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Mycoplasmas -general structure

The mycoplasmas are the smallest (0.15-0.30m) and the


simplest self replicating prokaryotes. Mycoplasma is a genus of
over 100 species of cell wall lacking bacteria. Each mycoplasmal
cell is externally bounded by a trilaminar membrane, rich in
cholesterol, which gives rigidity to the membrane. This
membrane is composed of approximately two-third protein and
one-third lipid as in other prokaryotes. The membrane lipids
resemble those of the other bacteria, apart from the large
quantities of cholesterol in the sterol-requiring mycoplasmas.
Inside the cell membrane, the mycoplasmal cell contains
ribosomes and the nucleoid, the genetic material which is a
double stranded circular DNA molecule. Mycoplasmas are the
organisms having the smallest genomes, with a total of 500-1000
genes. The mesosomes are not found in the Mycoplasma. The
wall-less mycoplasma are pleomorphic, with multiple shapes,
commonly with granular and filamentous forms. The coccus is the
basic form of all mycoplasmas in culture, with filamentous and
elongated forms also occurring frequently. The name Mycoplasma
has been derived from the tendency of filaments to branch truly
to produce myceloid structure (myces-fungus, plasma- form).
Some mycoplasmas possess unique attachment structures as
bulbous enlargement, or a tapered tip to adhere to the host
surfaces as in M. pneumoniae and M. genetalium. The
mycoplasms are generally non-motile, non-sporing and non-
fimbriated, however gliding motility is common in them. The lack
of a cell wall makes the mycoplasmal cell flexible in form and,
thus, they are not retained by the bacterial filters and can pass
through them readily like viruses. Mycoplasmas are osmotically
labile and grow best in hypertonic and isotonic conditions. They
experience osmotic burst or osmotic shocks in hypotonic
conditions. Most species of Mycoplasma are facultative
anaerobes, except M. pneumoniae, which is a strict aerobe.

Cells of Mycoplasma pneumoniae


Electron micrograph of thin-sectioned mycoplasma
cells

Pathogenicity

Mycoplasmas are parasitic on a wide range of hosts,


including humans, plants, animals and insects, causing diseases
in them. In humans Mycoplasma exists in parasitic association
with ciliated epithelial cells of genital and respiratory tracts,
causing surface infections and diseases in these organs. M.
pneumoniae infects the respiratory tract and causes
Pneumonia. Mycoplasma hominis causes Pyelonephritis, Pelvic
inflammatory disease; whereas M. genitalium causes urinary
tract infections and Non-gonococcal urethritis. Mastitis in cows is
also caused by many species of Mycoplasma - M. bovis, M.
bovihirnis, etc. In plants, pathogenic mycoplasmas are
responsible for several hundred diseases, and belong to two
groups-phytoplasmas and spiroplasmas. The mycoplasmas
cause Witches broom diseases, which are characterised by
hormonal disorders - witches broom growth, virescence,
phyllody, intensified negative geotropism, and disturbed
periodicity. Mycoplasmas also cause phloem degeneration, which
leads to a typical growth reduction, discoloration, wilting and
premature death. The diseases aster yellow, pear decline, and
elm necrosis, are also ascribed to mycoplasmas.
The three pathogenic spiroplasmas known to cause diseases
in plants are Spiroplasma citri, S. kunkelii, and S. phoeniceum.
Spiroplasma citri causes citrus stubborn, S. kunkelii is the causal
agent of corn stunt, whereas S. phoeniceum is responsible for
periwinkle yellows. In plants, phytopathogenic mycoplasmas are
restricted to the phloem sieve-tubes that contain photosynthetic
sap. In tissue culture, mycoplasma prove as serious
contaminants, causing diseases in the culture cells, tissues,
organs and plants.

Transmission of mycoplasmas

As we know, the plant pathogenic mycoplasmas are


restricted to phloem sieve- tubes, which are enriched in
photosynthetic sap, the food for many phloem-feeding insects,
viz. aphids, leaf-hoppers, psyllids, etc. Therefore, these insects,
after feeding on the infected phloem, carry these organisms in
their proboscis, and specifically transmit these phytopathogenic
mycoplasmas to other plants. In humans, the pathogenic
Mycoplasma pneumoniae is transmitted by inhalation of
respiratory droplets, and M. genetalium is transmitted by sexual
contact. M. pneumoniae is transmissible by respiratory droplets
during close contact with a symptomatic person.

Treatment of mycoplasmas

Mycoplasmas, unlike viruses, are susceptible to antibiotics


and are, therefore, treated by them. However, since these
smallest prokaryotes lack a cell wall, they are not killed by
antibiotics, such as penicillin and sulpha drugs, which have a
wall-degrading mechanism of action. Mycoplasmas are resistant
to antibacterial agents that target cell wall. They are inhibited by
antibiotics, such as tetracycline and erythromycin, which have
different mode of action (inhibition of protein synthesis) than
penicillin, which targets cell wall.

Taxonomy of mycoplasmas

The mycoplasmas have been placed in division Firmicutes on the


basis of 16s rRNA gene analysis. The bacteria in Firmicutes are
gram positive and have low G-C concentration in their genomes.
Mycoplasmas are also having low G-C concentration in their
genomes. The wall-less bacteria of division Firmicutes are placed
in the class Mollicutes (which stands for soft skin) and since
mycoplasmas lack a cell wall, they too are placed in the class
Mollicutes. The order Mycoplasmatales has a single family
Mycoplasmataceae, which contains two genera- Mycoplasma and
Ureaplasma. The mycoplasmas require sterols for their growth,
while as the ureaplasma depend on urea as metabolite. The
systematic position of the genus Mycoplasma is shown below:

Kingdom: Bacteria

Division: Firmicutes

Class: Mollicutes

Order: Mycoplasmatales

Family: Mycoplasmataceae

Genus: Mycoplasma

Cultural characteristics

The mycoplasmas depend on their host for diverse


nutritional requirements and, thus, require complex media for in-
vitro cultivation. This is the reason why a small number of
mycoplasma existing in nature have been so far cultivated in
laboratory. The cultivable mycoplasmas, viz. Mycoplasma
pneumonia and M. genetalium also grow very slowly and poorly
in-vitro. The media for mycoplasma culture contain serum (5-
20%)-which provides cholesterol and fatty acid for membrane
synthesis, heart infusion, peptone, yeast extract, salts, glucose or
arginine as source of energy. For culturing ureaplasmas, the
medium is supplemented with urea, possibly as source of energy.
In order to exclude and prevent contamination of fast growing
bacteria, Penicillin or Thallium acetate, or both, is added to the
medium.

Fried egg colonies of


Mycoplasma sp.
Metabolism

The reduced, minimal genome in mycoplasmas results


in poor metabolic and biosynthetic systems. Mycoplasmas do
have truncated respiratory system. The electron transport system
lacks quinones and cytochromes. Enough evidence about the
simplicity and incompleteness of the electron transport chain is
the fact that the reduced nicotinamide adenine dinucleotide
(NADH) oxidase activity is cytoplasmic. Therefore, mycoplasmas
lack a well-organised and complex electron transport system in
their membranes. Putting forth the above mentioned facts,
possibility of oxidative phosphorylation as an ATP-generating
system in mycoplasmas is out rightly ruled out. The two proven
ways of ATP generation in mycoplasmas are both based on the
substrate level phosphorylation.
The major source of ATP is the Arginine dihydrolase pathway.
Arginine deaminase
Arginine + H2O Citrulline + NH3

Ornithine carbamoyltransferase
Citrulline + Inorganic orthophosphate
Ornithine + CarbamoyIPO4

Carbamate kinase
Carbamoyl PO4 + ADP ATP + CO2 + NH3
In another mechanism of ATP generation, Acetyl coA
produced by the oxidative decarboxylation of pyruvate,
undergoes the following reactions to yield ATP by substrate-level
phosphorylation.
Phosphate acetyltransferase

Acetyl CoA + Inorganic orthophosphate


Acetyl PO4 + CoA
Acetate kinase
Acetyl PO4+ ADP Acetate + ATP
There are some species which derive their energy by
the hydrolysis of urea, or from the degradation of glucose. The
mycoplasmas are known to synthesise DNA, RNA, lipids and
proteins, but not amino acids, which they obtain from the host.
Sterol requiring mycoplasmas incorporate sterols,mainly
cholesterol, into the cell membrane up to concentrations of 65%
(for stabilizing their membranes).

Molecular biology and mode of nutrition in mycoplasmas

The genome of mycoplasmas is characteristically


prokaryotic, consisting of a circular, double-stranded DNA
molecule. Mycoplasmas represent a minimal life-form, having
yielded to selective pressures to reduce gene number and
genome size. Thus, their genomes are the smallest recorded for
any self-reproducing prokaryote. Because of their small size,
mycoplasmal genomes carry small amount of genes. Mycoplasma
genetalium has the smallest known genome of any free- living
organism, comprising of 580,070 base pairs with a total of 470
coding regions, which include genes required for DNA replication,
transcription and translation, DNA repair, cellular transport, and
energy metabolism. This gene content is about one-sixth the
number of genes in Escherichia coli. Mycoplasmas, accordingly,
express a small number of cell proteins, and lack many enzymatic
activities and metabolic pathways. They possess no complete
pathways for amino acids synthesis and degradation, implying
that these monomers must be obtained either from their hosts, or
from a culture medium. Their nutritional requirements are
correspondingly complex, and they are dependent on a parasitic
mode of life. There is an apparent correlation between
mycoplasmal growth rate and genome size: under optimal
conditions, M. pneumoniae M129 (genome size 816,394 bp)
grows twice as fast as M. genitalium G-37 (genome size 580,074
bp).
The mycoplasmal genome is characterized by a low guanine-
plus-cytosine content (18-40%); in M. genetalium the G + C
content is 32%, and in M. mycoides subsp. mycoides SC, it is
24%. Mycoplasmas are also characterised by preferential
utilization of codons containing adenine and uracil, particularly in
the third position. The universal stop codon UGA encodes for the
amino acid tryptophan in many mycoplasmas, an interesting
feature found so far only in mycoplasmas and in non plant
mitochondria. One more property which distinguishes
mycoplasmas from the conventional eubacteria is resistance of
mycoplasmal RNA polymerase to rifampicin. However, the
mycoplasmas are susceptible to antibiotics, such as tetracyclines
and chloramphenicol, which inhibit protein synthesis on
prokaryotic ribosomes.

Reproduction in Mycoplasma

Mycoplasma reproduces by binary fission in which the parent


normally splits into two cells. However, the cytokinesis frequently
lags behind the genome replication, resulting in multi-nucleoid
filaments. These multi-nucleoid filaments subsequently break into
coccoid bodies, resulting in enhanced multiplication as shown in
Fig. 1

Fig. 1 - Binary fission in mycoplasmas


Phylogeny of mycoplasmas

As far as phylogeny of Mycoplasmas is concerned, it poses


some exciting questions, for example
(i) Do they represent the descendents of primitive bacteria that
existed before the development of a peptidoglycan based wall, or
(ii) Do they represent the evolutionarily reduced forms, which
have lost their cell wall?
The comparison of base sequence analysis of conserved
ribosomal RNA molecules, particularly of the 16S rRNA type,
favours the degenerative evolution hypothesis. This analysis
serves as a strong molecular evidence, suggesting that
mycoplasmas represent evolutionarily degenerate eubacterial
forms, that have lost their cell wall. According to Woese et al.,
the mycoplasmas have evolved as a branch of the low-guanine-
plus-cytosine containing gram positive bacteria, and are
phylogenetically most closely related to two clostridia, Clostridium
innocuum and C. ramosum. During their evolution, the
mycoplasmas lost genes of many biosynthetic pathways, resulting
in minimal genomes. Their genomes dont carry genes for
peptidoglycan-synthesis and amino-acid metabolism. However,
there is another school of evolutionary thought, which believe
that mycoplasmas evolved from a variety of walled-bacteria and,
thus, have a polyphyletic origin. Their hypothesis is based on the
evidence of marked phenotypic and genotypic variability among
mycoplasmas. Woese et al. upholds the monophyletic origin of
mycoplasmas and attributes their great variety to the process of
rapid evolution, characteristic of the group.