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Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Biosynthetic characterization and biochemical features of the third natural nisin variant, nisin Q, produced by Lactococcus lactis 61-14 F. Yoneyama1, M. Fukao1, T. Zendo1, J. Nakayama1 and K. Sonomoto1,2 1 Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan 2 Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, Fukuoka, Japan

Keywords bacteriocin, lactic acid bacteria, Lactococcus lactis, lantibiotic, nisin. Correspondence Takeshi Zendo, Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. E-mail: [email protected]

2007 ⁄ 1925: received 28 November 2007, revised 9 May 2008 and accepted: 4 June 2008 doi:10.1111/j.1365-2672.2008.03958.x

Abstract Aims: To characterize the genetic and biochemical features of nisin Q. Methods and Results: The nisin Q gene cluster was sequenced, and 11 putative orfs having 82% homology with the nisin A biosynthesis gene cluster were identified. Nisin Q production was confirmed from the nisQ-introduced nisin Z producer. In the reporter assay, nisin Q exhibited an induction level that was threefold lower than that of nisin A. Nisin Q demonstrated an antimicrobial spectrum similar to those of the other nisins. Under oxidizing conditions, nisin Q retained a higher level of activity than nisin A. This higher oxidative tolerance could be attributed to the presence of only one methionine residue in nisin Q, in contrast to other nisins that contain two. Conclusions: The 11 orfs of the nisin producers were identical with regard to their functions. The antimicrobial spectra of the three natural nisins were similar. Nisin Q demonstrated higher oxidative tolerance than nisin A. Significance and Impact of the Study: Genetic and biochemical features of nisin Q are similar to those of other variants. Moreover, owing to its higher oxidative tolerance, nisin Q is a potential alternative for nisin A.

Introduction Several strains of lactic acid bacteria (LAB) secrete antimicrobial peptides termed bacteriocins (Cotter et al. 2005). In particular, nisin, the most studied bacteriocin, is used as a food preservative in more than 50 countries owing to its strong antimicrobial activity and high stability (Delves-Broughton et al. 1996). It has high stability in low-pH environment, and exhibits selective toxicity, which is quite effective (at concentrations in the range of nanomolar) against gram-positive bacteria. Additionally, Lactococcus lactis, a nisin-producing LAB species, has been well utilized in fermented foods. Hence, L. lactis-derived nisin has been extensively studied as a model bacteriocin in several applications. In general, small (10 >10 >10 >10

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Figure 2 Liquid chromatography-mass spectrometry analysis of partially purified nisin Z and nisin Q from Lactococcus lactis B48 harbouring pNZnisQ. (a) Mass chromatogram ranging from 1000 to 3000 m ⁄ z. Peak 1 was identified as oxidized nisin Z. Mass spectra corresponding to peaks 2 and 3 are illustrated in (b) and (c), respectively.

Reporter assay of nisin Q and nisin A by applying the NICE system As shown before, the biosynthesis proteins of nisin Q were compatible with those of nisin Z. However, it was unclear whether nisin Q could activate the two-component system of nisin A or nisin Z. Therefore, we adopted a reporter assay for detecting b-galactosidase activity controlled by the NICE system to investigate stimulation by nisin Q against NisK of L. lactis NZ9000. Lactococcus lactis NZ9000 is a NisR- and NisK-inserted mutant, and an exogenously introduced gene into the strain using pNZ8048 is expressed by the stimulation of nisin. Consequently, pNZlacZ was introduced into L. lactis NZ9000 to compare the differences in the stimulation levels between nisin Q and nisin A, which were expressed in terms of gene expression and were found to be 73Æ6 and 242Æ6 Miller units, respectively. Thus, stimulation of the twocomponent system of nisin A by nisin Q was observed, which was at a markedly lower level than that by nisin A. 1986

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Antimicrobial activity and stability of natural nisin variants The antimicrobial spectra of the three natural nisins were examined by the turbidimetric assay. All three natural nisins exhibited considerably similar spectra against grampositive bacteria (Table 3). No effects were observed against nisin A, nisin Z and nisin Q producers and E. coli, even upon exposure to a nisin concentration of 10 lg ml)1. Compared with the other nisins, nisin Q was predicted to be a more stable variant under oxidizing conditions because the methionine at position 21 of nisin A is substituted by leucine in nisin Q. In the purification steps, we confirmed that oxidized nisin A contained an additional oxygen atom, but this was absent in oxidized nisin Q (data not shown). We verified the activity of oxidized nisin A, which was separated and confirmed by HPLC and MS. Oxidized nisin A was found to possess c. 75% activity compared with intact nisin A. Next, nisin Q and nisin A were exposed to hydrogen peroxide to confirm the difference in their oxidative tolerance. Nisin Q and nisin A incubated in the buffer in the absence of hydrogen peroxide for 72 h were used as controls, and no changes were detected in their antimicrobial activity. The activity of the controls was considered to be 100%. The activity of nisin A treated with hydrogen peroxide for 72 h decreased to 40% of that of the control (Fig. 3). On the other hand, the activity of hydrogen

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Biosynthetic and biochemical characterization of nisin Q

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Figure 3 Antimicrobial activity of nisin Q and nisin A by exposure to 0Æ02% hydrogen peroxide at 4C. Open and filled circles indicate the residual activities of nisin Q and nisin A, respectively. Positive controls indicate 100% activity and represent the activities of nisin A and nisin Q after the indicated time in the absence of hydrogen peroxide. Controls exhibited no change in activity for 3 days. Similar results were observed in two independent experiments (data not shown).

peroxide-treated nisin Q retained 64% of that of the control. LC-MS analysis was then performed to evaluate the molecular weights of oxidized nisin A and nisin Q (Fig. 4). The mass chromatogram shows the derivative peaks of oxidized nisin A (Fig. 4a). Figure 4b and c represent the respective mass spectra derived from peaks 1 and 2 of Fig. 4a. In peak 1, we can observe ions corresponding to nisin A incorporated with two oxygen atoms (Fig. 4b). Nisin A incorporated with three oxygen atoms was observed in peak 2 (Fig. 4c). However, no intact nisin A was detected under this oxidation condition. Similarly, nisin Q exposed to identical conditions was separated into a few peaks (Fig. 4d). Peak 3 was the highest peak and mainly comprised ions derived from nisin Q incorporated with an oxygen atom (Fig. 4e). Unoxidized nisin Q was also confirmed in peak 4 (Fig. 4f). When nisins were incubated without hydrogen peroxide for 72 h, only the original molecular weights corresponding to nisin Q and nisin A were observed (data not shown). Discussion In this report, we focused on differences in the biosynthesis and molecular features of the three natural nisin variants. First, we analysed the nisin Q gene cluster and characterized it by BLAST. The nisin Q gene cluster exhibited 82% homology with the nisin A cluster; this homology was significantly lower than that between the nisin A and nisin Z clusters (99%). The most catalytic and substrate recognition domains of lantibiotic biosynthesis proteins have not been identified (Siezen et al. 1996; Li et al. 2006). However, amino acid residues conserved in lantibiotic-related proteins were identified in nisin Q biosynthesis proteins (data not shown; Siezen

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Figure 4 Liquid chromatography-mass spectrometry analysis of oxidized nisin A and nisin Q after exposure to 0Æ02% hydrogen peroxide at 4C for 72 h. (a) Mass chromatogram of nisin A ranging from 1000 to 3000 m ⁄ z. (b) Mass spectrum of peak 1 in (a). (c) Mass spectrum of peak 2 in (a). (d) Mass chromatogram of nisin Q, ranging from 1000 to 3000 m ⁄ z. (e) Mass spectrum of peak 3 in (d). (f) Mass spectrum of peak 4 in (d).

et al. 1996), and the altered, inserted and deleted residues would not be essential moieties for the production of nisin Q. This assumption was strongly supported by nisin Q production from pNZnisQ-introduced L. lactis B48. There are some successful precedents for introduction of unusual amino acids into numerous peptides (Xie et al. 2004; Nagao et al. 2005), including non-lantibiotic peptides (Rink et al. 2005). Nisin Q production by a non-cognate biosynthesis apparatus performed in this study was reasonable.

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Previously, only two naturally occurring nisin variants (A and Z) had been identified, and it was confirmed that the change was caused by a single nucleotide substitution corresponding to the amino acid at position 27 (Gross and Morell 1971; Mulders et al. 1991). Moreover, the GC contents and promoters were almost identical between the nisin A and Z gene clusters (Immonen et al. 1995). However, the nisin Q gene cluster had a lower level of similarity with the other two variants as compared with that between the other variants. The GC contents of the nisin Q (34Æ5%) and nisin A gene clusters (31Æ5%) were also demonstrated to be different. These results indicate that the nisin Q locus is at a greater genetic distance from the nisin A locus than the nisin Z locus. The reporter assay revealed that the level of stimulation exerted by nisin Q on the regulation system of nisin A is lower than that by nisin A. The nisin recognition mechanisms by NisK (histidine kinase) are almost unknown, whereas the DNA regions recognized by NisR have been well characterized (Kleerebezem 2004). Plantaricin A (PlnA, an antimicrobial peptide pheromone) and its receptor PlnB (histidine kinase) comprise a quorum-sensing system that controls LAB-bacteriocin gene expression, which is the only precedent that has been well characterized by mutational analogues and receptors (Johnsborg et al. 2004; Kristiansen et al. 2005). In contrast to the equal antimicrobial activities of the l- and d-enantiomeric forms of PlnA, only the l-form could function as a pheromone molecule towards PlnB. Similarly, nisins Q and A exhibit equal antimicrobial activity; however, they exhibit different pheromone activity towards NisK. The antimicrobial and pheromone activities of bacteriocins appear to be unrelated. Structure–activity studies of nisin as a pheromone molecule using nisin variants and mutants are required. Recently, a detection system has been developed for detection of nisin from cultures or food models using the NICE system with fluorescence probes (Reunanen and Saris 2003; Hakovirta et al. 2006). This system might facilitate the detection of nisin Q. However, the sensitivity might be lower than that for nisin A or nisin Z. Based on our results, we conclude that the biosynthesis proteins of nisin Q are interchangeable with other nisin-related proteins. The substitutions among the three nisins might be optimized for production in the original assortment. The antimicrobial mechanisms of lantibiotics have been well studied recently because of their possible applications and prevention of the emergence of drug-resistant bacteria (Bauer and Dicks 2005; Breukink and de Kruijff 2006). The three nisins exhibit considerably similar patterns of antimicrobial spectra (Table 3), and we hypothesize that the mode of action of nisin Q is similar to that of the other nisins. The essential components for binding the 1988

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pyrophosphate of lipid II are 11 N-terminal residues of nisin mainly comprising two thioether rings (Hsu et al. 2004). In this region, no substituted residues were confirmed among the three natural nisins (Fig. 1). Similar to the other nisins, nisin Q probably also binds to lipid II. As lipid II was considered to be one of the most important factors for the antimicrobial mechanism of nisins, it is understandable that the three nisins had similar antimicrobial spectra. On the other hand, the roles of the other three thioether rings and hinge regions in the nisins after binding to lipid II are questions that need to be answered in the future. No significant chemical shifts in the C-terminal half of nisin were observed even when constructing a complex with lipid II in sodium dodecyl sulfate (SDS) micelles (Hsu et al. 2002) and dimethyl sulfoxide (Hsu et al. 2004). The C-termini of nisins probably play an important role in pore formation (Wiedemann et al. 2001) and septum segregation (Hasper et al. 2006), which ensue after binding to lipid II. All the substituted residues were located at the C-terminus of nisin Q, and the effects of the substitutions were unknown. All the nisin producer strains tested in this study showed cross-immunity against all the nisins (Table 3). This cross-immunity is reasonable because nisin U from S. uberis strain 42, a nisin variant other than nisin Q that is more structurally different from nisins A and Z, also showed cross-immunity against nisin A and Z producers (Wirawan et al. 2006). In addition, all our stock of the three nisin A and four nisin Z producer strains exerted cross-immunity against nisin Q tested by the spot-on-lawn method. While nisins Q and A have similar biochemical features (Zendo et al. 2003), nisin Q was more stable under oxidative conditions (Fig. 3). Nisin A harbours two methionine residues at positions 17 and 21, while nisin Q has only one methionine at position 17. Previously, it has been demonstrated that nisin A is easily oxidized at a low pH or by freeze drying, and that oxidized methionine is located at position 21 in the hinge region, whereas no additional oxygen was incorporated at position 17 on the third ring from the N-terminus (Rollema et al. 1996). In the purification steps, we could confirm the presence of an additional oxygen atom in oxidized nisin A but not in oxidized nisin Q. As previously reported, these findings indicate that methionine at position 21 of nisins A and Z is more easily oxidized than that at position 17, which exists in the rigid ring structure. As a methionine residue can receive two oxygen atoms in an oxidizing environment, nisins A and Q can accept a minimum of four and two oxygen atoms, respectively. Our data indicates that the degree of oxidization in nisin molecules leads to the reduction of antimicrobial activity (Figs 3 and 4). Nisin Q is anticipated to inhibit the growth of gram-positive food pathogens for a longer time than nisin A, which

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easily reduces the activity. Molecular characterization of bacteriocins in various environments is required for optimal selection for various applications. In this study, we investigated the biosynthetic and biochemical characterization of the third natural nisin variant, nisin Q, which is produced by L. lactis 61-14. Based on the following information, we conclude that the three natural nisins share an identical biosynthetic pathway: (i) the nisin Q gene cluster exhibited 82% homology with that of nisin A; (ii) nisin Q was produced by the nisin Z-producing strain; (iii) the two-component system of nisin A was stimulated by nisin Q. However, nisins Q and A were not completely identical, as the stimulation level of the two-component system of nisin A by nisin Q was actually lower than that by nisin A. Additionally, the three natural nisins also exhibited similar antimicrobial spectra. Nisin Q demonstrated higher stability under oxidizing condition such as a 0Æ02% hydrogen peroxide solution. We could conclude that, similar to other nisins, nisin Q has a high potential for application as an antimicrobial agent; moreover, the possibility of more useful applications in oxidizing environments has also been suggested. Acknowledgements We thank C.-B. Hu and Y. Aso for providing strains. This work was supported in part by Regional Industry Revitalization Project of the Ministry of Economy, Trade and Industry of Japan, and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS). References Aso, Y., Nagao, J., Koga, H., Okuda, K., Kanemasa, Y., Sashihara, T., Nakayama, J. and Sonomoto, K. (2004) Heterologous expression and functional analysis of the gene cluster for the biosynthesis of and immunity to the lantibiotic, nukacin ISK-1. J Biosci Bioeng 98, 429–436. Bauer, R. and Dicks, L.M. (2005) Mode of action of lipid IItargeting lantibiotics. Int J Food Microbiol 101, 201–216. Bolotin, A., Wincker, P., Mauger, S., Jaillon, O., Malarme, K., Weissenbach, J., Ehrlich, S.D. and Sorokin, A. (2001) The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res 11, 731– 753. Breukink, E. and de Kruijff, B. (2006) Lipid II as a target for antibiotics. Nat Rev Drug Discov 5, 321–332. Chakicherla, A. and Hansen, J.N. (1995) Role of the leader and structural regions of prelantibiotic peptides as assessed by expressing nisin-subtilin chimeras in Bacillus subtilis 168, and characterization of their physical, chemical, and antimicrobial properties. J Biol Chem 270, 23533–23539.

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