Mutacin Production Research Articles

LiaS Regulates Virulence Factor Expression in Streptococcus mutans

Streptococcus mutans, a major oral pathogen responsible for dental caries formation, possesses a variety of mechanisms for survival in the human oral cavity, where the conditions of the external environment are diverse and in a constant state of flux. The formation of biofilms, survival under conditions of acidic pH, and production of mutacins are considered to be important virulence determinants displayed by this organism. Biofilm formation is facilitated by the production of GbpC, an important cell surface-associated protein that binds to glucan, an adhesive polysaccharide produced by the organism itself. To better understand the nature of the environmental cues that induce GbpC production, we examined the roles of 14 sensor kinases in the expression of gbpC in S. mutans strain UA159. We found that only the LiaS sensor kinase regulates gbpC expression, while the other sensor kinases had little or no effect on gbpC expression. We also found that while LiaS negatively regulates gbpC expression, the inactivation of its cognate response regulator, LiaR, does not appear to affect the expression of gbpC. Since both gbpC expression and mutacin IV production are regulated by a common regulatory network, we also tested the effect of the liaS mutation on mutacin production and found that LiaS positively regulates mutacin IV production. Furthermore, reverse transcription-PCR analysis suggests that LiaS does so by regulating the expression of nlmA, which encodes a peptide component of mutacin IV, and nlmT, which encodes an ABC transporter. As with the expression of gbpC, LiaR did not have any apparent effect on mutacin IV production. Based on the results of our study, we speculate that LiaS is engaged in cross talk with one or more response regulators belonging to the same family as LiaR, enabling LiaS to regulate the expression of several genes coding for virulence factors.

The response regulator ComE in Streptococcus mutans functions both as a transcription activator of mutacin production and repressor of CSP biosynthesis

In Streptococcus pneumoniae, competence and bacteriocin genes are controlled by two two-component systems, ComED and BlpRH, respectively. In Streptococcus mutans, both functions are controlled by the ComED system. Recent studies in S. mutans revealed a potential ComE binding site characterized by two 11 bp direct repeats shared by each of the bacteriocin genes responsive to the competence-stimulating peptide (CSP). Interestingly, this sequence was not found in the upstream region of the CSP structural gene comC. Since comC is suggested to be part of a CSP-responsive and ComE-dependent autoregulatory loop, it was of interest to determine how it was possible that the ComED system could simultaneously regulate bacteriocin expression and natural competence. Using the intergenic region IGS1499, shared by the CSP-responsive bacteriocin nlmC and comC, it was demonstrated that both genes are likely to be regulated by a bifunctional ComE. In a comE null mutant, comC gene expression was increased similarly to a fully induced wild-type. In contrast, nlmC gene expression was nearly abolished. Deletion of ComD exerted a similar effect on both genes to that observed with the comE null mutation. Electrophoretic mobility shift assays (EMSAs) with purified ComE revealed specific shift patterns dependent on the presence of one or both direct repeats in the nlmC-comC promoter region. The two direct repeats were also required for the promoter activity of both nlmC and comC. These results suggest that gene regulation of comC in S. mutans is fundamentally different from that reported for S. pneumoniae, which implicates a unique regulatory mechanism that allows the coordination of bacteriocin production with competence development.

Mutacin H-29B is identical to mutacin II (J-T8)

BackgroundStreptococcus mutans produces bacteriocins named mutacins. Studies of mutacins have always been hampered by the difficulties in obtaining active liquid preparations of these substances. Some of them were found to be lantibiotics, defined as bacterial ribosomally synthesised lanthionine-containing peptides with antimicrobial activity. The goal of this study was to produce and characterize a new mutacin from S. mutans strain 29B, as it shows a promising activity spectrum against current human pathogens.ResultsMutacin H-29B, produced by S. mutans strain 29B, was purified by successive hydrophobic chromatography from a liquid preparation consisting of cheese whey permeate (6% w/v) supplemented with yeast extract (2%) and CaCO3 (1%). Edman degradation revealed 24 amino acids identical to those of mutacin II (also known as J-T8). The molecular mass of the purified peptide was evaluated at 3246.08 ± 0.1 Da by MALDI-TOF MS.ConclusionA simple procedure for production and purification of mutacins along with its characterization is presented. Our results show that the amino acid sequence of mutacin H-29B is identical to the already known mutacin II (J-T8) over the first 24 residues. S. mutans strains of widely different origins may thus produce very similar bacteriocins.

LuxS controls bacteriocin production in Streptococcus mutans through a novel regulatory component

The oral pathogen Streptococcus mutans employs a variety of mechanisms to maintain a competitive advantage over many other oral bacteria which occupy the same ecological niche. Production of the bacteriocin, mutacin I, is one such mechanism. However, little is known about the regulatory mechanisms associated with mutacin I production. Previous work has demonstrated that the production of mutacin I greatly increased with cell density. In this study, we found that high cell density also triggered high level mutacin I gene transcription. However, this response was abolished upon deletion of luxS. Further analysis using real-time reverse transcription polymerase chain reaction (RT-PCR) demonstrated that in the luxS mutant transcription of both the mutacin I structural gene mutA and the mutacin I transcriptional activator mutR was impaired. Through microarray analysis, a putative transcription repressor annotated as Smu1274 in the Los Alamos National Laboratory Oral Pathogens Sequence Database was identified, which was strongly induced in the luxS mutant. Characterization of Smu1274, which we referred to as irvA, suggested that it may act as an inducible repressor to suppress mutacin I gene expression. A luxS and irvA double mutant regained the ability to produce mutacin I; whereas a constitutive irvA-producing strain was impaired in mutacin I production. These findings reveal a novel regulatory pathway for mutacin I gene expression, which may provide clues to the regulatory mechanisms of other cellular functions regulated by luxS in S. mutans.

Co‐ordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species

It is important to ensure DNA availability when bacterial cells develop competence. Previous studies in Streptococcus pneumoniae demonstrated that the competence-stimulating peptide (CSP) induced autolysin production and cell lysis of its own non-competent cells, suggesting a possible active mechanism to secure a homologous DNA pool for uptake and recombination. In this study, we found that in Streptococcus mutans CSP induced co-ordinated expression of competence and mutacin production genes. This mutacin (mutacin IV) is a non-lantibiotic bacteriocin which kills closely related Streptococcal species such as S. gordonii. In mixed cultures of S. mutans and S. gordonii harbouring a shuttle plasmid, plasmid DNA transfer from S. gordonii to S. mutans was observed in a CSP and mutacin IV-dependent manner. Further analysis demonstrated an increased DNA release from S. gordonii upon addition of the partially purified mutacin IV extract. On the basis of these findings, we propose that Streptococcus mutans, which resides in a multispecies oral biofilm, may utilize the competence-induced bacteriocin production to acquire transforming DNA from other species living in the same ecological niche. This hypothesis is also consistent with a well-known phenomenon that a large genomic diversity exists among different S. mutans strains. This diversity may have resulted from extensive horizontal gene transfer.

Mutacin production in Streptococcus mutans genotypes isolated from caries‐affected and caries‐free individuals

Relationships between genetic diversity and mutacin production in Streptococcus mutans were evaluated in 319 clinical isolates from eight caries-affected and eight caries-free individuals. The isolates were submitted to mutacin typing and AP-PCR (arbitrarily primed polymerase chain reaction) assay. The mutacin production was detected for 12 Streptococcus sp. indicator strains. Results showed significant variations in the mutacin production profiles and the inhibitory spectra of both groups. A possible association was seen between mutacin activity and the distinct patterns of Streptococcus sp. colonization in the two groups. Genotyping by AP-PCR using the primers OPA-02 and OPA-13 revealed 101 distinct genotypes against 48 phenotypes identified by mutacin typing. No correlation was observed between the inhibitory spectra of mutacin and genotypic similarities based on AP-PCR analyses. According to our results, strains of the same S. mutans genotype showed different mutacin profiles, suggesting a high degree of interstrain diversity. In conclusion, mutacin production seems to be of clinical importance in the colonization of S. mutans and is highly diversified in the S. mutans species.

Improved methods for mutacin detection and production

Studies of mutacins have always been hampered by the difficulties in obtaining active liquid preparations of these substances. In order to be commercially produced, good mutacin yields have to be obtained, preferably in inexpensive media. The results presented here indicate that mutacins can be produced in supplemented cheese whey permeate. The influence of carbon and nitrogen supplements on mutacin production varied according to the producer strain. The use of CaCO 3 as a buffer in batch cultures resulted in improved yields of mutacin in the supernatants. Antimicrobial activity assays were improve by acidification of the diluent (pH 2) and were less variable in peptone water (0.5%). The culture medium consisting of cheese whey permeate (6% w/v), yeast extract (2% w/v) and CaCO 3 (1% w/v) was found to be an inexpensive medium for the efficient production of mutacins.

Inactivation of the ciaH Gene in Streptococcus mutans diminishes mutacin production and competence development, alters sucrose-dependent biofilm formation, and reduces stress tolerance.

Many clinical isolates of Streptococcus mutans produce peptide antibiotics called mutacins. Mutacin production may play an important role in the ecology of S. mutans in dental plaque. In this study, inactivation of a histidine kinase gene, ciaH, abolished mutacin production. Surprisingly, the same mutation also diminished competence development, stress tolerance, and sucrose-dependent biofilm formation.

Determination of mutacin activity and detection of mutA genes in Streptococcus mutans genotypes from caries-free and caries-active children.

Relationships between genetic diversity, mutacin production and sensitivity to mutacins in Streptococcus mutans were evaluated in 19 clinical isolates from caries-free and caries-active children. Mutacin production was tested against 30 indicator strains; results showed significant variations in the inhibitory spectra of the clinical isolates. There was no association between the inhibitory spectrum of the infecting strain and the caries experience or the level of mutans streptococci infection of the host. Homology to the mutA gene coding for mutacin II was detected in one clinical isolate; none of the clinical isolates showed homology to the mutA genes coding for mutacins I or III. Genotyping by random amplified polymorphic DNA (RAPD) reactions grouped the isolates into three clusters, but no correlation was found between any of the clusters and mutacin activity, caries experience or level of mutans streptococci in the host.

Effect of amino acid substitutions in conserved residues in the leader peptide on biosynthesis of the lantibiotic mutacin II

The lantibiotic mutacin II, produced by Streptococcus mutans T8, is a ribosomally synthesized peptide antibiotic that contains thioether amino acids such as lanthionine and methyllanthionine as a result of post-translational modifications. The mutacin II leader peptide sequence shares a number of identical amino acid residues with class AII lantibiotic leader peptides. To study the role of these conservative residues in the production of active antimicrobial mutacin, 15 mutations were generated by site-directed mutagenesis. The effects of these substitutions vary from no effect to complete block-out. Mutations G-1A, G-2A, I-4D, and L-7K completely blocked the production of mature mutacin. Other mutations (I-4V, L-7M, E-8D, S-11T/A, V-12I/A, and E-13D) had no detectable effect on mutacin production. The changes of Glu-8 to Lys, Val-12 to Leu, Glu-13 to Lys reduced the mutacin production level to about 75%, 50%, and 10% of the wild-type, respectively. Thus, our data indicated that some of these conserved residues are essential for the mutacin biosynthesis, whereas others are important for optimal biosynthesis rates.

Effect of amino acid substitutions in conserved residues in the leader peptide on biosynthesis of the lantibiotic mutacin II.

The lantibiotic mutacin II, produced by Streptococcus mutans T8, is a ribosomally synthesized peptide antibiotic that contains thioether amino acids such as lanthionine and methyllanthionine as a result of post-translational modifications. The mutacin II leader peptide sequence shares a number of identical amino acid residues with class AII lantibiotic leader peptides. To study the role of these conservative residues in the production of active antimicrobial mutacin, 15 mutations were generated by site-directed mutagenesis. The effects of these substitutions vary from no effect to complete block-out. Mutations G-1A, G-2A, I-4D, and L-7K completely blocked the production of mature mutacin. Other mutations (I-4V, L-7M, E-8D, S-11T/A, V-12I/A, and E-13D) had no detectable effect on mutacin production. The changes of Glu-8 to Lys, Val-12 to Leu, Glu-13 to Lys reduced the mutacin production level to about 75%, 50%, and 10% of the wild-type, respectively. Thus, our data indicated that some of these conserved residues are essential for the mutacin biosynthesis, whereas others are important for optimal biosynthesis rates.

The group I strain of Streptococcus mutans, UA140, produces both the lantibiotic mutacin I and a nonlantibiotic bacteriocin, mutacin IV.

Strains of Streptococcus mutans produce at least three mutacins, I, II, and III. Mutacin II is a member of subgroup AII in the lantibiotic family of bacteriocins, and mutacins I and III belong to subgroup AI in the lantibiotic family. In this report, we characterize two mutacins produced by UA140, a group I strain of S. mutans. One is identical to the lantibiotic mutacin I produced by strain CH43 (F. Qi et al., Appl. Environ. Microbiol. 66:3221-3229, 2000); the other is a nonlantibiotic bacteriocin, which we named mutacin IV. Mutacin IV belongs to the two-peptide, nonlantibiotic family of bacteriocins produced by gram-positive bacteria. Peptide A, encoded by gene nlmA, is 44 amino acids (aa) in size and has a molecular mass of 4,169 Da; peptide B, encoded by nlmB, is 49 aa in size and has a molecular mass of 4,826 Da. Both peptides derive from prepeptides with glycines at positions -2 and -1 relative to the processing site. Production of mutacins I and IV by UA140 appears to be regulated by different mechanisms under different physiological conditions. The significance of producing two mutacins by one strain under different conditions and the implication of this property in terms of the ecology of S. mutans in the oral cavity are discussed.

Functional analyses of the promoters in the lantibiotic mutacin II biosynthetic locus in Streptococcus mutans.

The lantibiotic bacteriocin mutacin II is produced by the group II Streptococcus mutans. The mutacin II biosynthetic locus consists of seven genes, mutR, -A, -M, -T, -F, -E, and -G, organized as two operons. The mutAMTFEG operon is transcribed from the mutA promoter 55 bp upstream of the translation start codon for MutA, while the mutR promoter is 76 bp upstream of the mutR structural gene. Expression of the mutA promoter is regulated by the components of the growth medium, while the mutR promoter activity does not seem to be affected by these conditions. Inactivation of mutR abolishes transcription of the mutA operon but does not affect its own promoter activity. The expressions of both mutA and mutR promoters are independent of the growth stage, while the production of mutacin II is only elevated at the early stationary phase. Taken together, these results suggest that expression of the mutacin operon is regulated by a complex system involving transcriptional and posttranscriptional or posttranslational controls.

Mutacin production by Streptococcus mutans may promote transmission of bacteria from mother to child.

The production of bacteriocin-like inhibitory substances, mutacins, by mutans streptococci varies among isolates. To find if the degree of mutacin activity of an isolate was related to its transmission between mother and her child, 19 mothers and their 18-month- to 3-year-old children were sampled for their oral mutans streptococci. In addition, the stability of mutacin activity was studied with isolates from the mothers and with isolates from five unrelated 5-year-old children in 5- to 7-year follow-up studies. A total of 145 oral mutans streptococcal isolates were serotyped by immunodiffusion, ribotyped, and mutacin typed by the stab culture technique. Mutacin was produced by 88% of the strains against more than 1 of the 14 indicator strains, representing mutans streptococci, Streptococcus sanguis, Streptococcus salivarius, Streptococcus oralis, Streptococcus gordonii, and Streptococcus pyogenes. Streptococcus mutans isolates showed more inhibitory activity than did Streptococcus sobrinus isolates. Identical ribotypes had similar mutacin activity profiles within a subject, initially and in the follow-up studies, in all but two cases. The mothers harbored a total of 37 different mutans streptococcal ribotypes. Six children were negative for mutans streptococci. Transmission was probable in 9 of 20 mother-child pairs on the basis of the presence of identical strains, as determined by ribotyping and bacteriocin (mutacin) typing. S. mutans strains shared between a mother and her child showed a broader spectrum of inhibitory activity than did nontransmitted strains. In conclusion, the mutacin activity of clinical isolates is reasonably stable, and this virulence factor seems to be of clinical importance in early colonization by S. mutans.

Diacylglycerol kinase is involved in regulation of expression of the lantibiotic mutacin II of Streptococcus mutans.

Genetic characterization of a Tn916 transposon mutant, Streptococcus mutans T8-1, defective in mutacin II production, revealed that the transposon was inserted into the 3' region of a diacylglycerol kinase (dgk) gene. The insertion occurred in the same region as described for another S. mutans mutant, GS5Tn1, which was altered in its ability to respond to environmental stress (Y. Yamashita, T. Takehara, and H. K. Kuramitsu, J. Bacteriol. 175:6220-6228, 1993). Quantitative primer extension from the mutacin structural gene mutA showed a decreased level (about eightfold) of mutA transcription for mutant T8-1. Mutacin production was restored by transforming mutant T8-1 with integration vector pVA891 containing an intact dgk gene. These data indicated that the full-length dgk gene product along with the mutacin biosynthetic operon are required for the production of the mutacin II lantibiotic.

Sequence analysis of mutA and mutM genes involved in the biosynthesis of the lantibiotic mutacin II in Streptococcus mutans

Streptococcus mutans, along with many other Gram-positive bacteria produce small antibacterial peptides called bacteriocins. Bacteriocins elaborated by S. mutans, termed mutacins, may provide a selective force necessary for initial or sustained colonization in dental plaque by this major dental pathogen. Previously, we purified and characterized mutacin II, the first lantibiotic found in S. mutans. Specific oligonucleotides designed according to the N-terminal amino acid sequence permitted amplification of 0.7 kb upstream and 2.1 kb downstream of the N-terminus, using single-specific-primer PCR (SSP–PCR). The gene encoding the mutacin II prepeptide, mutA, was subsequently cloned and sequenced. The complete prepeptide consists of 53 amino acids, including the 26 amino acid amphipathic leader peptide with the Gly −2–Gly −1 sequence at the processing site. The prepeptide showed similarity to the lantibiotics lacticin 481, variacin, salivaricin and streptococcin A-FF22. A 3 kb open reading frame immediately downstream of mutA, denoted mutM, showed sequence similarities to LCNDR2 from Lactococcus lactis. By analogy, mutM is probably involved in post-translational modification of the mutacin prepeptide. Gene disruption with an insertional vector pVA891 showed that intact copies of mutA and mutM are required for production of mutacin II.

Mutacin activity of strains isolated from children with varying levels of mutans streptococci and caries

A total of 157 isolates of mutans streptococci from plaque and saliva of 94 children were studied for their serotypes, mutacin production, frequency and spectrum of activity. Of these isolates 71% were identified as serotype c and 22% as serotype e. Serotypes f, d and g, and one untypable strain made up about 7% of the isolates. More than one serotype was found in 13% of the children. Mutacin was produced by 83% of the isolates against one or more of the 14 indicator strains representing mutans streptococci, Streptococcus sanguis, Strep. oralis, Strep. gordonii, Strep. salivarius and Strep. pyogenes. Isolates that had a broad inhibitory spectrum also produced larger inhibition zones than isolates that inhibited fewer strains. When evaluating the effect of mutacin in vivo on plaque ecology, it was found that the counts of mutans streptococci or the proportion of mutans streptococci in the total streptococcal count of plaque did not differ between plaques containing strains that produced much mutacin and those with little production. The findings also failed to reveal an association between caries experience and mutacin activity.

Use of transposon Tn916 to inactivate and isolate a mutacin-associated gene from Streptococcus mutans

Among the attributes thought to contribute to the virulence of Streptococcus mutans is its ability to elaborate bacteriocinlike substances, which may provide a selective force enhancing its colonization potential. One such inhibitory substance, mutacin II, is produced by certain plasmid-containing strains of S. mutans. We introduced insertional mutations into a mutacin II-producing strain of S. mutans (UA96) by transformation with a plasmid carrying Tn916, resulting in transformants bearing single inserts of the transposon at different sites within the chromosome. The insertions identify five different EcoRI fragments required for production of mutacin II (Bac phenotype; bac-1 to bac-5 genotypes). The EcoRI fragments, containing bac-1::Tn916 was ligated into a cosmid vector, pJC74, and transduced into Escherichia coli DH1, where Tn916 is known to be unstable. The loss of Tn916 resulted in a 30-kb plasmid, pPC974, containing approximately 15 kb of S. mutans DNA. A Bac-associated DNA fragment was then subcloned into the streptococcus-E. coli shuttle vector pVA838 and transformed into S. mutants, where it was capable of complementing the bac mutation in the Bac- parent. These findings suggest that we have isolated at least one gene associated with mutacin production.

Evidence that mutacin II production is not mediated by a 5.6-kb plasmid in Streptococcus mutans

Here we present evidence that the cryptic 5.6-kb plasmid found in certain strains of Streptococcus mutans is not involved in mutacin production. This evidence comes from demonstrating similarities between a plasmid-less strain T8 and a group II plasmid strain UA96. Both produce what appears to be an identical mutacin based on spectrum of activity and physiological properties. Also, T8 and UA96 are members of the same immunity group (group II). Genotypically, both strains appear similar except for plasmid content based on DNA fingerprinting profiles. T8 and UA96 exhibit identical hybridization patterns following transformation of T8 with a mutacin-negative ( bac-1::Tn 916) sequence from a Tn 916-insertionally inactivated mutant of UA96. This transformation also resulted in the mutacin-negative phenotype in T8 transformants, showing recombination between a mutacin-associated gene in UA96 and its apparent homologous sequence in T8. Moreover, when a plasmid containing a putative repeat element from UA96 (pPC264) was used as a probe, it hybridized to the same five EcoRI fragments in both T8 and UA96. Collectively, these data, coupled with data from other sources, indicate that the plasmid resident in mutacin II strains is not involved in mutacin production.

Production and properties of bacteriocins (Mutacins) from Streptococcus Mutans

Studies using the stab culture technique determined that almost all of the reference Streptococcus mutans strains of serotypes c and e produced bacteriocins (mutacins) against more than one of the 10 indicator organisms which included strains of Strep. mutans, Strep. salivarius, Strep. sanguis and group A Strep. pyogenes. However, reference strains of Strep. mutans types a and d never elaborated mutacins against more than one indicator strain. Among 113 clinical isolates of Strep. mutans obtained from Japanese children, 84 strains (74 per cent) inhibited at least one of the indicator strains. Type c strains accounted for 85 of the 113 clinical isolates, and these strains produced mutacins more frequently than strains of other serotypes. Very few strains produced a cell-free form of mutacin in Trypticase Soy broth enriched with 2 per cent yeast extract. Mutacins were generally heat-stable, but some were destroyed by treatment with proteolytic and/or lipolytic enzymes. Part of the mutacin activity diffused through cellophane paper. These results suggest that mutacins contain at least 2 kinds of inhibitory substances ranging from low molecular weight materials to higher molecular weight, protein-lipid complexes. Mutacinogeny was not due to the accumulation of hydrogen peroxide. Production of mutacin was influenced markedly by the culture media employed.