Alternative TSS use is widespread in Cryptococcus fungi in response to environmental cues and regulated genome-wide by the transcription factor Tur1.

Alternative transcription start site (TSS) usage regulation has been identified as a major means of gene expression regulation in metazoans. However, in fungi, its impact remains elusive as its study has thus far been restricted to model yeasts. Here, we first re-analyzed TSS-seq data to define genuine TSS clusters in 2 species of pathogenic Cryptococcus. We identified 2 types of TSS clusters associated with specific DNA sequence motifs. Our analysis also revealed that alternative TSS usage regulation in response to environmental cues is widespread in Cryptococcus, altering gene expression and protein targeting. Importantly, we performed a forward genetic screen to identify a unique transcription factor (TF) named Tur1, which regulates alternative TSS (altTSS) usage genome-wide when cells switch from exponential phase to stationary phase. ChiP-Seq and DamID-Seq analyses suggest that at some loci, the role of Tur1 might be direct. Tur1 has been previously shown to be essential for virulence in C. neoformans. We demonstrated here that a tur1Δ mutant strain is more sensitive to superoxide stress and phagocytosed more efficiently by macrophages than the wild-type (WT) strain.

The oxidative stress response of pathogenic Leptospira is controlled by two peroxide stress regulators which putatively cooperate in controlling virulence.

Pathogenic Leptospira are the causative agents of leptospirosis, the most widespread zoonotic infectious disease. Leptospirosis is a potentially severe and life-threatening emerging disease with highest burden in sub-tropical areas and impoverished populations. Mechanisms allowing pathogenic Leptospira to survive inside a host and induce acute leptospirosis are not fully understood. The ability to resist deadly oxidants produced by the host during infection is pivotal for Leptospira virulence. We have previously shown that genes encoding defenses against oxidants in L. interrogans are repressed by PerRA (encoded by LIMLP_10155), a peroxide stress regulator of the Fur family. In this study, we describe the identification and characterization of another putative PerR-like regulator (LIMLP_05620) in L. interrogans. Protein sequence and phylogenetic analyses indicated that LIMLP_05620 displayed all the canonical PerR amino acid residues and is restricted to pathogenic Leptospira clades. We therefore named this PerR-like regulator PerRB. In L. interrogans, the PerRB regulon is distinct from that of PerRA. While a perRA mutant had a greater tolerance to peroxide, inactivating perRB led to a higher tolerance to superoxide, suggesting that these two regulators have a distinct function in the adaptation of L. interrogans to oxidative stress. The concomitant inactivation of perRA and perRB resulted in a higher tolerance to both peroxide and superoxide and, unlike the single mutants, a double perRAperRB mutant was avirulent. Interestingly, this correlated with major changes in gene and non-coding RNA expression. Notably, several virulence-associated genes (clpB, ligA/B, and lvrAB) were repressed. By obtaining a double mutant in a pathogenic Leptospira strain, our study has uncovered an interplay of two PerRs in the adaptation of Leptospira to oxidative stress with a putative role in virulence and pathogenicity, most likely through the transcriptional control of a complex regulatory network.

Role of the major determinant of polar flagellation FlhG in the endoflagella-containing spirochete Leptospira.

Spirochetes can be distinguished from other bacteria by their spiral-shaped morphology and subpolar periplasmic flagella. This study focused on FlhF and FlhG, which control the spatial and numerical regulation of flagella in many exoflagellated bacteria, in the spirochete Leptospira. In contrast to flhF which seems to be essential in Leptospira, we demonstrated that flhG- mutants in both the saprophyte L. biflexa and the pathogen L. interrogans were less motile than the wild-type strains in gel-like environments but not hyperflagellated as reported previously in other bacteria. Cryo-electron tomography revealed that the distance between the flagellar basal body and the tip of the cell decreased significantly in the flhG- mutant in comparison to wild-type and complemented strains. Additionally, comparative transcriptome analyses of L. biflexa flhG- and wild-type strains showed that FlhG acts as a negative regulator of transcription of some flagellar genes. We found that the L. interrogans flhG- mutant was attenuated for virulence in the hamster model. Cross-species complementation also showed that flhG is not interchangeable between species. Our results indicate that FlhF and FlhG in Leptospira contribute to governing cell motility but our data support the hypothesis that FlhF and FlhG function differently in each bacterial species, including among spirochetes.

Transcytosis subversion by M cell-to-enterocyte spread promotes Shigella flexneri and Listeria monocytogenes intracellular bacterial dissemination.

Microfold (M) cell host-pathogen interaction studies would benefit from the visual analysis of dynamic cellular and microbial interplays. We adapted a human in vitro M cell model to physiological bacterial infections, expression of fluorescent localization reporters and long-term three-dimensional time-lapse microscopy. This approach allows following key steps of M cell infection dynamics at subcellular resolution, from the apical onset to basolateral epithelial dissemination. We focused on the intracellular pathogen Shigella flexneri, classically reported to transcytose through M cells to initiate bacillary dysentery in humans, while eliciting poorly protective immune responses. Our workflow was critical to reveal that S. flexneri develops a bimodal lifestyle within M cells leading to rapid transcytosis or delayed vacuolar rupture, followed by direct actin motility-based propagation to neighboring enterocytes. Moreover, we show that Listeria monocytogenes, another intracellular pathogen sharing a tropism for M cells, disseminates in a similar manner and evades M cell transcytosis completely. We established that actin-based M cell-to-enterocyte spread is the major dissemination pathway for both pathogens and avoids their exposure to basolateral compartments in our system. Our results challenge the notion that intracellular pathogens are readily transcytosed by M cells to inductive immune compartments in vivo, providing a potential mechanism for their ability to evade adaptive immunity.

The transcriptional response of pathogenic Leptospira to peroxide reveals new defenses against infection-related oxidative stress.

Pathogenic Leptospira spp. are the causative agents of the waterborne zoonotic disease leptospirosis. Leptospira are challenged by numerous adverse conditions, including deadly reactive oxygen species (ROS), when infecting their hosts. Withstanding ROS produced by the host innate immunity is an important strategy evolved by pathogenic Leptospira for persisting in and colonizing hosts. In L. interrogans, genes encoding defenses against ROS are repressed by the peroxide stress regulator, PerR. In this study, RNA sequencing was performed to characterize both the L. interrogans response to low and high concentrations of hydrogen peroxide and the PerR regulon. We showed that Leptospira solicit three main peroxidase machineries (catalase, cytochrome C peroxidase and peroxiredoxin) and heme to detoxify oxidants produced during peroxide stress. In addition, canonical molecular chaperones of the heat shock response and DNA repair proteins from the SOS response were required for Leptospira recovering from oxidative damage. Identification of the PerR regulon upon exposure to H2O2 allowed to define the contribution of this regulator in the oxidative stress response. This study has revealed a PerR-independent regulatory network involving other transcriptional regulators, two-component systems and sigma factors as well as non-coding RNAs that putatively orchestrate, in concert with PerR, the oxidative stress response. We have shown that PerR-regulated genes encoding a TonB-dependent transporter and a two-component system (VicKR) are involved in Leptospira tolerance to superoxide. This could represent the first defense mechanism against superoxide in L. interrogans, a bacterium lacking canonical superoxide dismutase. Our findings provide an insight into the mechanisms required by pathogenic Leptospira to overcome oxidative damage during infection-related conditions. This will participate in framing future hypothesis-driven studies to identify and decipher novel virulence mechanisms in this life-threatening pathogen.

4-Methylcytosine DNA modification is critical for global epigenetic regulation and virulence in the human pathogen Leptospira interrogans.

In bacteria, DNA methylation can be facilitated by 'orphan' DNA methyltransferases lacking cognate restriction endonucleases, but whether and how these enzymes control key cellular processes are poorly understood. The effects of a specific modification, 4-methylcytosine (4mC), are even less clear, as this epigenetic marker is unique to bacteria and archaea, whereas the bulk of epigenetic research is currently performed on eukaryotes. Here, we characterize a 4mC methyltransferase from the understudied pathogen Leptospira spp. Inactivating this enzyme resulted in complete abrogation of CTAG motif methylation, leading to genome-wide dysregulation of gene expression. Mutants exhibited growth defects, decreased adhesion to host cells, higher susceptibility to LPS-targeting antibiotics, and, importantly, were no longer virulent in an acute infection model. Further investigation resulted in the discovery of at least one gene, that of an ECF sigma factor, whose transcription was altered in the methylase mutant and, subsequently, by mutation of the CTAG motifs in the promoter of the gene. The genes that comprise the regulon of this sigma factor were, accordingly, dysregulated in the methylase mutant and in a strain overexpressing the sigma factor. Our results highlight the importance of 4mC in Leptospira physiology, and suggest the same of other understudied species.

Rae1/YacP, a new endoribonuclease involved in ribosome-dependent mRNA decay in Bacillus subtilis.

The PIN domain plays a central role in cellular RNA biology and is involved in processes as diverse as rRNA maturation, mRNA decay and telomerase function. Here, we solve the crystal structure of the Rae1 (YacP) protein of Bacillus subtilis, a founding member of the NYN (Nedd4-BP1/YacP nuclease) subfamily of PIN domain proteins, and identify potential substrates in vivo Unexpectedly, degradation of a characterised target mRNA was completely dependent on both its translation and reading frame. We provide evidence that Rae1 associates with the B. subtilis ribosome and cleaves between specific codons of this mRNA in vivo Critically, we also demonstrate translation-dependent Rae1 cleavage of this substrate in a purified translation assay in vitro Multiple lines of evidence converge to suggest that Rae1 is an A-site endoribonuclease. We present a docking model of Rae1 bound to the B. subtilis ribosomal A-site that is consistent with this hypothesis and show that Rae1 cleaves optimally immediately upstream of a lysine codon (AAA or AAG) in vivo.

Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages.

Free-living amoebae are thought to represent an environmental niche in which amoeba-resistant bacteria may evolve towards pathogenicity. To get more insights into factors playing a role for adaptation to intracellular life, we characterized the transcriptomic activities of the emerging pathogen Mycobacterium abscessus in amoeba and murine macrophages (Mϕ) and compared them with the intra-amoebal transcriptome of the closely related, but less pathogenic Mycobacterium chelonae. Data on up-regulated genes in amoeba point to proteins that allow M. abscessus to resist environmental stress and induce defense mechanisms, as well as showing a switch from carbohydrate carbon sources to fatty acid metabolism. For eleven of the most upregulated genes in amoeba and/or Mϕ, we generated individual gene knock-out M. abscessus mutant strains, from which ten were found to be attenuated in amoeba and/or Mϕ in subsequence virulence analyses. Moreover, transfer of two of these genes into the genome of M. chelonae increased the intra-Mϕ survival of the recombinant strain. One knock-out mutant that had the gene encoding Eis N-acetyl transferase protein (MAB_4532c) deleted, was particularly strongly attenuated in Mϕ. Taken together, M. abscessus intra-amoeba and intra-Mϕ transcriptomes revealed the capacity of M. abscessus to adapt to an intracellular lifestyle, with amoeba largely contributing to the enhancement of M. abscessus intra-Mϕ survival.

Insights into amebiasis using a human 3D-intestinal model.

Entamoeba histolytica is the causative agent of amebiasis, an infectious disease targeting the intestine and the liver in humans. Two types of intestinal infection are caused by this parasite: silent infection, which occurs in the majority of cases, and invasive disease, which affects 10% of infected persons. To understand the intestinal pathogenic process, several in vitro models, such as cell cultures, human tissue explants or human intestine xenografts in mice, have been employed. Nevertheless, our knowledge on the early steps of amebic intestinal infection and the molecules involved during human-parasite interaction is scarce, in part due to limitations in the experimental settings. In the present work, we took advantage of tissue engineering approaches to build a three-dimensional (3D)-intestinal model that is able to replicate the general characteristics of the human colon. This system consists of an epithelial layer that develops tight and adherens junctions, a mucus layer and a lamina propria-like compartment made up of collagen containing macrophages and fibroblast. By means of microscopy imaging, omics assays and the evaluation of immune responses, we show a very dynamic interaction between E. histolytica and the 3D-intestinal model. Our data highlight the importance of several virulence markers occurring in patients or in experimental models, but they also demonstrate the involvement of under described molecules and regulatory factors in the amoebic invasive process.

checkMyIndex: a web-based R/Shiny interface for choosing compatible sequencing indexes.

SUMMARY: When sequencing several libraries simultaneously, the selection of compatible combinations of indexes is critical for ensuring that the sequencer will be able to decipher the short, sample-specific barcodes added to each fragment. However, researchers have few tools to help them choose optimal indexes. Here, we present checkMyIndex, an online R/Shiny application that facilitates the selection of the right indexes as a function of the experimental constraints. AVAILABILITY AND IMPLEMENTATION: checkMyIndex is available free of charge at https://checkmyindex.pasteur.fr as an online, web-based R/Shiny application. The source code is available on GitHub at https://github.com/PF2-pasteur-fr/checkMyIndex.

Enzymatic synthesis of random sequences of RNA and RNA analogues by DNA polymerase theta mutants for the generation of aptamer libraries.

Nucleic acid aptamers, especially RNA, exhibit valuable advantages compared to protein therapeutics in terms of size, affinity and specificity. However, the synthesis of libraries of large random RNAs is still difficult and expensive. The engineering of polymerases able to directly generate these libraries has the potential to replace the chemical synthesis approach. Here, we start with a DNA polymerase that already displays a significant template-free nucleotidyltransferase activity, human DNA polymerase theta, and we mutate it based on the knowledge of its three-dimensional structure as well as previous mutational studies on members of the same polA family. One mutant exhibited a high tolerance towards ribonucleotides (NTPs) and displayed an efficient ribonucleotidyltransferase activity that resulted in the assembly of long RNA polymers. HPLC analysis and RNA sequencing of the products were used to quantify the incorporation of the four NTPs as a function of initial NTP concentrations and established the randomness of each generated nucleic acid sequence. The same mutant revealed a propensity to accept other modified nucleotides and to extend them in long fragments. Hence, this mutant can deliver random natural and modified RNA polymers libraries ready to use for SELEX, with custom lengths and balanced or unbalanced ratios.

The Legionella pneumophila genome evolved to accommodate multiple regulatory mechanisms controlled by the CsrA-system.

The carbon storage regulator protein CsrA regulates cellular processes post-transcriptionally by binding to target-RNAs altering translation efficiency and/or their stability. Here we identified and analyzed the direct targets of CsrA in the human pathogen Legionella pneumophila. Genome wide transcriptome, proteome and RNA co-immunoprecipitation followed by deep sequencing of a wild type and a csrA mutant strain identified 479 RNAs with potential CsrA interaction sites located in the untranslated and/or coding regions of mRNAs or of known non-coding sRNAs. Further analyses revealed that CsrA exhibits a dual regulatory role in virulence as it affects the expression of the regulators FleQ, LqsR, LetE and RpoS but it also directly regulates the timely expression of over 40 Dot/Icm substrates. CsrA controls its own expression and the stringent response through a regulatory feedback loop as evidenced by its binding to RelA-mRNA and links it to quorum sensing and motility. CsrA is a central player in the carbon, amino acid, fatty acid metabolism and energy transfer and directly affects the biosynthesis of cofactors, vitamins and secondary metabolites. We describe the first L. pneumophila riboswitch, a thiamine pyrophosphate riboswitch whose regulatory impact is fine-tuned by CsrA, and identified a unique regulatory mode of CsrA, the active stabilization of RNA anti-terminator conformations inside a coding sequence preventing Rho-dependent termination of the gap operon through transcriptional polarity effects. This allows L. pneumophila to regulate the pentose phosphate pathway and the glycolysis combined or individually although they share genes in a single operon. Thus the L. pneumophila genome has evolved to acclimate at least five different modes of regulation by CsrA giving it a truly unique position in its life cycle.

Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and Regulation of Steroid Metabolism.

BAHD1 is a vertebrate protein that promotes heterochromatin formation and gene repression in association with several epigenetic regulators. However, its physiological roles remain unknown. Here, we demonstrate that ablation of the Bahd1 gene results in hypocholesterolemia, hypoglycemia and decreased body fat in mice. It also causes placental growth restriction with a drop of trophoblast glycogen cells, a reduction of fetal weight and a high neonatal mortality rate. By intersecting transcriptome data from murine Bahd1 knockout (KO) placentas at stages E16.5 and E18.5 of gestation, Bahd1-KO embryonic fibroblasts, and human cells stably expressing BAHD1, we also show that changes in BAHD1 levels alter expression of steroid/lipid metabolism genes. Biochemical analysis of the BAHD1-associated multiprotein complex identifies MIER proteins as novel partners of BAHD1 and suggests that BAHD1-MIER interaction forms a hub for histone deacetylases and methyltransferases, chromatin readers and transcription factors. We further show that overexpression of BAHD1 leads to an increase of MIER1 enrichment on the inactive X chromosome (Xi). In addition, BAHD1 and MIER1/3 repress expression of the steroid hormone receptor genes ESR1 and PGR, both playing important roles in placental development and energy metabolism. Moreover, modulation of BAHD1 expression in HEK293 cells triggers epigenetic changes at the ESR1 locus. Together, these results identify BAHD1 as a core component of a chromatin-repressive complex regulating placental morphogenesis and body fat storage and suggest that its dysfunction may contribute to several human diseases.

Expansion of the SOS regulon of Vibrio cholerae through extensive transcriptome analysis and experimental validation.

BACKGROUND: The SOS response is an almost ubiquitous response of cells to genotoxic stresses. The full complement of genes in the SOS regulon for Vibrio species has only been addressed through bioinformatic analyses predicting LexA binding box consensus and in vitro validation. Here, we perform whole transcriptome sequencing from Vibrio cholerae treated with mitomycin C as an SOS inducer to characterize the SOS regulon and other pathways affected by this treatment. RESULTS: Comprehensive transcriptional profiling allowed us to define the full landscape of promoters and transcripts active in V. cholerae. We performed extensive transcription start site (TSS) mapping as well as detection/quantification of the coding and non-coding RNA (ncRNA) repertoire in strain N16961. To improve TSS detection, we developed a new technique to treat RNA extracted from cells grown in various conditions. This allowed for identification of 3078 TSSs with an average 5'UTR of 116 nucleotides, and peak distribution between 16 and 64 nucleotides; as well as 629 ncRNAs. Mitomycin C treatment induced transcription of 737 genes and 28 ncRNAs at least 2 fold, while it repressed 231 genes and 17 ncRNAs. Data analysis revealed that in addition to the core genes known to integrate the SOS regulon, several metabolic pathways were induced. This study allowed for expansion of the Vibrio SOS regulon, as twelve genes (ubiEJB, tatABC, smpA, cep, VC0091, VC1190, VC1369-1370) were found to be co-induced with their adjacent canonical SOS regulon gene(s), through transcriptional read-through. Characterization of UV and mitomycin C susceptibility for mutants of these newly identified SOS regulon genes and other highly induced genes and ncRNAs confirmed their role in DNA damage rescue and protection. CONCLUSIONS: We show that genotoxic stress induces a pervasive transcriptional response, affecting almost 20% of the V. cholerae genes. We also demonstrate that the SOS regulon is larger than previously known, and its syntenic organization is conserved among Vibrio species. Furthermore, this specific co-localization is found in other γ-proteobacteria for genes recN-smpA and rmuC-tatABC, suggesting SOS regulon conservation in this phylum. Finally, we comment on the limitations of widespread NGS approaches for identification of all RNA species in bacteria.

GH16 and GH81 family ß-(1,3)-glucanases in Aspergillus fumigatus are essential for conidial cell wall morphogenesis.

The fungal cell wall is a rigid structure because of fibrillar and branched ß-(1,3)-glucan linked to chitin. Softening of the cell wall is an essential phenomenon during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosylhydrolases. During the search for glycosylhydrolases acting on ß-(1,3)-glucan, we identified seven genes in the Aspergillus fumigatus genome coding for potential endo-ß-(1,3)-glucanase. ENG1 (previously characterized and named ENGL1, Mouyna et al., ), belongs to the Glycoside-Hydrolase 81 (GH81) family, while ENG2 to ENG7, to GH16 family. ENG1 and four GH16 genes (ENG2-5) were expressed in the resting conidia as well as during germination, suggesting an essential role during A. fumigatus morphogenesis. Here, we report the effect of sequential deletion of AfENG2-5 (GH16) followed by AfENG1 (GH81) deletion in the Δeng2,3,4,5 mutant. The Δeng1,2,3,4,5 mutant showed conidial defects, with linear chains of conidia unable to separate while the germination rate was not affected. These results show, for the first time in a filamentous fungus, that endo ß-(1,3)-glucanases are essential for proper conidial cell wall assembly and thus segregation of conidia during conidiation.

Repressor activity of the RpoS/σS-dependent RNA polymerase requires DNA binding.

The RpoS/σ(S) sigma subunit of RNA polymerase (RNAP) activates transcription of stationary phase genes in many Gram-negative bacteria and controls adaptive functions, including stress resistance, biofilm formation and virulence. In this study, we address an important but poorly understood aspect of σ(S)-dependent control, that of a repressor. Negative regulation by σ(S) has been proposed to result largely from competition between σ(S) and other σ factors for binding to a limited amount of core RNAP (E). To assess whether σ(S) binding to E alone results in significant downregulation of gene expression by other σ factors, we characterized an rpoS mutant of Salmonella enterica serovar Typhimurium producing a σ(S) protein proficient for Eσ(S) complex formation but deficient in promoter DNA binding. Genome expression profiling and physiological assays revealed that this mutant was defective for negative regulation, indicating that gene repression by σ(S) requires its binding to DNA. Although the mechanisms of repression by σ(S) are likely specific to individual genes and environmental conditions, the study of transcription downregulation of the succinate dehydrogenase operon suggests that σ competition at the promoter DNA level plays an important role in gene repression by Eσ(S).

Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

RNase J depletion leads to massive changes in mRNA abundance in Helicobacter pylori.

Degradation of RNA as an intermediate message between genes and corresponding proteins is important for rapid attenuation of gene expression and maintenance of cellular homeostasis. This process is controlled by ribonucleases that have different target specificities. In the bacterial pathogen Helicobacter pylori, an exo- and endoribonuclease RNase J is essential for growth. To explore the role of RNase J in H. pylori, we identified its putative targets at a global scale using next generation RNA sequencing. We found that strong depletion for RNase J led to a massive increase in the steady-state levels of non-rRNAs. mRNAs and RNAs antisense to open reading frames were most affected with over 80% increased more than 2-fold. Non-coding RNAs expressed in the intergenic regions were much less affected by RNase J depletion. Northern blotting of selected messenger and non-coding RNAs validated these results. Globally, our data suggest that RNase J of H. pylori is a major RNase involved in degradation of most cellular RNAs.

Starvation, together with the SOS response, mediates high biofilm-specific tolerance to the fluoroquinolone ofloxacin.

High levels of antibiotic tolerance are a hallmark of bacterial biofilms. In contrast to well-characterized inherited antibiotic resistance, molecular mechanisms leading to reversible and transient antibiotic tolerance displayed by biofilm bacteria are still poorly understood. The physiological heterogeneity of biofilms influences the formation of transient specialized subpopulations that may be more tolerant to antibiotics. In this study, we used random transposon mutagenesis to identify biofilm-specific tolerant mutants normally exhibited by subpopulations located in specialized niches of heterogeneous biofilms. Using Escherichia coli as a model organism, we demonstrated, through identification of amino acid auxotroph mutants, that starved biofilms exhibited significantly greater tolerance towards fluoroquinolone ofloxacin than their planktonic counterparts. We demonstrated that the biofilm-associated tolerance to ofloxacin was fully dependent on a functional SOS response upon starvation to both amino acids and carbon source and partially dependent on the stringent response upon leucine starvation. However, the biofilm-specific ofloxacin increased tolerance did not involve any of the SOS-induced toxin-antitoxin systems previously associated with formation of highly tolerant persisters. We further demonstrated that ofloxacin tolerance was induced as a function of biofilm age, which was dependent on the SOS response. Our results therefore show that the SOS stress response induced in heterogeneous and nutrient-deprived biofilm microenvironments is a molecular mechanism leading to biofilm-specific high tolerance to the fluoroquinolone ofloxacin.

Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile.

Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA-seq and differential 5'-end RNA-seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA-based regulation of gene expression in this emergent enteropathogen.