Peptide-Regulated Gene Depletion System Developed for Use in Streptococcus pneumoniae

Abstract

To facilitate the study of pneumococcal genes that are essential for viability or normal cell growth, we sought to develop a tightly regulated, titratable gene depletion system that interferes minimally with normal cellular functions. A possible candidate for such a system is the recently discovered signal transduction pathway regulating competence for natural transformation in Streptococcus thermophilus. This pathway, which is unrelated to the ComCDE pathway used for competence regulation in Streptococcus pneumoniae, has not been fully elucidated, but it is known to include a short unmodified signaling peptide, ComS*, an oligopeptide transport system, Ami, and a transcriptional activator, ComR. The transcriptional activator is thought to bind to an inverted repeat sequence termed the ECom box. We introduced the ComR protein and the ECom box into the genome of S. pneumoniae R6 and demonstrated that addition of synthetic ComS* peptide induced the transcription of a luciferase gene inserted downstream of the ECom box. To determine whether the ComRS system could be used for gene depletion studies, the licD1 gene was inserted behind the chromosomally located ECom box promoter by using the Janus cassette. Then, the native versions of licD1 and licD2 were deleted, and the resulting mutant was recovered in the presence of ComS*. Cultivation of the licD1 licD2 double mutant in the absence of ComS* gradually affected its ability to grow and propagate, demonstrating that the ComRS system functions as intended. In the present study, the ComRS system was developed for use in S. pneumoniae. In principle, however, it should work equally well in many other Gram-positive species.Gene disruption studies have shown that the genome of Streptococcus pneumoniae R6 contains at least 133 essential genes, 32 of which have no known function (23, 25). As these studies were carried out with laboratory-grown pneumococci, it is reasonable to assume that additional genes are essential for survival under natural conditions. For obvious reasons, functional studies of essential genes are experimentally demanding. The best approach is probably to express essential genes ectopically under the control of a tightly regulated, titratable promoter. This allows deletion of the native gene, while the level of transcription of the ectopically expressed gene can be manipulated to gain insight into its function. The same technique ought to be applied to studies of growth-defective genes whose absence affects bacterial growth and proliferation. In this way it should be possible to avoid the selection pressure exerted by deletion of growth-defective genes that gives rise to suppressor mutations which mask or distort the real phenotype of the mutant.Ideally, gene expression/depletion systems should not interfere with the normal physiology of the host bacterium. The ComRS signal transduction pathway, which regulates competence for natural transformation in Streptococcus thermophilus (9), has no close homologs in S. pneumoniae. We therefore considered it a pro...

Structural basis of PcsB-mediated cell separation in Streptococcus pneumoniae

“…These two fragments were fused in an overlap extension PCR with the flanking primers ds95 and ds98 resulting in a lytF-CHAP pcsB chimera. LytF-CHAP PcsB was expressed in S. pneumoniae by using the ComRS system described by Berg et al 30 The 5 0 and 3 0 regions of the Janus cassette in strain SPH131 (see above) were amplified by the primer pairs khb31/khb36 and khb33/khb34, respectively, and then fused to the 5 0 and 3 0 ends of the lytF-CHAP pcsB fragment by overlap extension PCR using the primers khb31 and khb34. The resulting fragment was transformed into strain SPH131, replacing the Janus cassette and giving rise to strain SPH252.…”

“…As pcsB is an essential gene in S. pneumoniae R6, an extra copy of the wild-type gene had to be expressed ectopically before the native pcsB gene could be removed and replaced by a Janus cassette 42 . For ectopic expression of pcsB, we employed the R6 derivative SPH131, which has the ComRS expression/depletion system integrated in its genome 30 . First, the pcsB gene was amplified with the primers 35 and 36 and DNA from strain RH1 as template.…”

“…30 . Extracts from pneumococcal cultures grown in the presence of 0, 0.01, 0.2 and 2 mM ComS* were subjected to zymography with S. gordonii cells incorporated in the separating gel (Fig.…”

Cross-feeding and interkingdom communication in dual-species biofilms of Streptococcus mutans and Candida albicans

“…To protect themselves against their own fratricins, S. pneumoniae cells produce an immunity protein termed ComM (Berg et al, 2011). In S. mutans the two genes murN (SMU.716) and murM (SMU.717) show homology to ComM but they were not upregulated.…”

The function of the transmembrane and cytoplasmic domains of pneumococcal penicillin-binding proteins 2x and 2b extends beyond that of simple anchoring devices

“…After incubation at 37 uC for 120 min, cells were spread on Todd-Hewitt (TH) agar plates supplemented with kanamycin (400 mg ml 21 ), streptomycin (200 mg ml 21 ) or spectinomycin (200 mg ml 21 ) to select for transformants. When required, the inducer peptide ComS* (NH 2 -LPYFAGCL-COOH) was added to the medium during transformation and selection on agar plates to induce ectopic gene expression from the P comX promoter (Berg et al, 2011.…”