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Taxonomy

 

The first classification attempts of halophiles were based on cell morphology, salt requirement and gram staining pigmentation [1]. Originally systematists struggled to determine evolutionary relationships among halophiles because they had to rely on these phenotypic features to determine such associations. However starting in the 1960s with the advent of protein sequencing, PCR and electrophoresis (among other techniques), molecular phylogenetics led the way to understanding the interrelatedness of the ambiguous microscopic world and necessitated various taxonomic revisions [2].

 

Evidence of such taxonomic revisions is clear when considering the taxa that Halobacterium belongs. The suffix of its order through genus is derived from the Eubacteria with which it was incorporated, prior to the tripartite view of life proposed by Woese and colleagues [3]. Archaea are now considered distinct from all other organisms at the highest taxon. Initial recognition of Archaea as a discrete domain owed to sequence analysis of the semantide 16S rRNA and this notion was advanced with subsequent sequencing of other semantides (5S rRNA, 23S rRNA) and episemantides (DNA polymerases, ATPases) amongst other contributions such as the complete genome sequencing of the archaea Methanocaldococcus jannaschii [4]. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Studying Taxonomy

Aiming to minimize errors in taxonomy of halophiles a document published in 1997 proposed minimal standards for description of new taxa in the order Halobacteriales [7]. It promoted the polyphasic approach, a method combining a range of phenotypic, chemotaxonomic and genotypic properties to classify species. The document is still relevant today. The table (below) describes some of these techniques.

 

 

 

 

 

 

 

 

 

 

 

 

 

A more current form of genotypic classification popular today is a method known as multilocus sequence typing (MLST) which characterises cultured halophile strains by sequence analysis of multiple housekeeping genes [5].

 

 

Diversity

Halophiles are found in each domain of life [8] and although they are predominantly unicellular prokaryotes, multicellular eukaryotic halophiles do exist, two examples being brine shrimp and the larvae of brine flies [5].

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Archaea

Halophilic Archaea exist as a variety of heterotrophic, phototrophic and methanogenic species [5]. They exist only in the Euryarchaeota phylum and are found within two classes, Methanomicrobia & Halobacteria that encompass three orders: Methanomicriobiales, Methanosarcinales derived from Methanomicrobia and Halobacteriales from Halobacteria. The two former contain the families Methanospirillaceae and Methanosarcinaceae respectfully [8]. Therein lie organisms capable of living in hypersaline conditions as well as seawater and non-halophilic (less than 1% NaCl) environments. Whereas the Halobacteriales order contains one family, Halobacteriaceae and comprises entirely of halophiles including those that are the most salt-requiring and salt tolerant microorganisms known [8]. Of this family the genus Halobacterium currently contains the most extensively studied halophiles, Halobacterium salinarum (H. salinarum) and Halobacterium sp. NRC-1 because they are both easy to culture and manipulate genetically. Examples of phenotypes that distinguish Halobacterium as an Archaea would be the lack of peptidoglycan in its cell wall, unlike Bacteria and also its inclusion of phytanyl-based hydrophobic chains in its membrane, unlike both Bacteria and Eukarya [4]. Following is the derivation of H. salinarum's taxonomy:

 

Domain: Archaea

Phylum: Euryarchaeota

Class: Halobacteria

Order: Halobacteriales

Family: Halobacteriacaea

Genus: Halobacterium

Species: Halobacterium salinarum

 

According to the National Centre for Biotechnological Information (NCBI) there are currently 48 named genera that belong to the family Halobacteriaceae (including Halobacterium). At this level the genera and species are determined via the polyphasic approach described earlier.

 

Classifying Halobacterium strains into species has been a troublesome task. For example, the origin of the Halobacterium sp. NRC-1 is unclear though various literature had categorized it as a strain of either H. halobium, H. cutirubrum, H. salinarium or H. salinarum (the last two being the same species but renamed because of language issues). However in 2000 it became the first Halobacterium strain to have its whole genome sequenced and the authors of the relevant publication reframed from determining its ultimate taxonomy and instead reverted to the label of Halobacterium sp. NRC-1. Then in 2004 an article reclassified the wild-type isolate as a strain of H. salinarum although various evidence challenged this conclusion. For example an unmistakable phenotypic difference between Halobacterium sp. NRC-1 and the “H. salinarum” strain R-1 is the lack of gas vesicles in H. salinarum str. R-1, a feature that is considered to confer phototaxis, thermotaxis and chemotaxis and is of significant importance to Halobacterium sp. NRC-1’s survival [10], [11]. Considerable overreliance on phylogenetic trees produced from 16S rRNA comparisons has contributed to the misrepresentation of species because these sequences change in most of the participants of Halobacterium [11]. 

 

 

 

 

 

 

 

 

This graph shows the effects of increasing salt (sodium chloride) concentration on the growth rate of various microorganisms of differing salt tolerances.

Graph from 'Brock Biology of Microorganisms' [6].

The phylogenetic tree (left) is the product of 16S rRNA sequence comparisons. The bold branches within each domain indicate branches that contain halophilic microorganisms. The names given represent the type genus of that branch. 

Diagram from 'Halophilic Microorganisms and Their Environments' [8].

 

 

 

The picture (right) is of Artemia salina, a species of brine shrimp, a primitive group of anthropods, that can be found in the Great Salt Lake among other hypersaline habitats. They are multicellular Eukarya capable of living in almost saturated waters (50% salt concentration) as well as seawater levels of salinty (2.9 - 3.5% dissolved salts) [9].  

 

On a side note, a certain variety of these brine shrimp are the organisms that you can see in the 'Sea-Monkeys' kits!

Categorization of Halophiles

Halophiles are placed into 3 categories:

 

  • ​Slight halophiles - 1-5% NaCl

  • Moderate halophiles - 5-20% NaCl

  • Extreme halophiles - 20-30% NaCl

 

These groups are determined by the salt concentration in which the halophile can survive [5]. The graph (right) represents this point.

Bacteria

Halophilic Bacteria exist as a variety of phototrophic, lithotrophic and heterotrophic species. Cyanobacteria are the most common planktonic group in many hypersaline environments [5]. They form microbial mats, a cross section of which has been shown (right). The top brown layer of this mat contains species such as Aphanothece halophytica, which is an extreme halophile with an optimum NaCl concentration of 3.5M. Below the brown layer is the green layer that comprises various filamentous cyanobacteria such as Oscillatoria neglecta and they are more moderate halophiles and they are known to fix nitrogen and form heterocysts - specialised nitrogen fixing cells - when starved of nitrogen [5]. The purple layer below contains other phototrophic Bacteria including the green and purple sulfur and nonsulfur bacteria. These usually grow anaerobically via photosynthesis but retain the ability to live aerobically as heterotrophs. At the bottom of the microbial mat you have a

 

 

This diagram displays the structure of a microbial mat found in hypersaline environments, for example the Great Salt Lake. 

Diagram from 'Halophiles', [5].

Eukarya

Halophilic Eukarya comprise a variety of photosynthetic, lithotrophic and heterotrophic species [5]. There are the moderately halophilic green algae, a key example being the red Dunaliella salina shown (below left) that account for the red colour of certains areas of San Francsico Bay and the Red Sea. Diatoms have also been reportedly found at 2M salt concentration however this is rare. In contrast various protozoa thrive in hypersaline environments, one example being the moderately halophilic Fabrea salina [5]. Beyond protozoa there are yeasts and filamentous fungi that are capable of surviving hypersaline conditions

a large diversity of facultative and obligate anaerobic Archaea and Bacteria, including methanogenic Archaeon species of the Methanosarcinacaea family mentioned earlier and sulfate-reducing Bacteria such as Desulfohalobium retbaense. Besides the bacteria found in microbial mats there are the gram negative bacteria that are mostly moderately halophilic and belong to the two genera Halomonas and Chromohalobacter. There are also gram positive bacteria such as Halobacillus, also moderately halophilic [5]. 

This picture shows Dunaliella salina cells sampled from the coast of Israel. 

Image from 'A Hundred Years of Dunaliella Research: 1905-2005', [12].

conditions, for example Hortaea werneckii which can live in near saturated waters, making it one of the most halophilic fungi known. Finally there are a astonishingly wide variety of multicellular halophilic Eukarya, all invertebrates though a fish species, Tilapia, has been seen in moderately saturated environments [5]. 

References

 

  1. Oren, A. (2011). Taxonomy of the family Halobacteriaceae: a paradigm for changing concepts in prokaryote systematics. International Journal of Systematic and Evolutionary Microbiology, 62(2), pp.263-271.

  2. Stackebrandt, E. (2001). Phylogeny Based on 16S rRNA/DNA. Encyclopedia of Life Sciences.

  3. Woese, C. and Fox, G. (1977). Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proceedings of the National Academy of Sciences, 74(11), pp.5088-5090.

  4. Oren, A. (2001). Archaea. Encyclopedia of Life Sciences.

  5. DasSarma, S. and DasSarma, P. (2001). Halophiles. eLS.

  6. Madigan, M., Martinko, J. and Parker, J. (2003). Brock biology of microorganisms. Upper Saddle River, NJ: Prentice Hall/Pearson Education.

  7. Oren, A., Ventosa, A. and Grant, W. (1997). Proposed Minimal Standards for Description of New Taxa in the Order Halobacteriales. International Journal of Systematic Bacteriology, 47(1), pp.233-238.

  8. Oren, A. (2002). Halophilic microorganisms and their environments. Dordrecht: Kluwer Academic.

  9. Wikipedia, (2014). Artemia salina. [online] Available at: http://en.wikipedia.org/wiki/Artemia_salina [Accessed 19 Nov. 2014].

  10. Ng, W., Kennedy, S., Mahairas, G., Berquist, B., Pan, M., Shukla, H., Lasky, S., Baliga, N., Thorsson, V., Sbrogna, J., Swartzell, S., Weir, D., Hall, J., Dahl, T., Welti, R., Goo, Y., Leithauser, B., Keller, K., Cruz, R., Danson, M., Hough, D., Maddocks, D., Jablonski, P., Krebs, M., Angevine, C., Dale, H., Isenbarger, T., Peck, R., Pohlschroder, M., Spudich, J., Jung, K., Alam, M., Freitas, T., Hou, S., Daniels, C., Dennis, P., Omer, A., Ebhardt, H., Lowe, T., Liang, P., Riley, M., Hood, L. and DasSarma, S. (2000). Genome sequence of Halobacterium species NRC-1. Proceedings of the National Academy of Sciences, 97(22), pp.12176-12181.

  11. DasSarma, P. and DasSarma, S. (2008). On the origin of prokaryotic "species": the taxonomy of halophilic Archaea. Saline Syst, 4(1), p.5.

  12. Oren, A. (2005). Saline Syst, 1(1), p.2.

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