User:Ku Itoi/Microbial community of Brine Pools

Metagenomics Analysis
Metagenomics is a powerful genomic analysis to identify the microbiome communities in a variety of environments. One problem of previous gene analysis, which requires culturing microorganisms, is that most microorganisms present in nature are not cultivable. Unlike the old gene analysis, metagenomics allows researchers to directly sample and analyze the microbe community from the desired environment. Despite the harsh environment for living organisms, metagenomics has revealed the presence of previously unknown microbiome communities in multiple Brine pools. Common procedures of marine microbe metagenomics include sampling, filtration and extraction, DNA sequencing, and database analysis. Shotgun sequencing and 454 pyrosequencing are typically used to sequence genes, and 16S rRNA is used for the identification of species.

Main clades
The taxonomy of the microbe community found in Brine Pools is listed below. Unknown groups and minor groups are excluded from the list. Samples were collected from Atlantis II and Discovery. Identification of microbes and construction of phylogeny is controversial, and because of an increased amount of gene sequence data, phylogeny has been reconstructed. The list below is based on the data provided from the primary articles, which might not be up to date.


 * Domain
 * Phylum
 * Class
 * Order
 * Family
 * Genus


 * Bacteria
 * Actinobacteria (Actinomycetota)
 * Actinomycetia
 * Actinomycetales
 * Corynebacteriales (Mycobacteriales)
 * Mycobacteriaceae
 * Mycobacterium
 * Micrococcales
 * Microbacteriaceae
 * Acidobacteria
 * Bacillota
 * Bacilli
 * Bacillales
 * Bacillaceae
 * Bacillus [6]
 * Bacteroidetes [7]
 * Flavobacteria [5]
 * Candidatus division od1
 * Chloroflexi [7]
 * Anaerolineae [5]
 * SAR202 clade [5
 * Cyanobacteria
 * Deinococcota [7]
 * Deinococci
 * Thermales
 * Thermaceae
 * Meiothermus [6]
 * Deferribacterota
 * Deferribacteres [5]
 * Em19
 * Eurybacteria
 * Thermotogota
 * Thermotoaea [7]
 * Firmicutes [7]
 * Planctomycetes
 * Candidatus Scalindua [5]
 * Candidatus Brocadiales [5]
 * Proteobacteria (Pseudomonadota) ***
 * Alphaproteobacteria [5,7] **
 * Hyphomicrobiales
 * Phyllobacteriaceae
 * Phyllobacterium [6]
 * Nitrobacteraceae
 * Afipia [6]
 * Bradyrhizobium [6]
 * SAR11(Pelagibacterales) [5]
 * Betaproteobacteria [5,7]**
 * Burkholderiales
 * Comamonadaceae
 * Rhodoferax [6]
 * Malikia [6]
 * Burkholderiaceae
 * Cupriavidus [6]
 * Ralstonia [6]
 * Deltaproteobacteria [5,7]
 * Gammaproteobacteria [7] **
 * Alteromonadales [5]
 * Oceanospirillales [5]
 * Pseudomonadales
 * Moraxellaceae
 * Acinetobacter [6]
 * Alkanindiges [6]
 * Alkanindiges
 * Acinetobacter
 * Xanthomonadales
 * Xanthomonadaceae
 * Stenotrophomonas [6]
 * Archaea
 * Crenarchaeota ***
 * Group c3 [5]
 * Marine benthic group a (MBG-A) [5]
 * Marine benthic group b (MBG-B)[5]
 * Marine group I (MGI) [5]
 * Misc crenarchaeotic group [5]
 * Psl12 [5]
 * Thermoprotei
 * Desulfurococcales [7]
 * Sulfolobales [7]
 * Thermoproteales [7]
 * Euryarchaeota [7]***
 * Archaeoglobi [5]
 * Archaeoglobales [7]
 * Halobacteria [5]
 * Halobacteriales [7]
 * Methanomicrobia [5]
 * Methanobacteriales [7]
 * Methanocellales [7]
 * MEthanococcales [7]
 * Methanomicrobiales [7]
 * Methanopyrales [7]
 * Methanosarcinales [7]
 * Thermoplasmata [5]
 * Korarchaeota
 * Korarchaeia (Xenarchaeota)
 * Korachaeales
 * Korarchaeaceae
 * Candidatus Korarchaeum cryptofilum [7]
 * Thaumarchaeota
 * Candidatus Caldiarchaeum [7]

Environmental challenges and adaptations
The lack of mixing of the water column in combination with the extreme conditions of salinity, anoxia, temperature and hydrostatic pressure, has resulted in an environment which has been separated from the upper water column. there is a long prolonged historical separation of the brines from the upper water column (Cita et. al., 2016). These unique environmental characteristics promote unique extreme challenges and selective pressures on the organisms that live in these ecosystems, and promoting unique microbial assemblages that were able to adapt to the environment.

Challenges
Due to high levels of salinity of the environment, cell membranes cannot avoid the rapid loss of intracellular water, and thus need to develop extreme strategies to decrease their metabolic activity as much as possible, impairing cell turgor and functioning (Mei et. al., 2017). Brine pools also exert ionic, kosmotropic and chaotropic effects on the cells, which also causes additional challenges for the organisms to survive these extreme environments.

Adaptations
Different organisms developed a number of strategies to solve the challenges imposed by these extreme environments, such as the high levels of salinity. In order to decrease the risk of chaotropic effects on the cells, organisms like halophilic archaea successfully developed the "salt-in" and the "compatible-solute" strategy, which increases intracellular ionic concentration (mostly K+) to decrease the osmotic pressure, and thus forcing these organisms to adapt their entire metabolic machinery to increase salt concentration inside of their cells to ensure their survival.

In addition, in order to survive the extreme water temperatures and hydrostatic pressures, piezophilic microorganisms that synthesized thermoprotective molecules (e.g. hydroxyectoine) were selected for, and thus successfully avoiding denaturation of their proteins, decrease of fitness due to extreme hydrostatic pressures, decrease the risk of desiccation imposed by the environment, and developing novel characteristics in the microorganisms present in the brine pools.

Lastly, another important adaptation is the usage of alternative electron acceptors molecules to yield energy, such as iron, manganese, sulfate, elemental sulfur, carbon dioxide, nitrite and nitrate, which are abundantly available in these deep water layers.

Animals are have also been found living in these anaerobic brine pools, such as the first ever described metazoan by Danovaro et. al., in 2010., even though much research still has to be done about their entire life cycle. Many novel undefined taxa that live in these extreme environments are still unknown.

Chemical composition and metabolic significance
As the name suggests, Brine pools, or Deep Hypersaline Anoxic Basins (DHABs), are characterized by a very high salt concentration and suboxic to anoxic conditions. Sodium, chloride, magnesium, potassium, and calcium ion concentration are all extremely elevated in brine pools. And, due to low mixing rates between the above seawater and the brine water, brine pool water becomes anoxic within the first ten centimeters or so ( https://academic.oup.com/femsec/article/94/7/fiy085/4995905 ). While there are large variations in the geochemical composition of individual pools ( https://academic.oup.com/femsec/article/94/7/fiy085/4995905 ), as well as extreme chemical stratification within the same pool ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3648036/ ), conserved chemical trends are present. Deeper layers of DHABs will be saltier, hotter, more acidic and more anaerobic than the preceding layers ( https://www.sciencedirect.com/science/article/pii/0012821X96000982 ), ( https://www.sciencedirect.com/science/article/pii/0304420390900317 ). The concentration of heavy metals (Fe, Mn, Si, Cu) and certain nutrients (NO2-, NH4+, NO3-, and PO4-) will tend to increase with depth, while the concentration of SO4- and both organic and inorganic carbon decrease with depth ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3648036/ ). While these trends are all observed in DHABs, the intensity and distance over which these trends take effect can vary in depth from one meter, to tens of meters ( https://academic.oup.com/femsec/article/94/7/fiy085/4995905 ).

The heavy stratification within DHABs has led to increased microbial metabolic diversity and varying cell concentrations between layers. The majority of cell biomass has been found at the interfaces between the distinct chemical layers (with the highest concentrations of cells located at the brine-surface interface) ( https://pubmed.ncbi.nlm.nih.gov/15637281/ ). Microbes exploit the sharp chemical gradients between the layers to make their metabolisms more thermodynamically favorable ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685740/ ).

Four of heavily studied DHABs are Urania, Bannock, L’Atalante, and Discovery. All four of these Brine pools are located in the Mediterranean sea, yet they all exhibit distinct chemical properties:

Description: Urania has the highest concentration of Sulfuric Acid observed (at ~16mM)--compared to normal sea water (2.6 x 10^-6mM) or the next highest [HS-] in the Bannock basin (~3mM) (file:///C:/Users/dmile/OneDrive/Desktop/College/Wterm%202021-2022/EOSC%20475/vanderwielen.som.pdf), ( https://pubmed.ncbi.nlm.nih.gov/21518212/ ). Discovery has an extremely low concentration of Na+ (68mM) and an extremely high concentration of Mg2+ (4995mM)--compared to the surrounding seawater with concentrations of 528mM and 60mM respectively ( https://pubmed.ncbi.nlm.nih.gov/15637281/ ), ( https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/1462-2920.12587 ). The L’Atalante basin has a high [SO4 2-] compared to the other pools.

Carbon cycling
While it was initially thought that particulate organic matter (POM) was an important source of carbon for DHABs, due to their depth the concentration of POM reaching the pools was not significant as originally thought ( https://academic.oup.com/femsec/article/94/7/fiy085/4995905 ). The majority of fixed carbon is now thought to come from autotrophy, specifically methanogenesis. Direct measurements of methane production in DHABs have provided extensive molecular evidence of methanogenesis in these environments ( https://www.science.org/doi/10.1126/science.1103569 ). Proteomic analysis further support the presence of methanogenesis by identifying the enzyme RuBisCo in various DHABs ( https://academic.oup.com/femsle/article/259/2/326/585450?login=true ). Interestingly, it has been suggested that instead of CO2 or acetoclastic methanogenesis, prokaryotes in DHABs use methylotrophic methanogenesis as it allows for a higher energy yield ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC98969/ ) and the intermediates can be used for osmoprotectants ( https://www.nature.com/articles/srep03554 ).

Nitrogen Cycling
One of the key metabolic features of DHABs is the dissimilatory reduction of nitrogen. In Bannock basin and L’Atalante basin anammox and denitrification pathways have been identified using a combination of transcriptomics and direct isotope tracking ( https://pubmed.ncbi.nlm.nih.gov/23340764/ ). Other DHABs have been analyzed for anammox pathways using metatranscriptomic techniques with little positive results, which may be due to the limitations of transcriptomic sensitivity. In deeper DHAB layers, nitrogen fixation and ammonium assimilation has been observed. These reductive pathways are mainly performed by methanogens to synthesize osmoprotectants. ( https://www.nature.com/articles/ismej2014100 )

Sulfur Cycling
Due to the high concentration of sulfate (especially in the Uranian Basin), sulfate reduction is extremely important in the biogeochemical cycling of DHABs. The highest rates of sulfate reduction tend to be found in the deepest DHAB layers, where redox potential is lowest ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685740/ ). Sulfate reducing bacteria have been found in the brines of Kebrit deep, Nereus Deep, Erba deep, Atlantis II deep, and Discovery Deep ( https://www.sciencedirect.com/science/article/pii/S0923250815001175?via%3Dihub ). Oxidative sulfur pathways help close the biogeochemical sulfur loops within the DHABs. There are three main sulfur oxidizing pathways which are likely found in DHABs: 1) sulfur-oxidizing multienzyme complex which can oxidize sulfide or thiosulfate to sulfate (w/ elemental sulfur or sulfite as an intermediate), 2) a sulfide:quinone complex which oxidizes hydrogen sulfide to elemental sulfur, 3) polysulfide reductase, which reduces precipitate sulfur to sulfide. A combination between the second and third pathway would allow for increased energetic yield ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC92956/ ). Novel groups have been isolated from saline lakes which can anaerobically respire sulfur using acetate, pyruvate, formate, or hydrogen as a sole electron donors ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437934/ ).

Microbial Symbiosis
Symbiosis is a long term and close interaction and relationship between two or more organisms that are from different species (ex. microbes and its host species like clams). Symbiotic relationships entail both sides of the partnership benefiting from their relationship and interaction.

Reference:

Dimijian G. G. (2000). Evolving together: the biology of symbiosis, part 1. Proceedings (Baylor University. Medical Center), 13(3), 217–226.

There are symbiotic relationships between bivalves, such as brine clams and the microbial bacteria contents in nature.

Reference:

Roeselers, G., & Newton, I. L. (2012). On the evolutionary ecology of symbioses between chemosynthetic bacteria and bivalves. Applied microbiology and biotechnology, 94(1), 1–10. https://doi.org/10.1007/s00253-011-3819-9

The interactions between a microbe and its host such as clams have had the following exchanges within their relationship. Certain studies have demonstrated that the complex relationships between microbes and their host species can have great consequences with great disturbances and of either partner’s health. Furthermore, hosts like clams and microbes evolve together and interact intensely in their respective environments. Host-associated microbiota has a role in the host’s development, metabolism, behavior, adaptation, reproduction, environmental sensing, immunity as well as protection from pathogens, and organ morphogenesis. The microbiota also has a significant contribution regarding nutrition of the hosts. Many microbes synthesize the organic matter from carbon dioxide which are the primary nutrition source for many hosts such as clams. In exchange for the synthesis of nutrients for the hosts, clams for example will provide the microbes (symbionts) a habitat where the microbes then have access to synthesize organic matter.

Microbial organisms are an essential part of the ecosystem, and they inhabit many animals and plants as well. Microbes affect the overall health, development, and fitness of their hosts. This symbiotic interaction between the host and the microbes creates complex interactions between the microbes and the host as well as their respective microbial environment and communities. There are behavioral benefits between these relationships, and the disturbance of these interactions (i.e., absence of microbes on the host) can result in the development of diseases or improper development, and consequences to the host’s health and fitness.

Microbes also have a symbiotic relationship with algae, where studies have found that algae will grow abnormally with the absence of required bacteria. Without certain bacteria, the algae will miss-develop, but once the algae are set back to its’ appropriate marine environment and bacteria, it will eventually return to its’ typical morphology.