Superantigen-like gene(s) in human pathogenic Streptococcus dysgalactiae,subsp equisimilis: genomic localisation of the gene encoding streptococcal pyrogenic exotoxin G (speG(dys))

Streptococcus pyogenes (GAS) causes about 90% of streptococcal human infections while group C (GCS) and G (GGS) streptococci can be pathogenic for different mammalians. Especially the human pathogenic GCS and GGS, Streptococcus dysgalactiae, subsp. equisimilis, account for 5-8% of the human streptococcal diseases like wound infections, otitis media, purulent pharyngitis and also streptococcal toxic shock syndrome. A defined superantigen so far was not identified in GCS and GGS strains. In the present investigation we screened DNA of GCS and GGS human isolates for the presence of genes for streptococcal pyrogenic exotoxins (spe) by hybridisation with probes that stand for the GAS genes speA, speC, speZ (smeZ), speH, speG, speI, speJ and ssa. In many GCS and GGS strains we found positive reactions with the probes speG, speJ and ssa, but not with the probes for the remaining genes under investigation. PCR amplification with subsequent sequence analysis of the PCR fragments revealed only the presence of the gene speG in GCS and GGS strains, while no DNA fragments specific for speJ and ssa could be amplified. Additionally, the upstream and downstream regions flanking speG in GGS strain 39072 were sequenced. Remarkable differences were found in the neighbourhood of speG between GAS and GGS sequences. Downstream of speG we identified in strain GGS 39072 two new open reading frames encoding proteins with no similarity to protein sequences accessible in the databases so far. In the compared GAS strains SF370 and MGAS8232, this segment, apart from some small fragments, had been deleted. Our analysis suggests that a gene transfer from GGS to GAS has preceded following deletion of the two genes orf1 and orf2 in GAS.     

Inactivation of the streptokinase gene prevents Streptococcus equisimilis H46A from acquiring cell-associated plasmin activity in the presence of plasminogen

Abstract The effects of some physico-chemical parameters on production of extracellular α-L-arabinofuranosidase by Aspergillus nidulans were examined. Highest levels of α-L-arabinofuranosidase were generated with cultures grown on 1% (w/v) purified beet pulp arabinan at 30°C and at an initial pH of 7.0. The enzyme was shown to be very sensitive to the action of proteases. Zymogram overlay of a protein profile obtained by SDS-PAGE revealed the occurrence of a band ( M _r 36 000) exhibiting α-L-arabinofuranosidase activity. The isoelectric pH of the enzyme lay near 4.3. Temperature and pH optima for the activity of crude α-L-arabinofuranosidase preparations were 55°C and 5.5, respectively. Enzyme activity was greatly reduced by thiol reagents such as Hg²⁺ and p -hydroxymercuribenzoate and showed a K _m value of 2.7 mM on p -nitrophenyl α-L-arabinofuranoside as substrate.     

Severe invasive streptococcal infection by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

Streptococcus pyogenes, a group A Streptococcus (GAS), has been recognized as the causative pathogen in patients with severe invasive streptococcal infection with or without necrotizing fasciitis. In recent epidemiological studies, Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been isolated from severe invasive streptococcal infection. Complete genome sequence showed that SDSE is the closest bacterial species to GAS, with approximately 70% of genome coverage. SDSE, however, lacks several key virulence factors present in GAS, such as SPE‐B, the hyaluronan synthesis operon and active superantigen against human immune cells. A key event in the ability of GAS to cause severe invasive streptococcal infection was shown to be the acquisition of novel genetic traits such as phages. Strikingly, however, during severe invasive infection, GAS destroys its own covRS two‐component system, which negatively regulates many virulence factor genes, resulting in a hyper‐virulent phenotype. In contrast, this phenomenon has not been observed in SDSE. The present review describes the epidemiology of severe invasive streptococcal infection and the detailed pathogenic mechanisms of GAS and SDSE, emphasizing findings from their genome sequences and analyses of gene expression.     

Electrotransformation of Streptococcus pyogenes with plasmid and linear DNA

Electrotransformation was used to introduce both plasmid and linear DNA into Streptococcus pyogenes. The method was optimized using strain NZ131, for which transformation frequencies up to 10(7) per micrograms of plasmid DNA were obtained. A linear fragment of DNA, containing the streptokinase gene (ska) in which an internal fragment had been replaced with an erythromycin resistance gene (erm), was transformed into strain NZ131 with a frequency of 10(3) per micrograms DNA. The introduction of linear DNA into S. pyogenes by electrotransformation should be useful for future genetic analyses as well as targeted gene replacement.     

Production of acidocin B, a bacteriocin of Lactobacillus acidophilus M46 is a plasmid-encoded trait: Plasmid curing, genetic marking by in vivo plasmid integration, and gene transfer

Abstract Plasmid-curing studies suggest that acidocin B production is encoded by the 14-kb plasmid pCV461 in Lactobacillus acidophilus M46. Loss of pCV461 from the original producer strain M46 did not coincide with loss of immunity to acidocin B. Bacteriocin activity determination after SDS-PAGE showed that a substance of 2.4 kDa, absent in the culture supernatant of the mutant strain M46A2, lacking pCV461, represented acidocin B activity. In order to introduce a positive selection criterion, pCV461 was marked in vivo by the erythromycin resistance marker of pE194, present on pUC19 containing a 1.4-kb Hin dIII fragment of pCV461, after plasmid integration. Introduction of this recombinant plasmid into the mutant strain M46A2 or Lactobacillus plantarum resulted in erythromycin-resistant, acidocin B-producing transformants, showing unambiguously that acidocin B is encoded by pCV461.     

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).     

Allelic variation of the streptokinase gene in β-hemolytic streptococci group C and G isolates of human origin

Abstract Genetic diversity of the streptokinase gene ( sk ) from 36 strains of S. equisimilis and 54 strains of group G streptococci was examined. The strains were isolated from patients with various streptococcal disease manifestations and healthy carriers. The region of the gene that corresponds to amino acid residues 174–244, was PCR amplified. The amplified product was subjected to Mlu I, Pvu II, Dra I and Dde I digestion. Based on the restriction enzyme digestion patterns nine sk alleles were recognized. There was no correlation between the various sk gene alleles and streptococcal disease manifestations. Three of the nine sk gene alleles, sk 4, sk 7, and sk 8, were detected earlier among group A streptococci. The other six alleles were unique to S. equisimilis and group G streptococci. The most common alleles were sk 5, found in 21/90 (23%) and sk 10 detected in 43/90 (47%) of the strains. Alleles sk 1 and sk 2, the most frequent among group A streptococci, were not found among the strains in the present investigation. Thus, it appears that the sk gene has been evolving in line with other species distinguishing features of the streptococci.     

Construction of Streptococcus mutans glucosyltransferase mutants utilizing a cloned gene fragment

Abstract An internal 1.6-kb BAM HI DNA fragment of the previously cloned Streptococcus mutans GS-5 gtfB gene was utilized to construct plasmids capable of insertion in toto into the GS-5 chromosome. The resultant insertions primarily yielded mutants defective in glucosyltransferase-I activity. These mutants were also defective in sucrose-dependent colonization of smooth surfaces.     

儿童感染肺炎链球菌的青霉素结合蛋白基因突变分析

目的 分析儿童感染肺炎链球菌的青霉素结合蛋白基因突变与青霉素耐药水平之间的关系。方法 自2012年1月至2014年12月期间分离的1 317株肺炎链球菌中随机抽取出青霉素MIC=2.0 µg/mL、4.0 µg/mL、≥8.0 µg/mL各20株共60株作为实验菌株,采用PCR方法对实验菌株进行青霉素结合蛋白PBP1a、PBP1b、PBP2a、PBP2b、PBP2x、PBP3的基因扩增,扩增产物进一步纯化和测序,测序结果与青霉素敏感肺炎链球菌R6就国际上公认的PBPs保守序列进行比对分析。结果 60株肺炎链球菌的PBP2b、PBP1a、PBP2x、PBP2a基因的保守区或保守区附件均发现氨基酸突变,未发现PBP3与PBP1b突变。中介与耐药菌株基因突变位点存在重合,主要出现在单一的PBP1a序列的370STMK模体元件内Thr371Ser置换突变或伴有PBP2b序列的Thr451Ala/Ser和Ala624Gly置换突变,同时PBP2a序列的465SLN模体元件前置位发生Ser461Ala的置换突变。结论 肺炎链球菌对青霉素中、高水平耐药菌株绝大部分合并有不同PBP序列中4~6个氨基酸的置换突变,但合并多个氨基酸置换突变并非必然引起耐药水平相应升高。中、高水平耐药与PBP1a、PBP2b、PBP2a的变异关系密切,其中PBP1a的STMK保守区域Thr371Ser置换是引起耐药的主要因素之一。     

Purification, characterisation, cloning and sequencing of the gene encoding oligopeptidase PepO from Streptococcus thermophilus A

The oligopeptidase PepO from Streptococcus thermophilus A was purified to protein homogeneity by a five-step chromatography procedure. It was estimated to be a serine metallopeptidase of 70 kDa, with maximal activity at pH 6.5 and 41 degrees C. PepO has endopeptidase activity on oligopeptides composed of between five and 30 amino acids. PepO was demonstrated to be active and stable at the pH, temperature and salt concentrations found in Swiss-type cheese during ripening. Using a battery of PCR techniques, the pepO gene was amplified, subcloned and sequenced, revealing an open reading frame of 1893 nucleotides. The amino acid sequence analysis of the pepO gene-translation product shows homology with PepO enzymes from other lactic acid bacteria and contains the signature sequence of the metallopeptidase family.