Steady-State Growth under Inorganic Carbon Limitation Conditions Increases Energy Consumption for Maintenance and Enhances Nitrous Oxide Production in Nitrosomonas europaea

Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH₃) to obtain energy for growth on carbon dioxide (CO₂) and can also produce nitrous oxide (N₂O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to conditions of replete (>5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na₂CO₃) supplemented with atmospheric CO₂. IC-limited cultures oxidized 25 to 58% of available NH₃ to nitrite, depending on the dilution rate and Na₂CO₃ concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to those for NH₃-limited cultures. Rates of N₂O production increased 2.5- and 6.3-fold under the two IC-limited conditions, increasing the percentage of oxidized NH₃-N that was transformed to N₂O-N from 0.5% (replete) up to 4.4% (0.2 mM Na₂CO₃). Transcriptome analysis showed differential expression (P ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in a decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits and increased the expression of genes involved in C₁ metabolism, including the genes for RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C₁ moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924 to NE0927) correlated with increased production of N₂O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady-state growth, which leads to the uncoupling of NH₃ oxidation from growth and increased N₂O production. IMPORTANCE: Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, is an important process in the global nitrogen cycle. This process is generally dependent on ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria. Most nitrifiers are chemolithoautotrophs that fix inorganic carbon (CO₂) for growth. Here, we investigate how inorganic carbon limitation modifies the physiology and transcriptome of Nitrosomonas europaea, a model ammonia-oxidizing bacterium, and report on increased production of N₂O, a potent greenhouse gas. This study, along with previous work, suggests that inorganic carbon limitation may be an important factor in controlling N₂O emissions from nitrification in soils and wastewater treatment

Bacterial Resistance to Antisense Peptide-Phosphorodiamidate Morpholino Oligomers

Peptide phosphorodiamidate morpholino oligomers (PPMO) are synthetic DNA mimics that bind complementary RNA and inhibit bacterial gene expression. (RFF)₃RXB- AcpP PPMO (R, arginine; F, phenylalanine; X, 6-aminohexanoic acid; B, β-alanine) is complementary to 11 bases of the essential gene acpP (encodes acyl carrier protein). The MIC of (RFF)₃RXB-AcpP was 2.5 μM (14 μg/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF)₃RXB-AcpP was 4 x 10⁻⁷ mutations/cell division. A spontaneous (RFF)₃RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)₃RXB-AcpP was 40 μM (224 μg/ml) in PR200.1. The MICs of standard antibiotics were identical in PR200.1 and W3110. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)₃RXB or peptides with a similar composition or pattern of cationic and non-polar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF)₃RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmA restored susceptibility to (RFF)₃RXB-AcpP. Deletion of sbmA caused resistance to (RFF)₃RXB-AcpP. We conclude that resistance to (RFF)₃RXB-AcpP was linked to the peptide and not the PMO, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF)₃R-XB PPMOs may be transported across the plasma membrane by SbmA

Targeting RNA Polymerase Primary σ70 as a Therapeutic Strategy against Methicillin-Resistant Staphylococcus aureus by Antisense Peptide Nucleic Acid

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) causes threatening infection-related mortality worldwide. Currently, spread of multi-drug resistance (MDR) MRSA limits therapeutic options and requires new approaches to "druggable" target discovery, as well as development of novel MRSA-active antibiotics. RNA polymerase primary σ⁷⁰ (encoded by gene rpoD) is a highly conserved prokaryotic factor essential for transcription initiation in exponentially growing cells of diverse S. aureus, implying potential for antisense inhibition. METHODOLOGY/PRINCIPAL FINDINGS: By synthesizing a serial of cell penetrating peptide conjugated peptide nucleic acids (PPNAs) based on software predicted parameters and further design optimization, we identified a target sequence (234 to 243 nt) within rpoD mRNA conserved region 3.0 being more sensitive to antisense inhibition. A (KFF)₃K peptide conjugated 10-mer complementary PNA (PPNA2332) was developed for potent micromolar-range growth inhibitory effects against four pathogenic S. aureus strains with different resistance phenotypes, including clinical vancomycin-intermediate resistance S. aureus and MDR-MRSA isolates. PPNA2332 showed bacteriocidal antisense effect at 3.2 fold of MIC value against MRSA/VISA Mu50, and its sequence specificity was demonstrated in that PPNA with scrambled PNA sequence (Scr PPNA2332) exhibited no growth inhibitory effect at higher concentrations. Also, PPNA2332 specifically interferes with rpoD mRNA, inhibiting translation of its protein product σ⁷⁰ in a concentration-dependent manner. Full decay of mRNA and suppressed expression of σ⁷⁰ were observed for 40 µM or 12.5 µM PPNA2332 treatment, respectively, but not for 40 µM Scr PPNA2332 treatment in pure culture of MRSA/VISA Mu50 strain. PPNA2332 (≥1 µM) essentially cleared lethal MRSA/VISA Mu50 infection in epithelial cell cultures, and eliminated viable bacterial cells in a time- and concentration- dependent manner, without showing any apparent toxicity at 10 µM. CONCLUSIONS: The present result suggested that RNAP primary σ⁷⁰ is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections

En kunnskapsbasert finansnæring

Prosjektet ”En kunnskapsbasert finansnæring” inngår som delprosjekt i det store nasjonale forskningsprosjektet ”Et kunnskapsbasert Norge” som gjennomføres ved Handelshøyskolen BI under ledelse av professor Torger Reve. Norsk næringsliv er avhengig av en velfungerende finansnæring. Denne næringen fungerer på mange måter som et nav for de andre næringene i økonomien. For at næringslivet skal kunne skape vekst, må det kanaliseres kapital til prosjekter som har høy lønnsomhet og evne til verdiskaping. Dersom bedriftene selv ikke har denne kapitalen tilgjengelig er det nødvendig med ekstern kapitaltilførsel. Det er her finansnæringen bidrar, både med kapital og kompetanse om hvor kapitalen bør kanaliseres. Finansmarkedet fungerer også som et korrektiv til bedriftenes atferd. Bedrifter som gjør det bra blir premiert gjennom høyere verdsetting på børs, mens de som gjør det dårlig opplever fallende kurser. På denne måten får eierne et tydelig signal om hvor godt bedriften er styrt og kan på denne bakgrunn legge opp til endringer i bedriftens strategi. Videre spiller finansmarkedet en viktig rolle gjennom å ivareta både næringslivets, husholdningenes og offentlig sektors behov for likviditet gjennom å tilby regulære banktjenester. Næringen har også en sentral oppgave gjennom å avdempe risiko gjennom ulike forsikringsprodukter. Sist, men ikke minst, har finansnæringen en formidlerrolle gjennom å bistå aktører i økonomien i å flytte verdier seg imellom. Vi snakker med andre ord om en næring med mange aktivitetsområder og som i all hovedsak har forankret sine aktiviteter i andre aktørers økonomiske aktivitet

Quorum Quenching of Nitrobacter winogradskyi Suggests that Quorum Sensing Regulates Fluxes of Nitrogen Oxide(s) during Nitrification

Quorum sensing (QS) is a widespread process in bacteria used to coordinate gene expression with cell density, diffusion dynamics, and spatial distribution through the production of diffusible chemical signals. To date, most studies on QS have focused on model bacteria that are amenable to genetic manipulation and capable of high growth rates, but many environmentally important bacteria have been overlooked. For example, representatives of proteobacteria that participate in nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, produce QS signals called acyl-homoserine lactones (AHLs). Nitrification emits nitrogen oxide gases (NO, NO2, and N2O), which are potentially hazardous compounds that contribute to global warming. Despite considerable interest in nitrification, the purpose of QS in the physiology/ecology of nitrifying bacteria is poorly understood. Through a quorum quenching approach, we investigated the role of QS in a well-studied AHL-producing nitrite oxidizer, Nitrobacter winogradskyi. We added a recombinant AiiA lactonase to N. winogradskyi cultures to degrade AHLs to prevent their accumulation and to induce a QS-negative phenotype and then used mRNA sequencing (mRNA-Seq) to identify putative QS-controlled genes. Our transcriptome analysis showed that expression of nirK and nirK cluster genes (ncgABC) increased up to 19.9-fold under QS-proficient conditions (minus active lactonase). These data led to us to query if QS influenced nitrogen oxide gas fluxes in N. winogradskyi. Production and consumption of NOx increased and production of N2O decreased under QS-proficient conditions. Quorum quenching transcriptome approaches have broad potential to identify QS-controlled genes and phenotypes in organisms that are not genetically tractable