Science on Display | Bellevue University 
Fabiola Aviles, one of our former Biology majors, is the lead author on an article describing the discovery of a new bacterial species. The species was isolated from the San Elijo Lagoon near San Diego, where I took some samples while attending a science conference in 2019. After returning back to the lab, one of the samples gave bright yellow colonies on the plates. Fabiola extracted its DNA and sequenced the genome of this bacterium to find out its identity and characteristics.
Fabiola Aviles running the Illumina Miniseq sequencer at the BU labs.
At first glance we didn’t think we had anything unique there, because it looked like other soil bacteria, however Fabiola insisted on including it in her thesis project at BU, which made us look a little closer at the genome, and we were surprised to find it was a rather unique species.
As it turns out, the bacterium has the ability to decompose chitin (which is the hard outer shell of lobsters, shrimp and crabs, and insects), and cellulose, but can also feed on an unusual sugar called fucose. Therefore, the species was named Cellulosimicrobium fucosivorans (which literally means ‘fucose eating’ microbe). Fucose is found in insect and mammalian guts, but also in plant and algal polysaccharides. The ability to metabolize fucose gives certain bacteria in the gut flora a competitive advantage for colonization and invasion, making them pathogenic. Little is known about the ability to metabolize this sugar in nature.
Cellulosimicrobium fucosivorans cultivated on fucose and glucose.
It turned out to be even more surprising when we found out that the ability to feed on fucose was linked to the yellow color of the bacteria. Without the fucose, the bacteria are a pale white-pink color, but produce yellow carotenoids when fucose is present. These carotenoids are known to protect species from UV radiation damage from the sun (kind of like an internal sunscreen).
If you think about the environment this bacterium lives in, this starts making sense. There is plenty of algal and plant material, and if a bacterium can take the advantage of feeding on a component that others don’t use (for example fucose), then it has a competitive advantage. In addition, the coastal lagoons in San Diego gets lots of sun exposure and producing your own sun protection (carotenoids) seems a necessity for survival. So, this species turns out to be well adapted to living in the coastal sunny lagoon area.
The findings and a full detailed description were recently published in Archives of Microbiology and can be accessed here: https://rdcu.be/cmTYq
This is not Fabiola’s first scientific achievement. During her undergraduate studies at BU she also published a genome paper on Thiorhodococcus bacteria and recently completed a metagenomic study of the Piñones Lagoon in her home state of Puerto Rico. Fabiola plans to continue her studies in the field of marine biology, and these publications will undoubtedly help her in building out a successful scientific career.
This is project is a perfect example of how it is important to have an understanding of the different scientific disciplines, like microbiology, biochemistry, genomics, evolution, and bring them together to make unique discoveries.
You may not realize that, over 100 million years ago, Nebraska was covered by a large sea. This covered an estimated 20,000 acres, but now all that is left is salt marshes that are widespread around the state.
Sampling sites at the Eastern NE Salt Marshes.
These salt marshes are part of a rare wetland type that occurs in the Sandhills, the North Platte Valley and the valley of the Salt Creek and Little Salt creek. These saline wetlands are in danger of disappearing and several conservation efforts are ongoing to preserve this unique ecosystem.
Restoration and conservation efforts of native ecosystems for the most part focus on the insect and plant community, however the underlying microbial community is crucial to any preservation of marsh vegetation. Little is known about the bacteria and nutrient metabolism that occurs in these inland salt marshes, that are thousands of miles away from any coast, and have not been part of a larger saline body of water for apparently millions of years.
“Microbes are responsible for cycling and balancing the nutrients in any environmental ecosystem. So if we try to study and preserve these unique wetlands, we should start at the core, and look at what microorganisms are present and how they metabolize nutrients” says Dr. John Kyndt, who led the current research effort.
The BU team set out to investigate the algal and bacterial composition (the so-called ‘microbiome’) of these Salt Marshes. Samples were taken from the Salt Creek marshes near Lincoln last fall and analyzed in the BU labs. Biology undergraduate student Sierra Athen extracted DNA and used Illumina sequencing to produce the bacterial signatures. Together with BU employee Shivanghi Dubey, they used bioinformatic analyses to identify and compare the algae and bacteria in these samples. The results were recently published in the journal Life:
https://www.mdpi.com/2075-1729/11/5/446/htm
“These marshes are one of the only places in the United States where the naturally occurring water is saline. Realizing that this study could help in conservation of these endangered salt marches was not only motivating but also quite intriguing”, says Shivangi Dubey.
The team found that all these bacteria are well adapted to the high saline and alkaline conditions of the marshes, but some rare bacteria are present as well. For example, Rubribacterium was previously only isolated from an Eastern Siberian soda lake, but appears to be one of the most abundant bacteria present in the Nebraska Salt Marsh samples.
Microbial sulfur metabolism at the NE Salt Marsh ecosystem.
The microbial ecosystem appears to be well-balanced as far as sulfur and other nutrients, nevertheless, urban development, agricultural runoff, and the growth of invasive plant species are some of the current threats to maintaining these vital ecosystems. This current study establishes a good baseline for further studies on microbiological diversity, nutrient cycling, and ecological impacts in this locally important watershed.
“These land-locked saltmarshes are a relic of ancient oceans and, in some way, studying these salty microbes that live there, takes you back to the last ages of dinosaurs, which can be quite fascinating”, says Dr. Kyndt.
It is not every day that an entire bacterial genus is discovered and reclassified. We are lucky when occasionally we find a new species, but that alone certainly does not redefine an entire genus. It took the sequencing and comparative analysis of eight new genomes, in addition to the three known genomes of heliobacteria, to come to this discovery.
Dayana Montano Salama, an undergraduate research student at BU, sequenced the Heliomicrobium genomes using the in-house Illumina MiniSeq.
The family of heliobacteria are a small group of phototropic bacteria that were serendipitously found while Dr. Howard Gest was teaching a lab class in microbiology some 38 years ago. It is therefore suiting that an undergraduate student, Dayana Montano Salama, was a key player in this new research that led to new genus of Heliomicrobium.
“A combination of genome sequencing, whole genome comparison and detailed genetic analysis of the 11 species was performed to reclassify them into the Heliobacterium and Heliomicrobium genera.” says Dr. John Kyndt, who was the lead on this research project. “In the end, the new genus Heliomicrobium includes four unique species, although more will undoubtedly be discovered in the future with growing metagenomic efforts.”
The new genus name comes from the Greek words helios sun; micros small; bios life, so Heliomicrobium literally means ‘sun microbe’. Heliobacteria have been recognized as the ancestors of photosynthesis in the evolution of photosynthetic bacteria. All heliobacteria are strictly anaerobic, endospore-forming bacteria, and the ability to form endospores is unlike all other phototrophic bacteria, but similar to species of Clostridium and Bacillus. An improved understanding of the taxonomic classification of these bacteria will ultimately lead to a better understanding of the evolution of photosynthesis in bacteria in general.
The research was an international collaboration with Dr. Johannes Imhoff from the GEOMAR institute in Germany, and was recently published in the International Journal of Systematic and Evolutionary Microbiology, which is the flagship scientific journal for bacterial taxonomy.
A link to the publication can be found here: https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijsem.0.004729
Mutualistic relationships that involve close cell–cell interactions are most studied between bacterial and eukaryotic interactions, for example between pathogens and eukaryotic hosts (plant or animal). However symbiotic relationships between bacteria themselves have only been studied more recently. They can be found amongst archaea and bacteria in microbial mats where nutrient exchange and waste removal roles are crucial, in anaerobic methane-oxidizing communities of marine environments, or even in human digestive systems.
Microbial mats contain many bacterial species living in close interactions.
Although these syntrophic cocultures appear to be more widespread than commonly expected, very little is known about the physical interaction that are formed in the cell–cell interactions and the specific chemistry involved to establish a mutually beneficial relationship.
The clue is in the genomes…
By sequencing the genomes of two different species of a symbiotic mixture, and comparing the unique features of these genomes to other free living species, we were able to identify specific, unique interactions that can be formed within this syntrophy. The study showed several types of pili (hair-like appendages found on the surface of many bacteria) and so-called close-adhesion proteins present in one of the partners, while unique e-pili (electron-transporting pili) are formed by the other partner. The details of this are described in a publication by Kyndt et. al., just released this month in Microorganisms: https://www.mdpi.com/2076-2607/8/12/1939
One interesting technical aspect of the study was that the sequencing of both species in the symbiosis was performed simultaneously (without separating the species). However, we were able to separate and assemble the genomes by a process called metagenomic binning. This was made possible by our in-house Illumina sequencing and the publicly available PATRIC bioinformatics platform.
One cell’s trash is another cell’s treasure.
You may wonder what the benefit is for each of these cells to form this symbiosis. In the studied model, the green sulfur bacteria (Prosthecochloris) grow by photosynthesis. This is similar to the photosynthesis you may know from plants and algae, however it occurs anaerobic (without oxygen) and uses hydrogen sulfide (H2S) instead of H2O, and deposits elemental sulfur globules (S0) outside the cells, as a waste product. The partner bacteria in the mixture (Desulfuromonas) loves this, and uses this sulfur as an electron acceptor, converting it to sulfide. This sulfide in turn is used by the green sulfur bacteria to performs its photosynthesis, thereby completing the cycle.
Overview of interactions between Prosthecochloris ethyilca (green) and Desulfuromonas acetoxidans (brown/red). Image created in BioRender.com
Close interactions are preferred.
Thanks to the recent study, we now have a model by which so-called ‘tight adhesion’ (Tad) pili and large agglutination proteins from the green sulfur Prosthecochloris are key elements in the formation of the syntrophic complex. Once close cell–cell interactions are formed, the closed sulfur cycle can be established by electron transfer through specialized e-pili and several cytochromes produced by the Desulfuromonas component.
In addition to the sulfur cycling, another benefit of this relationship is that the colorless bacterium has flagella. This gives the entire complex the advantage of mobility and allows it to swim towards light and nutrients.
It is interesting that both the Tad pili and the adhesion proteins are best known from studies of bacterial virulence factors. However, based on their presence in other nonpathogenic species, they seem to be more widespread amongst bacteria and are likely involved in many environmentally important symbiotic interactions between bacteria.
What do you get when you put a project manager with an electrical & electronics engineering degree, and a biochemist together in a lab? It sounds like the beginning of a bad joke, but nothing is further from the truth. Sometimes unusual interdisciplinary collaborations can turn out to be very productive.
Shivangi Dubey – Manager, Project Management Office and Software Development at Bellevue University
This is exactly what happened when Shivangi Dubey, a BU employee in the IT Department, reached out to Dr. John Kyndt at the Science Department, and expressed that she had an interest in working on some real-life science projects. Together they started working on some of the ongoing project of bacterial genome sequencing at the BU Science Labs.
The collaboration turned out to be fruitful very fast. “Shivangi showed an honest interest and curiosity in the project and was very driven to accomplish results” says Dr. Kyndt. “From the start she described herself as a science enthusiast and a constant leaner. That made it real easy to teach her basic lab and bioinformatic skills”.
In less than two months after the start of the project, the team was able to publish a new article on the genome analysis of an unusual bacterial species. The publication was just released online in Microbiology Resource Announcements this week: Genome Sequence of the Unusual Purple Photosynthetic Bacterium Phaeovibrio sulfidiphilus, Only Distantly Related to Rhodospirillaceae, Reveals Unique Genes for Respiratory Nitrate Reduction and Glycerol Metabolism
Not only did they complete the genome of this species, the genomic analysis and comparisons also revealed the genetic reason behind the strict anaerobic nature of this bacteria and revealed unique metabolic pathways that will be the basis for further physiological studies.
“It all started when a research article on “Photoactive Proteins” by Dr. Kyndt caught my attention and out of curiosity I reached out to Dr. Kyndt to learn more about his research. He not only addressed my curiosity but also provided me an opportunity to learn from him and partner with him in his next research project. Dr. Kyndt has been very supportive and provided guidance throughout this research. I look forward to many more such opportunities to learn from him” Shivangi.
In an effort of continuous education, both Shivangi and Dr. Kyndt are currently taken an online course in Bacterial Bioinformatics, and are hoping to complete their Certificate in Bioinformatics from the University of Virginia in a couple of weeks.
The giant phototrophic bacterium Thiospirillum jenense was first discovered in 1838. Its large size (up to 100μm), spiral shape, orange-brown color, and formation of sulfur globules visible under the light microscope, made it an interesting study object for several renowned microbiologists throughout the 20th century.
Microscopic image of Thiospirillum jenense.
Although a very intriguing organism, it was proven to be extremely difficult to cultivate in the lab and even to date, no pure culture has been obtained. For a long time, the only source of this bacterium was a pond in former East Germany where it grew April to July, and bacterial cultures had to be smuggled across the then closed borders to West Germany. For more than five decades, the problems in cultivation of Tsp. jenense and later also the missing availability of cultures, have disabled further detailed studies.
Even though the growth and phototactic behavior was studied, nothing was known about the molecular genetics of this intriguing species, not even 16S rRNA sequences, so no complete taxonomic classification could be performed. Up until now.
Illumina-based sequencing was performed at Bellevue University on an enriched sample of Thiospirillum jenense, obtained from Dr. Johannes Imhoff from the GEOMAR Ocean Research Center in Kiel (Germany). Genome sequencing and metagenomic binning analysis now provided the full genome of Thiospirillum jenense which was published just this week in Archives of Microbiology. It showed the unique placement of this species amongst the purple sulfur bacteria and, in addition, potentially resolved some of the genetic reasons behind the challenges of cultivating Tsp. jenense that have been limiting further experiments in the past.
One key component appears to be lack of a high-affinity oxidase (FixNOP), which presumably renders the cells highly sensitive to oxygen damage. In addition, the two sequenced contaminant species, Rhodopseudomonas palustris and a new species Rhodoferax jenense, might help with removal of oxygen in the cultures. These results likely explain the difficulties with obtaining pure cultures of Tsp. jenense as described in the paper, and opens up the doors for new cultivation methods.
Having genomic and genetic data available for Tsp. jenense has widened our understanding of the microbial diversity and will undoubtedly help to further identify similarly unique species in environmental samples, where they play an important role in the sulfur cycle and other nutrient recycling in the environment.
With the recent growth in Natural Science courses and rapid development of the sustainability outdoor lab, the time was ripe to expand on the biology instructors for the science department.
Dr. Sarah Gaughan has a Ph.D. in Natural Resources (from UNL) and a background in aquatic ecology, fishes and genome sequencing. Her research has been focused on applying novel techniques to facilitate conservation of native species. She also focuses on population management and finding new ways to control invasive species.
Dr. Sarah Gaughan and Justin Haas with Nebraska Game and Parks doing Pallid Sturgeon gut microbiome research.
This provides a perfect fit for the growing science program and at the same time expands the experience and knowledge of the overall science faculty at BU. Having someone with a diverse ecology background will not only be beneficial for the outdoor lab projects, but also opens up the possibility for students that have an interest in environmental biology and ecology research projects or careers.
Dr. Gaughan will be teaching a variety of natural science courses for the general education but will also be available to mentor students in various research projects in the Biology or Sustainability program throughout the year.
To find out more about Dr. Gaughan’s research projects please check out our Faculty and Staff page, or feel free to contact her directly (sgaughan@bellevue.edu) if you have any environmental or ecology questions or project suggestions.
Welcome to the science team Sarah!
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