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Primitive thermophilic Bacteria

These two phyla represent the best known of several small groups of organisms that are both primitive and deeply branching in the bacterial tree.

They are primitive in that they have changed less than other organisms since their common ancestry, at least in terms of 16S rRNA sequences; the distance from the common ancestor to the modern sequences is shorter for them than for other Bacteria. This does not mean that they are the ancestors of other Bacteria, or that they are any less complex.

They are deeply branching in that their branches connect to the bacterial tree closer to the root than do the other branches. This statement, that these groups are “deeply branching”, comes with two caveats. The first is that it presumes that the bacterial phyla represent distinct “kinds” of creatures regardless of how these phyla are related to one another, in the same way that animal phyla are different “kinds” (evidenced by differences in basic body plan) regardless of the details of how these phyla are related one to another. This is related to the difference between evolutionary rates and modes, and implies that each phylum emerged during a distinct transition. If this is not the case, then “deeply branching” becomes a relative term only; Aquifex and Thermotoga are deeply branching with respect to, for examples, the cyanobacteria, proteobacteria, or Gram-positive Bacteria, but E. coli could be viewed as a deeply-branching mesophile from the perspective of Aquifex and its relatives.

The second caveat to the view that these phyla are deeply branching comes from uncertainties inherent in phylogenetic trees, particularly as the result of unequal evolutionary rates. This can result in an artifact in trees known as “long branch attraction”, in which long branches (groups with higher evolutionary rates) are “pushed” toward each other as the result (in part) of underestimating the actual evolutionary distance between them. In the case of the bacterial tree, this may result in the artifactual clustering of the longer branches, excluding the shortest branches into the deeper parts of the tree. One observation in favor of this view is that as more and more diverse sequences are included in the bacterial tree, the more all of the bacterial phyla come closer to seeming to emerge from a single point of radiation.

Even with these caveats in mind, however, it does seem that the deepest branches of the bacterial tree are both thermophilic and primitive. The fact that they are primitive with respect to 16S rRNA sequences may represent only a lower tolerance at high temperatures for the transient non-Watson-Crick base pairs that are the inevitable intermediates of evolutionary change in RNA structures, and may not represent primitiveness in other features of these organisms.

Phylum Aquificae (Aquifex and relatives)

Taxonomy

Phylum Aquificae

• Class Aquificae
  • Order Aquificales
    • Family Aquificaceae
      • Genus Aquifex
      • Genus Calderobacterium
      • Genus Hydrogenivirga
      • Genus Hydrogenobaculum
      • Genus Hydrogenobacter
      • Genus Hydrogenothermus
      • Genus Persephonella
      • Genus Sulfurihydrogenibium
      • Genus Thermocrinus
      • Genus Venenivibrio
  • Incertae sedis
• Genus Balnearium
• Genus Desulfurobacterium
• Genus Thermovibrio

General characteristics of the Aquificae

Diversity
Less than 2 dozen species have been described in this group, only one or a few species in each genus, and they are relatively homogenous in phenotype, especially among the Aquificales (the genera that are currently incertae sedis are more phenotypically distinct).

Metabolism
These organisms are all thermophilic or extremely thermophilic, and obtain energy by respirative hydrogen oxidation. This reaction is known as the “Knallgas” reaction. They are obligate aerobes, generally microaerophilic (the solubility of oxygen in water at the temperatures these organisms grow at is very low in any case). Reduced sulfur compounds such as sulfide, thiosulfate (S2O3 2-), or elemental sulfur can generally replace hydrogen, but nitrate cannot replace oxygen as the terminal electron acceptor in most species. These organisms are autotrophic, fixing carbon dioxide via the reverse TCA cycle. Few have been grown heterotrophically; they are generally considered to be obligate autotrophs.

Morphology
These organisms are rod-shaped (ca. 0.5 x 2-8μm) to filamentous, with a typical Gram-negative type envelop, some with an external crystalline protein “S-layer”. Some are motile and flagellated, but other appendages have not been seen. During growth on sulfide or thiosulfate, elemental sulfur granules can appear, but no storage granules or internal membranous structures been observed. Some produce carotenoid pigments.

Habitat
These organisms are common inhabitants of near-neutral pH, high temperature geothermal springs, including hot springs and submarine vents.

Example species

Aquifex pyrophilus

A. pyrophilus is, like the other members of this group, a thermophilic hydrogen oxidizer. A. pyrophilus is an extreme thermophile, growing optimally at 85°C and maximally at 95°C. This makes it the most extremely thermophilic isolated and characterized bacterium. It is unusual amongst its relatives in that it can use nitrate as a terminal electron acceptor, and so can grow anaerobically. It is also more sensitive to oxygen than most members of this group, and when grown without nitrate is an obligate microaerophile.

A. pyrophilus was isolated in 1992 from a deep sea hydrothermal vent on the Kolbeinsey Ridge North of Iceland (which is an outcropping of the Mid-atlantic ridge, where the two large plates underlying the ever-enlarging Atlantic Ocean come together, or more properly are coming apart).

The phylum is named after this organism because it was the first of its members to be characterized phylogenetically and discovered to represent a distinct, deeply-branching and primitive branch of the Bacteria. It was only later discovered that previously isolated species (Hydrogenobacter and Calderobacterium) were related to A. pyrophilus.

Thermocrinis ruber

T. ruber is also a thermophilic hydrogen oxidizer, growing optimally at 80°C but up to 89°C. Grows either as individual rod-shaped cells (0.5 x 1-3μm) that are motile by monopolar polytrichous flagella (multiple flagella at one end of the cell only), or as filaments. T. ruber can grow heterotrophically using formate or formamide, as well as autotrophically using hydrogen and reduced sulfur compounds, as electron donors, but cannot replace oxygen with nitrate as an alternative electron acceptor, and so it is an obligate microaerophile.

T. ruber was isolated from pink filamentous growth in Octopus Spring, Yellowstone National Park. This pink filamentous growth is common in the 80-90°C temperature zones of neutral to slightly alkaline hot springs throughout the park, and has been described since the early work of Thomas Brock in the 1960’s. Numerous attempts to cultivate the pink filamentous organism failed, although Thermus aquaticus (the source of Taq polymerase, that made PCR amplification a reasonable technology) was isolated as a by-product of these attempts.

Ultimately, of course, T. ruber was isolated from the pink filaments of Octopus Spring, using insight gained by molecular phylogenetic analysis of the organism prior to cultivation. This story is described in detail in Chapter 18.

Phylum Thermotogae (Thermotoga and relatives)

• Phylum Thermotogae
  • Order Thermotogales
    • Family Thermotogaceae
    • Genus Fervidobacterium
    • Genus Geotoga
    • Genus Marinitoga
    • Genus Petrotoga
    • Genus Thermopallium
    • Genus Thermosipho
    • Genus Thermotoga

General characteristics of the Thermotogae

Diversity
The less than 3 dozen described species in a single Family.

Metabolism
The members of this phylum are thermophilic (65°C to 90°C), anaerobic fermentative organisms that grow on a wide range or organic compounds, using protons or often elemental sulfur as the terminal electron acceptor. Ferric ion (Fe+++) can serve as an electron acceptor in some isolates. The products of the oxidation of organic material are typically CO2 and organic acids such as acetate and lactate. When grown in the absence of sulfur, hydrogen is the waste product of proton reduction, and hydrogen is a strong inhibitor of growth; hydrogen removal is required to maintain the energetic favorability if this reaction.

Morphology
The distinguishing feature of this phylum is the presence of a loose sheath, the “toga”, covering the rod-shaped cells. This sheath is typically snug over the sides of the cells but balloons out at each end of the cell. The toga is the outer membrane of an otherwise typical Gram-negative-type envelop, and so the space captured by the toga is conceptually periplasm. The toga is rich in porin-like proteins, arrayed in a regular pattern over the entire surface of the toga. The function of this unusual toga structure is unknown. Most members of this group are flagellated.

Habitat
These organisms have been isolated primarily from geothermally heated soils and sediments, including solfataras, the soil surrounding hot springs, and hot sediments in the vicinity of deep sea hydrothermal vents.

Example species

Thermotoga maritima

By far the best-studied member of this phylum is T. maritima, and for many years it was the only known member of the group. It was isolated from a heated submarine sediment off of the island of Vulcano, Italy. Cells are ca. 0.6 x 2-5μm rods with pronounced togas. It is an extreme thermophile, growing in a temperature range of 50-90°C, optimally at 80°C. This specie is motile via terminal flagella, but many related species of Thermotoga are non-motile. They are able to fix nitrogen (N2), but cannot use nitrate nor sulfur compounds other than elemental sulfur as terminal electron acceptors. T. maritima lacks genes for the electron transport chain, and so must generate its proton motive force (proton gradient) using either ATPase (run in the reverse of the usual reaction) or by a membrane-associated pyrophosphatase proton pump.

T. maritima is easy to grow, and the complete genome sequence was one of the earliest available from a thermophilic bacterium, following only that of Aquifex aeolicus, which is notoriously difficult to handle. Combined with the facts that proteins from extreme thermophiles often form high-quality crystals more readily than do those of mesophiles (perhaps because they are more rigid and so more uniform in structure than are the homologous proteins from mesophilies), and are usually much easier to express in recombinant form in E. coli than are the proteins of Archaea, make this organism an attractive source of proteins for structural examination by X-ray diffraction.

The genome sequence of T. maritima provided the best evidence available for the large-scale horizontal transfer of genes across large phylogenetic distances. It has been argued that up to 1/4th of the genome of T. maritima may have been acquired from an archaeal source recently enough that it can still be identified as foreign; others would revise this figure downward, to about 5%. Regardless, it is clear that this organism has acquired a great deal of genetic potential from outside sources.

Thermosipho africanus

T. africanus was isolated from hot, sandy sediment off the coast of Obock, Dijouti. This specie is morphologically much like Thermotoga, but grows in chains up to 12 cells long. Cells within chains are connected and separated by their togas. T. africanus is also a thermophile, but grows at lower temperatures than other members of this phyla; 35-77°C, optimally at 75°C.

Fervidobacterium islandicum

F. islandicum was the second member of this phylum to be isolated, after T. maritima, but is much less well-characterized. It was isolated from a solfatara in the Hveragerdi geothermal fields of Iceland. In this specie, and its close relative F. nodusum, the toga forms a large spheroid “nodule” at one end of the cell only. F. islandicum grows at temperatures ranging from 40-80°C, optimally at 65-70°C.

Other primitive thermophiles

There are a number of other groups of primitive thermophiles that are even less well-understood than are the Aquificae and Thermotogae. Most are known only from 16S rRNA sequences obtained directly from thermal environments, and have not been cultivated. Others are either know from single isolates, or their placement in the tree is uncertain (or both).

Thermodesulfobacterium

The phylum Thermodesulfobacteriales consists of only six named isolates in three genera: Geothermobacterium, Thermodesulfatator, and Thermodesulfobacterium. The best (bit still poorly) characterized member of this group is Thermodesulfobacterium hydrogenophilum (pictured above). T. hydrogenophilum is a thermophilic (50-80°C, optimum 75°C) anaerobic sulfate reducer isolated from the a deep sea hydrothermal vent in the Guaymus Basin. It is a small (0.4-0.5 x 0.5-0.8μm) motile rod with a single, polar flagellum. In stationary-phase culture, cell tend to form short chains of longer cells, and sometimes cyst-like cells appear. This organism grows by sulfate reduction to sulfide, using hydrogen (H2) as the only electron donor (other species in this genus use organics acids). Unlike other species of this genus, T. hydrogenophilum cannot use sulfite or thiosulfate as an electron acceptor. It is an obligate chemolithoautotroph.

Thermomicrobium

The phylum Thermomicrobia consists of only a single characterized specie, Thermomicrobium roseum, a potential second uncharacterized isolate, and some environmental sequences. This phylum may be a distant but specific relative of the Chloroflexi. T. roseum was isolated from Toadstool Spring in Yellowstone National Park, and grows optimally at 75°C. It forms irregular to pleomorphic short rods ca. 1.5 x 3-6μm in size, is non-motile, and pink colonies on plates. It is an obligately aerobic heterotroph, but it’s respiratory pathway is unknown and, as it is cyanide resistant, probably unique.

Chloroflexi and Deinococcus-Thermus

Although these phyla will be discussed in a future lecture, they are also largely thermophilic and perhaps deeply branching as well. However, these phyla do contain many mesophilic species, and, except perhaps for Thermus and its relatives, are not strikingly primitive. It is not clear whether or not these should be included amongst the primitive thermophiles and/or the ancestral thermophiles (see below).

Thermophilic ancestry of Bacteria

The fact that the deepest branches in the bacterial tree are thermophilic, and often extremely thermophilic, implies that the ancestors of Bacteria were also thermophilic, and that mesophily arose later in the tree, approximately at the main evolutionary radiation where most of the non-thermophilic phyla originate. The possibility of a thermophilic ancestry for Bacteria is strengthened by the fact that the deeply-branching and primitive Archaea are likewise thermophilic, and so probably both the Bacteria and Archaea share a thermophilic ancestry.

But not all bacterial thermophiles are primitive and related only to other thermophiles; many are close relatives of otherwise mesophilic species. This means that there are 2 types of fundamentally-different thermophiles:

1. Ancestral thermophiles (e.g. Thermotoga and Aquifex) have only thermophilic ancestors. These organisms have been thermophiles as far back as we can observe phylogenetically, & so seem never to have adapted to mesophily - they evolved from scratch in thermophilic environments and thermophily is built into them from the basics up.
2. Adapted thermophiles (e.g. thermophilic Bacillus and Clostridium species) are related to & evolved from mesophiles, (re)adapting to thermophily more recently.

This is a critical difference for those interested in examining how life is possible at high temperatures.

Life at high temperatures

How are these organisms capable of growth at such high temperatures? What is the upper limit of life? The answers to these questions aren't entirely clear, and high temperature may not really be an extreme condition except from our anthropocentric point of view. Nevertheless, here are some of the issues of life an very high temps:

Membrane fluidity and integrity
As temperature increases, the fluidity of the cells lipoprotein membranes increases. This fluidity must be balanced. In general, the membrane lipids of thermophiles have a higher melting point that those of mesophiles, so the the fluidity of the membranes of these organisms is appropriate at their optimum growth temperature. Organisms can also change their mix of membrane lipids in response to changes in temperature.

DNA structure
The two strands of typical DNA in solution separate at about 70°C. Although increasing the fraction of G=C pairs increases this melting point somewhat, there is no correlation between the growth temperature of an organism and the G+C content if its DNA. It turns out that in the cell, the DNA is inherently resistant to denaturation because of the high ionic strength and low water activity (i.e. most of the water is already tied up in hydration shells) of the cytoplasm. Most organisms negatively-supercoil their DNA, which makes it more easily denatured, but extremely thermophilic Archaea & Bacteria positively supercoil their DNA. DNA-binding proteins such as histones or histone-like proteins also stabilize DNA to thermal denaturation.

RNA structure
The folded structure of non-mRNAs (e.g. rRNA, tRNA, RNase P RNAs, snRNAs, snoRNAs, tmRNA, &c) can be denatured just like the strands of DNA. However, modest changes in the sequences and structures of RNAs can stabilize the structure. Most thermophilic RNAs are rich in G-C basepairs, and more importantly very low in G-U pairs, mismatches, bulges, and other irregularities that, in mesophiles, lead to flexibility in the RNA. The RNAs are also usually short, with no extra sequences; shorter sequences have fewer nonfunctional folding possibilities. In addition, base modifications and changes in protein binding can stabilize RNAs.

Protein structure
The denaturation of proteins from mesophiles at high temperature is dramatic - that's what happens in an egg when you boil it. However, stabilizing a protein for function at high temperatures seems to be relatively easy by organisms in evolution, although our understanding of these changes is poor at best.

Enzymatic function
The function of an enzyme is tuned to the organisms growth temperature. Mesophilic enzymes work best at 20-40°C and denature at higher temperatures, but enzymes from thermophiles work best at their growth temperature and denature when heated further. Thermophilic enzymes work slowly if at all at mesophilic temperatures; they are too rigid at these temps and are essentially 'frozen'. In other words, enzymes are tuned for optimal flexibility at the temperature they need to function at.

A more complex issue is balancing catalytic function in the cell. Different reactions will increase in speed at different rates as the temperature goes up, and these must be held in balance by homeostatic mechanisms.

Small molecule stability
This may be a more difficult problem for thermophiles. The half-life of GTP at 100°C is measured in seconds, and yet even organisms that grow at this temperature use GTP for translation, RNA synthesis, and many other processes. Many other small molecules are not very heat resistant - ATP, UTP, NAD, FAD, &c. These may be synthesized on a just-in-time basis so that their degradation isn't too great a loss for the cell; another way to put this is that the flux of reactions through these intermediates may be very high even though their steady-state concentration may be low.

The highest temperature of cultivated species is about 118°C, but there is good evidence for life growing at up to about 135-140°C in hydrothermal environments. This may represent the upper limit for life, because at this temperature amino acids become racemized (flip from L to D) at significant rates. This is independent of any of the normally stabilizing mechanisms, and the flipping of an amino-acid in a protein would potentially lead to its irreversible denaturation. However, one temperature after another has been suggested to be the limit for life, for seemingly very good reasons, and been proven wrong by biology. So who knows?

Questions for thought

• The Aquificae and Thermotogae are both primitive and deep-branching. Can you draw a tree with deep branches that are not primitive, and with primitive branches that are not deep? Do you know of any examples of either?
  
  
• Organisms such as Thermotoga maritima that reduce protons to generate hydrogen can only do so if very low ambient hydrogen concentrations make it energetically favorable. How do you suppose this hydrogen is gotten rid of so efficiently in their native environment?
  
  
• What do you imagine the absolute limiting issue for high temperature growth might be? What do you imagine the highest temperature any familiar type of organism could grow at?
  
  
• Can you think of any opportunities that extreme thermophiles might have that are not available to mesophiles? In other words, what might be some advantages of life in high temperature environments?
  
  
• Thermocrinus ruber is found in Yellowstone hot springs separated by many miles of inhospitable (for them) territory. How do you suppose they colonize new hot springs when the emerge? Would you predict the same organisms to exists in similar hot springs in other parts of the world?
  
  
• How many continuously or intermittently high-temperature microbial environments can you identify close to where you are right now. Do you think you could isolate thermophiles from these environments? How would yo go about it?

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