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1.
Nucleic Acids Res ; 2024 Jun 13.
Article in English | MEDLINE | ID: mdl-38869061

ABSTRACT

Pathogenic bacteria employ complex systems to cope with metal ion shortage conditions and propagate in the host. IsrR is a regulatory RNA (sRNA) whose activity is decisive for optimum Staphylococcus aureus fitness upon iron starvation and for full virulence. IsrR down-regulates several genes encoding iron-containing enzymes to spare iron for essential processes. Here, we report that IsrR regulates the tricarboxylic acid (TCA) cycle by controlling aconitase (CitB), an iron-sulfur cluster-containing enzyme, and its transcriptional regulator, CcpE. This IsrR-dependent dual-regulatory mechanism provides an RNA-driven feedforward loop, underscoring the tight control required to prevent aconitase expression. Beyond its canonical enzymatic role, aconitase becomes an RNA-binding protein with regulatory activity in iron-deprived conditions, a feature that is conserved in S. aureus. Aconitase not only negatively regulates its own expression, but also impacts the enzymes involved in both its substrate supply and product utilization. This moonlighting activity concurrently upregulates pyruvate carboxylase expression, allowing it to compensate for the TCA cycle deficiency associated with iron scarcity. These results highlight the cascade of complex posttranscriptional regulations controlling S. aureus central metabolism in response to iron deficiency.

2.
Nat Commun ; 15(1): 2787, 2024 Mar 30.
Article in English | MEDLINE | ID: mdl-38555352

ABSTRACT

In all living cells, genomic DNA is compacted through interactions with dedicated proteins and/or the formation of plectonemic coils. In bacteria, DNA compaction is achieved dynamically, coordinated with dense and constantly changing transcriptional activity. H-NS, a major bacterial nucleoid structuring protein, is of special interest due to its interplay with RNA polymerase. H-NS:DNA nucleoprotein filaments inhibit transcription initiation by RNA polymerase. However, the discovery that genes silenced by H-NS can be activated by transcription originating from neighboring regions has suggested that elongating RNA polymerases can disassemble H-NS:DNA filaments. In this study, we present evidence that transcription-induced counter-silencing does not require transcription to reach the silenced gene; rather, it exerts its effect at a distance. Counter-silencing is suppressed by introducing a DNA gyrase binding site within the intervening segment, suggesting that the long-range effect results from transcription-driven positive DNA supercoils diffusing toward the silenced gene. We propose a model wherein H-NS:DNA complexes form in vivo on negatively supercoiled DNA, with H-NS bridging the two arms of the plectoneme. Rotational diffusion of positive supercoils generated by neighboring transcription will cause the H-NS-bound negatively-supercoiled plectoneme to "unroll" disrupting the H-NS bridges and releasing H-NS.


Subject(s)
Chromatin , DNA-Binding Proteins , Chromatin/metabolism , DNA-Binding Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacteria/genetics , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , DNA/metabolism , Gene Silencing , Gene Expression Regulation, Bacterial , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Transcription, Genetic
3.
Proc Natl Acad Sci U S A ; 119(30): e2203011119, 2022 07 26.
Article in English | MEDLINE | ID: mdl-35858437

ABSTRACT

In Escherichia coli and Salmonella, many genes silenced by the nucleoid structuring protein H-NS are activated upon inhibiting Rho-dependent transcription termination. This response is poorly understood and difficult to reconcile with the view that H-NS acts mainly by blocking transcription initiation. Here we have analyzed the basis for the up-regulation of H-NS-silenced Salmonella pathogenicity island 1 (SPI-1) in cells depleted of Rho-cofactor NusG. Evidence from genetic experiments, semiquantitative 5' rapid amplification of complementary DNA ends sequencing (5' RACE-Seq), and chromatin immunoprecipitation sequencing (ChIP-Seq) shows that transcription originating from spurious antisense promoters, when not stopped by Rho, elongates into a H-NS-bound regulatory region of SPI-1, displacing H-NS and rendering the DNA accessible to the master regulator HilD. In turn, HilD's ability to activate its own transcription triggers a positive feedback loop that results in transcriptional activation of the entire SPI-1. Significantly, single-cell analyses revealed that this mechanism is largely responsible for the coexistence of two subpopulations of cells that either express or do not express SPI-1 genes. We propose that cell-to-cell differences produced by stochastic spurious transcription, combined with feedback loops that perpetuate the activated state, can generate bimodal gene expression patterns in bacterial populations.


Subject(s)
Bacterial Proteins , DNA-Binding Proteins , Gene Expression Regulation, Bacterial , Promoter Regions, Genetic , Salmonella , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , Gene Silencing , Salmonella/genetics , Salmonella/pathogenicity , Single-Cell Analysis , Transcription, Genetic , Virulence/genetics
4.
Nucleic Acids Res ; 50(15): 8529-8546, 2022 08 26.
Article in English | MEDLINE | ID: mdl-35904807

ABSTRACT

Staphylococcus aureus, a human opportunist pathogen, adjusts its metabolism to cope with iron deprivation within the host. We investigated the potential role of small non-coding RNAs (sRNAs) in dictating this process. A single sRNA, named here IsrR, emerged from a competition assay with tagged-mutant libraries as being required during iron starvation. IsrR is iron-repressed and predicted to target mRNAs expressing iron-containing enzymes. Among them, we demonstrated that IsrR down-regulates the translation of mRNAs of enzymes that catalyze anaerobic nitrate respiration. The IsrR sequence reveals three single-stranded C-rich regions (CRRs). Mutational and structural analysis indicated a differential contribution of these CRRs according to targets. We also report that IsrR is required for full lethality of S. aureus in a mouse septicemia model, underscoring its role as a major contributor to the iron-sparing response for bacterial survival during infection. IsrR is conserved among staphylococci, but it is not ortholog to the proteobacterial sRNA RyhB, nor to other characterized sRNAs down-regulating mRNAs of iron-containing enzymes. Remarkably, these distinct sRNAs regulate common targets, illustrating that RNA-based regulation provides optimal evolutionary solutions to improve bacterial fitness when iron is scarce.


Subject(s)
RNA, Bacterial , RNA, Small Untranslated , Animals , Bacteria/genetics , Gene Expression Regulation, Bacterial , Humans , Iron/metabolism , Mice , RNA, Bacterial/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Untranslated/genetics , RNA, Small Untranslated/metabolism , Staphylococcus/genetics , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism
5.
Antimicrob Agents Chemother ; 66(5): e0243521, 2022 05 17.
Article in English | MEDLINE | ID: mdl-35389235

ABSTRACT

Bacterial small RNAs (sRNAs) contribute to a variety of regulatory mechanisms that modulate a wide range of pathways, including metabolism, virulence, and antibiotic resistance. We investigated the involvement of sRNAs in rifampicin resistance in the opportunistic pathogen Staphylococcus aureus. Using a competition assay with an sRNA mutant library, we identified 6S RNA as being required for protection against low concentrations of rifampicin, an RNA polymerase (RNAP) inhibitor. This effect applied to rifabutin and fidaxomicin, two other RNAP-targeting antibiotics. 6S RNA is highly conserved in bacteria, and its absence in two other major pathogens, Salmonella enterica and Clostridioides difficile, also impaired susceptibility to RNAP inhibitors. In S. aureus, 6S RNA is produced from an autonomous gene and accumulates in stationary phase. In contrast to what was reported for Escherichia coli, S. aureus 6S RNA does not appear to play a critical role in the transition from exponential to stationary phase but affects σB-regulated expression in prolonged stationary phase. Nevertheless, its protective effect against rifampicin is independent of alternative sigma factor σB activity. Our results suggest that 6S RNA helps maintain RNAP-σA integrity in S. aureus, which could in turn help bacteria withstand low concentrations of RNAP inhibitors.


Subject(s)
Rifampin , Staphylococcus aureus , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , RNA, Bacterial/genetics , RNA, Untranslated , Rifampin/pharmacology , Sigma Factor/genetics , Sigma Factor/metabolism , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Transcription, Genetic
6.
Nucleic Acids Res ; 49(18): 10644-10656, 2021 10 11.
Article in English | MEDLINE | ID: mdl-34554192

ABSTRACT

Staphylococcus aureus is an opportunistic human and animal pathogen with an arsenal of virulence factors that are tightly regulated during bacterial infection. The latter is achieved through a sophisticated network of regulatory proteins and regulatory RNAs. Here, we describe the involvement of a novel prophage-carried small regulatory S. aureus RNA, SprY, in the control of virulence genes. An MS2-affinity purification assay reveals that SprY forms a complex in vivo with RNAIII, a major regulator of S. aureus virulence genes. SprY binds to the 13th stem-loop of RNAIII, a key functional region involved in the repression of multiple mRNA targets. mRNAs encoding the repressor of toxins Rot and the extracellular complement binding protein Ecb are among the targets whose expression is increased by SprY binding to RNAIII. Moreover, SprY decreases S. aureus hemolytic activity and virulence. Our results indicate that SprY titrates RNAIII activity by targeting a specific stem loop. Thus, we demonstrate that a prophage-encoded sRNA reduces the pathogenicity of S. aureus through RNA sponge activity.


Subject(s)
RNA, Bacterial/metabolism , RNA, Small Untranslated/metabolism , Staphylococcus aureus/genetics , Staphylococcus aureus/pathogenicity , Animals , Female , Gene Expression Regulation, Bacterial , Hemolysis , Mice , RNA, Bacterial/chemistry , RNA, Small Untranslated/chemistry , RNA, Small Untranslated/genetics , Staphylococcal Infections/microbiology , Staphylococcus aureus/growth & development , Staphylococcus aureus/metabolism , Virulence/genetics
7.
mSphere ; : e0013421, 2021 Jun 30.
Article in English | MEDLINE | ID: mdl-34190585

ABSTRACT

Vsr217 is a small RNA from Vibrio tasmaniensis LGP32, a pathogen associated with mortality events affecting juvenile oysters. The vsr217 gene is located within the 5' untranslated region (UTR) of malK, encoding the ATPase component of the maltose importer, and is conserved within the genus Vibrio. In the presence of maltose, vsr217 is regulated by MalT, the positive regulator of the maltose regulon. vsr217 is required in cis for the full expression of malK. In addition, Vsr217 acts in trans to downregulate the expression of fbp encoding fructose-1,6-bisphosphatase, an enzyme involved in gluconeogenesis. Thus, in the presence of maltose, the induction of Vsr217 is expected to promote glycolysis by negatively regulating the expression of a key enzyme of gluconeogenesis. IMPORTANCE Juvenile pacific oysters have been subject in recent years to summer mortality episodes with deep economic consequences. The pathogen Vibrio tasmaniensis has been associated with such mortality events. For bacterial pathogens, survival within the host requires profound metabolic adaptations according to available resources. All kinds of regulatory elements, including noncoding RNAs, orchestrate this response. Oysters are rich in glycogen, a precursor of maltose, and we previously reported that V. tasmaniensis maltose-regulated genes are strongly induced during oyster infection. Here, we report the dual mechanism by which a small regulatory RNA, generated from the 5' untranslated region of a gene belonging to the maltose regulon, acts both in cis and trans. In cis, it stimulates growth on maltose, and in trans, it downregulates the expression of a gene associated with gluconeogenesis, thus coordinating maltose utilization with central carbon metabolism.

8.
Biochim Biophys Acta Gene Regul Mech ; 1863(7): 194546, 2020 07.
Article in English | MEDLINE | ID: mdl-32217107

ABSTRACT

The largest and best studied group of regulatory small RNAs (sRNAs) in bacteria act by modulating translation or turnover of messenger RNAs (mRNAs) through base-pairing interactions that typically take place near the 5' end of the mRNA. This allows the sRNA to bind the complementary target sequence while the remainder of the mRNA is still being made, creating conditions whereby the action of the sRNA can extend to transcriptional steps, most notably transcription termination. Increasing evidence corroborates the existence of a functional interplay between sRNAs and termination factor Rho. Two general mechanisms have emerged. One mechanism operates in translated regions subjected to sRNA repression. By inhibiting ribosome binding co-transcriptionally, the sRNA uncouples translation from transcription, allowing Rho to bind the nascent RNA and promote termination. In the second mechanism, which functions in 5' untranslated regions, the sRNA antagonizes termination directly by interfering with Rho binding to the RNA or the subsequent translocation along the RNA. Here, we review the above literature in the context of other mechanisms that underlie the participation of Rho-dependent transcription termination in gene regulation. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.


Subject(s)
Gene Expression Regulation, Bacterial , RNA, Small Untranslated/metabolism , Rho Factor/metabolism , Transcription Termination, Genetic , Bacteria/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Untranslated/genetics , Rho Factor/genetics
9.
BMC Res Notes ; 13(1): 63, 2020 Feb 07.
Article in English | MEDLINE | ID: mdl-32033621

ABSTRACT

OBJECTIVE: The golden color of Staphylococcus aureus is due to the synthesis of carotenoid pigments. In Gram-negative bacteria, Hfq is a global posttranscriptional regulator, but its function in S. aureus remains obscure. The absence of Hfq in S. aureus was reported to correlate with production of carotenoid pigment leading to the conclusion that Hfq was a negative regulator of the yellow color. However, we reported the construction of hfq mutants in several S. aureus strains and never noticed any color change; we therefore revisited the question of Hfq implication in S. aureus pigmentation. RESULTS: The absence or accumulation of Hfq does not affect S. aureus pigmentation.


Subject(s)
Gene Expression Regulation, Bacterial/genetics , Host Factor 1 Protein/physiology , Pigmentation/genetics , Staphylococcus aureus/genetics
10.
RNA ; 26(4): 382-395, 2020 04.
Article in English | MEDLINE | ID: mdl-31992590

ABSTRACT

Transcription initiation and RNA processing govern gene expression and enable bacterial adaptation by reshaping the RNA landscape. The aim of this study was to simultaneously observe these two fundamental processes in a transcriptome responding to an environmental signal. A controlled σE system in E. coli was coupled to our previously described tagRNA-seq method to yield process kinetics information. Changes in transcription initiation frequencies (TIF) and RNA processing frequencies (PF) were followed using 5' RNA tags. Changes in TIF showed a binary increased/decreased pattern that alternated between transcriptionally activated and repressed promoters, providing the bacterial population with transcriptional oscillation. PF variation fell into three categories of cleavage activity: (i) constant and independent of RNA levels, (ii) increased once RNA has accumulated, and (iii) positively correlated to changes in TIF. This work provides a comprehensive and dynamic view of major events leading to transcriptomic reshaping during bacterial adaptation. It unveils an interplay between transcription initiation and the activity of specific RNA cleavage sites. This study utilized a well-known genetic system to analyze fundamental processes and can serve as a blueprint for comprehensive studies that exploit the RNA metabolism to decipher and understand bacterial gene expression control.


Subject(s)
Adaptation, Physiological , RNA, Bacterial/genetics , RNA/genetics , Transcription Initiation, Genetic , Escherichia coli , RNA/metabolism , RNA Processing, Post-Transcriptional , RNA Stability , RNA, Bacterial/metabolism
11.
Appl Environ Microbiol ; 85(19)2019 10 01.
Article in English | MEDLINE | ID: mdl-31324637

ABSTRACT

Bacteria synthesize amino acids according to their availability in the environment or, in the case of pathogens, within the host. We explored the regulation of the biosynthesis of branched-chain amino acids (BCAAs) (l-leucine, l-valine, and l-isoleucine) in Vibrio alginolyticus, a marine fish and shellfish pathogen and an emerging opportunistic human pathogen. In this species, the ilvGMEDA operon encodes the main pathway for biosynthesis of BCAAs. Its upstream regulatory region shows no sequence similarity to the corresponding region in Escherichia coli or other Enterobacteriaceae, and yet we show that this operon is regulated by transcription attenuation. The translation of a BCAA-rich peptide encoded upstream of the structural genes provides an adaptive response similar to the E. coli canonical model. This study of a nonmodel Gram-negative organism highlights the mechanistic conservation of transcription attenuation despite the absence of primary sequence conservation.IMPORTANCE This study analyzes the regulation of the biosynthesis of branched-chain amino acids (leucine, valine, and isoleucine) in Vibrio alginolyticus, a marine bacterium that is pathogenic to fish and humans. The results highlight the conservation of the main regulatory mechanism with that of the enterobacterium Escherichia coli, suggesting that such a mechanism appeared early during the evolution of Gram-negative bacteria, allowing adaptation to a wide range of environments.


Subject(s)
Amino Acids, Branched-Chain/biosynthesis , Gene Expression Regulation, Bacterial , Operon , Transcription, Genetic , Vibrio alginolyticus/genetics , Acetolactate Synthase/metabolism , Aquatic Organisms , Escherichia coli/genetics , Isoleucine/biosynthesis , Leucine/biosynthesis , Regulatory Sequences, Nucleic Acid , Valine/biosynthesis
12.
mSphere ; 3(6)2018 11 28.
Article in English | MEDLINE | ID: mdl-30487156

ABSTRACT

CsrBs are bacterial highly conserved and multiple-copy noncoding small RNAs (sRNAs) that play major roles in cell physiology and virulence. In the Vibrio genus, they are known to be regulated by the two-component system VarS/VarA. They modulate the well-characterized quorum sensing pathway controlling virulence and luminescence in Vibrio cholerae and Vibrio harveyi, respectively. Remarkably, Vibrio tasmaniensis LGP32, an oyster pathogen that belongs to the Splendidus clade, was found to have four copies of csrB, named csrB1-4, compared to two to three copies in other Vibrio species. Here, we show that the extra csrB4 copy results from a csrB3 gene duplication, a characteristic of the Splendidus clade. Interestingly, csrB genes are regulated in different ways in V. tasmaniensis, with csrB1 expression being independent of the VarS/VarA system. We found that a complex regulatory network involving CsrBs, quorum sensing, and the stationary-phase sigma factor σS redundantly but differentially controls the production of two secreted metalloproteases, Vsm and PrtV, the former being a major determinant of the V. tasmaniensis extracellular product toxicity. In particular, we identified a novel VarS/VarA-dependent but CsrB-independent pathway that controls positively both Vsm production and PrtV production as well as rpoS expression. Altogether, our data show that a csrB gene duplication event in V. tasmaniensis supported the evolution of the regulatory network controlling the expression of major toxic secreted metalloproteases, thereby increasing redundancy and enabling the integration of additional input signals.IMPORTANCE The conserved CsrB sRNAs are an example of sibling sRNAs, i.e., sRNAs which are present in multiple copies in genomes. This report illustrates how new copies arise through gene duplication events and highlights two evolutionary advantages of having such multiple copies: differential regulation of the multiple copies allows integration of different input signals into the regulatory network of which they are parts, and the high redundancy that they provide confers a strong robustness to the system.


Subject(s)
Gene Duplication , Gene Expression Regulation, Bacterial , Metalloproteases/biosynthesis , RNA, Untranslated/genetics , Vibrio/enzymology , Vibrio/genetics , Quorum Sensing , Vibrio/metabolism
13.
Nucleic Acids Res ; 46(17): 8803-8816, 2018 09 28.
Article in English | MEDLINE | ID: mdl-29986060

ABSTRACT

RsaE is a regulatory RNA highly conserved amongst Firmicutes that lowers the amount of mRNAs associated with the TCA cycle and folate metabolism. A search for new RsaE targets in Staphylococcus aureus revealed that in addition to previously described substrates, RsaE down-regulates several genes associated with arginine catabolism. In particular, RsaE targets the arginase rocF mRNA via direct interactions involving G-rich motifs. Two duplicated C-rich motifs of RsaE can independently downregulate rocF expression. The faster growth rate of ΔrsaE compared to its parental strain in media containing amino acids as sole carbon source points to an underlying role for RsaE in amino acid catabolism. Collectively, the data support a model in which RsaE acts as a global regulator of functions associated with metabolic adaptation.


Subject(s)
Arginine/metabolism , RNA, Bacterial/physiology , Regulatory Sequences, Ribonucleic Acid , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Amino Acids/metabolism , Amino Acids/pharmacology , Conserved Sequence , Culture Media/chemistry , Culture Media/pharmacology , Down-Regulation/drug effects , Down-Regulation/genetics , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Bacterial/genetics , Metabolic Networks and Pathways/drug effects , Metabolic Networks and Pathways/genetics , Organisms, Genetically Modified , Regulatory Sequences, Ribonucleic Acid/genetics , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development
14.
Front Microbiol ; 9: 228, 2018.
Article in English | MEDLINE | ID: mdl-29515534

ABSTRACT

Bacterial regulatory RNAs have been extensively studied for over a decade, and are progressively being integrated into the complex genetic regulatory network. Transcriptomic arrays, recent deep-sequencing data and bioinformatics suggest that bacterial genomes produce hundreds of regulatory RNAs. However, while some have been authenticated, the existence of the others varies according to strains and growth conditions, and their detection fluctuates with the methodologies used for data acquisition and interpretation. For example, several small RNA (sRNA) candidates are now known to be parts of UTR transcripts. Accurate annotation of regulatory RNAs is a complex task essential for molecular and functional studies. We defined bona fide sRNAs as those that (i) likely act in trans and (ii) are not expressed from the opposite strand of a coding gene. Using published data and our own RNA-seq data, we reviewed hundreds of Staphylococcus aureus putative regulatory RNAs using the DETR'PROK computational pipeline and visual inspection of expression data, addressing the question of which transcriptional signals correspond to sRNAs. We conclude that the model strain HG003, a NCTC8325 derivative commonly used for S. aureus genetic regulation studies, has only about 50 bona fide sRNAs, indicating that these RNAs are less numerous than commonly stated. Among them, about half are associated to the S. aureus sp. core genome and a quarter are possibly expressed in other Staphylococci. We hypothesize on their features and regulation using bioinformatic approaches.

15.
Nucleic Acids Res ; 46(7): 3400-3411, 2018 04 20.
Article in English | MEDLINE | ID: mdl-29474582

ABSTRACT

Transcription termination by Rho is essential for viability in various bacteria, including some major pathogens. Since Rho acts by targeting nascent RNAs that are not simultaneously translated, it also regulates antisense transcription. Here we show that RNase H-deficient mutants of Escherichia coli exhibit heightened sensitivity to the Rho inhibitor bicyclomycin, and that Rho deficiency provokes increased formation of RNA-DNA hybrids (R-loops) which is ameliorated by expression of the phage T4-derived R-loop helicase UvsW. We also provide evidence that in Rho-deficient cells, R-loop formation blocks subsequent rounds of antisense transcription at more than 500 chromosomal loci. Hence these antisense transcripts, which can extend beyond 10 kb in their length, are only detected when Rho function is absent or compromised and the UvsW helicase is concurrently expressed. Thus the potential for antisense transcription in bacteria is much greater than hitherto recognized; and the cells are able to retain viability even when nearly one-quarter of their total non-rRNA abundance is accounted for by antisense transcripts, provided that R-loop formation from them is curtailed.


Subject(s)
Genome, Bacterial/genetics , Rho Factor/genetics , Transcription Termination, Genetic , Transcription, Genetic , Bacteriophage T4/genetics , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Chromosomes/genetics , DNA Helicases/genetics , DNA Replication/genetics , DNA, Antisense/genetics , Escherichia coli/drug effects , Escherichia coli/genetics , Genes, rRNA/genetics , Genome, Bacterial/drug effects , Rho Factor/antagonists & inhibitors , Ribonuclease H/genetics , Viral Proteins/genetics
16.
Science ; 357(6347): 208-211, 2017 Jul 14.
Article in English | MEDLINE | ID: mdl-28596309

ABSTRACT

DNA damage induced by reactive carbonyls (mainly methylglyoxal and glyoxal), called DNA glycation, is quantitatively as important as oxidative damage. DNA glycation is associated with increased mutation frequency, DNA strand breaks, and cytotoxicity. However, in contrast to guanine oxidation repair, how glycated DNA is repaired remains undetermined. Here, we found that the parkinsonism-associated protein DJ-1 and its bacterial homologs Hsp31, YhbO, and YajL could repair methylglyoxal- and glyoxal-glycated nucleotides and nucleic acids. DJ-1-depleted cells displayed increased levels of glycated DNA, DNA strand breaks, and phosphorylated p53. Deglycase-deficient bacterial mutants displayed increased levels of glycated DNA and RNA and exhibited strong mutator phenotypes. Thus, DJ-1 and its prokaryotic homologs constitute a major nucleotide repair system that we name guanine glycation repair.


Subject(s)
DNA Damage , DNA Repair , Escherichia coli Proteins/metabolism , Guanine/metabolism , Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Protein Deglycase DJ-1/metabolism , Ribosomal Proteins/metabolism , Gene Knockdown Techniques , Glycosylation , HeLa Cells , Humans , Protein Deglycase DJ-1/genetics
18.
Methods ; 117: 21-27, 2017 03 15.
Article in English | MEDLINE | ID: mdl-27916561

ABSTRACT

Bacteria optimize their fitness in response to a changing environment by tight regulation of gene expression. Regulation can be controlled at both transcriptional and post-transcriptional levels via key players such as sigma factors, regulatory proteins and regulatory RNAs. The identification of phenotypes associated with gene deletions is the established method for finding gene functions but may require testing many conditions for each studied mutant. As regulatory RNAs often contribute to fine-tuning gene expression, phenotypes associated with their inactivation are often weak and difficult to detect. Nevertheless, minor phenotypes conferring modest advantages, may allow bacteria to emerge after some generations under selective pressure. A strategy employing DNA barcodes can be used to perform competition experiments between mutants and to monitor fitness associated with mutations in different growth conditions. We combined this strategy with deep sequencing to study regulatory RNAs in Staphylococcus aureus, a major opportunistic pathogen.


Subject(s)
Biological Assay , Gene Expression Regulation, Bacterial , Microbial Interactions/genetics , RNA, Bacterial/genetics , RNA, Small Untranslated/genetics , Staphylococcus aureus/genetics , DNA Barcoding, Taxonomic , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Fitness , Mutation , Phenotype , Plasmids/chemistry , Plasmids/metabolism , RNA, Bacterial/metabolism , RNA, Small Untranslated/metabolism , Sequence Analysis, RNA , Sigma Factor/genetics , Sigma Factor/metabolism , Staphylococcus aureus/metabolism , Transcription, Genetic , Transformation, Bacterial
19.
Curr Opin Microbiol ; 30: 30-35, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26773797

ABSTRACT

Bacterial regulatory RNAs have been defined as diverse classes of cis and trans elements that may intervene at each step of gene expression, from RNA and protein synthesis to degradation. Here, we report on a few examples from Gram-positive bacteria that extend the definition of regulatory RNAs to include 5' and 3' UTRs that also act as cis and trans regulators. New examples unveil the existence of cis and trans acting regulatory RNAs on a single molecule. Also, we highlight data showing that a key RNA chaperone in Enterobacteriaceae, Hfq, does not fulfill the same role in Gram-positive Firmicutes.


Subject(s)
Gene Expression Regulation, Bacterial , Gram-Positive Bacteria/metabolism , RNA, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gram-Positive Bacteria/chemistry , Gram-Positive Bacteria/genetics , RNA, Bacterial/chemistry , RNA, Bacterial/genetics
20.
J Biol Methods ; 3(1): e35, 2016.
Article in English | MEDLINE | ID: mdl-31453204

ABSTRACT

In bacteria, DNA is tightly compacted in a supercoiled organization, which is mediated in part by nucleoid-associated proteins (NAPs). NAPs are well characterized for their ability to bind nucleic acids and for their involvement in gene regulation. A method commonly used to study protein-nucleic acid interactions involves immunoprecipitation of the protein of interest which is subsequently incubated with nucleic acids. A common cause of artifact is due to nucleic acids that remains bound to the protein of interest during the whole purification process. We developed an optimized method for the purification of tagged NAPs on affinity columns. The combination of three known methods allows removal of most of the nucleic acids bound to proteins during the purification process. This protocol is designed to improve the quality and specificity of results of in vitro experiments involving nucleic acid binding tests on purified NAPs. It can be used for in vitro studies of other RNA/DNA binding proteins.

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