Efficient and specific gene knockdown by small interfering RNAs produced in bacteria

Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells1,2 and may be used for therapeutic purposes to knockdown genes implicated in disease3. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in E. coli. This method relies on ectopic expression of p19, a siRNA-binding protein found in a plant RNA virus4, 5. When expressed in E. coli, p19 stabilizes ~21 nt siRNA-like species produced by bacterial RNase III. Transfection of mammalian cells with siRNAs, generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene, at low nanomolar concentrations reproducibly knocks down gene expression by ~90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes

Systematic Overexpression of Genes Encoded By Mycobacteriophage Waterfoul Reveals Novel Inhibitors of Mycobacterial Growth

Bacteriophages represent an enormous reservoir of novel genes, many of which are unrelated to existing entries in public databases and cannot be assigned a predicted function. Characterization of these genes can provide important insights into the intricacies of phage–host interactions and may offer new strategies to manipulate bacterial growth and behavior. Overexpression is a useful tool in the study of gene-mediated effects, and we describe here the construction of a plasmid-based overexpression library of a complete set of genes for Waterfoul, a mycobacteriophage closely related to those infecting clinically important strains of Mycobacterium tuberculosis and/or Mycobacterium abscessus. The arrayed Waterfoul gene library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 32 Waterfoul gene products capable of inhibiting the growth of the host Mycobacterium smegmatis and providing a first look at the frequency and distribution of cytotoxic products encoded within a single mycobacteriophage genome. Several of these Waterfoul gene products were observed to confer potent anti-mycobacterial effects, making them interesting candidates for follow-up mechanistic studies

Control of a Programmed Cell Death Pathway in Pseudomonas aeruginosa by an Antiterminator

In Pseudomonas aeruginosa the alp system encodes a programmed cell death pathway that is switched on in a subset of cells in response to DNA damage and is linked to the virulence of the organism. Here we show that the central regulator of this pathway, AlpA, exerts its effects by acting as an antiterminator rather than a transcription activator. In particular, we present evidence that AlpA positively regulates the alpBCDE cell lysis genes, as well as genes in a second newly identified target locus, by recognizing specific DNA sites within the promoter, then binding RNA polymerase directly and allowing it to bypass intrinsic terminators positioned downstream. AlpA thus functions in a mechanistically unusual manner to control the expression of virulence genes in this opportunistic pathogen

StpA and the regulation of OmpF porin expression in Escherichia coli

THESIS 6485When the proteomic profile of a wild-type strain of Escherichia coli and its stpA hns mutant derivative were compared by two-dimensional gel electrophoresis, the levels of expression of several proteins were altered. One of these, which was strongly down-regulated in the absence of the regulatory proteins StpA and H-NS, was identified as the outer membrane porin protein, OmpF. In this study, the molecular details of how StpA and H-NS control OmpF porin expression were examined

Three-way interactions among the Sfh, StpA and H-NS nucleoid-structuring proteins of Shigella flexneri 2a strain 2457T.

International audienceShigella flexneri 2a strain 2457T has been found to express Sfh, a new member of the H-NS-like family of nucleoid-structuring proteins. With H-NS and its paralogue, StpA, this brings to three the number of these proteins expressed in this bacterium. This raises the possibility that three-way interactions may occur in S. flexneri among these proteins and between the proteins and each other's genes. Such three-way interactions among H-NS-like proteins have not been described previously. The expression of the sfh, stpA and hns genes was studied at the transcriptional and post-transcriptional levels. The Sfh protein displays growth phase-dependent regulation that distinguishes it from both H-NS and StpA. Like H-NS and StpA, Sfh can bind to its own promoter region, it negatively autoregulates transcription of its own gene, and when overexpressed all three proteins cross-repress transcription of each other's genes. The presence of highly conserved oligomerization domains within these molecules suggested the possibility of protein-protein interactions. Like H-NS and StpA, the purified Sfh protein forms homodimers in solution. Using the yeast two-hybrid assay we show that each of the three proteins also forms homodimers in vivo and, additionally, each protein can form heterodimers with either of its homologues. This raises the possibility that Sfh may modulate the activities of H-NS and StpA, and vice versa

Characterization of the Detachable Rho-Dependent Transcription Terminator of the fimE Gene in Escherichia coli K-12

The fim genetic switch in the chromosome of Escherichia coli K-12 is an invertible DNA element that harbors the promoter for transcription of the downstream fim structural genes and a transcription terminator that acts on the upstream fimE regulatory gene. Switches oriented appropriately for structural gene transcription also allow fimE mRNA to read through, whereas those in the opposite orientation terminate the fimE message. We show here that termination is Rho dependent and is suppressed in a rho mutant or by bicyclomycin treatment when fimE mRNA is expressed by the fimE gene, either from a multicopy recombinant plasmid or in its native chromosomal location. Two cis-acting elements within the central portion of the 314-bp invertible DNA switch were identified as contributors to Rho-dependent termination and dissected. These fim sequence elements show similarities to well-characterized Rho utilization (rut) sites and consist of a boxA motif and a C-rich and G-poor region of approximately 40 bp. Deletion of the boxA motif alone had only a subtle negative effect on Rho function. However, when this element was deleted in combination with the C-rich, G-poor region, Rho function was considerably decreased. Altering the C-to-G ratio in favor of G in this portion of the switch also strongly attenuated transcription termination. The implications of the existence of a fimE-specific Rho-dependent terminator within the invertible switch are discussed in the context of the fim regulatory circuit

A regulator from Chlamydia trachomatis modulates the activity of RNA polymerase through direct interaction with the β subunit and the primary σ subunit

The obligate intracellular human pathogen Chlamydia trachomatis undergoes a complex developmental program involving transition between two forms: the infectious elementary body (EB), and the rapidly dividing reticulate body (RB). However, the regulators controlling this development have not been identified. To uncover potential regulators of transcription in C. trachomatis, we screened a C. trachomatis genomic library for sequences encoding proteins that interact with RNA polymerase (RNAP). We report the identification of one such protein, CT663, which interacts with the β and σ subunits of RNAP. Specifically, we show that CT663 interacts with the flap domain of the β subunit (β-flap) and conserved region 4 of the primary σ subunit (σ66 in C. trachomatis). We find that CT663 inhibits σ66-dependent (but not σ28-dependent) transcription in vitro, and we present evidence that CT663 exerts this effect as a component of the RNAP holoenzyme. The analysis of C. trachomatis-infected cells reveals that CT663 begins to accumulate at the commencement of the RB-to-EB transition. Our findings suggest that CT663 functions as a negative regulator of σ66-dependent transcription, facilitating a global change in gene expression. The strategy used here is generally applicable in cases where genetic tools are unavailable

Tombusvirus p19 Captures RNase III-Cleaved Double-Stranded RNAs Formed by Overlapping Sense and Antisense Transcripts in Escherichia coli

Antisense transcription is widespread in bacteria. By base pairing with overlapping sense RNAs, antisense RNAs (asRNA) can form double-stranded RNAs (dsRNA), which are cleaved by RNase III, a dsRNA endoribonuclease. The ectopic expression of plant Tombusvirus p19 in Escherichia coli stabilizes ∼21-nucleotide (nt) dsRNA RNase III decay intermediates, which enabled us to characterize otherwise highly unstable asRNA by deep sequencing of p19-captured dsRNA. RNase III-produced small dsRNA were formed at most bacterial genes in the bacterial genome and in a plasmid.Antisense transcription is widespread in bacteria. By base pairing with overlapping sense RNAs, antisense RNAs (asRNA) can form double-stranded RNAs (dsRNA), which are cleaved by RNase III, a dsRNA endoribonuclease. The ectopic expression of plant Tombusvirus p19 in Escherichia coli stabilizes ∼21-nucleotide (nt) dsRNA RNase III decay intermediates, which enabled us to characterize otherwise highly unstable asRNA by deep sequencing of p19-captured dsRNA. RNase III-produced small dsRNA were formed at most bacterial genes in the bacterial genome and in a plasmid. We classified the types of asRNA in genomic clusters producing the most abundant p19-captured dsRNA and confirmed RNase III regulation of asRNA and sense RNA decay at three type I toxin-antitoxin loci and at a coding gene, rsd. Furthermore, we provide potential evidence for the RNase III-dependent regulation of CspD protein by asRNA. The analysis of p19-captured dsRNA revealed an RNase III sequence preference for AU-rich sequences 3 nucleotides on either side of the cleavage sites and for GC-rich sequences in the 2-nt overhangs. Unexpectedly, GC-rich sequences were enriched in the middle section of p19-captured dsRNA, suggesting some unexpected sequence bias in p19 protein binding. Nonetheless, the ectopic expression of p19 is a sensitive method for identifying antisense transcripts and RNase III cleavage sites in dsRNA formed by overlapping sense and antisense transcripts in bacteria