The Essential<i>tacF</i>Gene Is Responsible for the Choline-Dependent Growth Phenotype of<i>Streptococcus pneumoniae</i>

Damjanović, Marlen; Kharat, Arun S.; Eberhardt, Alice; Tomasz, Alexander; Vollmer, Waldemar

doi:10.1128/jb.00681-07

Cited by 52 publications

(61 citation statements)

References 30 publications

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“…These polymers or their repeat units are assembled on a polyisoprenol-phosphate carrier molecule (typically undecaprenylphosphate) and ultimately flipped to the extracytoplasmic face of the plasma membrane before their assembly and/or final localization. In Gram-positive bacteria, this model also holds true for the biosynthesis of poly(glycerophosphate) teichoic acids (Bhavsar & Brown, 2006;Damjanovic et al, 2007) and various capsular polysaccharides (Bentley et al, 2006). This strategy may facilitate co-ordination between peptidoglycan assembly and the attachment of polymers that are covalently linked to the glycan repeat units, which includes the capsular polysaccharide and GBC in S. agalactiae (Deng et al, 2000).…”

Section: Initiation Of Gbc Synthesismentioning

confidence: 99%

“…Significantly, SAG1412 belongs to the Wzx (RfbX) family of flippases involved in capsule and lipopolysaccharide O-antigen biosynthesis (Liu et al, 1996;Marolda et al, 2006;Whitfield, 2006), members of which are also predicted to translocate pneumococcal teichoic acid (Damjanovic et al, 2007) and most capsular polysaccharides of the different S. pneumoniae serotypes (Bentley et al, 2006). The presence of NAG as the reducing terminal sugar is apparently important for the activity of Wzx flippases and thus it is significant that this sugar is present at the terminal structure of Unit IV (Fig.…”

Section: Translocation Of Gbc To the Cell Envelope And Cross-linkage mentioning

confidence: 99%

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Bioinformatic insights into the biosynthesis of the Group B carbohydrate in Streptococcus agalactiae

2008

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Section: Initiation Of Gbc Synthesismentioning

confidence: 99%

Section: Translocation Of Gbc To the Cell Envelope And Cross-linkage mentioning

confidence: 99%

Bioinformatic insights into the biosynthesis of the Group B carbohydrate in Streptococcus agalactiae

2008

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“…The licD genes are located on a transcriptional unit consisting of tacF, licD1, and licD2. The first gene in the operon, tacF, encodes a transporter that is required for the transport of teichoic acid subunits (or polymerized chains) across the cytoplasmic membrane (1,5). In S. pneumoniae, the chemical structure of the repeating units in cell wall teichoic acid (WTA) and lipoteichoic acid (LTA) are identical.…”

mentioning

confidence: 99%

“…In general, each repeat unit contains two phosphoryl choline residues, one attached to the central N-acetyl-D-galactosamine residue and the other to the ribitol-linked N-acetyl-D-galactosamine residue (8). LicD1 and LicD2 attach phosphoryl choline residues to the teichoic acid subunits on the cytoplasmic side of the membrane, before they are exported by TacF (5). As TacF has a strict specificity for subunits containing phosphoryl choline, extracellular synthesis of WTA and LTA will probably be arrested in the absence of LicD1 and LicD2.…”

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confidence: 99%

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

et al. 2011

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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...

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“…To engineer this mutant, we used DNA prepared from an S. mitis strain (SK598) reported to possess ethanolamine-containing TAs irrespective of the incubation medium (3). During the course of our investigation and having isolated the novel Choind pneumococcal strain P072, a report was published ascribing to spr1150, an essential gene (31) located in the lic region, a role in TA metabolism and the Cho-ind phenotype of S. pneumoniae (5). In this report, Damjanovic et al (5) noted that a single-point mutation in the spr1150 gene of the spontaneous mutant strain R6Chi was sufficient to generate a Cho-ind phenotype.…”

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confidence: 99%

Mutations in thetacFGene of Clinical Strains and Laboratory Transformants ofStreptococcus pneumoniae:Impact on Choline Auxotrophy and Growth Rate

et al. 2008

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The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in the tacF gene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype of S. pneumoniae (M. Damjanovic, A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer, J. Bacteriol. 189:7105-7111, 2007). We now report that Streptococcus mitis can grow in choline-free medium, as previously illustrated for Streptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation of S. pneumoniae R6 by using S. mitis SK598 DNA and several PCR-amplified tacF fragments suggested that a minimum of two mutations were required to confer improved fitness to choline-independent S. pneumoniae mutants. This conclusion is supported by sequencing results also reported here that indicate that a spontaneous mutant of S. pneumoniae (strain JY2190) able to proliferate in the absence of choline (or analogs) is also a double mutant for the tacF gene. Microscopic observations and competition experiments during the cocultivation of choline-independent strains confirmed that a minimum of two amino acid changes were required to confer improved fitness to choline-independent pneumococcal strains when growing in medium lacking any aminoalcohol. Our results suggest complex relationships among the different regions of the TacF teichoic acid repeat unit transporter.

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The EssentialtacFGene Is Responsible for the Choline-Dependent Growth Phenotype ofStreptococcus pneumoniae

Cited by 52 publications

References 30 publications

Bioinformatic insights into the biosynthesis of the Group B carbohydrate in Streptococcus agalactiae

Bioinformatic insights into the biosynthesis of the Group B carbohydrate in Streptococcus agalactiae

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

Mutations in thetacFGene of Clinical Strains and Laboratory Transformants ofStreptococcus pneumoniae:Impact on Choline Auxotrophy and Growth Rate

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