Background

Sulfate reducing bacteria are becoming increasingly significant to our ever growing problem of heavy metal contamination and lake pollution. One of the best organisms for modeling sulfate reduction is Desulfovibrio vulgaris Hildenborough. This family of bacteria makes use of sulfate as its primary electron acceptor for respiration in anoxic environments. In the past several years, scientists have discovered that many sulfate reducing bacteria survive in mutualistic associations with other microorganisms. One form of mutualism executed by D. vulgaris is known as syntrophy. Syntrophy is a phenomenon in which two species live off of the byproducts of one another. In the case of D. vulgaris, researchers have successfully paired this sulfate reducer with a methanogen called Methanococcus maripaludis.

Methanococcus maripaludis is an anaerobic Archaea that goes through the process of methanogenesis, giving off methane as a byproduct. To create methane, M. maripaludis must have a steady supply of hydrogen to consume. In a syntrophic relationship between M. maripaludis and D. vulgaris, D. vulgaris utilizes lactate in the environment to undergo respiration and produce hydrogen.  M. maripaludis then consumes this hydrogen to produce methane, allowing partial pressure in the media to be decreased so that D. vulgaris can continue to convert lactate to hydrogen. This creates a situation where these two microorganisms can live off of each other with minimal outside resources.

Adaptations Related to Environmental Stress

Transcription factors can be thought of as switches which on or off regulate genes. As a part of this experiment, six transcription factor mutant D. vulgaris strains were partnered in co-cultures. The strains were studied under two different resource environments, one containing solely lactate, and the other both lactate and sulfate. In the lactate sulfate media, D. vulgaris undergoes sulfate respiration and is considered metabolically independent. In the lactate only media, D. vulgaris must rely on its syntrophic relationship with M. maripaludis in order to survive.

In this experiment, D. vulgaris co-cultured with M. maripaludis is transitioned between these resource environments in order to study adaptations related to environmental stress. These co-cultures are transferred during the logarithmic growth phase into the alternate metabolic state. In previous studies, scientists have discovered that these co-cultures collapse after a certain number of transitions between resource environments. Cultures collapse due to the dilution of essential transcripts throughout the transitions and too much energy being expended on up and down regulating genes as the environment fluctuates.

Scientists have discovered that some transcription factor mutant co-cultures survive through more transitions than the wild type co-cultures. In a previous study, it was discovered that one mutant, DVU0744, did not collapse after many transitions. Although mutant co-cultures often grow more slowly than the wild type, some mutations appear to be beneficial to survival through stress in transitions. For example, a mutation that stops the regulatory network from functioning saves a culture undergoing metabolic transfers from specialization and unneeded energy usage. Studying how these mutations impact survival reveals valuable information about adaptations in evolution.

Experimental Setup

Georgia and Lauren both inoculated three mutant strains from the mutant library. These mutants were genetically engineered to have an insertion mutation within a specific transcription factor. These transcription factors are thought to regulate a variety of possible functions related to metabolism. The mutant strains studied by Lauren and Georgia were DVU0621, DVU0653, DVU0946, DVU2394, DVU2894, and DVU3142. After establishment, three replicates of each strain were created, in order to avoid experimental error. When the experiment began, all strains were co-cultured in lactate media with wild type M. maripaludis to establish a syntrophic relationship. Upon reaching an optical density (600 nm) reflecting growth in the logarithmic phase, transition experiments began.

RNA samples were collected at relevant points throughout the experiment to determine what genes were up and down regulated by the transcription factor mutants. Samples taken after every four rounds of transitions were purified and sent for RNA analysis.

Results

Research conducted yielded the following learned information:

DVU0621 has a mutation in the transcription factor regulating the nitrate stress response and lactate metabolism. It is a fis-amily transcription factor regulating eleven genes and two operons including two cytochrome C nitrate reductases. Additionally, DVU0621 is related to the protein families NtrC and AAA-type ATPase. This transcription factor can also be found in Acinetobacter sp ADP1, E. coli, and Salmonella enterica CT18.

DVU0653 has a mutation in the transcription factor involved in post transcriptional regulation. It is a sigma-54 regulator, meaning it allows the polymerase to recognize the promoter. Additionally, it is related to CheY chemotaxis proteins involved in transmitting sensory signals to the flagellar motors and arginine fingers which stabilize substrate atoms. This transcription factor is also found in Acinetobacter sp ADP1, E. coli, and Salmonella enterica CT18.

DVU0946 has a mutation in the transcription factor involved in lactate metabolism and transmembrane transport. It is a member of the fis-type family. Fis-type proteins were found in a study in 2013 to be vital for cell replication at the right time. This transcription factor regulates twelve genes and four operons, ranging in function from phosphorelay kinases to transferases. This transcription factor can be found in Bos Taurus, Chlamydomonas Reinhardtii, E. coli, and Salmonella enterica CT18.

DVU2394 has a mutation in the transcription factor involved in energy metabolism. It is involved with the protein families sigma-54 and CheY. It controlls one gene and one operon. The gene it controls is an alcohol dehydrogenase necessary for growth on ethanol. This transcription factor can also be found in Acinetobacter sp ADP1, E. coli, and Salmonella enterica CT18.

DVU2894 has a mutation in the transcription factor regulating flagellar functions. Unique from the other mutants, DVU2894 is related to phage shock protein transcriptional activators. Phage shock proteins are activated in stressful conditions such as environmental changes to help the organism survive. This transcription is involved with the protein families sigma-54, helix-turn-helix, and fis-family. This transcription factor regulates eight genes and three operons, one of which involves flagellar biosynthesis proteins, and a flagellar motility switch.

DVU3142 has a mutation in the transcription factor regulating energy metabolism. Along with being a member of the fis-family, sigma-54, and AAA-type ATPase, DVU3142 is a member of the PAS protein domain. PAS proteins facilitate the binding of small molecules. This transcription factor regulates three genes and one operon, involving iron sulfur cluster binding and cytrochrome proteins. Additionally, DVU3142 is a single component regulatory system, meaning it lacks phospho-transfer domains.

Impacts of Research

Studying adaptations to environmental stress provides valuable information about genetic diversity, resistance, resilience, and survival of the fittest. Understanding sequencing results from this investigation unveils exciting new knowledge about the function of certain transcription factors.

The study of D. vulgaris reveals insight into solving issues related to pollution created by human activities. Specifically investigating syntrophy may lead us to a better understanding of how biomass turnover in natural habitats occurs. Along with improving our knowledge of waste degradation control, studying the syntrophic relationship between D. vulgaris and M. maripaludis improves the perception of the ability to convert renewable resources into methane.