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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