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Papers by Anna Brandi

Research paper thumbnail of Transcriptional and post-transcriptional events trigger de novo infB expression in cold stressed Escherichia coli

Nucleic Acids Research, Mar 27, 2019

After a 37 to 10 • C temperature downshift the level of translation initiation factor IF2, like t... more After a 37 to 10 • C temperature downshift the level of translation initiation factor IF2, like that of IF1 and IF3, increases at least 3-fold with respect to the ribosomes. To clarify the mechanisms and conditions leading to cold-stress induction of infB expression, the consequences of this temperature shift on infB (IF2) transcription, infB mRNA stability and translation were analysed. The Escherichia coli gene encoding IF2 is part of the metY-nusA-infB operon that contains three known promoters (P-1, P0 and P2) in addition to two promoters P3 and P4 identified in this study, the latter committed to the synthesis of a monocistronic mRNA encoding exclusively IF2. The results obtained indicate that the increased level of IF2 following cold stress depends on three mechanisms: (i) activation of all the promoters of the operon, P-1 being the most cold-responsive, as a likely consequence of the reduction of the ppGpp level that follows cold stress; (ii) a large increase in infB mRNA half-life and (iii) the cold-shock induced translational bias that ensures efficient translation of infB mRNA by the translational apparatus of cold shocked cells. A comparison of the mechanisms responsible for the cold shock induction of the three initiation factors is also presented.

Research paper thumbnail of Criss-cross regulation of the expression of genes encoding nucleoid proteins

Research paper thumbnail of The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending

The EMBO Journal, 1997

resulting in cell death (Spurio et al., 1992). To serve Camerino, 62032 Camerino (MC), Italy as a... more resulting in cell death (Spurio et al., 1992). To serve Camerino, 62032 Camerino (MC), Italy as a control for these studies, with the hope of obtaining a protein molecule inactive in DNA binding, we con-1 Corresponding author structed an hns mutant lacking four in-frame codons (hence the name hnsΔ12) corresponding to the tetrapeptide Escherichia coli hns, encoding the abundant nucleoid protein H-NS, was subjected to site-directed muta-G112-R113-T114-P115, which is close to the conserved Trp108 residue involved in protein-DNA interaction genesis either to delete Pro115 or to replace it with alanine. Unlike the wild-type protein, hyperproduction (Friedrich et al., 1988; Tippner and Wagner, 1995). Indeed, overproduction of this protein proved to be non-toxic and of the mutant proteins did not inhibit macromolecular syntheses, was not toxic to cells and caused a less did not perturb the above-mentioned macromolecular synthesis. Nevertheless, hyperproduction of the mutant drastic compaction of the nucleoid. Gel shift and ligasemediated circularization tests demonstrated that the protein resulted in a less pronounced, yet clearly detectable, condensation of the nucleoid and the resulting protein mutant proteins retained almost normal affinity for non-curved DNA, but lost the wild-type capacity to could be retained by matrix-bound DNA up to a fairly high concentration of NaCl. These findings suggested that, recognize preferentially curved DNA and to actively bend non-curved DNA, a property of wild-type H-NS although somewhat different from that of the wild-type, the DNA binding capacity of the mutant protein was not, demonstrated here for the first time. DNase I footprinting and in vitro transcription experiments showed or at least not entirely, lost (Spurio et al., 1992) and prompted us to pursue further the study of H-NSΔ12 and that the mutant proteins also failed to recognize the intrinsically bent site of the hns promoter required of similar mutants with the hope of shedding light on the mechanism by which H-NS specifically recognizes for H-NS transcription autorepression and to inhibit transcription from the same promoter. The failure of curved DNA. In this article we present data indicating that, in addition the Pro115 mutant proteins to recognize curved DNA and to bend DNA despite their near normal affinity to recognizing curved DNA, wild-type H-NS can also induce curvature in non-bent DNA and that both functions for non-curved DNA can be attributed to a defect in protein-protein interaction resulting in a reduced are lost in H-NSΔ12 and in mutants in which Pro115 is either deleted or replaced by alanine. These mutants were capacity to form oligomers observed in vitro and by a new in vivo test based on functional replacement by shown to have preserved an intact basal DNA binding activity but to have an impaired capacity to oligomerize H-NS of the oligomerization domain (C-domain) of bacteriophage λ cI repressor. in vitro and in vivo. Our results lead to the conclusion that the recognition of curved DNA and the bending of Keywords: curved DNA/DNA bending/DNA binding proteins/protein-protein interaction in vivo/ non-curved DNA depend on the quaternary structure of H-NS. transcriptional repression Results

Research paper thumbnail of Massive presence of the Escherichia coli 'major cold-shock protein' CspA under non-stress conditions

The EMBO Journal, 1999

The most characteristic event of cold-shock activation in Escherichia coli is believed to be the ... more The most characteristic event of cold-shock activation in Escherichia coli is believed to be the de novo synthesis of CspA. We demonstrate, however, that the cellular concentration of this protein is ജ ജ50 μM during early exponential growth at 37°C; therefore, its designation as a major cold-shock protein is a misnomer. The cspA mRNA level decreases rapidly with increasing cell density, becoming virtually undetectable by mid-tolate exponential growth phase while the CspA level declines, although always remaining clearly detectable. A burst of cspA expression followed by a renewed decline ensues upon dilution of stationary phase cultures with fresh medium. The extent of cold-shock induction of cspA varies as a function of the growth phase, being inversely proportional to the pre-existing level of CspA which suggests feedback autorepression by this protein. Both transcriptional and post-transcriptional controls regulate cspA expression under non-stress conditions; transcription of cspA mRNA is under the antagonistic control of DNA-binding proteins Fis and H-NS both in vivo and in vitro, while its decreased half-life with increasing cell density contributes to its rapid disappearance. The cspA mRNA instability is due to its 5Ј untranslated leader and is counteracted in vivo by the cold-shock DeaD box RNA helicase (CsdA).

Research paper thumbnail of Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS

Proceedings of the National Academy of Sciences, 1991

The hns (27 min) gene encoding the 15.4-kDa nucleoid protein H-NS was shown to belong to the cold... more The hns (27 min) gene encoding the 15.4-kDa nucleoid protein H-NS was shown to belong to the cold shock regulon of Escherichia coli, its expression being enhanced 3- to 4-fold during the growth lag that follows a shift from 37 degrees C to 10 degrees C. A 110-base-pair (bp) DNA fragment containing the promoter of hns fused to a promoterless cat gene (hns-cat fusion) conferred a similar cold shock response to the expression of chloramphenicol acetyltransferase (CAT) activity in vivo and in coupled transcription-translation systems prepared with extracts of cold-shocked cells. Extracts of the same cells produce a specific gel shift of the 110-bp DNA fragment and this fragment, immobilized on a solid support, specifically retains a single 7-kDa protein present only in cold-shocked cells that was found to be identical to F10.6 (CS7.4), the product of cspA. This purified protein, which is homologous to human DNA-binding protein YB-1, recognizes some feature of the 110-bp promoter region ...

Research paper thumbnail of A novel antisense RNA regulates at transcriptional level the virulence gene icsA of Shigella flexneri

Nucleic Acids Research, 2010

The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host coloniz... more The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host colonization by pathogenic bacteria. Within the intergenic region virA-icsA, we have discovered a new gene that encodes a non-translated antisense RNA (named RnaG), transcribed in cis on the complementary strand of icsA. In vitro transcription assays show that RnaG promotes premature termination of transcription of icsA mRNA. Transcriptional inhibition is also observed in vivo by monitoring the expression profile in Shigella by real-time polymerase chain reaction and when RnaG is provided in trans. Chemical and enzymatic probing of the leader region of icsA mRNA either free or bound to RnaG indicate that upon hetero-duplex formation an intrinsic terminator, leading to transcription block, is generated on the nascent icsA mRNA. Mutations in the hairpin structure of the proposed terminator impair the RnaG mediated-regulation of icsA transcription. This study represents the first evidence of transcriptional attenuation mechanism caused by a small RNA in Gram-negative bacteria. We also present data on the secondary structure of the antisense region of RnaG. In addition, alternatively silencing icsA and RnaG promoters, we find that transcription from the strong RnaG promoter reduces the activity of the weak convergent icsA promoter through the transcriptional interference regulation.

Research paper thumbnail of A multifactor regulatory circuit involving H-NS, VirF and an antisense RNA modulates transcription of the virulence gene icsA of Shigella flexneri

Nucleic Acids Research, 2011

The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal... more The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal mucosa by bacteria. This gene is expressed upon invasion of the host and is controlled by a complex regulatory circuit involving the nucleoid protein H-NS, the AraC-like transcriptional activator VirF, and a 450 nt antisense RNA (RnaG) acting as transcriptional attenuator. We investigated on the interplay of these factors at the molecular level. DNase I footprints reveal that both H-NS and VirF bind to a region including the icsA and RnaG promoters. H-NS is shown to repress icsA transcription at 30 C but not at 37 C, suggesting a significant involvement of this protein in the temperatureregulated expression of icsA. We also demonstrate that VirF directly stimulates icsA transcription and is able to alleviate H-NS repression in vitro. According to these results, icsA expression is derepressed in hns-background and overexpressed when VirF is provided in trans. Moreover, we find that RnaG-mediated transcription attenuation depends on 80 nt at its 5 0-end, a stretch carrying the antisense region. Bases engaged in the initial contact leading to sense-antisense pairing have been identified using synthetic RNA and DNA oligonucleotides designed to rebuild and mutagenize the two stem-loop motifs of the antisense region.

Research paper thumbnail of Post-transcriptional regulation of CspA expression in Escherichia coli

Molecular Microbiology, 1996

The Escherichia coli cspA gene, encoding the major cold-shock protein CspA, was deprived of its n... more The Escherichia coli cspA gene, encoding the major cold-shock protein CspA, was deprived of its natural promoter and placed in an expression vector under the control of the inducible k PL promoter. After induc-tion of transcription by thermal inactivation of the k ts repressor, ...

Research paper thumbnail of Reinitiation of protein synthesis in Escherichia coli can be induced by mRNA cis -elements unrelated to canonical translation initiation signals

FEBS Letters, 2000

In Eubacteria, de novo translation of some internal cistrons may be inefficient or impossible unl... more In Eubacteria, de novo translation of some internal cistrons may be inefficient or impossible unless the 5P P neighboring cistron is also translated (translational coupling). Translation reinitiation is an extreme case of translational coupling in which translation of a message depends entirely on the presence of a nearby terminating ribosome. In this work, the characteristics of mRNA cis-elements inducing the reinitiation process in Escherichia coli have been investigated using a combinatorial approach. A number of novel translational reinitiation sequences (TRSs) were thus identified, which show a wide range of reinitiation activities fully dependent on a translational coupling event and unrelated to the presence/absence of secondary structure or mRNA stability. Moreover, some of the isolated TRSs are similar to intercistronic sequences present in the E. coli genome.

Research paper thumbnail of Cold-stress-induced de novo expression of infC and role of IF3 in cold-shock translational bias

RNA, 2007

Expression of Escherichia coli infC, which encodes translation initiation factor IF3 and belongs ... more Expression of Escherichia coli infC, which encodes translation initiation factor IF3 and belongs to a transcriptional unit containing several promoters and terminators, is enhanced after cold shock, causing a transient increase of the IF3/ribosomes ratio. Here we show that after cold shock the two less used promoters (PT and PI1) remain active and/or are activated, resulting in de novo infC transcription and IF3 synthesis. These two events are partly responsible for the stoichiometric imbalance of the IF3/ribosomes ratio that contributes to establishing the cold-shock translational bias whereby cold-shock mRNAs are preferentially translated by cold-stressed cells while bulk mRNAs are discriminated against. Analysis of the IF3 functions at low temperature sheds light on the molecular mechanism by which IF3 contributes to the cold-shock translational bias. IF3 was found to cause a strong rate increase of fMet-tRNA binding to ribosomes programmed with cold-shock mRNA, an activity essen...

Research paper thumbnail of Preferential translation of cold-shock mRNAs during cold adaptation

RNA, 2004

Upon temperature downshift below the lower threshold of balanced growth (~20°C), the Escherichia ... more Upon temperature downshift below the lower threshold of balanced growth (~20°C), the Escherichia coli translational apparatus undergoes modifications allowing the selective translation of the transcripts of cold shock-induced genes, while bulk protein synthesis is drastically reduced. Here we were able to reproduce this translational bias in E. coli cell-free extracts prepared at various times during cold adaptation which were found to display different capacities to translate different types of mRNAs as a function of temperature. Several causes were found to contribute to the cold-shock translational bias: Cold-shock mRNAs contain cis-elements, making them intrinsically more prone to being translated in the cold, and they are selective targets for trans-acting factors present in increased amounts in the translational apparatus of cold-shocked cells. CspA was found to be among these trans-acting factors. In addition to inducing a higher level of CspA, cold shock was found to cause a...

Research paper thumbnail of Translation initiation factor IF2 contributes to ribosome assembly and maturation during cold adaptation

Research paper thumbnail of The Sub-Chromosomic Macronuclear Pheromone Genes of the Ciliate Euplotes raikovi: Comparative Structural Analysis and Insights into the Mechanism of Expression

The Journal of eukaryotic microbiology, Jan 4, 2018

In Euplotes raikovi, we have determined the full-length sequences of a family of macronuclear gen... more In Euplotes raikovi, we have determined the full-length sequences of a family of macronuclear genes that are the transcriptionally active versions of codominant alleles inherited at the mating-type (mat) locus of the micronuclear genome, and encode cell type-distinctive signaling pheromones. These genes include a 225 231-bp coding region flanked by a conserved 544-bp 5'-leader region and a more variable 3'-trailer region. Two transcription initiation start sites and two polyadenylation sites associated with non-conventional signals cooperate with a splicing phenomenon of a 326-bp intron residing in the 5'-leader region in the generation of multiple transcripts from the same gene. In two of them, the synthesis of functional products depends on the reassignment to a sense codon, or readthrough of a strictly conserved leaky UAG stop codon. That this reassignment may take place is suggested by the position this codon occupies in the transcripts, close to the transcript extre...

Research paper thumbnail of Screening an Archetypal Collection of Microorganisms for the Presence of Unexplored Antimicrobial Compounds

BioDiscovery, 2017

Background WHO estimates that more than 700.000 people die every year as a result of drug-resista... more Background WHO estimates that more than 700.000 people die every year as a result of drug-resistant infections. To tackle this problem and change the current trend, it is necessary to design advanced strategies for drug discovery and to promote early-stage research activities finalized at the development of new drugs (World Health Organization 2015). New information In this study, we present the results of the preliminary screening of a library of microorganisms, collected from different environmental settings. Approximately 300 strains of the culture collection were tested on solid medium for inhibition of growth of three tester ‡ ‡ ‡ § ‡ ‡

Research paper thumbnail of De novo Synthesis and Assembly of rRNA into Ribosomal Subunits during Cold Acclimation in Escherichia coli

Journal of Molecular Biology, 2016

During the cold adaptation that follows a cold stress, bacterial cells undergo many physiological... more During the cold adaptation that follows a cold stress, bacterial cells undergo many physiological changes and extensive reprogramming of their gene expression pattern. Bulk gene expression is drastically reduced, while a set of cold shock genes is selectively and transiently expressed. The initial stage of cold acclimation is characterized by the establishment of a stoichiometric imbalance of the translation initiation factors (IFs)/ribosomes ratio that contributes to the preferential translation of cold shock transcripts. Whereas de novo synthesis of the IFs following cold stress has been documented, nothing was known concerning the activity of the rrn operons during the cold acclimation period. In this work, we focus on the expression of the rrn operons and the fate of rRNA after temperature downshift. We demonstrate that in Escherichia coli, rRNA synthesis does not stop during the cold acclimation phase, but continues with greater contribution of the P2 compared to the P1 promoter and all seven rrn operons are active, although their expression levels change with respect to pre-stress conditions. Eight hours after the 37°→10 °C temperature downshift, the newly transcribed rRNA represents up to 20% of total rRNA and is preferentially found in the polysomes. However, with respect to the de novo synthesis of the IFs, both rRNA transcription and maturation are slowed down drastically by cold stress, thereby accounting in part for the stoichiometric imbalance of the IFs/ribosomes. Overall, our data indicate that new ribosomes, which are possibly suitable to function at low temperature, are slowly assembled during cold acclimation.

Research paper thumbnail of Translation initiation at the root of the cold-shock translational bias

Research paper thumbnail of Molecular Tools for the Selective Detection of Nine Diatom Species Biomarkers of Various Water Quality Levels

International Journal of Environmental Research and Public Health, 2015

Our understanding of the composition of diatom communities and their response to environmental ch... more Our understanding of the composition of diatom communities and their response to environmental changes is currently limited by laborious taxonomic identification procedures. Advances in molecular technologies are expected to contribute more efficient, robust and sensitive tools for the detection of these ecologically relevant microorganisms. There is a need to explore and test phylogenetic markers as an alternative to the use of rRNA genes, whose limited sequence divergence does not allow the accurate discrimination of diatoms at the species level. In this work, nine diatom species belonging to eight genera, isolated from epylithic environmental samples collected in central Italy, were chosen to implement a panel of diatoms covering the full range of ecological status of freshwaters. The procedure described in this work relies on the PCR amplification of specific regions in two conserved diatom genes, elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), as a first step to narrow down the complexity of the targets, followed by microarray hybridization experiments. Oligonucleotide probes with the potential to discriminate closely related species were designed taking into account the genetic

Research paper thumbnail of Translation initiation at the root of the cold-shock translational bias

Research paper thumbnail of Antagonistic involvement of FIS and H-NS proteins in the transcriptional control of hns expression

Molecular Microbiology, 1996

Gel shift and DNase I footprinting experiments showed that Escherichia coli FIS (factor for inver... more Gel shift and DNase I footprinting experiments showed that Escherichia coli FIS (factor for inversion stimulation) protein binds to at least seven sites in the promoter region of hns. These sites extend from -282 to +25 with two sites, closely flanking the DNA bend located at -150 from the transcriptional startpoint, partly overlapping the H-NS binding sites involved in the transcriptional autorepression of hns. The interplay between FIS, H-NS and the hns promoter region were studied by examining the effects of FIS and H-NS on in vitro transcription of hns-cat fusions, as well as looking at the effect of FIS on preformed complexes containing H-NS and a DNA fragment derived from the hns promoter region. Taken together, our data suggest that in the cell, FIS and H-NS interact with the promoter region of hns and influence their respective interactions (possibly competing for the same binding site), eliciting antagonistic effects so that an interplay between these proteins might contribute to the transcriptional control of hns.

Research paper thumbnail of Cold-shock-induced de novo transcription and translation of infA and role of IF1 during cold adaptation

Molecular Microbiology, 2007

Escherichia coli infA is transcribed from two promoters, P1 and P2, into a longer and a shorter m... more Escherichia coli infA is transcribed from two promoters, P1 and P2, into a longer and a shorter mRNA encoding translation initiation factor IF1. Although P1 is intrinsically stronger than P2, the shorter half-life of its transcripts causes the steady-state level of the P2 transcript to be substantially higher than that of P1 during growth at 37°C. After cold-shock, de novo transcription and translation of infA contribute to the transient increase of the IF1/ribosomes ratio, which is partially responsible for translational bias consisting in the preferential translation of cold-shock mRNAs in the cold. Cold-stress induction of infA expression is mainly due to the high activity of P1 at low temperature, which is further increased by transcriptional stimulation by CspA and by an increased transcript stability. Furthermore, the longer infA mRNA originating from P1 is preferentially translated at low temperature by the translational machinery of coldshocked cells. The increased level of IF1 during cold adaptation is essential for overcoming the higher stability of the 70S monomers at low temperature and for providing a sufficient pool of dissociated 30S subunits capable of initiating translation.

Research paper thumbnail of Transcriptional and post-transcriptional events trigger de novo infB expression in cold stressed Escherichia coli

Nucleic Acids Research, Mar 27, 2019

After a 37 to 10 • C temperature downshift the level of translation initiation factor IF2, like t... more After a 37 to 10 • C temperature downshift the level of translation initiation factor IF2, like that of IF1 and IF3, increases at least 3-fold with respect to the ribosomes. To clarify the mechanisms and conditions leading to cold-stress induction of infB expression, the consequences of this temperature shift on infB (IF2) transcription, infB mRNA stability and translation were analysed. The Escherichia coli gene encoding IF2 is part of the metY-nusA-infB operon that contains three known promoters (P-1, P0 and P2) in addition to two promoters P3 and P4 identified in this study, the latter committed to the synthesis of a monocistronic mRNA encoding exclusively IF2. The results obtained indicate that the increased level of IF2 following cold stress depends on three mechanisms: (i) activation of all the promoters of the operon, P-1 being the most cold-responsive, as a likely consequence of the reduction of the ppGpp level that follows cold stress; (ii) a large increase in infB mRNA half-life and (iii) the cold-shock induced translational bias that ensures efficient translation of infB mRNA by the translational apparatus of cold shocked cells. A comparison of the mechanisms responsible for the cold shock induction of the three initiation factors is also presented.

Research paper thumbnail of Criss-cross regulation of the expression of genes encoding nucleoid proteins

Research paper thumbnail of The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending

The EMBO Journal, 1997

resulting in cell death (Spurio et al., 1992). To serve Camerino, 62032 Camerino (MC), Italy as a... more resulting in cell death (Spurio et al., 1992). To serve Camerino, 62032 Camerino (MC), Italy as a control for these studies, with the hope of obtaining a protein molecule inactive in DNA binding, we con-1 Corresponding author structed an hns mutant lacking four in-frame codons (hence the name hnsΔ12) corresponding to the tetrapeptide Escherichia coli hns, encoding the abundant nucleoid protein H-NS, was subjected to site-directed muta-G112-R113-T114-P115, which is close to the conserved Trp108 residue involved in protein-DNA interaction genesis either to delete Pro115 or to replace it with alanine. Unlike the wild-type protein, hyperproduction (Friedrich et al., 1988; Tippner and Wagner, 1995). Indeed, overproduction of this protein proved to be non-toxic and of the mutant proteins did not inhibit macromolecular syntheses, was not toxic to cells and caused a less did not perturb the above-mentioned macromolecular synthesis. Nevertheless, hyperproduction of the mutant drastic compaction of the nucleoid. Gel shift and ligasemediated circularization tests demonstrated that the protein resulted in a less pronounced, yet clearly detectable, condensation of the nucleoid and the resulting protein mutant proteins retained almost normal affinity for non-curved DNA, but lost the wild-type capacity to could be retained by matrix-bound DNA up to a fairly high concentration of NaCl. These findings suggested that, recognize preferentially curved DNA and to actively bend non-curved DNA, a property of wild-type H-NS although somewhat different from that of the wild-type, the DNA binding capacity of the mutant protein was not, demonstrated here for the first time. DNase I footprinting and in vitro transcription experiments showed or at least not entirely, lost (Spurio et al., 1992) and prompted us to pursue further the study of H-NSΔ12 and that the mutant proteins also failed to recognize the intrinsically bent site of the hns promoter required of similar mutants with the hope of shedding light on the mechanism by which H-NS specifically recognizes for H-NS transcription autorepression and to inhibit transcription from the same promoter. The failure of curved DNA. In this article we present data indicating that, in addition the Pro115 mutant proteins to recognize curved DNA and to bend DNA despite their near normal affinity to recognizing curved DNA, wild-type H-NS can also induce curvature in non-bent DNA and that both functions for non-curved DNA can be attributed to a defect in protein-protein interaction resulting in a reduced are lost in H-NSΔ12 and in mutants in which Pro115 is either deleted or replaced by alanine. These mutants were capacity to form oligomers observed in vitro and by a new in vivo test based on functional replacement by shown to have preserved an intact basal DNA binding activity but to have an impaired capacity to oligomerize H-NS of the oligomerization domain (C-domain) of bacteriophage λ cI repressor. in vitro and in vivo. Our results lead to the conclusion that the recognition of curved DNA and the bending of Keywords: curved DNA/DNA bending/DNA binding proteins/protein-protein interaction in vivo/ non-curved DNA depend on the quaternary structure of H-NS. transcriptional repression Results

Research paper thumbnail of Massive presence of the Escherichia coli 'major cold-shock protein' CspA under non-stress conditions

The EMBO Journal, 1999

The most characteristic event of cold-shock activation in Escherichia coli is believed to be the ... more The most characteristic event of cold-shock activation in Escherichia coli is believed to be the de novo synthesis of CspA. We demonstrate, however, that the cellular concentration of this protein is ജ ജ50 μM during early exponential growth at 37°C; therefore, its designation as a major cold-shock protein is a misnomer. The cspA mRNA level decreases rapidly with increasing cell density, becoming virtually undetectable by mid-tolate exponential growth phase while the CspA level declines, although always remaining clearly detectable. A burst of cspA expression followed by a renewed decline ensues upon dilution of stationary phase cultures with fresh medium. The extent of cold-shock induction of cspA varies as a function of the growth phase, being inversely proportional to the pre-existing level of CspA which suggests feedback autorepression by this protein. Both transcriptional and post-transcriptional controls regulate cspA expression under non-stress conditions; transcription of cspA mRNA is under the antagonistic control of DNA-binding proteins Fis and H-NS both in vivo and in vitro, while its decreased half-life with increasing cell density contributes to its rapid disappearance. The cspA mRNA instability is due to its 5Ј untranslated leader and is counteracted in vivo by the cold-shock DeaD box RNA helicase (CsdA).

Research paper thumbnail of Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS

Proceedings of the National Academy of Sciences, 1991

The hns (27 min) gene encoding the 15.4-kDa nucleoid protein H-NS was shown to belong to the cold... more The hns (27 min) gene encoding the 15.4-kDa nucleoid protein H-NS was shown to belong to the cold shock regulon of Escherichia coli, its expression being enhanced 3- to 4-fold during the growth lag that follows a shift from 37 degrees C to 10 degrees C. A 110-base-pair (bp) DNA fragment containing the promoter of hns fused to a promoterless cat gene (hns-cat fusion) conferred a similar cold shock response to the expression of chloramphenicol acetyltransferase (CAT) activity in vivo and in coupled transcription-translation systems prepared with extracts of cold-shocked cells. Extracts of the same cells produce a specific gel shift of the 110-bp DNA fragment and this fragment, immobilized on a solid support, specifically retains a single 7-kDa protein present only in cold-shocked cells that was found to be identical to F10.6 (CS7.4), the product of cspA. This purified protein, which is homologous to human DNA-binding protein YB-1, recognizes some feature of the 110-bp promoter region ...

Research paper thumbnail of A novel antisense RNA regulates at transcriptional level the virulence gene icsA of Shigella flexneri

Nucleic Acids Research, 2010

The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host coloniz... more The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host colonization by pathogenic bacteria. Within the intergenic region virA-icsA, we have discovered a new gene that encodes a non-translated antisense RNA (named RnaG), transcribed in cis on the complementary strand of icsA. In vitro transcription assays show that RnaG promotes premature termination of transcription of icsA mRNA. Transcriptional inhibition is also observed in vivo by monitoring the expression profile in Shigella by real-time polymerase chain reaction and when RnaG is provided in trans. Chemical and enzymatic probing of the leader region of icsA mRNA either free or bound to RnaG indicate that upon hetero-duplex formation an intrinsic terminator, leading to transcription block, is generated on the nascent icsA mRNA. Mutations in the hairpin structure of the proposed terminator impair the RnaG mediated-regulation of icsA transcription. This study represents the first evidence of transcriptional attenuation mechanism caused by a small RNA in Gram-negative bacteria. We also present data on the secondary structure of the antisense region of RnaG. In addition, alternatively silencing icsA and RnaG promoters, we find that transcription from the strong RnaG promoter reduces the activity of the weak convergent icsA promoter through the transcriptional interference regulation.

Research paper thumbnail of A multifactor regulatory circuit involving H-NS, VirF and an antisense RNA modulates transcription of the virulence gene icsA of Shigella flexneri

Nucleic Acids Research, 2011

The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal... more The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal mucosa by bacteria. This gene is expressed upon invasion of the host and is controlled by a complex regulatory circuit involving the nucleoid protein H-NS, the AraC-like transcriptional activator VirF, and a 450 nt antisense RNA (RnaG) acting as transcriptional attenuator. We investigated on the interplay of these factors at the molecular level. DNase I footprints reveal that both H-NS and VirF bind to a region including the icsA and RnaG promoters. H-NS is shown to repress icsA transcription at 30 C but not at 37 C, suggesting a significant involvement of this protein in the temperatureregulated expression of icsA. We also demonstrate that VirF directly stimulates icsA transcription and is able to alleviate H-NS repression in vitro. According to these results, icsA expression is derepressed in hns-background and overexpressed when VirF is provided in trans. Moreover, we find that RnaG-mediated transcription attenuation depends on 80 nt at its 5 0-end, a stretch carrying the antisense region. Bases engaged in the initial contact leading to sense-antisense pairing have been identified using synthetic RNA and DNA oligonucleotides designed to rebuild and mutagenize the two stem-loop motifs of the antisense region.

Research paper thumbnail of Post-transcriptional regulation of CspA expression in Escherichia coli

Molecular Microbiology, 1996

The Escherichia coli cspA gene, encoding the major cold-shock protein CspA, was deprived of its n... more The Escherichia coli cspA gene, encoding the major cold-shock protein CspA, was deprived of its natural promoter and placed in an expression vector under the control of the inducible k PL promoter. After induc-tion of transcription by thermal inactivation of the k ts repressor, ...

Research paper thumbnail of Reinitiation of protein synthesis in Escherichia coli can be induced by mRNA cis -elements unrelated to canonical translation initiation signals

FEBS Letters, 2000

In Eubacteria, de novo translation of some internal cistrons may be inefficient or impossible unl... more In Eubacteria, de novo translation of some internal cistrons may be inefficient or impossible unless the 5P P neighboring cistron is also translated (translational coupling). Translation reinitiation is an extreme case of translational coupling in which translation of a message depends entirely on the presence of a nearby terminating ribosome. In this work, the characteristics of mRNA cis-elements inducing the reinitiation process in Escherichia coli have been investigated using a combinatorial approach. A number of novel translational reinitiation sequences (TRSs) were thus identified, which show a wide range of reinitiation activities fully dependent on a translational coupling event and unrelated to the presence/absence of secondary structure or mRNA stability. Moreover, some of the isolated TRSs are similar to intercistronic sequences present in the E. coli genome.

Research paper thumbnail of Cold-stress-induced de novo expression of infC and role of IF3 in cold-shock translational bias

RNA, 2007

Expression of Escherichia coli infC, which encodes translation initiation factor IF3 and belongs ... more Expression of Escherichia coli infC, which encodes translation initiation factor IF3 and belongs to a transcriptional unit containing several promoters and terminators, is enhanced after cold shock, causing a transient increase of the IF3/ribosomes ratio. Here we show that after cold shock the two less used promoters (PT and PI1) remain active and/or are activated, resulting in de novo infC transcription and IF3 synthesis. These two events are partly responsible for the stoichiometric imbalance of the IF3/ribosomes ratio that contributes to establishing the cold-shock translational bias whereby cold-shock mRNAs are preferentially translated by cold-stressed cells while bulk mRNAs are discriminated against. Analysis of the IF3 functions at low temperature sheds light on the molecular mechanism by which IF3 contributes to the cold-shock translational bias. IF3 was found to cause a strong rate increase of fMet-tRNA binding to ribosomes programmed with cold-shock mRNA, an activity essen...

Research paper thumbnail of Preferential translation of cold-shock mRNAs during cold adaptation

RNA, 2004

Upon temperature downshift below the lower threshold of balanced growth (~20°C), the Escherichia ... more Upon temperature downshift below the lower threshold of balanced growth (~20°C), the Escherichia coli translational apparatus undergoes modifications allowing the selective translation of the transcripts of cold shock-induced genes, while bulk protein synthesis is drastically reduced. Here we were able to reproduce this translational bias in E. coli cell-free extracts prepared at various times during cold adaptation which were found to display different capacities to translate different types of mRNAs as a function of temperature. Several causes were found to contribute to the cold-shock translational bias: Cold-shock mRNAs contain cis-elements, making them intrinsically more prone to being translated in the cold, and they are selective targets for trans-acting factors present in increased amounts in the translational apparatus of cold-shocked cells. CspA was found to be among these trans-acting factors. In addition to inducing a higher level of CspA, cold shock was found to cause a...

Research paper thumbnail of Translation initiation factor IF2 contributes to ribosome assembly and maturation during cold adaptation

Research paper thumbnail of The Sub-Chromosomic Macronuclear Pheromone Genes of the Ciliate Euplotes raikovi: Comparative Structural Analysis and Insights into the Mechanism of Expression

The Journal of eukaryotic microbiology, Jan 4, 2018

In Euplotes raikovi, we have determined the full-length sequences of a family of macronuclear gen... more In Euplotes raikovi, we have determined the full-length sequences of a family of macronuclear genes that are the transcriptionally active versions of codominant alleles inherited at the mating-type (mat) locus of the micronuclear genome, and encode cell type-distinctive signaling pheromones. These genes include a 225 231-bp coding region flanked by a conserved 544-bp 5'-leader region and a more variable 3'-trailer region. Two transcription initiation start sites and two polyadenylation sites associated with non-conventional signals cooperate with a splicing phenomenon of a 326-bp intron residing in the 5'-leader region in the generation of multiple transcripts from the same gene. In two of them, the synthesis of functional products depends on the reassignment to a sense codon, or readthrough of a strictly conserved leaky UAG stop codon. That this reassignment may take place is suggested by the position this codon occupies in the transcripts, close to the transcript extre...

Research paper thumbnail of Screening an Archetypal Collection of Microorganisms for the Presence of Unexplored Antimicrobial Compounds

BioDiscovery, 2017

Background WHO estimates that more than 700.000 people die every year as a result of drug-resista... more Background WHO estimates that more than 700.000 people die every year as a result of drug-resistant infections. To tackle this problem and change the current trend, it is necessary to design advanced strategies for drug discovery and to promote early-stage research activities finalized at the development of new drugs (World Health Organization 2015). New information In this study, we present the results of the preliminary screening of a library of microorganisms, collected from different environmental settings. Approximately 300 strains of the culture collection were tested on solid medium for inhibition of growth of three tester ‡ ‡ ‡ § ‡ ‡

Research paper thumbnail of De novo Synthesis and Assembly of rRNA into Ribosomal Subunits during Cold Acclimation in Escherichia coli

Journal of Molecular Biology, 2016

During the cold adaptation that follows a cold stress, bacterial cells undergo many physiological... more During the cold adaptation that follows a cold stress, bacterial cells undergo many physiological changes and extensive reprogramming of their gene expression pattern. Bulk gene expression is drastically reduced, while a set of cold shock genes is selectively and transiently expressed. The initial stage of cold acclimation is characterized by the establishment of a stoichiometric imbalance of the translation initiation factors (IFs)/ribosomes ratio that contributes to the preferential translation of cold shock transcripts. Whereas de novo synthesis of the IFs following cold stress has been documented, nothing was known concerning the activity of the rrn operons during the cold acclimation period. In this work, we focus on the expression of the rrn operons and the fate of rRNA after temperature downshift. We demonstrate that in Escherichia coli, rRNA synthesis does not stop during the cold acclimation phase, but continues with greater contribution of the P2 compared to the P1 promoter and all seven rrn operons are active, although their expression levels change with respect to pre-stress conditions. Eight hours after the 37°→10 °C temperature downshift, the newly transcribed rRNA represents up to 20% of total rRNA and is preferentially found in the polysomes. However, with respect to the de novo synthesis of the IFs, both rRNA transcription and maturation are slowed down drastically by cold stress, thereby accounting in part for the stoichiometric imbalance of the IFs/ribosomes. Overall, our data indicate that new ribosomes, which are possibly suitable to function at low temperature, are slowly assembled during cold acclimation.

Research paper thumbnail of Translation initiation at the root of the cold-shock translational bias

Research paper thumbnail of Molecular Tools for the Selective Detection of Nine Diatom Species Biomarkers of Various Water Quality Levels

International Journal of Environmental Research and Public Health, 2015

Our understanding of the composition of diatom communities and their response to environmental ch... more Our understanding of the composition of diatom communities and their response to environmental changes is currently limited by laborious taxonomic identification procedures. Advances in molecular technologies are expected to contribute more efficient, robust and sensitive tools for the detection of these ecologically relevant microorganisms. There is a need to explore and test phylogenetic markers as an alternative to the use of rRNA genes, whose limited sequence divergence does not allow the accurate discrimination of diatoms at the species level. In this work, nine diatom species belonging to eight genera, isolated from epylithic environmental samples collected in central Italy, were chosen to implement a panel of diatoms covering the full range of ecological status of freshwaters. The procedure described in this work relies on the PCR amplification of specific regions in two conserved diatom genes, elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), as a first step to narrow down the complexity of the targets, followed by microarray hybridization experiments. Oligonucleotide probes with the potential to discriminate closely related species were designed taking into account the genetic

Research paper thumbnail of Translation initiation at the root of the cold-shock translational bias

Research paper thumbnail of Antagonistic involvement of FIS and H-NS proteins in the transcriptional control of hns expression

Molecular Microbiology, 1996

Gel shift and DNase I footprinting experiments showed that Escherichia coli FIS (factor for inver... more Gel shift and DNase I footprinting experiments showed that Escherichia coli FIS (factor for inversion stimulation) protein binds to at least seven sites in the promoter region of hns. These sites extend from -282 to +25 with two sites, closely flanking the DNA bend located at -150 from the transcriptional startpoint, partly overlapping the H-NS binding sites involved in the transcriptional autorepression of hns. The interplay between FIS, H-NS and the hns promoter region were studied by examining the effects of FIS and H-NS on in vitro transcription of hns-cat fusions, as well as looking at the effect of FIS on preformed complexes containing H-NS and a DNA fragment derived from the hns promoter region. Taken together, our data suggest that in the cell, FIS and H-NS interact with the promoter region of hns and influence their respective interactions (possibly competing for the same binding site), eliciting antagonistic effects so that an interplay between these proteins might contribute to the transcriptional control of hns.

Research paper thumbnail of Cold-shock-induced de novo transcription and translation of infA and role of IF1 during cold adaptation

Molecular Microbiology, 2007

Escherichia coli infA is transcribed from two promoters, P1 and P2, into a longer and a shorter m... more Escherichia coli infA is transcribed from two promoters, P1 and P2, into a longer and a shorter mRNA encoding translation initiation factor IF1. Although P1 is intrinsically stronger than P2, the shorter half-life of its transcripts causes the steady-state level of the P2 transcript to be substantially higher than that of P1 during growth at 37°C. After cold-shock, de novo transcription and translation of infA contribute to the transient increase of the IF1/ribosomes ratio, which is partially responsible for translational bias consisting in the preferential translation of cold-shock mRNAs in the cold. Cold-stress induction of infA expression is mainly due to the high activity of P1 at low temperature, which is further increased by transcriptional stimulation by CspA and by an increased transcript stability. Furthermore, the longer infA mRNA originating from P1 is preferentially translated at low temperature by the translational machinery of coldshocked cells. The increased level of IF1 during cold adaptation is essential for overcoming the higher stability of the 70S monomers at low temperature and for providing a sufficient pool of dissociated 30S subunits capable of initiating translation.