culture conditions s anginosus  (ATCC)

Journal: Virulence

Article Title: The stress of carrying CRISPR-Cas

doi: 10.1080/21505594.2025.2541701

Figure Lengend Snippet: Expression and functionality of CRISPR-Cas system in S. anginosus . (a) RT-PCR performed with 20 ng isolated RNA of S. anginosus SK52 (WT), cas9 deletion (∆ cas9 ), cas9 complementation (∆ cas9:cas9 ) and CRISPR array deletion (∆CRISPR) strain verifying expression of cas9 using primer 13/41. Complete DNase digestion was controlled by conducting PCR without RT step. Controls with genomic DNA of S. anginosus SK52 are indicated by “+” and negative controls without nucleic acid by “-“. The 1 kb plus DNA ladder (Invitrogen) served as molecular weight marker. (b) Confirming the functionality of S. anginosus CRISPR-Cas system by interference with plasmid transformation. Relative transformation efficiencies of created plasmids normalized to the empty vector pAT28 were obtained via competence-based transformation assay. Vectors were designed to contain an endogenous spacer of S. anginosus SK52 CRISPR-Cas system followed by a functional or non-functional PAM sequence, resulting in the CRISPR-targeted plasmid pAT28_SP1_TGG and the non-targeted plasmid pAT28_SP1_AAA. Mean values and standard deviations of six independent experiments are shown. Mann-Whitney-U test was performed to illustrate significant differences as indicated (** p < 0.01).

Article Snippet: Streptococcus anginosus SK 52 , S. anginosus type strain, ATCC 33397, Hly+ , ATCC.

Techniques: Expressing, CRISPR, Reverse Transcription Polymerase Chain Reaction, Isolation, Molecular Weight, Marker, Plasmid Preparation, Transformation Assay, Functional Assay, Sequencing, MANN-WHITNEY

Journal: Virulence

Article Title: The stress of carrying CRISPR-Cas

doi: 10.1080/21505594.2025.2541701

Figure Lengend Snippet: Effects of UV irradiation on viability of S. anginosus SK52 and mutant strains ∆ cas9 , ∆ cas9:cas9 and ∆CRISPR. (a) Strains on this agar plate were exposed to UV irradiation for 43 s − 96 s on THY agar followed by 24 h of growth at 37°C. Shown is one representative plate out of five independently conducted experiments. (b) Results of (a) are represented as lethal irradiation time from which less than five CFUs could grow. (c) S. anginosus SK52, ∆ cas9 and clinical isolates naturally carrying (yellow n = 5) or lacking (orange n = 4) a type II-A CRISPR-Cas system were exposed to UV irradiation on THY agar followed by 24 h of growth at 37°C. Results are presented as percentage of the lethal irradiation time of strain SK52. (b) and (c) shown is the mean ± standard deviation of five independent experiments. Significant differences were calculated with Mann-Whitney-U test (** p < 0.01).

Article Snippet: Streptococcus anginosus SK 52 , S. anginosus type strain, ATCC 33397, Hly+ , ATCC.

Techniques: Irradiation, Mutagenesis, CRISPR, Standard Deviation, MANN-WHITNEY

Journal: Virulence

Article Title: The stress of carrying CRISPR-Cas

doi: 10.1080/21505594.2025.2541701

Figure Lengend Snippet: CRISPR-Cas related stress response to high temperature and oxidative stress. (a) Survival of S. anginosus SK52 and mutant strains after exposure to 50°C temperature stress for 10 min −120 min. Results represent mean CFU and standard deviations of five independent experiments conducted with the S. anginosus SK52 and mutant strains. Survival was determined as percentage of viable counts of heat-treated bacteria compared to the untreated bacteria. (b) Effect of oxidative stress to the survival of S. anginosus SK52 and mutant strains. S. anginosus SK52, as well as ∆ cas9 , ∆ cas9:cas9 and ∆CRISPR strains were subjected to 2 mM −3.5 mM hydrogen peroxide (H 2 O 2 ) for 24 h. Results were obtained during four independent experiments and represent mean values and standard deviations. Differences were statistically significant (* p < 0.05, ** p < 0.01; Mann-Whitney-U test), unless otherwise indicated it refers to wild-type strain.

Article Snippet: Streptococcus anginosus SK 52 , S. anginosus type strain, ATCC 33397, Hly+ , ATCC.

Techniques: CRISPR, Mutagenesis, Bacteria, MANN-WHITNEY

Journal: Virulence

Article Title: The stress of carrying CRISPR-Cas

doi: 10.1080/21505594.2025.2541701

Figure Lengend Snippet: Growth behaviour of S. anginosus influenced by Cas9. (a) Differences in colony size of S. anginosus SK52 (WT), cas9 deletion mutant (∆ cas9 ), cas9 complementation mutant (∆ cas9:cas9 ) and CRISPR array deletion mutant (∆CRISPR). 10 µl droplets of indicated strains were incubated for 24 h on THY agar. (b) Growth of S. anginosus SK52 wild-type and mutant strains over time. Each point represents the average of five independent optical density values per sample including standard deviation. Differences were statistically significant (* p < 0.05, ** p < 0.01; Mann-Whitney-U test). When indicated on left side of the ∆ cas9 growth curve it refers to wild-type strain and on right side it refers to ∆ cas9:cas9 .

Article Snippet: Streptococcus anginosus SK 52 , S. anginosus type strain, ATCC 33397, Hly+ , ATCC.

Techniques: Mutagenesis, CRISPR, Incubation, Standard Deviation, MANN-WHITNEY

Journal: Virulence

Article Title: The stress of carrying CRISPR-Cas

doi: 10.1080/21505594.2025.2541701

Figure Lengend Snippet: Metabolic activity related to the expression of cas9 with and without environmental stress. (a) Effect of 50°C temperature stress on the metabolic activity of S. anginosus SK52 and mutant strains within 4 h in a resazurin assay. (b) Measurement of the metabolic activity of S. anginosus SK52 and mutant strains within 2 h in a resazurin assay. Results represent the mean absorbance ratio 572 nm/600 nm and standard deviations of five independent experiments conducted with the S. anginosus SK52 and mutant strains. Differences were statistically significant (* p < 0.05, ** p < 0.01; Mann-Whitney-U test), unless otherwise indicated it refers to wild-type strain.

Article Snippet: Streptococcus anginosus SK 52 , S. anginosus type strain, ATCC 33397, Hly+ , ATCC.

Techniques: Activity Assay, Expressing, Mutagenesis, Resazurin Assay, MANN-WHITNEY