Biotransformations Of Fluoroaromatic Compounds: Accumulation Of Hydroxylated Products From 3-Fluorophthalic Acid Using Mutant Strains Of Pseudomonas Testosteroni

Biotransformations of 3-fluorophthalic acid have been investigated using blocked mutants of Pseudomonas testosteroni that are defective in the metabolism of phthalic acid (benzene-1,2-dicar-boxyfic acid). Mutant strains were grown with L-glutamic acid in the presence of 3-fluorophthalic acid as inducer of phthalic acid catabolic enzymes. Products that accumulated in the medium were isolated, purified and identified as the fluoroanalogues of those produced from phthalic acid by the same strains. The previously undescribed fluorochemicals cis-3-fluoro-4,5-dihydro-4,5-dihydroxyphthalic acid (VI) and 3-fluoro-4,5-dihydroxyphthalic acid (VII) have been obtained by biotransformation of 3-fluorophthalic acid, and 3-fluoro-5-hydroxyphthalic acid (X) from (VI) by freeze drying. In addition, samples of 2-fluoro-3,4-dihydroxybenzoic acid (2-fluoroprotocatechuic acid, VIII) and 3-fluoro-4,Sdi-hydroxybenzoic acid (5-fluoroprotocatechuic acid, IX) were obtained with a mutant deficient in the ring-fission enzyme, showing that the fluorine substituent in their precursor substrate (VII) is not recognized by the decarboxylase of the pathway, which shows no preference for which carboxyl group is removed. These studies of 3-fluorophthalic acid catabolism demonstrate the opportunities available for the production of novel fluorochemicals in reasonable yields by microbial transformations.     

Phthalate and 4-hydroxyphthalate metabolism in Pseudomonas testosteroni: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Phthalate is degraded through 4,5-dihydroxyphthalate and protocatechuate in Pseudomonas testosteroni NH1000. The ezyme 4,5-dihydroxyphthalate decarboxylase, catalyzing the conversion of 4,5-dihydroxyphthalate to protocatechuate and carbon dioxide, was purified approximately 130-fold from phthalate-induced cells of a protocatechuate 4,5-dioxygenase-deficient mutant of P. testosteroni. The most purified preparation showed a single protein band on sodium dodecyl sulfate-acrylamide disc gel electrophoresis with a molecular weight of 38,000. The apparent molecular weight of the native enzyme determined by Sephadex G-200 column chromatography was 150,000. Among the substrate analogs tested, only 4-hydroxyphthalate served as a substrate, which was decarboxylated to form m-hydroxybenzoate. The apparent Km values for 4,5-dihydroxyphthalate and 4-hydroxyphthalate were estimated to be 10.5 micrometer and 1.25 mM, respectively, and the Vmax for the former was 10 times larger than that for the latter. Whereas the wild-type strain could utilize 4-hydroxyphthalate as a sole source of carbon, none of the following could grow with the compound: 4,5-dihydroxyphthalate decarboxylase-deficient, m-hydroxybenzoate-nondegradable, and protocatechuate 4,5-dioxygenase-deficient mutants. Since one-step revertants of these mutants could utilize 4-hydroxyphthalate, the compound appears to be metabolized through m-hydroxybenzoate and protocatechuate in P. testosteroni NH1000.     

Phthalate and 4-hydroxyphthalate metabolism in Pseudomonas testosteroni: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Phthalate is degraded through 4,5-dihydroxyphthalate and protocatechuate in Pseudomonas testosteroni NH1000. The ezyme 4,5-dihydroxyphthalate decarboxylase, catalyzing the conversion of 4,5-dihydroxyphthalate to protocatechuate and carbon dioxide, was purified approximately 130-fold from phthalate-induced cells of a protocatechuate 4,5-dioxygenase-deficient mutant of P. testosteroni. The most purified preparation showed a single protein band on sodium dodecyl sulfate-acrylamide disc gel electrophoresis with a molecular weight of 38,000. The apparent molecular weight of the native enzyme determined by Sephadex G-200 column chromatography was 150,000. Among the substrate analogs tested, only 4-hydroxyphthalate served as a substrate, which was decarboxylated to form m-hydroxybenzoate. The apparent Km values for 4,5-dihydroxyphthalate and 4-hydroxyphthalate were estimated to be 10.5 micrometer and 1.25 mM, respectively, and the Vmax for the former was 10 times larger than that for the latter. Whereas the wild-type strain could utilize 4-hydroxyphthalate as a sole source of carbon, none of the following could grow with the compound: 4,5-dihydroxyphthalate decarboxylase-deficient, m-hydroxybenzoate-nondegradable, and protocatechuate 4,5-dioxygenase-deficient mutants. Since one-step revertants of these mutants could utilize 4-hydroxyphthalate, the compound appears to be metabolized through m-hydroxybenzoate and protocatechuate in P. testosteroni NH1000.     

The properties of syringyl, guaiacyl and p-hydroxyphenyl artificial lignins

1. Artificial lignins have been produced on potato parenchyma. 2. The methoxyl-free lignin and 4-hydroxy-3-methoxy (guaiacyl) lignins could be estimated by the sulphuric acid method but the 4-hydroxy-3,5-dimethoxy (syringyl) lignins could not. 3. Permanganate oxidation of isolated p-coumaric lignin gave 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid and small amounts of hydroxytrimesic acid and 4-hydroxyphthalic acid. Ferulic lignin gave vanillic acid and 5-carboxyvanillic acid and also small amounts of 4-hydroxybenzoic acid and dehydrodivanillic acid. The sinapic lignin gave traces of syringic acid and of 4-hydroxybenzoic acid. 4. The p-coumaric lignin is a highly condensed polymer. The ferulic lignin is partly uncondensed and partly condensed through the 5-position like gymnosperm lignin. The sinapic lignin shows no evidence of condensation and is probably an ether-linked polymer.     

Prevalence of fungi during Skylab missions.

Di-n-butyl phthalate and other dialkyl phthalates are used as carbon sources by three Nocardia sp. isolates; mono-n-butyl phthalate is used as a carbon source by an Arthrobacter sp. isolate and a Pseudomonas sp. isolate. The compounds were metabolized in these organisms by hydrolysis to the corresponding monoesters and free phthalic acid. Phthalic acid was then metabolized via protocatechuic acid by 3,4-dioxygenative ring cleavage.     

A simple method for detection of enzyme activities involved in the initial step of phthalate degradation in microorganisms

We have developed a simple method for the detection of phthalate 4,5-dioxygenase and 4,5-dihydro-4,5-dihydroxyphthalate dehydrogenase activities in the initial step of phthalate degradation in bacteria. It was found that cells of a Pseudomonas putida strain adapted for phthalate could convert quinolinic acid to a hydroxylated product having λ_max at 315 nm. The occurrence of this compound was visualized by reaction with diazotized p-nitroaniline with which a red compound having λ_max at 512 nm was produced. In practice, if cells in colonies developed on an agar plate containing mineral salt medium supplemented with 0.4% of disodium phthalate and 0.1% of quinolinic acid are active with respect to the 4,5-dihydroxyphthalate pathway, then the colonies would be colored red immediately upon spraying with the diazotized p-nitroaniline reagent. The method was used to identify the phthalate degradative pathway for 27 phthalate-utilizing strains of the genera Pseudomonas (18 strains), Agrobacterium (3 strains), Alcaligenes (5 strains), and Micrococcus (1 strain). It was found that 24 of the 26 Gram-negative bacteria have the 4,5-dihydroxyphthalate pathway and that the remaining two strains of Pseudomonas sp. may metabolize via an unidentified pathway other than the dihydroxyphthalate pathways, and the Gram-positive strain of Micrococcus sp. metabolizes phthalate via the 3,4-dihydroxyphthalate pathway.     

Isolation and Characterization of Thermophilic Bacilli Degrading Cinnamic, 4-Coumaric, and Ferulic Acids

Thirty-four thermophilic Bacillus sp. strains were isolated from decayed wood bark and a hot spring water sample based on their ability to degrade vanillic acid under thermophilic conditions. It was found that these bacteria were able to degrade a wide range of aromatic acids such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(p-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids. The metabolic pathways for the degradation of these aromatic acids at 60°C were examined by using one of the isolates, strain B1. Benzoic and 4-hydroxybenzoic acids were detected as breakdown products from cinnamic and 4-coumaric acids, respectively. The β-oxidative mechanism was proposed to be responsible for these conversions. The degradation of benzoic and 4-hydroxybenzoic acids was determined to proceed through catechol and gentisic acid, respectively, for their ring fission. It is likely that a non-β-oxidative mechanism is the case in the ferulic acid catabolism, which involved 4-hydroxy-3-methoxyphenyl-β-hydroxypropionic acid, vanillin, and vanillic acid as the intermediates. Other strains examined, which are V0, D1, E1, G2, ZI3, and H4, were found to have the same pathways as those of strain B1, except that strains V0, D1, and H4 had the ability to transform 3-hydroxybenzoic acid to gentisic acid, which strain B1 could not do.     

Isolation from a Shea Cake Digester of a Tannin-Tolerant Escherichia coli Strain Decarboxylating p-Hydroxybenzoic and Vanillic Acids

A facultatively anaerobic, mesophilic, Gram-negative, non-motile, non-sporulated bacterium, designated strain C2, was isolated from an anaerobic digester fed with shea cake rich in tannins and aromatic compounds and previously inoculated with anaerobic sludge from the pit of a slaughterhouse, after enrichment on tannic acid. The straight rods occurred singly or in pairs. Strain C2 fermented numerous carbohydrates (fructose, galactose, glucose, lactose, mannose, maltose, melibiose, raffinose, rhamnose, ribose, saccharose, sorbitol, trehalose, and xylose) and peptides (Biotrypcase, Casamino acids, and yeast extract), producing acid and gas, and had a G + C content of 51.6 ± 0.1 mol %. Strain C2 was very closely related to Escherichia coli (= DSM 30083^T) phylogenetically (similarity of 99%), genotypically (DNA homology of 79%), and phenotypically. The isolate tolerated tannic acid (hydrolyzable tannin) and decarboxylated by non-oxidative decarboxylation only p-hydroxybenzoic and vanillic acids to their corresponding phenol and guaicol, under anaerobic and aerobic conditions without further degradation. Adding glucose increased growth and the rate of conversion. High concentrations of p-hydroxybenzoic acid or vanillic acid inhibited growth, and decarboxylation could not occur completely, suggesting phenol toxicity. In contrast, the type strain of E. coli cannot metabolize p-hydroxybenzoic and vanillic acids, anaerobically or aerobically, with or without glucose added. Received: 30 July 2001 / Accepted: 17 August 2001     

Biodegradation of p-Cresol by Bacillus sp. Strain PHN 1

A bacterium capable of utilizing p-cresol as sole source of carbon and energy was isolated from soil and identified as a Bacillus species. The organism also utilized phenol, o-cresol, m-cresol, 4-hydroxybenzoic acid, and gentisic acid as growth substrates. The organism degraded p-cresol to 4-hydroxybenzoic acid, which was further metabolized by a gentisate pathway, as evidenced by isolation and identification of metabolites and enzyme activities in the cell-free extract. Such a bacterial strain can be used for bioremediation of environments contaminated with phenolic compounds.     

Production of 4,5-dihydro-4,5-dihydroxyphthalate from phthalate by a mutant strain of Pseudomonas testosteroni M4-1

Summary Pseudomonas testosteroni M4-1, capable of using phthalate as the sole carbon and energy source, was isolated. Tn5 mutagenesis using pSUP2021 yielded mutant strains of M4-1 that are defective in phthalate metabolism and produce a dihydrodiol compound. The dihydrodiol compound produced by mutant strain M4-122 was isolated and identified as 4,5-dihydro-4,5-dihydroxyphthalate (DDP) by elementary analysis, mass analysis and nuclear magnetic resonance. Various conditions to increase the yield of DDP from phthalate were examined for mutant strain M4-122. With resting cells 6 g DDP/1 were produced. The additional of ethanol to the resting-cell reaction mixture enhanced DDP production and 10 g DDP/1 was produced from 8.3 g/1 of phthalate. Offprint requests to: T. Omori     

Aerobic and anaerobic catabolism of phthalic acid by a nitrate-respiring bacterium

A Bacillus sp., isolated by anaerobic enrichment on a o-phthalic acid-nitrate medium, grew either aerobically or anaerobically on phthalic acid. Cells grown anaerobically on phthalate immediately oxidized phthalate and benzoate with nitrate, whereas aerobic oxidation only occurred after a lag period and was inhibited by chloramphenicol. 2-Fluoro-and 3-fluorobenzoate were formed from 3-fluorophthalate by cells grown anaerobically on phthalate. Aerobically grown cells immediately oxidized phthalate, benzoate, 3-hydroxybenzoate and gentisate with oxygen. The aerobic and anaerobic route of catabolism of phthalate may thus share an initial decarboxylation to benzoate. This is the first report of the anaerobic dissimilation of phthalic acid by a pure bacterial culture.     

Identification of a Novel Metabolite in the Degradation of Pyrene by Mycobacterium sp. Strain AP1: Actions of the Isolate on Two- and Three-Ring Polycyclic Aromatic Hydrocarbons

Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans- or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6′-dihydroxy-2,2′-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and ortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of ortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures.     

Assimilation of aromatic compounds by Comamonas testosteroni: characterization and spreadability of protocatechuate 4,5-cleavage pathway in bacteria

Comamonas testosteroni strain CNB-1 was isolated from activated sludge and has been investigated for its ability to degrade 4-chloronitrobenzene. Results from this study showed that strain CNB-1 grew on phenol, gentisate, vanillate, 3-hydroxybenzoate (3HB), and 4-hydroxybenzoate (4HB) as carbon and energy sources. Proteomic data and enzyme activity assays suggested that vanillate, 3HB, and 4HB were degraded in strain CNB-1 via protocatechuate (PCA) 4,5-cleavage pathway. The genetics and biochemistry of the PCA 4,5-cleavage pathway were investigated. Results showed that the 4-oxalomesaconate (OMA) hydratase from C. testosteroni takes only enol-OMA as substrate. A previously functionally unknown gene pmdU encodes an OMA tautomerase and catalyzes conversion of OMA_keto into OMA_enol. The 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase is encoded by pmdF and catalyzes the last step of the PCA 4,5-cleavage pathway. We explored the 1,183 microbial genomes at GenBank for potential PCA 4,5-cleavage pathways, and 33 putative pmd clusters were found. Results suggest that PCA 4,5-cleavage pathways are mainly distributed in α- and β-Proteobacteria.     

Degradation of Phthalic Acids by Denitrifying, Mixed Cultures of Bacteria

Mixed cultures of bacteria, enriched from aquatic sediments, grew anaerobically on all three isomers of phthalic acid. Each culture grew anaerobically on only one isomer and also grew aerobically on the same isomer. Pure cultures were isolated from the phthalic acid (o-phthalic acid) and isophthalic acid (m-phthalic acid) enrichments that grew aerobically on phthalic and isophthalic acids. Cell suspension experiments indicated that protocatechuate is an intermediate of aerobic catabolism. Pure cultures which grew aerobically on terephthalic acid (p-phthalic acid) could not be isolated from the enrichments, and neither could pure cultures that grew anaerobically on any of the isomers. Cell suspension experiments suggested that separate pathways exist for the aerobic and anaerobic oxidation of phthalic acids. Each enrichment culture used only one phthalic acid isomer under anaerobic conditions, but all isomers were simultaneously adapted for the anaerobic catabolism of benzoate. Cells grown anaerobically on a phthalic acid immediately attacked the isomer under anaerobic conditions, whereas there was a lag before aerobic breakdown occurred, and, for phthalic and terephthalic acids, chloramphenicol stopped aerobic adaptation but had no effect on anaerobic catabolism. This work suggests that phthalic acids are biodegradable in anaerobic environments.     

Hydroxylated Metabolites of 2,4-Dichlorophenol Imply a Fenton-Type Reaction in Gloeophyllum striatum

While degrading 2,4-dichlorophenol, two strains of Gloeophyllum striatum, a basidiomycetous fungus causing brown rot decay of wood, simultaneously produced 4-chlorocatechol and 3,5-dichlorocatechol. These metabolites were identified by comparing high-performance liquid chromatography retention times and mass spectral data with those of chemically synthesized standards. Under similar conditions, 3-hydroxyphthalic hydrazide was generated from phthalic hydrazide, a reaction assumed to indicate hydroxyl radical formation. Accordingly, during chemical degradation of 2,4-dichlorophenol by Fenton's reagent, identical metabolites were formed. Both activities, the conversion of 2,4-[U-¹⁴C]dichlorophenol into ¹⁴CO₂ and the generation of 3-hydroxyphthalic hydrazide, were strongly inhibited by the hydroxyl radical scavenger mannitol and in the absence of iron. These results provide new evidence in favor of a Fenton-type degradation mechanism operative in Gloeophyllum.     

Metabolism of hydroxybiphenyl and chloro-hydroxybiphenyl by biphenyl/chlorobiphenyl degradingPseudomonas testosteroni,strain B-356

Summary A biphenyl (BP) and chlorobiphenyl (CBP) metabolizingPseudomonas testosteroni, strain B-356 was also capable of utilizing 2-, 3-, and 4-hydroxybiphenyl. Data presented here suggest that utilization of biphenyl and mono-subtituted biphenyls involves the enzymes of the same pathway. Chloro-hydroxybiphenyls were also metabolized by strain B-356. The unsubstituted ring is first hydroxylated in position 2 and 3 and then cleaved in ameta 1, and 2, position to ultimately generate the benzoic acid derivatives. Since strain B-356 was capable of utilizing benzoic acid and mono-hydroxybenzoic acids, the utilization of biphenyl, 2-, 3-, and 4-hydroxybiphenyl is complete at non-toxic concentrations of the substrates. Chlorobenzoic acids and chloro-hydroxybenzoic acids were not metabolized further by this strain. Studies usingPseudomonas putida, strain KT2440 carrying cloned BP/CBP genes from strain B-356 provided further evidence for the presence of a common pathway for the metabolism of the above compounds inP. testosteroni, strain B-356. Suggestions are made on significance of the broad substrate specificity of the enzymes of biphenyl/chlorobiphenyl pathway in regard to their possible origin and in relation to PCB mixture degradation.     

Mutational biosynthesis of tacrolimus analogues by fkbO deletion mutant of Streptomyces sp. KCTC 11604BP

Tacrolimus (FK506) is an important macrocyclic polyketide showing antifungal and immunosuppressive activities, as well as neuroregenerative properties. Tacrolimus biosynthetic machinery should incorporate the shikimate-derived 4,5-dihydroxycyclohex-1-enecarboxylic acid (DHCHC) as a biosynthetic starter unit into the biosynthetic line of tacrolimus. fkbO is a homologue of rapK encoding chorismatase related to the biosynthesis of starter unit DHCHC from chorismate in the rapamycin biosynthetic gene cluster. FkbO and RapK are good targets for mutational biosynthesis to produce novel analogues of tacrolimus, ascomycin, and rapamycin, which could be important drugs for clinical application in the treatment of cancer and immune and neurodegenerative diseases. To make novel tacrolimus analogues, we prepared an fkbO in-frame deletion mutant, Streptomyces sp. GT110507, from a tacrolimus high producer. We scrutinized the cyclic carboxylic acids that were possibly incorporated instead of DHCHC by precursor-directed mutasynthesis using Streptomyces sp. GT110507 to lead tacrolimus analogues. Among them, trans-4-hydroxycyclohexanecarboxylic acid and 3-hydroxybenzoic acid were successfully incorporated into the tacrolimus backbone, which led to the production of 31-desmethoxytacrolimus and TC-225, respectively. Especially, adding of trans-4-hydroxycyclohexanecarboxylic acid produced a high amount (55 mg/L) of 31-desmethoxytacrolimus. Interestingly, in the rapK mutant, it has been reported that the incorporation of cyclohexanecarboxylic acid (CHC) led to 39-desmethoxy rapamycin. However, in Streptomyces sp. GT110507, CHC is not successfully incorporated. This discrepancy should reflect the differences in the DHCHC biosynthesis mechanism and/or substrate specificity of starter unit loading machineries (FkbP and RapP) of tacrolimus and rapamycin.     

Vanillic acid metabolism by the white-rot fungus Sporotrichum pulverulentum

Vanillic acid metabolism was studied in wild-type Sporotrichum pulverulentum and three different mutants. Vanillic acid was found to be oxidatively decarboxylated to methoxyhydroquinone (MHQ) and simultaneously reduced to vanillin and vanillyl alcohol to different degrees depending upon the cultivation conditions. The reducing pathway cannot be utilized unless the fungus has access to an easily metabolized carbon source such as glucose or cellobiose, while decarboxylation takes place in cultures with only vanillic acid present. Polymerization reactions also occurred in the culture solutions. Some evidence for reoxidation of vanillin and vanillyl alcohol was obtained in vivo, and in vitro experiments using horseradish peroxidase.Using vanillic acids labelled in the carboxyl, methoxyl and the aromatic ring it was shown that decarboxylation occures before ring-cleavage, which in turn takes place earlier than the release of ¹⁴CO₂ from O¹⁴CH₃-vanillate. The ¹⁴CO₂ evolution from the methoxyl group is repressed by 1% cellobiose as compared to 0.25% cellobiose, but is stimulated by 26 mM nitrogen (as asparagine plus NH₄NO₃) compared to 2.6 mM nitrogen. Since S. pulverulentum appears to require three hydroxyl groups attached to the benzene ring before ring-cleavage can occur, preparation for ring-cleavage is apparently achieved by hydroxylation rather than by demethylation.A scheme for metabolism of vanillic acid by S. pulverulentum based upon these results is proposed.Non-Standard Abbreviations WT wild type Sporotrichum pulverulentum - MHQ methoxyhydroquinone - MQ methoxyquinone - NKM Norkrans medium - DMS dimethylsuccinate - DHP dehydropolymer of coniferyl alcohol     

Hydrolysis of benzonitrile herbicides by soil actinobacteria and metabolite toxicity

The soil actinobacteria Rhodococcus rhodochrous PA-34, Rhodococcus sp. NDB 1165 and Nocardia globerula NHB-2 grown in the presence of isobutyronitrile exhibited nitrilase activities towards benzonitrile (approx. 1.1–1.9 U mg^?1 dry cell weight). The resting cell suspensions eliminated benzonitrile and the benzonitrile analogues chloroxynil (3,5-dichloro-4-hydroxybenzonitrile), bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile) (0.5 mM each) from reaction mixtures at 30°C and pH 8.0. The products were isolated and identified as the corresponding substituted benzoic acids. The reaction rates decreased in the order benzonitrile ? chloroxynil > bromoxynil > ioxynil in all strains. Depending on the strain, 92–100, 70–90 and 30–51% of chloroxynil, bromoxynil and ioxynil, respectively, was hydrolyzed after 5 h. After a 20-h incubation, almost full conversion of chloroxynil and bromoxynil was observed in all strains, while only about 60% of the added ioxynil was converted into carboxylic acid. The product of ioxynil was not metabolized any further, and those of the other two herbicides very slowly. None of the nitrilase-producing strains hydrolyzed dichlobenil (2,6-dichlorobenzonitrile). 3,5-Dibromo-4-hydroxybenzoic acid exhibited less inhibitory effect than bromoxynil both on luminescent bacteria and germinating seeds of Lactuca sativa. 3,5-Diiodo-4-hydroxybenzoic acid only exhibited lower toxicity than ioxynil in the latter test.