The Antibiotic Dosage of Fastest Resistance Evolution: Gene Amplifications Underpinning the Inverted-U

To determine the dosage at which antibiotic resistance evolution is most rapid, we treated Escherichia coli in vitro, deploying the antibiotic erythromycin at dosages ranging from zero to high. Adaptation was fastest just below erythromycin’s minimal inhibitory concentration (MIC) and genotype-phenotype correlations determined from whole genome sequencing revealed the molecular basis: simultaneous selection for copy number variation in three resistance mechanisms which exhibited an “inverted-U” pattern of dose-dependence, as did several insertion sequences and an integron. Many genes did not conform to this pattern, however, reflecting changes in selection as dose increased: putative media adaptation polymorphisms at zero antibiotic dosage gave way to drug target (ribosomal RNA operon) amplification at mid dosages whereas prophage-mediated drug efflux amplifications dominated at the highest dosages. All treatments exhibited E. coli increases in the copy number of efflux operons acrAB and emrE at rates that correlated with increases in population density. For strains where the inverted-U was no longer observed following the genetic manipulation of acrAB, it could be recovered by prolonging the antibiotic treatment at subMIC dosages.     

Syntrophic interactions improve power production in formic acid fed MFCs operated with set anode potentials or fixed resistances

Formic acid is a highly energetic electron donor but it has previously resulted in low power densities in microbial fuel cells (MFCs). Three different set anode potentials (-0.30, -0.15, and +0.15 V; vs. a standard hydrogen electrode, SHE) were used to evaluate syntrophic interactions in bacterial communities for formic acid degradation relative to a non-controlled, high resistance system (1,000 Ω external resistance). No current was generated at -0.30 V, suggesting a lack of direct formic acid oxidation (standard reduction potential: -0.40 V). More positive potentials that allowed for acetic acid utilization all produced current, with the best performance at -0.15 V. The anode community in the -0.15 V reactor, based on 16S rDNA clone libraries, was 58% Geobacter sulfurreducens and 17% Acetobacterium, with lower proportions of these genera found in the other two MFCs. Acetic acid was detected in all MFCs suggesting that current generation by G. sulfurreducens was dependent on acetic acid production by Acetobacterium. When all MFCs were subsequently operated at an external resistance for maximum power production (100 Ω for MFCs originally set at -0.15 and +0.15 V; 150 Ω for the control), they produced similar power densities and exhibited the same midpoint potential of -0.15 V in first derivative cyclic voltammetry scans. All of the mixed communities converged to similar proportions of the two predominant genera (ca. 52% G. sulfurreducens and 22% Acetobacterium). These results show that syntrophic interactions can be enhanced through setting certain anode potentials, and that long-term performance produces stable and convergent communities.     

Establishment and characterization of an epithelial cell line from thymus of Catla catla (Hamilton, 1822)

A cell line, CTE, derived from catla (Catla catla) thymus has been established by explant method and subcultured for more than 70 passages over a period of 400 days. The cell line has been maintained in L-15 (Leibovitz) medium supplemented with 10% fetal bovine serum. CTE cell line consists of homogeneous population of epithelial-like cells and grows optimally at 28 °C. Karyotype analysis revealed that the modal chromosome number of CTE cells was 50. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CTE cell line originated from catla. Significant green fluorescent signals were observed when the cell line was transfected with phrGFP II-N mammalian expression vector, indicating its potential utility for transgenic and genetic manipulation studies. The CTE cells showed strong positivity for cytokeratin, indicating that cell line was epithelial in nature. The flow cytometric analysis of cell line revealed a higher number of cells in S-phase at 48 h, suggesting a high growth rate. The extracellular products of Vibrio cholerae MTCC 3904 were toxic to the CTE cells. This cell line was not susceptible to fish betanodavirus, the causative agent of viral nervous necrosis in a large variety of marine fish.     

High-throughput isotopic analysis of RNA microarrays to quantify microbial resource use

Most microorganisms remain uncultivated, and typically their ecological roles must be inferred from diversity and genomic studies. To directly measure functional roles of uncultivated microbes, we developed Chip-stable isotope probing (SIP), a high-sensitivity, high-throughput SIP method performed on a phylogenetic microarray (chip). This approach consists of microbial community incubations with isotopically labeled substrates, hybridization of the extracted community rRNA to a microarray and measurement of isotope incorporation—and therefore substrate use—by secondary ion mass spectrometer imaging (NanoSIMS). Laboratory experiments demonstrated that Chip-SIP can detect isotopic enrichment of 0.5 atom % ¹³C and 0.1 atom % ¹⁵N, thus permitting experiments with short incubation times and low substrate concentrations. We applied Chip-SIP analysis to a natural estuarine community and quantified amino acid, nucleic acid or fatty acid incorporation by 81 distinct microbial taxa, thus demonstrating that resource partitioning occurs with relatively simple organic substrates. The Chip-SIP approach expands the repertoire of stable isotope-enabled methods available to microbial ecologists and provides a means to test genomics-generated hypotheses about biogeochemical function in any natural environment.     

Physico-chemical characteristics of a permanent Spanish hypersaline lake: La Salada de Chiprana (NE Spain)

La Salada de Chiprana Lake, located in the Ebro River basin, northeastern Spain, is the only permanent and deep water hypersaline ecosystem in all of western Europe. With a total surface of 31 ha and a maximum depth of 5.6 m, it has several basins bounded by elongated sandstone-bodies or ribbons which are paleochannels of Miocene age. Its salinity varied from 30 to 73 g 1^–1 during the 1989 hydrological cycle and the most abundant ions were magnesium and sulphate. Depth-time distributions of major physico-chemical variables demonstrated that the lake was stratified in two distinctive layers during most of the year. The chemocline disappeared only in October, with the complete overturn of the water column. In the deep water, three conditions occurred which allowed development of green sulphur bacteria populations: (1) oxygen depletion, (2) presence of hydrogen sulphide and (3) presence of light. Benthic microbial mats covered the sediments of shallow shores of moderate slope.     

Modulation of N-type calcium channel activity by G-proteins and protein kinase C

N-type voltage-gated calcium channel activity in rat superior cervical ganglion neurons is modulated by a variety of pathways. Activation of heterotrimeric G-proteins reduces whole-cell current amplitude, whereas phosphorylation by protein kinase C leads to an increase in current amplitude. It has been proposed that these two distinct pathways converge on the channel's pore-forming alpha(1B) subunit, such that the actions of one pathway can preclude those of the other. In this study, we have characterized further the actions of PKC on whole-cell barium currents in neonatal rat superior cervical ganglion neurons. We first examined whether the effects of G-protein-mediated inhibition and phosphorylation by PKC are mutually exclusive. G-proteins were activated by including 0.4 mM GTP or 0.1 mM GTP-gamma-S in the pipette, and PKC was activated by bath application of 500 nM phorbol 12-myristate 13-acetate (PMA). We found that activated PKC was unable to reverse GTP-gamma-S-induced inhibition unless prepulses were applied, indicating that reversal of inhibition by phosphorylation appears to occur only after dissociation of the G-protein from the channel. Once inhibition was relieved, activation of PKC was sufficient to prevent reinhibition of current by G-proteins, indicating that under phosphorylating conditions, channels are resistant to G-protein-mediated modulation. We then examined what effect, if any, phosphorylation by PKC has on N-type barium currents beyond antagonizing G-protein-mediated inhibition. We found that, although G-protein activation significantly affected peak current amplitude, fast inactivation, holding-potential-dependent inactivation, and voltage-dependent activation, when G-protein activation was minimized by dialysis of the cytoplasm with 0.1 mM GDP-beta-S, these parameters were not affected by bath application of PMA. These results indicate that, under our recording conditions, phosphorylation by PKC has no effect on whole-cell N-type currents, other than preventing inhibition by G-proteins.     

VOCs-mediated hormonal signaling and crosstalk with plant growth promoting microbes

In the natural environment, plants communicate with various microorganisms (pathogenic or beneficial) and exhibit differential responses. In recent years, research on microbial volatile compounds (MVCs) has revealed them to be simple, effective and efficient groups of compounds that modulate plant growth and developmental processes. They also interfere with the signaling process. Different MVCs have been shown to promote plant growth via improved photosynthesis rates, increased plant resistance to pathogens, activated phytohormone signaling pathways, or, in some cases, inhibit plant growth, leading to death. Regardless of these exhibited roles, the molecules responsible, the underlying mechanisms, and induced specific metabolic/molecular changes are not fully understood. Here, we review current knowledge on the effects of MVCs on plants, with particular emphasis on their modulation of the salicylic acid, jasmonic acid/ethylene, and auxin signaling pathways. Additionally, opportunities for further research and potential practical applications presented.     

Relative importance of the effect of 2,4-D,glyphosate, and environmental variables on the soil microbial biomass

Two post-emergence herbicides (glyphosate and 2,4-D) were applied at field application levels to tilled field plots in a mixed cropping area in south-central Alberta. The effects of these chemicals on certain variables associated with microbial biomass and activity were monitored in these plots (as well as corresponding control plots) for 45 days. Glyphosate did not influence any of the microbial variables tested. Addition of 2,4-D significantly influenced all microbial variables investigated but these effects were transient, being detectable only within the first 1–5 days of herbicide addition. The effects of 2,4-D addition on the microbial variables tested, even when significant, were typically small and probably of little ecological consequence especially when spatial and temporal variation in these variables is taken into account.     

Effects of fire on soil respiration,ATP content and enzyme activities in Mediterranean maquis

Question: Do low or high intensity fires affect micro‐organism activity in the upper soil layer of Mediterranean maquis? Location: 600 m from the sea in the Nature Reserve of Castel Volturno (Campania, southern Italy, 40°57’N; 13°55’E). Methods: Soil respiration was measured in situ on intact soil; enzyme activity (cellulase, xylanase, invertase, trehalase and protease) and ATP content were measured on soil samples collected under three species of maquis vegetation: Phillyrea angustifolia L., Myrtus communis L. and Cistus incanus L. Results: Soil microbial respiration showed no significant differences in CO₂ flux in treated and untreated plots, but the ATP content in the soil under C. incanus and M. communis was lower in the treated plots for most of the study period. In the soil under Ph. angustifolia, ATP content was low only for one week after fire. The reduction was more marked in the samples from ‘high fire intensity’ than from ‘low fire intensity’ plots. Soil respiration and ATP content exhibited seasonal variations linked to soil water content. Among the enzyme activity measured in the soil under the three plant covers, only invertase declined in burned plots throughout the study period, particularly in the ‘high fire intensity’ plots. Activity of the enzymes cellulase, xylanase, trehalase and protease had a different sensitivity depending on the respective shrub cover. Conclusions: Impact of fire on soil microbial activity is largely dependent on vegetation mosaic and species identity.     

Suppression of Pythium ultimum by Biowaste Composts in Relation to Compost Microbial Biomass, Activity and Content of Phenolic Compounds

Seventeen composts from separately collected organic household waste plus one bark compost and one compost from grape marc were analysed for suppression of Pythium ultimum, phytotoxicity, microbial biomass and activity, substrate-induced respiration, extractible phenolic compounds and other physical and chemical parameters. Nine of the samples were mildly suppressive to P. ultimum, the others were conducive. The bark compost sample was strongly suppressive. Therefore of the examined composts, only the bark could be used to exert an economically relevant control of P. ultimum in horticultural media. A large part of the compost samples was slightly phytotoxic. Microbial biomass and SIR had only weak correlations with disease incidence. Microbial activity and content of extractible phenolics were positively correlated with disease incidence. None of the tested parameters were therefore suitable as a predictive test for suppression of P. ultimum with the compost samples used in this study.