Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis

Nisin is a small gene-encoded antimicrobial protein produced by Lactococcus lactis that contains unusual dehydroalanine and dehydrobutyrine residues. The reactivity of these residues toward nucleophiles was explored by reacting nisin with a variety of mercaptans. The kinetics of reaction with 2-mercaptoethane-sulfonate and thioglycolate indicated that the reaction pathway includes a binding step. Reaction of nisin at high pH resulted in the formation of multimeric products, apparently as a result of intramolecular and intermolecular reactions between nucleophilic groups and the dehydro residues. One of the nucleophiles had a pKa of about 9.8. The unique vinyl protons of the dehydro residues that give readily identifiable proton nuclear magnetic resonances were used to observe the addition of nucleophiles to the dehydro moiety. After reaction with nucleophiles, nisin lost its antibiotic activity and no longer showed the dehydro resonances, indicating that the dehydro groups had been modified. The effect of pH on the solubility of nisin was determined; the solubility was quite high at low pH (57 mg/ml at pH 2) and was much lower at high pH (0.25 mg/ml at pH 8 to 12), as measured before significant pH-induced chemical modification had occurred. High-performance liquid chromatography on a C18 column was an effective technique for separating unmodified nisin from its reaction products. The cyanogen bromide cleavage products of nisin were about 90% less active toward inhibition of bacterial spore outgrowth than was native nisin. These results are consistent with earlier observations, which suggested that the dehydro residues of nisin have a role in the mechanism of antibiotic action, in which they act as electrophilic Michael acceptors toward nucleophiles in the cellular target.     

Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis.

Nisin is a small gene-encoded antimicrobial protein produced by Lactococcus lactis that contains unusual dehydroalanine and dehydrobutyrine residues. The reactivity of these residues toward nucleophiles was explored by reacting nisin with a variety of mercaptans. The kinetics of reaction with 2-mercaptoethane-sulfonate and thioglycolate indicated that the reaction pathway includes a binding step. Reaction of nisin at high pH resulted in the formation of multimeric products, apparently as a result of intramolecular and intermolecular reactions between nucleophilic groups and the dehydro residues. One of the nucleophiles had a pKa of about 9.8. The unique vinyl protons of the dehydro residues that give readily identifiable proton nuclear magnetic resonances were used to observe the addition of nucleophiles to the dehydro moiety. After reaction with nucleophiles, nisin lost its antibiotic activity and no longer showed the dehydro resonances, indicating that the dehydro groups had been modified. The effect of pH on the solubility of nisin was determined; the solubility was quite high at low pH (57 mg/ml at pH 2) and was much lower at high pH (0.25 mg/ml at pH 8 to 12), as measured before significant pH-induced chemical modification had occurred. High-performance liquid chromatography on a C18 column was an effective technique for separating unmodified nisin from its reaction products. The cyanogen bromide cleavage products of nisin were about 90% less active toward inhibition of bacterial spore outgrowth than was native nisin. These results are consistent with earlier observations, which suggested that the dehydro residues of nisin have a role in the mechanism of antibiotic action, in which they act as electrophilic Michael acceptors toward nucleophiles in the cellular target.     

NisinZ的定点突变及突变体性质的研究

以本实验室构建的含nisZ基因的质粒pHJ2 0 1为模板 ,采用定点突变技术将乳链菌肽Z分子中B环第 8位Thr突变为Ser(T8S)、将第 2位Dhb突变为Dha和第 31位His突变为Lys(T2S H31K)以及将第 2 7位Asn突变为Lys和第 31位His突变为Lys(N2 7K H31K) ,以pMG36e为载体 ,电击转化乳酸乳球菌 (L .lactis)NZ980 0进行表达。对表达产物性质的研究结果表明 ,3个突变体的抑菌谱和溶解度未发生变化 ,其抑菌活性略有下降 ,但它们的稳定性表现各不相同 :N2 7K H31K的稳定性与NisinZ几乎一致 ,而T8S和T2S H31K的稳定性有明显提高 ,在pH9条件下10 0℃加热 5min仍不丧失抑菌活性。     

Dissection and Modulation of the Four Distinct Activities of Nisin by Mutagenesis of Rings A and B and by C-Terminal Truncation

Nisin A is a pentacyclic antibiotic peptide produced by various Lactococcus lactis strains. Nisin displays four different activities: (i) it autoinduces its own synthesis; (ii) it inhibits the growth of target bacteria by membrane pore formation; (iii) it inhibits bacterial growth by interfering with cell wall synthesis; and, in addition, (iv) it inhibits the outgrowth of spores. Here we investigate the structural requirements and relevance of the N-terminal thioether rings of nisin by randomization of the ring A and B positions. The data demonstrate that: (i) mutation of ring A results in variants with enhanced activity and a modulated spectrum of target cells; (ii) for the cell growth-inhibiting activity of nisin, ring A is rather promiscuous with respect to its amino acid composition, whereas the bulky amino acid residues in ring B abolish antimicrobial activity; (iii) C-terminally truncated nisin A mutants lacking rings D and E retain significant antimicrobial activity but are unable to permeabilize the target membrane; (iv) the dehydroalanine in ring A is not essential for the inhibition of the outgrowth of Bacillus cells; (v) some ring A mutants have significant antimicrobial activities but have decreased autoinducing activities; (vi) the opening of ring B eliminates antimicrobial activity while retaining autoinducing activity; and (vii) some ring A mutants escape the nisin immune system(s) and are toxic to the nisin-producing strain NZ9700. These data demonstrate that the various activities of nisin can be engineered independently and provide a basis for the design and synthesis of tailor-made analogs with desired activities.     

Mode of Action of Nisin Z against Listeria monocytogenes Scott A Grown at High and Low Temperatures

Nisin Z, a natural nisin variant, was recently isolated from Lactococcus lactis subspecies lactis NIZO 22186. The gene for this lantibiotic, designated nisZ, has been cloned, and its nucleotide sequence was found to be identical to that of the precursor nisin gene with the exception of a single mutation resulting in the substitution of Asn-27 for His-27 in the mature polypeptide (J. W. M. Mulders, I. J. Boerrigter, H. S. Rollema, R. J. Siezen, and W. M. de Vos, Eur. J. Biochem. 201:581-584, 1991). A K⁺ electrode was used to investigate the effect of various environmental parameters on the action of nisin Z against Listeria monocytogenes. Addition of nisin Z resulted in immediate loss of cell K⁺, depolarization of the cytoplasmic membrane, inhibition of respiratory activity, and hydrolysis and partial efflux of cellular ATP. The action of nisin Z was optimal at pH 6.0 and was significantly reduced by di- and trivalent cations. The lanthanide gadolinium (Gd³⁺) was an efficient inhibitor and prevented nisin Z activity completely at a concentration of 0.2 mM. Nisin Z-induced loss of cell K⁺ was reduced at low temperatures, presumably as a result of the increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane. In cells grown at 30°C, the action of nisin Z was prevented below 7°C, whereas in cells grown at 4°C nisin Z was able to induce K⁺ leakage at this low temperature.     

Gene encoded antimicrobial peptides, a template for the design of novel anti-mycobacterial drugs

Nisin A is the most widely characterized lantibiotic investigated to date. It represents one of the many antimicrobial peptides which have been the focus of much interest as potential therapeutic agents. This has resulted in the search for novel lantibiotics and more commonly, the engineering of novel variants from existing peptides with a view to increasing their activity, stability and solubility.The aim of this study was to compare the activities of nisin A and novel bioengineered hinge derivatives, nisin S, nisin T and nisin V. The microtitre alamar blue assay (MABA) was employed to identify the enhanced activity of these novel variants against M. tuberculosis (H37Ra), M. kansasii (CIT11/06), M. avium subsp. hominissuis (CIT05/03) and M. avium subsp. paratuberculosis (MAP) (ATCC 19698). All variants displayed greater anti-mycobacterial activity than nisin A. Nisin S was the most potent variant against M. tuberculosis, M. kansasii and M. avium subsp. hominissuis, retarding growth by a maximum of 29% when compared with nisin A. Sub-species variations of inhibition were also observed with nisin S reducing growth of Mycobacterium avium subsp. hominissuis by 28% and Mycobacterium avium subsp. paratuberculosis by 19% and nisin T contrastingly reducing growth of MAP by 27% and MAC by 16%.Nisin S, nisin T and nisin V are potent novel anti-mycobacterial compounds, which have the capacity to be further modified, potentially generating compounds with additional beneficial characteristics. This is the first report to demonstrate an enhancement of efficacy by any bioengineered bacteriocin against mycobacteria.     

Impact of pectin esterification on the antimicrobial activity of nisin‐loaded pectin particles

The relationship between pectin structure and the antimicrobial activity of nisin‐loaded pectin particles was examined. The antimicrobial activity of five different nisin‐loaded pectin particles, i.e., nisin‐loaded high methoxyl pectin, low methoxyl pectin, pectic acid, dodecyl pectin with 5.4 and 25% degree of substitution were tested in the pH range of 4.0–7.0 by agar‐diffusion assay and agar plate count methods. It was found that the degree of esterification of carboxyl group of galacturonic acid in pectin molecule is important for the antimicrobial activity of nisin‐loaded pectin particles. Nisin‐loaded particles prepared using pectic acid or the pectin with low degree of esterification exhibit higher antimicrobial activity than nisin‐loaded high methoxyl pectin particles. Pectins with free carboxyl groups or of low degree of esterification are the most suitable for particles preparation. Moreover, nisin‐loaded pectin particles were active at close to neutral or neutral pH values. Therefore, they could be effectively applied for food preservation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:245–251, 2017     

Site-directed mutagenesis of the hinge region of nisinZ and properties of nisinZ mutants

To study the role of the hinge region in nisin and to obtain mutants that exhibit altered or new biological activities and functional properties, we changed certain amino acids in the hinge region by performing site-directed mutagenesis with the nisinZ structural gene (nisZ). The results showed that the nisinZ mutants had decreased antimicrobial activities against Micrococcus flavus NCIB8166 and Streptococcus thermophilus. Interestingly, compared with wild nisinZ, mutant N20K nisinZ and M21K nisinZ displayed antimicrobial activity against gram-negative Shigella, Pseudomonas and Salmonella; and they had a higher solubility than wild-type nisinZ. At pH 8, the solubilities of N20K nisinZ and M21K nisinZ were, respectively, three-fold higher and five-fold higher than that of nisinZ. Mutant N20Q nisinZ and M21G nisinZ were considerably more stable than nisinZ at higher temperatures and neutral or alkaline pH. These mutants provided information that the central hinge region in nisinZ plays an important role in providing the conformational flexibility required for the antimicrobial activity on the membrane. Our finding documented that it may well be worth considering the construction of the new nisin mutants with changed inhibitory activity against a wide range of gram-negative bacteria and the improvement of functional properties by site-directed mutagenesis.     

Biosynthetic characterization and biochemical features of the third natural nisin variant,nisin Q,produced by Lactococcus lactis 61‐14

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.     

Stereoselective disulfide formation stabilizes the local peptide conformation in nisin mimics

Nisin is a polymacrocyclic peptide antimicrobial with high activity against Gram-positive bacteria. Lanthionine and methyllanthionine bridges, closing the macrocycles, are stabilized by thioether bonds, formed between cysteines and dehydrated serine or threonine. The role of polypeptide backbone conformation in the formation of macrocycles A and B within cysteine mutants of nisin residues 1?12 is investigated here by molecular dynamics simulations. Enantiomeric combinational space of Cys3 and Cys7 and of Cys8 and Cys11 is examined for the preference of disulfide bond formation over helical turn formation within this region. A clear preference for spontaneous disulfide formation and closure of rings 3,7 and 8,11 is demonstrated for the D-Cys3, D-Cys7, L-Cys8, L-Cys11 nisin homologue, while interlinked rings A and B are obtained through disulfide bridges between L-Cys3 and D-Cys8 and between D-Cys7 and D-Cys11. This study offers a simple designer approach to solid phase synthesis of macrocyclic peptides and lantibiotic analogues.     

Role of transmembrane pH gradient and membrane binding in nisin pore formation.

Nisin is a cationic antimicrobial peptide that belongs to the group of lantibiotics. It is thought to form oligomeric pores in the target membrane by a mechanism that requires the transmembrane electrical potential delta psi and that involves local pertubation of the lipid bilayer structure. Here we show that nisin does not form exclusively voltage-dependent pores: even in the absence of a delta psi, nisin is able to dissipate the transmembrane pH gradient (delta pH) in sensitive Lactococcus lactis cells and proteoliposomes. The rate of dissipation increases with the magnitude of the delta pH. Nisin forms pores only when the delta pH is inside alkaline. The efficiency of delta psi-induced pore formation is strongly affected by the external pH, whereas delta pH-induced pore formation is rather insensitive to the external pH. Nisin(1-12), an amino-terminal fragment of nisin, and (des-deltaAla5)-(nisin(1-32) amide have a strongly reduced capacity to dissipate the delta psi and delta pH in cytochrome c oxidase proteoliposomes and L. lactis cells. Both variants bind with reduced efficiency to liposomes containing negatively charged phospholipids, suggesting that both ring A and rings C to E play a role in membrane binding. Nisin(1-12) competes with nisin for membrane binding and antagonizes pore formation. These findings are consistent with the wedge model of nisin-induced pore formation.     

Antimicrobial action of nisin against Salmonella typhimurium lipopolysaccharide mutants.

The antimicrobial activity of nisin against outer membrane lipopolysaccharide mutants of Salmonella typhimurium LT2 was investigated. Nisin sensitivity was associated with the extent of saccharide deletions from the outer membrane core oligosaccharide. The results indicated that the core oligosaccharide in lipopolysaccharide plays a role in nisin sensitivity.     

Antimicrobial action of nisin against Salmonella typhimurium lipopolysaccharide mutants.

The antimicrobial activity of nisin against outer membrane lipopolysaccharide mutants of Salmonella typhimurium LT2 was investigated. Nisin sensitivity was associated with the extent of saccharide deletions from the outer membrane core oligosaccharide. The results indicated that the core oligosaccharide in lipopolysaccharide plays a role in nisin sensitivity.     

Engineering dehydrated amino acid residues in the antimicrobial peptide nisin.

The small antimicrobial peptide nisin, produced by Lactococcus lactis, contains the uncommon amino acid residues dehydroalanine and dehydrobutyrine and five thio ether bridges. Since these structures are posttranslationally formed from Ser, Thr, and Cys residues, it is feasible to study their role in nisin function and biosynthesis by protein engineering. Here we report the development of an expression system for mutated nisin Z (nisZ) genes, using nisin A producing L. lactis as a host. Replacement by site-directed mutagenesis of the Ser-5 codon in nisZ by a Thr codon, led to a mutant with a dehydrobutyrine instead of a dehydroalanine residue at position 5, as shown by NMR. Its antimicrobial activity was 2-10-fold lower relative to wild-type nisin Z, depending on the indicator strain used. In another mutagenesis study a double mutation was introduced in the nisZ gene by replacing the codons for Met-17 and Gly-18 by codons for Gln and Thr, respectively, as in the third lanthionine ring of the related antimicrobial peptide subtilin from Bacillus subtilis. This resulted in the simultaneous production of two mutant species, one containing a Thr residue and the other containing a dehydrobutyrine residue at position 18, both having different bacteriocidal properties.     

Engineering a disulfide bond and free thiols in the lantibiotic nisin Z.

The antimicrobial peptide nisin contains the uncommon amino acid residues lanthionine and methyl-lanthionine, which are post-translationally formed from Ser, Thr and Cys residues. To investigate the importance of these uncommon residues for nisin activity, a mutant was designed in which Thr13 was replaced by a Cys residue, which prevents the formation of the thioether bond of ring C. Instead, Cys13 couples with Cys19 via an intramolecular disulfide bridge, a bond that is very unusual in lantibiotics. NMR analysis of this mutant showed a structure very similar to that of wild-type nisin, except for the configuration of ring C. The modification was accompanied by a dramatic reduction in antimicrobial activity to less than 1% of wild-type activity, indicating that the lanthionine of ring C is very important for this activity. The nisin Z mutants S5C and M17C were also isolated and characterized; they are the first lantibiotics known that contain an additional Cys residue that is not involved in bridge formation but is present as a free thiol. Secretion of these peptides by the lactococcal producer cells, as well as their antimicrobial activity, was found to be strongly dependent on a reducing environment. Their ability to permeabilize lipid vesicles was not thiol-dependent. Labeling of M17C nisin Z with iodoacetamide abolished the thiol-dependence of the peptide. These results show that the presence of a reactive Cys residue in nisin has a strong effect on the antimicrobial properties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.     

Depletion of proton motive force by nisin in Listeria monocytogenes cells.

The basal proton motive force (PMF) levels and the influence of the bacteriocin nisin on the PMF were determined in Listeria monocytogenes Scott A. In the absence of nisin, the interconversion of the pH gradient (Z delta pH) and the membrane potential (delta psi) led to the maintenance of a fairly constant PMF at -160 mV over the external pH range 5.5 to 7.0. The addition of nisin at concentrations of greater than or equal to 5 micrograms/ml completely dissipated PMF in cells at external pH values of 5.5 and 7.0. With 1 microgram of nisin per ml, delta pH was completely dissipated but delta psi decreased only slightly. The action of nisin on PMF in L. monocytogenes Scott A was both time and concentration dependent. Valinomycin depleted only delta pH, whereas nigericin and carbonyl cyanide m-chlorophenylhydrazone depleted only delta psi, under conditions in which nisin depleted both. Four other L. monocytogenes strains had basal PMF parameters similar to those of strain Scott A. Nisin (2.5 micrograms/ml) also completely dissipated PMF in these strains.     

Frequency of bacteriocin resistance development and associated fitness costs in Listeria monocytogenes

Bacteriocin-producing starter cultures have been suggested as natural food preservatives; however, development of resistance in the target organism is a major concern. We investigated the development of resistance in Listeria monocytogenes to the two major bacteriocins pediocin PA-1 and nisin A, with a focus on the variations between strains and the influence of environmental conditions. While considerable strain-specific variations in the frequency of resistance development and associated fitness costs were observed, the influence of environmental stress seemed to be bacteriocin specific. Pediocin resistance frequencies were determined for 20 strains and were in most cases ca. 10(-6). However, two strains with intermediate pediocin sensitivity had 100-fold-higher pediocin resistance frequencies. Nisin resistance frequencies (14 strains) were in the range of 10(-7) to 10(-2). Strains with intermediate nisin sensitivity were among those with the highest frequencies. Environmental stress in the form of low temperature (10 degrees C), reduced pH (5.5), or the presence of NaCl (6.5%) did not influence the frequency of pediocin resistance development; in contrast, the nisin resistance frequency was considerably reduced (<5 x 10(-8)). Pediocin resistance in all spontaneous mutants was very stable, but the stability of nisin resistance varied. Pediocin-resistant mutants had fitness costs in the form of reduction down to 44% of the maximum specific growth rate of the wild-type strain. Nisin-resistant mutants had fewer and less-pronounced growth rate reductions. The fitness costs were not increased upon applying environmental stress (5 degrees C, 6.5% NaCl, or pH 5.5), indicating that the bacteriocin-resistant mutants were not more stress sensitive than the wild-type strains. In a saveloy-type meat model at 5 degrees C, however, the growth differences seemed to be negligible. The applicational perspectives of the results are discussed.     

Salt effect of nisin Z isolated from a marine fish on the growth inhibition of Streptococcus iniae, a pathogen of streptococcosis

A bacteriocin-producing Lactococcus lactis subsp. lactis was isolated from the intestine of olive flounder. The bacteriocin was identified as nisin Z. It was active against Gram-positive bacteria. Nisin Z at 3,200 arbitrary units (AU) was more effective in seawater than in PBS; growth of Streptococcus iniae was completely inhibited within 3 h. Nisin Z preparations with 3.5% (w/v) NaCl was the most effective against S. iniae being similar to nisin Z in seawater. Nisin Z is thus a good alternative to antibiotics to prevent streptococcosis caused by S. iniae aquaculture systems.     

Controlling Wine Malolactic Fermentation with Nisin and Nisin-Resistant Strains of Leuconostoc oenos

The polypeptide nisin (100 U/ml) prevented malolactic fermentation in wines by indigenous or intentionally added lactic acid bacteria. Nisin (100 U/ml)-resistant mutants of Leuconostoc oenos were obtained and used with nisin in wine to carry out a pure-culture malolactic fermentation in the presence or absence of other lactic acid bacteria. Nisin degradation by mutants was not observed, and residual nisin was detectable in wines 4 months after it was added. Results indicated that nisin or nisin with resistant bacterial starter cultures can be used to control malolactic fermentation in wines.