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.     

Elucidation of the antimicrobial mechanism of mutacin 1140

Mutacin 1140 and nisin A are peptide antibiotics that belong to the lantibiotic family. N-Terminal rings A and B of nisin A and mutacin 1140 (lipid II-binding domain) share many structural and sequence similarities. Nisin A binds lipid II and thus disrupts cell wall synthesis and also forms transmembrane pores. Very little is known about mutacin 1140 in this regard. We performed fluorescence-based studies using a bacteria-mimetic membrane system. The results indicated that lipid II monomers are arranged differently in the mutacin 1140 complex than in the nisin A complex. These differences in complex formation may be attributed to the fact that nisin A uses lipid II to form a distinct pore complex, while mutacin 1140 does not form pores in this membrane system. Further experiments demonstrated that the mutacin 1140-lipid II and nisin A-lipid II complexes are very stable and capable of withstanding competition from each other. Transmembrane electrical potential experiments using a Streptococcus rattus strain, which is sensitive to mutacin 1140, demonstrated that mutacin 1140 does not form pores in this strain even at a concentration 8 times higher than the minimum inhibitory concentration (MIC). Circular complexes of mutacin 1140 and nisin A were observed by electron microscopy, providing direct evidence for a lateral assembly mechanism for these antibiotics. Mutacin 1140 did exhibit a membrane disruptive function in another commonly used artificial bacterial membrane system, and its disruptive activity was enhanced by increasing amounts of anionic phospholipids.     

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.     

Covalent adducts of lactate oxidase. Photochemical formation and structure identification.

Lactate oxidase forms tight complexes with a variety of mono- and dicarboxylic acids. Most of these undergo facile photoreactions involving decarboxylation of the carboxylic acid and formation of covalent adducts at position N(5) of the flavin, characterized by absorption maxima from 325 to 365 nm and fluorescence emission in the range 440 to 490 nm. The properties of the adducts are strongly dependent on the nature of the substituent. Enzyme-bound N(5)-acyl adducts and N(5)-CH2-R derivatives are stable in the dark. Glycollyl- and alpha-lactyl adducts, however, decay to oxidized enzyme with half-lives in the order of minutes. Upon denaturation of the enzyme, the N(5)-alkyl adducts decay rapidly or are oxidized by oxygen. Reduced lactate oxidase is also photoalkylated in the presence of halogenated carboxylic acids. Bromoacetate yields an N(5)-carboxymethyl adduct; with beta-bromopropionate, a C(4a)-beta-propionyl derivate is formed. The N(5) adduct is identical with that from the photochemical reaction of oxidized enzyme and malonic acid. When the native coenzyme FMN is substituted by 2-S-FMN, qualitatively the same photoproducts are formed. The adducts obtained with the 2-S-FMN enzyme show the expected bathochromic shifts in absorption spectra. The results indicate that the photoreactivity of the enzyme is restricted to the positions C(4a) and N(5) of the flavin.     

LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila

Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59% (98/107) and 93.33% (56/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100% (51/51) and 100% (18/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.     

Isolation and biochemical characterization of a novel lantibiotic mutacin from Streptococcus mutans.

Certain members of the indigenous biota of humans produce antimicrobial substances called bacteriocins, which inhibit other bacteria, including members of their own species. One of these substances, mutacin, is made by Streptococcus mutans, a member of the oral biota. Mutacin inhibits other mutans streptococci as well as many gram-positive exogenous pathogens. Here, we report for the first time the purification and partial biochemical characterization of a lanthionine-containing mutacin peptide from S. mutants T8. The biologically active peptide was isolated from the broth cultures by ultrafiltration and differential precipitation. The final mutacin preparation was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal amino acid sequencing. A molecular mass of the peptide was estimated by electrospray ionization mass spectroscopy to be 3,244.64 +/- 1.15 Da. Its amino acid composition indicates the presence of lanthionine and likely beta-methyllanthionine in a total of about 25 amino acids. Because alpha,beta-unsaturated amino acids, the precursors of lanthionine residues, are often found in lantibiotics, we carried out the addition reaction of the mutacin with N-(methyl)mercaptoacetamide. The subsequent electrospray ionization mass spectroscopy analysis indicated the presence of two reaction products with M(r)s of 3,350.45 and 3,456.0. These are interpreted as the mutacin molecule with the addition of one and two molecules of reagent to the unsaturated amino acids, respectively. Sequencing of the peptide revealed an N-terminal amino acid sequence of Asn-Arg-Trp-Trp-Gln-Gly-Val-Val.     

Fourier-transform infrared microspectroscopy,a novel and rapid tool for identification of yeasts

Fourier-transform infrared (FT-IR) microspectroscopy was used in this study to identify yeasts. Cells were grown to microcolonies of 70 to 250 micro m in diameter and transferred from the agar plate by replica stamping to an IR-transparent ZnSe carrier. IR spectra of the replicas on the carrier were recorded using an IR microscope coupled to an IR spectrometer, and identification was performed by comparison to reference spectra. The method was tested by using small model libraries comprising reference spectra of 45 strains from 9 genera and 13 species, recorded with both FT-IR microspectroscopy and FT-IR macrospectroscopy. The results show that identification by FT-IR microspectroscopy is equivalent to that achieved by FT-IR macrospectroscopy but the time-consuming isolation of the organisms prior to identification is not necessary. Therefore, this method also provides a rapid tool to analyze mixed populations. Furthermore, identification of 21 Debaryomyces hansenii and 9 Saccharomyces cerevisiae strains resulted in 92% correct identification at the strain level for S. cerevisiae and 91% for D. hansenii, which demonstrates that the resolution power of FT-IR microspectroscopy may also be used for yeast typing at the strain level.     

A new method for rapid identification of influenza virus isolates.

With the use of bacteria sensitized by influenza virus strain-specific antisera, virus isolates can be identified rapidly. One drop of virus suspension is mixed with one drop of sensitized bacteria on a slide that is then agitated; reaction occurs within 10 minutes. The test is subtype-specific. The mehod is based on the fact that the cell wall of the Cowan type 1 strain of Staphylococcus aureus contains abundant quantities of an antigen, known as protein A, that reacts with the IgG molecule by binding it in such a manner that the antibody-combining sites remain free. If an antigen homologous to the antibody coated on the surface of the bacteria is added to the suspension of sensitized staphylococci, agglutination occurs.     

Development of a rapid identification method for Aeromonas species by multiplex-PCR

Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.     

FT-IR microspectroscopy: a promising method for the rapid identification of Listeria species

This work presents a pilot study to investigate the potential of fourier transform infrared (FT-IR) microspectroscopy for rapid identification of Listeria at the species level. Using this technique, FT-IR spectra were acquired from 30 strains from five Listeria species. The FT-IR spectra were analysed using stepwise canonical discriminant analysis and partial least-squares regression in a stepwise identification scheme. The results showed that 93% of all the samples were assigned to the correct species, and that 80% of the Listeria monocytogenes strains were correctly identified. In comparison, 100% of the samples, including the L. monocytogenes samples, were correctly identified using spectra acquired by FT-IR macrospectroscopy. The results show that FT-IR microspectroscopy has potential as a rapid screening method for Listeria, which is especially valuable for the food industry.     

A method for the rapid identification of pathogenic yeast-like microorganisms

Summary A new rapid diagnostic method for the identification of pathogenic yeast-like microorganisms is described. By this method in the course of one week these genera can be identified:Candida, Torulopsis, Rhodotorula, Trichosporon, Geotrichum and seven species often found in infectious material, of the genusCandida: C. albicans, C. tropicalis, C. pseudotropicalis, C. krusei, C. guilliermondii, C. pelliculosa andC. parapsilosis.