2002 Progress Report: Cold-Induced Hibernation of Marine Vibrios in the Gulf of Mexico: A Study of Cell-Cell Communication and Dormancy in Vibrio vulnificus

EPA Grant Number: R827072C025
Subproject: this is subproject number 025 , established and managed by the Center Director under grant R827072
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Alabama Center For Estuarine Studies (ACES)
Center Director: Shipp, Robert L.
Title: Cold-Induced Hibernation of Marine Vibrios in the Gulf of Mexico: A Study of Cell-Cell Communication and Dormancy in Vibrio vulnificus
Investigators: Foster, John W.
Institution: University of South Alabama
EPA Project Officer: Packard, Benjamin H
Project Period: January 1, 2002 through December 31, 2004
Project Period Covered by this Report: January 1, 2002 through December 31, 2003
RFA: Alabama Center For Estuarine Studies (ACES) (1999) RFA Text |  Recipients Lists
Research Category: Targeted Research


The objective of this research project is to better understand factors that influence entry and exit from dormancy as well as the physiology of dormancy itself. Vibrio vulnificus is a human pathogen commonly found in marine and brackish waters. It is responsible for causing serious life-threatening diseases associated with the ingestion of shellfish. Under low temperature conditions, V. vulnificus enters an unusual and poorly understood physiological state called dormancy (also known as viable-but-nonculturable). In this state, cells appear alive but incapable of replication. Aside from being a fascinating biological phenomenon, the dormant state may result in underestimating the contamination hazards of oysters.

Progress Summary:

We have made significant progress over the past 4 months and our results have revealed that progression into dormancy is, at a minimum, a two-stage process. The first stage is a pseudodormant state in which cells become ultra-sensitive to oxygen radicals naturally present in plating media. Pseudodormant cells are easily recoverable by plating on media containing catalase or sodium pyruvate. Catalase will consume hydrogen peroxide, whereas sodium pyruvate scavenges oxygen radicals. Annotation of the V. vulnificus genome reveals this organism possesses two catalase genes. We now are examining extracts of log phase and dormant cells for the presence of these enzymes. We also are planning experiments to determine if more stable catalases introduced into V. vulnificus by genetic engineering will delay or prevent entry into dormancy.

Following the pseudodormant state, V. vulnificus enters a second stage where the cells are not recoverable by catalase. This occurs approximately 30 days after initial cold induction. These dormant cells still appear alive based on their ability to maintain an energized membrane. We have subjected log phase and dormant cells to SDS-PAGE analysis and have identified significant proteomic differences between these two physiological states. Five proteins were observed to decrease or completely disappear in the dormant cells. Preliminary identification of these proteins by mass spectral analysis indicate they are DnaK, an essential chaperone protein, ribosomal proteins S1 and L1, glutamine synthetase, and alanine dehydrogenase. More definitive two-dimensional separations of the proteomes are underway. The confirmed loss of two key ribosomal proteins, however, could explain why dormant cells fail to divide.

We also are investigating whether V. vulnificus secretes autoinducer molecules that influence entry or exit from the dormant state. Initial experiments suggest that dormant cells do not produce a factor that triggers dormancy. We now are using a drug resistant strain of V. vulnificus to determine if coculturing log phase drug-sensitive cells with drug-resistant dormant cells will encourage exit from dormancy, a result that would indicate the presence of a secreted rescue molecule.

Another aspect of our work centers on the effect that salt concentration has on the synthesis of an apparent autoinducer molecule. As a marine microbe, V. vulnificus is a halophile that grows best at high-salt concentrations (approximately 1-1.5 percent). We have discovered that this organism produces a factor at low-salt concentrations (less than or equal to 0.5 percent) that facilitates growth when these cells are diluted to low cell density in low salt media. Experiments are planned to determine if this growth factor will influence the dormant state.

The physiological changes associated with dormancy represent a fascinating biological program that enables cells to resist death under environmental stress. Learning how this program works will impact our general understanding of how marine microbes cope with their unique ecological niche and will aid in the safety assessment of shellfish contaminated with this pathogen.

Future Activities:

We will examine the genome for additional proteins that may substitute for these key protein synthesis factors. Why V. vulnificus discards these proteins, whereas other organisms that lack a dormancy program do not, is a critical question that we will address. We will continue using a drug resistant strain of V. vulnificus to determine if co-culturing log phase drug-sensitive cells with drug-resistant dormant cells will encourage exit from dormancy, a result that would indicate the presence of a secreted rescue molecule. Experiments also are planned to determine if the growth factor produced at low-salt concentrations will influence the dormant state.

Supplemental Keywords:

Vibrio vulnificus, human pathogen, marine, brackish waters, life-threatening diseases, ingestion of shellfish, contamination hazards, oysters, genetic engineering, SDS-PAGE analysis, DnaK, chaperone protein, ribosomal proteins, S1, L1, glutamine synthetase, alanine dehydrogenase, halophile, ecosystem, ecosystem protection, ecology, ecological effects, ecological indicators, environmental exposure, geographic area, water, aquatic ecosystem, coastal ecosystem, coastal environments, estuary, estuaries, estuarine research, estuarine waters, environmental chemistry, chemistry, chemical engineering, risk, assessment, indicators, human health, Alabama, AL, human modifications, fishery sampling., RFA, Scientific Discipline, ECOSYSTEMS, Geographic Area, Water, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, Ecology, estuarine research, Ecosystem/Assessment/Indicators, exploratory research environmental biology, Chemistry, State, Restoration, Aquatic Ecosystem, Aquatic Ecosystems, Terrestrial Ecosystems, Ecological Monitoring, Ecology and Ecosystems, Aquatic Ecosystem Restoration, coastal ecosystem, water use, endangered species, estuaries, watersheds, Alabama (AL), predators, marine vibrios, coastal environments, Alabama estuaries, ecosystem, environmental indicators, water quality, estuarine waters, human modifications, contaminated oysters

Relevant Websites:

http://www.southalabama.edu/aces/ Exit

Progress and Final Reports:

Original Abstract
  • 2003
  • Final

  • Main Center Abstract and Reports:

    R827072    Alabama Center For Estuarine Studies (ACES)

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R827072C001 Fluorescent Whitening Agents As Facile Pollution Markers In Shellfishing Waters
    R827072C002 Red Snapper Demographics on Artificial Reefs: The Effect of Nearest-Neighbor Dynamics
    R827072C003 Stabilization of Eroding Shorelines in Estuarine Wave Eliminates with Constructed Fringe Wetlands Incorporating Offshore Breakwaters
    R827072C004 Interaction Between Water Column Structure and Reproduction in Jellyfish Populations Of Mobile Bay (SGER)
    R827072C005 Effects of Variation in River Discharge and Wind-Driven Resuspension on Higher Trophic Levels in the Mobile Bay Ecosystem
    R827072C006 Results of Zooplankton Component
    R827072C007 Benthic Study Component
    R827072C008 A Preliminary Survey of Macroalgal and Aquatic Plant Distribution in the Mobile Tensaw Delta
    R827072C009 Fisheries-induced changes in the structure and function of shallow water "nursery habitats": an experimental assessment
    R827072C010 Effects Of Variation in River Discharge and Wind-Driven Resuspension on Lower Trophic Levels of the Mobile Bay Ecosystem
    R827072C011 Evaluation of Alabama Estuaries as Developmental Habitat for Juvenile Sea Turtles
    R827072C012 Effects of Salinity Stress on Natural and Anthropogenically-Derived Bacteria in Estuarine Environments
    R827072C013 The Role of Land-Use/Land-Cover and Sub-estuarine Ecosystem Nitrogen Cycling in the Regulation of Nitrogen Delivery to a River Dominated Estuary; Mobile Bay, Alabama
    R827072C014 Environmental Attitudes of Alabama Coastal Residents: Public Opinion Polls and Environmental Policy
    R827072C015 Synthesis and Characterization of an Electrochemical Peptide Nucleic Acid Probe
    R827072C016 Determinants of Small-Scale Variation in the Abundance of the Blue Crab Callinectes Sapidus
    R827072C017 Effects of Estrogen Pollution on the Reproductive Fitness of the Gulf Pipefish, Syngnathus scovelli
    R827072C019 A Model for Genetic Diversity Aquatic Insects of the Mobile/Tensaw River Delta
    R827072C020 Evaluating Trophic Processes as Indicators of Anthropogenic Eutrophication in Coastal Ecosystems: An Exploratory Analysis
    R827072C021 Effects of Anthropogenic Eutrophication on the Magnitude and Trophic Fate of Microphytobenthic Production in Estuaries
    R827072C022 Characteristics of Ship Waves and Wind Waves in Mobile Bay
    R827072C023 Methods Comparison Between Stripping Voltammetry and Plasma Emission Spectroscopy for Metals in Mobile Bay
    R827072C024 Changes in Water Conditions and Sedimentation Rates Associated With Construction of the Mobile Bay Causeway
    R827072C025 Cold-Induced Hibernation of Marine Vibrios in the Gulf of Mexico: A Study of Cell-Cell Communication and Dormancy in Vibrio vulnificus
    R827072C026 Holocene Sedimentary History of Weeks Bay, AL: Human and Natural Impacts on Deposition in a Gulf Coast Estuary
    R827072C027 Shelter Bottlenecks and Self-Regulation in Blue Crab Populations: Assessing the Roles of Nursery Habitats and Juvenile Interactions for Shelter Dependent Organisms
    R827072C028 Predicting Seagrass Survival in Nutrient Enriched Waters: Toward a New View of an Existing Paradigm
    R827072C029 DMSP and its Role as an Antioxidant in the Salt Marsh Macrophyte Spartina alterniflora
    R827072C030 A Preliminary Survey of Aerial and Ground-Dwelling Insects of the Mobile/Tensaw Delta
    R827072C031 Natural Biogeochemical Tags of Striped Mullet, Mugil cephalus, Estuarine Nursery Areas in the North Central Gulf of Mexico
    R827072C032 Resolution of Sedimentation Rates in Impacted Coastal Environments Using 137Cs and 210Pb Markers: Dog River and Fowl River Embayments
    R827072C033 Investigation of the Use of Pulse Amplitude Modulated (PAM) Fluorometry as an Indicator of Submerged Aquatic Vegetation Health in Mobile Bay
    R827072C034 Influence of Invasive Plant Species in Determining Diversity of Aquatic Vegetation in the Mobile-Tensaw Delta
    R827072C035 The Influence of Shallow Water Hydrodynamics on the Importance of Seagrass Detritus in Estuarine Food Webs
    R827072C036 Food Web Interactions, Spatial Subsidies and the Flow of Energy Between the Mobile Bay Delta and Offshore Waters: A SGER Proposal to the Alabama Center for Estuarine Studies
    R830651C001 Meteorological Modeling of Hurricanes and Coastal Interactions: A Stability Study For Vertical Pressure Levels
    R830651C002 Characterization of Glycoprotein Cues Used by the Parasitic Rhizocephalan Barnacle Loxothylacus texanus To Identify Its Blue Crab Host, Callinectes sapidus
    R830651C003 Survey of Diamondback Terrapin Populations in Alabama Estuaries
    R830651C004 An Assessment of Environmental Contaminant Levels in Water and Dragonfly Larvae Tissues from the Mobile/Tensaw Delta