Novel series of plasmid vectors for gene inactivation and expression analysis in group A streptococci (GAS)

Podbielski, Andreas; Spellerberg, Barbara; Woischnik, Markus; Pohl, Barbara; Lütticken, Rudolf

doi:10.1016/0378-1119(96)84178-3

Cited by 191 publications

(179 citation statements)

References 41 publications

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“…A fragment including the rny gene together with the upstream region and the TT3 transcriptional terminator [45] was generated by PCR with OLEC3584/OLEC3579. The downstream region of rny and the lox71- ermAM/B -lox66 cassette were amplified by PCR with OLEC3480/OLEC3572 and OLEC2000/OLEC3585, respectively.…”

Section: Methodsmentioning

confidence: 99%

RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B

et al. 2018

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Endoribonuclease Y (RNase Y) is a crucial regulator of virulence in Gram-positive bacteria. In the human pathogen Streptococcus pyogenes, RNase Y is required for the expression of the major secreted virulence factor streptococcal pyrogenic exotoxin B (SpeB), but the mechanism involved in this regulation remains elusive. Here, we demonstrate that the 5′ untranslated region of speB mRNA is processed by several RNases including RNase Y. In particular, we identify two RNase Y cleavage sites located downstream of a guanosine (G) residue. To assess whether this nucleotide is required for RNase Y activity in vivo, we mutated it and demonstrate that the presence of this G residue is essential for the processing of the speB mRNA 5′ UTR by RNase Y. Although RNase Y directly targets and processes speB, we show that RNase Y-mediated regulation of speB expression occurs primarily at the transcriptional level and independently of the processing in the speB mRNA 5′ UTR. To conclude, we demonstrate for the first time that RNase Y processing of an mRNA target requires the presence of a G. We also provide new insights on the speB 5′ UTR and on the role of RNase Y in speB regulation.

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Section: Methodsmentioning

confidence: 99%

RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B

et al. 2018

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“…The flanking sequences were ligated by PCR, generating an amplimer (comCDEflank) with a central unique BamHI site that was cloned into pGEM-T (Promega) in E. coli JM109. The aad9 cassette with its own promoter and transcription terminator (1082 bp) was amplified from pFW5 (Podbielski et al, 1996) with terminal BamHI sites and cloned into the unique BamHI site within the vector pGEM-T+comCDEflank. The resulting construct (pGEM-T+comCDEflank-aad9; 2272 bp) was confirmed by restriction enzyme digestion (SacI and PstI) and DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

Streptococcus gordonii comCDE (competence) operon modulates biofilm formation with Candida albicans

et al. 2015

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“…The region 59 to CRISPR1 was amplified by PCR with primers CR1 and CR2, using genomic DNA from S. mutans OMZ 1001 as template (OMZ 1001 is a derivative of UA159 with a high transformation frequency). The product was digested with BamHI and HindIII and cloned into plasmid pFW15 (Podbielski et al, 1996) to create plasmid pOMZ357. Subsequently, the region 39 to CRISPR1 was amplified with primers CR3 and CR4, digested with EcoRI and NcoI, and cloned into pOMZ357 to create pOMZ358.…”

Section: Methodsmentioning

confidence: 99%

Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages

Ploeg

2009

Microbiology

102

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Clustered regularly interspaced short palindromic repeats (CRISPR) consist of highly conserved direct repeats interspersed with variable spacer sequences. They can protect bacteria against invasion by foreign DNA elements. The genome sequence of Streptococcus mutans strain UA159 contains two CRISPR loci, designated CRISPR1 and CRISPR2. The aims of this study were to analyse the organization of CRISPR in further S. mutans strains and to investigate the importance of CRISPR in acquired immunity to M102-like phages. The sequences of CRISPR1 and CRISPR2 arrays were determined for 29 S. mutans strains from different persons. More than half of the CRISPR1 spacers and about 35 % of the CRISPR2 spacers showed sequence similarity with the genome sequence of M102, a virulent siphophage specific for S. mutans. Although only a few spacers matched the phage sequence completely, most of the mismatches had no effect on the amino acid sequences of the phage-encoded proteins. The results suggest that S. mutans is often attacked by M102-like bacteriophages, and that its acquisition of novel phage-derived CRISPR sequences goes along with the presence of S. mutans phages in the environment. Analysis of CRISPR1 of M102-resistant mutants of S. mutans OMZ 381 showed that some of them had acquired novel spacers, and the sequences of all but one of these matched the phage M102 genome sequence. This suggests that the acquisition of the spacers contributed to the resistance against phage infection. However, since not all resistant mutants had new spacers, and since the removal of the CRISPR1 array in one of the mutants and in wild-type strains did not lead to loss of resistance to infection by M102, the acquisition of resistance must be based on further elements as well.

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Novel series of plasmid vectors for gene inactivation and expression analysis in group A streptococci (GAS)

Cited by 191 publications

References 41 publications

RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B

RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B

Streptococcus gordonii comCDE (competence) operon modulates biofilm formation with Candida albicans

Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages

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