Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

Evolution of Protein Molecules

This book is written as a reference on organic substances in natural waters and as a supplementary text for graduate students in water chemistry. The chapters address five topics: amount, origin, nature, geochemistry, and characterization of organic carbon. Of these topics, the main themes are the amount and nature of dissolved organic carbon in natural waters (mainly fresh water, although seawater is briefly discussed). It is hoped that the reader is familiar with organic chemistry, but it is not necessary. The first part of the book is a general overview of the amount and general nature of dissolved organic carbon. Over the past 10 years there has been an exponential increase in knowledge on organic substances in water, which is the result of money directed toward the research of organic compounds, of new methods of analysis (such as gas chromatography and mass spectrometry), and most importantly, the result of more people working in this field. Because of this exponential increase in knowledge, there is a need to pull together and summarize the data that has accumulated from many disciplines over the last decade.

Organic geochemistry of natural waters

Ribosomal DNA (rDNA) sequences have been aligned and compared in a number of living organisms, and this approach has provided a wealth of information about phylogenetic relationships. Studies of rDNA sequences have been used to infer phylogenetic history across a very broad spectrum, from studies among the basal lineages of life to relationships among closely related species and populations. The reasons for the systematic versatility of rDNA include the numerous rates of evolution among different regions of rDNA (both among and within genes), the presence of many copies of most rDNA sequences per genome, and the pattern of concerted evolution that occurs among repeated copies. These features facilitate the analysis of rDNA by direct RNA sequencing, DNA sequencing (either by cloning or amplification), and restriction enzyme methodologies. Constraints imposed by secondary structure of rRNA and concerted evolution need to be considered in phylogenetic analyses, but these constraints do not appear to impede seriously the usefulness of rDNA. An analysis of aligned sequences of the four nuclear and two mitochondrial rRNA genes identified regions of these genes that are likely to be useful to address phylogenetic problems over a wide range of levels of divergence. In general, the small subunit nuclear sequences appear to be best for elucidating Precambrian divergences, the large subunit nuclear sequences for Paleozoic and Mesozoic divergences, and the organellar sequences of both subunits for Cenozoic divergences. Primer sequences were designed for use in amplifying the entire nuclear rDNA array in 15 sections by use of the polymerase chain reaction; these "universal" primers complement previously described primers for the mitochondrial rRNA genes. Pairs of primers can be selected in conjunction with the analysis of divergence of the rRNA genes to address systematic problems throughout the hierarchy of life.

Ribosomal DNA: molecular evolution and phylogenetic inference.

Food’s environmental impacts are created by millions of diverse producers. To identify solutions that are effective under this heterogeneity, we consolidated data covering five environmental indicators; 38,700 farms; and 1600 processors, packaging types, and retailers. Impact can vary 50-fold among producers of the same product, creating substantial mitigation opportunities. However, mitigation is complicated by trade-offs, multiple ways for producers to achieve low impacts, and interactions throughout the supply chain. Producers have limits on how far they can reduce impacts. Most strikingly, impacts of the lowest-impact animal products typically exceed those of vegetable substitutes, providing new evidence for the importance of dietary change. Cumulatively, our findings support an approach where producers monitor their own impacts, flexibly meet environmental targets by choosing from multiple practices, and communicate their impacts to consumers.

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Reducing food’s environmental impacts through producers and consumers

The methanotroph Methylosinus trichosporium OB3b, a type II methanotroph, degraded trichloroethylene at rates exceeding 1.2 mmol/h per g (dry weight) following the appearance of soluble methane monooxygenase in continuous and batch cultures. Cells capable oxidizing trichloroethylene contained components of soluble methane monooxygenase as demonstrated by Western blot (immunoblot) analysis with antibodies prepared against the purified enzyme. Growth of cultures in a medium containing 0.25 microM or less copper sulfate caused derepression of the synthesis of soluble methane monooxygenase. In these cultures, the specific rates of methane and methanol oxidation did not change during growth, while trichloroethylene oxidation increased with the appearance of soluble methane monooxygenase. M. trichosporium OB3b cells that contained soluble methane monooxygenase also degraded vinyl chloride, 1,1-dichloroethylene, cis-1,2-dichloroethylene, and trans-1,2-dichloroethylene. Images

Biodegradation of trichloroethylene by Methylosinus trichosporium OB3b.

Microorganisms that biosynthesize broad-specificity oxygenases to initiate metabolism of linear and branched-chain alkanes, nitroalkanes, cyclic ketones, alkenoic acids, and chromenes were surveyed for the ability to biodegrade trichloroethylene (TCE). The results indicated that TCE oxidation is not a common property of broad-specificity microbial oxygenases. Bacteria that contained nitropropane dioxygenase, cyclohexanone monooxygenase, cytochrome P-450 monooxygenases, 4-methoxybenzoate monooxygenase, and hexane monooxygenase did not degrade TCE. However, one new unique class of microorganisms removed TCE from incubation mixtures. Five Mycobacterium strains that were grown on propane as the sole source of carbon and energy degraded TCE. Mycobacterium vaccae JOB5 degraded TCE more rapidly and to a greater extent than the four other propane-oxidizing bacteria. At a starting concentration of 20 microM, it removed up to 99% of the TCE in 24 h. M. vaccae JOB5 also biodegraded 1,1-dichloroethylene, trans-1,2-dichloroethylene, cis-1,2-dichloroethylene, and vinyl chloride.

Survey of microbial oxygenases: trichloroethylene degradation by propane-oxidizing bacteria.

Competition experiments were performed in a continuous-flow reactor using Methylosinus trichosporium OB3b, a type II methanotroph, and Methylomonas albus BG8, a type I methanotroph. The experiments were designed to establish conditions under which type II methanotrophs, which have significant cometabolic potential, prevail over type I strains. The primary determinants of species selection were dissolved methane, copper, and nitrate concentrations. Dissolved oxygen and methanol concentrations played secondary roles. M. trichosporium OB3b proved dominant under copper and nitratelimited conditions. The ratio of M. trichosporium to M. albus in the reactor increased ten-fold in less than 100 hours following the removal of copper from the reactor feed. Numbers of M. albus declined to levels that were below detection limits (<106/ml) under nitrogen-limited conditions. In the latter experiment, the competitive success of M. trichosporiumdepended on the maintenance of an ambient dissolved oxygen level below about 7.5 × 10(-5) M, or 30% of saturation with air. The ability of M. trichosporium to express soluble methane monooxygenase under copper limitation and nitrogenase under nitrate limitation was very significant. M. albus predominated under methane-limited conditions, especially when low levels of methanol were simultaneously added with methane to the reactor. The results imply that nitrogen limitation can be used to select for type II strains such as M. trichosporium OB3b.

Factors affecting competition between type I and type II methanotrophs in two-organism, continuous-flow reactors

Methylosinus trichosporium OB3b biosynthesizes a broad specificity soluble methane monooxygenase that rapidly oxidizes trichloroethylene (TCE). The selective expression of the soluble methane monooxygenase was followed in vivo by a rapid colorimetric assay. Naphthalene was oxidized by purified soluble methane monooxygenase or by cells grown in copper-deficient media to a mixture of 1-naphthol and 2-naphthol. The naphthols were detected by reaction with tetrazotized o-dianisidine to form purple diazo dyes with large molar absorptivities. The rate of color formation with the rapid assay correlated with the velocity of TCE oxidation that was determined by gas chromatography. Both assays were used to optimize conditions for TCE oxidation by M. trichosporium OB3b and to test several methanotrophic bacteria for the ability to oxidize TCE and naphthalene.

Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity

A new genus and a new species of methane-oxidizing bacteria are described. The colonies produced by these bacteria are pink, circular, and convex with entire margins. Cells are gram negative and are normally found singularly with some rosettes. Negative stains indicate polar flagellation. In thin sections, intracytoplasmic membranes, similar to those described as type II in other methylotrophs, were present when the cells were grown with methane. No such membranes were apparent when the cells were grown with the other carbon and energy sources tested. The serine pathway for formaldehyde incorporation is the pathway of C1 metabolism. The deoxyribonucleic acid base composition is 66 mol% guanine plus cytosine. Methylobacterium is proposed as the name for this new genus of rod-shaped, methane-oxidizing bacteria. The specific epithet in the name of the type species, Methylobacterium organophilum sp. nov., denotes the preference of this organism for organic carbon and energy sources more complex than methane. The type strain of M. organophilum is XX (= ATCC 27886). This bacterium differs from all previously described genera and species of methane-oxidizing bacteria in its ability to utilize a variety of organic substrates with carbon-carbon bonds as sources of carbon and energy. The pathways for methane oxidation and the assimilation of one-carbon units are repressed during growth on complex organic substrates.

Richard S. Hanson

Papers

Biodegradation of trichloroethylene by Methylosinus trichosporium OB3b.

Survey of microbial oxygenases: trichloroethylene degradation by propane-oxidizing bacteria.

Factors affecting competition between type I and type II methanotrophs in two-organism, continuous-flow reactors

Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity

Methylobacterium, a New Genus of Facultatively Methylotrophic Bacteria