Don L. Crawford

Novel Plant-Microbe Rhizosphere Interaction Involving Streptomyces lydicus WYEC108 and the Pea Plant (Pisum sativum)

ABSTRACT A previously undescribed plant-microbe interaction between a root-colonizing Streptomyces species, S. lydicus WYEC108, and the legume Pisum sativum is described. The interaction is potentially of great importance to the health and growth in nature of this nodulating legume. The root-colonizing soil actinomycete S. lydicus WYEC108 influences pea root nodulation by increasing root nodulation frequency, possibly at the level of infection by Rhizobium spp. S. lydicus also colonizes and then sporulates within the surface cell layers of the nodules. Colonization leads to an increase in the average size of the nodules that form and improves the vigor of bacteroids within the nodules by enhancing nodular assimilation of iron and possibly other soil nutrients. Bacteroid accumulation of the carbon storage polymer, poly-β-hydroxybutyrate, is reduced in colonized nodules. Root nodules of peas taken from agricultural fields in the Palouse hills of northern Idaho were also found to be colonized by actinomycete hyphae. We hypothesize that root and nodule colonization is one of several mechanisms by which Streptomyces acts as a naturally occurring plant growth-promoting bacterium in pea and possibly other leguminous plants.

Microbial degradation of lignin

Abstract The transformations of lignin that occur during its biodegradation are complex and incompletely understood. Certain fungi of the white-rot group, and possibly other fungi and bacteria, completely decompose lignin to carbon dioxide and water. Other fungi and bacteria apparently degrade lignin incompletely. Differences in lignin-degrading abilities observed for different organisms may result from differences in the completeness of their ligninolytic enzyme systems. Not all lignin components may be attacked by a particular organism. Alternatively, different organisms may differ in their basic mechanisms of attack on lignin. The basic pathways of lignin degradation have been elucidated only for certain representatives of the white-and brown-rot fungi. Although it is known that each of the principal structural components of lignin is attacked by other fungi and bacteria, the biochemistry of that attack has not been elucidated. Work with low molecular weight lignin models has provided only limited information on possible pathways of lignin degradation by microorganisms. There is little evidence to suggest a correlation between abilities to degrade single-ring aromatic or lignin model compounds and the ability to degrade polymeric lignin. More evidence has come from analysis of spent culture media for lignin breakdown products and from comparative chemical analyses of sound lignins versus decayed lignin residues. Accumulated evidence with the most thoroughly studied white-rot fungi suggests that with these fungi lignin degradation proceeds by way of extracellular mixed-function oxygenases and dioxygenases, which catalyse demethylations, hydroxylations and ring-fission reactions within a largely intact polymer, concomitant with some release of low molecular weight lignin fragments. There are also apparent relationships between lignin, carbohydrate and nitrogen metabolism for some organisms, but the relationships may vary from one organism to another. Although research is now mostly at a basic level, industrial applications may result from lignin degradation research. Considerable potential exists for the development of bioconversions which might produce low molecular weight chemicals from waste lignins, and thereby reduce our dependence on petroleum as a source of these chemicals. Alternatively, such bioconversions might produce chemically altered forms of polymeric lignin that may be valuable industrially.

Cellulose and lignin degradation in forest soils: Response to moisture, temperature, and acidity.

The concentration of lignin in plant tissue is a major factor controlling organic matter degradation rates in forest ecosystems. Microbial biomass and lignin and cellulose decomposition were measured for six weeks in forest soil microcosms in order to determine the influence of pH, moisture, and temperature on organic matter decomposition. Microbial biomass was determined by chloroform fumigation; lignin and cellulose decomposition were measured radiometrically. The experiment was designed as a Latin square with soils of pH of 4.5, 5.5, and 6.5 adjusted to 20, 40, or 60% moisture content, and incubated at temperatures of 4, 12, or 24°C. Microbial biomass and lignin and cellulose decomposition were not significantly affected by soil acidity. Microbial biomass was greater at higher soil moisture contents. Lignin and cellulose decomposition significantly increased at higher soil temperatures and moisture contents. Soil moisture was more important in affecting microbial biomass than either soil temperature or soil pH.

Properties of the chitinase of the antifungal biocontrol agent Streptomyces lydicus WYEC108

Abstract An extracellular chitinase from culture filtrates of Streptomyces lydicus WYEC108, a broad spectrum antifungal biocontrol agent, was characterized and purified. Its role in the antifungal activity of this actinomycete was studied. Low constitutive levels of the enzyme were observed when cultures were grown with both simple and complex carbon substrates. The optimal temperature and substrate concentration for maximal chitinase production were 25–30°C and 0.4–0.8 g ml −1 chitin, respectively. High chitinase production was obtained when 1% colloidal chitin was present in the medium as a growth substrate. Activity was induced by n -acetylglucosamine or n,n′ -diacetylchitobiose (GlcNac) 2 and repressed by glucose, xylose, arabinose, raffinose, and carboxymethyl cellulose. Strong catabolite repression of the chitinase was observed. Addition of pectin, laminarin, starch, or β-glucan to the chitin-containing medium, however, increased chitinase production. Probing the S. lydicus genomic DNA with the chi A gene from S. lividans has localized the gene to a 2.5 kb DNA fragment of genomic DNA. The chitinase appears to play a role in the antifungal activities of S. lydicus WYEC108. Production was greatly enhanced when cells were grown in a medium containing colloidal chitin supplemented with certain fungal cell wall preparations, in particular those from Pythium or Aphanomyces species. Crude fungal cell walls were lysed by partially purified chitinase. While S. lydicus also produces one or more antifungal antibiotics, its chitinase probably plays a significant role in the in vivo antifungal biocontrol activity of this rhizosphere-colonizing actinomycete.

Recent advances in studies of the mechanisms of microbial degradation of lignins

Abstract Major advances in our understanding of the biochemical and enzymological mechanisms of lignin biodegradation have been made in the past three years. Research has principally involved two ligninolytic microorganisms, the white rot fungus Phanerochaete chrysosporium and the actinomycete Streptomyces viridosporus. Research has been centred on attempts to identify the microbial catalysts that mediate lignin decay in these two microbes. Emphasis has been on studies concerned with isolating specific lignin catabolic enzymes and/or reduced forms of oxygen involved in attacking the lignin polymer. The possibility that lignin degradation might be non-enzymatic and mediated by extracellular reduced oxygen species such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2 ∪c-|_.), hydroxyl radical (·OH) or singlet oxygen ( 1 O 2 ) has been investigated with both microorganisms. Using methods which have not always been unequivocal, the question of involvement of reduced oxygen species in lignin degradation by P. chrysosporium has been examined exhaustively. Evidence for the involvement of H 2 O 2 is conclusive. However, there is little evidence to support the involvement of other extracellular reduced oxygen species, including ·OH, directly in the process of lignin degradation. Scavenger studies have been inconclusive because of questions of their specificity. If activated oxygen species are involved, the activated oxygen is probably held within the active site of an enzyme molecule. With S. viridosporus , scavenger studies also strongly indicate that extracellular reduced oxygen species are not involved in lignin degradation since scavengers generally do not significantly affect the ligninolytic system. The involvement of specific enzymes in lignin degradation by both P. chrysosporium and S. viridosporus has now been confirmed. With P. chrysosporium, ligninolytic enzymes recently discovered include extracellular non-specific peroxidases and oxygenases. They show numerous activities including dehydrogenative, peroxidatic, oxygenative and C α −C β cleavages of lignin side chains. At least one P. chrysosporium enzyme, a unique H 2 O 2 -requiring oxygenase, has been purified to homogeneity. Evidence has been presented to show that S. viridosporus also produces a ligninolytic enzyme complex involved in demethylation of lignins aromatic rings and in the oxidation of lignin side chains and cleavage of β-tether linkages within the polymer. The combined activites of these enzymes generate water-soluble polymeric modified lignin fragments, which are then slowly degraded further by S. viridosporus. The β-ether cleaving enzyme complex is probably membrane associated, but it is not extracellular. These first isolations of ligninolytic enzymes have changed the course of basic research on lignin biodegradation. New research priorities are already emerging and include enzyme purifications, kinetic studies, enzyme reaction mechanism studies and screenings for more enzymes. In addition, genetic studies are being carried out with both P. chrysosporium and S. viridosporus. Genetic manipulations include not only classical mutagenesis techniques, but also recombinant DNA techniques such as protoplast fusion. This latter technique has already been used to generate overproducers of the ligninolytic enzyme complex in S. viridosporus and it has been successfully used to recombine mutant strains of P. chrysosporium.

Evidence that microbially produced alkaline materials are involved in coal biosolubilization

The biosolubilities (susceptibility to being attacked and solubilized by microbes) of 17 coals by 9 microbes were evaluated, as were solubilities of these coals in dilute alkaline buffers. A direct relationship between coal alkali- and biosolubilities was observed suggesting a common mechanism of coal solubilization. These data are consistent with the hypothesis that a mechanism by which microorganisms solubilize coal is by production of alkaline materials that raise medium pH and effect solubilization.

Chemistry of softwood lignin degradation byStreptomyces viridosporus

Polymeric lignin isolated from ground spruce phloem/bark tissue following decay by the actinomyceteStreptomyces viridosporus (T7A) was characterized chemically and compared to undergraded lignin from the same source. The chemical transformations resulting from degradation were compared to those that result from fungal degradation of softwood lignins by brown- and white-rot fungi. Degradative chemical analyses showed thatS. viridosporus-degraded lignin was significantly altered in structure. Much of the integrity of the basic 4-hydroxy-3-methoxyphenylpropane subunit structure was lost. Actinomycete-decayed lignin was decreased in carbon and enriched in oxygen and hydrogen contents. It also had been extensively demethylated. Chemical analysed indicated that phenylpropanoid side-chains had been oxidized by introduction of α-carbonyls and by side-chain shortening reactions. Although the degraded lignin remained polymeric, it was significantly dearomatized. These changes are similar to those previously reported for white-rotted lignins, except for the increased hydrogen content. The evidence indicated that lignin degradation byS. viridosporus is oxidative and involves demethylations, ring cleavage reactions, and oxidative attack on phenylpropanoid side-chains. Also, some reduced structures accumulate in the polymer and some low molecular weight intermediates are released into the growth medium.

Oxidation of phenolic and non-phenolic substrates by the lignin peroxidase of Streptomyces viridosporus T7A

SummaryNumerous single-ring, aromatic, phenolic and non-phenolic compounds were tested as substrates of Streptomyces viridosporus T7A extracellular lignin peroxidase. Oxidations were monitored by spectroscopy, with and without 4-aminoantipyrine (4-AAP) as a color-forming reagent. The oxidation of phenols containing one or no carbon groups in the para position resulted in coupling with 4-AAP to form a red color. Thin layer chromatography and mass spectroscopy showed that the oxidation of vanillic acid (4-hydroxy-3-methoxybenzoic acid) and syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid) resulted in a direct coupling between 4-AAP and the phenol ring to form a quinone structure. In the reaction with vanillyl acetone (4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one) and 4-AAP, 4-AAP coupled to Á-carbon of vanillyl acetone. As shown by UV-visible spectroscopy, S. viridosporus T7A peroxidase oxidized phenolic compounds, but was unable to oxidize non-phenolic ones.

Activities of cellulase and other extracellular enzymes during lignin solubilization by Streptomyces viridosporus

SummaryTwo mutant strains of the lignin degrading bacterium Streptomyces viridosporus strain T7A with enhanced abilities to produce a soluble lignin degradation intermediate, acid-precipitable polymeric lignin (APPL) and several mutants derepressed for cellulase production were compared with the wild type to examine the roles of cellulase and selected other extracellular enzymes in lignin solubilization by S. viridosporus. The two APPL-overproducing mutants, T-81 and T-138, had higher cellulase activities than the wild type. Mutants specifically derepressed for cellulase were also isolated and were found to produce more APPL than the wild type. The results are indicative of some involvement of cellulase in the lignin solubilization process. The lignin solubilized from corn (Zea mays) lignocellulose by the mutants was slightly different chemically as compared to wild type solubilized lignin in that it had a higher coumaric acid ester content. The production of extracellular coumarate ester esterase, aromatic aldehyde oxidase, and xylanase was also examined in the mutants. Xylanase and aromatic aldehyde oxidase production did not differ significantly between the mutants and the wild type. Mutant T-81 was found to have a slightly lower activity for esterase as compared with the wild type. It was concluded that xylanase, oxidase and esterase are not the enzymes directly responsible for enhanced lignin solubilization. The results, however, do implicate cellulase in the process.

Production of an aromatic aldehyde oxidase by Streptomyces viridosporus

Streptomyces viridosporus strain T7A, when grown in liquid media containing yeast extract and aromatic aldehydes, oxidized the aromatic aldehydes to the corresponding aromatic acids. Benzaldehyde, m-hydroxybenzaldehyde, p-hydroxybenzaldehyde, and protocatechualdehyde were catabolized further via the β-ketoadipate and gentisate pathways. Dehydrodivanillin, isophthalaldehyde, salicylaldehyde, syringaldehyde, terephthalaldehyde, vanillin, and veratraldehyde were oxidized only as far as the corresponding aromatic acids. Phthalaldehyde and aliphatic aldehydes were not oxidized. The aromatic aldehyde oxidase, which was produced by cultures grown in either the presence or absence of aromatic aldehydes, was partially purified by ammonium sulfate precipitation and ion-exchange chromatography. It consumed molecular oxygen, oxidized aromatic aldehydes to aromatic acids, and produced hydrogen peroxide all in equimolar amounts.