skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Molecular Approaches to Understanding C & N Dynamics in MArine Sediments

Abstract

Continental margin sediments constitute only about 10% of the total sediment surface area in the world’s oceans, nevertheless they are the dominant sites of nitrogen (N) cycling. Recent studies suggest that the oceanic nitrogen budget is unbalanced, primarily due to a higher nitrogen removal rate in contrast to the fixation rate, and it has been suggested that denitrification activity contributes significantly to this imbalance. Although denitrification in marine environments has been studied intensively at the process level, little is known about the species abundance, composition, distribution, and functional differences of the denitrifying population. Understanding the diversity of microbial populations in marine environments, their responses to various environmental factors such as NO3-, and how this impact the rate of denitrification is critical to predict global N dynamics. Environmental Microbiology has the prompt to study the influence of each microbial population on a biogeochemical process within a given ecosystem. Culture-dependent and –independent techniques using nucleic acid probes can access the identity and activity of cultured and uncultured microorganisms. Nucleic acid probes can target distintict genes which set phylogenetic relationships, such as rDNA 16S, DNA gyrase (gyrB) and RNA polymerase sigma 70 factor (rpoD). In the other hand, the genetic capabilities and theirmore » expression could be tracked using probes that target several functional genes, such as nirS, nirK, nosZ, and nifH, which are genes involved in denitrification. Selective detection of cells actively expressing functional genes within a community using In Situ Reverse Transcription-PCR (ISRT-PCR) could become a powerful culture-independent technique in microbial ecology. Here we describe an approach to study the expression of nirS genes in denitrifying bacteria. Pure cultures of Pseudomonas stutzeri and Paracoccus denitrificans, as well as co-cultures with non-denitrifying populations were used to optimize the ISRT-PCR protocol. Cells grown on nitrate broth were harvested and fixed at both logarithmic (24-48 h) and stationary phase (7 days). Fixed and RNA protectedTMcc cells were spotted on microscope slides to optimize cell wall permeabilization conditions with lyzozyme and proteinase K. Subsequently, ISRT-PCR was performed with NirS 1F and NirS 6R primers using the QIAGEN® OneStep RT-PCR Kit. Amplification products within the cell were detected by Fluorescent In Situ Hybridization (FISH) at 40ºC overnight using a Cy3 labeled internal probe, specifically designed to detect the nirS gene. After hybridization, the cells were counterstained with DAPI and examined by confocal fluorescence microscopy. P. stutzeri cells treated with RNase and Pseudomonas G179 (a nirK denitrifying strain) were used as negative controls. Optimal cell permeabilization was achieved using 1 mg ml-1 lyzozyme for 30 min and 2 µg ml-1 Proteinase K. RNase treated cells did not fluoresce after FISH, but were detectable by DAPI. Only nirS-type denitrifying cells in log phase (80-95% of total direct cell counts) were detected by this approach while fewer cells (5-10%) were detectable after 7 days in stationary phase. Co-cultures of P. denitrificans with a non-denitrifying isolate resulted in selective identification of target cells, thus supporting the potential use of this approach for gene expression analysis at the community level.« less

Authors:
; ; ;
Publication Date:
Research Org.:
University of Puerto Rico - Mayaguez
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
903396
Report Number(s):
DOE G 241.1-1A
TRN: US200722%%368
DOE Contract Number:
FG02-04ER63738
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
54 ENVIRONMENTAL SCIENCES; CELL WALL; CONTINENTAL MARGIN; DENITRIFICATION; GENES; NITROGEN; NUCLEIC ACIDS; PROBES; RNA POLYMERASES; SEDIMENTS; SURFACE AREA; MOLECULES; In situ RT-PCR, denitrification, marine sediments, nitrogen cycle

Citation Formats

Arturo Massol, James Tiedje, Jizhong Zhou, and Allan Devol. Molecular Approaches to Understanding C & N Dynamics in MArine Sediments. United States: N. p., 2007. Web. doi:10.2172/903396.
Arturo Massol, James Tiedje, Jizhong Zhou, & Allan Devol. Molecular Approaches to Understanding C & N Dynamics in MArine Sediments. United States. doi:10.2172/903396.
Arturo Massol, James Tiedje, Jizhong Zhou, and Allan Devol. Wed . "Molecular Approaches to Understanding C & N Dynamics in MArine Sediments". United States. doi:10.2172/903396. https://www.osti.gov/servlets/purl/903396.
@article{osti_903396,
title = {Molecular Approaches to Understanding C & N Dynamics in MArine Sediments},
author = {Arturo Massol and James Tiedje and Jizhong Zhou and Allan Devol},
abstractNote = {Continental margin sediments constitute only about 10% of the total sediment surface area in the world’s oceans, nevertheless they are the dominant sites of nitrogen (N) cycling. Recent studies suggest that the oceanic nitrogen budget is unbalanced, primarily due to a higher nitrogen removal rate in contrast to the fixation rate, and it has been suggested that denitrification activity contributes significantly to this imbalance. Although denitrification in marine environments has been studied intensively at the process level, little is known about the species abundance, composition, distribution, and functional differences of the denitrifying population. Understanding the diversity of microbial populations in marine environments, their responses to various environmental factors such as NO3-, and how this impact the rate of denitrification is critical to predict global N dynamics. Environmental Microbiology has the prompt to study the influence of each microbial population on a biogeochemical process within a given ecosystem. Culture-dependent and –independent techniques using nucleic acid probes can access the identity and activity of cultured and uncultured microorganisms. Nucleic acid probes can target distintict genes which set phylogenetic relationships, such as rDNA 16S, DNA gyrase (gyrB) and RNA polymerase sigma 70 factor (rpoD). In the other hand, the genetic capabilities and their expression could be tracked using probes that target several functional genes, such as nirS, nirK, nosZ, and nifH, which are genes involved in denitrification. Selective detection of cells actively expressing functional genes within a community using In Situ Reverse Transcription-PCR (ISRT-PCR) could become a powerful culture-independent technique in microbial ecology. Here we describe an approach to study the expression of nirS genes in denitrifying bacteria. Pure cultures of Pseudomonas stutzeri and Paracoccus denitrificans, as well as co-cultures with non-denitrifying populations were used to optimize the ISRT-PCR protocol. Cells grown on nitrate broth were harvested and fixed at both logarithmic (24-48 h) and stationary phase (7 days). Fixed and RNA protectedTMcc cells were spotted on microscope slides to optimize cell wall permeabilization conditions with lyzozyme and proteinase K. Subsequently, ISRT-PCR was performed with NirS 1F and NirS 6R primers using the QIAGEN® OneStep RT-PCR Kit. Amplification products within the cell were detected by Fluorescent In Situ Hybridization (FISH) at 40ºC overnight using a Cy3 labeled internal probe, specifically designed to detect the nirS gene. After hybridization, the cells were counterstained with DAPI and examined by confocal fluorescence microscopy. P. stutzeri cells treated with RNase and Pseudomonas G179 (a nirK denitrifying strain) were used as negative controls. Optimal cell permeabilization was achieved using 1 mg ml-1 lyzozyme for 30 min and 2 µg ml-1 Proteinase K. RNase treated cells did not fluoresce after FISH, but were detectable by DAPI. Only nirS-type denitrifying cells in log phase (80-95% of total direct cell counts) were detected by this approach while fewer cells (5-10%) were detectable after 7 days in stationary phase. Co-cultures of P. denitrificans with a non-denitrifying isolate resulted in selective identification of target cells, thus supporting the potential use of this approach for gene expression analysis at the community level.},
doi = {10.2172/903396},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Wed May 16 00:00:00 EDT 2007},
month = {Wed May 16 00:00:00 EDT 2007}
}

Technical Report:

Save / Share:
  • The ocean plays an important role in regulating the earth`s climate, sustains a large portion of the earth`s biodiversity, is a tremendous reservoir of commercially important substances, and is used for a variety of often conflicting purposes. In recent decades marine scientists have discovered much about the ocean and its organisms, yet many important fundamental questions remain unanswered. Human populations have increased, particularly in coastal regions. As a result, the marine environment in these areas is increasingly disrupted by human activities, including pollution and the depletion of some ecologically and commercially important species. There is a sense of urgency aboutmore » reducing human impacts on the ocean and a need to understand how altered ecosystems and the loss of marine species and biodiversity could affect society. During the past two decades, the development of sophisticated technologies and instruments for biomedical research has resulted in significant advances in the biological sciences. While some of these technologies have been readily incorporated into the study of marine organisms as models for understanding basic biology, the value of molecular techniques for addressing problems in marine biology and biological oceanography has only recently begun to be appreciated. This report defines critical scientific questions in marine biology and biological oceanography, describes the molecular technologies that could be used to answer these questions, and discusses some of the implications and economic opportunities that might result from this research which could potentially improve the international competitive position of the United States in the rapidly growing area of marine biotechnology. The committee recommends that the federal government provide the infrastructure necessary to use the techniques of molecular biology in the marine sciences.« less
  • This report describes molecular techniques that could be invaluable in addressing process-oriented problems in the ocean sciences that have perplexed oceanographers for decades, such as understanding the basis for biogeochemical processes, recruitment processes, upper-ocean dynamics, biological impacts of global warming, and ecological impacts of human activities. The coupling of highly sophisticated methods, such as satellite remote sensing, which permits synoptic monitoring of chemical, physical, and biological parameters over large areas, with the power of modern molecular tools for ``ground truthing`` at small scales could allow scientists to address questions about marine organisms and the ocean in which they live thatmore » could not be answered previously. Clearly, the marine sciences are on the threshold of an exciting new frontier of scientific discovery and economic opportunity.« less
  • Traditionally, the importance of inorganic nitrogen (N) for the nutrition and growth of marine phytoplankton has been recognized, while inorganic N utilization by bacteria has received less attention. Likewise, organic N has been thought to be important for heterotrophic organisms but not for phytoplankton. However, accumulating evidence suggests that bacteria compete with phytoplankton for nitrate (NO3-) and other N species. The consequences of this competition may have a profound effect on the flux of N, and therefore carbon (C), in ocean margins. Because it has been difficult to differentiate between N uptake by heterotrophic bacterioplankton versus autotrophic phytoplankton, the processesmore » that control N utilization, and the consequences of these competitive interactions, have traditionally been difficult to study. Significant bacterial utilization of DIN may have a profound effect on the flux of N and C in the water column because sinks for dissolved N that do not incorporate inorganic C represent mechanisms that reduce the atmospheric CO2 drawdown via the ?biological pump? and limit the flux of POC from the euphotic zone. This project was active over the period of 1998-2007 with support from the DOE Biotechnology Investigations ? Ocean Margins Program (BI-OMP). Over this period we developed a tool kit of molecular methods (PCR, RT-PCR, Q-PCR, QRT-PCR, and TRFLP) and combined isotope mass spectrometry and flow-cytometric approaches that allow selective isolation, characterization, and study of the diversity and genetic expression (mRNA) of the structural gene responsible for the assimilation of NO3- by heterotrophic bacteria (nasA). As a result of these studies we discovered that bacteria capable of assimilating NO3- are ubiquitous in marine waters, that the nasA gene is expressed in these environments, that heterotrophic bacteria can account for a significant fraction of total DIN uptake in different ocean margin systems, that the expression of nasA is differentially regulated in genetically distinct NO3- assimilating bacteria, and that the best predictors of nasA gene expression are either NO3- concentration or NO3- uptake rates. These studies provide convincing evidence of the importance of bacterial utilization of NO3-, insight into controlling processes, and provide a rich dataset that are being used to develop linked C and N modeling components necessary to evaluate the significance of bacterial DIN utilization to global C cycling. Furthermore, as a result of BI-OMP funding we made exciting strides towards institutionalizing a research and education based collaboration between the Skidaway Institute of Oceanography (SkIO) and Savannah State University (SSU), an historically black university within the University System of Georgia with undergraduate and now graduate programs in marine science. The BI-OMP program, in addition to supporting undergraduate (24) graduate (10) and postdoctoral (2) students, contributed to the development of a new graduate program in Marine Sciences at SSU that remains an important legacy of this project. The long-term goals of these collaborations are to increase the capacity for marine biotechnology research and to increase representation of minorities in marine, environmental and biotechnological sciences.« less
  • The ocean plays an important role in regulating the earth`s climate, sustains a large portion of the earth`s biodiversity, is a tremendous reservoir of commercially important substances, and is used for a variety of often conflicting purposes. In recent decades marine scientists have discovered much about the ocean and its organisms, yet many important fundamental questions remain unanswered. Human populations have increased, particularly in coastal regions. As a result, the marine environment in these areas is increasingly disrupted by human activities, including pollution and the depletion of some ecologically and commercially important species. There is a sense of urgency aboutmore » reducing human impacts on the ocean and a need to understand how altered ecosystems and the loss of marine species and biodiversity could affect society. This report describes molecular techniques that could be invaluable in addressing process-oriented problems in the ocean sciences that have perplexed oceanographers for decades, such as understanding the basis for biogeochemical processes, recruitment processes, upper-ocean dynamics, biological impacts of global warming, and ecological impacts of human activities. The coupling of highly sophisticated methods, such as satellite remote sensing, which permits synoptic monitoring of chemical, physical, and biological parameters over large areas, with the power of modern molecular tools for ground truthing at small scales could allow scientists to address questions about marine organisms and the ocean in which they live that could not be answered previously.« less