Efficient electrotransformation system and gene targeting in pyogenic streptococci

Hemolytic streptococci are lacking in natural competence for uptake of DNA, and existing electrotransformation methods are still ineffective for most strains. By optimizing biological and electric parameters of electroporation, we established a simple, efficient, and reproducible transformation method for streptococcal cells. The major factor was an increase in the electric field strength. All tested streptococci (6 group A strains and one group C strain) were successfully transformed, and the maximal efficiency was higher than 1 x 10(7) transformants per mug of plasmid DNA. Targeted inactivation of the chromosomal genes of group A and C streptococci was achieved, using the electrotransformation method. The slo- or sagB- mutants constructed by the gene-targeting showed elevated competence for electrotransformation. Availability of the electrotransfer system for cloning and analysis of streptococcal genes is discussed.     

Preparation of Protoplasts of Group H Streptococci (Streptococcus sanguis)

Stable protoplasts of several strains of group H streptococci (Streptococcus sanguis) can be prepared by use of group C streptococcal phage-associated lysin in the presence of 30% raffinose. Sucrose cannot be substituted for raffinose. Protoplast formation did not require the addition of Mg²⁺; however, this cation enhanced their stability. Some other strains, also presumptive group H streptococci, were not sensitive to phage-associated lysin.     

A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA

An improved method for the electrotransformation of wild-type Corynebacterium glutamicum (ATCC 13032) is described. The two crucial alterations to previously developed methods are: cultivation of cells used for electrotransformation at 18 °C instead of 30 °C, and application of a heat shock immediately following electrotransformation. Cells cultivated at sub optimal temperature have a 100-fold improved transformation efficiency (10⁸ cfu μg⁻¹) for syngeneic DNA (DNA isolated from the same species). A heat shock applied to these cells following electroporation improved the transformation efficiency for xenogeneic DNA (DNA isolated from a different species). In combination, low cultivation temperature and heat shock act synergistically and increased the transformation efficiency by four orders of magnitude to 2.5 × 10⁶ cfu μg⁻¹ xenogeneic DNA. The method was used to generate gene disruptions in C. glutamicum. Received: 26 March 1999 / Received revision: 9 June 1999 / Accepted: 11 June 1999     

Pulsed-Field Gel Electrophoresis Is More Discriminating Than Multilocus Enzyme Electrophoresis and Random Amplified Polymorphic DNA Analysis for Typing Pyogenic Streptococci

The SmaI restriction endonuclease digestion patterns of chromosomal DNAs from 99 pyogenic streptococci belonging to Lancefield group A (41 Streptococcus pyogenes), group C (seven S. dysgalactiae, 11 \QS. equisimilis\W, three S. equi, eight S. zooepidemicus) and group G (25 human group G Streptococcus, four S. canis) were analyzed by pulsed-field gel electrophoresis (PFGE), and the results were compared with those previously obtained by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA analysis (RAPD). PFGE revealed 93 distinct types among the 99 strains, and no patterns were common to strains of different species. The discriminatory power of PFGE was greater than that of MLEE and RAPD for groups A and G streptococci. The polymorphism among group C streptococci was similar with the three techniques. PFGE is, therefore, the most efficacious method for epidemiological typing of pyogenic streptococci. Received: 31 July 1996 / Accepted: 17 August 1996     

Horizontal gene transfer in the evolution of group A streptococcal emm-like genes: gene mosaics and variation in Vir regulons

Most M type 5 group A streptococcal strains were found to contain a single emm-like gene between virR and scpA (the Vir reguion), but two distinct emm-like genes were identified in the Vir regulon of the MS strain NCTC8193. The complete sequences of both of these genes were determined. One, called emm5.8193, was shown to be a minor variant of the previously described emm5 gene from strain Manfredo. The second, designated enn5.8193, expresses an IgG-binding protein when cloned in Escherichia coli. A comparison of enn5.8193 with emm-like gene sequences from other strains indicated that it has a mosaic structure, consisting of distinct segments originating from emm-like genes in different OF⁺ and OF⁻strains. These data provide the first clear evidence that the horizontal transfer of emm-like sequences between distinct strains contributes to the evolution of group A streptococcal emm-like genes and Vir regulons.     

Rapid Identification of Mutans Streptococcal Species

Mutans streptococci are considered the predominant pathogens in dental caries. Three methods, i.e. dot blot hybridization analysis, PCR analysis and SDS-blue dextran-PAGE, were examined for identifying mutans streptococcal species. In dot blot hybridization, DNA probe derived from the dextranase gene (dexA) of Streptococcus mutans hybridized with different intensities under the condition of low stringency against each species of mutans streptococci although the dexA probe was specific for S. mutans under the condition of high stringency. Oligonucleotide primers for polymerase chain reaction (PCR) were designed on the basis of the dexA DNA sequence. The primers amplified species-specific PCR products in the reference species (15 strains of 5 species) of mutans streptococci. An electrophoretic profile of dextranases from the mutans streptococci on SDS-blue dextran-PAGE also showed species-specific behavior. These results suggest that the three identification methods examined here are useful for distinguishing the species of mutans streptococci and also indicate that PCR analysis is suitable for simple, rapid and reliable identification of mutans streptococcal species.     

Competence for Natural Genetic Transformation in the Streptococcus bovis Group Streptococci S. infantarius and S. macedonicus

Natural genetic transformation is common among many species of the genus Streptococcus, but it has never, or rarely, been reported for the Streptococcus pyogenes and S. bovis groups of species, even though many streptococcal competence genes and the competence regulators SigX, ComR, and ComS are well conserved in both groups. To explore the incidence of competence in the S. bovis group, 25 isolates of S. infantarius and S. macedonicus were surveyed by employing culture in chemically defined media devoid of peptide nutrients and treatment with synthetic candidate pheromone peptides predicted from the sequence of the gene comS. Approximately half of strains examined were transformable, many transforming at high rates comparable to those for the well-characterized streptococcal natural transformation systems. In S. infantarius, nanomolar amounts of the synthetic pheromone LTAWWGL induced robust but transient competence in high-density cultures, but mutation of the ComRS locus abolished transformation. We conclude that at least these two species of the S. bovis group retain a robust system of natural transformation regulated by a ComRS pheromone circuit and the alternative sigma factor SigX and infer that transformation is even more common among the streptococci than has been recognized. The tools presented here will facilitate targeted genetic manipulation in this group of streptococci.     

High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media

An efficient method for genetic transformation of lactococci by electroporation is presented. Highly competent lactococci for electrotransformation were obtained by growing cells in media containing high concentrations of glycine and 0.5 M sucrose as the osmotic stabilizers. These cells could be stored at −85°C without loss of competence. With Lactococcus lactis subsp. cremoris BC101, a transformation frequency of 5.7 × 10⁷ transformants per μg of pIL253 DNA was obtained, which represents 5% of the surviving cells. All the lactococcal strains tested could be transformed by the present method.     

Two-dimensional gel electrophoresis analysis of the abundance of virulent exoproteins of group A streptococcus caused by environmental changes

Group A streptococci regulate the expression of virulence factors in response to environmental change. In order to investigate this mechanism, the growth of group A streptococci and the abundance of virulent exoprotein production in culture supernatant were analyzed by two-dimensional gel electrophoresis (2-D electrophoresis) under several culture conditions. Judging from alterations in their growth, group A streptococci were affected by various environmental stresses. Under high O₂ and low CO₂ concentrations, streptococcal pyrogenic exotoxin B (SpeB) and streptococcal pyrogenic exotoxin F (SpeF) significantly decreased, and the streptococcal inhibitor of complement (Sic) increased . At 30 °C, increases in endo--N-acetylglucosaminidase (EndoS) and -amylase were also detected, while at 41 °C EndoS became undetectable and SpeB and SpeF decreased. Sic, SpeF and mitogenic factor 3 (Mf3) decreased when cells were cultured in higher NaCl concentrations, and EndoS disappeared following culture of the cells in high glucose concentration. An increase in acid phosphatase and a decrease in several other proteins were detected when the cells were cultivated in high iron concentrations. These results suggest that group A streptococci have a versatile adaptation system that responds to several environmental stresses by altering the level of exoprotein production.Abbreviations SpeB Streptococcal pyrogenic exotoxin B - SpeF Streptococcal pyrogenic exotoxin F - Sic Streptococcal inhibitor of complement - EndoS Endo--N-acetylglucosaminidase     

Prevalence and genomic characterization of Group A Streptococcus dysgalactiae subsp. equisimilis isolated from patients with invasive infections in Toyama prefecture,Japan

Streptococcus dysgalactiae subsp. equisimilis (SDSE) causes severe invasive streptococcal infections, especially in elderly people. Between 2013 and 2018, 88 streptococci were isolated from clinical blood culture in a hospital in Toyama prefecture, Japan. The collection included six Group A SDSE (ASD) strains, which are rarely isolated. Multilocus sequence typing categorized five of the six strains into ST128 and the remaining strain into a new type. Maximum-likelihood phylogenetic analysis revealed that the six ASD strains had highly similar genome sequences. Bayesian analysis indicated that the most recent common ancestor of the strains appeared 39 years ago. The ASD strains possessed carbohydrate synthase genes that are conserved in Streptococcus pyogenes strains, whereas one strain featured a different arrangement of the gene cluster. The carbohydrate synthase genes varied by Lancefield type (A, C, and G).     

The action of nitrosoguanidine and other DNA-inactivating agents on Streptococcus pyogenes K56 strains with normal and reduced dark repair ability

Summary The response pattern for ultraviolet light, nitrogen mustard, and ethyl methane sulphonate of Hcr⁺ and Hcr^- strains ofStreptococcus pyogenes K 56 is similar to that observed for analogous strains ofE. coli, whereas repair-apt streptococcal strains are much more sensitive to nitrosoguanidine and mitomycin C thanE. coli. Theuvr gene(s) appear(s) to be without effect upon survival, prophage induction, and mutation to streptomycin resistance caused by nitrosoguanidine and only of little influence on repair of mitomycin C damage.     

Electroporation and conjugal plasmid transfer to members of the genus Aquaspirillum

Electroporation methods and conjugal matings were used to transfer several plasmid vectors to Aquaspirillum dispar and Aquaspirillum itersonii. The incompatibility P class plasmid RP4 was conjugally transferred from Escherichia coli HB101 to these spirilla, and the transconjugants subsequently donated the molecule to plasmid-free E. coli and A. dispar strains via conjugal matings. High-voltage electrotransformation was used to transfer plasmids pUCD2, pSa151 and RP4 to A. dispar and A. itersonii, at efficiencies as high as 3×10⁴ transformants per μg plasmid DNA. RP4 DNA isolated from spirillum hosts, but not RP4 from E. coli cells was successfully transferred to A. dispar and A. itersonii by electrotransformation, suggesting that modification and/or restriction activity may be present in these Aquaspirillum species.     

Electrotransformation of Clostridium thermocellum

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 ± 0.5) × 10⁵ transformants per μg of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 ± 1.8) × 10⁴ transformants per μg of plasmid DNA for strain ATCC 27405 and ~1 × 10³ transformants per μg of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was ~50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.     

Detection of cholera (ctx) and zonula occludens (zot) toxin genes in Vibrio cholerae O1, O139 and non-O1 strains

Vibrio cholerae O1 and V. cholerae non-O1 strains isolated from environmental samples collected in São Paulo, Brazil, during cholera epidemics and pre-epidemic periods were examined for the presence of toxin genes. V. cholerae O1 strains isolated from clinical samples in Peru and Mexico, and V. cholerae O139 strains from India were also examined for the presence of ctx (cholera toxin gene) and zot (zonula occludens toxin gene) by polymerase chain reaction (PCR). A modified DNA-extraction method applied in this study yielded satisfactory recovery of genomic DNA from vibrios. Results showed that strains of V. cholerae O1 isolated during the preepidemic period were ctx ^-/zot ^- whereas strains isolated during the epidemic were ctx ⁺/zot ⁺. All V. cholerae non-O1 strains tested in the study were ctx ^-/zot ^-, whereas all V. cholerae O139 strains were ctx ⁺/zot ⁺. Rapid detection of the virulence genes (ctx and zot) can be achieved by PCR and this can serve as an important tool in the epidemiology and surveillance of V. cholerae.     

Translocation of antibiotic resistance markers of a plasmid-free Streptococcus pyogenes (group A) strain into different streptococcal hemolysin plasmids

Summary Wild-type strain A454 (Streptococcus pyogenes) transferred en bloc its erythromycin (Em) and tetracycline (Tc) resistance markers into several plasmid-free streptococcal recipients. No plasmid DNA was detected in either the wild-type or the transconjugant strains. Crosses were performed between A454 and S. faecalis Rec⁺ or Rec^- recipients carrying hemolysin-bacteriocin plasmids, pIP964 or pAD1. The Em Tc-resistant transconjugants obtained harbored either the parental plasmid or an Em Tc resistance plasmid derived from pIP964 or pAD1. The restriction endonuclease analysis of 12 derivative plasmids showed insertions of various sizes into different fragments of pIP964 or pAD1. A454 and the Em Tc-resistant plasmid-free transconjugants were found to contain two EcoRI DNA fragments, that shared homology with ³²P-labeled pIP1077, one of the Em Tc resistance derivative plasmids, but not with ³²P-labeled pIP964. No homology was detected between pIP1077 and the cellular DNA of the antibiotic-susceptible recipients.Previously Thea Horodniceanu     

Electrotransformation of Streptococcus agalactiae with plasmid DNA

Abstract A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli - Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-μl aliquot containing about 5×10⁹ colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 μg. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2×10⁴ cfu μg⁻¹. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae .     

Glycoconjugates as possible receptors forStreptococcus pyogenes

The adherence capacities of M-protein-positive (M+) and M-protein-negative (M-) strains ofStreptococcus pyogenes were compared in human epithelial cells obtained from the pharynx (PEC) or from the buccal mucosa (BEC). Adherence to PEC was related to the presence of M protein (40.5±1.1 M+ and 17.8±0.6 M–S. pyogenes per PEC), whereas BEC showed adherence equally for M+ and M– strains. Different receptor sites may thus be involved on the two cell types. Preincubation of the bacteria with disialogangliosides (1 mg/ml), orN-acetylgalactosamine, ord-galactose (10 g/ml) resulted in diminished adherence of M+ strains to PEC but not to BEC. Chromatography ond-galactose-Sepharose 6B showed specific binding only of M+ group A streptococcal strains to gel beads. M– group A, and groups C and G streptococci did not bind. These observations suggest that the receptors on PEC for group A streptococci are distinct from those on BEC, and that most probably the attachment ofS. pyogenes to human pharyngeal cells occurs by specific, lectin-like binding to galactose residues on epithelial cells.