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Microbially mediated nitrification plays an important role in the nitrogen (N) cycle, and rates of activity have been shown to change significantly with temperature. Despite this, the substrate affinities of nitrifying bacteria and archaea have not been comprehensively measured and are often assumed to be static in mathematical models of environmental systems. In this study, we measured the oxidation kinetics of ammonia- (NH3) oxidizing archaea (AOA), NH3-oxidizing bacteria (AOB), and two distinct groups of nitrite (NO2–)-oxidizing bacteria (NOB), of the genera Nitrobacter and Nitrospira , by measuring the maximum rates of apparent activity (V max(app)), the apparent half-saturation constant (K m (app)), and the overall catalytic efficiency (V max(app) / K m (app)) over a range of temperatures. Changes in V max(app) and K m (app) with temperature were different between groups, with V max(app) and catalytic efficiency increasing with temperature in AOA, while V max(app) , K m (app), and catalytic efficiency increased in AOB. In Nitrobacter NOB, V max(app) and K m (app) increased, but catalytic efficiency decreased significantly with temperature. Nitrospira NOB were variable, but V max(app) increased while catalytic efficiency and K m (app) remained relatively unchanged. Michaelis–Menten (MM) and Haldane (H) kinetic models of NH3 oxidation and NO2– oxidation based on the collected data correctly predict nitrification potential in some soil incubation experiments, but not others. Despite previous observations of coupled nitrification in many natural systems, our results demonstrate significant differences in response to temperature strategies between the different groups of nitrifiers; and indicate the need to further investigate the response of nitrifiers to environmental changes. [ABSTRACT FROM AUTHOR]

The factors influencing how soil nitrite (NO2-)- and ammonia (NH3)-oxidizing activities remain coupled are unknown. A short-term study (<48 h) was conducted to examine the dynamics of NO2--oxidizing activity and the accumulation of NO2- in three Oregon soils stimulated by the addition of 1 mM NH4+ in soil slurry. Nitrite initially accumulated in all three soils; its subsequent decline or slowing of the accumulation of the NO2- pool by 24 h was accompanied by an increase in the size of the nitrate (NO3-) pool, indicating a change in NO2- oxidation kinetics. Bacterial protein synthesis inhibitors prevented the NO2- pool decline, resulting in a larger accumulation in all three soils. Although no significant increases in NO2--oxidizing bacteria nxrA (Nitrobacter) and nxrB (Nitrospira) gene abundances were detected over the time course, maximum NO2- consumption rates increased 2-fold in the treatment without antibiotics compared to no change with antibiotics. No changes were observed in the apparent half saturation constant (Km) values for NO2- consumption. This study demonstrates phenotypic flexibility among soil NO2- oxidizers, which can undergo protein synthesis-dependent increases in NO2- consumption rates to match NH3 oxidation rates and recouple nitrification. [ABSTRACT FROM AUTHOR]

The genomes of many bacteria that participate in nitrogen cycling through the process of nitrification contain putative genes associated with acylhomoserine lactone (AHL) quorum sensing (QS). AHL QS or bacterial cell-cell signaling is a method of bacterial communication and gene regulation and may be involved in nitrogen oxide fluxes or other important phenotypes in nitrifying bacteria. Here, we carried out a broad survey of AHL production in nitrifying bacteria in three steps. First, we analyzed the evolutionary history of AHL synthase and AHL receptor homologs in sequenced genomes and metagenomes of nitrifying bacteria to identify AHL synthase homologs in ammonia-oxidizing bacteria (AOB) of the genus Nitrosospira and nitrite-oxidizing bacteria (NOB) of the genera Nitrococcus, Nitrobacter, and Nitrospira. Next, we screened cultures of both AOB and NOB with uncharacterized AHL synthase genes and AHL synthase-negative nitrifiers by a bioassay. Our results suggest that an AHL synthase gene is required for, but does not guarantee, cell density-dependent AHL production under the conditions tested. Finally, we utilized mass spectrometry to identify the AHLs produced by the AOB Nitrosospira multiformis and Nitrosospira briensis and the NOB Nitrobacter vulgaris and Nitrospira moscoviensis as N-decanoyl-L-homoserine lactone (C10-HSL), N-3-hydroxy-tetradecanoyl-L-homoserine lactone (3-OH-C14-HSL), a monounsaturated AHL (C10:1-HSL), and N-octanoyl-L-homoserine lactone (C8-HSL), respectively. Our survey expands the list of AHL-producing nitrifiers to include a representative of Nitrospira lineage II and suggests that AHL production is widespread in nitrifying bacteria. IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite by nitrifying microorganisms, plays an important role in environmental nitrogen cycling from agricultural fertilization to wastewater treatment. The genomes of many nitrifying bacteria contain genes associated with bacterial cell-cell signaling or quorum sensing (QS). QS is a method of bacterial communication and gene regulation that is well studied in bacterial pathogens, but less is known about QS in environmental systems. Our previous work suggested that QS might be involved in the regulation of nitrogen oxide gas production during nitrite metabolism. This study characterized putative QS signals produced by different genera and species of nitrifiers. Our work lays the foundation for future experiments investigating communication between nitrifying bacteria, the purpose of QS in these microorganisms, and the manipulation of QS during nitrification. [ABSTRACT FROM AUTHOR]

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 1.0mMor 0.2mMsodium 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. [ABSTRACT FROM AUTHOR]

Nitrobacter winogradskyi is a chemolithotrophic bacterium that plays a role in the nitrogen cycle by oxidizing nitrite to nitrate. Here, we demonstrate a functional N-acyl-homoserine lactone (acyl-HSL) synthase in this bacterium. The N. winogradskyi genome contains genes encoding a putative acyl-HSL autoinducer synthase (nwi0626, nwiI) and a putative acyl-HSL autoinducer receptor (nwi0627, nwiR) with amino acid sequences 38 to 78% identical to those in Rhodopseudomonas palustris and other Rhizobiales. Expression of nwiI and nwiR correlated with acyl-HSL production during culture. N. winogradskyi produces two distinct acyl-HSLs, N-decanoyl-L-homoserine lactone (C10-HSL) and a monounsaturated acyl-HSL (C10:1-HSL), in a cell-densityand growth phase-dependent manner, during batch and chemostat culture. The acyl-HSLs were detected by bioassay and identified by ultraperformance liquid chromatography with information-dependent acquisition mass spectrometry (UPLC-IDA-MS). The C=C bond in C10:1-HSL was confirmed by conversion into bromohydrin and detection by UPLC-IDA-MS. [ABSTRACT FROM AUTHOR]

Here, we present the 3.9-Mb draft genome sequence of Nitrobacter vulgaris strain Ab 1 , which was isolated from a sewage system in Hamburg, Germany. The analysis of its genome sequence will contribute to our knowledge of nitrite-oxidizing bacteria and acyl-homoserine lactone quorum sensing in nitrifying bacteria., (Copyright © 2017 Mellbye et al.)

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, NO 2 , and N 2 O), 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 NO x increased and production of N 2 O 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., Importance: Bacterial cell-cell signaling, or quorum sensing (QS), is a method of bacterial communication and gene regulation that is well studied in bacteria. However, little is known about the purpose of QS in many environmentally important bacteria. Here, we demonstrate quorum quenching coupled with mRNA-Seq to identify QS-controlled genes and phenotypes in Nitrobacter winogradskyi, a nitrite-oxidizing bacterium. Nitrite oxidizers play an important role in the nitrogen cycle though their participation in nitrification, the aerobic oxidation of ammonia to nitrate via nitrite. Our quorum quenching approach revealed that QS influences production and consumption of environmentally important nitrogen oxide gases (NO, NO 2 , and N 2 O) in N. winogradskyi This study demonstrated a novel technique for studying QS in difficult-to-work-with microorganisms and showed that nitrite oxidizers might also contribute to nitrification-dependent production of nitrogen oxide gases that contribute to global warming., (Copyright © 2016 Mellbye et al.)

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

Two types of phosphorodiamidate morpholino oligomers (PMOs) were tested for inhibition of growth of Salmonella enterica serovar Typhimurium. Both PMOs have the same 11-base sequence that is antisense to the region near the start codon of acpP, which is essential for lipid biosynthesis and viability. To the 3' end of each is attached the membrane-penetrating peptide (RXR)4XB (R, X, and B indicate arginine, 6-aminohexanoic acid, and beta-alanine, respectively). One peptide-PMO (AcpP PPMO) has no charge on the PMO moiety. The second PPMO has three cations (piperazine) attached to the phosphorodiamidate linkages (3+Pip-AcpP PPMO). A scrambled-sequence PPMO (Scr PPMO) was synthesized for each type of PMO. The MICs of AcpP PPMO, 3+Pip-AcpP PPMO, and either one of the Scr PPMOs were 1.25 microM (7 microg/ml), 0.156 microM (0.94 microg/ml), and >160 microM (>900 microg/ml), respectively. 3+Pip-AcpP PPMO at 1.25 or 2.5 microM significantly reduced the growth rates of pure cultures, whereas AcpP PPMO or either Scr PPMO had no effect. However, the viable cell count was significantly reduced at either concentration of 3+Pip-AcpP PPMO or AcpP PPMO, but not with either Scr PPMO. In other experiments, macrophages were infected intracellularly with S. enterica and treated with 3 microM 3+Pip-AcpP PPMO. Intracellular bacteria were reduced >99% with 3+Pip-AcpP PPMO, whereas intracellular bacteria increased 3 orders of magnitude in untreated or Scr PPMO-treated cultures. We conclude that either AcpP PPMO or 3+Pip-AcpP PPMO inhibited growth of S. enterica in pure culture and that 3+Pip-AcpP PPMO reduced intracellular viability of S. enterica in macrophages.

The potency of antisense peptide-phosphorodiamidate morpholino oligomers (PPMOs) was improved by varying the peptide composition. An antisense phosphorodiamidate morpholino oligomer (PMO) complementary to the mRNA of the essential gene acpP (which encodes the acyl carrier protein required for lipid biosynthesis) in Escherichia coli was conjugated to the 5' ends of various cationic membrane-penetrating peptides. Each peptide had one of three repeating sequence motifs: C-N-N (motif 1), C-N (motif 2), or C-N-C (motif 3), where C is a cationic residue and N is a nonpolar residue. Variations in the cationic residues included arginine, lysine, and ornithine (O). Variations in the nonpolar residues included phenylalanine, valine, beta-alanine (B), and 6-aminohexanoic acid (X). The MICs of the PPMOs varied from 0.625 to >80 microM (about 3 to 480 microg/ml). Three of the most potent were the (RX)(6)B-, (RXR)(4)XB-, and (RFR)(4)XB-AcpP PMOs, which were further tested in mice infected with E. coli. The (RXR)(4)XB-AcpP PMO was the most potent of the three conjugates tested in mice. The administration of 30 microg (1.5 mg/kg of body weight) (RXR)(4)XB-AcpP PMO at 15 min postinfection reduced CFU/ml in blood by 10(2) to 10(3) within 2 to 12 h compared to the numbers in water-treated controls. All mice treated with 30 microg/dose of (RXR)(4)XB-AcpP PMO survived infection, whereas all water-treated mice died 12 h postinfection. The reduction in CFU/ml in blood was proportional to the dose of PPMO from 30 to 300 microg/ml. In summary, the C-N-C motif was more effective than the other two motifs, arginine was more effective than lysine or ornithine, phenylalanine was more effective than 6-aminohexanoic acid in vitro but not necessarily in vivo, and (RXR)(4)XB-AcpP PMO reduced bacterial infection and promoted survival at clinically relevant doses.