regulatory mechanisms of LuxS/AI-2-mediated quorum sensing system on the bacteriocin synthesis of L. plantarum NMD-17 in mono-cultivation and co-cultivation.

Strains, media, culture conditions, and plasmids
Bacterial strains and plasmids used in this experiment are listed in Table 1.The plantaricin MX-producer L. plantarum NMD-17 was separated from "koumiss" of Inner Mongolia.L. reuteri NMD-86, which could not have the ability to inhibit Salmonella typhimurium ATCC14028, could induce the bacteriocin synthesis of L. plantarum NMD-17 in cocultivation.L. plantarum NMD-17 or L. reuteri NMD-86 were incubated in De Man-Rogosa-Sharp (MRS) (HKM, China) broth at 37 °C.Salmonella typhimurium ATCC14028 was used as indicator strain to test the antimicrobial activity, and cultivated in nutrient broth medium (NB) (HKM, China) at 37 °C.Vibrio harveyi BB120 or V. harveyi BB170 were oscillated (160 rpm) in Zobell 2216 broth at 30 °C for 24 h; Zobell 2216 broth was prepared as described by Man et al. (2014).Escherichia coli DH5α was cultured in

DNA isolation, amplification of corresponding genes for construction of recombinant plasmids
Total genomic DNA of L. plantarum NMD-17 was abstracted by Takara MiniBEST Bacteria Genomic DNA Extraction Kit (Takara, China), and used as template to amplify the gene fragments used for construction of recombinant plasmids.
As shown in Table 2, primers luxS-up-F and luxS-up-R and luxS-down-F and luxS-down-R were applied to amplify the upstream fragment and downstream fragment for luxS gene knockout, respectively.Both upstream and downstream fragments include partial sequences of luxS gene and its flanking sequences.Primers plnB-plnD-F and plnB-plnD-R were applied to amplify the plnB-plnD gene with corresponding restriction sites for gene knockout.Plasmid pBR322 was applied as template; the tetracycline genes for luxS gene and plnB-pinD gene knockout were amplified by primer tet-s-F and tet-s-R and tet-BD-F and tet-BD-R, respectively.The amplification procedure was as below: 94 °C for 3 min, 30 cycles of 94 °C for 30 s, annealing temperature for 1 min, and 72 °C for 1 min.The amplified products were detected by agarose gel electrophoresis and sequenced.

Construction the luxS gene knockout mutant of L. plantarum NMD-17
This suicide plasmid, which is unable to replicate in lactic acid bacteria, contains an upstream fragment and a downstream fragment of luxS gene.Both genes, which are flanking the tetracycline gene, were used as homologous DNA for allelic exchange the corresponding sequences from the chromosome of L. plantarum NMD-17 to achieve the luxS gene knockout.The suicide plasmid pUC18-UF-tet-DF was electrotransformed into L. plantarum NMD-17 in the circular state or linearized state cut by EcoR I and Hind III.luxS gene knockout mutants were screened by MRS agar plate with tetracycline.Tetracycline-resistant strains were further identified by PCR using primers luxS-Y-F and luxS-Y-R.

Construction the plnB-plnD gene knockout mutant of L. plantarum NMD-17
The sequence of plnB-plnD with corresponding restriction sites was inserted into plasmid pMD18-T simple, then an internal fragment was removed by EcoR I to inactivate plnB-plnD genes, and the tetracycline gene was inserted between two EcoR I sites to construct the suicide plasmid pMD18-T simple-plnB-tet-plnD.The suicide plasmid pMD18-T simple-plnB-tet-plnD was electroconverted into L. plantarum NMD-17 in the circular state or linearized state with Xba I and kpn I. plnB-plnD gene knockout mutants were screened by MRS agar plate with tetracycline.Tetracycline-resistant strains were further identified by PCR using primers BD-Y-F and BD-Y-R.

Preparation of mono-cultivation culture and co-cultivation culture
Mono-cultivation culture was obtained as follows: fresh MRS broth was inoculated with 1% of an overnight culture of luxS knockout mutant or plnB-plnD knockout mutant (about 10 9 CFU/mL), and then grown for 30 h at 30 °C.Inhibition diameters, cell numbers, and AI-2 activity were tested every 3 h.Wild-type strain cultured in mono-cultivation was applied as the control.Co-cultivation culture was obtained as follows: fresh MRS broth was inoculated with 1% of an overnight culture of luxS gene mutant or plnB-plnD knockout mutant (approximately 10 9 CFU/mL) plus 0.5% of an overnight culture of L. reuteri NMD-86 (about 10 8 CFU/mL), and then grown for 30 h at 30 °C.Inhibition diameters, cell numbers, and AI-2 activity were tested every 3 h.Wild-type strain cultured in co-cultivation was applied as the control.

Measurement of antibacterial activity, cell numbers, and AI-2 activity
The antibacterial activity, cell numbers, and AI-2 activity were detected as reported by Man et al. (2014).Agar well diffusion assay (AWDA) was applied to test the inhibition diameters, and S. typhimurium ATCC14028 was used as indicator strain.Plate colony counting method was used to measure the cell numbers.V. harveyi BB170 was used as reporter strain, and AI-2 activity was shown as the foldinduction of V. harveyi BB120 over background.

Statistical analysis
The results were shown as the mean values ± standard deviation of three parallel experiments.Statistical analysis was carried out using SPSS 19.0.

Cloning of upstream fragment and downstream fragment for luxS gene knockout
Primers luxS-up-F and luxS-up-R and luxS-down-F and luxS-down-R were used to amplify upstream and downstream fragments of luxS genes with corresponding restriction sites by PCR (Table 2).The agarose gel electrophoresis and sequencing demonstrated that specific bands of 645 bp (Fig. 1A) and 634 bp (Fig. 1B) were obtained, and the upstream and downstream fragments of luxS gene were successfully amplification.Both upstream and downstream fragments include partial sequences of luxS gene and its flanking sequences, respectively.

Cloning of plnB-plnD gene
The fragment of plnB-plnD gene with corresponding restriction sites was amplified by PCR using primers plnB-plnD-F and plnB-plnD-R (Table 2).The results of agarose gel electrophoresis and sequencing showed that only one specific band of 2102 bp was acquired (Fig. 2); the twocomponent regulatory system coding gene plnB-plnD was successfully amplification.

Cloning of tetracycline gene
Plasmid pBR322 was applied as template; the primers tets-F and tet-s-R and tet-BD-F and tet-BD-R were used to amplify the tetracycline resistance gene fragments with corresponding restriction sites by PCR (Table 2), which were used to construct the recombinant plasmids for luxS gene and plnB-plnD gene knockout.The results of agarose gel electrophoresis and sequencing indicated that specific bands of 1405 bp (Fig. 3A) and 1408 bp (Fig. 3B) were obtained and sequenced correctly.To further analyze the function of luxS gene on bacteriocin synthesis in L. plantarum NMD-17, the recombinant plasmid and its linearized state were electroconverted into L. plantarum NMD-17 for homologous recombination.luxS gene knockout mutants were screened by tetracycline-resistant plates and PCR identification using primers luxS-Y-F and luxS-Y-R.The specific bands of about 2.7 kb (Fig. 5A) or 1.4 kb (Fig. 5B) were amplified by PCR to identify luxS gene knockout mutant or unhomologous recombinant strain, respectively.The PCR analysis and sequencing result showed that the luxS gene knockout mutant had been successfully constructed to better understand the effect of luxS gene on bacteriocin production of L. plantarum NMD-17.To study the role of plnB-plnD gene on bacteriocin synthesis of L. plantarum NMD-17, the recombinant plasmid and its linearized state were electrotransformed into L. plantarum NMD-17.Tetracycline-resistant strains were obtained and identified by tetracycline-resistant plates and PCR using primers BD-Y-F and BD-Y-R.The specific bands of about 3.0 kb (Fig. 6A) or 2.0 kb (Fig. 6B) were amplified by PCR to identify plnB-plnD gene knockout mutant or unhomologous recombinant strain, respectively.The sequencing results demonstrated that the plnB-plnD knockout mutant had been successfully constructed.

Comparative bacteriocin synthesis, cell numbers, and AI-2 activity studies in luxS gene knockout mutant and wild-type strain
Figure 7A demonstrates the antibacterial activity of luxS gene knockout mutant and wild-type strain in mono-cultivation and in co-cultivation.Inhibition diameters of luxS gene knockout mutant in mono-cultivation had no significant difference with that of wild-type strain in mono-cultivation (P > 0.05), indicating that the luxS gene was not an essential gene for the bacteriocin synthesis of L. plantarum NMD-17.Inhibition diameters of luxS gene knockout mutant in co-cultivate with L. reuteri NMD-86 were obviously lower than that shown by wild-type strain in co-cultivation during 6-30 h (P < 0.05).The result indicated that luxS gene had an important effect on bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation.
As shown in Fig. 7B, cell numbers of luxS gene knockout mutant in mono-cultivation were lower than those shown by wild-type strain in mono-cultivation, but the difference was not significant (P > 0.05).The growth kinetics of luxS gene knockout mutant approximately kept consistency with that of wild-type strain, showing that deletion of luxS gene has little effect on the growth of L. plantarum NMD-17.When luxS gene mutant was co-cultured with L. reuteri NMD-86, cell numbers of luxS gene knockout mutant were lower than those shown by wild-type strain in co-cultivation during 6-18 h (P < 0.01).
As shown in Fig. 7C, none of AI-2 activity was detected in luxS gene knockout mutant in mono-cultivation, indicating that luxS gene is an essential gene for the AI-2 synthesis of L. plantarum NMD-17.AI-2 activity of luxS gene knockout mutant in co-culture with L. reuteri NMD-86 was significantly lower than that shown by wild-type strain in co-cultivation (P < 0.01), and AI-2 activity of luxS gene knockout mutant in co-cultivation reached maximum value 6 h later than that of wild-type strain in co-cultivation.Figure 8A shows the antibacterial activity of plnB-plnD gene knockout mutant and wild-type strain in mono-cultivation and in co-cultivation.When L. plantarum NMD-17 cocultivated with L. reuteri NMD-86, bacteriocin synthesis was higher than that of L. plantarum NMD-17 in monocultivation, and induction effect was most obvious in 6-9 h (P < 0.01).No antibacterial activity was detected when the plnB-plnD gene knockout mutant was cultivated alone, indicating that plnB-plnD gene was an essential gene for bacteriocin synthesis in L. plantarum NMD-17.Inhibition diameters of plnB-plnD mutant in co-cultivate with L. reuteri NMD-86 were significantly lower than that shown by wild-type strain in co-cultivation during 6-30 h (P < 0.01), demonstrating that plnB-plnD gene had an important effect on the bacteriocin synthesis in co-cultivation.As shown in Fig. 8B, there was no significant difference in the cell numbers of plnB-plnD mutant in mono-cultivation compared with the cell numbers of wild-type strain in monocultivation (P > 0.05).The growth kinetics curves of plnB-plnD gene knockout mutant and wild-type strain were consistent, indicating that the deletion of plnB-plnD did not affect the growth of L. plantarum NMD-17.Cell numbers of plnB-plnD gene knockout mutant in co-cultivation with L. reuteri NMD-86 were significantly lower than those shown by wild-type strain in co-cultivation at 6-9 h (P < 0.01).
As shown in Fig. 8C, there was no significant difference between the AI-2 activity of plnB-plnD gene knockout mutant and wild-type strain in mono-cultivation (P > 0.05).AI-2 activity of plnB-plnD gene knockout mutant in cocultivation with L. reuteri NMD-86 was significantly lower than that of wild-type strain in co-cultivation at 6-12 h (P < 0.01), and the time required for the former to achieve maximum AI-2 activity is 3 h later than the latter.

Discussion
Our previous studies indicated that the bacteriocin synthesis of L. plantarum NMD-17 was remarkably increased in co-cultivation with L. reuteri NMD-86.The induction of bacteriocin synthesis had very significant positive correlation with cell numbers of L. plantarum NMD-17 and AI-2 activity in co-cultivation, which is in accord with the density-dependent mode of QS regulatory system.The results of quantitative real-time PCR further demonstrated that the bacteriocin synthesis might be regulated by LuxS/AI-2-mediated quorum sensing system during co-cultivation.LuxS/AI-2-mediated quorum sensing system is an interspecies quorum sensing system, which is composed of three-component regulatory system (3CRS) involving signal molecule (AI-2), histidine protein kinase (HPK), and response regulator (RR).HPK and RR are called two-component regulatory system (2CRS) (Gobbetti et al. 2007;Han and Lu 2009).Similar to the L. plantarum JDM1, L. plantarum UCMA3037, and L. plantarum 16 (Li et al. 2016), 2CRS was consisted of PlnB (a HPK) and PlnD (a RR) in L. plantarum NMD-17.The 2CRS of L. plantarum NMD-17 represented significant differences with regard to that of L. plantarum ZJ316, L. plantarum STIII, L. plantarum ATCC14917, L. plantarum J51, L. plantarum UL4, and L. plantarum BFE5092 (Navarro et al. 2008;Tai et al. 2015).
In our previous studies, PlnB protein including six transmembrane helices at the N-terminal was a cytoplasmic membrane protein by CDD analysis; the results demonstrated that PlnB protein possessed the ability to complete the signal transduction.PlnD protein having DNA-binding domains and signal receiver domain pertained to the LytTR/ AlgR family; the results showed that it possessed the ability to receive signals from PlnB, and activate the genes involved in bacteriocin production (Man and Xiang 2021).
AI-2 is formed from 4,5-dihydroxy-2,3-pentanedione that is generated by the action of LuxS AI-2 synthase encoded by luxS gene on S-ribosylhomocysteine (Ascenso et al. 2011;Wu et al. 2020).The intermediate process of AI-2 synthesis was interrupted by the knockout of luxS gene.The previous study of QS system focused on pathogenic bacteria (Kozlova et al. 2008;Han and Lu 2009;Li et al. 2018a, b;Wang et al. 2018;Sun et al. 2020); the effect of luxS gene on bacteriocin synthesis of L. plantarum was not fully understood.A mutation in luxS gene produced no detectable AI-2 activity; this result demonstrated that luxS gene is required for AI-2 production in L. plantarum NMD-17.The deletion of luxS gene in L. plantarum NMD-17 did not result in noticeable changes in growth kinetics and antibacterial activity in mono-cultivation, implying that luxS gene does not play a significant role in the growth and bacteriocin synthesis of L. plantarum NMD-17 in mono-cultivation.
Bacteriocin synthesis, cell numbers, and AI-2 activity of L. plantarum NMD-17 in co-cultivation indicated positive relations during 6-9 h; the above results were consistent with the definition of quorum sensing named cell population density-dependent regulation of gene expression (Bowdish et al. 2005;Funck et al. 2020;Kareb and Aïder 2020).Similar to previously described for L. plantarum NC8 and L. plantarum J23, the induction of bacteriocin synthesis happened at 6-9 h in co-cultivation with inducing strain.Cell numbers, AI-2 activity, and antibacterial activity of luxS gene knockout mutant in co-cultivation were obviously lower than that shown by wild-type strain in co-cultivation during 6-9 h.Similar to Lactobacillus sakei Lb706 (Axelsson and Holck 1995) and L. plantarum KLDS1.0391(Man et al. 2014), the strains lost their ability to synthesize bacteriocin because of the deletion of 2CRS encoding genes; plnB-plnD gene knockout mutant failed to produce detectable antimicrobial activity against Salmonella typhimurium ATCC14028, suggesting that plnB-plnD gene is essential for bacteriocin synthesis of L. plantarum NMD-17.Cell numbers, AI-2 activity, and antibacterial activity of plnB-plnD gene knockout mutant in co-cultivation were significantly lower than that shown by wild-type strain in co-cultivation at 6-9 h.The above results further testified that the bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation was regulated by luxS/AI-2-mediated quorum sensing system.
In summary, the mechanism of induction of bacteriocin synthesis in co-cultivation might be that the presence of bacteriocin-inducing strain L. reuteri NMD-86 was recognized as an external stimulus by L. plantarum NMD-17, and brought about the accumulation of signaling molecule AI-2 as the cell density of L. plantarum NMD-17 increases.When the concentration of AI-2 reached the threshold in the external environment, PlnB protein served as a membrane-located sensor for receiving signal molecules and transmitted the signal by phosphorylation to PlnD protein, which conversely bound DNA to trigger the transcription of corresponding genes causing the enhancement of bacteriocin synthesis (Fig. 9).Nevertheless, when luxS gene knockout mutant co-cultivated with L. reuteri NMD-86, the concentration of signal molecular AI-2 could not reach the threshold to induce bacteriocin synthesis.When plnB-plnD gene knockout mutant co-cultivated with L. reuteri NMD-86, transport pathway of signaling molecule AI-2 was cut by the deletion of plnB-plnD gene.The regulatory mechanism of LuxS/AI-2-mediated quorum sensing system in bacteriocin synthesis of L. plantarum NMD-17 will be further investigated by proteomic and transcriptomics.original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Function
GAT TAC CTG GGG TGA AC CCC AAG CTT CCA ACT AAT TGC GAC CGA Pst I Hind III Amplification for downstream fragment of luxS gene plnB-plnD-F plnB-plnD-R GCTCT AGA ATG CTG GAT TTT GGT GTT GTT GAT CGG GGT ACC CTA GTT GTC TCT CAA CAA CTT ATT C Xba I kpn I Amplification for plnB-pinD gene tet-s-F tet-s-R CGAG CTC TTC TCA TGT TTG ACA GCT T AACTG CAG TAA TAG ATA TGT TCT GCC A Sac I Pst I Tetracycline gene used for luxS gene mutation tet-BD-F tet-BD-R CCG GAA TTC TTC TCA TGT TTG ACA GCT TAT CAT C CCG GAA TTC TAA TAG ATA TGT TCT GCC AAG GGT T EcoR I EcoR I Tetracycline gene used for plnB-plnD gene mutation luxS-Y-F luxS-Y-R CGA CGG AAA CTA CAG ACC TTG GTC GA CCA ACT AAT TGC GAC CGA CGT ATC Identification of luxS gene mutant BD-Y-F BD-Y-R GAT AAA TTA GGT GGT GTC CCGC GTC AAA TAA CAC TGA CAG AAG CTC T Identification of plnB-plnD gene mutant 1 3

Fig. 1
Fig. 1 Analysis of the PCR products of upstream fragments (A) and downstream fragments (B) of luxS gene used for construction of recombinant plasmid by agarose gel electrophoresis.M, marker DL2000.Lane 1, amplicon

Figure
Figure 4A demonstrates the construction of the recombinant plasmid for luxS gene knockout.The recombinant plasmid pUC18-UF-tet-DF includes an upstream fragment of 645 bp, a downstream fragment of 634 bp, and a tetracycline gene fragment of 1405 bp between the upstream and downstream fragments, applied as homologous DNA for allelic exchange the luxS gene in L. plantarum NMD-17.To further analyze the function of luxS gene on bacteriocin synthesis in L. plantarum NMD-17, the recombinant plasmid and its linearized state were electroconverted into L. plantarum NMD-17 for homologous recombination.luxS gene knockout mutants were screened by tetracycline-resistant plates and PCR identification using primers luxS-Y-F and luxS-Y-R.The specific bands of about 2.7 kb (Fig.5A) or 1.4 kb (Fig.5B) were amplified by PCR to identify luxS gene knockout mutant or unhomologous recombinant strain, respectively.The PCR analysis and sequencing result showed that the luxS gene knockout mutant had been successfully constructed to better understand the effect of luxS gene on bacteriocin production of L. plantarum NMD-17.

FigureFig. 2 Fig. 3
Figure4Bshows the construction of the recombinant plasmid for plnB-plnD gene knockout.The sequence of plnB-plnD with the restriction sites of Xba I and kpn I was inserted into plasmid pMD18-T simple, then an inner fragment of around 560 bp was dislodged by EcoR I, and the tetracycline gene of 1408 bp was inserted between two EcoR I sites to construct the recombinant plasmid pMD18-T simple-plnB-tet-plnD.

Fig. 4
Fig. 4 Schematic illustration of construction of luxS (A) or plnB-plnD (B) recombinant plasmids.A The recombinant plasmid pUC18-UF-tet-DF includes an upstream fragment, a downstream fragment, and a tetracycline gene fragment between the upstream and downstream fragments, used as homologous DNA for allelic exchange the luxS gene sequences in L. plantarum NMD-17.B The sequence of plnB-plnD was inserted into plasmid pMD18-T simple, then an inner fragment was dislodged by EcoR I to inactivate plnB-plnD genes, and the tetracycline gene was inserted between two EcoR I sites to construct the recombinant plasmid pMD18-T simple-plnB-tet-plnD

Table 1
Bacterial strains and plasmids used in this experiment

Table 2
PCR primers used in this experiment