Effects of dietary carbohydrates on mutacin production and activity

Although mutacins (bacteriocins produced by Streptococcus mutans) were shown to be active in vivo, their ecological role in the oral cavity is still controversial. In the present paper, the effect of dietary carbohydrates, one of the ecological parameters which influences oral bacterial populations, on the activity and the production of mutacins from four S. mutans strains (C67-1, Ny257, Ny266 and T8) is described. Results obtained by the deferred antagonism test in solid media and by the mixed cultivation of the mutacinogenic strains with a sensitive indicator strain in liquid batch cultures, indicate that a minimal fermentable sugar concentration is needed for mutacin production. Among all the fermentable carbohydrates tested (fructose, glucose, lactose, mannitol and sucrose), none significantly affected the production and the activity of the four mutacinogenic strains used, in concentrations up to 5%. Although the results do not discount the possibility of mutacin inactivation in vivo, they indicate that they are not affected by dietary carbohydrates.     

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.     

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.     

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.     

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.     

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.     

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.     

Enzyme-labeled oligonucleotide probes for detection of the genes for thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus.

Alkaline phosphatase conjugated oligonucleotide probes were developed to detect the genes (tdh and trh) coding for the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus. Using dot blot hybridization, probes were tested with 94 clinical isolates of V. parahaemolyticus. Results agreed well with those obtained using radio-labeled recombinant DNA probes for the genes tdh and trh. Specificity and sensitivity of enzyme tdh probes for detection of the trh gene were 100 and 93%, respectively, and those of the trh probes for trh gene detection were 93 and 86%, respectively. The tdh probes also hybridized with tdh-like genes processed by all strains of V. hollisae, and some strains of V. mimicus and V. cholerae non-O1, but neither tdh nor trh probes reacted with other bacterial species isolated from diarrheal stools. However, some V. parahaemolyticus strains that were negative with the enzyme trh probe hybridized weakly with a radio-labeled trh DNA fragment probe at medium stringency, and a few strains that were negative in high stringency conditions with a radio-labeled trh DNA fragment probe hybridized with the enzyme trh probe. This suggests that some strains of V. parahaemolyticus may carry another gene resembling trh.     

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.     

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.     

Structure and dynamics of the lantibiotic mutacin 1140

Mutacin 1140 is a member of a family of ribosomally synthesized peptide bacteriocins called lantibiotics (lanthionine-containing antibiotics) and is produced by the Gram-positive bacterium Streptococcus mutans. Mutacin 1140 has been shown to be effective against a broad array of Gram-positive bacteria. Chromatography and mass spectroscopy data suggested that mutacin 1140 forms a small compact structure. Nuclear magnetic resonance (NMR) data and restrained molecular dynamics simulations showed that mutacin 1140 interconverts between multiple structures. Calculations of scalar (J) coupling constants showed the best agreement with experimental values when the entire population-weighted ensemble of structures was used, providing independent support for the ensemble. Representative structures from each major group in the ensemble had a common feature in which they are all kinked around the hinge region forming a horseshoe-like shape, and the regions of flexibility of the molecule were limited and well-defined. The structures determined in this study provide a starting point for modeling the mutacin 1140-membrane interactions and pore formation.     

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.     

应用生物素标记DNA探针检测伪狂犬病病毒的研究

应用生物素标记ＤＮＡ探针检测伪狂犬病病毒的研究王琴,郭万柱,阴文奇（四川农业大学动物科技学院,四川雅安,６２５５０１４）关键词伪狂犬病毒,核酸,杂交,检测核酸杂交技术已广泛用于检测畜禽疱疹病毒和其它动物病毒,已报道ＰＲＶ－ＤＮＡ的探针方法有ＲＮＡ－Ｄ...     

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.     

Multiple rpoD-related genes of cyanobacteria.

Genomes of many eubacterial strains have been shown to encode for multiple rpoD-related genes. In this report, we describe the identification of the multiple rpoD-related genes of cyanobacterial strains. DNAs of three cyanobacterial strains, Anabaena sp. PCC7120, Synechococcus sp. PCC7942, and Synechocystis sp. PCC6803, were examined by Southern hybridization, using a synthetic probe designed for detecting rpoD or rpoD-related genes. Four or five hybridization signals were found in each DNA. Four DNA regions of Synechococcus sp. PCC7942 corresponding to the hybridization signals were cloned and partially sequenced. The sequence data indicate the presence of genes, named rpoD1, rpoD2, rpoD3, and rpoD4, whose products are highly similar to the basic structure of the principal sigma factors of eubacterial strains. The rpoD1 gene showed the greatest similarity to the sigA gene of Anabaena sp. PCC7120.     

Hybridization analysis of the gene encoding a hemolysin (suilysin) of Streptococcus suis type 2: evidence for the absence of the gene in some isolates

A hemolysin gene was cloned from a virulent strain of Streptococcus suis type 2 strain 1933. Analysis of the gene and its product revealed that it is identical to a previously reported hemolysin (suilysin) of S. suis type 2. Southern hybridization analysis of the digested total genomic DNA from S. suis with the cloned hemolysin DNA sequences as probe indicated that the hemolysin gene is present as a single copy on the genome. Genomic DNA of 63 isolates of S. suis encompassing all known serotypes were examined by DNA hybridization and polymerase chain reaction (PCR) studies for the presence of the hemolysin gene homolog. The results of both techniques were identical and demonstrated the absence of the hemolysin gene in some isolates. In DNA hybridization studies, three DNA probes derived from the hemolysin encoding gene were used. Results showed that sequences encoding the C-terminal 257 amino acid residues (Probe 1) were the most conserved and hybridized to a 1.2 kb fragment in 32 (51%) strains and a 4.0 kb fragment in 23 (36%) strains respectively. Thus, Probe 2 hybridized to the DNA of 55 (87%) of the isolates tested. The first probe (Probe 1) comprising almost the entire hemolysin gene and the third probe (Probe 3) which consisted of the N-terminal sequences hybridized only to a 4.0 kb fragment in 23 (36%) of the strains tested. Eight (13%) of the strains tested were hybridization and PCR negative. The hybridization of the C-terminal end sequences (Probe 2) to the 1.2 kb fragment in 32 (51%) of the strains and the lack of hybridization of the probes to eight (13%) strains may suggest the presence of different types of hemolysin molecule in S. suis strains.     

Site-Directed Mutations in the Lanthipeptide Mutacin 1140

The oral bacterium Streptococcus mutans, strain JH1140, produces the antibiotic mutacin 1140. Mutacin 1140 belongs to a group of antibiotics called lanthipeptides. More specifically, mutacin 1140 is related to the epidermin type A(I) lanthipeptides. Mutagenesis experiments of this group of lanthipeptides have been primarily restricted to the posttranslationally modified meso-lanthionine and 3-methyllanthionine residues. Site-directed mutagenesis of the core peptide of mutacin 1140 was performed using the suicide vector pVA891. Substitutions of the N-terminal residue, the charged residue in the hinge region, and residues in ring A and intertwined rings C and D were investigated. A truncation and insertion of residues in ring A and intertwined rings C and D were also performed to determine whether or not they would alter the antimicrobial activity of the producing strain. Bioassays revealed that five of 14 mutants studied had improved antimicrobial activity against the indicator strain Micrococcus luteus ATCC 10240. MICs against Streptococcus mutans UA159, Streptococcus pneumoniae ATCC 27336, Staphylococcus aureus ATCC 25923, Clostridium difficile UK1, and Micrococcus luteus ATCC 10240 were determined for three mutacin 1140 variants that had the most significant increases in bioactivity in the M. luteus bioassay. This mutagenesis study of the epidermin group of lanthipeptides shows that antimicrobial activity can be significantly improved.     

Nucleotide sequence of the type A staphylococcal enterotoxin gene.

We determined the nucleotide sequence of the gene encoding staphylococcal enterotoxin A (entA). The gene, composed of 771 base pairs, encodes an enterotoxin A precursor of 257 amino acid residues. A 24-residue N-terminal hydrophobic leader sequence is apparently processed, yielding the mature form of staphylococcal enterotoxin A (Mr, 27,100). Mature enterotoxin A has 82, 72, 74, and 34 amino acid residues in common with staphylococcal enterotoxins B and C1, type A streptococcal exotoxin, and toxic shock syndrome toxin 1, respectively. This level of homology was determined to be significant based on the results of computer analysis and biological considerations. DNA sequence homology between the entA gene and genes encoding other types of staphylococcal enterotoxins was examined by DNA-DNA hybridization analysis with probes derived from the entA gene. A 624-base-pair DNA probe that represented an internal fragment of the entA gene hybridized well to DNA isolated from EntE+ strains and some EntA+ strains. In contrast, a 17-base oligonucleotide probe that encoded a peptide conserved among staphylococcal enterotoxins A, B, and C1 hybridized well to DNA isolated from EntA+, EntB+, EntC1+, and EntD+ strains. These hybridization results indicate that considerable sequence divergence has occurred within this family of exotoxins.