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.     

Bacteriocin (mutacin) production by Streptococcus mutans genome sequence reference strain UA159: elucidation of the antimicrobial repertoire by genetic dissection

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic mutacin activity. In this study, bioinformatic and mutational analyses were employed to demonstrate that the antimicrobial repertoire of strain UA159 includes mutacin IV (specified by the nlm locus) and a newly identified bacteriocin, mutacin V (encoded by SMU.1914c).     

Purification of Mutacin III from Group III Streptococcus mutans UA787 and Genetic Analyses of Mutacin III Biosynthesis Genes

Previously, members of our group reported the isolation and characterization of mutacin II from Streptococcus mutans T8 and the genetic analyses of the mutacin II biosynthesis genes (J. Novak, P. W. Caufield, and E. J. Miller, J. Bacteriol. 176:4316–4320, 1994; F. Qi, P. Chen, and P. W. Caufield, Appl. Environ. Microbiol. 65:652–658, 1999; P. Chen, F. Qi, J. Novak, and P. W. Caufield, Appl. Environ. Microbiol. 65:1356–1360, 1999). In this study, we cloned and sequenced the mutacin III biosynthesis gene locus from a group III strain of S. mutans, UA787. DNA sequence analysis revealed eight open reading frames, which we designated mutR, -A, -A′, -B, -C, -D, -P, and -T. MutR bears strong homology with MutR of mutacin II, while MutA, -B, -C, -D, -P, and -T are counterparts of proteins in the lantibiotic epidermin group. MutA′ has 60% amino acid identity with MutA and therefore appears to be a duplicate of MutA. Insertional inactivation demonstrated that mutA is an essential gene for mutacin III production, while mutA′ is not required. Mutacin III was purified to homogeneity by using reverse-phase high-pressure liquid chromatography. N-terminal peptide sequencing of the purified mutacin III determined mutA to be the structural gene for prepromutacin III. The molecular mass of the purified peptide was measured by laser disorption mass spectrophotometry and found to be 2,266.43 Da, consistent with our supposition that mutacin III has posttranslational modifications similar to those of the lantibiotic epidermin.     

Genome Sequence of the Lantibiotic Bacteriocin Producer Streptococcus salivarius Strain K12

Streptococcus salivarius is a prevalent commensal species of the oropharyngeal tract. S. salivarius strain K12 is an isolate from the saliva of a healthy child, used as an oral probiotic. Here, we report its genome sequence, i.e., the full sequence of the 190-kb megaplasmid pSsal-K12 and a high-quality draft 2.2-Gb chromosomal sequence.     

Interactions between Oral Bacteria: Inhibition of Streptococcus mutans Bacteriocin Production by Streptococcus gordonii

Streptococcus mutans has been recognized as an important etiological agent in human dental caries. Some strains of S. mutans also produce bacteriocins. In this study, we sought to demonstrate that bacteriocin production by S. mutans strains GS5 and BM71 was mediated by quorum sensing, which is dependent on a competence-stimulating peptide (CSP) signaling system encoded by the com genes. We also demonstrated that interactions with some other oral streptococci interfered with S. mutans bacteriocin production both in broth and in biofilms. The inhibition of S. mutans bacteriocin production by oral bacteria was stronger in biofilms than in broth. Using transposon Tn916 mutagenesis, we identified a gene (sgc; named for Streptococcus gordonii challisin) responsible for the inhibition of S. mutans bacteriocin production by S. gordonii Challis. Interruption of the sgc gene in S. gordonii Challis resulted in attenuated inhibition of S. mutans bacteriocin production. The supernatant fluids from the sgc mutant did not inactivate the exogenous S. mutans CSP as did those from the parent strain Challis. S. gordonii Challis did not inactivate bacteriocin produced by S. mutans GS5. Because S. mutans uses quorum sensing to regulate virulence, strategies designed to interfere with these signaling systems may have broad applicability for biological control of this caries-causing organism.     

Effects of mutating putative two-component systems on biofilm formation by Streptococcus mutans UA159

Streptococcus mutans has at least six pairs of open reading frames that are homologous to bacterial two-component regulatory systems. Putative response regulators from five out of six of these pairs were successfully mutated by insertion of a kanamycin resistance marker and the effects of inactivation of the genes on the ability of the cells to form biofilms in an in vitro model were assessed. Disruption of the response regulators of four systems had no effect on biofilm formation, whereas disruption of one response regulator caused a substantial decrease in biofilm formation as compared to the wild-type S. mutans.     

Complete Genome Sequence of Streptococcus mutans GS-5, a Serotype c Strain

Streptococcus mutans, a principal causative agent of dental caries, is considered to be the most cariogenic among all oral streptococci. Of the four S. mutans serotypes (c, e, f, and k), serotype c strains predominate in the oral cavity. Here, we present the complete genome sequence of S. mutans GS-5, a serotype c strain originally isolated from human carious lesions, which is extensively used as a laboratory strain worldwide.     

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.     

Effect of the Medium on Bacteriocin Production Among Strains of Streptococcus mutans

Bacteriocin production by Streptococcus mutans was shown to be influenced by the medium on which the strains were grown, and it is suggested that selected media might be useful in bacteriocin typing.     

Genome Sequence of the Swine Pathogen Streptococcus suis Serotype 2 Strain S735

Streptococcus suis is a major swine pathogen responsible for significant, worldwide economic losses in the swine industry, in addition to being an emerging zoonotic agent. Strains of serotype 2 are the most commonly associated with infections causing meningitis, endocarditis, and septicemia. Here we present the genome sequence of S. suis serotype 2 strain S735.     

Structure-Activity Study of the Lantibiotic Mutacin II from Streptococcus mutans T8 by a Gene Replacement Strategy

Mutacin II, elaborated by group II Streptococcus mutans, is a ribosomally synthesized and posttranslationally modified polypeptide antibiotic containing unusual thioether and didehydro amino acids. To ascertain the role of specific amino acid residues in mutacin II antimicrobial activity, we developed a streptococcal expression system that facilitates the replacement of the mutA gene with a single copy of a mutated variant gene. As a result, variants of mutacin II can be designed and expressed. The system was tested by constructing the following mutant peptides: ΔN1, V7A, P9A, T10A, T10S, C15A, C26A, and C27A. All of these mutacin II variants except ΔN1 and T10A, which were not secreted, were isolated, and their identities were verified by mass spectrometry. Variants P9A, C15A, C26A, and C27A failed to exert antimicrobial activity. Because the P9A and T10A variants comprise the “hinge” region of mutacin II, these observations suggest that in addition to the thioether and didehydro amino acids, the hinge region is essential for biological activity and biosynthesis or export of the peptide. Tandem mass spectrometry of the N-terminal part of the wild-type molecule and its C15A variant confirmed that the threonine at position 10 is dehydrated and present as a didehydrobutyrine residue. This analysis of the active T10S variant further suggested that a didehydro amino acid at this position is specific for antimicrobial activity and that the biosynthetic machinery does not discriminate between threonine and serine. In contrast, the lack of production of mutacin variants with alanine substituted for threonine at position 10, as well as the deletion of asparagine at the N terminus (ΔN1), indicates that specific residues in the propeptide may be crucial for certain steps in the biosynthetic pathway of this lantibiotic.     

iTRAQ-based quantitative analysis reveals the mechanism underlying the changes in physiological activity in a glutamate racemase mutant strain of Streptococcus mutans UA159

Molecular Biology Reports - Streptococcus mutans UA159 is responsible for human dental caries with robust cariogenic potential. Our previous study noted that a glutamate racemase (MurI) mutant...     

Draft Genome Sequence of the Reference Strain of the Korean Medicinal Mushroom Wolfiporia cocos KMCC03342

Wolfiporia cocos is a wood-decay brown rot fungus belonging to the family Polyporaceae. While the fungus grows, the sclerotium body of the strain, dubbed Bokryeong in Korean, is formed around the roots of conifer trees. The dried sclerotium has been widely used as a key component of many medicinal recipes in East Asia. Wolfiporia cocos strain KMCC03342 is the reference strain registered and maintained by the Korea Seed and Variety Service for commercial uses. Here, we present the first draft genome sequence of W. cocos KMCC03342 using a hybrid assembly technique combining both short- and long-read sequences. The genome has a total length of 55.5 Mb comprised of 343 contigs with N50 of 332 kb and 95.8% BUSCO completeness. The GC ratio was 52.2%. We predicted 14,296 protein-coding gene models based on ab initio gene prediction and evidence-based annotation procedure using RNAseq data. The annotated genome was predicted to have 19 terpene biosynthesis gene clusters, which was the same number as the previously sequenced W. cocos strain MD-104 genome but higher than Chinese W. cocos strains. The genome sequence and the predicted gene clusters allow us to study biosynthetic pathways for the active ingredients of W. cocos.     

Two‐dimensional gel‐based proteomic of the caries causative bacterium Streptococcus mutans UA159 and insight into the inhibitory effect of carolacton

Streptococcus mutans is considered to be the most cariogenic organism. Carolacton, isolated from the myxobacterium Sorangium cellulosum, shows the ability to disturb S. mutans biofilm viability that makes it a potential anti‐biofilm drug. However, the molecular mechanism of carolacton remains to be elucidated. In order to use proteomics to characterize the effect of carolacton, we constructed a 2DE‐based proteome reference map of the cytoplasmic and extracellular proteins for S. mutans in the present study. In total, 239 protein spots representing 192 different cytoplasmic proteins were identified by MALDI‐TOF MS and PMF. This represents the highest number of identified proteins so far for S. mutans UA159 in the pI range of 4–7 and would benefit further research on the physiology and pathogenicity of this strain. Based on the constructed reference map, the inhibitory effects of carolacton on S. mutans biofilm and planktonic‐growing cells were investigated. The results of the comparative proteome analysis indicate that carolacton exerts its inhibitory effects by disturbing the peptidoglycan biosynthesis and degradation and thereby causes damages to the integrity of the cell envelope, leading ultimately to cell death.     

Draft Genome Sequence of the Flocculating Zymomonas mobilis Strain ZM401 (ATCC 31822)

Zymomonas mobilis ZM401 is a flocculating strain which can be self-immobilized within fermentors for a high-cell-density culture to improve ethanol productivity, as well as high-gravity fermentation to increase ethanol titer, due to its improved ethanol tolerance associated with the morphological change. Here, we report its draft genome sequence.     

Complete Genome Sequence of Streptococcus agalactiae ZQ0910, a Pathogen Causing Meningoencephalitis in the GIFT Strain of Nile Tilapia (Oreochromis niloticus)

Streptococcus agalactiae (group B streptococcus [GBS]) is a pathogen that causes meningoencephalitis in Nile tilapia (Oreochromis niloticus). Here, we reported the complete genome sequence of S. agalactiae strain ZQ0910, which was isolated from the GIFT strain of Nile tilapia in Guangdong, China.     

Cloning,Nucleotide Sequence,and Disruption of Streptococcus mutans Glutathione Reductase Gene (gor)

We cloned and sequenced the glutathione reductase gene (gor) of an oxygen-tolerant Streptococcus mutans, and constructed a gor-disruption mutant by homologous recombination. The gor gene consisted of 1,350 bp, coding for a protein of 450 amino acid residues. The deduced amino acid sequence of the S. mutans gor gene product showed extensive similarity with those of glutathione reductases from prokaryotes and eukaryotes. Although the mutant could grow aerobically, it showed no growth in the presence of 2 mM diamide, a thiol-specific oxidant. In contrast, growth of the wild-type strain was not significantly inhibited by 2 mM diamide, and glutathione reductase activity was increased 2.2-fold under these conditions. In addition, the level of glutathione reductase activity in the wild-type strain was increased 3.6-fold upon exposure to air, and the elevated level of the enzyme was retained throughout the aerobic growth. Thus, glutathione reductase may be important in protection of S. mutans against oxidative stress.