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Bacterial phyla with few or no cultivated species

The 13 phyla of Bacteria described in the previous lectures constitute the classical main phylogenetic groups, as defined by Carl Woese's 1987 review of bacterial diversity, with the more recent addition of Aquifex. As more sequences become available from cultivated species and from surveys of natural populations, it has become increasingly clear that the main radiation of bacterial phyla contains many more branches than originally thought; over a hundred contain at least two established sequences. Several of these groups contain only a few cultivated species, but in most cases contain none at all. These groups are known mainly or exclusively from ssu-rRNA sequences cloned from DNA extracted directly from environmental samples (see below).

This being said, don't forget that most of the cultivated, characterized Bacteria and most ssu-rRNA sequences from environmental samples fall into only 5 bacterial phyla: the Proteobacteria, the Gram-positive Bacteria (Firmicutes and Actinobacteria), the Bacteriodes and the Cyanobacteria. The other groups, the Aquificae, Thermotogae, Green sulfur and Green non-sulfur Bacteria, Planctomycetes, Chlamydiae, Deinococci, and even the Spirochaetes, qualify in some sense as “Phyla with few cultivated species”.

The observation that most cultivated species of Bacteria come from only a small number of phyla is similar to the situation in animals; the vast majority of animal diversity belongs to only 9 of the ca. 35 animal phyla: mollusks, sponges, cnidarians (coelenterates), flatworms, nematodes, annelids, arthropods, echinoderms, and chordates contain approximately 95% of animal species. The remaining 5%, most of animal diversity, are mostly obscure.

How do we know about these organisms?

The knowledge that these scattered species belong to novel bacterial phyla comes from their ssu-rRNA sequence analysis. Some of these are species isolated long ago that have only recently had their ssu-rRNA sequences determined; both the ATCC (American Type Culture Collection) and DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, i.e. German Collection of Microorganisms and Cell Cultures) are involved in efforts to sequence ssu-rRNAs from their collections. Others are newly discovered organisms; obtaining ssu-rRNA sequence information is now one of the first steps in the characterization of new isolates.

However, most of the these odd phyla are best, or even entirely, represented by ssu-rRNA sequences extracted directly from environmental samples; so-called molecular phylogenetic surveys. In a typical molecular phylogenetic analysis, the ssu-rRNA sequence is obtained by PCR amplification from DNA isolated from a pure culture of the organisms of interest. The resulting sequence is used to determined the place of that organism in the 'big tree'. It is also possible, however, to start a molecular phylogenetic analysis with DNA extracted directly from an environmental sample instead of a pure culture. The collection of sequences obtained from such PCR amplification products (hopefully) represent the population of organisms in the original sample. This gives us phylogenetic information about organisms in an environment regardless of whether they can be cultivated or not. This approach is far superior to the traditional cultivation-based approaches, and is described in detail (including its weaknesses) later in the class.

Summary of molecular phylogenetic surveys

In a 1998 review (J. Bacteriol. 180:4765), Philip Hugenholtz, Brett Goebel and Norman Pace summarized the results of 86 ssu-rRNA molecular phylogenetic surveys of microbial populations from a wide range of environments: geothermal sites, soils, fresh and saltwater environments, wastewater, &c. The final distillation of these surveys was summarized in the following Table:

(Note that in this table, the proteobacteria are so numerous that they are divided into their 5 sub-branches.)

They found that nearly 3000 bacterial sequences were reported, 90% of which fell into the proteobacteria, Gram-positive Bacteria (Firmicutes and Actinobaceria), or Cytophagales (Bacteroides). The remaining were widely scattered amongst the other groups and many were not related to any known cultivated specie. There were 9 groups of such sequences that did not fall into any of the standard bacterial groups. One of these groups (called OP11, from the first such sequence reported, originating from Obsidian Pool) seems to a a major constituent of subsurface environments and common in most other places, too.

Unfortunately, no more recent such compilation has been published, but this early work is probably representative of what would be found today in a compilation of the hundreds of thousands of environmental su-rRNA sequences now available from environmental surveys. However, the table below tabulates the numbers of ssu-rRNA sequences in release 10 of the Ribosomal database Project, divided into type strains, isolates with varying degrees of characterization, and sequences from uncultivated organisms. These numbers are largely consistent with those of the previous Table.

Phyla with few cultivated species

There are several bacterial phlya that have only a few cultivated, well-known species. Several have been described in previous chapters, such as the Thermotogae, Aquificae, Cloroflexi, Chlorobi, &c, &c. The dividing lines between the classical 13 bacterial phyla and the phyla describes below are, of course, arbitrary and historical. In reality, some of these groups are apparently far more abundant in the environment and now have many more species known (although not as well known) as the groups already discussed.

Example phylum : Verrucomicrobia

This phylum contains a couple of appendaged Bacteria, Verrucomicrobium and Prosthecobacter. These were previously though to be related to the appendaged α-proteobacteria. The remaining cultivated members of this phylum are poorly-characterized. The uncultivated species in this group are apparently abundant in the soil and subsurface, making up significant fractions of the total number of sequences isolated. Fluorescent probes targeting EA25 (which appeared frequently in an analysis of soil) indicate that the organism it comes from can make up 1-10% of soil microbes; 10^7 - 10^8 per gram of soil. This phylum is probably related to the Chlamydiae.

Example species:

	Verrucomicrobium spinosum is a mesophilic non-motile heterotrophic aerobe isolated from a small eutrophic lake (Lake Pluβsee). Unlike the appendaged α-proteobacteria, the appendages of V. spinosum have bundles of fimbriae at their tips (a bit like the stereotypical hairs from the nose warts of a witch). It is covered in many short (ca. 0.5μm long) appendages that appear conical under phase-contrast microscopy, often with a single large polar appendage.
	Prosthecobacter fusiformis is a heterotrophic mesophile, obligate aerobe isolated from freshwater contact slides. Generally resemble Caulobacter, but are more fusiform, and dividing cells have a stalk on both cells. They do not have a dimorphic life cycle, and are non-motile throughout their life cycle.
	Opitutus terrae is a heterotrophic, motile (monotrichous), obligately anaerobic coccus isolated from rice paddy soil. Cells are very small, only ca. 0.5μm in diameter; this organism and its relatives were previously informally known as Ultramicrobium. Pairs or longer chains of cells can be mistaken for rod-shaped cells.

Example phylum : Acidobacteria

This group contains only 6 cultivated species, none of them well-known, but a large number of ssu-rRNA sequences from environmental samples, including mostly soils, but other from a wide variety of habitats, including a jet-fuel contaminated aquifer, a hot spring,a sponge symbiont, and freshwater and marine environments. Fluorescently-labeled oligonucleotide probes specific for one subgroup of this phylum (subgroup 6) hybridize to cells of all shapes & sizes, suggesting a broad phenotypic range to match the broad phylogenetic diversity of this group.

Example specie: Acidobacterium capsulatum

A. capsulatum is an acidophilic aerobic heterotroph isolated from acid mine drainage. It is heavily encapsulated and saccharolytic, rod-shaped and non-motile. Acidobacterium is related to a number of rRNA sequences isolated from acidic environments (e.g. peat bog, acid mine drainage), consistent with its acidophilic phenotype.

Example phylum : Nitrospira

This phylum contains only 5 cultivated species in 4 genera, all very different phenotypically and not well characterized. Nevertheless, members of this phylum are apparently common in acidic and nitrogen-cycling environments and anaerobic marine sediments.

Example species:

	Nitrospira marina is, together with other species of this genus, the predominant nitrite oxidizer (to nitrate) in marine environments and aquariums. The species in this genus are poorly distinguished, and many of the original species have been lost. Nevertheless, mixtures of Nitrospira and Nitrobacter (another nitrite oxidizer) and Nitrosomonas (an ammonia oxidizer, produces nitrite) are sold commercially to help start a productive nitrogen cycle in aquariums.
	Leptospirillum ferrooxidans is also a chemolithoautotroph, oxidizing Fe++ to Fe+++ in mine tailings and contributing to acid mine drainage, perhaps the pollution “worst case scenario”. The natural habitat for this organism is presumably the deep aquifers infusing iron-rich ores.
	Magnetobacterium bavaricum is a magnetotactic organism, that swims North following the Earths magnetic field, directed by internal magnetite beads. These are anaerobic heterotrophs that live in aquatic sediments; in the Northern hemisphere, the magnetic field lines lead both North and down, toward their desired habitat. Other magnetotactic Bacteria (e.g. Magnetospirillum) are Proteobacteria, but few are cultivated.

Example phylum : Fusobacteria

Most of the few cultivated members of this group are animal symbionts, probably opportunistic rather than serious pathogens. By far the best studied genus of this Phylum is Fusobacterium. Some are also soil organisms, and environmental sequences have come primarily from oral samples (where Fusobacterium is common), fecal samples, soil and sediments.

Example specie : Fusobacterium nucleatum

F. nucleatum is part of the normal flora of the oral cavity, and is particularly abundant in dental plaque, where it plays a central role in nucleating the accumulation of various types of Bacteria, including organisms such as Porphyromonas that can lead to periodontal disease. It is an anaerobic heterotroph, fermenting primarily sugars to butyric acid. F. nucleatum is spindle-shaped to filamentous with tapered ends, and so is easily mistaken for oral Bacteroids.

Other examples of Phyla with few cultivated species

• Dehalococcoides - Dehalococcoides ethenogenes and relatives
• Chrysiogenetes - Chrysiogenes arsenatis and relatives
• Deferribacteres - Deferribacter and relatives
• Fibrobacteres - Fibrobacter succiogenes, F. intestinales, and relatives
• Dictyoglomi- Dictyoglomus thermophilum and relatives
• Gemmatimonadetes - Gemmatimonas aurantiaca and relatives
• Lentisphaerae - Lentisphaera araneosa, Victivallis vadensis, and relatives

Phyla with no cultivated species

The extreme case of a phylum with few known cultivated species is, of course, a phylum with no known cultivated members. There are many of these; some are large groups that are commonly seen in microbial surveys (e.g. OP10, TM7), but most are small groups, and many are only one or a few sequences that are not specifically related to any bacterial phylum. Some of these will be found to be deep branches in known phyla once additional related sequences are obtained. Most, however, probably represent the hidden bulk of bacterial diversity.

Example phylum : OP11

This is a large group containing bout 100 unique sequences (only 43 are nearly full-length) from a very wide range of environments - but there are no known cultivated species in this group, so nothing is know anything about their phenotype. This phylum is particularly interesting because of its high evolutionary rate, reflected in its long branch length; this is an unusually 'advanced' group of Bacteria. Despite years of attempts to cultivate something, anything, from this group, so far none are in culture.

Like most of these phyla, OP11 gets its name from a sequence designation of the original sequence identified in this group. OP11 was sequence number 11 from a collection of cloned bacterial ssu-rRNA sequences from Obsidian Pool (OP).

Example phylum : SR1

A large number of environmental ssu-rRNA sequences do not fall into any of the recognized bacterial Phyla; conceptually, each group of these is a cryptic phylum. Even if you demand that at least 2 related, non-identical, nearly full-length sequences be identified before describing them as a new phylum, there are several hundred such cryptic Phyla. The phylum “SR1” is an example, composed of only 2 nearly full-length and 23 shorter sequences. (SR is from Sulfur River)

Phylogenetic groups at all levels are dominated by uncultivated sequences

Although it is easy to think of sequences from uncultivated organisms only in terms of the unusual phyla described in this chapter, in reality sequences from uncultivated organisms fall into phylogenetic groups at all levels. Even in well-studied phyla, there are Orders, Classes, Families, and Genera with large numbers of species represented only or largely by sequences from uncultivated organisms. The tree below was generated from an arbitrarily-selected collection of sequences from cultivated and uncultivated organisms within the Family Enterobacteriaceae. These organisms are familiar to ay microbiologist, but there are any number of uncultivated species hidden even within this family.

How much of the microbial world do we know about?

This is a difficult question to answer; in fact, it cannot be realistically answered at this time within even a factor of a thousand. Some believe that bacteria may consist of a few thousands, or maybe tens of thousands, of species. This is ridiculous. There are over 350,000 described species of beetles, and even if there is only a single specific bacterial symbiont for each of these beetle species, that would imply at least 10 times more species of bacteria than these folks would believe just among these species. If you plate a typical environmental sample of onto rich media after counting cells microscopically, you typically see that less than 1 observable bacterium in a thousand grows to produce a colony (averaging about 1 in a million), and of course these are only from the most abundant species. As poorly characterized microbiologically as the world around us is, we know nothing at all about some very large microbial habitats: the subsurface world, the deep aquifer world, the hydrothermal field world, &c, &c.

Another problem is that we really don't have a very good idea of what a bacterial "species" is. This is a general problem with asexual organisms; species in plants and animals are defined in terms of breeding populations, and so this only applied to organisms that ‘breed’. The concept of a species is critical to biology; this is part of why "The Origin of Species" was so important, and most of this book was spent creating a rational description of a ‘species’ in the plant and animal worlds. (Actually, most of this is spent showing how indistinct the divisions really are between species.) A rational "concept of a bacterial species" does not yet exist. This is probably the most important open question in microbiology. 70% DNA:DNA hybridization is sometimes used as an operational definition of a species, but this is an arbitrary definition, without a theoretical underpinning. Until we have a meaningful definition of a species, how can we count them? In fact, it has ben argued that asexual organisms don’t have ‘species’, in which case some other term (and definition) might be needed.

Questions for thought

• How many species of Bacteria would you predict there are? Why not more?
  
  
• What do you think defines a bacterial Phylum? The question is, is a phylum just any coherent deep branch? How deep does it have to be to be a distinct phylum? If it’s more than just branch depth, then what is it that defines a phylum?
  
  
• Opitutus terrae has a volume of only about 0.1 cubic micron, less that 5% that of E. coli. What do you think would limit how small a free-living organism could be?
  
  
• “Kirk” is a graduate student in a famous lab at a big university, and the project he and his faculty advisor & committee agreed on is to obtain a pure culture of a specie in the OP11 group, for further microbiological characterization. How might Kirk go about trying to do this?

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