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Gram-positive Bacteria

Traditional taxonomy divides all Bacteria into two groups: Gram-positive and Gram-negative. In phylogenetic terms, this division is false. As can be seen in the tree above, the Gram-positive Bacteria do, indeed, form a coherent phylogenetic group. This single group is now commonly divided into two related phyla, the Firmicutes (a.k.a. the low G+C Gram-positive Bacteria) and the Actinobacteria (a.k.a. the high G+C Gram-positive Bacteria). However, the remaining bacterial phyla have a generally Gram-negative cell envelop. And so, like the terms “prokaryote” and “invertebrate”, the term Gram-negative tells you what an organism isn’t but not what an organism is.

Nevertheless, the use of the Gram stain as a starting point in the identification of Bacteria has been an incredibly important tool. Perhaps this is because, although the Gram-positive Bacteria represent only a fraction of the immense phylogenetic diversity of Bacteria in nature, this group contains more than its share of the most important human and animal pathogens.

The original names for the Firmicutes (low G+C Gram-positive Bacteria) and Actinobacteria (high G+C Gram-positive Bacteria) came from the fact that many of the most familiar Firmicutes have relatively low genomic G+C contents, whereas many of the familiar Actinobacteria have relatively high genomic G+C contents. This rule is by no means universal, nor particularly meaningful.

Given the incredible diversity of organisms in the phyla Firmicutes and Actinobacteria, we will only be able to touch on a few examples, focusing on less familiar representatives rather than the well-known pathogens.

What does being Gram-positive mean?

The structural distinction between Gram-positive and Gram-negative Bacteria lies in the structure of the cell envelop. Gram-positive Bacteria have no phospholipid outer membrane; they are bound by the cytoplasmic membrane and usually a very thick cell wall (& so stain Gram-positive). There is therefore no periplasmic space, that Gram-negative Bacteria use as an environmental buffer. But they do have some control of the conditions in the spongy thick wall, and so this may serve a purpose similar to that of the periplasmic space. Other Bacteria (Gram-negative Bacteria) have both an outer & inner membrane, sandwiching a thin cell wall, and therefore stain Gram-negative. Like many other Bacteria and Archaea, some Gram-positive Bacteria also have an outer protein coat called the S-layer.

However, a wide range of factors, in addition to the outer membrane and cell wall thickness, determine whether or not a culture stains Gram-positive or negative. Even in the same species, cells may stain differently in different stages of the growth cycle or when grown under different conditions.

In addition, some members of the Gram-positive phylogenetic group do have an outer membrane. Some Actinobacteria have an outer membrane composed of mycolic acids rather than phospholipids. These organisms are sometimes considered neither Gram-positive nor Gram-negative, but “acid fast”. Some Firmicutes have a traditional Gram-negative envelop, complete with phospholipid outer membrane and lipopolysaccharide. It is very important, therefore, when using the terms “Gram-positive” or “Gram-negative”, to be clear about whether this refers to how the cells stain, the structure of their envelop, or a phylogenetic group.

An alternative view of Gram-positive Bacteria

Some molecular phylogenists argue, primarily on the basis of conserved insertions and deletions (indels) in protein-coding genes, that the Gram-positive Bacteria are more closely related to Archaea and eukaryotes than are any other group of Bacteria. This view is not supported by analyses of conserved protein or RNA gene sequences, but it does separate organisms into those with 2-membrane envelops (Gram-negative Bacteria), which in this scheme are termed “Diderms”, and those with single-membrane envelops (Gram-positive Bacteria, Archaea and eukaryotes), termed “Monoderms”. This issue remains a matter of contention.

Phylum Firmicutes (Low G+C Gram-positive Bacteria)

Taxonomy

• phylum Firmicutes
  • class Bacilli
    • order Bacillales
      • family Bacillaceae (e.g. Bacillus, Geobacillus, Halobacillus)
      • family Alicyclobacillaceae (Alicyclobacillus)
      • family Listeriaceae (e.g. Listeria, Aneurinibacillus)
      • family Paenibacillaceae (e.g. Paenibacillus, Brevibacillus)
      • family Pasteuriaceae (Pasteuria)
      • family Planococcaceae (e.g. Planococcus, Kurthia)
      • family Sporolactobacillaceae (e.g. Sporolactobacillus)
      • family Staphylococcaceae (e.g.Staphylococcus,Salinicoccus)
      • family Thermoactinomycetaceae (e.g. Thermoactinomyces)
    • order Lactobacillales
      • family Lactobacillaceae (e.g. Lactobacillus, Pediococcus)
      • family Aerococcaceae (e.g. Aerococcus, Facklamia)
      • family Carnobacteriaceae (e.g.Carnobacterium, Aptobacter)
      • family Enterococcaceae (e.g. Enterococcus, Vagococcus)
      • family Leuconostocaceae (e.g. Leuconostoc, Weissella)
      • family Streptococcaceae (e.g. Streptococcus, Lactococcus)
  • class Clostridia
    • order Clostridiales
      • family Clostridiaceae (e.g. Clostridium, Sarcina)
      • family Eubacteriaceae (e.g. Eubacterium, Acetobacterium)
      • family Gracilibacteraceae (Gracilobacter)
      • family Heliobacteriaceae (e.g.Heliobacterium,Heliophilum)
      • family Lachnospiraceae (e.g. Butyrovibrio, Coprococus)
      • family Peptococcaceae (e.g. Peptococcus,Desulfotomculum)
      • family Peptostreptococcaceae (e.g. Peptostreptococcus)
      • family Ruminococcaceae (e.g. Ruminococcus, Acetivibrio)
      • family Syntrophomonadaceae (e.g. Syntrophomonas)
      • family Veillonellaceae (e.g. Sporomusa, Megasphaera)
    • order Halanaerobiales
      • family Halanaerobiaceae (E.g. Haloanaerobium, Halocella)
      • family Halobacteroidaceae (e.g. Halanerobacter, Orenia)
    • order Thermoanaerobacterales
      • family Thermoanaerobacteraceae (e.g.Thermoanaerobacter)
      • family Thermodesulfobiaceae (e.g. Coprothermobacter)
  • class Erysipelotrichi
    • order Erysipelotrichales
      • family Erysipelotrichaceae (e.g. Erysipelothrix)
  • class Mollicutes
    • order Mycoplasmatales
      • family Mycoplasmataceae (e.g. Mycoplasma, Ureaplasma)
    • order Entomoplasmatales
    • family Entomoplasmataceae (Entomoplasma, Mesoplasma)
    • family Spiroplasmataceae (Spiroplasma)
    • order Acholeplasmatales
      • family Acholeplasmataceae (Acholeplasma, Phytoplasma)
    • order Anaeroplasmatales
      • family Anaeroplasmataceae (Anaeroplasma,Asteroleplasma)

About this phylum

Diversity
The Firmicutes are a large and diverse group of organisms, encompassing 4 Classes, 11 Orders, 35 Families, and more than 240 species. Included in the Firmicutes are the Mollicutes (a.k.a. Tenericutes), the Mycoplasma and relatives. Although not usually considered Gram-positive - they entirely lack the peptidoglycan cell wall - they are members of this phylogenetic group and like other Gram-positive Bacteria lack the outer membrane. Also amongst the Firmicutes are the members of the Family Veillonellaceae (Veillonella, Dialister, Megasphaera, and Sporomusa), that have traditional Gram-negative cell envelops (complete with outer membrane) despite being members of a Gram-positive phylogenetic group. Anaerobic, endospore-forming rods (“Clostridia”) form several deep lineages in the Firmicutes, and so this probably represents the primitive phenotype of this group.

Metabolism
These organisms are heterotrophic, except for the photosynthetic heliobacteria, but have a wide range of heterotrophic lifestyles. The Bacilli are generally obligate aerobes, other Firmicutes are usually anaerobic, although often aerotolerant. Anaerobic metabolism is usually by substrate-level phosphorylation rather than anaerobic respiration; these organisms often lack a complete electron transport chain. Most are mesophilic, although a few psychrophilic or moderately thermophilic species exist. A wide range of carbon/energy sources are used by members of this group, and result in a similarly wide range of fermentation products.

Morphology
Familiar members of this group are either rod-shaped or cocci; these sometimes form nearly filamentous chains, as in some species of Bacillus and Streptococcus. Individual cells or pairs are also very common. Endospores are a common unique feature of this group. Morphology varies more widely in the Mollicutes, but their small size and lack of peptidoglycen means that these morphologies are less easily observed.

Habitat
Both Firmicutes and Actinobacteria are abundant in most soil and sediment communities. Firmicutes are also the predominant symbionts of the skin, mucous membranes, and gut of animals; perhaps this helps explain the large number of human and animal pathogens that also are members of this group.

Aerobic endospore-forming rods (Bacillus and relatives)

These are generally aerobic (some grow anaerobically by nitrate reduction) rod-shaped endospore-forming heterotrophs that obtain energy and carbon by respiration. These are common soil inhabitants. A few are opportunistically pathogenic to humans, and a very few are bona fide pathogens, Bacillus anthracis being the most important. Listeria and Staphylococcus, although not endospore-forming rods, are members of this phylogenetic group. Most of the endospore-forming aerobic rods were originally considered to be species of the genus Bacillus, but the size of this genus and phylogenetic considerations lead to its division into a number of new genera, most of which retain the -bacillus suffix (e.g. Paenibacillus, Brevibacillus, Geobacillus, &c)

Example : Bacillus cereus

B. cereus is a very close relative of the human pathogen B. anthracis, the insect pathogen B. thuringensis (the source of the widely used Bt insecticide), B. mycoides, B. pseudomycoides and B. weihenstephanensis; these may more appropriately be considered to be different strains of the same species. B. cereus is easily isolated from soil and soil has traditionally been considered to be the natural habitat of this organism. However, B. cereus is abundant in the guts of a wide range of arthropods, in which it is usually filamentous rather than the individual cells or short chains seen in cultivation. Cells shed in the arthropod feces sporulate, awaiting ingestion by a new arthropod host. This life cycle is very similar to that of B. anthracis and B. thuringensis, except that the host is not (apparently) harmed. B. cereus is often considered to be a food-born opportunistic pathogen, although it is unclear whether these are typically infections or reactions to Bt-like toxin.

Anaerobic endospore-forming rods (Clostridium and relatives)

These organisms, and their non-spore-forming relatives (Eubacterium) are abundant in anaerobic soils and sediments. They constitute the bulk of the Bacteria in the gut contents (and feces) of humans, and are particularly important in animal decay. Many species are opportunistically pathogenic, and some are well known pathogens, for example C. tetani (the causative agent of tetanus) and C. perfringens (the causative agent of gas-gangrene). Clostridia lack the electron transport chain, and obtain energy (ATP) from a wide variety of substrate-level phosphorylation reactions. The proton gradient, which is required to drive many active transport pumps, seems to be maintained by a traditional ATPase run in reverse; protons are pumped from inside to outside at the expense of the hydrolysis of ATP.

Example : Clostridium botulinum

C. botulinum produces a potent neurotoxin (sometimes said to be the most deadly toxin known); it is this neurotoxin, rather than the organism itself, that produces the disease Botulism. The most common form of botulism is infantile botulism, in which C. botulinum colonizes the gut amongst the wide range of other normal Clostridium species; the toxin is then absorbed through the gut mucosa. Sudden Infant Death Syndrome (SIDS) is sometimes (and probably incorrectly) attributed to C. botulinum acquired from honey. In wound botulism, C. botulinum grows in a gangrenous wound; the toxin is absorbed directly into the surrounding tissue and circulation. This is in many ways analogous to tetanus, caused by a related specie Clostridium tetani. The most well-known form of botulism, however, is the food-borne type. The organism grows in anaerobic canned food or sausage; the toxin is absorbed through the gut mucosa upon ingestion. The botulism toxin inhibits neurotransmission, resulting in flaccid paralysis. The botulism toxin (Botox) has important medical uses in a wide variety of conditions in which muscles contract inappropriately.

Lactic Acid Bacteria

The lactic acid Bacteria (commonly know by the acronym LAB) are acid-tolerant, non-sporulating relatives of the Bacilli and Clostridia. Like the Clostridia, the LAB lack the electron transport chain, and so generate ATP by substrate-level phosphorylation. Unlike Clostridia, most are aerotolerant. In nature, these organisms are usually associated with the decomposition of plant material, but are also important human and animal symbionts. Most grow on simple carbohydrates (sugars), either producing lactic acid alone via glycolysis (homofermentation) or lactic acid, ethanol, and CO2 via the pentose-phosphate pathway (heterofermentation). The LAB are widely used in the food industry for the production of fermented vegetable (pickles, sauerkraut), dairy (yogurt, cheese), and meat (fermented sausages). Although generally nonpathogenic, and very often beneficial to humans, some members of the genus Streptococcus are harmful (S. mutans causes tooth decay) or pathogenic (S. pyogenes causes Strep throat and scarlet fever).

Example : Leuconostoc mesenteroides

Species of the genus Leuconostoc are strictly heterofermentative LAB with distinctly oval cells that grow in chains. L. mesenteroides produces dextran slime when grown on sucrose; colonies will become so slimy that they will drip onto the lid of culture plates. L. mesenteroides predominates most lactic fermentations in the initial stages; they are succeeded in later stages of these fermentations by the more acidophilic Lactobacillus species.

Mollicutes (Tenericutes; Mycoplasma and relatives)

The Mollicutes are not often considered to be Gram-positive Bacteria; lacking peptidoglycan, they stain Gram-negative. Historically, they were often considered with the viruses, because of their small size and obligately parasitic lifestyle. But they are phylogenetic members of the Firmicutes, being an offshoot of the Clostridium innocuum branch, and like other Gram-positive Bacteria lack an outer membrane. The mollicutes are obligate extracellular symbionts or parasites of plants and animals. Most are unusual in that they have sterols in their membrane in addition to the usual fatty-acid esters; they don't make sterols themselves, it is acquired from its eukaryotic host. They are very small, typically about 0.25um in diameter, amongst the smallest known cellular organisms with the smallest genomes. Genomes lack the genes for the Krebs cycle, animo acid biosynthesis, purine and pyrimidine biosynthesis, and many other metabolic pathways. Most do contain the genes for the glycolytic pathway, which they use for energy production by substrate-level phosphorylation. They are often motile by gliding. Members of this group have a wide range of complex morphologies, but are usually described as amorphous or pleomorphous because of their size (at or below the resolving power of light microscopy) and the fact that their relatively non-rigid envelop cannot withstand traditional fixing treatments.

Example : Mycoplasma hominis

Species of the genus Mycoplasma are obligate parasites of animals, usually infecting the lung (e.g. M. pneumoniae) or urogenital tract (e.g. M. genitalium). M. hominis is an opportunistic pathogen; it seems to reside asymptomatically in the vagina of healthy women, but is also one of many causes of bacterial vaginitis and pelvic inflammatory disease. Disease is usually associated either with invasive surgical procedures or coinfection with the obligately parasitic protist Trichomonas vaginalis. In the former case, the parasite proliferates in deep surgical wounds and can be difficult to identify or treat; lacking peptoglycan cell walls, they are resistant to many commonly-used post-surgical broad-spectrum antibiotics. In the latter case, there is a symbiosis between T. vaginalis and M. hominis, and most infected women are co-infected. Although typically an extracellular parasite, M. hominis can reside and replicate in the cytoplasm of T. vaginalis; this may facilitate both the transfer of M. hominis to new human hosts and the resistance of M. hominis to antibiotics. In both females and males, M. hominis is associated with reduced fertility. M. hominis cells can attach to and invade sperm cells, suggesting that the ability to persist intracellularly may be a general mechanism for infecting new hosts. M. hominis is generally spherical, lacking the elongated “flask” shape of most other species. Also unlike other Mycoplasma species, M. hominis is not saccharolytic, but instead uses only arginine for both carbon and energy.

Heliobacteria (Green photosynthetic Gram-positives)

The heliobacteria are the only phototrophic Gram-positive Bacteria. They carry out cyclic photophosphorylation, growing photoheterotrophically using pyruvate or similar organic acids as their sole carbon sources. Heliobacteria use a chlorophyl g, that absorbs wavelengths in the range of 790nm not utilized by other photopigments and so can avoid competition for light with other phototrophs. Unlike other phototrophs, heliobacteria house their photosynthetic complexes in the cytoplasmic membrane; no internal membranous structures or membranous invaginations are present. However, the photo reaction cycle of the heliobacteria is very similar to that of the Chlorobi. The heliobacteria are rod-shaped (sometimes slightly helical) anaerobic endospore-formers, motile by gliding or flagella. They lack the typical thick Gram-positive cell wall; the thin peptidoglycan layer is covered in a regular array of 11nm protein beads. The heliobacteria are commonly found in soil, and are very efficient nitrogen fixers.

Example : Heliobacterium chlorum

Heliobacterium chlorum is a long rod-shaped organism (1 x 6-10μm), forming green-brown to emerald green colonies due to the presence of both chlorophyl g and the green accessory carotinoid neurosporene. H, chlorum is motile by gliding rather than the flagella present in other heliobacteria. The only carbon sources known to support its photoheterotrophic lifestyle are pyruvate and lactate. Sporulation is rarely observed; older cultures usually degenerate into spheroplasts and lyse.

Veillonelli (Firmicutes with Gram-negative envelops)

This little-known group of organisms is unique amongst the Gram-positive phylogenetic group of Bacteria; they have Gram-negative envelops, including an outer membrane and (generally) lipopolysaccharide layer. It is unclear whether this represents a re-acquisition of the Gram-negative envelop by an Gram-positive ancestor of this group, or if this branch separated from the Gram-positive phylogenetic group prior to the loss in the remainder of the group of the outer membrane. Members of this group are anaerobic heterotrophs, and are common symbionts of the GI tract, except for Pectinatus and Megasphaera, which are found in spoiled unpasteurized beer. Pectinatus and Selenomonas are rod-shaped organisms motile by flagella that occur only on one side of the cell; they swim sideways in a distinct tumbling “X” shape. Sporomusa and Sporohalobacter produce endospores similar to those of other Firmicutes. Syntrophomonas is an gut-dwelling obligate syntroph, growing only in association methanogenic Archaea.

Example : Veillonella atypica

Veillonella are the most numerous cultivable anaerobes in human saliva. These small (ca. 0.5m) cocci grow by decarboxylation of lactate and other organic acids produced by primary oral colonizers such as Streptococcus salivarius, fusobacteria, and actinomycetes, to which they adhere. Species of Veillonella are specialized to adhere to the primary colonizers of specific portions of the oral cavity; V. atypica adheres specifically to Streptococcus salivarius and Fusobacterium nucleatum, inhabitants of the saliva and upper surface of the tongue.

Phylum Actinobacteria (high G+C Gram-positive Bacteria)

• phylum Actinobacteria
  • class Actinobacteria
    • order Acidimicrobiales
      • family Acidimicrobiaceae (Acidimicrobium)
    • order Rubrobacterales
    • family Rubrobacteraceae (e.g.Rubrobacter,Thermoleophilum)
    • family Patulibacteraceae (Patulibacter)
    • order Coriobacteriales
      • family Coriobacteriaceae (e.g. Atopobium, Slakia)
    • order Sphaerobacterales
      • family Sphaerobacteraceae (Sphaerobacter)
    • order Actinomycetales
      • family Actinomycetaceae (e.g. Actinomyces, Arcanobacterium)
      • family Micrococcaceae (e.g. Micrococcus, Arthrobacter)
      • family Bogoriellaceae (Bogoriella)
      • family Rarobacteraceae (Rarobacter)
      • family Sanguibacteraceae (Sanguibacter)
      • family Brevibacteriaceae (Brevibacterium)
      • family Cellulomonadaceae (e.g. Cellulomonas, Oerskovia)
      • family Dermabacteraceae (Dermabacter, Brachybacterium)
      • family Dermatophilaceae (Dermatophilus, Kineosphaera)
      • family Dermacoccaceae(Dermococcus,Demetria,Kytococus)
      • family Intrasporangiaceae (e.g. Janibacter, Tetrasphaera)
      • family Jonesiaceae (Jonesia)
      • family Microbacteriaceae (e.g. Microbacterium, Agromyces)
      • family Beutenbergiaceae (Beutenbergia, Georgenia, Salana)
      • family Promicromonosporaceae (e.g. Promicromonospora)
      • family Catenulisporaceae (Actinospica, Catenulispora)
      • family Corynebacteriaceae (e.g. Corynebacterium)
      • family Dietziaceae (Dietzia)
      • family Gordoniaceae (Gordonia, Skermania, Millisia)
      • family Mycobacteriaceae (Mycobacterium)
      • family Nocardiaceae (e.g. Nocardia, Rhodococcus)
      • family Tsukamurellaceae (Tsukamurella)
      • family Williamsiaceae (Williamsia)
      • family Segniliparaceae (Segniliparus)
      • family Micromonosporaceae (e.g. Micromonospora)
      • family Propionibacteriaceae (e.g. Propionibacterium)
      • family Nocardioidaceae (e.g. Nocardoides, Aeromicrobium)
      • family Pseudonocardiaceae (e.g. Pseudonocardia)
      • family Actinosynnemataceae (e.g. Lentzea, Saccharothrix)
      • family Streptomycetaceae (e.g.Streptomyces,Kitasatospora)
      • family Streptosporangiaceae (e.g. Streptosporangium)
      • family Nocardiopsaceae (e.g. Nocardopsis, Thermobifida)
      • family Thermomonosporaceae (e.g. Thermomonospora)
      • family Frankiaceae (Frankia)
      • family Geodermatophilaceae (e.g. Blastococcus)
      • family Microsphaeraceae (Microsphaera)
      • family Sporichthyaceae (Sporichthya)
      • family Acidothermaceae (Acidothermus)
      • family Kineosporiaceae (e.g.Kineosporia,Cryptosporangium)
      • family Nakamurellaceae (Quadrasphaera, Nakamurella)
      • family Glycomycetaceae (Glycomyces, Stackebrandtia)
    • order Bifidobacteriales
      • family Bifidobacteriaceae (e.g. Bifidobacterium)

About this phylum

Diversity
Familiar actinobacteria, such as Mycobacterium, Corynebacterium, Micrococcus and Streptomyces, are members of a single Order, the Actinobacteriales, which span a relatively small phylogenetic range, but a large number of Families, genera and species. The outlying branch containing of Sphaerobacter, Thermoleophilum, Acidimicrobium, and relatives is generally considered part of the actinobacteria (as shown here), but there are contrary data suggesting instead that these organisms might instead be members of the Thermomicrobium branch of the Chloroflexi.

Metabolism
The actinobacteria are generally aerobic respirers. A few exceptions, such as Propionobacterium and Bifidobacterium, are anaerobic or aerotolerant. A few are moderately thermophilic (up to ca. 60°C), but most are mesophilic. Chemoorganotrophic, but growth substrates vary widely. Members of this group (most notably Streptomyces and Actinomyces) are well known for their ability to produce antibiotics; these and Bacillus are probably the most common bacterial sources of antibiotics.

Morphology
These organisms are typically nonmotile rods, filaments, or sometimes cocci. Rod-shaped cells are usually uneven, irregular, or club-shaped (coryneform). Endospores are not produced, but filamentous species often form spores (sometimes called “arthrospores” to distinguish them from endospores) that are not particularly resistant but rather are reproductive and important in dispersal.

Many Actinobacteria, including the mycobacteria, corynebacteria, nocardia, and rhodococci, have a mycolic acid outer membrane. This is not the typical outer membrane seen in Gram-negative Bacteria; typical membrane lipids and lipopolysaccharides are not present. Instead, the typically thick Gram-positive-type cell wall is covered in an arabinogalactan polysaccharide layer, which in turn is covered by a mycolic acid bilayer. This is a true lipid outer membrane, not a “waxy coating”, as it is often described. As in Gram-negative Bacteria, this outer membrane incorporates a variety of proteins, including porins. The mycolic acid outer membrane is a potent permeability barrier, even more so that the outer phospholipid membrane of Gram-negative Bacteria. It is not known whether this mycolic acid outer membrane represents a highly-altered descendent of an ancestral Gram-negative outer membrane or an independently-derived addition to an ancestral Gram-positive envelop.

Habitat
Most actinobacteria are soil organisms; one genus, Streptomyces, forms a white growth commonly seen in decaying wood (easily mistaken for fungus) and gives good soil its rich earthy odor. A few are symbionts or pathogens of plants and animals, including some notorious examples; Mycobacterium tuberculosis and M. leprae, Corynebacterium diphtheria. More common than pathogens are the commensals, such as Bifidobacterium, which is common in the gut and beneficial, and is often used in probiotics.

Coryneform actinobacteria

These organisms are typified by their club-like or irregular rod-shaped cells. Pairs of cells after division are angled or V-shaped; this is referred to as either “snapping” or “Chinese letter” division. This is caused by asymmetric fracturing of an outer layer of the cell wall after cytokinesis. These organisms are common symbionts of the skin and mucous membranes of animals, and the surfaces of plants, as well as being abundant in the soil. The most well-known genera in this group are Corynebacterium and Arthrobacter. Arthrobacteria are very common soil and root surface inhabitants that are typically pleomorphic; small cocci in stationary phase, and irregular rods with jointed or V-shaped pairs during rapid growth. Corynebacteria are common symbionts of animals; a few species are pathogens, including of course C. diphtheriae, the causative agent of Whooping cough. Members of this genus are typically club-shaped, and also often pleomorphic.

Example : Arthrobacter globiformis

A. globiformis is one of the most numerically abundant easily cultivated inhabitants of neutral pH or alkaline soils, and is also abundant on the aerial surfaces of plants. This genus is easily recognized morphologically, but distinguishing species requires analysis of the cell wall sugars and amino acids, or rRNA sequence analysis. Stationary phase cells are small cocci; upon transfer to typical rich media, these cocci swell, then produce outgrowths to generate irregular rods-shaped cells, which divide by “snapping” division, producing V-shaped pairs of cells or sometime longer pseudo-hyphae. As cells enter stationary phase, divisions continue as growth slows, resulting in the formation of cocci. Colonies on plates will contain both coyneform/rod-shaped cells and cocci, and when examined microscopically so are often mistakenly thought to be impure or contaminated. These organisms can use a remarkably wide range of organic substrates for growth and energy, including nicotine, the antibiotic puromycin, and a range of herbicides. Most are also nitrogen fixers.

Filamentous actinobacteria

Streptomyces and related genera form branched filamentous hyphae, and although usually much thinner, otherwise resemble the filamentous fungi. This is no coincidence, but it represents an evolutionary convergence because of their common habitat and lifestyle rather than any specific evolutionary relationship.

The filamentous actinobacteria have a complex life cycle that includes programmed cell death and cellular differentiation; these are truly multicellular Bacteria. Initial growth from spores on solid media is in the form of branching vegetative hyphae. These hyphae are mostly non-septated; DNA replication produces new nucleoids, but no cytokinesis occurs, and so the filaments share a common syncytial cytoplasm. Filament growth occurs only at the tips; branching is required to allow logarithmic growth because, of course, individual hyphal tips have a limit to their growth rate. Vegetative hyphae give rise to waxy aerial hyphae that grow upward away from the growth substrate. This growth is at the expense of the underlying vegetative hyphae, which undergo programmed cells death (although their cell walls remain largely intact and serve as a supporting structure for the aerial hyphae). The growth tips of the aerial hyphae then undergo cytokinesis to create a series of individual cells, which develop into dormant spores. Again, this growth and development of spores is at the expense of the aerial hyphae.

It is important to remember that the “arthrospores” of these actinobacteria are distinct from the endospores of the firmicutes. Both are metabolically inactive resting stages of the life cycle, but arthrospores are produced in great numbers from each aerial hyphum (they are reproductive), are readily dispersed by the air or water, but are not particular resistant to harsh treatment. Endospores, on the other hand, are extremely resistant to heat and chemical assault, but are not reproductive (a mother cell produces a single spore) and are not readily dispersable.

Species of filamentous actinobacteria are distinguished morphologically, mostly on the basis of the structure and morphology of their spore-bearing hyphae. These organisms are metabolically diverse; most can use a very wide range of growth substrates. They produce a wide range of antibiotics, including aminoglycosides (e.g. streptomycin), macrolides (e.g. erythromycin), tetracycine and chloramphenicol, just to name a few. Interestingly, the filamentous actinobacteria have linear rather than circular chromosomes, with unique telomeres.

Streptomyces antibioticus

S. antibioticus is a typical member of the genus Streptomyces, and has long been used in the industrial production of the important antibiotic actinomycin. More recently, S. antibioticus is being used as a model system for more detailed examination of the life cycle of filamentous actinobacteria. Although still poorly understood, this life cycle is turning out to be far more complex than previously imagined, particularly in terms of the horizontal (as opposed to vertical) spatial organization, and waves of growth in specific spatial arrangements. For example, the initial germination hyphae is cellular rather than syncytial, and upon reaching a specified density, alternating cells in these filaments die. Vegetative, syncytial hyphae are the outgrowths of the surviving cells from these initial filaments.

Acid-fast Bacteria

The mycolic acids of the outer membrane of the genus Mycobacterium and relatives are much longer than those of other actinobacteria than contain mycolic acids. As a result, these species can be specifically stained using the “acid fast” stain first developed by Robert Koch during his work to identify the cause of tuberculosis. In culture, the mycobacteria typically grow as branched or unbranched filaments, but these filaments are chains of individual cells rather than syncytial hyphae. Most of the mycobacteria are soil inhabitants, and some are important in the bioremediation of pollutants that are otherwise recalcitrant. A few are human and animal pathogens, most notably Mycobacterium tuberculosis and M. leprae.

Mycobacterium ulcerans

M. ulcerans is the causative agent of Buruli (a.k.a. Barnsdale) ulcer, a necrotic disease of the skin and surrounding soft tissue and bone. Lesions are most common on the arms and legs, primarily in children, and although not generally fatal typically results in permanent disfiguration. Although not as well known (or understood) as leprosy or tuberculosis, which are caused by related species of Mycobacterium, Buruli ulcer has surpassed these diseases in frequency in some parts of impoverished central and western Africa. Unlike M. leprae or M. tuberculosis, M. ulcerans is an environmental pathogen, probably from transmitted to humans from aquatic insects.

Deeply-branching questionable members

There are some organisms that, although currently classified as actinobacteria, are on such deep branches that their placement amongst the actinobacteria are uncertain. One of these, Sphaerobacter, is almost certainly really a deep branch of the chloroflexi rather than the actinobacteria. The opposite may be the case for the genus Thermoleophilum; originally classified a member of the green non-sulfur Bacteria (chloroflexi), there is some evidence that it may instead be an actinobacterium, where it is currently classified as a relative of Rubrobacter. Alternatively, it may represent an independent phylum of Bacteria.

Thermoleophilum album

T. album is one of only two species of the genus Thermoleophilum (the other is T. minutum). These organisms are very small (ca. 0.5 x 1μm), obligately aerobic Gram-negative (in terms of staining) rods. All isolates of these species are thermophilic, growing optimally at about 60°C, and have been isolated from hot spring sediments and dark muds exposed to solar heating from a wide range of sites scattered around the United States. The unique feature of these organisms is that they can grow only on long-chain n-alkanes, i.e. wax. No other substrates can be used for either carbon or energy, not even the alcohol derivatives of growth substrates.

Bacterial development

Log phase cells are very different from stationary-phase cells: log cells are adapted for maximal growth rates in nutrient-saturated conditions, where growth rate is often limited only by the organisms physical ability to import nutrients, process them into cell material, and replicate. They are usually large cells with ability to replicate DNA & make RNA and proteins rapidly. Stationary cells are usually smaller, and are adapted for maximum competitiveness. In many species, the morphology of the cells types are strikingly different, such as Arthrobacter described above. Keep in mind, as well, that microbes generally spend most of their time in stationary phase in the environment.

The shift to stationary phase (& later back to log phase) is a complex developmental process, controlled by sigma-factor cascades (like phage infection or sporulation in Bacillus). In log-phase Bacteria, the “vegetative” sigma subunit (σ70) is the predominant sigma factor directly RNA polymerase promoter recognition. In late log phase, expression of the stationary-phase sigma (σS) is turned on. Because of its higher affinity for the core RNA polymerase, σS progressively replaces σ70. Genes needed during log phase growth require σ70-containing RNA polymerase for expression, and so these genes are progressively turned off as the concentration of σ70 declines. Genes for stationary phase growth (including the gene encoding σS) are expressed by σS, and so the expression of these genes increases until they reach normal levels for stationary phase. In the case of more complex developmental pathways, such as sporulation, heterocyst formation, or phage infection, many sigma factor transitions occur sequentially, driven by the fact that each sigma factor initiated expression of the sigma factor to follow. Each sigma factor directs expression of the genes required at that stage of the developmental pathway. These sigma factor directed developmental pathways are in many ways analogous to the homeo-box directed developmental pathways of animals.

Secondary metabolites

Many bacteria produce antibiotics and other antimicrobials, but it is very common in Streptomyces & Bacillus. Antibiotics are not produced during rapid growth, but in stationary phase, i.e. they are secondary metabolites. Secondary metabolites are compounds produced (and typically secreted into the environment) only during stationary phase, and so are not required for growth. Other secondary metabolites include iron-binding compounds (siderophores) and other compounds that help the organism compete for limited resources.

There are two ways an organism can increase its competitive fitness in a tight environment: increasing its own fitness (self-improvement), or decreasing the fitness of its competitors. Siderophores and high-affinity uptake mechanisms are examples of secondary metabolites that directly increase an organisms supply of nutrients; this is self-improvement. Antibiotics and bacteriocins are examples of secondary metabolites that provide a competitive advantage to an organism by crippling the competition, allowing the producing organism access to the resources that otherwise would have gone to the competition, or even providing the producer with the nutrients released by the extinguished competitor.

Bacterial multicellularity

We generally think of Bacteria (and Archaea and protists, for that matter) as being unicellular - every bug for itself. But this is not a very sophisticated viewpoint. It's true that Bacteria don't develop into large interdependent clusters that walk around talking to one another on cell phones, but multicellularity is a spectrum, not just an "is" or "is not". Most Bacteria communicate by secreting small compounds called phermones; cell-to-cell communication is a kind of multicellularity, in the sense that the cells act and react as a population rather than individually. Many Bacteria form aggregates of various kinds during their life cycle in which cells differentiate. In some cases, there is no doubt that the bacterium is multicellular, for example the filamentous cyanobacteria in which some cell undergo terminal differentiation in nitrogen-fixing heterocysts, or the colonies of Streptomyces described above that undergo complex morphological development and programmed cell death. Cells in a group specializing into different forms, and especially if that differentiation is terminal (those cells will not contribute to future generations) is the hallmark of multicellularity.

One distinction between multicellular behavior in Bacteria (and Archaea and unicellular eukaryotes) in contrast to plants/animals/fungi is that the groups of cells are often composed of more than one species; for example, the photosynthetic mats. Here we have complex layers of different kinds of cells, working together to make a living; a tissue, of a sort, made up of cells of various kinds specialized for different functions. Not much different than the tissue of a plant of animal, except that the cells that make it up are of different species.

Questions for thought

• Given that Gram-positive bacteria evolved from a Gram-negative ancestor, what might be the advantages, or disadvantages, of this change in cell envelop structure?
  
  
• The complete genome sequence of Mycoplasma genitalium is 0.6 Mbp - all of the genes it doesn't absolutely need for its parasitic lifestyle have been lost. What genes do you think remain? What genes would a minimal free-living heterotrophic specie need?
  
  
• Endospores of Bacillus or Clostridium seem to be able to wait more-or-less indefinitely for favorable conditions to germinate. However, how can the 'monitor' the environmental conditions without metabolic activity (and therefore a continuous energy cost)? What do you think might limit the lifespan of an endospore? Why not longer?
  
  
• What do you imagine to be some of the differences in metabolism and gene expression in log phase vs stationary cells? How might an organism evolve to grow faster - or subsist better without growth?
  
  
• How would you go about seeing if the mechanism underlying programmed cell death in Streptomyces is in any way related mechanistically to programmed cell death (apoptosis) in animals?
  
  
• Can you give any other examples of multicellularity in prokaryotes?

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