StaR Is a Positive Regulator of Topoisomerase I Activity Involved in Supercoiling Maintenance in Streptococcus pneumoniae.

The DNA topoisomerases gyrase and topoisomerase I as well as the nucleoid-associated protein HU maintain supercoiling levels in Streptococcus pneumoniae, a main human pathogen. Here, we characterized, for the first time, a topoisomerase I regulator protein (StaR). In the presence of sub-inhibitory novobiocin concentrations, which inhibit gyrase activity, higher doubling times were observed in a strain lacking staR, and in two strains in which StaR was over-expressed either under the control of the ZnSO4-inducible PZn promoter (strain ΔstaRPZnstaR) or of the maltose-inducible PMal promoter (strain ΔstaRpLS1ROMstaR). These results suggest that StaR has a direct role in novobiocin susceptibility and that the StaR level needs to be maintained within a narrow range. Treatment of ΔstaRPZnstaR with inhibitory novobiocin concentrations resulted in a change of the negative DNA supercoiling density (σ) in vivo, which was higher in the absence of StaR (σ = -0.049) than when StaR was overproduced (σ = -0.045). We have located this protein in the nucleoid by using super-resolution confocal microscopy. Through in vitro activity assays, we demonstrated that StaR stimulates TopoI relaxation activity, while it has no effect on gyrase activity. Interaction between TopoI and StaR was detected both in vitro and in vivo by co-immunoprecipitation. No alteration of the transcriptome was associated with StaR amount variation. The results suggest that StaR is a new streptococcal nucleoid-associated protein that activates topoisomerase I activity by direct protein-protein interaction.

Identification of 88 regulatory small RNAs in the TIGR4 strain of the human pathogen Streptococcus pneumoniae.

Streptococcus pneumoniae is the main etiological agent of community-acquired pneumonia and a major cause of mortality and morbidity among children and the elderly. Genome sequencing of several pneumococcal strains revealed valuable information about the potential proteins and genetic diversity of this prevalent human pathogen. However, little is known about its transcriptional regulation and its small regulatory noncoding RNAs. In this study, we performed deep sequencing of the S. pneumoniae TIGR4 strain RNome to identify small regulatory RNA candidates expressed in this pathogen. We discovered 1047 potential small RNAs including intragenic, 5'- and/or 3'-overlapping RNAs and 88 small RNAs encoded in intergenic regions. With this approach, we recovered many of the previously identified intergenic small RNAs and identified 68 novel candidates, most of which are conserved in both sequence and genomic context in other S. pneumoniae strains. We confirmed the independent expression of 17 intergenic small RNAs and predicted putative mRNA targets for six of them using bioinformatics tools. Preliminary results suggest that one of these six is a key player in the regulation of competence development. This study is the biggest catalog of small noncoding RNAs reported to date in S. pneumoniae and provides a highly complete view of the small RNA network in this pathogen.

Synergies between RNA degradation and trans-translation in Streptococcus pneumoniae: cross regulation and co-transcription of RNase R and SmpB.

BACKGROUND: Ribonuclease R (RNase R) is an exoribonuclease that recognizes and degrades a wide range of RNA molecules. It is a stress-induced protein shown to be important for the establishment of virulence in several pathogenic bacteria. RNase R has also been implicated in the trans-translation process. Transfer-messenger RNA (tmRNA/SsrA RNA) and SmpB are the main effectors of trans-translation, an RNA and protein quality control system that resolves challenges associated with stalled ribosomes on non-stop mRNAs. Trans-translation has also been associated with deficiencies in stress-response mechanisms and pathogenicity. RESULTS: In this work we study the expression of RNase R in the human pathogen Streptococcus pneumoniae and analyse the interplay of this enzyme with the main components of the trans-translation machinery (SmpB and tmRNA/SsrA). We show that RNase R is induced after a 37°C to 15°C temperature downshift and that its levels are dependent on SmpB. On the other hand, our results revealed a strong accumulation of the smpB transcript in the absence of RNase R at 15°C. Transcriptional analysis of the S. pneumoniae rnr gene demonstrated that it is co-transcribed with the flanking genes, secG and smpB. Transcription of these genes is driven from a promoter upstream of secG and the transcript is processed to yield mature independent mRNAs. This genetic organization seems to be a common feature of Gram positive bacteria, and the biological significance of this gene cluster is further discussed. CONCLUSIONS: This study unravels an additional contribution of RNase R to the trans-translation system by demonstrating that smpB is regulated by this exoribonuclease. RNase R in turn, is shown to be under the control of SmpB. These proteins are therefore mutually dependent and cross-regulated. The data presented here shed light on the interactions between RNase R, trans-translation and cold-shock response in an important human pathogen.

Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex.

RNA degradation is a determining factor in the control of gene expression. The maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. RNase II-like enzymes are found in all three kingdoms of life, but there are no structural data for any of the proteins of this family. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 A resolution) and RNA-bound (at 2.74 A resolution) forms of Escherichia coli RNase II. In contrast to sequence predictions, the structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented alphabeta-fold, and one S1 domain. The enzyme establishes contacts with RNA in two distinct regions, the 'anchor' and the 'catalytic' regions, which act synergistically to provide catalysis. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity. We propose a reaction mechanism for exonucleolytic RNA degradation involving key conserved residues. Our three-dimensional model corroborates all existing biochemical data for RNase II, and elucidates the general basis for RNA degradation. Moreover, it reveals important structural features that can be extrapolated to other members of this family.

Role of PatAB Transporter in Efflux of Levofloxacin in Streptococcus pneumoniae.

PatAB is an ABC bacterial transporter that facilitates the export of antibiotics and dyes. The overexpression of patAB genes conferring efflux-mediated fluoroquinolone resistance has been observed in several laboratory strains and clinical isolates of Streptococcus pneumoniae. Using transformation and whole-genome sequencing, we characterized the fluoroquinolone-resistance mechanism of one S. pneumoniae clinical isolate without mutations in the DNA topoisomerase genes. We identified the PatAB fluoroquinolone efflux-pump as the mechanism conferring a low-level resistance to ciprofloxacin (8 µg/mL) and levofloxacin (4 µg/mL). Genetic transformation experiments with different amplimers revealed that the entire patA plus the 5'-terminus of patB are required for levofloxacin-efflux. By contrast, only the upstream region of the patAB operon, plus the region coding the N-terminus of PatA containing the G39D, T43A, V48A and D100N amino acid changes, are sufficient to confer a ciprofloxacin-efflux phenotype, thus suggesting differences between fluoroquinolones in their binding and/or translocation pathways. In addition, we identified a novel single mutation responsible for the constitutive and ciprofloxacin-inducible upregulation of patAB. This mutation is predicted to destabilize the putative rho-independent transcriptional terminator located upstream of patA, increasing transcription of downstream genes. This is the first report demonstrating the role of the PatAB transporter in levofloxacin-efflux in a pneumoccocal clinical isolate.

A Small Non-Coding RNA Modulates Expression of Pilus-1 Type in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, and about 30% of the pneumococcal clinical isolates show type I pili-like structures. These long proteinaceous polymers extending from the bacterial surface are encoded by pilus islet 1 and play major roles in adhesion and host colonization. Pili expression is bistable and is controlled by the transcriptional activator RlrA. In this work, we demonstrate that the previously identified small noncoding RNA srn135 also participates in pilus regulation. Our findings show that srn135 is generated upon processing of the 5'-UTR region of rrgA messenger and its deletion prevents the synthesis of RrgA, the main pili adhesin. Moreover, overexpression of srn135 increases the expression of all pili genes and rises the percentage of piliated bacteria within a clonal population. This regulation is mediated by the stabilization of rlrA mRNA since higher levels of srn135 increase its half-life to 165%. Our findings suggest that srn135 has a dual role in pilus expression acting both in cis- (on the RrgA levels) and in trans- (modulating the levels of RlrA) and contributes to the delicate balance between pili expressing and non-expressing bacteria.

Determination of key residues for catalysis and RNA cleavage specificity: one mutation turns RNase II into a "SUPER-ENZYME".

RNase II is the prototype of a ubiquitous family of enzymes that are crucial for RNA metabolism. In Escherichia coli this protein is a single-stranded-specific 3'-exoribonuclease with a modular organization of four functional domains. In eukaryotes, the RNase II homologue Rrp44 (also known as Dis3) is the catalytic subunit of the exosome, an exoribonuclease complex essential for RNA processing and decay. In this work we have performed a functional characterization of several highly conserved residues located in the RNase II catalytic domain to address their precise role in the RNase II activity. We have constructed a number of RNase II mutants and compared their activity and RNA binding to the wild type using different single- or double-stranded substrates. The results presented in this study substantially improve the RNase II model for RNA degradation. We have identified the residues that are responsible for the discrimination of cleavage of RNA versus DNA. We also show that the Arg-500 residue present in the RNase II active site is crucial for activity but not for RNA binding. The most prominent finding presented is the extraordinary catalysis observed in the E542A mutant that turns RNase II into a "super-enzyme."

Boldine-Derived Alkaloids Inhibit the Activity of DNA Topoisomerase I and Growth of Mycobacterium tuberculosis.

The spread of multidrug-resistant isolates of Mycobacterium tuberculosis requires the discovery of new drugs directed to new targets. In this study, we investigated the activity of two boldine-derived alkaloids, seconeolitsine (SCN) and N-methyl-seconeolitsine (N-SCN), against M. tuberculosis. These compounds have been shown to target DNA topoisomerase I enzyme and inhibit growth of Streptococcus pneumoniae. Both SCN and N-SCN inhibited M. tuberculosis growth at 1.95-15.6 µM, depending on the strain. In M. smegmatis this inhibitory effect correlated with the amount of topoisomerase I in the cell, hence demonstrating that this enzyme is the target for these alkaloids in mycobacteria. The gene coding for topoisomerase I of strain H37Rv (MtbTopoI) was cloned into pQE1 plasmid of Escherichia coli. MtbTopoI was overexpressed with an N-terminal 6-His-tag and purified by affinity chromatography. In vitro inhibition of MtbTopoI activity by SCN and N-SCN was tested using a plasmid relaxation assay. Both SCN and N-SCN inhibited 50% of the enzymatic activity at 5.6 and 8.4 µM, respectively. Cleavage of single-stranded DNA was also inhibited with SCN. The effects on DNA supercoiling were also evaluated in vivo in plasmid-containing cultures of M. tuberculosis. Plasmid supercoiling densities were -0.060 in cells untreated or treated with boldine, and -0.072 in 1 × MIC N-SCN treated cells, respectively, indicating that the plasmid became hypernegatively supercoiled in the presence of N-SCN. Altogether, these results demonstrate that the M. tuberculosis topoisomerase I enzyme is an attractive drug target, and that SCN and N-SCN are promising lead compounds for drug development.

Characterization of the functional domains of Escherichia coli RNase II.

RNase II is a single-stranded-specific 3'-exoribonuclease that degrades RNA generating 5'-mononucleotides. This enzyme is the prototype of an ubiquitous family of enzymes that are crucial in RNA metabolism and share a similar domain organization. By sequence prediction, three different domains have been assigned to the Escherichia coli RNase II: two RNA-binding domains at each end of the protein (CSD and S1), and a central RNB catalytic domain. In this work we have performed a functional characterization of these domains in order to address their role in the activity of RNase II. We have constructed a large set of RNase II truncated proteins and compared them to the wild-type regarding their exoribonucleolytic activity and RNA-binding ability. The dissociation constants were determined using different single- or double-stranded substrates. The results obtained revealed that S1 is the most important domain in the establishment of stable RNA-protein complexes, and its elimination results in a drastic reduction on RNA-binding ability. In addition, we also demonstrate that the N-terminal CSD plays a very specific role in RNase II, preventing a tight binding of the enzyme to single-stranded poly(A) chains. Moreover, the biochemical results obtained with RNB mutant that lacks both putative RNA-binding domains, revealed the presence of an additional region involved in RNA binding. Such region, was identified by sequence analysis and secondary structure prediction as a third putative RNA-binding domain located at the N-terminal part of RNB catalytic domain.

Expression, purification, crystallization and preliminary diffraction data characterization of Escherichia coli ribonuclease II (RNase II).

RNA degradation is important in the post-transcriptional control of gene expression. The processing, degradation and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a 643-amino-acid enzyme that degrades single-stranded RNA from its 3'-end, releasing ribonucleoside 5'-monophosphates. RNase II was expressed both as the wild type and as a D209N mutant form. The latter was also produced as an SeMet derivative. The various protein forms were crystallized using the vapour-diffusion method. Wild-type RNase II was crystallized in two crystal forms, both of which belonged to space group P2(1). X-ray diffraction data were collected to 2.44 and 2.75 angstroms resolution, with unit-cell parameters a = 56.8, b = 125.7, c = 66.2 angstroms, beta = 111.9 degrees and a = 119.6, b = 57.2, c = 121.2 angstroms, beta = 99.7 degrees, respectively. The RNase II D209N mutant gave crystals that belonged to space group P6(5), with unit-cell parameters a = b = 86.3, c = 279.2 angstroms, and diffracted to 2.74 angstroms. Diffraction data from the mutant and its SeMet derivative enabled the determination of a partial Se-atom substructure by SIRAS.

Absence of tmRNA Has a Protective Effect against Fluoroquinolones in Streptococcus pneumoniae.

The transfer messenger RNA (tmRNA), encoded by the ssrA gene, is a small non-coding RNA involved in trans-translation that contributes to the recycling of ribosomes stalled on aberrant mRNAs. In most bacteria, its inactivation has been related to a decreased ability to respond to and recover from a variety of stress conditions. In this report, we investigated the role of tmRNA in stress adaptation in the human pathogen Streptococcus pneumoniae. We constructed a tmRNA deletion mutant and analyzed its response to several lethal stresses. The ΔssrA strain grew slower than the wild type, indicating that, although not essential, tmRNA is important for normal pneumococcal growth. Moreover, deletion of tmRNA increased susceptibility to UV irradiation, to exogenous hydrogen peroxide and to antibiotics that inhibit protein synthesis and transcription. However, the ΔssrA strain was more resistant to fluoroquinolones, showing twofold higher MIC values and up to 1000-fold higher survival rates than the wild type. Deletion of SmpB, the other partner in trans-translation, also reduced survival to levofloxacin in a similar extent. Accumulation of intracellular reactive oxygen species associated to moxifloxacin and levofloxacin treatment was also highly reduced (∼100-fold). Nevertheless, the ΔssrA strain showed higher intracellular accumulation of ethidium bromide and levofloxacin than the wild type, suggesting that tmRNA deficiency protects pneumococcal cells from fluoroquinolone-mediated killing. In fact, analysis of chromosome integrity revealed that deletion of tmRNA prevented the fragmentation of the chromosome associated to levofloxacin treatment. Moreover, such protective effect appears to relay mainly on inhibition of protein synthesis, since a similar effect was observed with antibiotics that inhibit that process. The emergence and spread of drug-resistant pneumococci is a matter of concern and these results contribute to a better comprehension of the mechanisms underlying fluoroquinolones action.

The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae.

The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation.

A single mutation in Escherichia coli ribonuclease II inactivates the enzyme without affecting RNA binding.

Exoribonuclease II (RNase II), encoded by the rnb gene, is a ubiquitous enzyme that is responsible for 90% of the hydrolytic activity in Escherichia coli crude extracts. The E. coli strain SK4803, carrying the mutant allele rnb296, has been widely used in the study of the role of RNase II. We determined the DNA sequence of rnb296 and cloned this mutant gene in an expression vector. Only a point mutation in the coding sequence of the gene was detected, which results in the single substitution of aspartate 209 for asparagine. The mutant and the wild-type RNase II enzymes were purified, and their 3' to 5' exoribonucleolytic activity, as well as their RNA binding capability, were characterized. We also studied the metal dependency of the exoribonuclease activity of RNase II. The results obtained demonstrated that aspartate 209 is absolutely essential for RNA hydrolysis, but is not required for substrate binding. This is the first evidence of an acidic residue that is essential for the activity of RNase II-like enzymes. The possible involvement of this residue in metal binding at the active site of the enzyme is discussed. These results are particularly relevant at this time given that no structural or mutational analysis has been performed for any protein of the RNR family of exoribonucleases.

Small regulatory RNAs in Streptococcus pneumoniae: discovery and biological functions.

Streptococcus pneumoniae is a prominent human pathogen responsible for many severe diseases and the leading cause of childhood mortality worldwide. The pneumococcus is remarkably adept at colonizing and infecting different niches in the human body, and its adaptation to dynamic host environment is a central aspect of its pathogenesis. In the last decade, increasing findings have evidenced small RNAs (sRNAs) as vital regulators in a number of important processes in bacteria. In S. pneumoniae, a small antisense RNA was first discovered in the pMV158 plasmid as a copy number regulator. More recently, genome-wide screens revealed that the pneumococcal genome also encodes multiple sRNAs, many of which have important roles in virulence while some are implicated in competence control. The knowledge of the sRNA-mediated regulation in pneumococcus remains very limited, and future research is needed for better understanding of functions and mechanisms. Here, we provide a comprehensive summary of the current knowledge on sRNAs from S. pneumoniae, focusing mainly on the trans-encoded sRNAs.

Isolation of a point mutation associated with altered expression of the CmeABC efflux pump in a multidrug-resistant Campylobacter jejuni population of poultry origin.

The objective of this study was to investigate the antibiotic resistance phenotype of Campylobacter jejuni isolates from a poultry flock of broiler production in Spain. Isolates were characterised by RFLP-PCR of the flaA gene and multilocus sequence typing. Minimum inhibitory concentrations of quinolones, aminoglycosides, ß-lactams, tetracyclines, phenicols, macrolides and lincosamides were determined by Etest. Determinants of resistance and the regulatory region of the cmeABC operon were investigated in all isolates by PCR detection and sequencing. Expression of the CmeABC efflux pump was investigated by quantitative RT-PCR and accumulation assay. Based on their molecular markers, two different populations of C. jejuni were identified: one resistant to quinolones, ß-lactams and tetracyclines, considered multidrug-resistant (MDR); and another resistant only to tetracyclines. Both populations possessed the tetO gene, previously associated with tetracycline resistance. The blaOXA-61 gene was also present in both populations, although only the MDR population showed ß-lactamase activity. In addition, MDR isolates possessed the Thr86Ile mutation in the gyrA gene responsible for quinolone resistance. Moreover, sequencing of the regulatory region of the cmeABC operon revealed the presence of the C-32→T mutation in the MDR isolates, which was accompanied by an increase in cmeA mRNA levels compared with the non-mutant population. In conclusion, this is the first report of the mutation C-32→T in the cmeABC operon in C. jejuni isolates of veterinary origin. This mutation is associated with overexpression of the CmeABC efflux pump in a MDR population and is possibly related to enhanced tolerance to antimicrobials that favours the development of resistance.

Characterizing ribonucleases in vitro examples of synergies between biochemical and structural analysis.

The contribution of RNA degradation to the posttranscriptional control of gene expression confers on it a fundamental role in all biological processes. Ribonucleases (RNases) are essential enzymes that process and degrade RNA and constitute one of the main groups of factors that determine RNA levels in the cells. RNase II is a ubiquitous, highly processive hydrolytic exoribonuclease that plays an important role in RNA metabolism. This ribonuclease can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. In this chapter, we explain the general procedures normally used for the characterization of ribonucleases, using as an example a study performed with Escherichia coli RNase II. We present the overexpression and purification of RNase II recombinant enzyme and of a large set of RNase II truncations. We also describe several methods that can be used for biochemically characterizing the exoribonucleolytic activity and studying RNA binding in vitro. Dissociation constants were determined by electrophoretic mobility shift assay (EMSA), surface plasmon resonance (SPR), and filter binding assays using different single- or double-stranded RNA substrates. We discuss the synergies among the biochemical analyses and the structural studies. These methods will be very useful for the study of other ribonucleases.

New insights into the mechanism of RNA degradation by ribonuclease II: identification of the residue responsible for setting the RNase II end product.

RNase II is a key exoribonuclease involved in the maturation, turnover, and quality control of RNA. RNase II homologues are components of the exosome, a complex of exoribonucleases. The structure of RNase II unraveled crucial aspects of the mechanism of RNA degradation. Here we show that mutations in highly conserved residues at the active site affect the activity of the enzyme. Moreover, we have identified the residue that is responsible for setting the end product of RNase II. In addition, we present for the first time the models of two members of the RNase II family, RNase R from Escherichia coli and human Rrp44, also called Dis3. Our findings improve the present model for RNA degradation by the RNase II family of enzymes.

The role of the S1 domain in exoribonucleolytic activity: substrate specificity and multimerization.

RNase II is a 3'-5' exoribonuclease that processively hydrolyzes single-stranded RNA generating 5' mononucleotides. This enzyme contains a catalytic core that is surrounded by three RNA-binding domains. At its C terminus, there is a typical S1 domain that has been shown to be critical for RNA binding. The S1 domain is also present in the other major 3'-5' exoribonucleases from Escherichia coli: RNase R and polynucleotide phosphorylase (PNPase). In this report, we examined the involvement of the S1 domain in the different abilities of these three enzymes to overcome RNA secondary structures during degradation. Hybrid proteins were constructed by replacing the S1 domain of RNase II for the S1 from RNase R and PNPase, and their exonucleolytic activity and RNA-binding ability were examined. The results revealed that both the S1 domains of RNase R and PNPase are able to partially reverse the drop of RNA-binding ability and exonucleolytic activity resulting from removal of the S1 domain of RNase II. Moreover, the S1 domains investigated are not equivalent. Furthermore, we demonstrate that S1 is neither responsible for the ability to overcome secondary structures during RNA degradation, nor is it related to the size of the final product generated by each enzyme. In addition, we show that the S1 domain from PNPase is able to induce the trimerization of the RNaseII-PNP hybrid protein, indicating that this domain can have a role in the biogenesis of multimers.