Nanoscale Carbonate Ion-Selective Amperometric/Voltammetric Probes Based on Ion-Ionophore Recognition at the Organic/Water Interface: Hidden Pieces of the Puzzle in the Nanoscale Phase.

Here, we report on the successful demonstration and application of carbonate (CO32-) ion-selective amperometric/voltammetric nanoprobes based on facilitated ion transfer (IT) at the nanoscale interface between two immiscible electrolyte solutions. This electrochemical study reveals critical factors to govern CO32--selective nanoprobes using broadly available Simon-type ionophores forming a covalent bond with CO32-, i.e., slow dissolution of lipophilic ionophores in the organic phase, activation of hydrated ionophores, peculiar solubility of a hydrated ion-ionophore complex near the interface, and cleanness at the nanoscale interface. These factors are experimentally confirmed by nanopipet voltammetry, where a facilitated CO32- IT is studied with a nanopipet filled with an organic phase containing the trifluoroacetophenone derivative CO32-ionophore (CO32-ionophore VII) by voltammetrically and amperometrically sensing CO32- in water. Theoretical assessments of reproducible voltammetric data confirm that the dynamics of CO32- ionophore VII-facilitated ITs (FITs) follows the one-step electrochemical (E) mechanism controlled by both water-finger formation/dissociation and ion-ionophore complexation/dissociation during interfacial ITs. The yielded rate constant, k0 = 0.048 cm/s, is very similar to the reported values of other FIT reactions using ionophores forming non-covalent bonds with ions, implying that a weak binding between CO32- ion-ionophore enables us to observe FITs by fast nanopipet voltammetry regardless of the nature of bondings between the ion and ionophore. The analytical utility of CO32--selective amperometric nanoprobes is further demonstrated by measuring the CO32- concentration produced by metal-reducing bacteria Shewanella oneidensis MR-1 as a result of organic fuel oxidation in bacterial growth media in the presence of various interferents such as H2PO4-, Cl-, and SO42-.

Birnessite films are sensitive indicators of microbial manganese reduction in soil.

Manganese (Mn) and Fe indicator of reduction in soils (IRIS) devices are low-cost, convenient tools for identifying reducing conditions in soils. Because Mn is reduced at similar redox potentials as nitrate, there is considerable interest in using Mn IRIS tools for understanding microbial reduction of Mn as a surrogate for processes such as denitrification. However, the sensitivity of these devices to differences in Mn-reducing capacity has not been empirically investigated. Here we have found that the rate of birnessite paint removal from Mn IRIS films exposed to a twofold dilution series of the Mn-reducing bacterium Shewanella oneidensis is directly proportional to the number of S. oneidensis cells added. Thus, regularly monitored birnessite IRIS sensors are capable of indicating twofold differences in Mn reduction in soil and can be used to measure relative Mn reduction rates over time in a single location or compare and contrast Mn reduction rates across soil types.

Shewanella oneidensis Hfq promotes exponential phase growth, stationary phase culture density, and cell survival.

BACKGROUND: Hfq is an RNA chaperone protein that has been broadly implicated in sRNA function in bacteria. Here we describe the construction and characterization of a null allele of the gene that encodes the RNA chaperone Hfq in Shewanella oneidensis strain MR-1, a dissimilatory metal reducing bacterium. RESULTS: Loss of hfq in S. oneidensis results in a variety of mutant phenotypes, all of which are fully complemented by addition of a plasmid-borne copy of the wild type hfq gene. Aerobic cultures of the hfq∆ mutant grow more slowly through exponential phase than wild type cultures, and hfq∆ cultures reach a terminal cell density in stationary phase that is ~2/3 of that observed in wild type cultures. We have observed a similar growth phenotype when the hfq∆ mutant is cultured under anaerobic conditions with fumarate as the terminal electron acceptor, and we have found that the hfq∆ mutant is defective in Cr(VI) reduction. Finally, the hfq∆ mutant exhibits a striking loss of colony forming units in extended stationary phase and is highly sensitive to oxidative stress induced by H2O2 or methyl viologen (paraquat). CONCLUSIONS: The hfq mutant in S. oneidensis exhibits pleiotropic phenotypes, including a defect in metal reduction. Our results also suggest that hfq mutant phenotypes in S. oneidensis may be at least partially due to increased sensitivity to oxidative stress.

Capicua regulates cell proliferation downstream of the receptor tyrosine kinase/ras signaling pathway.

Signaling via the receptor tyrosine kinase (RTK)/Ras pathway promotes tissue growth during organismal development and is increased in many cancers [1]. It is still not understood precisely how this pathway promotes cell growth (mass accumulation). In addition, the RTK/Ras pathway also functions in cell survival, cell-fate specification, terminal differentiation, and progression through mitosis [2-7]. An important question is how the same canonical pathway can elicit strikingly different responses in different cell types. Here, we show that the HMG-box protein Capicua (Cic) restricts cell growth in Drosophila imaginal discs, and its levels are, in turn, downregulated by Ras signaling. Moreover, unlike normal cells, the growth of cic mutant cells is undiminished in the complete absence of a Ras signal. In addition to a general role in growth regulation, the importance of cic in regulating cell-fate determination downstream of Ras appears to vary from tissue to tissue. In the developing eye, the analysis of cic mutants shows that the functions of Ras in regulating growth and cell-fate determination are separable. Thus, the DNA-binding protein Cic is a key downstream component in the pathway by which Ras regulates growth in imaginal discs.

A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.

Biofilms are surface-attached microbial communities with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts. One of the best-known of these biofilm-specific properties is the development of antibiotic resistance that can be up to 1,000-fold greater than planktonic cells. We report a genetic determinant of this high-level resistance in the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa. We have identified a mutant of P. aeruginosa that, while still capable of forming biofilms with the characteristic P. aeruginosa architecture, does not develop high-level biofilm-specific resistance to three different classes of antibiotics. The locus identified in our screen, ndvB, is required for the synthesis of periplasmic glucans. Our discovery that these periplasmic glucans interact physically with tobramycin suggests that these glucose polymers may prevent antibiotics from reaching their sites of action by sequestering these antimicrobial agents in the periplasm. Our results indicate that biofilms themselves are not simply a diffusion barrier to these antibiotics, but rather that bacteria within these microbial communities employ distinct mechanisms to resist the action of antimicrobial agents.

Genes affecting cell competition in Drosophila.

Cell competition is a homeostatic mechanism that regulates the size attained by growing tissues. We performed an unbiased genetic screen for mutations that permit the survival of cells being competed due to haplo-insufficiency for RpL36. Mutations that protect RpL36 heterozygous clones include the tumor suppressors expanded, hippo, salvador, mats, and warts, which are members of the Warts pathway, the tumor suppressor fat, and a novel tumor-suppressor mutation. Other hyperplastic or neoplastic mutations did not rescue RpL36 heterozygous clones. Most mutations that rescue cell competition elevated Dpp-signaling activity, and the Dsmurf mutation that elevates Dpp signaling was also hyperplastic and rescued. Two nonlethal, nonhyperplastic mutations prevent the apoptosis of Minute heterozygous cells and suggest an apoptosis pathway for cell competition . In addition to rescuing RpL36 heterozygous cells, mutations in Warts pathway genes were supercompetitors that could eliminate wild-type cells nearby. The findings show that differences in Warts pathway activity can lead to competition and implicate the Warts pathway, certain other tumor suppressors, and novel cell death components in cell competition, in addition to the Dpp pathway implicated by previous studies. We suggest that cell competition might occur during tumor development in mammals.

The Drosophila F-box protein Fbxl7 binds to the protocadherin fat and regulates Dachs localization and Hippo signaling.

The Drosophila protocadherin Fat (Ft) regulates growth, planar cell polarity (PCP) and proximodistal patterning. A key downstream component of Ft signaling is the atypical myosin Dachs (D). Multiple regions of the intracellular domain of Ft have been implicated in regulating growth and PCP but how Ft regulates D is not known. Mutations in Fbxl7, which encodes an F-box protein, result in tissue overgrowth and abnormalities in proximodistal patterning that phenocopy deleting a specific portion of the intracellular domain (ICD) of Ft that regulates both growth and PCP. Fbxl7 binds to this same portion of the Ft ICD, co-localizes with Ft to the proximal edge of cells and regulates the levels and asymmetry of D at the apical membrane. Fbxl7 can also regulate the trafficking of proteins between the apical membrane and intracellular vesicles. Thus Fbxl7 functions in a subset of pathways downstream of Ft and links Ft to D localization.

Reduced heme levels underlie the exponential growth defect of the Shewanella oneidensis hfq mutant.

The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA) function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA), the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

The Drosophila tumor suppressors Expanded and Merlin differentially regulate cell cycle exit, apoptosis, and Wingless signaling.

Mutations that inactivate either merlin (mer) or expanded (ex) result in increased cell growth and proliferation in Drosophila. Both Mer and Ex are members of the Band 4.1 protein superfamily, and, based on analyses of mer ex double mutants, they are proposed to function together in at least a partially redundant manner upstream of the Hippo (Hpo) and Warts (Wts) proteins to regulate cell growth and division. By individually analyzing ex and mer mutant phenotypes, we have found important qualitative and quantitative differences in the ways Mer and Ex function to regulate cell proliferation and cell survival. Though both mer and ex restrict cell and tissue growth, ex clones exhibit delayed cell cycle exit in the developing eye, while mer clones do not. Conversely, loss of mer substantially compromises normal developmental apoptosis in the pupal retina, while loss of ex has only mild effects. Finally, ex has a role in regulating Wingless protein levels in the eye that is not obviously shared by either mer or hpo. Taken together, our data suggest that Mer and Ex differentially regulate multiple downstream pathways.

The Drosophila homolog of the putative phosphatidylserine receptor functions to inhibit apoptosis.

Exposure of phosphatidylserine is a conserved feature of apoptotic cells and is thought to act as a signal for engulfment of the cell corpse. A putative receptor for phosphatidylserine (PSR) was previously identified in mammalian systems. This receptor is proposed to function in engulfment of apoptotic cells, although gene ablation of PSR has resulted in a variety of phenotypes. We examined the role of the predicted Drosophila homolog of PSR (dPSR) in apoptotic cell engulfment and found no obvious role for dPSR in apoptotic cell engulfment by phagocytes in the embryo. In addition, dPSR is localized to the nucleus, inconsistent with a role in apoptotic cell recognition. However, we were surprised to find that overexpression of dPSR protects from apoptosis, while loss of dPSR enhances apoptosis in the developing eye. The increased apoptosis is mediated by the head involution defective (Wrinkled) gene product. In addition, our data suggest that dPSR acts through the c-Jun-NH(2) terminal kinase pathway to alter the sensitivity to cell death.

Drosophila Omi, a mitochondrial-localized IAP antagonist and proapoptotic serine protease.

Although essential in mammals, in flies the importance of mitochondrial outer membrane permeabilization for apoptosis remains highly controversial. Herein, we demonstrate that Drosophila Omi (dOmi), a fly homologue of the serine protease Omi/HtrA2, is a developmentally regulated mitochondrial intermembrane space protein that undergoes processive cleavage, in situ, to generate two distinct inhibitor of apoptosis (IAP) binding motifs. Depending upon the proapoptotic stimulus, mature dOmi is then differentially released into the cytosol, where it binds selectively to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC. This interaction alone, however, is insufficient to promote apoptosis, as dOmi fails to displace the effector caspase DrICE from the BIR1 domain in DIAP1. Rather, dOmi alleviates DIAP1 inhibition of all caspases by proteolytically degrading DIAP1 and induces apoptosis both in cultured cells and in the developing fly eye. In summary, we demonstrate for the first time in flies that mitochondrial permeabilization not only occurs during apoptosis but also results in the release of a bona fide proapoptotic protein.

Exo-oligosaccharides of Rhizobium sp. strain NGR234 are required for symbiosis with various legumes.

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with beta-1,3, beta-1,4, beta-1,6, alpha-1,3, and alpha-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGROmegaexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGROmegaexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGROmegaexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, approximately 50 microg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-beta-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.

A LuxR homolog controls production of symbiotically active extracellular polysaccharide II by Sinorhizobium meliloti.

Production of complex extracellular polysaccharides (EPSs) by the nitrogen-fixing soil bacterium Sinorhizobium meliloti is required for efficient invasion of root nodules on the host plant alfalfa. Any one of three S. meliloti polysaccharides, succinoglycan, EPS II, or K antigen, can mediate infection thread initiation and extension (root nodule invasion) on alfalfa. Of these three polysaccharides, the only symbiotically active polysaccharide produced by S. meliloti wild-type strain Rm1021 is succinoglycan. The expR101 mutation is required to turn on production of symbiotically active forms of EPS II in strain Rm1021. In this study, we have determined the nature of the expR101 mutation in S. meliloti. The expR101 mutation, a spontaneous dominant mutation, results from precise, reading frame-restoring excision of an insertion sequence from the coding region of expR, a gene whose predicted protein product is highly homologous to the Rhizobium leguminosarum bv. viciae RhiR protein and a number of other homologs of Vibrio fischeri LuxR that function as receptors for N-acylhomoserine lactones (AHLs) in quorum-sensing regulation of gene expression. S. meliloti ExpR activates transcription of genes involved in EPS II production in a density-dependent fashion, and it does so at much lower cell densities than many quorum-sensing systems. High-pressure liquid chromatographic fractionation of S. meliloti culture filtrate extracts revealed at least three peaks with AHL activity, one of which activated ExpR-dependent expression of the expE operon.