Preliminary characterization of four bacteriocins from Streptococcus mutans

The various properties of the inhibitory substances produced by Streptococcus mutans strains C67-1, Ny257-S, Ny266, and T8, and the fact that inhibitory zones produced could not be associated with lactic acid (or other organic acids), bacteriophages, or hydrogen peroxide indicate that these substances can be classified as mutacins. The substances produced by strains C67-1, Ny266, and T8 possessed similar properties. They were shown to be thermoresistant (100 degrees C, 30 min), low molecular weight (less than 3500) substances sensitive to proteolytic enzymes (chymotrypsin, papain, pronase E, proteinase K, and trypsin) and they were active against most of the Gram-positive bacteria tested but not against most of the Gram-negative bacteria. The substance produced by strain Ny257-S differs from the other three by its narrower activity spectrum, its lower thermoresistance (80 degrees C, 30 min), and its higher molecular weight (8,000-14,000). The gene or the genes coding for the mutacins are probably located on the chromosome since no plasmid DNA could be detected in these four producing strains. Restriction-fragment patterns of C67-1 and T8 suggest that these strains are closely related, as supported by the strong similarity observed between the properties of their mutacins.     

变链素活性与变形链球菌基因多态性的关系研究

目的探讨变链素的活性与变形链球菌(MS)基因多态性的关系。方法在AP-PCR基因分型的基础上,选择来自单基因型定植的个体50株MS为单基因型组,另50株来自多基因型共同定植的个体为多基因型组,用平板法检测2组菌株产生变链素对10个指示株的抑制情况,T-检验比较2组菌株抑菌环和抑菌谱的均数差异。结果所有的实验株(100%)均可产生抑制6~8个指示株的变链素,抑菌环和抑菌谱在不同个体之间变异,组间均数的比较不具有显著性(P值分别是0.12,1.79)。结论多基因型MS定植的口腔,在产生变链素方面似乎不具有优势。     

SMU.152 acts as an immunity protein for mutacin IV

Streptococcus mutans, a principal causative agent of dental caries, secretes antimicrobial peptides known as mutacins to suppress the growth of competing species to establish a successful colonization. S. mutans UA159, a sequenced strain, produces at least two major mutacins, mutacins IV and V. Mutacin IV is a two-peptide mutacin encoded by nlmAB genes, which are mapped just upstream of a putative immunity-encoding gene SMU.152. Here we explored the function of SMU.152 as an immunity protein. We observed that overexpression of SMU.152 in two sensitive host strains converted the strains to become immune to mutacin IV. To identify the residues that are important for immunity function, we sequentially deleted residues from the C-terminal region of SMU.152. We observed that deletion of as few as seven amino acids, all of which are highly charged (KRRSKNK), drastically reduced the immunity function of the protein. Furthermore, we identified two other putative immunity proteins, SMU.1909 and SMU.925, which lack the last four charged residues (SKNK) that are present in SMU.152 but contain the KRR residues. Synthetic addition of SKNK residues to either SMU.1909 or SMU.925 to reconstitute the KRRSKNK motif and expressing these constructs in sensitive cells rendered the cells resistant to mutacin IV. We also demonstrated that deletion of Man-PTS system from a sensitive strain made the cells partially resistant to mutacin IV, indicating that the Man-PTS system plays a role in mutacin IV recognition.     

Mutacins from Streptococcus mutans UA159 are active against multiple streptococcal species

Streptococcus mutans UA159, whose genome is completely sequenced, produces two nonlantibiotic mutacins, mutacin IV (encoded by nlmAB) and mutacin V (encoded by nlmC). In this study, we investigated the contribution of nlmA and nlmB to mutacin IV activity and demonstrated by performing genetic studies as well as by using semipurified molecules that, in contrast to a previous report, both of these genes are required for optimum mutacin IV activity. We also showed that mutacin IV is active against multiple Streptococcus species. In contrast, mutacin V displayed a narrower inhibitory range than mutacin IV. Our results suggest that mutacin IV and mutacin V may act synergistically to inhibit various organisms.     

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 CaCO3 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 CaCO3 (1% w/v) was found to be an inexpensive medium for the efficient production of mutacins.     

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.     

The specific genes for lantibiotic mutacin II biosynthesis in Streptococcus mutans T8 are clustered and can be transferred en bloc

Mutacin II is a ribosomally synthesized peptide lantibiotic produced by group II Streptococcus mutans. DNA sequencing has revealed that the mutacin II biosynthetic gene cluster consists of seven specific open reading frames: a regulator (mutR), the prepromutacin structural gene (mutA), a modifying protein (mutM), an ABC transporter (mutT), and an immunity cluster (mutFEG). Transformations of a non-mutacin-producing strain, S. mutans UA159, and a mutacin I-producing strain, S. mutans UA140, with chromosomal DNA from S. mutans T8 with an aphIII marker inserted upstream of the mutacin II structural gene yielded transformants producing mutacin II and mutacins I and II, respectively.     

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.     

Streptococcus mutans strain N produces a novel low molecular mass non-lantibiotic bacteriocin

Streptococcus mutans strain N was shown to have bacteriocin production and immunity characteristics consistent with those of Group I mutacin-producing strains of S. mutans. The bacteriocin mutacin N was purified from agar cultures of S. mutans strain N using XAD andp6 reversed phase chromatography. The molecular mass of mutacin N was 4806 Da and the entire 49 amino acid sequence was determined by N-terminal sequencing. Database searches indicate that mutacin N is a novel bacteriocin, but with some homology to the protein IIC domain of a hypothetical sugar-phosphotransferase enzyme from Acholeplasma florum.     

液体培养基中天然变链素的纯化

目的 从变形链球菌临床株的液体培养基中分离纯化变链素,为进一步从分子水平研究变链素奠定基础.方法 通过抑菌活性检测,从变链临床株中选择出抑菌活性较强的菌株.用氯仿抽提法从该菌株的培养液中粗提变链素,经固相萃取和反相高效液相色谱(RP-HPLC)对粗提物进行纯化.结果 获得变链素活性较强的菌株"1G".从其200 ml液体培养基中粗提出变链素约15 μg,经固相萃取柱洗脱,再经过RP-HPLC 2次纯化,得到有抑菌活性的成分,此为纯化的变链素.结论 变链素分子量小,分离提纯步骤复杂,本实验得到纯化的变链素,为下一步研究变链素的氨基酸序列和基因序列奠定了基础.     

Production of mutacin-like substances by Streptococcus mutans

Production of inhibitory substances by strains of the Streptococcus mutans group is well documented, but the nature of the substances implied is often unknown. Of nine laboratory strains known to produce inhibitory substances, the optimal conditions for producing inhibition zones on solid media were found to vary between strains but good production was generally obtained on all-purpose media with Tween 80 at 37 degrees C after 2-4 days of aerobic incubation. Streptococcus sanguis Ny101 was found to be more sensitive than Streptococcus rattus LG-1 to all inhibitory substances produced by the S. mutans strains tested. While all strains showed some inhibition, only six showed inhibition after neutralization; arginine incorporated in agar at 0.75% completely eliminated all inhibition zones. However 1% arginine in the overlays did not affect the production of inhibition zones by strains of S. mutans C67-1, Ny257, Ny266, and T8. These strains were shown to elaborate (in a reproducible fashion) inhibitory substances which were not organic acids. Inhibitory activity was never obtained in liquid preparations, except for strains Ny257 and T8 where it was found to be very unstable.     

Purification and biochemical characterization of mutacin I from the group I strain of Streptococcus mutans, CH43, and genetic analysis of mutacin I biosynthesis genes

Previously, we reported isolation and characterization of mutacin III and genetic analysis of mutacin III biosynthesis genes from the group III strain of Streptococcus mutans, UA787 (F. Qi, P. Chen, and P. W. Caufield, Appl. Environ. Microbiol. 65:3880-3887, 1999). During the same process of isolating the mutacin III structural gene, we also cloned the structural gene for mutacin I. In this report, we present purification and biochemical characterization of mutacin I from the group I strain CH43 and compare mutacin I and mutacin III biosynthesis genes. The mutacin I biosynthesis gene locus consists of 14 genes in the order mutR, -A, -A', -B, -C, -D, -P, -T, -F, -E, -G, orfX, orfY, orfZ. mutA is the structural gene for mutacin I, while mutA' is not required for mutacin I activity. DNA and protein sequence analysis revealed that mutacins I and III are homologous to each other, possibly arising from a common ancestor. The mature mutacin I is 24 amino acids in size and has a molecular mass of 2, 364 Da. Ethanethiol modification and peptide sequencing of mutacin I revealed that it contains six dehydrated serines, four of which are probably involved with thioether bridge formation. Comparison of the primary sequence of mutacin I with that of mutacin III and epidermin suggests that mutacin I likely has the same bridging pattern as epidermin.     

Purification and properties of extracellular mutacin, a bacteriocin from Streptococcus sobrinus.

Mutacin MT6223, a cell-free bacteriocin produced by Streptococcus sobrinus MT6223, was purified by ammonium sulphate precipitation, chromatofocusing with PBE 94 and column chromatography on SP Sephadex C-25. The specific activity of the purified mutacin was increased 1950-fold with a recovery of 9.7%. The molecular mass of the purified mutacin preparation was estimated to be 6.5 kDa. The mutacin activity was stable from pH 2-7, and was resistant to treatment at 100 degrees C for 20 min. It was inactivated by papain or ficin digestion, and was partially inhibited by alpha-chymotrypsin. The mutacin was found to be active against strains of serotypes c, e and f of Streptococcus mutans and the addition of purified mutacin MT6223 to growing cells of S. mutans MT8148 resulted in a rapid inhibition of incorporation of [3H]thymidine, [3H]uracil or L-[3H]glutamic acid into DNA, RNA or protein, respectively. Specific pathogen-free Fischer rats fed diet 2000 and infected with S. mutans MT8148R showed significantly fewer caries and lower plaque scores when mutacin was administered through drinking water. The present study demonstrates that mutacin MT6223 inhibited the growth of mutans streptococci. Thus, mutacin MT6223 may be a candidate for use in dental caries prevention.     

Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans

Biofilms are polymicrobial, with diverse bacterial species competing for limited space and nutrients. Under healthy conditions, the different species in biofilms maintain an ecological balance. This balance can be disturbed by environmental factors and interspecies interactions. These perturbations can enable dominant growth of certain species, leading to disease. To model clinically relevant interspecies antagonism, we studied three well-characterized and closely related oral species, Streptococcus gordonii, Streptococcus sanguinis, and cariogenic Streptococcus mutans. S. sanguinis and S. gordonii used oxygen availability and the differential production of hydrogen peroxide (H(2)O(2)) to compete effectively against S. mutans. Interspecies antagonism was influenced by glucose with reduced production of H(2)O(2). Furthermore, aerobic conditions stimulated the competence system and the expression of the bacteriocin mutacin IV of S. mutans, as well as the H(2)O(2)-dependent release of heterologous DNA from mixed cultures of S. sanguinis and S. gordonii. These data provide new insights into ecological factors that determine the outcome of competition between pioneer colonizing oral streptococci and the survival mechanisms of S. mutans in the oral biofilm.     

Comparison of the activity spectra against pathogens of bacterial strains producing a mutacin or a lantibiotic

The increase of drug resistance among bacterial pathogens is currently a major threat in hospital settings. New and more efficient antibiotic compounds have to be developed to fight infectious diseases. In the present work, a deferred antagonism test was used to determine the activity of different bacterial strains producing either a mutacin or a lantibiotic against bacterial pathogens. The mutacins A, B, C, D, I, K, L, M, and nisins A and Z were active against all enterococci tested. Mutacins A and B, and nisins A and Z inhibited all the staphylococci tested. Except for the strains producing mutacins P, Q, and X, all the other producing strains inhibited the streptococci tested. Mutacins A, B, I, J, T, nisins A and Z, and epidermin inhibited the two antibiotic-resistant strains of Neisseria gonorrhoeae tested. Mutacins A, B, C, D, and nisins A and Z inhibited Campylobacter jejuni and Helicobacter pylori. Thus, the wide activity spectra of nisin A and Z are confirmed. These results also indicate that many of the mutacins, especially those of groups A, B, C, D, I, J, K, L, M, and T, could be candidates for further development as useful antibiotics.     

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::Tn916) sequence from a Tn916-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.     

变形链球菌中的次级代谢产物及其在口腔生物被膜中的生态功能

口腔生物被膜是由数百种微生物构成的复杂微生物群体。变形链球菌作为其中的重要一员,被认为是引起龋病的主要病原菌。变形链球菌在牙齿表面以生物被膜形式生长的能力和它产酸耐酸的特点赋予其致龋性。不同的变形链球菌菌株之间保留了多样的次级代谢形式,这是与宿主长期共进化的结果。迄今为止,变形链球菌中报道的次级代谢产物包括10种细菌素(又称变链素)和一种聚酮/非核糖体肽类化合物。这些化合物多样的活性形式暗示了它们参与口腔生物被膜中跨种间和跨界间的相互作用。未来随着变形链球菌菌株数目的增加和更多菌株全基因组序列的完成,可以预见会有更多新颖活性次级代谢产物的出现。对变形链球菌次级代谢的研究不仅有助于治疗和预防口腔疾病,而且新颖活性次级代谢产物的发现对新药的研发也具有重要意义。     

Identification of nlmTE, the locus encoding the ABC transport system required for export of nonlantibiotic mutacins in Streptococcus mutans

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic bacteriocin (mutacin) activity. In this study, we have combined bioinformatic and mutational analyses to identify the ABC transporter designated NlmTE, which is required for mutacin biogenesis in strain UA159 as well as in another mutacin producer, S. mutans N.