Introduction
The bacteria with the smallest known genomes are found among members of
the class Mollicutes. This class presently comprises the six eubacterial
genera Acholeplasma, Anaeroplasma, Asteroleplasma, Mycoplasma, Spiroplasma
and Ureaplasma (however, the term mycoplasma has been frequently used to
denote any species included in the class Mollicutes). The common characteristics
are the complete lack of a bacterial cell wall, osmotic fragility, colony
shape and filterability through 450-nm pore diameter membrane filters. The
relatively close phylogenetic relationship of these genera was measured
by comparative sequence analysis of the 5S and 16S ribosomal RNA (rRNA).
The rRNA sequence analyses also revealed that the Mollicutes are not at
the root of the bacterial phylogenetic tree, but rather developed by degenerate
evolution from gram-positive bacteria with a low mol% G+C (guanine plus
cytosine) content of DNA, the Lactobacillus group containing Lactobacillus,
Bacillus, Streptococcus and two Chlostridium species. The Mollicutes lost
during the process of evolution a substantial part of their genetic information.
This is reflected by significantly smaller genome sizes as low as 600 kbp
and extending to 2300 kbp as compared with 2500-5700 kbp long genomes of
their ancestor bacteria. The loss of coding capacity could probably be tolerated
because of the parasitic life style of the Mollicutes. They have never been
found as freely living organisms. In nature Mollicutes depend on a host
cell, respectively on a host organism. For instance, Mycoplasmas and Ureaplasmas
are parasites in different vertebrates, from which they obtain essential
compounds such as fatty acids, amino acids, precursors for nucleic acid
synthesis and cholesterol, a compound normally not found in bacteria. Only
Acholeplasma and Asteroleplasma do not require cholesterol for growth.
Mycoplasma pneumoniae, the subject of this study, is a human pathogenic
bacterium causing tracheobronchitis and primary atypical pneumonia. Associated
with the host cell, surface colonization of human respiratory tract epithelial
cells takes place. In the laboratory, M. pneumoniae can be grown without
a host cell in rich medium supplemented with 10-20% horse serum. The lack
of a cell wall most probably facilitates the close contact between M. pneumoniae
and its host cell and guarantees the exchange of compounds, which support
the growth of the bacterium. As a consequence of this bacterial surface-parasitism
the host cell is severely damaged. The exchange of toxic metabolic compounds
is discussed as a possible cause of cell damage, however, at this stage
not a single toxic compound has been identified as a causative agent of
cell damage.
M. pneumoniae has an exceptional position among the Mollicutes since its
DNA has the highest G+C content (41 mol%), whereas the genomes of most of
the other Mollicutes have a G+C content below 30 mol%. The genome size of
M. pneumoniae is about 800 kbp having a coding capacity for 700 proteins
assuming an average molecular mass of 40000 Da. Hence M. pneumoniae is among
the smallest self replicating cells known today.
Mainly for this reason it was selected as a model system for defining the
minimal genetic requirements of an autonomously reproducing cell. This can
be done by determining as many as possible genes and then classifying them
in essential and nonessential ones. Based on these results we should be
able to define a set of genes which are sufficient for the reproduction
of M. pneumoniae under defined laboratory conditions. Morowitz already proposed
several years ago that a mycoplasma species would be a suitable candidate
for defining the essentials of a self-replicating cell. Apart from this
model character as a genetically reduced self-replicating cell, M. pneumoniae
offers a number of interesting phenomena to analyze. For instance, studies
on the interaction between this prokaryotic surface parasite and its eukaryotic
host cell, including the host immune reaction, might help to reveal bacterial
pathomechanisms. Another promising area of research concerns the bacterial
cytoskeleton. Despite the lack of a cell wall and other cellular appendages,
M. pneumoniae exhibits a characteristic cell shape and motility. Both might
be correlated to a cytoskeleton-like structure. Last but not least the evolution
of the Mollicutes is, despite considerable progress in this field, still
left with many unanswered questions. The large body of DNA sequence data
from bacteria which are phylogenetically related to M. pneumoniae such as
Bacillus subtilis might allow to reconstruct the process of degenerate evolution
and to understand how Mollicutes genomes with different G+C contents, between
25 and 41mol%, developed.
Little is known about genetics, physiology and molecular biology of M. pneumoniae
in comparison to the relatively well studied bacteria E. coli and B. subtilis.
An efficient transformation system for M. pneumoniae comparable to the ones
for E. coli is missing, however transposon mutagenesis has been successfully
applied for the isolation of mutants. The dependence on rich medium for
growth prevents the isolation of auxotrophic mutants and the efficient incorporation
of labelled precursors. These disadvantages can be compensated to a large
extent by the methods of molecular biology, for example DNA cloning techniques,
expression of genes or parts of genes in E. coli, restriction analysis and
the construction of physical genome maps. Furthermore, combined with improved
DNA sequencing techniques, computer aided data collection and analysis and
a rapidly expanding source of information on genes and proteins in freely
accessible data banks allow genes to be proposed on basis of DNA or protein
sequence homology. At present approximately 50-70% of DNA sequences derived
from open reading frames can be defined by significant sequence homology
to known genes, gene products or conserved typical motifs in proteins or
DNA sequences. DNA sequence analysis is therefore the fastest way to identify
a large number of genes of a given genome
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