Activity and Function in Human Cells of the Evolutionary Conserved Exonuclease Polynucleotide Phosphorylase.

Polynucleotide phosphorylase (PNPase) is a phosphorolytic RNA exonuclease highly conserved throughout evolution. Human PNPase (hPNPase) is located in mitochondria and is essential for mitochondrial function and homeostasis. Not surprisingly, mutations in the PNPT1 gene, encoding hPNPase, cause serious diseases. hPNPase has been implicated in a plethora of processes taking place in different cell compartments and involving other proteins, some of which physically interact with hPNPase. This paper reviews hPNPase RNA binding and catalytic activity in relation with the protein structure and in comparison, with the activity of bacterial PNPases. The functions ascribed to hPNPase in different cell compartments are discussed, highlighting the gaps that still need to be filled to understand the physiological role of this ancient protein in human cells.

Mutation and Suppressor Analysis of the Essential Lipopolysaccharide Transport Protein LptA Reveals Strategies To Overcome Severe Outer Membrane Permeability Defects in Escherichia coli.

In Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane (OM), preventing the entry of toxic molecules, such as detergents and antibiotics. LPS is transported from the inner membrane (IM) to the OM by the Lpt multiprotein machinery. Defects in LPS transport compromise LPS assembly at the OM and result in increased antibiotic sensitivity. LptA is a key component of the Lpt machine that interacts with the IM protein LptC and chaperones LPS through the periplasm. We report here the construction of lptA41, a quadruple mutant in four conserved amino acids potentially involved in LPS or LptC binding. Although viable, the mutant displays increased sensitivity to several antibiotics (bacitracin, rifampin, and novobiocin) and the detergent SDS, suggesting that lptA41 affects LPS transport. Indeed, lptA41 is defective in Lpt complex assembly, and its lipid A carries modifications diagnostic of LPS transport defects. We also selected and characterized two phenotypic bacitracin-resistant suppressors of lptA41 One mutant, in which only bacitracin sensitivity is suppressed, harbors a small in-frame deletion in mlaA, which codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other mutant, in which bacitracin, rifampin, and SDS sensitivity is suppressed, harbors an additional amino acid substitution in LptA41 and a nonsense mutation in opgH, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharide synthesis. Characterization of the suppressor mutants highlights different strategies adopted by the cell to overcome OM defects caused by impaired LPS transport.IMPORTANCE Lipopolysaccharide (LPS) is the major constituent of the outer membrane (OM) of most Gram-negative bacteria, forming a barrier against antibiotics. LPS is synthesized at the inner membrane (IM), transported across the periplasm, and assembled at the OM by the multiprotein Lpt complex. LptA is the periplasmic component of the Lpt complex, which bridges IM and OM and ferries LPS across the periplasm. How the cell coordinates the processes involved in OM biogenesis is not completely understood. We generated a mutant partially defective in lptA that exhibited increased sensitivity to antibiotics and selected for suppressors of the mutant. The analysis of two independent suppressors revealed different strategies adopted by the cell to overcome defects in LPS biogenesis.

Human PNPase causes RNA stabilization and accumulation of R-loops in the Escherichia coli model system.

Polyribonucleotide phosphorylase (PNPase) is a phosphorolytic RNA exonuclease highly conserved throughout evolution. In Escherichia coli, PNPase controls complex phenotypic traits like biofilm formation and growth at low temperature. In human cells, PNPase is located in mitochondria, where it is implicated in the RNA import from the cytoplasm, the mitochondrial RNA degradation and the processing of R-loops, namely stable RNA-DNA hybrids displacing a DNA strand. In this work, we show that the human PNPase (hPNPase) expressed in E. coli causes oxidative stress, SOS response activation and R-loops accumulation. Hundreds of E. coli RNAs are stabilized in presence of hPNPase, whereas only few transcripts are destabilized. Moreover, phenotypic traits typical of E. coli strains lacking PNPase are strengthened in presence of the human enzyme. We discuss the hypothesis that hPNPase expressed in E. coli may bind, but not degrade, the RNA, in agreement with previous in vitro data showing that phosphate concentrations in the range of those found in the bacterial cytoplasm and, more relevant, in the mitochondria, inhibit its activity.

Suppressor Mutations in LptF Bypass Essentiality of LptC by Forming a Six-Protein Transenvelope Bridge That Efficiently Transports Lipopolysaccharide.

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of many Gram-negative bacteria, providing a barrier against the entry of toxic molecules. In Escherichia coli, LPS is exported to the cell surface by seven essential proteins (LptA-G) that form a transenvelope complex. At the inner membrane, the ATP-binding cassette (ABC) transporter LptB2FG associates with LptC to power LPS extraction from the membrane and transfer to the periplasmic LptA protein, which is in complex with the OM translocon LptDE. LptC interacts both with LptB2FG and LptADE to mediate the formation of the transenvelope bridge and regulates the ATPase activity of LptB2FG. A genetic screen has previously identified suppressor mutants at a residue (R212) of LptF that are viable in the absence of LptC. Here, we present in vivo evidence that the LptF R212G mutant assembles a six-protein transenvelope complex in which LptA mediates interactions with LptF and LptD in the absence of LptC. Furthermore, we present in vitro evidence that the mutant LptB2FG complexes restore the regulation of ATP hydrolysis as it occurs in the LptB2FGC complex to achieve wild-type efficient coupling of ATP hydrolysis and LPS movement. We also show the suppressor mutations restore the wild-type levels of LPS transport both in vivo and in vitro, but remarkably, without restoring the affinity of the inner membrane complex for LptA. Based on the sensitivity of lptF suppressor mutants to selected stress conditions relative to wild-type cells, we show that there are additional regulatory functions of LptF and LptC that had not been identified. IMPORTANCE The presence of an external LPS layer in the outer membrane makes Gram-negative bacteria intrinsically resistant to many antibiotics. Millions of LPS molecules are transported to the cell surface per generation by the Lpt molecular machine made, in E. coli, by seven essential proteins. LptC is the unconventional regulatory subunit of the LptB2FGC ABC transporter, involved in coordinating energy production and LPS transport. Surprisingly, despite being essential for bacterial growth, LptC can be deleted, provided that a specific residue in the periplasmic domain of LptF is mutated and LptA is overexpressed. Here, we apply biochemical techniques to investigate the suppression mechanism. The data produced in this work disclose an unknown regulatory function of LptF in the transporter that not only expands the knowledge about the Lpt complex but can also be targeted by novel LPS biogenesis inhibitors.

Different csrA Expression Levels in C versus K-12 E. coli Strains Affect Biofilm Formation and Impact the Regulatory Mechanism Presided by the CsrB and CsrC Small RNAs.

Escherichia coli C is a strong biofilm producer in comparison to E. coli K-12 laboratory strains due to higher expression of the pgaABCD operon encoding the enzymes for the biosynthesis of the extracellular polysaccharide poly-ß-1,6-N-acetylglucosamine (PNAG). The pgaABCD operon is negatively regulated at the post-transcriptional level by two factors, namely CsrA, a conserved RNA-binding protein controlling multiple pathways, and the RNA exonuclease polynucleotide phosphorylase (PNPase). In this work, we investigated the molecular bases of different PNAG production in C-1a and MG1655 strains taken as representative of E. coli C and K-12 strains, respectively. We found that pgaABCD operon expression is significantly lower in MG1655 than in C-1a; consistently, CsrA protein levels were much higher in MG1655. In contrast, we show that the negative effect exerted by PNPase on pgaABCD expression is much stronger in C-1a than in MG1655. The amount of CsrA and of the small RNAs CsrB, CsrC, and McaS sRNAs regulating CsrA activity is dramatically different in the two strains, whereas PNPase level is similar. Finally, the compensatory regulation acting between CsrB and CsrC in MG1655 does not occur in E. coli C. Our results suggest that PNPase preserves CsrA-dependent regulation by indirectly modulating csrA expression.

Sanguinarine Inhibits the 2-Ketogluconate Pathway of Glucose Utilization in Pseudomonas aeruginosa.

Interfering with the ability of pathogenic bacteria to import glucose may represent a new promising antibacterial strategy, especially for the treatment of infections occurring in diabetic and other hyperglycemic patients. Such patients are particularly susceptible to infections caused by a variety of bacteria, among which opportunistic pathogens like Pseudomonas aeruginosa. In P. aeruginosa, glucose can be directly imported into the cytoplasm or after its periplasmic oxidation into gluconate and 2-ketogluconate (2-KG). We recently demonstrated that a P. aeruginosa mutant lacking the 2-KG transporter KguT is less virulent than its kguT + parental strain in an insect infection model, pointing to 2-KG branch of glucose utilization as a possible target for anti-Pseudomonas drugs. In this work, we devised an experimental protocol to find specific inhibitors of the 2-KG pathway of P. aeruginosa glucose utilization and applied it to the screening of the Prestwick Chemical Library. By exploiting mutants lacking genes involved in the transport of glucose derivatives in the primary screening and in the secondary assays, we could identify sanguinarine as an inhibitor of 2-KG utilization. We also demonstrated that sanguinarine does not prevent 2-KG formation by gluconate oxidation or its transport, suggesting that either KguD or KguK is the target of sanguinarine in P. Aeruginosa.

Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A.

LpxT is an inner membrane protein that transfers a phosphate group from the essential lipid undecaprenyl pyrophosphate (C-55PP) to the lipid A moiety of lipopolysaccharide, generating a lipid A tris-phosphorylated species. The protein is encoded by the non-essential lpxT gene, which is conserved in distantly related Gram-negative bacteria. In this work, we investigated the phenotypic effect of lpxT ectopic expression from a plasmid in Escherichia coli. We found that lpxT induction inhibited cell division and led to the formation of elongated cells, mostly with absent or altered septa. Moreover, the cells became sensitive to detergents and to hypo-osmotic shock, indicating that they had cell envelope defects. These effects were not due to lipid A hyperphosphorylation or C-55PP sequestering, but most likely to defective lipopolysaccharide transport. Indeed, lpxT overexpression in mutants lacking the L,D-transpeptidase LdtD and LdtE, which protect cells with outer membrane defects from osmotic lysis, caused cell envelope defects. Moreover, we found that pyrophosphorylated lipid A was also produced in a lpxT deletion mutant, indicating that LpxT is not the only protein able to perform such lipid A modification in E. coli.

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. 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 (LptFSupC). In complementation tests, lptFSupC 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.