Natural Genetic Transformation: a Novel Tool for Efficient Genetic Engineering of the Dairy Bacterium<i>Streptococcus thermophilus</i>

Blomqvist, Trinelise; Steinmoen, Hilde; Håvarstein, Leiv Sigve

doi:10.1128/aem.01156-06

Cited by 72 publications

(82 citation statements)

References 29 publications

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“…Upon binding of ComS*, ComR is thought to undergo a conformational change that allows it to bind to the ECom box in the comX promoter and activate transcription of the comX gene. Previous studies have shown that the alternative sigma factor ComX controls the competence regulon in S. thermophilus, i.e., the genes involved in binding, uptake, and integration of exogenous DNA (2). How the ComS* precursor is processed and how the mature peptide pheromone is exported from the cell are not known.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

confidence: 99%

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

et al. 2011

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“…However, natural transformation was nevertheless never reported for these species (6,9,10,14,41). Furthermore, the chromosomal sequences near the attL site from the S. thermophilus ICEs, i.e., the sequence downstream from the fda gene in an empty attB site, are completely different in the various species used as recipient or donor.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, the integration by homologous recombination of a linear DNA fragment carrying an ICE could happen if this transforming fragment is internalized in the recipient. Nevertheless, no ICE transfer was recovered in liquid cultures, which are the conditions used for the artificial induction of S. thermophilus competence (9). Therefore, the acquisition of an S. thermophilus ICE by natural transformation is also very unlikely in intraspecies matings.…”

Section: Discussionmentioning

confidence: 99%

Conjugative Transfer of the Integrative Conjugative Elements ICE St1 and ICE St3 from Streptococcus thermophilus

et al. 2009

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Integrative and conjugative elements (ICEs), also called conjugative transposons, are genomic islands that excise, self-transfer by conjugation, and integrate in the genome of the recipient bacterium. The current investigation shows the intraspecies conjugative transfer of the first described ICEs in Streptococcus thermophilus, ICESt1 and ICESt3. Mitomycin C, a DNA-damaging agent, derepresses ICESt3 conjugative transfer almost 25-fold. The ICESt3 host range was determined using various members of the Firmicutes as recipients. Whereas numerous ICESt3 transconjugants of Streptococcus pyogenes and Enterococcus faecalis were recovered, only one transconjugant of Lactococcus lactis was obtained. The newly incoming ICEs, except the one from L. lactis, are site-specifically integrated into the 3 end of the fda gene and are still able to excise in these transconjugants. Furthermore, ICESt3 was retransferred from E. faecalis to S. thermophilus. Recombinant plasmids carrying different parts of the ICESt1 recombination module were used to show that the integrase gene is required for the site-specific integration and excision of the ICEs, whereas the excisionase gene is required for the site-specific excision only.Horizontal transfer of genomic islands plays a key role in bacterial evolution, leading to the acquisition of advantageous functions or to major modifications of the bacterial way of life (37, 38). However, the mechanism of their transfer and integration remains generally unknown. Recent in silico analyses revealed that numerous genomic islands could be integrative and conjugative elements (ICEs) or elements deriving from them (18,30,62,67) The ICEs, also called conjugative transposons, were defined as elements which encode their own excision, their transfer by conjugation, and their integration in a replicon of the recipient cell, whatever their specificity and their mechanism of integration and conjugation (18). Most of the ICEs encode a putative integrase belonging to the tyrosine recombinase family. This enzyme, generally associated with an excisionase, catalyzes the recombination between identical sequences carried by the attL and attR recombination sites flanking the element. This event leads to the excision of a circular form of the ICE harboring an attI site, resulting from the recombination between attL and attR sites, and to a chromosome carrying an empty attB site. After conjugative transfer, most of the ICEs integrate by site-specific recombination between identical sequences carried by the attI site and the attB site. The attB sites are generally located in the

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“…In 2006, Blomqvist et al (4) showed the functionality of natural transformation in S. thermophilus LMG18311 at a remarkably high frequency (10 Ϫ2 to 10 Ϫ3 ). The strategy was based on the overexpression of comX under the control of a BlpC-inducible promoter from S. thermophilus (4). Recently, Gardan et al (20) showed that competence development in S. thermophilus can spontaneously turn on during growth in chemically defined medium (CDM).…”

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confidence: 99%

A Novel Pheromone Quorum-Sensing System Controls the Development of Natural Competence in Streptococcus thermophilus and Streptococcus salivarius

et al. 2010

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In streptococcal species, the key step of competence development is the transcriptional induction of comX, which encodes the alternative sigma factor X , which positively regulates genes necessary for DNA transformation. In Streptococcus species belonging to the mitis and mutans groups, induction of comX relies on the activation of a three-component system consisting of a secreted pheromone, a histidine kinase, and a response regulator. In Streptococcus thermophilus, a species belonging to the salivarius group, the oligopeptide transporter Ami is essential for comX expression under competence-inducing conditions. This suggests a different regulation pathway of competence based on the production and reimportation of a signal peptide. The objective of our work was to identify the main actors involved in the early steps of comX induction in S. thermophilus LMD-9. Using a transcriptomic approach, four highly induced early competence operons were identified. Among them, we found a Rgg-like regulator (Ster_0316) associated with a nonannotated gene encoding a 24-amino-acid hydrophobic peptide (Shp0316). Through genetic deletions, we showed that these two genes are essential for comX induction. Moreover, addition to the medium of synthetic peptides derived from the C-terminal part of Shp0316 restored comX induction and transformation of a Shp0316-deficient strain. These peptides also induced competence in S. thermophilus and Streptococcus salivarius strains that are poorly transformable or not transformable. Altogether, our results show that Ster_0316 and Shp0316, renamed ComRS, are the two members of a novel quorum-sensing system responsible for comX induction in species from the salivarius group, which differs from the classical phosphorelay three-component system identified previously in streptococci.

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Natural Genetic Transformation: a Novel Tool for Efficient Genetic Engineering of the Dairy BacteriumStreptococcus thermophilus

Cited by 72 publications

References 29 publications

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

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

Conjugative Transfer of the Integrative Conjugative Elements ICE St1 and ICE St3 from Streptococcus thermophilus

A Novel Pheromone Quorum-Sensing System Controls the Development of Natural Competence in Streptococcus thermophilus and Streptococcus salivarius

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