Molecular and genetic characterization of a novel nisin variant produced by Streptococcus uberis

Streptococcus uberis is one of the principal causative agents of bovine mastitis. In this study, we report that S. uberis strain 42 produces a lantibiotic, nisin U, which is 78% identical (82% similar) to nisin A from Lactococcus lactis. The 15.6-kb nisin U locus comprises 11 open reading frames, similar in putative functionality but differing in arrangement from that of the nisin A biosynthetic cluster. The nisin U producer strain exhibits specific resistance (immunity) to nisin U and cross-resistance to nisin A, a finding consistent with the 55% sequence similarity of their respective immunity peptides. Homologues of the nisin U structural gene were identified in several additional S. uberis strains, and in each case cross-protective immunity was expressed to nisin A and to the other producers of nisin U and its variants. To our knowledge, this is the first report both of characterization of a bacteriocin by S. uberis, as well as of a member of the nisin family of peptides in a species other than L. lactis.     

Complete nisin A gene cluster from Lactococcus lactis M78 (HM219853) — obtaining the nucleic acid sequence and comparing it to other published nisin sequences

Nisin is an antimicrobial peptide produced by Lactococcus lactis. It has a long history of safe use, mainly in food production. This bacteriocin has been studied from many aspects of genetics, biosynthesis, immunity, regulation and mode of action. The strain Lac. lactis M78 has already been described in previous studies as a good nisin A producer with equally good potential to be used in food production. The main objective of the present study was to determine the complete nucleic acid sequence of the nisin A gene cluster from this strain. This is the first time that all 11 genes that form the nisin A gene cluster were sequenced. The obtained sequence (GenBank: HM219853) was compared to other known nucleic acid sequences of bacteriocin nisin. The results of the comparison showed certain differences in sequences that might influence the structure and function of proteins involved in nisin biosynthesis, immunity and regulation.     

Comparative genomic analysis shows that Streptococcus suis meningitis isolate SC070731 contains a unique 105 K genomic island

Streptococcus suis (SS) is an important swine pathogen worldwide that occasionally causes serious infections in humans. SS infection may result in meningitis in pigs and humans. The pathogenic mechanisms of SS are poorly understood. Here, we provide the complete genome sequence of S. suis serotype 2 (SS2) strain SC070731 isolated from a pig with meningitis. The chromosome is 2,138,568 bp in length. There are 1933 predicted protein coding sequences and 96.7% (57/59) of the known virulence-associated genes are present in the genome. Strain SC070731 showed similar virulence with SS2 virulent strains HA9801 and ZY05719, but was more virulent than SS2 virulent strain P1/7 in the zebrafish infection model. Comparative genomic analysis revealed a unique 105 K genomic island in strain SC070731 that is absent in seven other sequenced SS2 strains. Further analysis of the 105 K genomic island indicated that it contained a complete nisin locus similar to the nisin U locus in S. uberis strain 42, a prophage similar to S. oralis phage PH10 and several antibiotic resistance genes. Several proteins in the 105 K genomic island, including nisin and RelBE toxin–antitoxin system, contribute to the bacterial fitness and virulence in other pathogenic bacteria. Further investigation of newly identified gene products, including four putative new virulence-associated surface proteins, will improve our understanding of SS pathogenesis.     

Lipid-free NisI: interaction with nisin and contribution to nisin immunity via secretion

Nisin-producing Lactococcus lactis cells protect their own cytoplasmic membrane by specific immunity proteins, NisF/E/G and NisI, a transporter complex and a lipoprotein, respectively. A portion of NisI is secreted to the medium in a lipid-free form (LF-NisI). Here, kinetics of the interaction between nisin and LF-NisI was examined by surface plasmon resonance analysis. The affinity constant K_D for the interaction was calculated to be in the micromolar range. Contribution of the secreted LF-NisI to nisin immunity was studied by replacing the lipoprotein specific nisI signal sequence with a secretion signal of non-lipoprotein origin. Secretion of LF-NisI in NisF/E/G-expressing L. lactis strain NZ9840 increased significantly its nisin tolerance suggesting that the lipid-free form of NisI could have a supportive role in nisin immunity.     

Loss of IrpT Function in Lactococcus lactis subsp. lactis N8 Results in Increased Nisin Resistance

The antibiotic nisin, produced by Lactococcus lactis subsp. lactis N8, offers an extensive commercial prospect as natural food preservatives. The nisin immunity of the L. lactis strains is regulated by a variety of mechanisms. In this study, we isolated a L. lactis L31 strain with increased nisin resistance from a mini-Mu transposon mutant pool of strain N8. The single Mu insertion in strain L31 was in the irpT gene with unknown function. By comparing the proteomic profiles of L. lactis L31 and its parental strain, we found that changes occurred in the synthesis of a protein involved in cell wall biosynthesis (RmlD). Strain L31 had 13.7% higher content of rhamnose in the cell wall than the N8 strain. Overexpression of RmlD involved in the synthesis of dTDP-l-rhamnose in the nisin-sensitive MG1363 strain increased nisin resistance of the strain. The results indicate that these cellular proteins effected nisin resistance in L. lactis N8.     

Lantibiotic Immunity: Inhibition of Nisin Mediated Pore Formation by NisI

Nisin, a 3.4 kDa antimicrobial peptide produced by some Lactococcus lactis strains is the most prominent member of the lantibiotic family. Nisin can inhibit cell growth and penetrates the target Gram-positive bacterial membrane by binding to Lipid II, an essential cell wall synthesis precursor. The assembled nisin-Lipid II complex forms pores in the target membrane. To gain immunity against its own-produced nisin, Lactococcus lactis is expressing two immunity protein systems, NisI and NisFEG. Here, we show that the NisI expressing strain displays an IC₅₀ of 73±10 nM, an 8–10-fold increase when compared to the non-expressing sensitive strain. When the nisin concentration is raised above 70 nM, the cells expressing full-length NisI stop growing rather than being killed. NisI is inhibiting nisin mediated pore formation, even at nisin concentrations up to 1 µM. This effect is induced by the C-terminus of NisI that protects Lipid II. Its deletion showed pore formation again. The expression of NisI in combination with externally added nisin mediates an elongation of the chain length of the Lactococcus lactis cocci. While the sensitive strain cell-chains consist mainly of two cells, the NisI expressing cells display a length of up to 20 cells. Both results shed light on the immunity of lantibiotic producer strains, and their survival in high levels of their own lantibiotic in the habitat.     

Improving nisin production by increasing nisin immunity/resistance genes in the producer organism Lactococcus lactis

The effect on nisin production of increasing nisin immunity/resistance genes in Lactococcus lactis subsp. lactis MG1363 was investigated. The 60-kb nisin immunity/resistance plasmid pND300, which was isolated from a non-nisin-producing strain, encodes five genes involved in nisin immunity/resistance, which are very similar to those of the immunity/resistance system encoded by the nisin-production transposon. The introduction of pND300 into MG1363(TnNip) resulted in the construct being able to produce significantly more nisin than the parent MG1363(TnNip). The introduction of pND314, which contains the nisin immunity/resistance genes subcloned into pSA3, into MG1363(TnNip) allowed the strain to grow more rapidly than the parent MG1363(TnNip) with a concomitant increase in the rate of nisin production. This work illustrates that introduction of pND300 and a derivative containing the nisin immunity/resistance system of pND300 into MG1363 (TnNip) can result in significant alterations to the kinetics of nisin production. These observations indicate approaches that may be used successfully to improve the economics of nisin production. Received: 11 February 1998 / Received revision: 25 June 1998 / Accepted: 27 June 1998     

A Nisin Bioassay Based on Bioluminescence

A Lactococcus lactis subsp. lactis strain that can sense the bacteriocin nisin and transduce the signal into bioluminescence was constructed. By using this strain, a bioassay based on bioluminescence was developed for quantification of nisin, for detection of nisin in milk, and for identification of nisin-producing strains. As little as 0.0125 ng of nisin per ml was detected within 3 h by this bioluminescence assay. This detection limit was lower than in previously described methods.     

Nisin H Is a New Nisin Variant Produced by the Gut-Derived Strain Streptococcus hyointestinalis DPC6484

Accumulating evidence suggests that bacteriocin production represents a probiotic trait for intestinal strains to promote dominance, fight infection, and even signal the immune system. In this respect, in a previous study, we isolated from the porcine intestine a strain of Streptococcus hyointestinalis DPC6484 that displays antimicrobial activity against a wide range of Gram-positive bacteria and produces a bacteriocin with a mass of 3,453 Da. Interestingly, the strain was also found to be immune to a nisin-producing strain. Genome sequencing revealed the genetic determinants responsible for a novel version of nisin, designated nisin H, consisting of the nshABTCPRKGEF genes, with transposases encoded between nshP and nshR and between nshK and nshG. A similar gene cluster is also found in S. hyointestinalis LMG14581. Notably, the cluster lacks an equivalent of the nisin immunity gene, nisI. Nisin H is proposed to have the same structure as the prototypical nisin A but differs at 5 amino acid positions—Ile1Phe (i.e., at position 1, nisin A has Ile while nisin H has Phe), Leu6Met, Gly18Dhb (threonine dehydrated to dehydrobutyrine), Met21Tyr, and His31Lys—-and appears to represent an intermediate between the lactococcal nisin A and the streptococcal nisin U variant of nisin. Purified nisin H inhibits a wide range of Gram-positive bacteria, including staphylococci, streptococci, Listeria spp., bacilli, and enterococci. It represents the first example of a natural nisin variant produced by an intestinal isolate of streptococcal origin.     

Inhibition of Listeria innocua in Cheddar Cheese by Addition of Nisin Z in Liposomes or by In Situ Production in Mixed Culture

The effect of addition of purified nisin Z in liposomes to cheese milk and of in situ production of nisin Z by Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in the mixed starter on the inhibition of Listeria innocua in cheddar cheese was evaluated during 6 months of ripening. A cheese mixed starter culture containing Lactococcus lactis subsp. lactis biovar diacetylactis UL719 was selected for high-level nisin Z and acid production. Experimental cheddar cheeses were produced on a pilot scale, using the selected starter culture, from milk with added L. innocua (10⁵ to 10⁶ CFU/ml). Liposomes with purified nisin Z were prepared from proliposome H and added to cheese milk prior to renneting to give a final concentration of 300 IU/g of cheese. The nisin Z-producing strain and nisin Z-containing liposomes did not significantly affect cheese production and gross chemical composition of the cheeses. Immediately after cheese production, 3- and 1.5-log-unit reductions in viable counts of L. innocua were obtained in cheeses with encapsulated nisin and the nisinogenic starter, respectively. After 6 months, cheeses made with encapsulated nisin contained less than 10 CFU of L. innocua per g and 90% of the initial nisin activity, compared with 10⁴ CFU/g and only 12% of initial activity in cheeses made with the nisinogenic starter. This study showed that encapsulation of nisin Z in liposomes can provide a powerful tool to improve nisin stability and inhibitory action in the cheese matrix while protecting the cheese starter from the detrimental action of nisin during cheese production.     

Lysis of Lactococcus lactis subsp. cremoris SK110 and Its Nisin-Immune Transconjugant in Relation to Flavor Development in Cheese

To develop a nisin-producing cheese starter, Lactococcus lactis subsp. cremoris SK110 was conjugated with transposon Tn5276-NI, which codes for nisin immunity but not for nisin production. Cheese made with transconjugant SK110::Tn5276-NI as the starter was bitter. The muropeptide of the transconjugant contained a significantly greater amount of tetrapeptides than the muropeptide of strain SK110, which could have decreased the susceptibility of the cells to lysis and thereby the release of intracellular debittering enzymes.     

Effects of Lactococcus lactis on Composition of Intestinal Microbiota: Role of Nisin

This study examined the ability of (i) pure nisin, (ii) nisin-producing Lactococcus lactis strain CHCC5826, and (iii) the non-nisin-producing L. lactis strain CHCH2862 to affect the composition of the intestinal microbiota of human flora-associated rats. The presence of both the nisin-producing and the non-nisin-producing L. lactis strains significantly increased the number of Bifidobacterium cells in fecal samples during the first 8 days but decreased the number of enterococci/streptococci in duodenum, ileum, cecum, and colon samples as detected by selective cultivation. No significant changes in the rat fecal microbiota were observed after dosage with nisin. Pearson cluster analysis of denaturing gradient gel electrophoresis profiles of the 16S rRNA genes present in the fecal microbial population revealed that the microbiota of animals dosed with either of the two L. lactis strains were different from that of control animals dosed with saline. However, profiles of the microbiota from animals dosed with nisin did not differ from the controls. The concentrations of nisin estimated by competitive enzyme-linked immunosorbent assay (ELISA) were approximately 10-fold higher in the small intestine and 200-fold higher in feces than the corresponding concentrations estimated by a biological assay. This indicates that nisin was degraded or inactivated in the gastrointestinal tract, since fragments of this bacteriocin are detected by ELISA while an intact molecule is needed to retain biological activity.     

Production of Dehydroamino Acid-Containing Peptides by Lactococcus lactis

Nisin is a pentacyclic peptide antibiotic produced by some Lactococcus lactis strains. Nisin contains dehydroresidues and thioether rings that are posttranslationally introduced by a membrane-associated enzyme complex, composed of a serine and threonine dehydratase NisB and the cyclase NisC. In addition, the transporter NisT is necessary for export of the modified peptide. We studied the potential of L. lactis expressing NisB and NisT to produce peptides whose serines and threonines are dehydrated. L. lactis containing the nisBT genes and a plasmid coding for a specific leader peptide fusion construct efficiently produced peptides with a series of non-naturally occurring multiple flanking dehydrobutyrines. We demonstrated NisB-mediated dehydration of serines and threonines in a C-terminal nisin(1-14) extension of nisin, which implies that also residues more distant from the leader peptide than those occurring in prenisin or any other lantibiotic can be modified. Furthermore, the feasibility and efficiency of generating a library of peptides containing dehydroresidues were demonstrated. In view of the particular shape and reactivity of dehydroamino acids, such a library provides a novel source for screening for peptides with desired biological and physicochemical properties.     

SmbFT,a Putative ABC Transporter Complex,Confers Protection against the Lantibiotic Smb in Streptococci

Streptococcus mutans, a dental pathogen, secretes different kinds of lantibiotic and nonlantibiotic bacteriocins. For self-protection, a bacteriocin producer strain must possess one or more cognate immunity mechanisms. We report here the identification of one such immunity complex in S. mutans strain GS-5 that confers protection against Smb, a two-component lantibiotic. The immunity complex that we identified is an ABC transporter composed of two proteins: SmbF (the ATPase component) and SmbT (the permease component). Both of the protein-encoding genes are located within the smb locus. We show that GS-5 becomes sensitized to Smb upon deletion of smbT, which makes the ABC transporter nonfunctional. To establish the role SmbFT in providing immunity, we heterologously expressed this ABC transporter complex in four different sensitive streptococcal species and demonstrated that it can confer resistance against Smb. To explore the specificity of SmbFT in conferring resistance, we tested mutacin IV (a nonlantibiotic), nisin (a single peptide lantibiotics), and three peptide antibiotics (bacitracin, polymyxin B, and vancomycin). We found that SmbFT does not recognize these structurally different peptides. We then tested whether SmbFT can confer protection against haloduracin, another two-component lantibiotic that is structurally similar to Smb; SmbFT indeed conferred protection against haloduracin. SmbFT can also confer protection against an uncharacterized but structurally similar lantibiotic produced by Streptococcus gallolyticus. Our data suggest that SmbFT truly displays immunity function and confer protection against Smb and structurally similar lantibiotics.     

A comparison of the properties of bacteriocins formed by Lactococcus lactis subsp. lactis strains of diverse origin

Bacteriocins formed by four strains of Lactococcus lactis subsp. lactis have been studied and compared: 729 (a natural strain isolated from milk), 1605 (a mutant of strain 729), F-116 (a recombinant obtained by fusing of protoplasts of the two related strain 729 and 1605), and a nisin-forming strain obtained by adaptive selection at Moscow State University. Antimicrobial activity studies revealed differences between the strains in the effects on individual groups of microorganisms; the activities of the strains were also distinct from that of Nisaplin (a commercial preparation of the bacteriocin nisin). Methods for isolation and purification of bacteriocins have been developed, making it possible to obtain individual components of antibiotic complexes as chromatographically pure preparations. Bacteriocins formed by the strains of Lactococcus lactis subsp. lactis have been identified and differences in their biological and physicochemical properties, established. A novel potent broad-spectrum antibiotic substance distinct from nisin has been isolated from the recombinant strain F-116.     

Transfer of Sucrose-Fermenting Ability and Nisin Production Phenotype among Lactic Streptococci

Transfer of sucrose fermentation ability, nisin production, and nisin resistance from Streptococcus lactis to S. lactis and Streptococcus lactis subsp. diacetylactis occurred between cells immobilized on nitrocellulose filters in the presence of DNase. Transconjugants were able to act as donors to transfer the Suc-Nis phenotype in subsequent mating. No changes in sensitivity to lytic phage c2 were noted in S. lactis transconjugants. However, temperature-independent restriction of lytic phage 18-16 was noted in transconjugants of S. lactis subsp. diacetylactis 18-16. Adsorption studies with phage-resistant transconjugants showed that resistance was not due to lack of adsorption by the lytic phage. Physical evidence for the presence of introduced plasmid DNA was not found in lysates of transconjugants.     

Promoting acid resistance and nisin yield of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> F44 by genetically increasing D-Asp amidation level inside cell wall

Nisin fermentation by Lactococcus lactis requires a low pH to maintain a relatively higher nisin activity. However, the acidic environment will result in cell arrest, and eventually decrease the relative nisin production. Hence, constructing an acid-resistant L. lactis is crucial for nisin harvest in acidic nisin fermentation. In this paper, the first discovery of the relationship between D-Asp amidation-associated gene (asnH) and acid resistance was reported. Overexpression of asnH in L. lactis F44 (F44A) resulted in a sevenfold increase in survival capacity during acid shift (pH 3) and enhanced nisin desorption capacity compared to F44 (wild type), which subsequently contributed to higher nisin production, reaching 5346 IU/mL, 57.0% more than that of F44 in the fed-batch fermentation. Furthermore, the engineered F44A showed a moderate increase in D-Asp amidation level (from 82 to 92%) compared to F44. The concomitant decrease of the negative charge inside the cell wall was detected by a newly developed method based on the nisin adsorption amount onto cell surface. Meanwhile, peptidoglycan cross-linkage increased from 36.8% (F44) to 41.9% (F44A), and intracellular pH can be better maintained by blocking extracellular H⁺ due to the maintenance of peptidoglycan integrity, which probably resulted from the action of inhibiting hydrolases activity. The inference was further supported by the acmC-overexpression strain F44C, which was characterized by uncontrolled peptidoglycan hydrolase activity. Our results provided a novel strategy for enhancing nisin yield through cell wall remodeling, which contributed to both continuous nisin synthesis and less nisin adsorption in acidic fermentation (dual enhancement).     

Characteristics and identification of bacteriocins produced by Lactococcus lactis subsp. lactis 194-K

The Lactococcus lactis subsp. lactis 194-K strain has been established to be able to produce two bacteriocins, one of which was identified as the known lantibiotic nisin A, and the other 194-D bacteriocin represents a polypeptide with a 2589-Da molecular mass and comprises 20 amino acid residues. Both bacteriocins were produced in varying proportions in all of the studied culture media, which support the growth of the producer. Depending on the cultivation medium, the nisin A content was 380- to 1123-fold lower in the 194-K stain culture broth than that of the 194-D peptide. In comparision to nisin A Bacteriocin 194-D possessed a wide range of antibacterial activity and suppressed the growth of both Gram-positive and Gram-negative bacteria. An optimal medium for 194-D bacteriocin synthesis was shown to be a fermentation medium which contained yeast extract, casein hydrolysate, and potassium phosphate. The biosynthesis of bacteriocin 194-D by the 194-K strain in these media occurred parallel to producer growth, and its maximal accumulation in the culture broth was observed at14–20 h of the strain’s growth.