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.     

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.     

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.     

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.     

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).     

SmbFT,a Putative ABC Transporter Complex,Confers Protection against the Lantibiotic Smb in Streptococci

Streptococcus mutans, a dental pathogen, secretes different kinds of lantibiotic and nonlantibiotic bacteriocins. For self-protection, a bacteriocin producer strain must possess one or more cognate immunity mechanisms. We report here the identification of one such immunity complex in S. mutans strain GS-5 that confers protection against Smb, a two-component lantibiotic. The immunity complex that we identified is an ABC transporter composed of two proteins: SmbF (the ATPase component) and SmbT (the permease component). Both of the protein-encoding genes are located within the smb locus. We show that GS-5 becomes sensitized to Smb upon deletion of smbT, which makes the ABC transporter nonfunctional. To establish the role SmbFT in providing immunity, we heterologously expressed this ABC transporter complex in four different sensitive streptococcal species and demonstrated that it can confer resistance against Smb. To explore the specificity of SmbFT in conferring resistance, we tested mutacin IV (a nonlantibiotic), nisin (a single peptide lantibiotics), and three peptide antibiotics (bacitracin, polymyxin B, and vancomycin). We found that SmbFT does not recognize these structurally different peptides. We then tested whether SmbFT can confer protection against haloduracin, another two-component lantibiotic that is structurally similar to Smb; SmbFT indeed conferred protection against haloduracin. SmbFT can also confer protection against an uncharacterized but structurally similar lantibiotic produced by Streptococcus gallolyticus. Our data suggest that SmbFT truly displays immunity function and confer protection against Smb and structurally similar lantibiotics.     

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.     

Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

A new Type III restriction endonuclease designated PstII has been purified from Providencia stuartii. PstII recognizes the hexanucleotide sequence 5′-CTGATG(N)_25-26/27-28-3′. Endonuclease activity requires a substrate with two copies of the recognition site in head-to-head repeat and is dependent on a low level of ATP hydrolysis (~40 ATP/site/min). Cleavage occurs at just one of the two sites and results in a staggered cut 25–26 nt downstream of the top strand sequence to generate a two base 5′-protruding end. Methylation of the site occurs on one strand only at the first adenine of 5′-CATCAG-3′. Therefore, PstII has characteristic Type III restriction enzyme activity as exemplified by EcoPI or EcoP15I. Moreover, sequence asymmetry of the PstII recognition site in the T7 genome acts as an historical imprint of Type III restriction activity in vivo. In contrast to other Type I and III enzymes, PstII has a more relaxed nucleotide specificity and can cut DNA with GTP and CTP (but not UTP). We also demonstrate that PstII and EcoP15I cannot interact and cleave a DNA substrate suggesting that Type III enzymes must make specific protein–protein contacts to activate endonuclease activity.     

Optimization of the production of the lantibiotic mutacin 1140 in minimal media

Mutacin 1140 is produced by Streptococcus mutans and belongs to the type A lantibiotic family. Experiments were done to optimize production of mutacin 1140 in minimal media enabling a more cost efficient downstream purification method. The development of a small volume fermentation method enabled a rapid screen of several variables in a standard shaking incubator. This method provided a fast approach for determining components that promote mutacin 1140 production in minimal media broth. Lactose was determined to be the optimal carbon source for mutacin 1140 production. High concentrations of CaCl₂ (0.3%, w/v) and MgSO₄ (0.77%, w/v) promoted an increase in mutacin 1140 production, while ZnCl₂ and FeCl₃ appeared to impair production. Optimization of mutacin 1140 production in minimal media resulted in more than a 100-fold increase in production compared to the base medium used to begin our optimizations. The yield has been estimated by RP-HPLC to be ∼10 mg/L.     

Isolation, Characterization, and Heterologous Expression of the Novel Lantibiotic Epicidin 280 and Analysis of Its Biosynthetic Gene Cluster

Epicidin 280 is a novel type A lantibiotic produced by Staphylococcus epidermidis BN 280. During C₁₈ reverse-phase high-performance liquid chromatography two epicidin 280 peaks were obtained; the two compounds had molecular masses of 3,133 ± 1.5 and 3,136 ± 1.5 Da, comparable antibiotic activities, and identical amino acid compositions. Amino acid sequence analysis revealed that epicidin 280 exhibits 75% similarity to Pep5. The strains that produce epicidin 280 and Pep5 exhibit cross-immunity, indicating that the immunity peptides cross-function in antagonization of both lantibiotics. The complete epicidin 280 gene cluster was cloned and was found to comprise at least five open reading frames (eciI, eciA, eciP, eciB, and eciC, in that order). The proteins encoded by these open reading frames exhibit significant sequence similarity to the biosynthetic proteins of the Pep5 operon of Staphylococcus epidermidis 5. A gene for an ABC transporter, which is present in the Pep5 gene cluster but is necessary only for high yields (G. Bierbaum, M. Reis, C. Szekat, and H.-G. Sahl, Appl. Environ. Microbiol. 60:4332–4338, 1994), was not detected. Instead, upstream of the immunity gene eciI we found an open reading frame, eciO, which could code for a novel lantibiotic modification enzyme involved in reduction of an N-terminally located oxopropionyl residue. Epicidin 280 produced by the heterologous host Staphylococcus carnosus TM 300 after introduction of eciIAPBC (i.e., no eciO was present) behaved homogeneously during reverse-phase chromatography.     

Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB

Previous work has demonstrated that iron-dependent variations in the steady-state concentration and translatability of sodB mRNA are modulated by the small regulatory RNA RyhB, the RNA chaperone Hfq and RNase E. In agreement with the proposed role of RNase E, we found that the decay of sodB mRNA is retarded upon inactivation of RNase E in vivo, and that the enzyme cleaves within the sodB 5′-untranslated region (5′-UTR) in vitro, thereby removing the 5′ stem–loop structure that facilitates Hfq and ribosome binding. Moreover, RNase E cleavage can also occur at a cryptic site that becomes available upon sodB 5′-UTR/RyhB base pairing. We show that while playing an important role in facilitating the interaction of RyhB with sodB mRNA, Hfq is not tightly retained by the RyhB–sodB mRNA complex and can be released from it through interaction with other RNAs added in trans. Unlike turnover of sodB mRNA, RyhB decay in vivo is mainly dependent on RNase III, and its cleavage by RNase III in vitro is facilitated upon base pairing with the sodB 5′-UTR. These data are discussed in terms of a model, which accounts for the observed roles of RNase E and RNase III in sodB mRNA turnover.     

Synthesis and characterization of oligonucleotides containing 2'-fluorinated thymidine glycol as inhibitors of the endonuclease III reaction

Endonuclease III (Endo III) is a base excision repair enzyme that recognizes oxidized pyrimidine bases including thymine glycol. This enzyme is a glycosylase/lyase and forms a Schiff base-type intermediate with the substrate after the damaged base is removed. To investigate the mechanism of its substrate recognition by X-ray crystallography, we have synthesized oligonucleotides containing 2′-fluorothymidine glycol, expecting that the electron-withdrawing fluorine atom at the 2′ position would stabilize the covalent intermediate, as observed for T4 endonuclease V (Endo V) in our previous study. Oxidation of 5′- and 3′-protected 2′-fluorothymidine with OsO₄ produced two isomers of thymine glycol. Their configurations were determined by NMR spectroscopy after protection of the hydroxyl functions. The ratio of (5R,6S) and (5S,6R) isomers was 3:1, whereas this ratio was 6:1 in the case of the unmodified sugar. Both of the thymidine glycol isomers were converted to the corresponding phosphoramidite building blocks and were incorporated into oligonucleotides. When the duplexes containing 2′-fluorinated 5R- or 5S-thymidine glycol were treated with Escherichia coli endo III, no stabilized covalent intermediate was observed regardless of the stereochemistry at C5. The 5S isomer was found to form an enzyme–DNA complex, but the incision was inhibited probably by the fluorine-induced stabilization of the glycosidic bond.     

Aspergillus parasiticus Cyclase Catalyzes Two Dehydration Steps in Aflatoxin Biosynthesis

In the aflatoxin biosynthetic pathway, 5′-oxoaverantin (OAVN) cyclase, the cytosolic enzyme, catalyzes the reaction from OAVN to (2′S,5′S)-averufin (AVR) (E. Sakuno, K. Yabe, and H. Nakajima, Appl. Environ. Microbiol. 69:6418-6426, 2003). Interestingly, the N-terminal 25-amino-acid sequence of OAVN cyclase completely matched an internal sequence of the versiconal (VHOH) cyclase that was deduced from its gene (vbs). The purified OAVN cyclase also catalyzed the reaction from VHOH to versicolorin B (VB). In a competition experiment using the cytosol fraction of Aspergillus parasiticus, a high concentration of VHOH inhibited the enzyme reaction from OAVN to AVR, and instead VB was newly formed. The recombinant Vbs protein, which was expressed in Pichia pastoris, showed OAVN cyclase activity, as well as VHOH cyclase activity. A mutant of A. parasiticus SYS-4 (= NRRL 2999) with vbs deleted accumulated large amounts of OAVN, 5′-hydroxyaverantin, averantin, AVR, and averufanin in the mycelium. These results indicated that the cyclase encoded by the vbs gene is also involved in the reaction from OAVN to AVR in aflatoxin biosynthesis. Small amounts of VHOH, VB, and aflatoxins also accumulated in the same mutant, and this accumulation may have been due to an unknown enzyme(s) not involved in aflatoxin biosynthesis. This is the first report of one enzyme catalyzing two different reactions in a pathway of secondary metabolism.     

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

Background  

Streptococcus 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.     

Small-Subunit rRNA Genotyping of Rhizobia Nodulating Australian Acacia spp.

The structure of rhizobial communities nodulating Acacia in southeastern Australia from south Queensland to Tasmania was investigated by a molecular approach. A total of 118 isolates from nodule samples from 13 different Acacia species collected at 44 sites were characterized by small-subunit (SSU) ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis. Nine rhizobial genomospecies were identified, and these taxa corresponded to previously described genomospecies (B. Lafay and J. J. Burdon, Appl. Environ. Microbiol. 64:3989–3997, 1998). Eight of these genomospecies belonged to the Bradyrhizobium lineage and accounted for 96.6% of the isolates. The remaining genomospecies corresponded to Rhizobium tropici. For analysis of geographic patterns, results were grouped into five latitudinal regions regardless of host origin. In each region, as observed previously for rhizobial isolates taken from non-Acacia legumes (Lafay and Burdon, Appl. Environ. Microbiol. 64:3989–3997, 1998), rhizobial communities were dominated by one or two genomospecies, the identities of which varied from place to place. Despite this similarity in patterns, the most abundant genomospecies for Acacia isolates differed from the genomospecies found in the non-Acacia-derived rhizobial collection, suggesting that there is a difference in nodulation patterns of the Mimosoideae and the Papilionoideae. Only two genomospecies were both widespread and relatively abundant across the range of sites sampled. Genomospecies A was found in all regions except the most northern sites located in Queensland, whereas genomospecies B was not detected in Tasmania. This suggests that genomospecies A might be restricted to the more temperate regions of Australia, whereas in contrast, genomospecies B occurs in different climatic and edaphic conditions across the whole continent. The latter hypothesis is supported by the presence of genomospecies B in southwestern Australia, based on partial SSU rDNA sequence data (N. D. S. Marsudi, A. R. Glenn, and M. J. Dilworth, Soil Biol. Biochem. 31:1229–1238, 1998).     

Epitope Identification for a Panel of Anti-Sinorhizobium meliloti Monoclonal Antibodies and Application to the Analysis of K Antigens and Lipopolysaccharides from Bacteroids

In two published reports using monoclonal antibodies (MAbs) generated against whole cells, Olsen et al. showed that strain-specific antigens on the surface of cultured cells of Sinorhizobium meliloti were diminished or absent in the endophytic cells (bacteroids) recovered from alfalfa nodules, whereas two common antigens were not affected by bacterial differentiation (P. Olsen, M. Collins, and W. Rice, Can. J. Microbiol. 38:506–509, 1992; P. Olsen, S. Wright, M. Collins, and W. Rice, Appl. Environ. Microbiol. 60:654–661, 1994). The nature of the antigens (i.e., the MAb epitopes), however, were not determined in those studies. For this report, the epitopes for five of the anti-S. meliloti MAbs were identified by polyacrylamide gel electrophoresis-immunoblot analyses of the polysaccharides extracted from S. meliloti and Sinorhizobium fredii. This showed that the strain-specific MAbs recognized K antigens, whereas the strain-cross-reactive MAbs recognized the lipopolysaccharide (LPS) core. The MAbs were then used in the analysis of the LPS and K antigens extracted from S. meliloti bacteroids, which had been recovered from the root nodules of alfalfa, and the results supported the findings of Olsen et al. The size range of the K antigens from bacteroids of S. meliloti NRG247 on polyacrylamide gels was altered, and the epitope was greatly diminished in abundance compared to those from the cultured cells, and no K antigens were detected in the S. meliloti NRG185 bacteroid extract. In contrast to the K antigens, the LPS core appeared to be similar in both cultured cells and bacteroids, although a higher proportion of the LPS fractionated into the organic phase during the phenol-water extraction of the bacteroid polysaccharides. Importantly, immunoblot analysis with an anti-LPS MAb showed that smooth LPS production was modified in the bacteroids.     

Identification of 16S Ribosomal DNA-Defined Bacterial Populations at a Shallow Submarine Hydrothermal Vent near Milos Island (Greece)

In a recent publication (S. M. Sievert, T. Brinkhoff, G. Muyzer, W. Ziebis, and J. Kuever, Appl. Environ. Microbiol. 65:3834–3842, 1999) we described spatiotemporal changes in the bacterial community structure at a shallow-water hydrothermal vent in the Aegean Sea near the isle of Milos (Greece). Here we describe identification and phylogenetic analysis of the predominant bacterial populations at the vent site and their distribution at the vent site as determined by sequencing of DNA molecules (bands) excised from denaturing gradient gels. A total of 36 bands could be sequenced, and there were representatives of eight major lineages of the domain Bacteria. Cytophaga-Flavobacterium and Acidobacterium were the most frequently retrieved bacterial groups. Less than 33% of the sequences exhibited 90% or more identity with cultivated organisms. The predominance of putative heterotrophic populations in the sequences retrieved is explained by the input of allochthonous organic matter at the vent site.     

Escherichia coli Cells Exposed to Streptomycin Display a Mutator Phenotype

Mistranslation mediated by the mutA and mutC tRNA alleles elicits a strong mutator phenotype (H. S. Murphy and M. Z. Humayun, J. Bacteriol. 179:7507–7514, 1997; M. M. Slupska, C. Baikalov, R. Lloyd, and J. H. Miller, Proc. Natl. Acad. Sci. USA 93:4380–4385, 1996). Here, we show that exposure to streptomycin, an antibiotic known to promote mistranslation, induces a recA- and umuDC-independent mutator phenotype detected as enhanced mutagenesis at a 3,N⁴-ethenocytosine lesion borne on transfected M13 single-stranded DNA.