Inhibition of Ammonium Oxidation by Nitrapyrin in Soil and Liquid Culture

Inhibition of growth of axenic cultures of Nitrosomonas europaea by nitrapyrin was investigated in liquid culture and in soil. In liquid culture, exponentially growing cells were more sensitive than stationary-phase cells, possibly due to a requirement for uptake of nitrapyrin, metabolism of nitrapyrin, or both before inhibition. Differences in sensitivity were observed between the parent strain and two strains, sp1 and sp2, that were selected through repeated subculturing. These differences were reflected in the length of the lag period induced by nitrapyrin and in the specific growth rate and were due to different bactericidal and bacteriostatic effects. Soil provided significant protection from inhibition, with concentrations of nitrapyrin approximately one order of magnitude greater than those required for equivalent inhibition in liquid culture. The data show that strain differences alone do not explain differences in sensitivity between nitrification in soil and in liquid culture and suggest that the inhibitor may be more effective against actively nitrifying soils.     

Production of Nitrous Oxide by Ammonia-Oxidizing Chemoautotrophic Microorganisms in Soil

Gas chromatographic studies showed that nitrous oxide was produced in each instance when sterilized (autoclaved) soil was incubated after treatment with ammonium sulfate and inoculation with pure cultures of ammonia-oxidizing chemoautotrophic microorganisms (strains of Nitrosomonas, Nitrosospira, and Nitrosolobus). Production of N₂O in ammonium-treated sterilized soil inoculated with Nitrosomonas europaea increased with the concentration of ammonium and the moisture content of the soil and was completely inhibited by both nitrapyrin and acetylene. Similar effects of nitrapyrin, acetylene, ammonium concentration, and soil moisture content were observed in studies of factors affecting N₂O production in nonsterile soil treated with ammonium sulfate. These observations support the conclusion that, at least under some conditions, most of the N₂O evolved from soils treated with ammonium or ammonium-producing fertilizers is generated by chemoautotrophic nitrifying microorganisms during oxidation of ammonium to nitrite.     

Inhibition of biofilm populations of Nitrosomonas europaea

The influence of surface attachment and growth on inhibition of the ammonia oxidizing bacterium, Nitrosomonas europaea, by nitrapyrin was investigated in liquid culture in the presence and absence of glass slides. Significant attachment to glass slides occurred in the absence of ammonia, but the extent of attachment was not affected by nitrapyrin, nor by previous culture of cells in medium containing nitrapyrin. The presence of glass slides affected neither the specific growth rate of N. europaea, measured by changes in nitrite concentration, nor inhibition by nitrapyrin. Inhibitory effects of nitrapyrin on increases in nitrite concentration and in free cell concentration were similar, but greater effects were observed on changes in attached cell concentration. Established biofilms on glass slides grew at a lower specific growth rate than freely suspended cells. Both biofilm cells, and those detached from the biofilm, were protected from inhibition. A mechanism for protection of biofilm populations is proposed involving reduced sensitivity of slowly growing cells producing extracellular polymeric material. Offprint requests to.: J. I. Prosser.     

Oxidation of Nitrapyrin to 6-Chloropicolinic Acid by the Ammonia-Oxidizing Bacterium Nitrosomonas europaea

Suspensions of Nitrosomonas europaea catalyzed the oxidation of the commercial nitrification inhibitor nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine]. Rapid oxidation of nitrapyrin (at a concentration of 10 μM) required the concomitant oxidation of ammonia, hydroxylamine, or hydrazine. The turnover rate was highest in the presence of 10 mM ammonia (0.8 nmol of nitrapyrin per min/mg of protein). The product of the reaction was 6-chloropicolinic acid. By the use of ¹⁸O₂, it was shown that one of the oxygens in 6-chloropicolinic acid came from diatomic oxygen and that the other came from water. Approximately 13% of the radioactivity of [2,6-¹⁴C]nitrapyrin was shown to bind to cells. Most (94%) of the latter was bound indiscriminately to membrane proteins. The nitrapyrin bound to membrane proteins may account for the observed inactivation of ammonia oxidation.     

Effects of field fertilizer practices on labeled ammonium-nitrogen transformations and its utilization by winter wheat

Summary Field experiments with winter wheat (Triticum aestivum L.) were conducted in two years at two locations using¹⁵N-enriched (NH₄)₂SO₄ on Smolan silt loam (Pachic Argiustoll) and Ost loam (Typic Arguistoll) soils. The objective was to relate differences in crop utilization of fertilizer to movement and transformations of the N in a complete factorial experiment having fall and spring applications, banded and broadcast, with and without nitrapyrin. Plant uptake of the 60 kg N/ha applied varied from 31% to 62% with greatest uptake when fertilizer was banded in the spring without nitrapyrin and least uptake from fall and spring broadcast treatments using nitrapyrin. Analysis of single factor effects showed greater crop contents of fertilizer N for spring than fall applications. That was related to immobilization of the applied N. Much more fertilizer N was in inorganic forms during the period of rapid wheat growth with spring applications than with fall. Banding the fertilizer at a depth of 0.05 m resulted in greater plant uptake than broadcasting or banding it on the soil surface. A significant portion of the applied N was immobilized near the point of application. That limited the downward movement of the N placed on the surface, making it less available to plant roots than the N placed 0.05 m deep where soil moisture was more favorable. Use of nitrapyrin resulted in lowered amounts of fertilizer N as NO₃-until mid-May for fall treatments and until harvest with spring treatments. That appeared to be the reason for lowered plant uptake when nitrapyrin was used. Published in memory of Professor R V Olson and over 40 years of contributions and service to agriculture and soil science (1919–1985).     

Epistatic interactions between four rad loci in yeast

Haploid yeast strains carrying mutations in two or more of four ad genes were contrusted by tetrad dissection, and the UV survival of these strains was measured. It was found that (with one exception) double mutant strains were not significantly more sensitive than the most sensitive single mutants, for strains involving mutant loci rad 1, rad 3 and rad 4. The exception was the double mutant rad 1–5 rad 4-4, but another double mutant involving different alleles of the the same loci did not show an enhanced UV sensitivity. Triple and quadruple mutants also failed to show a significantly increased UV sensitivity with respect to the single mutants. The results indicate that all these four mutant loci confer UV sensitivity by the same mechanism, and it is suggested that the wild-type alleles mediate excision-repair of UV-induced DNA lesions. Enhanced sensitivity of the genotype rad 1–5 rad 4-4 is attributed to leakiness of these alleles.     

Comparative phytotoxicity of nitrapyrin and ATC to several leguminous species

Summary The relative toxicity of nitrapyrin 2-chloro-6-(trichloromethyl) pyridine and ATC (4-amino-1, 2, 4-triazole) on the growth of chick peas (Cicer arietinum L.) cow peas (Vigna sinensis L.), green beans (Phaseolus vulgaris L.), green peas (Pisum sativum L.) and mung beans (Phaseolus aureus Roxb.) and their effectiveness as nitrification inhibitor were studied under greenhouse conditions. ATC produced no toxicity symptoms in green peas, whereas resulted in leaf chlorosis in cow peas, chick peas and green beans. However, nitrapyrin toxicity appeared as leaf chlorosis in cow peas, and interveinal chlorosis in chick peas. Moreover, nitrapyrin-treated green beans and peas developed leaf curling and cupping. Although ATC had no significant effect on growth, a suppression in plant growth was associated with nitrapyrin application. Furthermore, green beans was the most resistant and chick peas the most sensitive to nitrapyrin. Nitrapyrin was more effective nitrification inhibitor than ACT, especially at the lower rates.     

Toxicity of nitrapyrin to sunflower under different N nutrition regimes

Summary The purpose of this study was to investigate the phytotoxicity of nitrapyrin 2-chloro-6-(trichloromethyl)pyridine to sunflower (Helianthus annuus L.) under different N regimes and to see if N forms affect the phytotoxicity of nitrapyrin. Sunflower was grown in pot culture for 21 days and was fertilized with (NH₄)₂SO₄, NH₄NO₃ and NaNO₃ to provide 0, 100 and 200 ppm N and with nitrapyrin application of 0 and 20 ppm. All N-treated sunflower plants in all N regimes and regardless of titrapyrin treatment produced more root and shoot dry weights and contained a significantly higher N than untreated check. Nitrapyrin toxicity appeared as a curling of leaf margin and a tendril type of stem growth, the visible toxicity symptoms decreased in the order: (NH₄)₂SO₄>NH₄NO₃>NaNO₃. Furthermore nitrapyrin addition suppressed sunflower growth in each N regime, the suppressing effect being greater with (NH₄)₂SO₄ and NH₄NO₃ than as with NaNO₃. Although, shoot growth from plants receiving nitrapyrin was not significantly affected by any N regime, root growth of nitrapyrin-treated plants was somewhat restricted by NH₄ ⁺−N nutrition relative to NO₃ ⁻−N nutrition.     

Genetically Modified Vibrio harveyi Strains as Potential Bioindicators of Mutagenic Pollution of Marine Environments

For biodetection of mutagenic pollution of marine environments, an organism naturally occurring in these habitats should be used. We found that marine bacterium Vibrio harveyi may be an appropriate bioindicator of mutagenic pollution. For positive selection of mutants, we developed a simple method for isolation of V. harveyi mutants resistant to neomycin. We constructed genetically modified V. harveyi strains that produce significantly more neomycin-resistant mutants upon treatment with low concentrations of mutagens than the wild-type counterpart. The sensitivity of the mutagenicity test with the V. harveyi strains is at least comparable to (if not higher than) that of the commonly used Ames test, which uses Salmonella enterica serovar Typhimurium strains. Therefore, we consider that the V. harveyi strains described in this report could be used as potential bioindicators of mutagenic pollution of marine environments.     

Distinct signal transduction processes by IL-4 and IL-13 and influences from the Q551R variant of the human IL-4 receptor alpha chain

Background

Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia.

Methods

The PG-1 sensitivity/resistance and PG-1 binding properties of P. aeruginosa and B. cepacia were assessed using radial diffusion assays, radioiodinated PG-1, and surface plasmon resonance (BiaCore).

Results

The six P. aeruginosa strains examined were very sensitive to PG-1, exhibiting minimal active concentrations from 0.0625–0.5 μg/ml in radial diffusion assays. In contrast, all five B. cepacia strains examined were greater than 10-fold to 100-fold more resistant, with minimal active concentrations ranging from 6–10 μg/ml. When incubated with a radioiodinated variant of PG-1, a sensitive P. aeruginosa strain bound considerably more protegrin molecules per cell than a resistant B. cepacia strain. Binding/diffusion and surface plasmon resonance assays revealed that isolated lipopolysaccharide (LPS) and lipid A from the sensitive P. aeruginosa strains bound PG-1 more effectively than LPS and lipid A from resistant B. cepacia strains.

Conclusion

These findings support the hypothesis that the relative resistance of B. cepacia to protegrin is due to a reduced number of PG-1 binding sites on the lipid A moiety of its LPS.     

Effects of Nitrapyrin [2-Chloro-6-(Trichloromethyl) Pyridine] on the Obligate Methanotroph Methylosinus trichosporium OB3b

Nitrapyrin inhibited growth, CH₄ oxidation, and NH₄⁺ oxidation, but not the oxidation of CH₃OH, HCHO, or HCOONa, by Methylosinus trichosporium OB3b, suggesting that nitrapyrin acts against the methane monooxygenase enzyme system. The inhibition of CH₄ oxidation could be reversed by repeated washing of nitrapyrin-inhibited cells, indicating that its effect is bacteriostatic. The addition of Cu²⁺ did not release the inhibition. Methane oxidation was also inhibited by 6-chloro-2-picoline. These data suggest that the mode of action of nitrapyrin on M. trichosporium is different from that on chemoautotrophic NH₄⁺ oxidizers or methanogens.     

Isolation of mutants of Escherichia coli uncoupled in oxidative phosphorylation using hypersensitivity to streptomycin

Mutants of Escherichia coli, harbouring the uncA401 or uncB402 alleles, were found to take up streptomycin more rapidly than the coupled parent strains. The increased rate of uptake results in greater sensitivity of the uncoupled strains, compared to the parent strains, to low concentrations of streptomycin. Studies with unc⁺ revertants showed that hypersensitivity to streptomycin is attributable to the mutation causing uncoupling. The uptake of streptomycin in an unc^? strain is abolished by addition of the chemical uncoupler carbonylcyanide m-chlorophenylhydrazone. The phenotype of hypersensitivity to streptomycin can be used as a selection procedure for the isolation of uncoupled strains. In an experiment reported here, nine out of 12 strains isolated as being sensitive to streptomycin (at 2.5 μg/ml), were found to be unable to grow on succinate as a sole source of carbon. Five of the nine Suc^? strains were found to be uncoupled in oxidative phosphorylation, and two of the five uncoupled strains lacked Mg²⁺-ATPase activity. The mutations causing uncoupling were cotransducible with the ilv genes.     

Sensitivity to antibiotics of Clostridium difficile toxigenic nosocomial strains

Clostridium difficile is the etiological agent of diarrhoea and colitis, especially in elderly patients. The incidence of these diseases has increased during the last 10 years. Emergence of so-called hypervirulent strains is considered as one of the main factors responsible for the more severe disease and changed profile of sensitivity to antimicrobial agents. The aim of this work was to determine the sensitivity profile of toxigenic strains of C. difficile in the Czech Republic in 2011–2012 to selected antibiotics. The antibiotics clindamycin, metronidazole, vancomycin and amoxicillin with clavulanic acid were used for this purpose. Isolates cultured on Brazier's C. difficile selective agar were analysed for the presence of toxin genes using Xpert detection system. Xpert analysis revealed that 33 strains carried the genes for toxins tcdB, cdt and tcdCΔ117, thus showing characteristics typical for the hypervirulent ribotype 027/PFGE type NAP1/REA type B1. The remaining 29 strains carried only the gene for toxin B (tcdB) and not cdt and tcdCΔ117. Our results indicate the higher susceptibility of C. difficile hypertoxigenic strains to three out of four tested antibiotics (except vancomycin) than it is for the other toxigenic strains. We found that only 10.34 % of other toxigenic strains were resistant to clindamycin, and no resistance was found in all other cases. All the isolates were sensitive to amoxicillin/clavulanic acid in vitro. However, its use is not recommended for therapy of infections caused by C. difficile.     

Dic2 and Dic3 loci confer osmotic adaptation and fungicidal sensitivity independent of the HOG pathway in Cochliobolus heterostrophus

Previously, we identified three gene loci, Dic1, Dic2, and Dic3, that confer high-osmolarity adaptation and dicarboximide/phenylpyrrole fungicide sensitivity in Cochliobolus heterostrophus. Dic1 encoded a group III histidine kinase, but the other genes were not characterized. In the present study, we revealed that both Dic2 and Dic3 are involved in the Skn7 pathway. Dic2 encoded an Skn7-type response regulator, ChSkn7. Strain N4502 contained D359N in the response regulator domain of ChSkn7. Strain E4503 contained a deletion of 50 amino acids in the DNA-binding domain. Strain N4507 was a null mutant of the ChSkn7 gene. All of the dic2 mutant strains showed similar levels of sensitivity to high osmolarity and similar levels of resistance to fungicides. These results strongly suggested that both the DNA-binding domain and response regulator domain are essential for Skn7 function in osmotic adaptation and fungicide sensitivity. A western blot analysis revealed that Dic3 is not involved in the regulation of Hog1-type MAPKs. The Chssk1/dic3 double mutant strains clearly showed greater resistance to fungicides than the single mutant strains. An additive effect was also observed in the high-osmolarity experiments. On the other hand, the dic3/dic2 double mutant strains did not show higher levels of resistance to fungicides and greater sensitivity to KCl than the single mutant strains. These results strongly suggested that the dic3 locus confer high-osmolarity adaptation and fungicide sensitivity independently from Ssk1-Hog1 pathway, but not the Skn7 pathway. Moreover, the dic3 strain and all dic2 strains showed similar levels of sensitivity to high-osmolarity stress and similar levels of resistance to fungicides, suggesting Dic3 to have an essential role in the Skn7 pathway. Our results provide new insight into the functions of the Skn7 pathway in filamentous fungi.     

Mutagenicity of dichlorvos and methyl methanesulphonate for Escherichia coli WP2 and some derivatives deficient in DNA repair

The mutagenic and lethal action of methyl methanesulphonate (MMS) and dichlorvos (DDVP) has been studied on Escherichia coli WP2 and some derivatives deficient in DNA repair genes. The exrA⁺ and recA⁺ alleles were necessary for significant mutagenesis by either compound, and the uvrA gene affected neither the lethal nor mutagenic responses. Increased sensitivity to both compounds was shown by the exrA and uvrAexrA strains and in a more pronounced way by the uvrApolA, recA, and uvrAexrApolA strains.Bacteria deficient at the polA locus were 2 and 3 times more mutable by DDVP and MMS respectively, consistent with the hypothesis that the absence of the polA system for the repair of single-strand gaps results in a greater proportion of the total repair being channelled through the error-prone exrA⁺/recA⁺-dependent system. Single-strand breaks were detectable by alkaline sucrose gradient centrifugation after both MMS and DDVP treatment of polA bacteria. Thus in all the tests carried out, both compounds showed similar patterns of activity, and the results are consistent with their known ability to alkylate DNA. The chief differences were quantitative; sensitivity increases were far more pronounced with MMS which was also a far more potent mutagen than DDVP.     

Mortality of bifidobacteria in boiled yogurt

Bifidobacterial strains showed various mortalities during storage at 30°C in boiled yogurt prepared with Streptococcus sp. and Lactobacillus sp. Bifidobacterium breve 203 was most stable, maintaining its initial cell number for more than 5 days. It was also stable at 4 or 10°C in fresh yogurt. B. longum 401 rapidly lost viability in the boiled yogurt and was unstable in the fresh yogurt at any temperature. Lactobacillus sp. remained fully viable for one week or more at 4–30°C while Streptococcus sp. lost viability at 20°C or above. The difference in mortality between B. breve 203 and B. longum 401 was mainly due to their sensitivity to the acidic environment, with temperature during storage having a secondary effect. Effects of lysozyme, pepsin and bile acids on the two strains were also investigated.     

Effects of Streptococcus pneumoniae strain background on complement resistance

Background

Immunity to infections caused by Streptococcus pneumoniae is dependent on complement. There are wide variations in sensitivity to complement between S. pneumoniae strains that could affect their ability to cause invasive infections. Although capsular serotype is one important factor causing differences in complement resistance between strains, there is also considerable other genetic variation between S. pneumoniae strains that may affect complement-mediated immunity. We have therefore investigated whether genetically distinct S. pneumoniae strains with the same capsular serotype vary in their sensitivity to complement mediated immunity.

Methodology and Principal Findings

C3b/iC3b deposition and neutrophil association were measured using flow cytometry assays for S. pneumoniae strains with different genetic backgrounds for each of eight capsular serotypes. For some capsular serotypes there was marked variation in C3b/iC3b deposition between different strains that was independent of capsule thickness and correlated closely to susceptibility to neutrophil association. C3b/iC3b deposition results also correlated weakly with the degree of IgG binding to each strain. However, the binding of C1q (the first component of the classical pathway) correlated more closely with C3b/iC3b deposition, and large differences remained in complement sensitivity between strains with the same capsular serotype in sera in which IgG had been cleaved with IdeS.

Conclusions

These data demonstrate that bacterial factors independent of the capsule and recognition by IgG have strong effects on the susceptibility of S. pneumoniae to complement, and could therefore potentially account for some of the differences in virulence between strains.     

Effects of aflatoxin B1 and caffeine on viability in natural strains of Drosophila melanogaster

Thirty-five natural populations of Drosophila melanogaster from Virginia were tested for their degree of sensitivity to the carcinogen Aflatoxin B₁ (AFB₁) and to caffeine. Significant variations in sensitivity to each was demonstrated among these strains, but no correlation existed for relative degrees of sensitivity to AFB₁ or caffeine when strains were compared, although most strains were more severely affected by AFB₁. Eight of these strains were tested for the effects of simultaneous administration of AFB₁ and caffeine. Generally, caffeine demonstrated both a significant protective effect in preventing the high degree of egg-adult mortality produced by AFB₁ alone and a retardation of the significant decrease in body length shown by adults which survive in the AFB₁ treatments.     

Mutagen sensitivity of Drosophila melanogaster. I. Isolation and preliminary characterization of a methyl methanesulphonate-sensitive strain

Sensitivity to the monofunctional alkylating agent methyl methanesulfonate (MMS) has been tested as a selection technique to isolate mutant strains which can provide insights into the genetic control of DNA replication, DNA repair and recombination in the complex eucaryote, Drosophila melanogaster. The successful isolation of an X-linked MMS-sensitive strain, mut^s, has suggested that mutagen sensitivity is a feasible methodology for the selection of mutant strains of Drosophila which will be useful in the genetic and biochemical analysis of these cellular functions. Preliminary characterization of this mutant strain indicates that: (A) it is extremely sensitive to killing by MMS; (B) it is more mutable by MMS than the parent wildtype strain; and (C) it appears to possess mutator gene activity.     

Factors Influencing the Chlorine Susceptibility of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum

The susceptibility of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility. M. avium and M. intracellulare strains were more resistant to chlorine than were strains of M. scrofulaceum. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of M. avium cells grown at 30°C was increased when cells were exposed to chlorine at 40°C compared to susceptibility after exposure at 30°C. Cells of M. avium grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80, para-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.