Antibacterial Nucleoside-Analog Inhibitor of Bacterial RNA Polymerase.

Drug-resistant bacterial pathogens pose an urgent public-health crisis. Here, we report the discovery, from microbial-extract screening, of a nucleoside-analog inhibitor that inhibits bacterial RNA polymerase (RNAP) and exhibits antibacterial activity against drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a natural product comprising a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine.

Polynucleotide phosphorylase is implicated in homologous recombination and DNA repair in Escherichia coli.

Background: 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.

The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter.

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.

Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli.

Unlabelled: The assembly of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) requires the transenvelope Lpt (lipopolysaccharide transport) complex, made in Escherichia coli of seven essential proteins located in the inner membrane (IM) (LptBCFG), periplasm (LptA), and OM (LptDE). At the IM, LptBFG constitute an unusual ATP binding cassette (ABC) transporter, composed by the transmembrane LptFG proteins and the cytoplasmic LptB ATPase, which is thought to extract LPS from the IM and to provide the energy for its export across the periplasm to the cell surface. LptC is a small IM bitopic protein that binds to LptBFG and recruits LptA via its N- and C-terminal regions, and its role in LPS export is not completely understood.

Regulation and functions of bacterial PNPase.

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.

RNase III-Independent Autogenous Regulation of Escherichia coli Polynucleotide Phosphorylase via Translational Repression.

Unlabelled: 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.

Crystal structure of LptH, the periplasmic component of the lipopolysaccharide transport machinery from Pseudomonas aeruginosa.

Unlabelled: 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.

Tet-Trap, a genetic approach to the identification of bacterial RNA thermometers: application to Pseudomonas aeruginosa.

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.

Dissecting Escherichia coli outer membrane biogenesis using differential proteomics.

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.

Ribonuclease PH interacts with an acidic ribonuclease E site through a basic 80-amino acid domain.

In this work, we characterize the domains for the in vivo interaction between ribonuclease E (RNase E) and ribonuclease PH (RNase PH). We initially explored the interaction using pull-down assays with full wild-type proteins expressed from a chromosomal monocopy gene. Once the interaction was confirmed, we narrowed down the sites of interaction in each enzyme to an acidic 16-amino acid region in the carboxy-terminal domain of RNase E and a basic 80-amino acid region in RNase PH including an α3 helix.

A conserved loop in polynucleotide phosphorylase (PNPase) essential for both RNA and ADP/phosphate binding.

Polynucleotide phosphorylase (PNPase) reversibly catalyzes RNA phosphorolysis and polymerization of nucleoside diphosphates. Its homotrimeric structure forms a central channel where RNA is accommodated. Each protomer core is formed by two paralogous RNase PH domains: PNPase1, whose function is largely unknown, hosts a conserved FFRR loop interacting with RNA, whereas PNPase2 bears the putative catalytic site, ∼20 Å away from the FFRR loop.

The Escherichia coli Lpt transenvelope protein complex for lipopolysaccharide export is assembled via conserved structurally homologous domains.

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain.

The RNA processing enzyme polynucleotide phosphorylase negatively controls biofilm formation by repressing poly-N-acetylglucosamine (PNAG) production in Escherichia coli C.

Background: Transition from planktonic cells to biofilm is mediated by production of adhesion factors, such as extracellular polysaccharides (EPS), and modulated by complex regulatory networks that, in addition to controlling production of adhesion factors, redirect bacterial cell metabolism to the biofilm mode.

Results: Deletion of the pnp gene, encoding polynucleotide phosphorylase, an RNA processing enzyme and a component of the RNA degradosome, results in increased biofilm formation in Escherichia coli. This effect is particularly pronounced in the E.

Comparative profiling of Pseudomonas aeruginosa strains reveals differential expression of novel unique and conserved small RNAs.

Pseudomonas aeruginosa is a highly adaptable bacterium that thrives in a broad range of ecological niches and can infect multiple hosts as diverse as plants, nematodes and mammals. In humans, it is an important opportunistic pathogen. This wide adaptability correlates with its broad genetic diversity.

Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins.

Bacillus subtilis pnpA gene product, polynucleotide phosphorylase (PNPase), is involved in double-strand break (DSB) repair via homologous recombination (HR) or non-homologous end-joining (NHEJ). RecN is among the first responders to localize at the DNA DSBs, with PNPase facilitating the formation of a discrete RecN focus per nucleoid. PNPase, which co-purifies with RecA and RecN, was able to degrade single-stranded (ss) DNA with a 3' → 5' polarity in the presence of Mn(2+) and low inorganic phosphate (Pi) concentration, or to extend a 3'-OH end in the presence dNDP · Mn(2+).

Isolation of conditional expression mutants in Mycobacterium tuberculosis by transposon mutagenesis.

In Mycobacterium tuberculosis identification of essential genes has been hampered by the scarcity of suitable genetic tools for genome wide screenings. We constructed two Himar1 transposon derivatives in which the Streptomyces pristinamycin I-inducible ptr promoter was inserted at one transposon end in outward orientation. These transposons, Tn-pip/pptr (which harbours the promoter and its repressor pip gene) and Tn-pptr (which depends on a host expressing the pip gene), were inserted in the thermosensitive mycobacteriophage phAE87.

S1 ribosomal protein and the interplay between translation and mRNA decay.

S1 is an 'atypical' ribosomal protein weakly associated with the 30S subunit that has been implicated in translation, transcription and control of RNA stability. S1 is thought to participate in translation initiation complex formation by assisting 30S positioning in the translation initiation region, but little is known about its role in other RNA transactions. In this work, we have analysed in vivo the effects of different intracellular S1 concentrations, from depletion to overexpression, on translation, decay and intracellular distribution of leadered and leaderless messenger RNAs (mRNAs).

Complex transcriptional organization regulates an Escherichia coli locus implicated in lipopolysaccharide biogenesis.

The Escherichia coli yrbG-lptB locus (yrbG kdsD kdsC lptC lptA lptB) encodes genes for outer membrane biogenesis, namely, kdsC and kdsD for biosynthesis of the lipopolysaccharide inner core sugar Kdo, and lptA, lptB, and lptC for lipopolysaccharide transport to the outer membrane. Three promoters (yrbGp, kdsCp and the σ(E)-dependent lptAp) have been previously identified by genetic analysis. In this work, we show that transcription of this locus generates an array of overlapping mRNAs and we characterize the two intralocus promoter regions.

Probing the active site of the sugar isomerase domain from E. coli arabinose-5-phosphate isomerase via X-ray crystallography.

Lipopolysaccharide (LPS) biosynthesis represents an underexploited target pathway for novel antimicrobial development to combat the emergence of multidrug-resistant bacteria. A key player in LPS synthesis is the enzyme D-arabinose-5-phosphate isomerase (API), which catalyzes the reversible isomerization of D-ribulose-5-phosphate to D-arabinose-5-phosphate, a precursor of 3-deoxy-D-manno-octulosonate that is an essential residue of the LPS inner core. API is composed of two main domains: an N-terminal sugar isomerase domain (SIS) and a pair of cystathionine-β-synthase domains of unknown function.

The lipopolysaccharide transport system of Gram-negative bacteria.

The cell envelope of Gram-negative bacteria consists of two distinct membranes, the inner (IM) and the outer membrane (OM) separated by the periplasm. The OM contains in the outer leaflet the lipopolysaccharide (LPS), a complex lipid with important biological activities. In the host it elicits the innate immune response whereas in the bacterium it is responsible for the peculiar permeability barrier properties exhibited by the OM.

A proteomic approach to the analysis of RNA degradosome composition in Escherichia coli.

The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli, it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components: the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. The variable presence of additional proteins, however, suggests that the degradosome is a flexible machine that may vary its composition in response to different conditions.

Autogenous regulation of Escherichia coli polynucleotide phosphorylase expression revisited.

The Escherichia coli polynucleotide phosphorylase (PNPase; encoded by pnp), a phosphorolytic exoribonuclease, posttranscriptionally regulates its own expression at the level of mRNA stability and translation. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double-strand cleavage about in the middle of a long stem-loop in the 5'-untranslated region. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner.

Polynucleotide phosphorylase hinders mRNA degradation upon ribosomal protein S1 overexpression in Escherichia coli.

The exoribonuclease polynucleotide phosphorylase (PNPase, encoded by pnp) is a major player in bacterial RNA decay. In Escherichia coli, PNPase expression is post-transcriptionally regulated at the level of mRNA stability. The primary transcript is very efficiently processed by the endonuclease RNase III at a specific site and the processed pnp mRNA is rapidly degraded in a PNPase-dependent manner.