Mineralization of 4-fluorocinnamic acid by a Rhodococcus strain

A bacterial strain capable of aerobic degradation of 4-fluorocinnamic acid (4-FCA) as the sole source of carbon and energy was isolated from a biofilm reactor operating for the treatment of 2-fluorophenol. The organism, designated as strain S2, was identified by 16S rRNA gene analysis as a member of the genus Rhodococcus. Strain S2 was able to mineralize 4-FCA as sole carbon and energy source. In the presence of a conventional carbon source (sodium acetate [SA]), growth rate of strain S2 was enhanced from 0.04 to 0.14 h^?1 when the culture medium was fed with 0.5 mM of 4-FCA, and the time for complete removal of 4-FCA decreased from 216 to 50 h. When grown in SA-supplemented medium, 4-FCA concentrations up to 1 mM did not affect the length of the lag phase, and for 4-FCA concentrations up to 3 mM, strain S2 was able to completely remove the target fluorinated compound. 4-Fluorobenzoate (4-FBA) was transiently formed in the culture medium, reaching concentrations up to 1.7 mM when the cultures were supplemented with 3.5 mM of 4-FCA. Trans,trans-muconate was also transiently formed as a metabolic intermediate. Compounds with molecular mass compatible with 3-carboxymuconate and 3-oxoadipate were also detected in the culture medium. Strain S2 was able to mineralize a range of other haloorganic compounds, including 2-fluorophenol, to which the biofilm reactor had been exposed. To our knowledge, this is the first time that mineralization of 4-FCA as the sole carbon source by a single bacterial culture is reported.     

Biodegradation kinetics of 4-fluorocinnamic acid by a consortium of Arthrobacter and Ralstonia strains

Arthrobacter sp. strain G1 is able to grow on 4-fluorocinnamic acid (4-FCA) as sole carbon source. The organism converts 4-FCA into 4-fluorobenzoic acid (4-FBA) and utilizes the two-carbon side-chain for growth with some formation of 4-fluoroacetophenone as a dead-end side product. We also have isolated Ralstonia sp. strain H1, an organism that degrades 4-FBA. A consortium of strains G1 and H1 degraded 4-FCA with Monod kinetics during growth in batch and continuous cultures. Specific growth rates of strain G1 and specific degradation rates of 4-FCA were observed to follow substrate inhibition kinetics, which could be modeled using the kinetic models of Haldane–Andrew and Luong–Levenspiel. The mixed culture showed complete mineralization of 4-FCA with quantitative release of fluoride, both in batch and continuous cultures. Steady-state chemostat cultures that were exposed to shock loadings of substrate responded with rapid degradation and returned to steady-state in 10–15 h, indicating that the mixed culture provided a robust system for continuous 4-FCA degradation.     

Characterization and molecular mechanism of a naturally occurring metsulfuron-methyl resistant strain of Pseudomonas aeruginosa

Strain L36, naturally resistant to the herbicide metsulfuron-methyl (SM), was isolated and characterized with respect to the molecular mechanism of resistance. The isolate was identified as Pseudomonas aeruginosa based on bacterial morphology, physiology, cellular fatty acid, and 16S rRNA gene sequence. Minimal inhibitory concentrations of metsulfuron-methyl against the growth of L36 and wild type isolate PAO1 were 6.03 and 1.33 mM, respectively. L36 carried a nucleotide base change in the acetolactate synthase (ALS) gene that coded for a single amino acid mutation (Ala29 → Val29). The mutated ilvIH gene was functionally expressed, purified, and the kinetic properties of the purified ALS were tested. The mutant enzyme had K _m for pyruvate fourfold higher than the wild type enzyme, and K _i ^app for sulfonylureas some 30-fold higher. The A29 V mutation in the ALS resulted in the resistance of P. aeruginosa to sulfonylurea herbicides but not to imidazolinone herbicides.     

Simulation and experimental analysis of the influence of herbicides on soil nitrification

The effect of 35 herbicides on the nitrification process was tested both by experiment, and by simulation of possible mechanisms of inhibition in a mathematical model. The model consists of nine equations with six coordinated constant and seven measurable parameters (or initial values), depending on the specific soil. The only free parameters are the initial values of the oxidative enzyme systems, and the parameters which determine the course of possible inhibition effects. For the majority of the herbicides, the inhibitory effects on the NH₄ ⁺ or NO₂ ^- oxidation were found negligible in the range of practical application. Hypotheses of a completely reversible or partially reversible inhibition of the oxidase systems gave the best correspondence between the model and reality, while an alteration of the growth parameters of the nitrifying populations in the model (death rate, proliferation rate, initial kill) due to the application of herbicides led to strong contrasts between simulated and experimental curves. Significant inhibitory effects became evident only when the hydrogen ion concentration in the soil fell below pH 7. Results with several herbicides indicated that the process of NO₂ ^- oxidation was more sensitive than that of NH₄ ⁺ oxidation. With a number of herbicides, an accumulation of NO₂ ^- ions was noticed during the course of soil percolation. In consideration of the buffering capacity, the model is applicable to other soils.     

Resistance mechanism to acetyl coenzyme A carboxylase inhibiting herbicides in Phalaris paradoxa collected in Mexican wheat fields

Background and aims

In this study, we describe the molecular, physiological and agronomic aspects involved in the resistance to acetyl coenzyme A carboxylase inhibiting herbicides (ACCase) observed in one biotype of Phalaris paradoxa from Mexico.

Methods

Dose–response Assays: The herbicide rate inhibiting plant growth of each biotype by 50% with respect to the untreated control, ED₅₀. Enzyme purification and ACCase assays to determine herbicide rate inhibiting the enzyme of each biotype by 50% with respect to the untreated control, I₅₀. Absorption and Translocation Assays with [¹⁴C]diclofop-methyl. Metabolism of diclofop-methyl and its metabolites were identified by thin-layer chromatography. Study of target site resistance mechanism at enzyme and molecular levels.

Results

In this work, it has been studied the whole-plant response of Phalaris paradoxa biotypes from Mexico resistant (R) and susceptible (S) to ACCase-inhibiting herbicides: aryloxyphenoxypropionate (APP), cyclohexanedione (CHD) and phenylpyrazoline (PPZ), and the mechanism behind their resistance were studied. To analyse the resistance mechanism, the enzyme ACCase activity was investigated. Results from biochemical assays indicated a target-site resistance as the cause of reduced susceptibility to ACCase inhibitors. The absorption, translocation and metabolism were similar between R and S biotypes. A point mutation never described before was detected within the triplet of glycine at the amino acid position 2096 (referring to EMBL accession no. AJ310767) and resulted in the triplet of serine. This new mutation could explain the loss of affinity for the ACCase-inhibiting herbicides.

Conclusions

We found a new mutation, which had never been described before. This mutation was detected within the triplet of glycine at the amino acid position 2096. This new mutation confers cross-resistance to three different chemical groups of ACCase-inhibiting herbicides.     

Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf Weed, Kochia scoparia

Selection of kochia (Kochia scoparia) biotypes resistant to the sulfonylurea herbicide chlorsulfuron has occurred through the continued use of this herbicide in monoculture cereal-growing areas in the United States. The apparent sulfonylurea resistance observed in kochia was confirmed in greenhouse tests. Fresh and dry weight accumulation in the resistant kochia was 2- to >350-fold higher in the presence of four sulfonylurea herbicides as compared to the susceptible biotype. Acetolactate synthase (ALS) activity isolated from sulfonylurea-resistant kochia was less sensitive to inhibition by three classes of ALS-inhibiting herbicides, sulfonylureas, imidazolinones, and sulfonanilides. The decrease in ALS sensitivity to inhibition (as measured by the ratio of resistant I₅₀ to susceptible I₅₀) was 5- to 28-fold, 2- to 6-fold, and 20-fold for sulfonylurea herbicides, imidazolinone herbicides, and a sulfonanilide herbicide, respectively. No differences were observed in the ALS-specific activities or the rates of [¹⁴C]chlorsulfuron uptake, translocation, and metabolism between susceptible and resistant kochia biotypes. The K_m values for pyruvate using ALS from susceptible and resistant kochia were 2.13 and 1.74 mm, respectively. Based on these results, the mechanism of sulfonylurea resistance in this kochia biotype is due solely to a less sulfonylurea-sensitive ALS enzyme.     

Studies on synthetic hybrids of british species of Senecio: I Senecio viscosus L. × S. vulgaris L.

Summary

Four aquatic hyphomycetes and one terrestrial fungus were examined for their responses to the phenoxy herbicides (±)-MCPP and 2,4-D as both single and binary preparations with respect to hyphal extension, sporulation and respiration. Hyphal extension of all species was unaffected at concentrations less than 100 mg l^?1. At higher concentrations there was a variable inhibitory response to the herbicides but no clear pattern was observed between the five fungi. The binary herbicide mixture had a weak synergistic effect on inhibition of growth rate. For the four aquatic hyphomycetes sporulation was reduced at several herbicide concentrations, but there was no consistent reduction over the experimental period. Flagellospora curvula and Clavariopsis aquatica showed increased sporulation at 100 and 1000 mg l^?1 for only some herbicide combinations. The respiration rates of the fungi varied with species and herbicide concentration and ranged between stimulation at 100 mg l^?1 to inhibition at 4000 mg l^?1. The results indicate that the five fungi are not likely to be severely effected by the phenoxy herbicides at concentrations normally occurring in the field. The possible effects of these herbicides on nutrient cycling are briefly discussed.     

Synthesis of potential herbicides designed to uncouple photophosphorylation

In a search for novel herbicides we attempted to make uncouplers of photophosphorylation. Good herbicides were discovered, but not all were good uncouplers and we present evidence which supports the view that their primary action is through inhibition of carotenoid biosynthesis. This paper describes the synthesis and in vivo and in vitro activities of 21 compounds of the series. The best herbicide was active enough to justify extensive field testing.     

Inhibition of auxin transport by isoquinolinedione herbicides

2-(p-carbethoxyphenyl)-1,3(2H,4H)-isoquinolinedione (CEPIQ), an experimental herbicide, caused effects on geotropism, which are often indicative of an effect on auxin transport, in a whole plant herbicidal screen. However, it showed little or no activity in an in vitro binding assay in corn coleoptiles for the auxin-transport inhibitor,N-1-naphthylphthalamic acid (NPA). Other active isoquinolinedione analogues of this compound did, however, exhibit significant in vitro activity. Direct measurements of auxin transport in corn coleoptiles were undertaken in an attempt to resolve the apparent discrepancy between herbicidal and binding activities. In all cases examined, compounds that were highly active on whole plants were good inhibitors of auxin transport, and compounds that were weak as herbicides showed little or no effect on auxin transport. Therefore, it is concluded that the mode of action of these isoquinolinedione herbicides is the inhibition of auxin transport. Ring-opened analogues of several isoquinolinediones were synthesized and assayed in both the transport and binding assays, in order to test whether compounds in this class express their herbicidal activity by undergoing ring-opening in vivo, yielding products that are more straightforward analogues of NPA with free carboxyl groups. The homophthalamic acids had little or no activity in both assays. On the other hand, thep-ethyl- andp-ethoxy-phenyl phthalamic acids showed auxin transport inhibition comparable to the parent isoquinolinediones, but with markedly increased binding activity. These results support the possible role of ring-opening in the generation of biological activity. However, thep-carbethoxyphenyl phthalamic acid, analogous to CEPIQ, was very weak in both assays. Thus, ring-opening in vivo cannot alone account for the biological activity of this class of compounds.     

Methods for Performing Crosses in Setaria viridis,a New Model System for the Grasses

Setaria viridis is an emerging model system for C₄ grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced ^1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production ³ and developed systems for both transient and stable transformation ⁴. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 °C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.     

Synthesis,antioxidant capacity,and structure–activity relationships of tri-O-methylnorbergenin analogues on tyrosinase inhibition

A series of tri-O-methylnorbergenin analogues 1–9 were synthesized and their antioxidant activities and inhibitory effects on tyrosinase were evaluated. Among tested analogues, compound 4 bearing cathechol moiety exhibited greater antioxidant activity and excellent inhibition on tyrosinase with IC₅₀ value of 9.1 μM, comparable to that of corresponding positive controls. The inhibition mechanism analysis of compound 4 demonstrated that it was a mixed-type inhibitor on tyrosinase. These results suggest that these compounds may serve as a useful clue for further designing and development of novel potential tyrosinase inhibitors.     

Effects of Photosystem II Herbicides on the Photosynthetic Membranes of the Cyanobacterium Aphanocapsa 6308

The effects of the photosystem II herbicides diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on the photosynthetic membranes of a cyanobacterium, Aphanocapsa 6308, were compared to the effects on a higher plant, Spinacia oleracea. The inhibition of photosystem II electron transport by these herbicides was investigated by measuring the photoreduction of the dye 2,6-dichlorophenol-indophenol spectrophotometrically using isolated membranes. The concentration of herbicide that caused 50% inhibition of electron transport (I₅₀ value) in Aphanocapsa membranes for diuron was 6.8 × 10⁻⁹ molar and the I₅₀ value for atrazine was 8.8 × 10⁻⁸ molar. ¹⁴C-labeled diuron and atrazine were used to investigate herbicide binding with calculated binding constants (K) being 8.2 × 10⁻⁸ molar for atrazine and 1.7 × 10⁻⁷ molar for diuron. Competitive binding studies carried out on Aphanocapsa membranes using radiolabeled [¹⁴C]atrazine and unlabeled diuron revealed that diuron competed with atrazine for the herbicide-binding site. Experiments involving the photoaffinity label [¹⁴C]azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-2-triazine) and autoradiography of polyacrylamide gels indicated that the herbicide atrazine binds to a 32-kilodalton protein in Aphanocapsa 6308 cell extracts.     

Setaria viridis: A Model for C4 Photosynthesis

C₄ photosynthesis drives productivity in several major food crops and bioenergy grasses, including maize (Zea mays), sugarcane (Saccharum officinarum), sorghum (Sorghum bicolor), Miscanthus x giganteus, and switchgrass (Panicum virgatum). Gains in productivity associated with C₄ photosynthesis include improved water and nitrogen use efficiencies. Thus, engineering C₄ traits into C₃ crops is an attractive target for crop improvement. However, the lack of a small, rapid cycling genetic model system to study C₄ photosynthesis has limited progress in dissecting the regulatory networks underlying the C₄ syndrome. Setaria viridis is a member of the Panicoideae clade and is a close relative of several major feed, fuel, and bioenergy grasses. It is a true diploid with a relatively small genome of ~510 Mb. Its short stature, simple growth requirements, and rapid life cycle will greatly facilitate genetic studies of the C₄ grasses. Importantly, S. viridis uses an NADP-malic enzyme subtype C₄ photosynthetic system to fix carbon and therefore is a potentially powerful model system for dissecting C₄ photosynthesis. Here, we summarize some of the recent advances that promise greatly to accelerate the use of S. viridis as a genetic system. These include our recent successful efforts at regenerating plants from seed callus, establishing a transient transformation system, and developing stable transformation.     

Herbicide binding and thermal stability of photosystem II isolated from Thermosynechococcus elongatus

Binding of herbicides to photosystem II inhibits the electron transfer from Q_A to Q_B due to competition of herbicides with plastoquinone bound at the Q_B site. We investigated herbicide binding to monomeric and dimeric photosystem II core complexes (PSIIcc) isolated from Thermosynechococcus elongatus by a combination of different methods (isothermal titration and differential scanning calorimetry, CD spectroscopy and measurements of the oxygen evolution) yielding binding constants, enthalpies and stoichiometries for various herbicides as well as information regarding stabilization/destabilization of the complex. Herbicide binding to detergent-solubilized PSIIcc can be described by a model of single independent binding sites present on this important membrane protein. Interestingly, binding stoichiometries herbicide:PSIIcc are lower than 1:1 and vary depending on the herbicide under study. Strong binding herbicides such as terbutryn stabilize PSIIcc in thermal unfolding experiments and endothermically binding herbicides like ioxynil probably cause large structural changes accompanied with the binding process as shown by differential scanning calorimetry experiments of the unfolding reaction of PSIIcc monomer in the presence of ioxynil. In addition we studied the occupancy of the Q_B sites with plastoquinone (PQ9) by measuring flash induced fluorescence relaxation yielding a possible explanation for the deviations of herbicide binding from a 1:1 herbicide/binding site model.     

Modulation by gibberellic acid and adenosine-3′,5′-cyclic monophosphate of starch hydrolysing activity of cowpea seedlings

In cowpea seedlings starch hydrolysing activity increases 35–50 fold on germination for 4 days. This increase in enzyme activity was inhibited by the in vivo addition of 1% glucose but this inhibition was completely overcome by the addition of gibberellic acid (GA₃) (10^?5 M) and adenosine-3′,5′-cyclic monophosphate (cAMP) (10^?5 M). At 5% glucose, GA₃ and cAMP were only partially effective. Structural analogues of cAMP failed to relieve the inhibitory effect of glucose. The inhibition by glucose is not direct but RNA and protein synthesis may be involved. Glucose appears to reduce the internal pool of cAMP which causes inhibition of RNA synthesis and decrease in starch hydrolysing activity. Exogenous application of cAMP may replenish the endogenous pool of cyclic nucleotide and thus overcome inhibition of RNA synthesis and enzyme activity.     

Inhibition of pea leaf glutamine synthetase by methionine sulphoximine,phosphinothricin and other glutamate analogues

The kinetics of the inhibition of glutamine synthetase from Pisum sativum leaves by l-methionine sulphoximine and dl-phosphinothricin were determined. Inhibition by both compounds was mixed-competitive, and apparent K_i values of 0.16 mM and 0.073 mM respectively were determined. dl-5-Hydroxylysine, dl-glutamate-4-tetrazole and l-4-methyleneglutamic acid were also strong inhibitors. Analogues of methionine sulphoximine, dl-ethionine sulphoximine and dl-prothionine sulphoximine were poor inhibitors of glutamine synthetase. Other glutamine and glutamate analogues e.g. azaserine, albizziine, asparagine and kainic acid had no inhibitory action.     

Comparison of 16S rRNA gene phylogeny and functional tfdA gene distribution in thirty-one different 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid degraders

31 different bacterial strains isolated using the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, were investigated for their ability to mineralize 2,4-D and the related herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA). Most of the strains mineralize 2,4-D considerably faster than MCPA. Three novel primer sets were developed enabling amplification of full-length coding sequences (CDS) of the three known tfdA gene classes known to be involved in phenoxy acid degradation. 16S rRNA genes were also sequenced; and in order to investigate possible linkage between tfdA gene classes and bacterial species, tfdA and 16S rRNA gene phylogeny was compared. Three distinctly different classes of tfdA genes were observed, with class I tfdA sequences further partitioned into the two sub-classes I-a and I-b based on more subtle differences. Comparison of phylogenies derived from 16S rRNA gene sequences and tfdA gene sequences revealed that most class II tfdA genes were encoded by Burkholderia sp., while class I-a, I-b and III genes were found in a more diverse array of bacteria.     

Synthesis and anti-CVB3 activity of 4-amino acid derivative substituted pyrimidine nucleoside analogues

Seven novel 4-amino acid derivative substituted pyrimidine nucleoside analogues were designed, synthesized, and tested for their anti-CVB3 activity. Initial biological studies indicated that among these 4-amino acid derivative substituted pyrimidine nucleoside analogues, 4-N-(2′-amino-glutaric acid-1′-methylester)-1-(2′- deoxy-2′-β-fluoro-4′-azido)-furanosyl-cytosine 2 exhibited the most potent anti-CVB activity (IC₅₀ = 9.3 μM). The cytotoxicity of these compounds has also been assessed. The toxicity of compound 2 was similar to that of ribavirin.     

A second mutation enhances resistance of a tobacco mutant to sulfonylurea herbicides

Summary Cultures of Nicotiana tabacum cells homozgous for a mutation (S4) at the SuRB locus that confers resistance to the sulfonylurea herbicides chlorsulfuron and sulfometuron methyl (Chaleff and Ray 1984; Chaleff and Bascomb 1987) were used to isolate a doubly mutant cell line (S4 Hra/S4+) resistant to even higher herbicide concentrations. Growth of cells homozygous for both the S4 and Hra mutations (S4 Hra/S4 Hra) was uninhibited by a herbicide concentration 500-fold higher than a concentration by which growth of S4+/S4+ callus was inhibited by 75%. Plants homozygous for both mutations were at least five-fold more resistant to foliar applications of chlorsulfuron than were singly mutant S4+/S4+ plants. This enhanced resistance was inherited as a single, semidominant, nuclear trait that is genetically linked to the S4 mutation. Acetolactate synthase (ALS) activity in extracts of leaves of doubly mutant (S4 Hra/S4 Hra) plants was approximately 20-fold more resistant to inhibition by chlorsulfuron and sulfometuron methyl than was ALS activity in singly mutant (S4+/ S4+) leaf extracts, which was in turn more resistant to inhibition by these compounds than was the normal enzyme. Extracts prepared from plants of these three genotypes possessed the same ALS specific activities. Therefore, Hra represents a second independent mutation at or near the SuRB locus that reduces the sensitivity of tobacco ALS activity to inhibition by sulfonylurea herbicides.