Construction of a ColD cda Promoter-Based SOS-Green Fluorescent Protein Whole-Cell Biosensor with Higher Sensitivity toward Genotoxic Compounds than Constructs Based on recA, umuDC, or sulA Promoters

Four different green fluorescent protein (GFP)-based whole-cell biosensors were created based on the DNA damage inducible SOS response of Escherichia coli in order to evaluate the sensitivity of individual SOS promoters toward genotoxic substances. Treatment with the known carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) revealed that the promoter for the ColD plasmid-borne cda gene had responses 12, 5, and 3 times greater than the recA, sulA, and umuDC promoters, respectively, and also considerably higher sensitivity. Furthermore, we showed that when the SOS-GFP construct was introduced into an E. coli host deficient in the tolC gene, the minimal detection limits toward mitomycin C, MNNG, nalidixic acid, and formaldehyde were lowered to 9.1 nM, 0.16 μM, 1.1 μM, and 141 μM, respectively, which were two to six times lower than those in the wild-type strain. This study thus presents a new SOS-GFP whole-cell biosensor which is not only able to detect minute levels of genotoxins but, due to its use of the green fluorescent protein, also a reporter system which should be applicable in high-throughput screening assays as well as a wide variety of in situ detection studies.     

Development of a Microbial Model for the Combined Effect of Temperature and pH on Spoilage of Ground Meat, and Validation of the Model under Dynamic Temperature Conditions

The changes in microbial flora and sensory characteristics of fresh ground meat (beef and pork) with pH values ranging from 5.34 to 6.13 were monitored at different isothermal storage temperatures (0 to 20°C) under aerobic conditions. At all conditions tested, pseudomonads were the predominant bacteria, followed by Brochothrix thermosphacta, while the other members of the microbial association (e.g., lactic acid bacteria and Enterobacteriaceae) remained at lower levels. The results from microbiological and sensory analysis showed that changes in pseudomonad populations followed closely sensory changes during storage and could be used as a good index for spoilage of aerobically stored ground meat. The kinetic parameters (maximum specific growth rate [μ_max] and the duration of lag phase [λ]) of the spoilage bacteria were modeled by using a modified Arrhenius equation for the combined effect of temperature and pH. Meat pH affected growth of all spoilage bacteria except that of lactic acid bacteria. The “adaptation work,” characterized by the product of μ_max and λ(μ_max × λ) was found to be unaffected by temperature for all tested bacteria but was affected by pH for pseudomonads and B. thermosphacta. For the latter bacteria, a negative linear correlation between ln(μ_max × λ) and meat pH was observed. The developed models were further validated under dynamic temperature conditions using different fluctuating temperatures. Graphical comparison between predicted and observed growth and the examination of the relative errors of predictions showed that the model predicted satisfactorily growth under dynamic conditions. Predicted shelf life based on pseudomonads growth was slightly shorter than shelf life observed by sensory analysis with a mean difference of 13.1%. The present study provides a “ready-to-use,” well-validated model for predicting spoilage of aerobically stored ground meat. The use of the model by the meat industry can lead to effective management systems for the optimization of meat quality.     

Scalable Production of Recombinant Membrane Active Peptides and Its Potential as a Complementary Adjunct to Conventional Chemotherapeutics

The production of short anticancer peptides in recombinant form is an alternative method for costly chemical manufacturing. However, the limitations of host toxicity, bioactivity and column purification have impaired production in mass quantities. In this study, short cationic peptides were produced in aggregated inclusion bodies by double fusion with a central protein that has anti-cancer activity. The anticancer peptides Tachiplicin I (TACH) and Latarcin 1 (LATA) were fused with the N- and C-terminus of the MAP30 protein, respectively. We successfully produced the recombinant TACH-MAP30-LATA protein and MAP30 alone in E. coli that represented 59% and 68% of the inclusion bodies. The purified form of the inclusion bodies was prepared by eliminating host cell proteins through multiple washing steps and semi-solubilization in alkaline buffer. The purified active protein was recovered by inclusive solubilization at pH 12.5 in the presence of 2 M urea and refolded in alkaline buffer containing oxides and reduced glutathione. The peptide-fusion protein showed lower CC₅₀ values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components. The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane. The cancer chemotherapy agent doxorubicin showed higher efficiency and selectivity against cancer cells in combination with the peptide- fusion protein. This study provides new data on the mass production of short anticancer peptides as inclusion bodies in E. coli by fusion with a central protein that has similar activity. The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.     

Inhibition of Photosynthesis in Certain Algae by Extreme Red Light

This paper shows that in Porphyridium cruentum and in Chlorella pyrenoidosa (but apparently not in Anacystis nidulans) “extreme red” light (> 720 mμ) can inhibit photosynthesis produced by “far red” light (up to 720 mμ). From the action spectrum of this phenomenon, it appears that an unknown pigment with an absorption band around 745 mμ must be responsible for it.     

Aerosols of Mycoplasmas, L Forms, and Bacteria: Comparison of Particle Size, Viability, and Lethality of Ultraviolet Radiation

Aerosols of microorganisms were tested for particle size by use of an Andersen sampler. Mycoplasma aerosols had an average count median diameter (CMD) of 2.1 ± 0.5 μ. Staphylococcus aureus L forms gave an average CMD of 4.6 ± 1.7 μ; the diphtheroid L form, a CMD of 3.4 ± 0.3 μ. Escherichia coli had a CMD of 5.4 ± 2.5 μ; Neisseria sicca, 3.3 ± 0.5 μ; N. meningitidis, 3.4 ± 0.2 μ. S. aureus ATCC 6538, the parent strain of the L form, yielded a CMD of 3.9 ± 1.2 μ. Candida albicans gave an average CMD of 5.9 ± 1.4 μ. All organisms tested survived aerosolizing and could be recovered in viable form for at least 1 hr. Ultraviolet radiation at 2,537 A destroyed the bacteria and mycoplasmas instantaneously, and destroyed 87% of the L forms of S. aureus, 69% of the diphtheroid L form, and 98% of the C. albicans cells. After irradiation, viable particles of the L form and C. albicans aerosols were consistently larger, indicating that clumping led to survival. Submicron size particles were found in aerosols of all species tested except C. albicans.     

Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant,Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T₁) and reached a maximum after the completion of CaDPA release (T_release) and spore cortex lysis (T_lysis).     

XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”

“Candidatus Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “Ca. Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from “Ca. Methylomirabilis oxyfera” that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of “Ca. Methylomirabilis oxyfera” cells and catalyzed methanol oxidation with appreciable specific activity and affinity (V_max of 10 μmol min⁻¹ mg⁻¹ protein, K_m of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from “Ca. Methylomirabilis oxyfera” is the first representative of type 1 XoxF proteins to be described.     

In glucose-limited continuous culture the minimum substrate concentration for growth,smin, is crucial in the competition between the enterobacterium Escherichia coli and Chelatobacter heintzii,an environmentally abundant bacterium

The competition for glucose between Escherichia coli ML30, a typical copiotrophic enterobacterium and Chelatobacter heintzii ATCC29600, an environmentally successful strain, was studied in a carbon-limited culture at low dilution rates. First, as a base for modelling, the kinetic parameters μ_max and K_s were determined for growth with glucose. For both strains, μ_max was determined in batch culture after different precultivation conditions. In the case of C. heintzii, μ_max was virtually independent of precultivation conditions. When inoculated into a glucose-excess batch culture medium from a glucose-limited chemostat run at a dilution rate of 0.075 h⁻¹ C. heintzii grew immediately with a μ_max of 0.17±0.03 h⁻¹. After five transfers in batch culture, μ_max had increased only slightly to 0.18±0.03 h⁻¹. A different pattern was observed in the case of E. coli. Inoculated from a glucose-limited chemostat at D=0.075 h⁻¹ into glucose-excess batch medium E. coli grew only after an acceleration phase of ∼3.5 h with a μ_max of 0.52 h⁻¹. After 120 generations and several transfers into fresh medium, μ_max had increased to 0.80±0.03 h⁻¹. For long-term adapted chemostat-cultivated cells, a K_s for glucose of 15 μg l⁻¹ for C. heintzii, and of 35 μg l⁻¹ for E. coli, respectively, was determined in ¹⁴C-labelled glucose uptake experiments. In competition experiments, the population dynamics of the mixed culture was determined using specific surface antibodies against C. heintzii and a specific 16S rRNA probe for E. coli. C. heintzii outcompeted E. coli in glucose-limited continuous culture at the low dilution rates of 0.05 and 0.075 h⁻¹. Using the determined pure culture parameter values for K_s and μ_max, it was only possible to simulate the population dynamics during competition with an extended form of the Monod model, which includes a finite substrate concentration at zero growth rate (s_min). The values estimated for s_min were dependent on growth rate; at D=0.05 h⁻¹, it was 12.6 and 0 μg l⁻¹ for E. coli and C. heintzii, respectively. To fit the data at D=0.075 h⁻¹, s_min for E. coli had to be raised to 34.9 μg l⁻¹ whereas s_min for C. heintzii remained zero. The results of the mathematical simulation suggest that it is not so much the higher K_s value, which is responsible for the unsuccessful competition of E. coli at low residual glucose concentration, but rather the existence of a significant s_min.     

Correlation of Intracellular Trehalose Concentration with Desiccation Resistance of Soil Escherichia coli Populations

Naturalized soil Escherichia coli populations need to resist common soil desiccation stress in order to inhabit soil environments. In this study, four representative soil E. coli strains and one lab strain, MG1655, were tested for desiccation resistance via die-off experiments in sterile quartz sand under a potassium acetate-induced desiccation condition. The desiccation stress caused significantly lower die-off rates of the four soil strains (0.17 to 0.40 day⁻¹) than that of MG1655 (0.85 day⁻¹). Cellular responses, including extracellular polymeric substance (EPS) production, exogenous glycine betaine (GB) uptake, and intracellular compatible organic solute synthesis, were quantified and compared under the desiccation and hydrated control conditions. GB uptake appeared not to be a specific desiccation response, while EPS production showed considerable variability among the E. coli strains. All E. coli strains produced more intracellular trehalose, proline, and glutamine under the desiccation condition than the hydrated control, and only the trehalose concentration exhibited a significant correlation with the desiccation-contributed die-off coefficients (Spearman''s ρ = −1.0; P = 0.02). De novo trehalose synthesis was further determined for 15 E. coli strains from both soil and nonsoil sources to determine its prevalence as a specific desiccation response. Most E. coli strains (14/15) synthesized significantly more trehalose under the desiccation condition, and the soil E. coli strains produced more trehalose (106.5 ± 44.9 μmol/mg of protein [mean ± standard deviation]) than the nonsoil reference strains (32.5 ± 10.5 μmol/mg of protein).     

Antischistosomal,antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents

BackgroundGhana is endemic for some neglected tropical diseases (NTDs) including schistosomiasis, onchocerciasis and lymphatic filariasis. The major intervention for these diseases is mass drug administration of a few repeatedly recycled drugs which is a cause for major concern due to reduced efficacy of the drugs and the emergence of drug resistance. Evidently, new treatments are needed urgently. Medicinal plants, on the other hand, have a reputable history as important sources of potent therapeutic agents in the treatment of various diseases among African populations, Ghana inclusively, and provide very useful starting points for the discovery of much-needed new or alternative drugs.Methodology/Principal findingsIn this study, extracts of fifteen traditional medicines used for treating various NTDs in local communities were screened in vitro for efficacy against schistosomiasis, onchocerciasis and African trypanosomiasis. Two extracts, NTD-B4-DCM and NTD-B7-DCM, prepared from traditional medicines used to treat schistosomiasis, displayed the highest activity (IC₅₀ = 30.5 μg/mL and 30.8 μg/mL, respectively) against Schistosoma mansoni adult worms. NTD-B2-DCM, also obtained from an antischistosomal remedy, was the most active against female and male adult Onchocera ochengi worms (IC₅₀ = 76.2 μg/mL and 76.7 μg/mL, respectively). Antitrypanosomal assay of the extracts against Trypanosoma brucei brucei gave the most promising results (IC₅₀ = 5.63 μg/mL to 18.71 μg/mL). Incidentally, NTD-B4-DCM and NTD-B2-DCM, also exhibited the greatest antitrypanosomal activities (IC₅₀ = 5.63 μg/mL and 7.12 μg/mL, respectively). Following the favourable outcome of the antitrypanosomal screening, this assay was selected for bioactivity-guided fractionation. NTD-B4-DCM, the most active extract, was fractionated and subsequent isolation of bioactive constituents led to an eupatoriochromene-rich oil (42.6%) which was 1.3-fold (IC₅₀ <0.0977 μg/mL) more active than the standard antitrypanosomal drug, diminazene aceturate (IC₅₀ = 0.13 μg/mL).Conclusion/SignificanceThese findings justify the use of traditional medicines and demonstrate their prospects towards NTDs drug discovery.     

Soil Nitrogen Status Modifies Rice Root Response to Nematode-Bacteria Interactions in the Rhizosphere

It has been hypothesized that faunal activity in the rhizosphere influences root growth via an auxin-dependent pathway. In this study, two methods were used to adjust nematode and bacterial populations within experimental soils. One is “exclusion”, where soil mixed with pig manure was placed in two bags with different mesh sizes (1mm and 5μm diameter), and then surrounded by an outer layer of unamended soil resulting in soil with a greater populations of bacterial-feeding nematodes (1mm) and a control treatment (5μm). The second method is “inoculation”, whereby autoclaved soil was inoculated with bacteria (E. coli and Pseudomonas) and Nematodes (Cephalobus and C. elegans). In order to detect the changes in the rice’s perception of auxin under different nutrient and auxin conditions in the presence of soil bacterial-feeding nematodes, responses of soil chemistry (NH₄⁺, NO₃^- and indole acetic acid (IAA)), rice root growth and the expression of an auxin responsive gene GH3-2 were measured. Results showed that, under low soil nutrient conditions (exclusion), low NO₃^- correlated with increased root branching and IAA correlated with increased root elongation and GH3-2 expression. However, under high soil nutrient conditions (inoculation), a high NH₄⁺ to NO₃^- ratio promoted an increase in root surface area and there was an additional influence of NH₄⁺ and NO₃^- on GH3-2 expression. Thus it was concluded that soil bacterial-feeding nematodes influenced soil nutritional status and soil IAA content, promoting root growth via an auxin dependent pathway that was offset by soil nitrogen status.     

Studies on the Second Emerson Effect in the Hill Reaction in Algal Cells

This paper shows that the “second Emerson effect”¹ exists not only in photosynthesis, but also in the quinone reduction (Hill reaction), in Chlorella pyrenoidosa and Anacystis nidulans. The peaks at 650 mμ, 600 mμ, 560 mμ, 520 mμ, and 480 mμ, observed in the action spectrum of this effect in the Hill reaction in Chorella, are attributable to chlorophyll b; the occurrence of an additional peak at 670 mμ, 620 mμ, and of two (or three) peaks in the blueviolet region suggests that (at least) one form of chlorophyll a contributes to it. In analogy to suggestions made previously in the interpretation of the Emerson effect in photosynthesis, these results are taken as indicating that excitation by light preferentially absorbed by one (or two) forms of chlorophyll a (Chl a 690 + 700), needs support by simultaneous absorption of light in another form of chlorophyll a (Chl a 670)—directly or via energy transfer from chlorophyll b—in order to produce the Hill reaction with its full quantum yield. In Anacystis, the participation of phycocyanin in the Emerson effect in the Hill reaction is revealed by the occurrence, in the action spectrum of this effect, of peaks at about 560 mμ, 610 mμ, and 640 mμ; a peak at 670 mμ, due to Chl a 670, also is present.     

Ochratoxin A Production and Amplified Fragment Length Polymorphism Analysis of Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger Strains Isolated from Grapes in Italy

Ochratoxin A is a potent nephrotoxin and a possible human carcinogen that can contaminate various agricultural products, including grapes and wine. The capabilities of species other than Aspergillus carbonarius within Aspergillus section Nigri to produce ochratoxin A from grapes are uncertain, since strain identification is based primarily on morphological traits. We used amplified fragment length polymorphisms (AFLPs) and genomic DNA sequences (rRNA, calmodulin, and β-tubulin genes) to identify 77 black aspergilli isolated from grape berries collected in a 2-year survey in 16 vineyards throughout Italy. Four main clusters were distinguished, and they shared an AFLP similarity of <25%. Twenty-two of 23 strains of A. carbonarius produced ochratoxin A (6 to 7,500 μg/liter), 5 of 20 strains of A. tubingensis produced ochratoxin A (4 to 130 μg/liter), 3 of 15 strains of A. niger produced ochratoxin A (250 to 360 μg/liter), and none of the 19 strains of Aspergillus “uniseriate” produced ochratoxin A above the level of detection (4 μg/liter). These findings indicate that A. tubingensis is able to produce ochratoxin and that, together with A. carbonarius and A. niger, it may be responsible for the ochratoxin contamination of wine in Italy.     

Determination of Bacterial Cell Dry Mass by Transmission Electron Microscopy and Densitometric Image Analysis

We applied transmission electron microscopy and densitometric image analysis to measure the cell volume (V) and dry weight (DW) of single bacterial cells. The system was applied to measure the DW of Escherichia coli DSM 613 at different growth phases and of natural bacterial assemblages of two lakes, Piburger See and Gossenköllesee. We found a functional allometric relationship between DW (in femtograms) and V (in cubic micrometers) of bacteria (DW = 435 · V^0.86); i.e., smaller bacteria had a higher ratio of DW to V than larger cells. The measured DW of E. coli cells ranged from 83 to 1,172 fg, and V ranged from 0.1 to 3.5 μm³ (n = 678). Bacterial cells from Piburger See and Gossenköllesee (n = 465) had DWs from 3 fg (V = 0.003 μm³) to 1,177 fg (V = 3.5 μm³). Between 40 and 50% of the cells had a DW of less than 20 fg. By assuming that carbon comprises 50% of the DW, the ratio of carbon content to V of individual cells varied from 466 fg of C μm⁻³ for Vs of 0.001 to 0.01 μm³ to 397 fg of C μm⁻³ (0.01 to 0.1 μm³) and 288 fg of C μm⁻³ (0.1 to 1 μm³). Exponentially growing and stationary cells of E. coli DSM 613 showed conversion factors of 254 fg of C μm⁻³ (0.1 to 1 μm³) and 211 fg of C μm⁻³ (1 to 4 μm³), respectively. Our data suggest that bacterial biomass in aquatic environments is higher and more variable than previously assumed from volume-based measurements.     

Isolation of a Multiheme Protein with Features of a Hydrazine-Oxidizing Enzyme from an Anaerobic Ammonium-Oxidizing Enrichment Culture

A multiheme protein having hydrazine-oxidizing activity was purified from enriched culture from a reactor in which an anammox bacterium, strain KSU-1, was dominant. The enzyme has oxidizing activity toward hydrazine but not hydroxylamine and is a 130-kDa homodimer composed of a 62-kDa polypeptide containing eight hemes. It was therefore named hydrazine-oxidizing enzyme (HZO). With cytochrome c as an electron acceptor, the V_max and K_m for hydrazine are 6.2 ± 0.3 μmol/min · mg and 5.5 ± 0.6 μM, respectively. Hydrazine (25 μM) induced an increase in the proportion of reduced form in the spectrum, whereas hydroxylamine (500 μM) did not. Two genes coding for HZO, hzoA and hzoB, were identified within the metagenomic DNA from the culture. The genes encode the same amino acid sequence except for two residues. The sequences deduced from these genes showed low-level identities (<30%) to those of all of the hydroxylamine oxidoreductases reported but are highly homologous to two hao genes found by sequencing the genome of “Candidatus Kuenenia stuttgartiensis” (88% and 89% identities). The purified enzyme might therefore be a novel hydrazine-oxidizing enzyme having a critical role in anaerobic ammonium oxidation.     

Action Spectrum for the Appearance of the 520 mμ Difference Band in Illuminated Chlorella Cells

An action spectrum of the 520 mμ difference band in Chlorella is determined using dim illumination. Pigment (or pigments) absorbing most strongly at and above 680 mμ, probably the so-called “long-wave forms” of chlorophyll a appear to be the primary sensitizer of the 520 mμ effect.