List of seven special groups of bacteria:- 1. Mollicutes 2. L-Forms 3. Rickettsia 4. Fastidious Vascular Bacteria 5. Chlamydias 6. Spirochaetes 7. Actinomycetes.
Special Group # 1. Mollicutes (Mycoplasmas):
The mollicutes represent a group of eubacteria. The common name for this group has traditionally been mycoplasmas. However, this usage invites confusion, since it is often not clear whether “mycoplasma” is being used to represent a genus or to refer to a member of the genus Mycoplasma.
To avoid this confusion, the more recently coined term “mollicutes” to designate the members of this eubacterial group is being adopted here.
The distinguishing property of the mollicutes is the lack of a definite cell wall in them. They all are sensitive to osmotic lysis, resistant to penicillin and other wall-synthesis attacking antibiotics, pleomorphic in shape to at least some extent and parasites to eukaryotic organisms. Their colonies that develop on solid culture media are often small, typically having a nipped or “fried-egg” appearance.
The mollicutes have unusually low guanine (G) + cytosine (C) values (usually 23 to 36%) and small genome size (MW of 0.5 x 10 to 1 0 x 109) 16S ribosomal RNA (rRNA) sequencing reveals that the mollicutes are a coherent polygenene group closely related to Clostridia. Six genera are recognised as mollicutes: Mycoplasma, Phytoplasma, Ureaplasma, Acholeplasma, Anaeroplasma, and Spiroplasma (Table 6.1).
The Mycoplasma (Fig. 6.1) is typical prokaryotes being the smallest known organisms capable of autonomous growth and reproduction. The smallest viable mycoplasmal cell known is that of Mycoplasma homonis H39 which is about 0.33 µm. This is only about two times larger than the theoretically considered smallest cell containing all informations.
A highly contageous diseases of cattle appeared in Germany and Switzerland in 1713 and it spread throughout Europe during the 18th century. This disease was named ‘Bovine Pleuropneumonia’ and its causative organism was thought to be “Pleuropneumonia-like Organism (PPLO)”.
This discovery was made by Nocard and Roux (1898) and the organism discovered was given the first binomial name as Asterococcus mycoides. Later, in the year 1929, Nowak replaced the generic name by ‘Mycoplasma’ and the latter is now generally used in the place of PPLO to represent these organisms.
Mycoplasma are non-motile and do not produce spores. They are important pathogens of animals though they may occur as saprophytes.
At present, more than 36 mycoplasmal representatives have been isolated; the most typical representatives of the pathogenic species are the causative agents of pleuropneumonia in cattle (Mycoplasma mycoides), acute respiratory infections (Mycoplasma hominis), and a typical pneumonia in humans (Mycoplasma pneumoniae).
Mycoplasmas lack cell wall, are highly pleomorphic varying in shape from spherical to branched filamentous structures, and multiply by binary fission. Organelles, viz. plasma membrane, ribosomes, and DNA and RNA are present in mycoplasmal cell. The plasma membrane, which binds mycoplasmal cell in absence of cell wall, is tri-layered and contains cholesterol.
The genome (DNA) of the known mycoplasms is large enough to code only for about 600 to 1,000 polypeptides. It is this limited information, which limits the ultimate complexity of mycoplasmas.
The latter are resistant to the wall-attacking antibiotics such as penicillin because, as we know, the mycoplasmas lack cell wall. The growth of mycoplasmas is inhibited by tetracyclines and similar antibiotics that act on the metabolic pathways.
Phytoplasms (= MLOs):
A group of Japanese workers (Doi et al., 1967; Ishii et al., 1967) found wall-less microorganisms in the phloem of plants affected with certain diseases and in the body of the insect vectors of these diseases. Such diseases, up to that moment, were thought to be caused by viruses.
Morphologically, these organisms resembled typical mycoplasmas found in animals and humans and those living saprophytically, but their genomes are only distantly related to true mycoplasmas and they cannot be grown on artificial nutrient media and. so far, no plant disease has been reproduced on healthy plants inoculated directly with them obtained from diseased plants.
Since these organisms did not satisfy Koch’s postulates (the rules of pathogenicity test), they were, therefore, called mycoplasma-like organisms (= MLOs) and later assigned the name ‘phytoplasmas’ .
Generally, phytoplasmas (= MLOs) are more related to Acholeplasma than to Mycoplasma. They lack cell walls are bounded by a ‘unit’ membrane and have cytoplasm, ribosomes and strands of nuclear material. Their shape is usually spheroidal to ovoid to irregularly tubular to filamentous, and their sizes are comparable to those of typical mycoplasma.
Phytoplasmas are generally present in the sap of phloem sieves tubes of the host plants. Most of them are transmitted by leaf hoppers but some by psyllids and plant-hoppers. In addition to the host plant, phytoplasmas also grow in alimentary canal, haemolymph, salivary glands, and intercellularly in different body organs of their insect vectors.
So far, more than two hundred distinct plant diseases affecting many hundred plant genera have been reported to be caused by Phytoplasmas (MLOs). Some important ones are coconut lethal yellowing, peach X- disease, little leaf of brinjal, grossy shoot of sugarcane, phylloidy of sesame, sandal spike, witche’s broom etc.
The Ureaplasma are the only nonfermentative mollicutes, i.e., they do not ferment the growth substrates such as carbohydrates and amino acids like other mollicutes but they depend on the hydrolysis of urea for their energy. Ureaplasma normally inhabit the mouth, respiratory tract, and genital tract of humans and animals. They lack motility and are coccoid in morphology.
They form tiny colonies less than 60 µm diameter on solid culture media and never grow in high cell densities. U. urealyticum, the only species of the genus, usually behaves as commensal in the genital tract but, rarely, appears to be pathogenic causing inflammatory disease (tuboovarian abscess, salpingitis).
The Acholeplasma usually inhabit the tissues of many vertebrate animals and also are found in plants or as saprobes. They sharply distinguish from other mollicutes by its ability to grow in the absence of sterols. Acholeplasma, like Mycoplasma, reproduce in two ways; unicellular coccoid cells may divide by fission, or they may elongate into branching filaments that then fragment into many coccoid cells.
Cultures of Acholeplasma thus may contain a mixture of coccoid cells, short filaments, and longer branched filaments. They become problematic in animal cell culture studies in vitro because they damage the cultured animal cells by producing H2O2 which is cytotoxic.
The Anaeroplasma are strictly anaerobic mollicutes recovered from the bovine and ovine rumen of cattle and sheep. They ferment carbohydrates to a mixture of acids (acetic acid, formic acid, succinic acid, lactic acid, propionic acid), ethanol and CO2. Some are bacteriolytic as they lyse walled-bacteria by excreting lytic enzymes.
The causative organisms of some plant diseases (e.g., citrus stubborn disease, Corn stunt disease) is not spherical like mycoplasmas but is rather a long, motile, helical filament which is completely different from the filamentous mycoplasmal structures. The name ‘Spiroplasma’ was suggested by Davis and Worley in 1973 for such organisms.
However, the genus Spiroplasma is based after Spiroplasma citri (Fig. 6.2), the causal organism of cirus stubborn disease. Spiroplasma have a wide host range extending from fungi to plants and arthropods to vertebrates. It has been found that the spiroplasmas, unlike phytoplasmas (MLOs), satisfy the Koch’s postulates.
Spiroplasmas lack cell wall and are surrounded by a tri-layered plasma membrane. They are motile in liquid culture; the motility of their helical filament is with a rapid rotatory screw-type motion and a slow undulation.
It is notable that no organs of locomotion like flagella, axial filaments or other organelles have been observed; the motility is probably due to the existence of a contractile mechanism that can operate only under optimal conditions of a liquid culture medium.
This organism requires cholesterol or possibly other sterols for growth, forms typical fried-egg colonies on agar medium and is completely inhibited by erythromycin, neomycin, tetracycline and thallium acetate.
Special Group # 2. L-Forms:
Several species of bacteria have been observed which undergo a transition from their normal morphological forms to very small bodies—the so-called “L-forms”. The latter were first discovered and investigated by Klieneberger-Novel in 1935, and were named after Lister Institute, London where the discoverer was working.
The L-forms, characteristic bacterial varients, may arise “spontaneously” during some state in the culture of the bacterial form, as its formation may be induced by an inhibiting agent (e.g., penicillin and other antibiotics, enzymes like Izsozyme and antibodies specific for the bacterial form).
L-forms are devoid of cell wall. They are normally non-pathogenic, resistant to phage infections, not capable of active motility, and are unstable structures very sensitive to the influence of osmotic pressure mechanical action and aeration. L-forms of bacteria grow on media containing serum; their growth is enhanced particularly in presence of penicillin.
However, when grown in a medium devoid of any inhibiting agent, L- forms revert back to their parental (bacterial) state. The exact relationship of L-forms is still a matter for speculation. L-forms are formed in Listeria monocytogenes, Mycobacterium tuberculosis, Streptococcus, and others.
Special Group # 3. Rickettsia (Rickettsiae):
Rickettsia (pl. rickettsias; also called rickettsiae) are abligate intracellular parasites of such arthropods as lice, fleas, mites, and ticks which they inhabit without injury and may be pathogenic to human being (Fig 6.3). The rickettsias have not yet been cultured in the absence of host cells. They are generally cultured either by inoculation into the yolk-sac of chicken eggs, or by infecting host cells in tissue culture.
Rickettsias were discovered at the end of the first decade of 20th century by an American medical microbiologist, H.T. Ricketts, while studying the Rocky Mountain spotted fever and were later named in honour of the discoverer. For many years these organisms were thought to be biologically intermediate between bacteria and viruses, they are now considered to be bacteria and thus included in bacterial classification.
The morphology of rickettsia varies from coccoid to rod-shaped to filamentous forms. They are small, non-motile, gram-negative, ranging 0.3 -0.7 µm wide and 1-2 µm long in size. They possess cell wall, cell membrane, both RNA and DNA and are pleomorphic. They multiply by normal binary fission with doubling times of about 8 hours.
They resemble viruses in living endoparasitically in their arthropod vectors and multiplying within their cell; they also multiply within the cells of infected animals and humans. The penetration of a host cell by a rickettsial cell is an active process requiring both host and parasite to be alive and metabolically active.
Once inside a host cell, the rickettsia multiply primarily in the cytoplasm and continues replicating until the host cell is loaded with its progeny. Finally, the host cell is lysed and the rickettsia are released into the surrounding fluid.
Rickettsias do not produce spores and capsules and stain well by Romanowsky Giemsa Stain and the Ziehl-Neelsen Stain. They are autonomous organisms and have the capacity to produce their own energy (ATP) through oxidative phosphorylation with the help of a cytochrome system.
Out of all known rickettsias, three genera named Rickettsia, Coxiella and Rochalimaea have been well studied. Several human diseases are caused by rickettsias.
Rickettsia prowazekii causes typhus fever; R. mooseri causes enemic fever; R. tsutsugamushi causes scrub typhus; and R. rickettsii causes Rocky Mountain spotted fever. Q-fever is caused by Coxiella burnettii. Rochalimaea quintana is the causative agent of trench fever, a disease that decimated troops in World War I.
Special Group # 4. Fastidious Vascular Bacteria (= RLOs):
The fastidious vascular bacteria (previously known as rickettsia-like organisms, or RLOs) are such forms of bacteria that have complex specific growth factor requirements and are able to reproduce only under greatly restricted conditions (hence called ‘fastidious’).
These were earlier believed to be biotrophs (obligate parasites) closely related to rickettsia but, now, it has been shown that these microbes are not related to rickettsia and they are apparently a new kind of parasitic bacteria which simply cannot be grown on simple culture media in the absence of host cells.
The fastidious vascular bacteria (Fig. 6.4) are confined either to phloem or xylem of the host plant but never to both and are usually transmitted by leafhoppers. They are generally rod-shaped (1-4 µm in length and 0.2-0.5 µm in diameter), aflagellate, bound by a cell membrane and a cell wall.
The outer layer of the cell wall is usually undulating or ripped. Almost all known fastidious vascular bacteria are gram-negative but two, causing Sugarcane ratoon stunting and Bermudagrass stunting disease. The gram-negative fastidious vascular bacteria have been placed in the recently created genus Xylella and the gram-positive ones in the genus Clavibacter.
The phloem-limited fastidious vascular bacteria were first observed in 1972 by Windsor and Black in clover plants affected with clover club leaf disease whereas those limited to xylem in 1973 in grape plants affected with pierce’s disease.
Some other plant diseases caused by these bacteria are: citrus greening, elm leaf scorch, potato leaflet stunt, etc. Fastidious vascular bacteria are sensitive to antibiotics such as tetracyclins and penicillin and to high temperatures.
Special Group # 5. Chlamydias (Chlamydiae):
Chlamydias or Chlamydiae (Sing. Chlamydia) are non-motile obligate intracellular parasitic gram-negative bacteria. They are coccoid in shape, measure 0.2 to 0.4 µm in diameter and reproduce only in the cytoplasmic vesicles of the living cells of their hosts, the vertebrates.
Their size ranges from 0.2-1.5 µm and the size of their genome is 4 to 6 x 108 daltons, one of the smallest of all prokaryotes and the G + C content in the genome is 41-44 per cent.
Growth reproduction and maturation of chlamydia’s is completed usually in 48 hours.
The life cycle alternates between two forms (Fig. 6.5):
(i) The elementary body (EB) small, rigid walled, infectious form which survives when released from the host cell, and
(ii) The reticulate body (RB) or initial body: larger, thin walled, non-infectious form that is divided by fission.
When the elementary body (also called chlamydospore) contacts a host cell and enters inside, it enlarges and loses its rigidity, and now is called reticulate body. The latter continue to grow in size and start dividing by binary fission 10-15 hours after the infection.
At 20-30 hours after the infection the reticulate bodies decrease in size and become typical elementary bodies. The infected host cells undergo lysis 40-60 hours after infection and release the elementary bodies which may cause a new infection in healthy cell of the host.
(i) Microbiologists call chlamydias an “energy parasite” as they believe that they obtain its energy (ATP) from the host cell and lack ATP-generating system of their own. However, it has been found recently that Chlamydias have the genes to make at least some ATP of its own.
(ii) They contain enzymes for the synthesis of peptidoglycan. Chlamydial cell walls lack peptidoglycan but the antibiotic penicillin that disrupts peptidoglycan syntnesis, is able to inhibit chlamydial growth. Though the occurrence of enzymes for the synthesis of peptidoglycan helps account for penicillin effect, but no one knows the purpose of peptidoglycan synthesis in this bacterium.
(iii) Chlamydias lack FtsZ gene in its genome. This gene is thought to be required by all bacteria and archaea (archaebacteria) for septum formation during the cell division. The absence of this essential gene makes one wonder how chlamydias divide.
3. Species Known and their Role:
Only three species of genus Chlamydia are recognized: C. trachomatis, C. psittaci and C. penumoniae. The former is the agent of two common human diseases the genitourinary tract disease called ‘‘lymphogranuloma venereum” and the conjectivital infection called ‘trachoma’. C. psittaci causes ‘psittacosis’ disease in birds that is occasionally transmitted to humans and causes pneumonia-like symptoms. C. penumoniae is the causal agent of a variety of respiratory syndromes in humans.
Special Group # 6. Spirochaetes:
Spirochaetes (G. spira = a coil; chaete = hair) are gram-negative motile, chemoheterotrophic, tightly coiled bacteria typically slender and flexous in morphology (Fig. 6.6A). Their size varies from 5 to 250 µm x 0.1 to 3.0 µm. These bacteria are widespread in aquatic environments and in animals and are distinguished by their structure and motility-mechanism.
Spirochaetes differ greatly from other bacteria with respect to motility as they easily move through very viscous solutions though they do not possess any external rotating flagellum.
The spirochaete cell consists of a ‘protoplasmic cylinder’ surrounded by plasma membrane and gram-negative type cell wall. Two to more than a hundred flagella called axial fibrils, periplasmic flagella or endoflagella are located in the periplasm. They extend from both ends of the protoplasmic cylinder and often overlap one another.
The whole complex of periplasmic flagella is called axial filament. However, both the axial fibrils and the protoplasmic cylinder are surrounded by a multi-layered but flexible cuter membrane or outer sheath (Fig. 6.6B) which contains lipids, protein and carbohydrate.
Each endoflagellum rotates like typical bacterial flagella. When all the endoflagella rotate in the same direction, the protoplasmic cylinder rotates in the opposite direction. This could cause the corkscrew-shaped outer sheath to rotate and move the cell through the surrounding liquid (Fig. 6.7). Flagellar rotation could also flex or bend the cell and account for the crawling movement on solid surfaces.
Special Group # 7. Actinomycetes (The Filamentous Bacteria):
The actinomycetes (sing, actinomycete) are a large group of aerobic, high GC percentage gram-positive bacteria that form branching filaments or hyphae and asexual spores. These bacteria closely resemble fungi in overall morphology. Presumably this resemblance results partly from adaptation to the same habitat.
When grown on agar-surface, the actinomycetes branch forming a network of hyphae growing both on the surface and under-surface of the agar. The on-the-surface hyphae are called aerial hyphae and the under- surface hyphae are called substrate hyphae (Fig. 6.8).
Septa normally divide the hyphae into long cells (20 µm and longer) possessing many bacterial chromosomes (nucleoids). These are the aerial hyphae that extend above the substratum and reproduce asexually. Most actinomycetes are non-motile. When motality is present, it is confined to flagellated spores.
(i) Cell Wall Composition:
The composition of cell wall in actinomycetes varies greatly among different groups and is of considerable taxonomic significance. Four major cell wall types are distinguished in these filamentous bacteria on the basis of the three features of peptidoglycan composition and structure.
These features are:
(i) Diaminopimelic acid isomer on tetrapeptide side chain position 3,
(ii) The presence of glycine in inter peptide bridges, and
(iii) Sugar content of peptidoglycan.
As is evident in Table 6.3, characteristic sugar patterns are present only in cell wall types II-IV of those actinomycetes with meso-diaminopimelic acid.
Actinomycetes, like other bacteria, reproduce only asexually; the asexual mode of reproduction are accomplished by arthrospore or conidia (conidiospores) formation. In this respect, however, they resemble fungi to some extent.
Arthrospores are formed in bacteria having fungus-like filamentous bodies (members of actinomycetes). The filamentous bodies of these bacteria break into rod-shaped smaller fragments called ‘arthrospores’ each capable of growing into a new filament (Fig. 6.9A).
Conidia formation is a common method of reproduction in some members of actinomycetes. These filamentous branched bacteria produce smaller, oval or rounded structures called conidia terminally on certain apical branches called conidiophores (Fig. 6.9B). Each conidium germinates giving rise to a bacterial cell.
(iii) Major Groups of Actinomycetes:
Most actinomycetes are spore-forming and the manner of spore formation varies among them hence used in separating groups as outlined in Table 6.4. The composition of bases in DNA of most of the members of these filamentous bacteria fall within the range of 54-75% GC and the members at the upper end of this range have the highest percentage of GC of any bacteria known.
(iv) Practical Significance:
(a) Actinomycetes are primarily soil-inhabitants and are very widely distributed.
(b) They usually degrade many a number and variety of organic compounds and play extremely important role in the mineralization of organic matter in soil.
(c) Actinomycetes are medically very significant as they produce most of the natural antibiotics.
(d) Some actinomycetes are pathogenic to humans, animals and even some plants.
An Important Actinomycete:
Streptomyces is a large genus consisting of about 500 species. They are strict aerobes having hyphae usually 0.5-1.0 µm in diameter. The hyphae are of indefinite length and often lack cross-walls (septa) in the vegetative phase. Growth takes place at the tips of the hyphae and is often accompanied by branching resulting in the formation of a compact colony.
As the colony matures, characteristic aerial filaments are formed. Most of the aerial filaments are called conidiophores as they project above the surface of the colony and give rise to conidiospores or conidia by the formation of cross-walls (septa) in the conidiophores followed by the separation of the individual cells directly into conidiospores.
The conidiophores and conidiospores (conidia) are usually pigmented and contribute a characteristic colour to the mature colony.
Streptomyces is soil-inhabitant. In fact, the characteristic earthy odour of moist soil is due to Streptomyces and other streptomycetes genera. The streptomycetes produce a series of volatile substances called geosmins which are sesquiterpenoid compounds, unsaturated ring compounds of carbon, oxygen and hydrogen.
A common geosmin is trans-1, 10-dimethyl-trans-9-decalol. Streptomyces significantly contribute in the process of mineralization in soil as they aerobically degrade resistant substances, e.g., pectin, chitin, lignin, keratin, latex and aromatic compounds. This filamentous bacterium, however, is best recognised for its contribution of large variety of antibiotics.
Few species of Streptomyces are pathogenic. Streptomyces scabies causes scab disease in potatoes and beets whereas S. somaliensis is associated with actinomycetoma in humans, an infection of subcutaneous tissues that produces lesions resulting in swelling, abscesses and even bone destruction if untreated.
Streptomyces can be isolated from soil relatively easily. For this purpose, a selective agar medium is prepared which contains the usual assortment of inorganic salts to which starch, asparagine or calcium malate is added as a source of carbon and undigested casein or potassium nitrate as source of nitrogen.
Then a suspension of soil in sterile water is diluted and spread on the selective medium taken in petri dish. The latter is now incubated at 25°C for 5-7 days.
After incubation, the plates are examined for the presence of characteristic colonies of Streptomyces which are opaque, rough, and of non-spreading morphology. The spores of interesting colonies can be collected and streaked on fresh media to isolate pure cultures.