Product Citations and Abstracts

This book chapter contains the following headings and subheadings: Introduction; Experimental Approach - Precautions, Template, Primers, Reaction Conditions, Enhancers, Post Amplification; Procedures - Template DNA, Basic PCR, Thermal Cycle Parameters, Enzyme Addition, Agarose Gel Analysis.

Devereux, Richard. 2005. Seagrass Rhizosphere Microbial Communities. Chapter 10. In: Interactions Between Macro- and Microorganisms in Marine Sediments. E. Kristense, J.E. Kostka and R.H. Haese, Editors. American Geophysical Union, Washington, DC. Pp. 199-216. (ERL,GB 1213).

Seagrasses are aquatic marine angiosperms that exert profound controls on sediment biogeochemistry. Oxygen and carbon formed in the leaves during photosynthesis are translocated below ground and released through the roots into the surrounding sediments. This daily pulse stimulates the rhizosphere microbial communities resulting in rates of bacterial production, nitrogen fixation and sulfate reduction being higher in the day than at night. Yet at the same time, end products of microbial respiration, in particular sulfide, can be toxic to the plants if they attain sufficiently high concentrations. Sulfate reduction is the dominant terminal electron accepting process and is tightly coupled with nitrogen fixation, pointing to the rhizosphere where a synergistic relationship between the plants and sulfate-reducing bacteria is played out. Bacterial numbers and activities are enriched in seagrass bed sediments over nearby non-vegetated sediments, and are even more so on and within the roots in comparison to bulk vegetated sediments. The distribution and activities of bacteria in seagrass bed sediments suggests a model that distinguishes rhizosphere and root-associated microbial communities responding to root exudates from microbial communities in bulk vegetated sediments degrading organic matter derived from benthic algae and detritus that settles on the surface of seagrass bed sediments.

Devereux, Richard, Parke Rublee, John H. Paul, Katharine G. Field and Jorge W. Santo Domingo. 2006. Development and Applications of Microbial Ecogenomic Indicators for Monitoring Water Quality: Report of a Workshop Assessing the State of the Science, Research Needs and Future Directions. Environ. Monit. Assess. 116(1-3):459-479. (ERL,GB 1217).

This article brings forth recommendations from a workshop sponsored by the U.S. Environmental Protection Agency's Science to Achieve Results (STAR) and Environmental Monitoring and Assessment (EMAP) Programs and by the Council of State Governments, held during May 2002 in Kansas City, Kansas. The workshop assembled microbial ecologists and environmental scientists to review available molecular biological methods, consider approaches arising from genomics level technologies, and determine the research and science needed to bring existing molecular biological approaches, and emerging technologies arising from genomic research with microorganisms, to environmental monitoring and water quality assessments. Research to develop genomics and proteomics technologies for environmental science is a very new area that holds great potential for improving environmental water quality assessments. The workshop participants noted that microbial ecologists are already using molecular biological methods well suited for monitoring and water quality assessments and anticipate that genomics-enabled technologies could be made available within a decade.

Devereux, Richard and Diane F. Yates. Unpublished. Optimization of the N,N-dimethyl-p-phenylenediamine Procedure to Measure Low Levels of Sulfide Obtained in Zinc Acetate Traps During Sulfate Reduction Rate Determinations. J. Microbiol. Methods. 10p. (ERL,GB 1252).

The Cline procedure to measure sulfide was optimized to measure acid volatile sulfide in solutions containing high zinc acetate concentrations. Addition of HCL eliminated interference from zinc acetate by lowering the pH to near the optimum for the procedure. Modification of the procedure enabled the measurement of low concentrations of acid volatile sulfide in seagrass bed sediments.

Devereux, Richard, Matthew D. Kane, Janet Winfrey and David A. Stahl. 1992. Genus- and Group-Specific Hybridization Probes for Determinative and Environmental Studies of Sulfate-Reducing Bacteria. EPA/600/J-93/064. Syst. Appl. Microbiol. 15(4):601-609. (ERL,GB 787). (Also avail. from NTIS, Springfield, VA: PB93-168987)

A set of six oligonucleotides, complementary to conserved tracts of 16S rRNA from phylogenetically-defined groups of sulfate-reducing bacteria, was characterized for use as hybridization probes in determinative and environmental microbiology. Four probes were genus specific and identified Desulfobacterium spp., Desulfobacter spp., Desulfobulbus spp., or Desulfovibrio spp. The other two probes encompassed more diverse assemblages. One probe was specific for the phylogenetic lineage composed of Desulfococcus multivorans, Desulfosarcina variabilis, and Desulfobotulus sapovorans. The remaining probe was specific for Desulfobacterium spp., Desulfobacter spp., D. multivorans, D. variabilis, and D. sapovorans. Temperature of dissociation was determined for each probe and the designed specificities of each were evaluated by hybridizations against closely related nontargeted species. In addition, each probe was screened by using a "phylogrid" membrane which consisted of nucleic acids from sixtyfour non-targeted organisms representing a diverse collection of eukarya, archaea, and bacteria. The value of these probes to studies in environmental microbiology was evaluated by hybridizations to 16S rRNAs of sulfate-reducing bacteria present in marine sediments.

Devereux, R. 1993. Prokaryotes (Book Review). BioScience. 43(3):182-184. (ERL,GB 806).

The author reviews the book, The Prokaryotes: a Handbook on the Biology of Bacteria: Ecophysiology, Identification, Applications, 2nd. edition, published by Springer-Verlag, New York. The book was edited by A. Balows, H.G. Truper, M. Dworkin, W. Harder, and K.-H. Schleifer and sells for $1,980.

Devereux, Richard and David Stahl. 1993. Phylogeny of Sulfate-Reducing Bacteria and a Perspective for Analyzing Their Natural Communities. In: Sulfate-Reducing Bacteria: Contemporary Perspectives. J.M. Odom and Rivers Singleton, Jr., Editors. Springer-Verlag, New York, NY. Pp. 131-160. (ERL,GB 807).

Authors summarize recent phylogenetic studies of sulfate-reducing bacteria and the application of 16S rRNA sequence information to environmental studies of these bacteria. A brief overview is provided on the use of 16S rRNA sequences to infer phylogenetic relationships. Where possible, some of the nutritional and biochemical characteristics of sulfate-reducing bacteria have been placed in an evolutionary context.

Devereux, Richard, Tamar Barkay and James Harvey. 1994. Application of Microbial Ecology Research to Environmental Problems. EPA/600/J-94/341. Water Report. 4(1):8-11. (ERL,GB 874). (Also avail. from NTIS, Springfield, VA: PB94-203783)

Authors describe the U.S. Environmental Protection Agency research program in biotechnology risk assessment to study fate and effects of nonindigenous microorganisms in aquatic and terrestrial environments. Information from the studies can be used by industries and regulatory agencies to assess the risk of biotechnological products (both genetically engineered or natural, but nonindigenous) in the environment. Examples of research at the U.S. EPA Gulf Breeze laboratory are cited.

Devereux, Richard and Gary W. Mundfrom. 1994. Phylogenetic Tree of 16S rRNA Sequences from Sulfate-Reducing Bacteria in a Sandy Marine Environment. EPA/600/J-94/436. Appl. Environ. Microbiol. 60(9):3437-3439. (ERL,GB 878). (Avail. from NTIS, Springfield, VA: PB95-111829)

The divergence of 16S rDNA sequences in marine sediment was investigated. Twenty unique partial sequences were found among 33 cloned following PCR. Thirteen shared 82 to 91% similarity with sequences of delta subclass sulfate-reducing bacteria. Three contained the target sequence for a sulfate-reducing bacterium-specific oligonucleotide probe designed from pure-culture studies.

Devereux, Richard and Stephanie G. Willis. 1995. Amplification of Ribosomal RNA Sequences. In: Molecular Microbial Ecology Manual. A.D.L. Akkermans, J.D. van Elsas, and F.J. DeBruin, Editors. Kluwer Academic Publishers, Norwell, MA. Pp. 3.3.1:1-11. (ERL,GB 881).

This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedures for the isolation of microbial nucleic acids from pure cultures and environmental samples and preparation of DNA templates.

Devereux, R., J. Kurtz and G. Mundfrom. 1993. Molecular Phylogenetic Explorations of Natural Microbial Community Composition and Diversity. In: Trends in Microbial Ecology. EPA/600/A-94/115. R. Guerrero and C. Pedros-Alio, Editors. Spanish Society for Microbiology, Barcelona, Spain. Pp. 387-390. (ERL,GB 886). (Also avail. from NTIS, Springfield, VA: PB94-190832)

Comparative sequence analysis of ribosomal RNA molecules has led to a phylogenetic-based approach to characterize natural microbial communities. The approach has been applied to study natural communities of sulfate-reducing bacteria. Hybridization probes were used to measure relative amounts of specific sulfate reducer rRNAs in an estuarine sediment. Selective amplification, cloning, and comparative sequence analysis of 16S ribosomal RNA gene sequences have revealed new diversity among sulfate-reducing bacteria.

Devereux, R., M.E. Hines and D.A. Stahl. 1996. S Cycling: Characterization of Natural Communities of Sulfate-Reducing Bacteria by 16S rRNA Sequence Comparisons. Microb. Ecol. 32(3):283-292. (ERL,GB 917).

Past studies of microbial communities responsible for geochemical transformations have been limited by an inability to representatively cultivate, and then identify, the constituent members. Ribosomal RNA sequences, particularly 16S-like rRNAs, provide a measure of phylogenetic relationship that can now be used to examine the structure and diversity of microbial communities. Sulfate-reducing bacteria (SRB) play an important role in the sulfur cycle and the terminal mineralization of organic matter in estuarine and marine environments. Because the Gram-negative mesophilic SRB comprise a phylogenetically coherent assemblage, their communities are well suited to explorations through rRNA sequence-based methodologies. In this study we related molecular biological methods using rRNA probes to geochemical measurements at two different sites. At a non-vegetated site in northwest Florida, rates of sulfate reduction were low and SRB rRNA comprised about 5% of the total rRNA extracted from the sediment. The other site, a salt marsh in New Hampshire, had higher rates of sulfate-reduction with SRB rRNA accounting for up to 30% of the total rRNA extracted from the sediment. SRB community structure differed dramatically between the two sites with Desulfobulbus rRNA much less abundant in the non-vegetated site than in the salt marsh. The differences in these SRB communities reflect differences in the ecology of their habitats.

Devereux, Richard, Michael R. Winfrey, Janet Winfrey and David A. Stahl. 1996. Depth Profile of Sulfate-Reducing Bacterial Ribosomal RNA and Mercury Methylation in an Estuarine Sediment. FEMS Microbiol. Ecol. 20(1):23-31. (ERL,GB 929).

The community structure of complex anaerobic microbial communities has been difficult to elucidate because of an inability to cultivate most of the contributing populations. In this study, the distribution of sulfate-reducing bacteria (SRB) in anaerobic sediments was determined using oligonucleotide probes complementary to the 16S ribosomal RNAs of major phylogenetic groups. Sediment cores were collected from Santa Rosa Sound in northwest Florida, and sectioned by depth into 1 to 2 cm fractions. Nucleic acids were extracted from each fraction and hybridized with the SRB-specific ribosomal RNA probes. SRB ribosomal RNAs accounted for almost 5% of the microbial community ribosomal RNA pool in the 3-4 cm depth fraction and were dominated by Desulfovibrionaceae ribosomal RNA. The SRB ribosomal RNA peak coincided with mercury methylation, an activity attributed to SRB. Profiles of the ribosomal RNAs indicate that SRB populations in sediments are stratified by depth.

Devereux, Richard, Stephanie G. Willis and Mark E. Hines. 1997. Genome Sizes of Desulfovibrio desulfuricans, Desulfovibrio vulgaris, and Desulfobulbus propionicus Estimated by Pulsed-Field Gel Electrophoresis of Linearized Chromosomal DNA. Curr. Microbiol. 34(6):337-339. (ERL,GB 983).

Pulsed-field gel electrophoresis (PFGE) of linearized, full-length chromosomal DNA was used to estimate the genome sizes of three species of sulfate-reducing bacteria. Genome sizes of Desulfovibrio desulfuricans, Desulfovibrio vulgaris, and Desulfobulbus propionicus were estimated to be 3.1, 3.6, and 3.7 Mb, respectively. These values are double the genome sizes previously determined for two Desulfovibrio species by two-dimensional agarose gel electrophoresis of DNA cut with restriction enzymes. PFGE of full-length chromosomal DNA could provide a generally applicable method to rapidly determine bacterial genome size and organization.

Devereux, Richard, Mary Delaney, Friedrich Widdel and David A. Stahl. 1989. Natural Relationships Among Sulfate-Reducing Eubacteria. EPA/600/J-89/424. J. Bacteriol. 171(12):6689-6695. (ERL,GB X654). (Avail. from NTIS, Springfield, VA: PB90-245739)

Phylogenetic relationships among 20 nonsporeforming and two endospore-forming species of sulfate-reducing eubacteria were inferred from comparative 16S rRNA sequencing. All genera of mesophilic sulfate-reducing eubacteria except the new genus Desulfomicrobium and the gliding Desulfonema species were included. The sporeforming species Defulfotomaculum ruminis and Desulfotomaculum orientis were found to be gram-positive organisms sharing 83% 16S rRNA sequence similarity, indicating that this genus is diverse. The gram-negative nonsporforming species could be divided into seven natural groups; group 1, Desulfovibrio desulfuricans and other species of this genus that do not degrade fatty acids (this group also included 'Desulfomonas' pigra); group 2, the fatty acid-degrading 'Desulfovibrio' sapovorans; group 3, Desulfobulbus species; group 4, Desulfobacter species; group 5, Desulfobacterium species and 'Desulfococcus' niacini; group 6, Desulfococcus multivorans and Desulfosarcina variabilis; and group 7, the fatty acid-oxidizing 'Desulfovibrio' baarsii. (The quotation marks are used to indicate the need for taxonomic revision.) Groups 1 to 3 are incomplete oxidizers that form acetate as an end product; groups 4 to 7 are complete oxidizers. The data were consistent with and refined relationships previously inferred by oligonucleotide catalogs of 16S rRNA. Although the determined relationships are generally consistent with the existing classification based on physiology and other characteristics, the need for some taxonomic revision is indicated.

Devereux, Richard, Shao-Hua He, Carolyn L. Doyle, Silvia Orkland, David A. Stahl, Jean LeGall and William B. Whitman. 1990. Diversity and Origin of Desulfovibrio Species: Phylogenetic Definition of a Family. EPA/600/J-90/372. J. Bacteriol. 172(7):3609-3619. (ERL,GB X705). (Avail. from NTIS, Springfield, VA: PB91-163857)

The different nutritional properties of several Desulfovibrio desulfuricans strains suggest that either the strains are misclassified or there is a high degree of phenotypic diversity within the genus Desulfovibrio. The results of partial 16S rRNA and 23S rRNA sequence determinations demonstrated that Desulfovibrio desulfuricans ATCC 27774 and 'Desulfovibrio multispirans' are closely related to the type strain (strain Essex 6) and that strains ATCC 7757, Norway 4, and El Agheila Z are not. Therefore, these latter three strains of Desulfovibrio desulfuricans are apparently misclassified. A comparative analysis of nearly complete 16S rRNA sequences in which we used a least-squares analysis method for evolutionary distances, an unweighted pair group method, a signature analysis method, and maximum parsimony was undertaken to further investigate the phylogeny of Desulfovibrio species. The species analyzed were resolved into two branches with origins deep within the beta subdivision of the purple photosynthetic bacteria. One branch contained five deep lineages, which were represented by (i) Desulfovibrio salexigens and Desulfovibrio desulfuricans El Agheila Z; (ii) Desulfovibrio africanus; (iii) Desulfovibrio desulfuricans ATCC 27774, Desulfomonas pigra, and Desulfovibrio vulgaris; (iv) Desulfovibrio gigas; and (v) Desulfomicrobium baculatus (Desulfovibrio baculatus) and Desulfovibrio desulfuricans Norway 4. A correlation between 16S rRNA sequence similarity and percentage of DNA relatedness showed that these five deep lineages are related at levels below the minimum genus level suggested by Johnson (in Bergey's Manual of Systematic Bacteriology, vol. 1, 1984). We propose that this branch should be grouped into a single family, the Desulfovibrionaceae. The other branch includes other genera of sulfate-reducing bacteria (e.g., Desulfobacter and Desulfococcus) and contains Desulfovibrio sapovorans and Desulfovibrio baarsii as separate, distantly related lineages.