Gianni Dehò

Polynucleotide phosphorylase (PNPase, encoded by pnp) is generally thought of as an enzyme dedicated to RNA metabolism. The pleiotropic effects of PNPase deficiency is imputed to altered processing and turnover of mRNAs and small RNAs, which in turn leads to aberrant gene expression. However, it has long since been known that this enzyme may also catalyze template-independent polymerization of dNDPs into ssDNA and the reverse phosphorolytic reaction. Recently, PNPase has been implicated in DNA recombination, repair, mutagenesis and resistance to genotoxic agents in diverse bacterial species, raising the possibility that PNPase may directly, rather than through control of gene expression, participate in these processes. In this work we present evidence that in Escherichia coli PNPase enhances both homologous recombination upon P1 transduction and error prone DNA repair of double strand breaks induced by zeocin, a radiomimetic agent. Homologous recombination does not require PNPase phosphorolytic activity and is modulated by its RNA binding domains whereas error prone DNA repair of zeocin-induced DNA damage is dependent on PNPase catalytic activity and cannot be suppressed by overexpression of RNase II, the other major enzyme (encoded by rnb) implicated in exonucleolytic RNA degradation. Moreover, E. coli pnp mutants are more sensitive than the wild type to zeocin. This phenotype depends on PNPase phosphorolytic activity and is suppressed by rnb, thus suggesting that zeocin detoxification may largely depend on RNA turnover. Our data suggest that PNPase may participate both directly and indirectly through regulation of gene expression to several aspects of DNA metabolism such as recombination, DNA repair and resistance to genotoxic agents.

There is an urgent need for new antibacterial drugs effective against bacterial pathogens resistant to current drugs ^1–2 . Nucleoside-analog inhibitors (NAIs) of viral nucleotide polymerases have had transformative impact in treatment of HIV ³ and HCV ⁴ . NAIs of bacterial RNA polymerase (RNAP) potentially could have major impact on treatment of bacterial infection, particularly because functional constraints on substitution of RNAP nucleoside triphosphate (NTP) binding sites ^4-5 could limit resistance emergence ^4-5 . Here we report the discovery, from microbial extract screening, of an NAI that inhibits bacterial RNAP and exhibits antibacterial activity against a broad spectrum of drug-sensitive and drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a novel microbial natural product consisting of a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, potently and selectively inhibits bacterial growth in culture, and potently clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the "i+1" NTP binding site) and mechanism (competition with UTP for occupancy of the "i+1" NTP binding site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. The results provide the first example of a selective NAI of bacterial RNAP, provide an advanced lead compound for antibacterial drug development, and provide structural information and synthetic routes that enable lead optimization for antibacterial drug development.

The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in γ-Proteobacteria. LptBFG constitute the IM ABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable ΔlptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptF^SupC). In complementation tests, lptF^SupC mutants suppress lethality of both ΔlptC and lptC conditional expression mutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine.

Polynucleotide phosphorylase (PNPase) is an exoribonuclease that catalyzes the processive phosphorolytic degradation of RNA from the 3′‐end. The enzyme catalyzes also the reverse reaction of polymerization of nucleoside diphosphates that has been implicated in the generation of heteropolymeric tails at the RNA 3′‐end. The enzyme is widely conserved and plays a major role in RNA decay in both Gram‐negative and Gram‐positive bacteria. Moreover, it participates in maturation and quality control of stable RNA. PNPase autoregulates its own expression at post‐transcriptional level through a complex mechanism that involves the endoribonuclease RNase III and translation control. The activity of PNPase is modulated in an intricate and still unclear manner by interactions with small molecules and recruitment in different multiprotein complexes. Not surprisingly, given the wide spectrum of PNPase substrates, PNPase‐defective mutations in different bacterial species have pleiotropic effects and perturb the execution of genetic programs involving drastic changes in global gene expression such as biofilm formation, growth at suboptimal temperatures, and virulence. WIREs RNA 2016, 7:241–258. doi: 10.1002/wrna.1328This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications RNA Turnover and Surveillance > Regulation of RNA Stability

The complex posttranscriptional regulation mechanism of the Escherichia coli pnp gene, which encodes the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase), involves two endoribonucleases, namely, RNase III and RNase E, and PNPase itself, which thus autoregulates its own expression. The models proposed for pnp autoregulation posit that the target of PNPase is a mature pnp mRNA previously processed at its 5′ end by RNase III, rather than the primary pnp transcript (RNase III-dependent models), and that PNPase activity eventually leads to pnp mRNA degradation by RNase E. However, some published data suggest that pnp expression may also be regulated through a PNPase-dependent, RNase III-independent mechanism. To address this issue, we constructed isogenic Δ pnp rnc ⁺ and Δ pnp Δ rnc strains with a chromosomal pnp-lacZ translational fusion and measured β-galactosidase activity in the absence and presence of PNPase expressed by a plasmid. Our results show that PNPase also regulates its own expression via a reversible RNase III-independent pathway acting upstream from the RNase III-dependent branch. This pathway requires the PNPase RNA binding domains KH and S1 but not its phosphorolytic activity. We suggest that the RNase III-independent autoregulation of PNPase occurs at the level of translational repression, possibly by competition for pnp primary transcript between PNPase and the ribosomal protein S1. IMPORTANCE In Escherichia coli , polynucleotide phosphorylase (PNPase, encoded by pnp ) posttranscriptionally regulates its own expression. The two models proposed so far posit a two-step mechanism in which RNase III, by cutting the leader region of the pnp primary transcript, creates the substrate for PNPase regulatory activity, eventually leading to pnp mRNA degradation by RNase E. In this work, we provide evidence supporting an additional pathway for PNPase autogenous regulation in which PNPase acts as a translational repressor independently of RNase III cleavage. Our data make a new contribution to the understanding of the regulatory mechanism of pnp mRNA, a process long since considered a paradigmatic example of posttranscriptional regulation at the level of mRNA stability.

Lipopolysaccharide (LPS) is the main glycolipid present in the outer leaflet of the outer membrane (OM) of Gram‐negative bacteria, where it modulates OM permeability, therefore preventing many toxic compounds from entering the cell. LPS biogenesis is an essential process in Gram‐negative bacteria and thus is an ideal target pathway for the development of novel specific antimicrobials. The lipopolysaccharide transport (Lpt) system is responsible for transporting LPS from the periplasmic surface of the inner membrane, where it is assembled, to the cell surface where it is then inserted in the OM. The Lpt system has been widely studied in Escherichia coli, where it consists of seven essential proteins located in the inner membrane (LptBCFG), in the periplasm (LptA) and in the OM (LptDE). In the present study, we focus our attention on the Pseudomonas aeruginosa PAO1 Lpt system. We identified an LptA orthologue, named LptH, and solved its crystal structure at a resolution of 2.75 Å. Using interspecies complementation and site‐directed mutagenesis of a conserved glycine residue, we demonstrate that P. aeruginosa LptH is the genetic and functional homologue of E. coli LptA, with whom it shares the β‐jellyroll fold identified also in other members of the canonical E. coli Lpt model system. Furthermore, we modeled the N‐terminal β‐jellyroll domain of P. aeruginosa LptD, based on the crystal structure of its homologue from Shigella flexneri, aiming to provide more general insight into the mechanism of LPS binding and transport in P. aeruginosa. Both LptH and LptD may represent new targets for the discovery of next generation antibacterial drugs, targeting specific opportunistic pathogens such as P. aeruginosa.Database: Coordinates and structure factors have been deposited in the Protein Data Bank under accession number PDB 4uu4.

Modulation of mRNA translatability either by trans-acting factors (proteins or sRNAs) or by in cis-acting riboregulators is widespread in bacteria and controls relevant phenotypic traits. Unfortunately, global identification of post-transcriptionally regulated genes is complicated by poor structural and functional conservation of regulatory elements and by the limitations of proteomic approaches in protein quantification. We devised a genetic system for the identification of post-transcriptionally regulated genes and we applied this system to search for Pseudomonas aeruginosa RNA thermometers, a class of regulatory RNA that modulates gene translation in response to temperature changes. As P. aeruginosa is able to thrive in a broad range of environmental conditions, genes differentially expressed at 37°C versus lower temperatures may be involved in infection and survival in the human host. We prepared a plasmid vector library with translational fusions of P. aeruginosa DNA fragments (PaDNA) inserted upstream of TIP2, a short peptide able to inactivate the Tet repressor (TetR) upon expression. The library was assayed in a streptomycin-resistant merodiploid rpsL⁺/rpsL31 Escherichia coli strain in which the dominant rpsL⁺ allele, which confers streptomycin sensitivity, was repressed by TetR. PaDNA fragments conferring thermosensitive streptomycin resistance (i.e., expressing PaDNA–TIP2 fusions at 37°C, but not at 28°C) were sequenced. We identified four new putative thermosensors. Two of them were validated with conventional reporter systems in E. coli and P. aeruginosa. Interestingly, one regulates the expression of ptxS, a gene implicated in P. aeruginosa pathogenesis.

The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality.

Ribonucleases E and PH have a role in tRNA metabolism in Escherichia coli; here we describe the domains that permit them to form a functional complex.