Unveiling the regulatory network controlling natural transformation in lactococci
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ABSTRACT:
ORGANISM(S): Lactococcus lactis
PROVIDER: GSE255719 | GEO | 2024/06/13
REPOSITORIES: GEO
Similar Datasets
Project description:Lactococcus lactis is one of the most important lactic acid bacterium used in the dairy industry. Activation of natural DNA transformation in this species would greatly improve the selection of novel strains with desired industrial traits. Here, we investigate the activation of natural transformation in L. lactis ssp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of essential late gene products required for natural transformation under its potential control. In absence of any information on the competence signaling system in this species, activation of natural DNA transformation by the constitutive production of ComX was attempted. Using a reporter strain of late competence phase activation and transcriptomic analyses, we show that all key genes for natural transformation can be induced in strain KW2. Then, we demonstrate that natural DNA transformation is functional in this strain and that this process requires the DNA uptake machinery dedicated to competence. Various chromosomal modifications such as point mutations or gene deletion/insertion could be quickly and efficiently achieved. These results are the first to report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of molecular mechanisms activating competence development in this species.
2017-06-29 | GSE86476 | GEO
Project description:Transcriptional profiling of the competence-stimulating peptide (CSP) response in Streptococcus mutans of FACS-separated subpopulations and mixed cells of a clonal culture. CSP-mediated competence development in S. mutans is a transient and biphasic process since only a subpopulation induces expression of ComX in the presence of CSP and activation of the DNA uptake machinery in this fraction shuts down ~3-4 hours post induction. Here we combine, for the first time in bacteria to our knowledge, flow cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and non-competent fractions of CSP-treated S. mutans cells. Sorting was guided by a ComX-GFP reporter and the transcriptome analysis demonstrated the successful combination of both methods because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, among them ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. By contrast, expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and mutacin V confirmed this expression pattern on the single cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, uptake of DNA could be shown on the single cell level. This study demonstrates that all cells in the population respond to CSP through activation of bacteriocin-related genes but that two subpopulations segregate, one becoming competent and another one that lyses, resulting in intrapopulation diversity of the clonal culture. Two conditions: induced and not induced with CSP. Three induced cell populations: competent subpopulation, incompetent subpopulation, all cells. Two biological replicates each population.
2011-02-01 | E-GEOD-25284 | biostudies-arrayexpress
Project description:Transcriptional profiling of the competence-stimulating peptide (CSP) response in Streptococcus mutans of FACS-separated subpopulations and mixed cells of a clonal culture. CSP-mediated competence development in S. mutans is a transient and biphasic process since only a subpopulation induces expression of ComX in the presence of CSP and activation of the DNA uptake machinery in this fraction shuts down ~3-4 hours post induction. Here we combine, for the first time in bacteria to our knowledge, flow cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and non-competent fractions of CSP-treated S. mutans cells. Sorting was guided by a ComX-GFP reporter and the transcriptome analysis demonstrated the successful combination of both methods because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, among them ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. By contrast, expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and mutacin V confirmed this expression pattern on the single cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, uptake of DNA could be shown on the single cell level. This study demonstrates that all cells in the population respond to CSP through activation of bacteriocin-related genes but that two subpopulations segregate, one becoming competent and another one that lyses, resulting in intrapopulation diversity of the clonal culture.
2011-02-01 | GSE25284 | GEO
Project description:BACKGROUND: Streptococcus mutans, the etiological agent of human dental caries, displays complex regulation of natural genetic competence. Competence development in S. mutans is controlled by a peptide derived from ComS (comX inducing peptide, XIP); which along with the cytosolic regulator ComR controls the expression of the alternative sigma factor comX, the master regulator of competence development. ComR-XIP controls the expression of both PcomX and PcomS, with the latter creating a positive feedback loop. Recently, a gene embedded within the coding region of comX was discovered and designated xrpA (comX regulatory peptide A). XrpA was found to be an antagonist of ComX, but the mechanism was not established. In this study, we began to dissect how XrpA exerts control over competence development in S. mutans. METHODS: RNA-Seq was utilized to compare the transcriptomes of S. mutans wild-type strain UA159 (3 replicates), UA159 with addition of 2 uM sXIP (3 replicates) and with a mutant of xrpA (comX::T162C; 3 replicates). Strains were grown to OD600 nm = 0.5 in FMC medium before harvest. sXIP was added at OD600 nm = 0.2. Deep sequencing was performed at the University of Florida ICBR facilities (Gainesville, FL). Approximately 20 million short-reads were obtained for each sample. After removing adapter sequences from each short-read and trimming of the 3’-ends by quality scores (59), the resulting sequences were mapped onto the reference genome of strain UA159 (GenBank accession no. AE014133) using the short-read aligner. Mapped short-read alignments were then converted into readable formats using SAMTOOLS. RESULTS: Using an optimzed data analysis workflow, we mapped 14-19 million reads per sample to the genome of UA159. For viewing of the mapped reads aligned to the genome, .bam files were uploaded into the Integrative Genomics Viewer (IGV – version 2.3.55) (61). A .csv file containing raw read counts for each replicate (3) was then uploaded to Degust (http://degust.erc.monash.edu/) and edgeR analysis performed to determine Log2 fold change and a false discovery rate (FDR). Comparison of the transcriptome by RNA-Seq of the wild-type with the mutant lacking XrpA revealed 56 differentially expressed genes, with 34 upregulated in xrpA and 22 downregulated. Many of the upregulated genes were competence-related genes, including comX and genes that are a part of the ComX regulon of S. mutans . Since loss of xrpA caused upregulation of competence genes, we also analyzed the transcriptome of UA159 treated with 2 uM sXIP to induce competence, with UA159 treated with vehicle (DMSO) as a control. Cells treated with sXIP had 137 genes differentially expressed compared to the DMSO control. CONCLUSIONS: Collectively, these data reveal that the novel regulator XrpA exerts its influence over competence development by impacting ComRS functions, resulting in decreased expression of comX and late com gene expression that negatively impact transformability. These results highlight XrpA as a new negative regulator of competence signaling as well as broaden our understanding of the complex regulatory mechanisms that modulate competence and virulence in S. mutans.
2018-02-13 | GSE110167 | GEO
Project description:To perform natural transformation, one of the three main Horizontal Gene Transfer mechanisms, bacteria need to enter a physiological differentiated state called genetic competence. Interestingly, new bacteria displaying such aptitude are often discovered, and one of the latest is the human pathogen Staphylococcus aureus. Here, we developed a new protocol, based on planktonic cells cultures, leading to a large percentage of the population activating competence development and a significant improvement of S. aureus natural transformation efficiencies. Taking advantage of these new conditions, we performed transcriptomics analyses to characterize each central competence regulators regulon. SigH and ComK1 were both found essential for activating natural transformation genes but also important for activation or repression of peripheral functions. Even though ComK2 was not found important for the control of transformation genes, its regulon showed an important overlap with that of SigH and ComK1. Finally, we showed how microaerobic conditions, sensed by the SrrAB two-component system, was key to activate competence in S. aureus.
2023-04-26 | PXD040550 | Pride
Project description:Transcriptional profiling of the M-NM-^T835np mutant vs the wild-type strain grown in BHI to OD600=0.5. An intimate linkage between the regulation of biofilm formation, stress tolerance and genetic competence exists in the dental caries pathogen Streptococcus mutans. We previously reported that the rcrRPQ genes, which encode ABC exporters (RcrPQ) and a MarR-family transcriptional regulator (RcrR) that represses the expression of the rcr operon, play a dominant role in regulation of development of genetic competence and connect competence with stress tolerance and (p)ppGpp production in S. mutans. Here we identify the region required for efficient RcrR binding to the rcr promoter region using purified recombinant RcrR (rRcrR) protein in electrophoretic mobility shift and fluorescence polarization assays. DNA fragments carrying mutations in the binding region resulted in decreased binding of rRcrR. Direct binding of RcrR to the comX gene promoter in vitro revealed that RcrR has the potential to directly regulate competence development. Likewise, direct interactions of rRcrR with the promoter region of relP, encoding a (p)ppGpp synthase, show the potential for RcrR-dependent modulation of (p)ppGpp pools. Introduction of mutations into the RcrR binding sites in the rcrR promoter region in vivo demonstrated that optimal RcrR binding is essential for proper expression of competence genes and optimal transformation efficiency. Transcription profiling by RNA-seq and mircroarrays of strains with mutations in rcrR showed that genes in the competence pathway were those most differentially expressed. Thus, here and in a accompanying paper, we establish mechanisms by which the products of the rcrRPQ operon integrate com gene expression, genetic transformation, ppGpp metabolism and stress tolerance in S. mutans Two-condition experiment, M-NM-^T835np mutant vs the wild-type strain. Biological replicates: 4 per strain, independently grown and harvested. One replicate per array
2015-02-01 | E-GEOD-52574 | biostudies-arrayexpress
Project description:Transcriptional profiling of the Δ835np mutant vs the wild-type strain grown in BHI to OD600=0.5. An intimate linkage between the regulation of biofilm formation, stress tolerance and genetic competence exists in the dental caries pathogen Streptococcus mutans. We previously reported that the rcrRPQ genes, which encode ABC exporters (RcrPQ) and a MarR-family transcriptional regulator (RcrR) that represses the expression of the rcr operon, play a dominant role in regulation of development of genetic competence and connect competence with stress tolerance and (p)ppGpp production in S. mutans. Here we identify the region required for efficient RcrR binding to the rcr promoter region using purified recombinant RcrR (rRcrR) protein in electrophoretic mobility shift and fluorescence polarization assays. DNA fragments carrying mutations in the binding region resulted in decreased binding of rRcrR. Direct binding of RcrR to the comX gene promoter in vitro revealed that RcrR has the potential to directly regulate competence development. Likewise, direct interactions of rRcrR with the promoter region of relP, encoding a (p)ppGpp synthase, show the potential for RcrR-dependent modulation of (p)ppGpp pools. Introduction of mutations into the RcrR binding sites in the rcrR promoter region in vivo demonstrated that optimal RcrR binding is essential for proper expression of competence genes and optimal transformation efficiency. Transcription profiling by RNA-seq and mircroarrays of strains with mutations in rcrR showed that genes in the competence pathway were those most differentially expressed. Thus, here and in a accompanying paper, we establish mechanisms by which the products of the rcrRPQ operon integrate com gene expression, genetic transformation, ppGpp metabolism and stress tolerance in S. mutans
2015-02-01 | GSE52574 | GEO
Project description:We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner-transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counter-selection of mutants in 21 different genes during the challenge. Eight of the counter-selected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants in which seven of the eight remaining genes, i.e., spr0459/spr0460, spr0777, spr0838, spr1259/spr1260, and spr1357, were deleted, displayed reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation of chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and DNA damage repair in S. pneumoniae. Keywords: GAF competence Selection for genes essential for transformation. Aliquots of a marinerT7 transposon library generated in S. pneumoniae R6 containing approximately 40,000 independent mutants were transformed with an antibiotic resistance marker and grown without (initial library) or with (challenged library) antibiotics for 25 generation until DNA extraction.
2007-09-04 | E-GEOD-8362 | biostudies-arrayexpress
Project description:We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner-transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counter-selection of mutants in 21 different genes during the challenge. Eight of the counter-selected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants in which seven of the eight remaining genes, i.e., spr0459/spr0460, spr0777, spr0838, spr1259/spr1260, and spr1357, were deleted, displayed reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation of chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and DNA damage repair in S. pneumoniae. Keywords: GAF competence
2007-09-05 | GSE8362 | GEO
Project description:Transcriptomic analysis of Lactococcus lactis ssp. cremoris KW2 after ComX constitutive production
| PRJNA342014 | ENA