Nisin E, a New Nisin Variant Produced by Streptococcus equinus MDC1
Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin 9054, New Zealand
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(2), 1186; https://doi.org/10.3390/app13021186
Submission received: 22 December 2022
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Revised: 10 January 2023
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Accepted: 12 January 2023
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Published: 16 January 2023
(This article belongs to the Section Applied Microbiology)
Abstract
:Members of the genus Streptococcus inhabit a variety of sites in humans and other animals and some species are prolific producers of proteinaceous antibiotics (bacteriocins). As little is known about (i) streptococci inhabiting domestic pets, and (ii) whether novel bacteriocin-producing streptococci can be isolated from domestic pets, the aim of this study is to address these gaps in the research literature. In this study, Streptococcus equinus MDC1, isolated from a healthy dog, was found to exhibit potent antibacterial activity against Micrococcus luteus in a simultaneous antagonism assay, suggesting that strain MDC1 produces a lantibiotic bacteriocin. The inhibitory activity spectrum of S. equinus MDC1, determined using agar-based deferred antagonism assays against >70 indicator strains, was found to be similar to that of nisin U (a lantibiotic produced by Streptococcus uberis). However, the spectra of the two bacteriocins differed by 23 strains, mainly with the MDC1 bacteriocin having no inhibitory activity towards certain streptococci of human origin (e.g., Streptococcus gordonii, Streptococcus anginosus, Streptococcus salivarius). The genome of S. equinus MDC1, which was sequenced completely using single-molecule real-time (SMRT) next-generation DNA sequencing technology, comprises a single 1,936,555-basepair chromosome containing seven copies of the ribosomal RNA operon, 69 tRNA genes and nearly 1900 putative coding sequences. Analysis of the MDC1 genome sequence using the bacteriocin detection algorithms BAGEL4 and antiSMASH revealed the location of a 13,164-basepair 11-gene locus, designated nmd, which encoded a mature nisin E peptide that differed from nisin U by only two amino acids (Ile15→Ala and Leu21→Ile) and an extra C-terminal asparagine residue, and the proteins required for post-translational modification of the bacteriocin, processing, export, and producer immunity. Despite the high homology (90.6% identity, 93.8% similarity) between nisin E and nisin U, there was considerably less homology (47.4–76.3% identity, 68.4–88.8% similarity) between the other proteins encoded by their respective biosynthetic loci. This new natural variant of nisin, called nisin E, represents the first nisin variant to be reported for S. equinus; additionally, its differences with nisin U may provide some insight into the amino acids that influence bacteriocin potency and killing spectrum.
1. Introduction
Bacteriocins are ribosomally synthesized proteinaceous substances produced by prokaryotes that usually kill or inhibit species or strains closely related to the producer organism [1]. Whilst some bacteriocins conform to this original definition, i.e., have a fairly narrow inhibitory spectrum, many bacteriocins exhibit broad inhibitory spectra and, as a result, are potentially useful as biocontrol agents in the food, agricultural and pharmaceutical industries [2,3]. Indeed, nisin (produced by Lactococcus lactis), one of the first bacteriocins to be studied extensively, has been used as a food preservative for several decades [3]. To date, the majority of known bacteriocins are produced by Gram-positive bacteria and these bacteriocins are generally divided into four classes [1]: (i) lantibiotics, which are post-translationally modified peptides containing amino acid derivatives such as lanthionine, (ii) small (<10 kilodalton (kDa)) unmodified peptides, (iii) large (>10 kDa) heat-labile proteins and (iv) circular (cyclic) bacteriocins.
Members of the genus Streptococcus can be found inhabiting a wide variety of habitats in humans and other animals [4]. The streptococci are also prolific producers of a wide range of bacteriocins spanning all four classes, e.g., the lantibiotic salivaricins [5,6], the non-lantibiotic mutacins [7], the Class III dysgalacticin [8] and the unusual cyclic peptide uberolysin [9]. Furthermore, several streptococcal species have been shown to produce natural variants of nisin (the prototype lantibiotic), e.g., nisin U (Streptococcus uberis), nisin H (Streptococcus hyointestinalis), nisin P (Streptococcus agalactiae) and suicin 90-1330 (Streptococcus suis) [10,11,12,13].
Although streptococci have been isolated from livestock (e.g., cattle and pigs), horses and domestic pets, primarily from dogs, much less is known about streptococci residing in other types of pets such as rabbits and guinea pigs, i.e., does the host range of the genus Streptococcus extend to the latter animal species? Furthermore, to the best of our knowledge, there are no reports of bacteriocin-producing streptococci originating from domestic pets (including dogs and cats), i.e., are there novel bacteriocins to be found in our domestic pets? With these gaps in the literature in mind, we conducted a science fair project to investigate (a) whether streptococci could be isolated from the oral cavities of domestic pets and (b) whether any streptococcal isolates produced bacteriocin-like inhibitory substances (BLIS) [14]. In order to accomplish this, and for sampling convenience, we sampled the oral cavities of five family pets (two guinea pigs, a rabbit, a cat and a dog), using Mitis-Salivarius agar as the isolation medium as it is selective for streptococci. We subsequently tested for antibacterial activity, employing a simultaneous antagonism method [6] against Micrococcus luteus T-18, an organism exquisitely sensitive to lantibiotic activity. Not only were presumptive streptococci isolated from all the domestic pets sampled (data not shown) but several α-hemolytic streptococcal colonies (isolated from the dog) exhibited potent BLIS activity against M. luteus [14]. One particular BLIS-positive isolate, MDC1, identified as Streptococcus equinus by 16S ribosomal RNA gene sequencing, was selected for further study.
The aim of the present study is to expand the scope of the original science fair project by (i) investigating the BLIS activity produced by S. equinus strain MDC1, particularly its inhibitory activity spectrum and (ii) identifying the genetic locus specifying the biosynthesis of the putative bacteriocin.
2. Materials and Methods
2.1. Bacterial Strains and Culture Conditions
The bacterial strains used in this study are listed in Table 1. The bacteria were routinely subcultured on Columbia sheep blood agar (Fort Richard Laboratories, Auckland, New Zealand). When liquid media was required, Todd-Hewitt broth (THB; Becton, Dickinson & Co., Sparks, MD, USA) was used. As the different species have different optimal growth requirements, the following incubation conditions were used: (i) 30 °C aerobically (Lactococcus strains), (ii) 37 °C under anaerobic conditions (Streptococcus, Enterococcus faecalis and Abiotrophia defectiva strains) and (iii) 37 °C aerobically (all other strains).
2.2. Testing of Bacteriocin Activity Using Deferred Antagonism Assays
The spectrum of inhibitory activity of S. equinus MDC1 was determined using the established deferred antagonism assay by Tagg and Bannister [15] with the only exception being that the producer bacteria were killed by ultraviolet irradiation (20 min) rather than with chloroform. The test agar was Columbia agar base supplemented with 5% human blood and 0.1% calcium carbonate (Fort Richard Laboratories). The bacteriocin-like activity produced by strain MDC1 was tested against a set of nine standard indicator bacteria [15] as well as a panel of bacterial strains from a range of genera (Table 1). A positive inhibitory result was defined as the complete absence of growth of the indicator organism within the area of the producer streak (containing the bacteriocin). In addition, the potency of inhibition was categorized semi-quantitatively by measuring the distance (in mm) from the border of the producer streak to where the growth of the indicator organism began, e.g., an inhibitory score of +++ was recorded if the zone of inhibition extended >15 mm beyond the border of the producer streak.
2.3. Genome Sequencing, Assembly and Sequence Analysis
The genome of Streptococcus equinus MDC1 was sequenced using the Pacific Biosciences single-molecule real-time (SMRT) next-generation DNA sequencing technology. Genomic DNA for whole-genome sequencing was purified from an overnight (18 h) 200 mL THB culture using the Genomic DNA Genomic DNA Maxi Kit (Geneaid Biotech Ltd., New Taipei City, Taiwan), and sequenced in an SMRT Cell 8Pac V3 (in combination with DNA/Polymerase Binding Kit P6 V2) on the Pacific Biosciences RS-II platform (Macrogen Inc., Seoul, South Korea). A total of 1,456,237,433 basepairs (bp) (>700-fold coverage), generated from 194,313 quality-filtered sequence subreads (average length of 7494 bp; N50 of 10,983 bp) was assembled using Canu version 1.7 [16], using the default parameters recommended by the software developers. The S. equinus MDC1 genome sequence was annotated using RAST [17] and PGAP [18], and subsequently analyzed for the presence of bacteriocin-encoding loci using BAGEL4 [19] and antiSMASH 6.0 [20]. The complete genome sequence of S. equinus MDC1 has been deposited in GenBank under accession number NZ_CP059471.
3. Results
3.1. S. equinus MDC1 Produces a Nisin U-like Bacteriocin
When the BLIS activity produced by Streptococcus equinus MDC1 was tested initially against the set of nine standard indicator bacteria [15], it was observed that the growth of Streptococcus uberis ATCC 27958 (standard indicator I4) was not inhibited. As S. uberis ATCC 27958 is a known producer of nisin U (and nisin U alone) [10], it was speculated that strain MDC1 could be producing a nisin-like lantibiotic bacteriocin. For that reason, we decided to compare the inhibitory activity spectra of the two strains.
The inhibitory activity of S. equinus MDC1 and S. uberis ATCC 27958 was tested by deferred antagonism assays against >70 indicator strains from a range of sources including oral bacteria (human and animal) and environmental isolates (Table 1). As shown in Table 2, the inhibitory spectra of both strains are largely similar but there are distinct differences. Whereas both bacteriocins were especially potent against strains of Micrococcus and Macrococcus, Streptococcus sanguinis, and Streptococcus pyogenes, the MDC1 BLIS exhibited a reduced inhibitory spectrum (i.e., by 23 fewer strains), particularly against strains of Lactococcus lactis (5 of 8), Streptococcus constellatus (3 of 8), Streptococcus oralis (1 of 5), Streptococcus gordonii (0 of 5), Streptococcus anginosus (0 of 4) and Streptococcus salivarius (0 of 3) (Table 2). Furthermore, there were noticeable differences in the potency of inhibitory activity between nisin U and the MDC1 BLIS with strains of Streptococcus mitis (Table 2). Both bacteriocins do not kill Staphylococcus aureus, Enterococcus faecalis and Escherichia coli, a finding consistent with previous experiments with nisin U [10]. Interestingly, whilst S. uberis ATCC 27958 was immune to the inhibitory effects of the MDC1 BLIS, the reverse was not true as partial inhibition of MDC1 was observed with nisin U. This unexpected result will be discussed below. Nevertheless, these results collectively support the hypothesis that the MDC1 BLIS is a new natural variant of the nisin family.
3.2. Genome Sequence Analysis and Identification of the Nisin E Biosynthetic Locus
The genome of Streptococcus equinus MDC1 comprises a single 1,936,555-bp chromosome containing seven copies of the ribosomal RNA (16S, 23S and 5S) operon, 69 tRNA genes and 1899 putative coding sequences (CDS). The overall G + C content of the genome is 37.4%, which is consistent with those obtained with other streptococcal genomes. When the MDC1 genome sequence was analyzed by BAGEL4 and antiSMASH, both algorithms detected the presence of a lantibiotic-encoding locus, specifically that of nisin U, thus confirming that strain MDC1 did indeed produce a nisin-like lantibiotic (now called nisin E).
The nisin E biosynthetic locus, nmd, in the same way as its nisin A and nisin U counterparts, comprises 11 genes that encode proteins in several functional modules (Figure 1): nmdA (nisin E precursor peptide), nmdBCT (modification and transport proteins), nmdPRK (peptide processing and regulation of synthesis), and nmdFEGI (producer immunity). Although the genetic loci of the various nisins are comparable in length (13,164 (nmd) to 13,540 bp (nsu)), the organization of each is different with respect to the juxtaposition of the functional modules (Figure 1). Whereas the nisin A locus is organized in the order nisABTCIPRKFEG, i.e., with the structural gene being the first, followed by the modification/transport, regulation and immunity genes [21], the nisin U locus appears to be swapped such that the processing/regulation and immunity genes are at the 5′-end with the structural gene in the middle of the cluster (Figure 1) [10]. The organization of the nmd locus essentially follows that of the nsu locus, except that nmdA is located between the regulation and immunity modules rather than being immediately upstream of the modification/transport genes (Figure 1). However, this unusual arrangement of genes obviously does not affect the overall expression of the nmd locus as functional nisin E is produced.
Whilst the NmdA and NsuA precursor peptides exhibit relatively high homology scores (83.9% identity, 92.9% similarity), mainly due to differences in their leader peptides, the mature nisin E and nisin U peptides differ by only two amino acids, with nisin E having one extra asparagine residue at its C-terminus (Figure 2). However, there is considerably less homology (47% to 76% identity, 68% to 89% similarity) between the other proteins of the biosynthetic machinery, e.g., the immunity protein NmdI displaying only 47.4% identity (68.4% similarity) to NsuI (Table 3).
4. Discussion
Bacteriocin production is a common occurrence among the streptococci, presumably as an anti-competitor mechanism, and a wide variety of bacteriocin types have been characterized from nearly every Streptococcus species. While some species, for example Streptococcus salivarius, produce a myriad of antibacterial agents including non-ribosomally synthesized peptides [22], other species have only been shown to produce a single type, e.g., Streptococcus dysgalactiae and Streptococcus agalactiae [8,23]. Streptococcus equinus may be in the latter category as only one other bacteriocin, namely bovicin HC5 (a member of the streptin class of lantibiotics), has been characterized to date [24]. In the present study, we identified a new natural nisin variant, nisin E, which is produced by Streptococcus equinus MDC1. We have also sequenced the genome of strain MDC1 and identified the 11-gene nmd locus which encodes not only the nisin E precursor peptide but also the protein machinery required for the post-translational modification, processing, and export of the bacteriocin and producer self-immunity. Despite the availability of three other S. equinus genome sequences in GenBank that contain nmd-like loci, i.e., those of strains GA-1, pR-5 and SI, this study is the first to demonstrate that nisin E is indeed biologically active, thus expanding the bacteriocin repertoire of the species.
In the present work, we have added several species to the screening regime originally reported by Wirawan et al. [10], mainly those belonging to the anginosus and mitis/oralis streptococcal groups, and the relatively new genus Macrococcus [25]. These additional indicator bacteria may be useful for the detection of subtle differences in the inhibitory spectra of bacteriocins of interest, as evidenced by the testing results listed in Table 2. Comparison of the inhibitory spectra of nisin E and its homolog nisin U revealed that not only fewer species were sensitive to the antibacterial effects of nisin E but also that the potency of nisin E appeared reduced against some species, e.g., Streptococcus mitis (Table 2). This could be explained by the amino acid differences between nisin E and nisin U (Figure 2). Although there are only two amino acid differences between the two peptides, isoleucine15→alanine and leucine21→isoleucine, of which the latter is a conserved substitution, nisin E has an additional asparagine residue at its C-terminus (Figure 2). As shown in Figure 2, these amino acid differences are not expected to affect the post-translational modification of the various residues essential in forming the modified amino acids didehydroalanine, didehydrobutyrine, lanthionine and methyl-lanthionine, along with the associated internal thioether bridges. However, the isoleucine15→alanine substitution and the presence of the additional asparagine might influence the overall structure of the nisin E propeptide, thus compromising its interaction with the cell envelopes of certain streptococcal species such as Streptococcus gordonii and Streptococcus anginosus. Unlike the unmodified class II bacteriocins which can be chemically synthesized and readily tested, nisin E requires extensive post-translational modification of the nmdA gene product, hence the requirement for at least the nmdBTC and nmdP genes to be present. Future work to explore engineered variants of nisin E and nisin U that either target specific pathogens or kill a wider range of bacteria may involve expression systems already established for the expression of lantibiotic gene clusters [26,27].
One of the unexpected findings during deferred antagonism testing was the partial inhibition of S. equinus MDC1 by nisin U. As both strain MDC1 and S. uberis ATCC 27958 produce nisin U-like bacteriocins, and their respective biosynthetic loci possessed the immunity genes (e.g., nmdFEG and nmdI), it was expected that both strains would be cross-immune to the other’s bacteriocin. This could be explained by the considerable differences in their putative immunity proteins (Table 3), which range from 47.1% identity (68.4% similarity) to 74.8% identity (85.2% similarity) for NmdI/NsuI and NmdF/NsuF, respectively. As producer self-immunity relies on the concerted interactions of the ABC transporter (comprising the NisFEG/NsuFEG/NmdFEG proteins) and the membrane-bound NisI/NsuI/NmdI proteins to pump out the bacteriocin [28], it is possible that the NmdFEGI complex is optimized for nisin E efflux and therefore unable to protect adequately against nisin U due to the inability to fully recognize nisin U. In contrast, either the NsuFEG and NsuI proteins are able to protect S. uberis ATCC 27958 against a range of nisin-like peptides (including nisin E). An alternative explanation is that nisin E itself is intrinsically unable to effect pore formation in S. uberis. Although beyond the scope of the present study, future work could be conducted to answer this intriguing question, perhaps by expressing nmdFEGI in S. uberis and examining its effect on immunity to nisin E.
During the course of this research project, the genome of S. equinus MDC1 was sequenced, primarily motivated by the aim of locating the bacteriocin-encoding biosynthetic locus. Indeed, the MDC1 genome sequence has served this purpose. However, S. equinus MDC1 has an unusual growth characteristic on mutans selective agar (a medium containing 20% sucrose and the antibiotic bacitracin) routinely used to cultivate the cariogenic pathogen Streptococcus mutans and related organisms [29]. Whilst MDC1 grows on a variety of solid media (e.g., Columbia sheep blood agar and Mitis Salivarius agar (which also contains 20% sucrose)) under both aerobic and anaerobic conditions, the strain is only able to grow on mutans selective agar under anaerobic conditions (Figure 3). In addition, the colonies appear very mucoid, a phenotype not observed with other media (data not shown), suggesting production of a polysaccharide capsule that is somehow regulated by anaerobiosis and possibly the presence of bacitracin (Figure 3). Mining the MDC1 genome sequence would therefore be a very useful step towards elucidating the basis of this interesting phenomenon.
5. Conclusions
We demonstrate, for the first time, that Streptococcus equinus MDC1 produces nisin E, a new natural variant of nisin more closely related to nisin U (produced by Streptococcus uberis). This expands the bacteriocin repertoire of S. equinus to include a member of the nisin class of lantibiotic bacteriocins. Furthermore, we have obtained the complete genome sequence of strain MDC1, which was not only essential for the identification of the nmd locus, but will also be invaluable for future studies, for example, in identifying the genes involved in the mucoid phenotype switching process. Future studies with nisin E may involve amino acid substitutions in the peptide in order to delineate those residues that may either adversely affect or expand its spectrum of inhibitory activity and potency.
Author Contributions
Conceptualization, M.C. and N.H.; methodology and investigation, L.H., M.C. and N.H.; project supervision and administration, resources and funding acquisition, N.H.; writing—original draft preparation, review and editing, N.H. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding but received internal financial assistance from the Sir John Walsh Research Molecular Biosciences Programme for the Streptococcus equinus MDC1 genome sequencing project. This financial assistance is gratefully acknowledged.
Institutional Review Board Statement
Although this study involved domestic pets, this statement is not applicable as the bacterial sampling was conducted at the pets’ homes, i.e., in their natural habitat, with their owners’ written and verbal consent; therefore, this component of the study did not require regulatory approval. The other aspects of the study, i.e., microbiological and genetic analyses, were subsequently conducted at the University of Otago laboratory.
Informed Consent Statement
Not applicable.
Data Availability Statement
The complete genome sequence of Streptococcus equinus MDC1 is available in GenBank under accession number NZ_CP059471.
Conflicts of Interest
The authors declare no conflict of interest.
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Figure 1.
Comparison of the nisin A (L. lactis) [21], nisin U (S. uberis) [10] and nisin E (S. equinus) biosynthetic loci.
Figure 2.
Predicted structures of the mature nisin U (adapted with permission from Ref. [10], 2006, John R. Tagg) and nisin E peptides. The post-translationally modified amino acids are highlighted in blue text, and the amino acid differences between nisin U and nisin E are highlighted in red text. Dha, 2,3-didehydroalanine; Dhb, 2,3-didehydrobutyrine; Lan, lanthionine; Mln, β-methyl-lanthionine.
Figure 2.
Predicted structures of the mature nisin U (adapted with permission from Ref. [10], 2006, John R. Tagg) and nisin E peptides. The post-translationally modified amino acids are highlighted in blue text, and the amino acid differences between nisin U and nisin E are highlighted in red text. Dha, 2,3-didehydroalanine; Dhb, 2,3-didehydrobutyrine; Lan, lanthionine; Mln, β-methyl-lanthionine.
Figure 3.
The unusual growth characteristics of Streptococcus equinus MDC1, observed only on mutans selective agar. (a) An absence of bacterial growth when incubated at 37 °C aerobically; (b) The appearance of mucoid colonies when incubated at 37 °C under anaerobic conditions.
Figure 3.
The unusual growth characteristics of Streptococcus equinus MDC1, observed only on mutans selective agar. (a) An absence of bacterial growth when incubated at 37 °C aerobically; (b) The appearance of mucoid colonies when incubated at 37 °C under anaerobic conditions.
Table 1.
Bacterial strains used in this study.
| Species | Strain(s) and/or Description(s) |
|---|---|
| Streptococcus equinus | MDC1; nisin E producer |
| Streptococcus uberis | ATCC 27958 (standard indicator I4); nisin U producer |
| Micrococcus luteus | T-18 (standard indicator I1), M6 (environmental isolate) |
| Streptococcus pyogenes | FF22 (standard indicator I2), 71-679 (standard indicator I5), 71-698 (standard indicator I7), W-1 (standard indicator I8), M57 |
| Streptococcus constellatus | T-29 (standard indicator I3), 963, DCCS3B, G16, H10, H19F, K-3, K4; all are human oral isolates |
| Lactococcus lactis | T-21 (standard indicator I6), 4862, LM0230, DRC2, 346, MG1614, 166, E8 |
| Streptococcus dysgalactiae | T-148 (standard indicator I9) |
| Streptococcus gordonii | NCTC 10558 (type strain), DL1, C219, Wicky, OB143; all human oral isolates |
| Streptococcus oralis | NCTC 10557 (type strain), 34, SK10, SK39, SK616; all human oral isolates |
| Other S. uberis | O140J (genome reference strain), E, Buddle, D528, D536 |
| Streptococcus anginosus | NCTC 10713 (type strain), OB98, OB107, OB108; all human oral isolates |
| Streptococcus mitis | OB638, OB711, OB721, OB722; all human oral isolates |
| Streptococcus salivarius | NCTC 8618, M18, JH |
| Streptococcus sanguinis | NCTC 10556 (type strain), OB633; both human oral isolates |
| Streptococcus australis | I18; human oral isolate |
| Streptococcus kieseri | CB1; oral isolate from a New Zealand brushtail possum |
| Abiotrophia defectiva | CW1; human oral isolate |
| Enterococcus faecalis | V583; genome reference strain and human clinical isolate |
| Bacillus tropicus | M11; environmental isolate |
| Macrococcus equipercicus | M21, M23; both environmental isolates |
| Micrococcus aloeverae | M27, M31, M32, M33, M34, M35, M36; all environmental isolates |
| Staphylococcus aureus | MChE1, S3, S9, S16; all environmental isolates |
| Escherichia coli | DH5α; Gram-negative species |
Table 2.
Comparison of the bacteriocin activity spectra of nisin U and the MDC1 BLIS (nisin E).
| Producer Strain (Bacteriocin Produced) | ||||
|---|---|---|---|---|
| S. uberis ATCC 27958 (Nisin U) | S. equinus MDC1 (Nisin E) | |||
| Indicator Bacteria (no. of Strains) | No. of Strains Inhibited | Potency of Inhibitory Activity 1 | No. of Strains Inhibited | Potency of Inhibitory Activity 1 |
| S. equinus MDC1 (1) | (1)/1 2 | (+) 2 | 0/1 | - |
| S. uberis ATCC 27958 (1) | 0/1 | - | 0/1 | - |
| Category I 3 | ||||
| Micrococcus aloeverae (7) | 7/7 | +++ | 7/7 | +++ |
| Streptococcus mitis (4) | 4/4 | ++ (2 strains) + (2 strains) | 4/4 | + |
| Micrococcus luteus (2) | 2/2 | +++ | 2/2 | +++ |
| Macrococcus equipercicus (2) | 2/2 | +++ | 2/2 | +++ |
| Streptococcus sanguinis (2) | 2/2 | +++ | 2/2 | +++ |
| Streptococcus kieseri (1) | 1/1 | ++ | 1/1 | ++ |
| Abiotrophia defectiva (1) | 1/1 | + | 1/1 | + |
| Streptococcus pyogenes (5) | 4/5 | ++ | 4/5 | ++ |
| Category II | ||||
| Lactococcus lactis (8) | 8/8 | +++ (E8 only) + | 5/8 | +++ (E8 only) + |
| Streptococcus constellatus (8) | 8/8 | + | 3/8 | + |
| Other S. uberis strains (5) | 4/5 | + | 2/5 | + |
| Streptococcus oralis (5) | 5/5 | + | 1/5 | + |
| Streptococcus gordonii (5) | 5/5 | + | 0/5 | - |
| Streptococcus anginosus (4) | 2/4 | + | 0/4 | - |
| Streptococcus salivarius (3) | 2/3 | + | 0/3 | - |
| Category III | ||||
| Streptococcus dysgalactiae (1) | 0/1 | - | 0/1 | - |
| Streptococcus australis (1) | 0/1 | - | 0/1 | - |
| Staphylococcus aureus (4) | 0/4 | - | 0/4 | - |
| Enterococcus faecalis (1) | 0/1 | - | 0/1 | - |
| Bacillus tropicus (1) | 0/1 | - | 0/1 | - |
| Escherichia coli (1) | 0/1 | - | 0/1 | - |
1 The potency of inhibitory activity is semi-quantitative and based on the width (in mm) of growth inhibition beyond the producer growth streak: +++, >15 mm; ++, >10 and up to 15 mm; +, up to 10 mm; -, no growth inhibition detected. 2 Partial growth inhibition of S. equinus MDC1 by nisin U was observed, i.e., there was some growth within the S. uberis ATCC 27958 producer streak. 3 For clarity, the indicator bacteria have been divided into three categories: (I) where both bacteriocins exhibit identical inhibitory activity spectra; (II) where both bacteriocins display different inhibitory activity spectra (including species that are not inhibited by nisin E); (III) strains not inhibited by both bacteriocins.
Table 3.
Proteins encoded by the nmd (nisin E) locus and comparison with its Nsu (nisin U) counterparts.
Table 3.
Proteins encoded by the nmd (nisin E) locus and comparison with its Nsu (nisin U) counterparts.
| Homology Scores | ||||
|---|---|---|---|---|
| Protein (Gene) 1 | Putative Function | Length in Amino Acids (Nsu Counterpart) | % Identity 2 | % Similarity |
| NmdP (nmdP) | Serine protease (removal of NmdA leader peptide) | 455 (461) | 58.9 | 75.3 |
| NmdR (nmdR) | Response regulator (regulation of synthesis) | 227 (232) | 76.3 | 88.8 |
| NmdK (nmdK) | Histidine kinase (regulation of synthesis | 446 (448) | 56.3 | 75.1 |
| NmdA (nmdA) | Nisin E precursor peptide | 56 (55) | 83.9 | 92.9 |
| Nisin E | Nisin E propeptide (active peptide) | 32 (31) | 90.6 | 93.8 |
| NmdF (nmdF) | Immunity protein | 230 (230) | 74.8 | 85.2 |
| NmdE (nmdE) | Immunity protein | 244 (253) | 61.3 | 76.3 |
| NmdG (nmdG) | Immunity protein | 216 (230) | 51.7 | 70.0 |
| NmdB (nmdB) | Modification protein (dehydratase) | 993 (991) | 56.7 | 76.1 |
| NmdT (nmdT) | Transport protein (bacteriocin export) | 598 (598) | 66.1 | 83.1 |
| NmdC (nmdC) | Modification protein (thiother formation) | 420 (425) | 52.1 | 71.3 |
| NmdI (nmdI) | Immunity protein | 245 (238) | 47.4 | 68.4 |
1 The genes are listed in order of their location in the genetic locus (5′ → 3′). 2 Homology scores were determined with sequence gaps taken into account.
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Christophers, M.; Heng, L.; Heng, N. Nisin E, a New Nisin Variant Produced by Streptococcus equinus MDC1. Appl. Sci. 2023, 13, 1186. https://doi.org/10.3390/app13021186
AMA Style
Christophers M, Heng L, Heng N. Nisin E, a New Nisin Variant Produced by Streptococcus equinus MDC1. Applied Sciences. 2023; 13(2):1186. https://doi.org/10.3390/app13021186
Chicago/Turabian StyleChristophers, Meg, Lauren Heng, and Nicholas Heng. 2023. "Nisin E, a New Nisin Variant Produced by Streptococcus equinus MDC1" Applied Sciences 13, no. 2: 1186. https://doi.org/10.3390/app13021186
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