Abundance and Diversity of Viruses in Six Delaware Soils

The importance of viruses in marine microbial ecology has been established over the past decade. Specifically, viruses influence bacterial abundance and community composition through lysis and alter bacterial genetic diversity through transduction and lysogenic conversion. By contrast, the abundance and distribution of viruses in soils are almost completely unknown. This study describes the abundance and diversity of autochthonous viruses in six Delaware soils: two agricultural soils, two coastal plain forest soils, and two piedmont forest soils. Viral abundance was measured using epifluorescence microscopy, while viral diversity was assessed from morphological data obtained through transmission electron microscopy. Extracted soil virus communities were dominated by bacteriophages that demonstrated a wide range of capsid diameters (20 nm to 160 nm) and morphologies, including filamentous forms and phages with elongated capsids. The reciprocal Simpson's index suggests that forest soils harbor more diverse assemblages of viruses, particularly in terms of morphological distribution. Repeated extractions of virus-like particles (VLPs) from soils indicated that the initial round of extraction removes approximately 70% of extractable viruses. Higher VLP abundances were observed in forest soils (1.31 × 10⁹ to 4.17 × 10⁹ g⁻¹ dry weight) than in agricultural soils (8.7 × 10⁸ to 1.1 × 10⁹ g⁻¹ dry weight). Soil VLP abundance was significantly correlated to moisture content (r = 0.988) but not to soil texture. Land use (agricultural or forested) was significantly correlated to both bacterial (r = 0.885) and viral (r = 0.812) abundances, as were soil organic matter and water content. Thus, land use is a significant factor influencing viral abundance and diversity in soils.     

Accumulation of Pharmaceuticals,Enterococcus, and Resistance Genes in Soils Irrigated with Wastewater for Zero to 100 Years in Central Mexico

Irrigation with wastewater releases pharmaceuticals, pathogenic bacteria, and resistance genes, but little is known about the accumulation of these contaminants in the environment when wastewater is applied for decades. We sampled a chronosequence of soils that were variously irrigated with wastewater from zero up to 100 years in the Mezquital Valley, Mexico, and investigated the accumulation of ciprofloxacin, enrofloxacin, sulfamethoxazole, trimethoprim, clarithromycin, carbamazepine, bezafibrate, naproxen, diclofenac, as well as the occurrence of Enterococcus spp., and sul and qnr resistance genes. Total concentrations of ciprofloxacin, sulfamethoxazole, and carbamazepine increased with irrigation duration reaching 95% of their upper limit of 1.4 µg/kg (ciprofloxacin), 4.3 µg/kg (sulfamethoxazole), and 5.4 µg/kg (carbamazepine) in soils irrigated for 19–28 years. Accumulation was soil-type-specific, with largest accumulation rates in Leptosols and no time-trend in Vertisols. Acidic pharmaceuticals (diclofenac, naproxen, bezafibrate) were not retained and thus did not accumulate in soils. We did not detect qnrA genes, but qnrS and qnrB genes were found in two of the irrigated soils. Relative concentrations of sul1 genes in irrigated soils were two orders of magnitude larger (3.15×10⁻³±0.22×10⁻³ copies/16S rDNA) than in non-irrigated soils (4.35×10⁻⁵±1.00×10⁻⁵ copies/16S rDNA), while those of sul2 exceeded the ones in non-irrigated soils still by a factor of 22 (6.61×10^–4±0.59×10⁻⁴ versus 2.99×10⁻⁵±0.26×10⁻⁵ copies/16S rDNA). Absolute numbers of sul genes continued to increase with prolonging irrigation together with Enterococcus spp. 23S rDNA and total 16S rDNA contents. Increasing total concentrations of antibiotics in soil are not accompanied by increasing relative abundances of resistance genes. Nevertheless, wastewater irrigation enlarges the absolute concentration of resistance genes in soils due to a long-term increase in total microbial biomass.     

Environmental Influences on Vibrio Populations in Northern Temperate and Boreal Coastal Waters (Baltic and Skagerrak Seas)

Even if many Vibrio spp. are endemic to coastal waters, their distribution in northern temperate and boreal waters is poorly studied. To identify environmental factors regulating Vibrio populations in a salinity gradient along the Swedish coastline, we combined Vibrio-specific quantitative competitive PCR with denaturant gradient gel electrophoresis-based genotyping. The total Vibrio abundance ranged from 4 × 10³ to 9.6 × 10⁴ cells liter⁻¹, with the highest abundances in the more saline waters of the Skagerrak Sea. Several Vibrio populations were present throughout the salinity gradient, with abundances of single populations ranging from 5 × 10² to 7 × 10⁴ cells liter⁻¹. Clear differences were observed along the salinity gradient, where three populations dominated the more saline waters of the Skagerrak Sea and two populations containing mainly representatives of V. anguillarum and V. aestuarianus genotypes were abundant in the brackish waters of the Baltic Sea. Our results suggest that this apparent niche separation within the genus Vibrio may also be influenced by alternate factors such as nutrient levels and high abundances of dinoflagellates. A V. cholerae/V. mimicus population was detected in more than 50% of the samples, with abundances exceeding 10³ cells liter⁻¹, even in the cold (annual average water temperature of around 5°C) and low-salinity (2 to 4‰) samples from the Bothnian Bay (latitude, 65°N). The unsuspected and widespread occurrence of this population in temperate and boreal coastal waters suggests that potential Vibrio pathogens may also be endemic to cold and brackish waters and hence may represent a previously overlooked health hazard.     

High Abundances of Aerobic Anoxygenic Photosynthetic Bacteria in the South Pacific Ocean

Little is known about the abundance, distribution, and ecology of aerobic anoxygenic phototrophic (AAP) bacteria, particularly in oligotrophic environments, which represent 60% of the ocean. We investigated the abundance of AAP bacteria across the South Pacific Ocean, including the center of the gyre, the most oligotrophic water body of the world ocean. AAP bacteria, Prochlorococcus, and total prokaryotic abundances, as well as bacteriochlorophyll a (BChl a) and divinyl-chlorophyll a concentrations, were measured at several depths in the photic zone along a gradient of oligotrophic conditions. The abundances of AAP bacteria and Prochlorococcus were high, together accounting for up to 58% of the total prokaryotic community. The abundance of AAP bacteria alone was up to 1.94 × 10⁵ cells ml⁻¹ and as high as 24% of the overall community. These measurements were consistent with the high BChl a concentrations (up to 3.32 × 10⁻³ μg liter⁻¹) found at all stations. However, the BChl a content per AAP bacterial cell was low, suggesting that AAP bacteria are mostly heterotrophic organisms. Interestingly, the biovolume and therefore biomass of AAP bacteria was on average twofold higher than that of other prokaryotic cells. This study demonstrates that AAP bacteria can be abundant in various oligotrophic conditions, including the most oligotrophic regime of the world ocean, and can account for a large part of the bacterioplanktonic carbon stock.     

Housefly Larva Vermicomposting Efficiently Attenuates Antibiotic Resistance Genes in Swine Manure,with Concomitant Bacterial Population Changes

Manure from swine treated with antimicrobials as feed additives is a major source for the expansion of the antibiotic resistance gene (ARG) reservoir in the environment. Vermicomposting via housefly larvae (Musca domestica) can be efficiently used to treat manure and regenerate biofertilizer, but few studies have investigated its effect on ARG attenuation. Here, we tracked the abundances of 9 ARGs and the composition and structure of the bacterial communities in manure samples across 6 days of full-scale manure vermicomposting. On day 6, the abundances of genes encoding tetracycline resistance [tet(M), tet(O), tet(Q), and tet(W)] were reduced (P < 0.05), while those of genes encoding sulfonamide resistance (sul1 and sul2) were increased (P < 0.05) when normalized to 16S rRNA. The abundances of tetracycline resistance genes were correlated (P < 0.05) with the changing concentrations of tetracyclines in the manure. The overall diversity and richness of the bacteria significantly decreased during vermicomposting, accompanied by a 100 times increase in the relative abundance of Flavobacteriaceae spp. Variations in the abundances of ARGs were correlated with the changing microbial community structure and the relative abundances of the family Ruminococcaceae, class Bacilli, or phylum Proteobacteria. Vermicomposting, as a waste management practice, can reduce the overall abundance of ARGs. More research is warranted to assess the use of this waste management practice as a measure to attenuate the dissemination of antimicrobial residues and ARGs from livestock production before vermicompost can be safely used as biofertilizer in agroecosystems.     

Chlorine Demand and Inactivation of Fungal Propagules

Conidia of filamentous fungi, vegetative yeast cells, and coliform bacteria were tested to determine their chlorine demand and their sensitivity to chlorine inactivation. Levels of chlorine demand for the various conidia, yeast, and coliforms were, respectively, 3.6 × 10⁻⁹ to 3.2 × 10⁻⁸, 1.2 × 10⁻⁹ to 8.0 × 10⁻⁹, and 2.5 × 10⁻¹¹ to 6.3 × 10⁻¹⁰ mg of chlorine per propagule. Preliminary evidence suggests that the chlorine demand per propagule increases as the number of propagules per milliliter decreases. In general, conidia showed greatest resistance to chlorine inactiviation, followed by the yeast and coliforms. Inactivation by chlorine was influenced by pH, with inactivation (chlorine activity) falling in the order pH 5 > 7 > 8.     

Horizontal and Vertical Distribution of Marine Virioplankton: A Basin Scale Investigation Based on a Global Cruise

Despite the fact that marine viruses have been increasingly studied in the last decade, there is little information on viral abundance and distribution on a global scale. In this study, we report on a global-scale survey covering the Pacific, Atlantic, and Indian Oceans on viral distribution using flow cytometry. Viruses were stained with the SYBR Green I, which targets only dsDNA viruses. The average viral abundance was 1.10±0.73×10⁷ ml⁻¹ in global surface oceans and decreased from the areas with high chlorophyll concentration (on average, 1.47±0.78×10⁷ ml⁻¹) to the oligotrophic subtropical gyres (on average, 6.34±2.18×10⁶ ml⁻¹). On a large-spatial-scale, viruses displayed significant relationships with both heterotrophic and autotrophic picoplankton abundance, suggesting that viral distribution is dependent on their host cell abundance. Our study provided a basin scale pattern of marine viral distributions and their relationship with major host cells, indicating that viruses play a significant role in the global marine ecosystem.     

Development and Application of a Real-Time PCR Approach for Quantification of Uncultured Bacteria in the Central Baltic Sea

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the “Epsilonproteobacteria” related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 × 10³ to 4.4 × 10⁹ copies ml⁻¹ or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 × 10¹ to 2.2 ×10⁶ copies ml⁻¹ or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml⁻¹. The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.     

High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10⁻⁸ to 2 × 10⁻⁶, 1 × 10⁻⁸ to 4 × 10⁻⁸, and <4 × 10⁻⁹ to 4 × 10⁻⁸ per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10⁻⁴ to 1 × 10⁻³, 9 × 10⁻⁴ to 3 × 10⁻³, and 5 × 10⁻⁴ to 4 × 10⁻³ for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6 × 10² to 2.3 × 10⁴ cell equivalents liter⁻¹, whereas GR-corrected abundances ranged from 4.7 × 10³ to 1.6 × 10⁶ cell equivalents liter⁻¹. GR-corrected qPCR results were in good agreement with an independent cell-based direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs.     

An Inexpensive,Accurate, and Precise Wet-Mount Method for Enumerating Aquatic Viruses

Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral abundance in both field and laboratory samples. One current, widely used method to accomplish this with aquatic samples is the “filter mount” method, in which samples are filtered onto costly 0.02-μm-pore-size ceramic filters for enumeration of viruses by epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold-lower materials cost) alternative virus enumeration method in which fluorescently stained samples are wet mounted directly onto slides, after optional chemical flocculation of viruses in samples with viral concentrations of <5 × 10⁷ viruses ml⁻¹. The concentration of viruses in the sample is then determined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus concentrations obtained by using this wet-mount method, with and without chemical flocculation, were significantly correlated with, and had precision equivalent to, those obtained by the filter mount method across concentrations ranging from 2.17 × 10⁶ to 1.37 × 10⁸ viruses ml⁻¹ when tested by using cultivated viral isolates and natural samples from marine and freshwater environments. In summary, the wet-mount method is significantly less expensive than the filter mount method and is appropriate for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations (≥1 × 10⁶ viruses ml⁻¹) encountered in field and laboratory samples.     

Abundance of Viruses in Marine Waters: Assessment by Epifluorescence and Transmission Electron Microscopy

Abundance of bacteria and tiny DNA-associated particles in the upper layer of Japanese coastal and offshore waters was evaluated by epifluorescence microscopy with 0.015-μm-pore-size Nuclepore filters. The number of tiny DNA-associated particles was compared with the abundance of virus particles estimated by transmission electron microscopy. Although a large variation in virus abundance (1.2 × 10⁶ to 35 × 10⁶ ml⁻¹) was obtained with the transmission electron microscopy method, the ratio of 4′,6-diamidino-2-phenylindole-reactive tiny particles to viruses was in a rather narrow range (1.0 to 1.6), indicating that the majority of the tiny DNA-associated particles identified by epifluorescence microscopy were actually virus particles. This result implies the possibility of using epifluorescence microscopy for the evaluation of virus abundance in marine environments.     

Probability Distribution of Drug-Resistant Mutants in Unselected Populations of Mycobacterium tuberculosis

The fluctuation test shows that Mycobacterium tuberculosis mutates to resistance to isoniazid, streptomycin, ethambutol and rifampin spontaneously and at random. The average mutation rates for the drugs, in the same order, were calculated to be 2.56 × 10⁻⁸, 2.95 × 10⁻⁸, 10⁻⁷, and 2.25 × 10⁻¹⁰ mutation per bacterium per generation. The relatively high mutation rate to ethambutol resistance and the low mutation rate to rifampin resistance were confirmed by analyzing the increase in the proportion of mutants with time in a growing population of the tubercle bacilli. The highest proportions of mutants to be expected in unselected populations of the tubercle bacilli were calculated from the results of fluctuation tests.     

Bacterioplankton in Antarctic Ocean Waters During Late Austral Winter: Abundance, Frequency of Dividing Cells, and Estimates of Production

Bacterioplankton productivity in Antarctic waters of the eastern South Pacific Ocean and Drake Passage was estimated by direct counts and frequency of dividing cells (FDC). Total bacterioplankton assemblages were enumerated by epifluorescent microscopy. The experimentally determined relationship between in situ FDC and the potential instantaneous growth rate constant (μ) is best described by the regression equation ln μ = 0.081 FDC − 3.73. In the eastern South Pacific Ocean, bacterioplankton abundance (2 × 10⁵ to 3.5 × 10⁵ cells per ml) and FDC (11%) were highest at the Polar Front (Antarctic Convergence). North of the Subantarctic Front, abundance and FDC were between 1 × 10⁵ to 2 × 10⁵ cells per ml and 3 to 5%, respectively, and were vertically homogeneous to a depth of 600 m. In Drake Passage, abundance (10 × 10⁵ cells per ml) and FDC (16%) were highest in waters south of the Polar Front and near the sea ice. Subantarctic waters in Drake Passage contained 4 × 10⁵ cells per ml with 4 to 5% FDC. Instantaneous growth rate constants ranged between 0.029 and 0.088 h⁻¹. Using estimates of potential μ and measured standing stocks, we estimated productivity to range from 0.62 μg of C per liter · day in the eastern South Pacific Ocean to 17.1 μg of C per liter · day in the Drake Passage near the sea ice.     

Viral Abundance, Decay, and Diversity in the Meso- and Bathypelagic Waters of the North Atlantic

To elucidate the potential importance of deep-water viruses in controlling the meso- and bathypelagic picoplankton community, the abundance, decay rate, and diversity of the virioplankton community were determined in the meso- and bathypelagic water masses of the eastern part of the subtropical North Atlantic. Viral abundance averaged 1.4 × 10⁶ ml⁻¹ at around 100 m of depth and decreased only by a factor of 2 at 3,000 to 4,000 m of depth. In contrast, picoplankton abundance decreased by 1 order of magnitude to the Lower Deep Water (LDW; 3,500- to 5,000-m depth). The virus-to-picoplankton ratio increased from 9 at about 100 m of depth to 110 in the LDW. Mean viral decay rates were 3.5 × 10⁻³ h⁻¹ between 900 m and 2,750 m and 1.1 × 10⁻³ h⁻¹ at 4,000 m of depth, corresponding to viral turnover times of 11 and 39 days, respectively. Pulsed-field gel electrophoresis fingerprints obtained from the viral community between 2,400 m and 4,000 m of depth revealed a maximum of only four bands from 4,000 m of depth. Based on the high viral abundance and the low picoplankton production determined via leucine incorporation, we conclude that the viral production calculated from the viral decay is insufficient to maintain the high viral abundance in the deep North Atlantic. Rather, we propose that substantial allochthonous viral input or lysogenic or pseudolysogenic production is required to maintain the high viral abundance detected in the meso- and bathypelagic North Atlantic. Consequently, deep-water prokaryotes are apparently far less controlled in their abundance and taxon richness by lytic prokaryotic phages than the high viral abundance and the virus-to-picoplankton ratio would suggest.     

Kinetics of Leptin Binding to the Q223R Leptin Receptor

Studies in human populations and mouse models of disease have linked the common leptin receptor Q223R mutation to obesity, multiple forms of cancer, adverse drug reactions, and susceptibility to enteric and respiratory infections. Contradictory results cast doubt on the phenotypic consequences of this variant. We set out to determine whether the Q223R substitution affects leptin binding kinetics using surface plasmon resonance (SPR), a technique that allows sensitive real-time monitoring of protein-protein interactions. We measured the binding and dissociation rate constants for leptin to the extracellular domain of WT and Q223R murine leptin receptors expressed as Fc-fusion proteins and found that the mutant receptor does not significantly differ in kinetics of leptin binding from the WT leptin receptor. (WT: k_a 1.76×10⁶±0.193×10⁶ M⁻¹ s⁻¹, k_d 1.21×10⁻⁴±0.707×10⁻⁴ s⁻¹, K_D 6.47×10⁻¹¹±3.30×10⁻¹¹ M; Q223R: k_a 1.75×10⁶±0.0245×10⁶ M⁻¹ s⁻¹, k_d 1.47×10⁻⁴±0.0505×10⁻⁴ s⁻¹, K_D 8.43×10⁻¹¹±0.407×10⁻¹¹ M). Our results support earlier findings that differences in affinity and kinetics of leptin binding are unlikely to explain mechanistically the phenotypes that have been linked to this common genetic variant. Future studies will seek to elucidate the mechanism by which this mutation influences susceptibility to metabolic, infectious, and malignant pathologies.     

Occurrence and Abundance of Antibiotics and Resistance Genes in Rivers,Canal and near Drug Formulation Facilities – A Study in Pakistan

Antibiotic resistance (AR) is a global phenomenon that has severe epidemiological ramifications world-wide. It has been suggested that antibiotics that have been discharged into the natural aquatic environments after usage or manufacture can promote the occurrence of antibiotic resistance genes (ARG). These environmental ARGs could serve as a reservoir and be horizontally transferred to human-associated bacteria and thus contribute to AR proliferation. The aim of this study was to investigate the anthropogenic load of antibiotics in Northern Pakistan and study the occurrence of ARGs in selected samples from this region. 19 sampling sites were selected; including six rivers, one dam, one canal, one sewage drain and four drug formulation facilities. Our results show that five of the rivers have antibiotic levels comparable to surface water measurements in unpolluted sites in Europe and the US. However, high levels of antibiotics could be detected in the downstream river in close vicinity of the 10 million city Lahore, 1100, 1700 and 2700 ng L⁻¹ for oxytetracycline, trimethoprim, and sulfamethoxazole respectively. Highest detected levels were at one of the drug formulation facilities, with the measured levels of 1100, 4100, 6200, 7300, 8000, 27000, 28000 and 49000 ng L⁻¹ of erythromycin, lincomycin, ciprofloxacin, ofloxacin, levofloxacin, oxytetracycline, trimethoprim and sulfamethoxazole respectively. ARGs were also detected at the sites and the highest levels of ARGs detected, sulI and dfrA1, were directly associated with the antibiotics detected at the highest concentrations, sulfamethoxazole and trimethoprim. Highest levels of both antibiotics and ARGs were seen at a drug formulation facility, within an industrial estate with a low number of local residents and no hospitals in the vicinity, which indicates that the levels of ARGs at this site were associated with the environmental levels of antibiotics.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Muramic Acid Measurements for Bacterial Investigations in Marine Environments by High-Pressure Liquid Chromatography

Muramic acid, a constituent of procaryotic cell walls, was assayed by high-pressure liquid chromatography in samples from several marine environments (water column, surface microlayer, and sediment) and a bacterial culture. It is used as a microbial biomass indicator. The method gave a good separation of muramic acid from interfering compounds with satisfactory reproducibility. A pseudomonad culture had a muramic acid content of 4.7 × 10⁻¹⁰ to 5.3 × 10⁻¹⁰ μg per cell during growth. In natural water samples, highly significant relationships were found between muramic acid concentrations and bacterial numbers for populations of 10⁸ to 10¹¹ cells per liter. The muramic acid content in natural marine water decreased from 5.3 × 10⁻¹⁰ to 1.6 × 10⁻¹⁰ μg per cell with increasing depth. In coastal sediments exposed to sewage pollution, concentrations of muramic acid, ATP, organic carbon, and total amino acids displayed a parallel decrease with increasing distance from the sewage outlet. Advantages of muramic acid measurement by high-pressure liquid chromatography are its high sensitivity and reduction of preparation steps, allowing a short time analysis.     

Evidence for the Cooccurrence of Nitrite-Dependent Anaerobic Ammonium and Methane Oxidation Processes in a Flooded Paddy Field

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two of the most recent discoveries in the microbial nitrogen cycle. In the present study, we provide direct evidence for the cooccurrence of the anammox and n-damo processes in a flooded paddy field in southeastern China. Stable isotope experiments showed that the potential anammox rates ranged from 5.6 to 22.7 nmol N₂ g⁻¹ (dry weight) day⁻¹ and the potential n-damo rates varied from 0.2 to 2.1 nmol CO₂ g⁻¹ (dry weight) day⁻¹ in different layers of soil cores. Quantitative PCR showed that the abundance of anammox bacteria ranged from 1.0 × 10⁵ to 2.0 × 10⁶ copies g⁻¹ (dry weight) in different layers of soil cores and the abundance of n-damo bacteria varied from 3.8 × 10⁵ to 6.1 × 10⁶ copies g⁻¹ (dry weight). Phylogenetic analyses of the recovered 16S rRNA gene sequences showed that anammox bacteria affiliated with “Candidatus Brocadia” and “Candidatus Kuenenia” and n-damo bacteria related to “Candidatus Methylomirabilis oxyfera” were present in the soil cores. It is estimated that a total loss of 50.7 g N m⁻² per year could be linked to the anammox process, which is at intermediate levels for the nitrogen flux ranges of aerobic ammonium oxidation and denitrification reported in wetland soils. In addition, it is estimated that a total of 0.14 g CH₄ m⁻² per year could be oxidized via the n-damo process, while this rate is at the lower end of the aerobic methane oxidation rates reported in wetland soils.