An
            <i>rpsL</i>
            Cassette, Janus, for Gene Replacement through Negative Selection in
            <i>Streptococcus pneumoniae</i>

Sung, Chang Kyoo; Li, H.; Claverys, Jean‐Pierre; Morrison, Donald A.

doi:10.1128/aem.67.11.5190-5196.2001

Cited by 373 publications

(416 citation statements)

References 29 publications

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“…The extended CC PcsB is composed of residues arranged in a continuous helical arrangement. Only the first four residues (41)(42)(43)(44)(45) and those of the short turns do not follow this pattern. All the helices within the CC domain are located in the same plane except for the a5 helix.…”

Section: Resultsmentioning

confidence: 96%

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Structural basis of PcsB-mediated cell separation in Streptococcus pneumoniae

et al. 2014

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Separation of daughter cells during bacterial cell division requires splitting of the septal cross wall by peptidoglycan hydrolases. In Streptococcus pneumoniae, PcsB is predicted to perform this operation. Recent evidence shows that PcsB is recruited to the septum by the transmembrane FtsEX complex, and that this complex is required for cell division. However, PcsB lacks detectable catalytic activity in vitro, and while it has been proposed that FtsEX activates PcsB, evidence for this is lacking. Here we demonstrate that PcsB has muralytic activity, and report the crystal structure of full-length PcsB. The protein adopts a dimeric structure in which the V-shaped coiled-coil (CC) domain of each monomer acts as a pair of molecular tweezers locking the catalytic domain of each dimeric partner in an inactive configuration. This suggests that the release of the catalytic domains likely requires an ATP-driven conformational change in the FtsEX complex, conveyed towards the catalytic domains through coordinated movements of the CC domain.

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Section: Resultsmentioning

confidence: 96%

“…Genomic DNA from strain SPH154 (ref. 43), which contains a Janus cassette inserted in the ectopic locus, served as template to amplify a PCR fragment consisting of the Janus cassette plus B1,000 bp flanking regions. The PCR reaction was carried out with the primers khb31 and khb34.…”

Section: Methodsmentioning

confidence: 99%

Structural basis of PcsB-mediated cell separation in Streptococcus pneumoniae

et al. 2014

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“…As CbpD and LytC both contain N-terminal signal peptides, the Histag was introduced immediately downstream of the predicted signal peptidase cleavage site. The wild-type genes were replaced with the corresponding mutant genes by double-crossover recombination using the counterselectable Janus cassette as described by Sung et al (2001). The activities of the CbpD-His, LytA-His and LytC-His proteins were found to be the same or somewhat reduced compared to their non-modified counterparts.…”

Section: Resultsmentioning

confidence: 99%

Fratricide in Streptococcus pneumoniae: contributions and role of the cell wall hydrolases CbpD, LytA and LytC

et al. 2009

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Pneumococci that have developed the competent state kill and lyse non-competent sister cells and members of closely related species during co-cultivation in vitro. The key component in this process, called fratricide, is the product of the late competence gene cbpD. In addition, the peptidoglycan hydrolases LytA and LytC are required for efficient lysis of target cells. Here, we have investigated the relative contribution and possible role of each of the proteins mentioned above. Previous studies have shown that CbpD is produced exclusively by competent cells, whereas LytA and LytC can be provided by the competent attackers as well as the non-competent target cells. By using an improved assay to compare the effect of cis-versus trans-acting LytA and LytC, we were able to show that target cells are lysed much more efficiently when LytA and LytC are provided in cis, i.e. by the target cells themselves. Western analysis demonstrated that considerable amounts of LytC are present in the growth medium. In contrast, we were not able to detect any extracellular LytA. This finding indicates that LytA-and LytC-mediated fratricide represent different processes. In the absence of LytA and LytC, only a tiny fraction of the target cells were lysed, demonstrating that CbpD does not function efficiently on its own. However, in the presence of 1 mM EDTA, the fraction of target cells lysed directly by CbpD increased dramatically, indicating that divalent cations are involved in the regulation of fratricide under natural conditions.

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“…The capsular variants of otherwise isogenic strains were constructed in three different genetic backgrounds-TIGR4 (originally of serotype 4) and 603 and 618 (both originally of serotype 6B)-by using a previously described method (48). In short, the parent strain was rendered unencapsulated by insertion of a cassette into the capsule locus using the transformation protocol published by Pozzi et al (37) and the bicistronic Janus cassette constructed by Sung et al (46). Encapsulated mutants were generated by replacing the cassettes with a cps locus from a donor strain.…”

Section: Methodsmentioning

confidence: 99%

The Capsular Serotype ofStreptococcus pneumoniaeIs More Important than the Genetic Background for Resistance to Complement

et al. 2010

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The polysaccharide capsule of Streptococcus pneumoniae inhibits phagocytic killing by innate immune mechanisms. Certain serotypes are associated with invasive disease while others with a nasopharyngeal carriage. The invasiveness of serotypes may partly be explained by ability to resist deposition of complement (C3) on the bacterial surface and consequent opsonophagocytic killing. In our previous studies, we observed that clinical isolates of serotypes 1 and 5, which are rarely detected in asymptomatic carriage, were resistant to complement deposition and opsonophagocytosis, whereas serotypes 6B and 23F, both common in carriage, were more sensitive to deposition of C3 and opsonophagocytic killing. However, presence of significant variation in C3 deposition between isolates of the same serotype indicated that factors other than the capsule also affect complement resistance. To distinguish the relative effect of the capsular serotype and other virulence factors on C3 deposition, we compared capsule-switched mutants prepared in genetic backgrounds of pneumococcal strains TIGR4, 603, and 618. Clinical isolates which had the same multilocus sequence type but expressed different serotypes were also compared. We found that the serotype had a significant impact on complement resistance and that the more resistant the strain was to complement, the higher was the concentration of polysaccharide-specific antibodies required for opsonophagocytic killing. Comparison of strains expressing the same capsular polysaccharides in the different genetic backgrounds and various capsular mutants of the same strain suggests that while the genotype affects complement resistance, the serotype is the most important determinant. Differences between serotypes were more significant than the differences between strains.

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An rpsL Cassette, Janus, for Gene Replacement through Negative Selection in Streptococcus pneumoniae

Cited by 373 publications

References 29 publications

Structural basis of PcsB-mediated cell separation in Streptococcus pneumoniae

Structural basis of PcsB-mediated cell separation in Streptococcus pneumoniae

Fratricide in Streptococcus pneumoniae: contributions and role of the cell wall hydrolases CbpD, LytA and LytC

The Capsular Serotype ofStreptococcus pneumoniaeIs More Important than the Genetic Background for Resistance to Complement

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