Lee A. Deobald

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.

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.

Depolymerization and chemical modification of lignite coal byPseudomonas cepacia strain DLC-07

Coal metabolizingPseudomonas cepacia DLC-07 was isolated from soil and shown to use soluble lignite coal as a carbon/energy source.Pseudomonas modified coal residues were characterized by FT-IR, HPLC, elemental analysis, and13C-NMR. P.cepacia DLC-07 depolymerized lignite, as shown by HPLC. FT-IR analysis showed fewer carbonyl and carboxyl groups, decreased etheric oxygen, and fewer aromatic and conjugated carbon-carbon double bonds in modified, compared to control coal.13C-NMR indicated a decrease in carbonyls and heteroaromatic carbons containing N, O, and S, a decrease in unsubstituted aromatic carbons, and an increase in long chain methylenes in treated coal. Elemental analysis supported the spectral data concerning metabolism of the coal polymer. P.cepacia cometabolized coal model compounds and utilizedp-hydroxy substituted benzoic or cinnamic acids or aldehydes as sole carbon and energy sources.

Proteogenomic and functional analysis of chromate reduction in Acidiphilium cryptum JF-5, an Fe(III)-respiring acidophile.

Acidiphilium cryptum JF-5, an acidophilic iron-respiring Alphaproteobacterium, has the ability to reduce chromate under aerobic and anaerobic conditions, making it an intriguing and useful model organism for the study of extremophilic bacteria in bioremediation applications. Genome sequence annotation suggested two potential mechanisms of Cr(VI) reduction, namely, a number of c-type cytochromes, and a predicted NADPH-dependent Cr(VI) reductase. In laboratory studies using pure cultures of JF-5, an NADPH-dependent chromate reductase activity was detected primarily in soluble protein fractions, and a periplasmic c-type cytochrome (ApcA) was also present, representing two potential means of Cr(VI) reduction. Upon further examination, it was determined that the NADPH-dependent activity was not specific for Cr(VI), and the predicted proteins were not detected in Cr(VI)-grown cultures. Proteomic data did show measureable amounts of ApcA in cells grown with Cr(VI). Purified ApcA is reducible by menadiol, and in turn can reduce Cr(VI), suggesting a means to obtain electrons from the respiratory chain and divert them to Cr(VI). Electrochemical measurements confirm that Cr reduction by ApcA is pH dependent, with low pH being favored. Homology modeling of ApcA and comparison to a known Cr(VI)-reducing c-type cytochrome structure revealed basic amino acids which could interact with chromate ion. From these studies, it can be concluded that A. cryptum has the physiologic and genomic capability to reduce Cr(VI) to the less toxic Cr(III). However, the expected chromate reductase mechanism may not be the primary means of Cr(VI) reduction in this organism.

C5‐Functionalized LNA: Unparalleled Hybridization Properties and Enzymatic Stability

Antisense oligonucleotides (ONs) are widely explored as fundamental research tools and therapeutic agents against diseases of genetic origin due to their ability to modulate gene expression by interfering with target RNA. Introduction of chemically modified nucleotides into antisense ONs is crucial to increase binding affinity toward RNA targets, improve discrimination of mismatched RNA to avoid off-target effects, and enhance stability against nucleases to slow down degradation. The use of conformationally restricted nucleotides and locked nucleic acids (LNAs, Scheme 1) in particular, has to some extent addressed these challenges. Antisense LNAs are accordingly evaluated in several clinical trials. Substantial efforts have been invested to develop LNA analogues with even more desirable biophysical properties and reduced hepatotoxicity. These studies have primarily focused on modification of the oxymethylene bridge spanning the C2’and C4’-positions and/or introduction of minor-groove-oriented substituents into the bridge. Improved enzymatic stability, e, f, j] altered biodistribution, or reduced hepatotoxicity has been reported for some of the analogues, but improvements in hybridization properties relative to LNA were generally not observed. Results from comparative in vivo antisense studies must be awaited to assess if the significantly increased synthetic complexity of these conformationally restricted nucleotides is justified. C5-functionalized pyrimidine DNA building blocks have attracted considerable attention due to their ability to accommodate functional entities in the major groove of nucleic acid duplexes and straightforward synthesis. Small C5-entities are generally well tolerated in duplexes and result in small increases in thermal affinity toward DNA/RNA complements. f] In light of this, we hypothesized that C5-alkynyl-functionalized LNA monomers would synergistically integrate beneficial Scheme 1. Synthetic outline of phosphoramidites 5 W–5 Z. CAN = ceric ammonium nitrate, DMTr = 4,4’-dimethoxytrityl, TBAF = tetrabutylammonium fluoride, PCl = 2-cyanoethyl N, N’diisopropylchlorophosphoramidite.

Optimized DNA-targeting using triplex forming C5-alkynyl functionalized LNA

Triplex forming oligonucleotides (TFOs) modified with C5-alkynyl functionalized LNA (locked nucleic acid) monomers display extraordinary thermal affinity toward double stranded DNA targets, excellent discrimination of Hoogsteen-mismatched targets, and high stability against 3?-exonucleases.

Proteomic detection of proteins involved in perchlorate and chlorate metabolism

Mass spectrometry and a time-course cell lysis method were used to study proteins involved in perchlorate and chlorate metabolism in pure bacterial cultures and environmental samples. The bacterial cultures used included Dechlorosoma sp. KJ, Dechloromonas hortensis, Pseudomonas chloritidismutans ASK-1, and Pseudomonas stutzeri. The environmental samples included an anaerobic sludge enrichment culture from a sewage treatment plant, a sample of a biomass-covered activated carbon matrix from a bioreactor used for treating perchlorate-contaminated drinking water, and a waste water effluent sample from a paper mill. The approach focused on detection of perchlorate (and chlorate) reductase and chlorite dismutase proteins, which are the two central enzymes in the perchlorate (or chlorate) reduction pathways. In addition, acetate-metabolizing enzymes in pure bacterial samples and housekeeping proteins from perchlorate (or chlorate)-reducing microorganisms in environmental samples were also identified.

Cadmium stress studies: Media development, enrichment, consortia analysis, and environmental relevance

The effects of Cadmium (Cd) toxicity on bacterial consortia originating from an-aerobic sewage sludge and cultivated under differing enrichment conditions were studied. Cultures were enriched in minimal media developed specifically for Cd stress studies. At inoculation all Cd was soluble in free ion or chelated form. Electron donors and acceptors were varied to obtain each physiological enrichment type. Adaptation leading to higher levels of Cd resistance of the consortia over time was observed under all physiological conditions. Initial and increased Cd tolerances were consistently greatest in multiphysiological enrichments (MPH). Sulfate reducing (SRB), methanogenic (MET), and fermentative (FRM) enrichments had less tolerance however, the level of tolerance to the Cd varied from one inoculation to the next. The Cd remained soluble as free Cd in MPH and FRM conditions and was precipitated significantly in SRB and moderately in MET conditions. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rRNA of the SRB, MPH, and FRM enrichments were followed over time. The consortia underwent succession under all physiological conditions when compared with the profile of the inoculum. Microbial population diversity decreased as the consortia were subcultured. The effects of chelators in the MPH medium were also evaluated. The addition of chelators transiently decreased toxicity. Effects of MPH medium on the Cd sorption capacity of soil were evaluated. Microbial growth decreased the amount of Cd left in solution.

First detection of microcystin in Puget Sound, Washington, mussels (Mytilus trossulus)

Abstract Presence of the cyanobacterial hepatotoxin microcystin was detected by liquid chromatography-mass spectrometry in mussels (Mytilus trossulus) from Puget Sound, Washington. Microcystin was found in all but one mussel exposed to inflows from a lake, with maximum concentrations of 52.4 ppb. This is the first known report of marine bivalves in Puget Sound, Washington, accumulating microcystin from freshwater sources.

Lignin biotransformations by an aromatic aldehyde oxidase produced byStreptomyces viridosporus T7A

Streptomyces viridosporus produces an intracellular aromatic aldehyde oxidase that oxidizes aromatic and α, β-unsaturated aromatic aldehydes to their corresponding acids. It also produces extracellular oxidase as shown by zones of clearing when grown on agar containing insoluble dehydrodivanillin (DHDV). This extracellular form may be responsible for oxidizing aldehyde groups in lignin. The extracellular oxidase was expressed maximally after 3 d growth in medium containing only yeast extract. However, higher levels were produced when lignocellulose was in the medium. The enzyme was partially purified and its molecular weight was approximated to be about 80,000 daltons. Mutant cultures that had lost the ability to produce zones of clearing on DHDV-containing agar solubilized smaller quantities of lignin as compared to the wild type, except for one strain. A partially purified oxidase preparation was shown to oxidize a natural lignocellulose substrate.