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Comparative genome analysis of proteases, oligopeptide uptake and secretion systems in Mycoplasma spp


Mycoplasmas are very fastidious in their nutritional requirements for in vitro growth and have limited biosynthetic capacity, a reflection of their reduced genomes. As a result, these bacteria depend upon external metabolites for nutrition and growth and have developed dependence on their hosts for survival and maintenance. Protein degradation and peptide importation play an important role in Mycoplasma spp. nutrition, and proteases can play a role in host adaptation and pathogenicity. Here, we present a general survey on the genes involved in protein degradation, secretion and importation, comparing all available Mollicute genomes.

Mycoplasma; proteases; minimal genomes; secretion systems


Comparative genome analysis of proteases, oligopeptide uptake and secretion systems in Mycoplasma spp

Charley Christian Staats; Juliano Boldo; Leonardo Broetto; Marilene Vainstein; Augusto Schrank

Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

Send correspondence to Send correspondence to Augusto Schrank Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul Caixa Postal 15005, 91501-970 Porto Alegre, RS, Brazil E-mail:


Mycoplasmas are very fastidious in their nutritional requirements for in vitro growth and have limited biosynthetic capacity, a reflection of their reduced genomes. As a result, these bacteria depend upon external metabolites for nutrition and growth and have developed dependence on their hosts for survival and maintenance. Protein degradation and peptide importation play an important role in Mycoplasma spp. nutrition, and proteases can play a role in host adaptation and pathogenicity. Here, we present a general survey on the genes involved in protein degradation, secretion and importation, comparing all available Mollicute genomes.

Key words:Mycoplasma, proteases, minimal genomes, secretion systems.


Mycoplasmas are considered the smallest cells capable of propagation in cell-free medium, and some species are pathogenic to humans, animals and plants. In spite of their reduced genomes, due to significant gene loss through evolution/adaptation, mycoplasmas are a very successful group of organisms, as judged by their large number of species and habitats (Razin et al., 1998). The 'minimum cell' life style of mycoplasmas became possible by the adoption of a parasitic mode of life, exploiting nutrients not synthesized by themselves, and evolving systems to invade and to persist in their hosts (van Ham et al., 2003).

Over the last few years, the genomes of nine Mycoplasma species were sequenced, reinforcing comparative genome studies that allow a better understanding of their metabolism and the relations with their hosts. Mycoplasmas evolved from gram-positive bacteria and, through evolution, lost the cell wall and many metabolic pathways for the synthesis of macromolecule building blocks. Mycoplasmas possess no complete routes for amino acids synthesis and degradation, implying that these monomers must be acquired either from their hosts or from a culture medium, depending upon membrane transporters (Vasconcelos et al., 2005). Exogenous peptides are an important source of amino acids. Indeed, bacteria have evolved peptide transport systems that also assist in responses to environmental changes, mediating functions such as quorum sensing, sporulation, pheromone transport, and chemotaxis (Wang et al., 2004).

Despite the presence of a complete set of genes responsible for essential cell activities such as replication, transcription and translation, genes involved in posttranslational protein modifications are not readily disclosed by the annotation of the mycoplasma genomes. Some of these processes, such as protein maturation and localization, are intrinsically dependent on proteases. Microbial proteases may also play important roles in pathogenicity and nutrition.

Bacterial development is also dependent on the secretion of proteins with a plethora of functions. One of the major transport routes, the so-called Sec pathway, is conserved in all domains of life and is the only system found in mycoplasmas by genome surveys (Stephenson, 2005).

In this work, we present a general survey on the genes involved in protein metabolism, based on the available mycoplasma genomes.

Material and Methods

The complete genome sequences of the Mycoplasma spp. used in this work were retrieved from the NCBI data base (, as available in October, 2005. Primary searches were conducted using BLAST search tools (Altschul et al., 1990), or based on annotated genome files. The search for Opp (oligopeptide transport genes) and secretion systems was conducted using InterPro entries for Bacillus subtilis components (, Mulder et al., 2005). The classification and analysis of proteases were done according to the MEROPS peptidase database (, Rawlings et al., 2004).

Results and Discussion

Oligopeptide importing

Mycoplasma genomes possess a diversity of ABC transporters predicted to be involved in the uptake of several inorganic and organic substrates. One class of ABC transporters, the peptide/opine/nickel uptake transporter family (3.A.1.5.1), is involved in oligopeptide uptake with high affinity for tripeptides (Transport Classification Database,

The known genomic organization of the Opp operon in Mollicutes is shown in Table 1. Proteins encoded by this operon are anchored in the cell membrane; they transport oligopeptides from the extracellular milieu and represent an important form of nutrition (Detmers et al., 1998). Mesoplasma florum is the only species with no sequences related to the Opp system, as far as predicted by the annotation. The remaining genomes vary from one to two copies of the operon and also scattered single cistron copies. This distribution does not follow the division hominis/ pneumoniae clades. The complete Opp operon (OppABCDF) was annotated only in M. mycoides and in Phytoplasma, and is present in two copies. It is noteworthy that in one of the M. mycoides operons the cistron order is altered from ABCDF to BCDFA. Moreover, in both species there are scattered copies of single components elsewhere in the genome. Two copies of the incomplete operon (lacking OppA) are present in three species (M. pulmonis, M. penetrans and M. hyopneumoniae). The same incomplete operon is present, as single copies, in all five genomes; however, in M. synoviae, the cistron order is different, and in M. mobile an extra copy of OppF is present.

The function of OppA as substrate-binding protein (oligopeptide recognition) is well recognized in bacteria (LeDeaux et al., 1997; Detmers et al., 1998). In Mycoplasma hominis (genome sequence not available), OppA functions as the P100 adherence-associated lipoprotein, and the operon is organized as OppABCDF (Henrich et al., 1999). Therefore, an important role for OppA in oligopeptide uptake could be expected in other Mollicutes. However, the OppA gene was found only in two Mollicute genome sequences (Table 1). This raises the question if OppA is really a necessary component of the oligopeptide uptake systems in these bacteria. Nevertheless, the low conservation of this protein could hinder its annotation. In addition, the habitat broadness of Mollicutes could result in strong selection/adaptation, especially for proteins involved in the recognition (binding) of oligopeptides, expected to be variable in different habitats. Also, lipoproteins are among the most prominent components of mycoplasma cell membranes (Razin et al., 1998), and the substrate recognition function of OppA could be fulfilled by one of these proteins.


Bacterial development is dependent on a plethora of proteolytic activities involved in diverse functions, such as protein homeostasis, pathogenicity and nutrient acquisition. Mycoplasma genomes analysis revealed a complex distribution of these enzymes (Table 2). ATP-dependent proteases, such as Lon and FtsH, that degrade aberrant proteins, were found in all genomes analyzed here. Lon is a DNA-binding protease that degrades regulatory and abnormal proteins and has both the proteolytic and the ATPase domains. FtsH is a membrane protease that degrades membrane and cytoplasmic proteins. However, other important proteases involved in abnormal protein degradation, such as ClpPX and HslUV, were not annotated in five Mycoplasma species analyzed previously. This wider in silico survey of 12 Mollicute genomes supports the hypothesis outlined by Wong and Houry (2004) that the protein homeostatic process in these organisms has shifted through evolution towards favoring protein degradation rather than protein folding. Lon-defective Escherichia coli mutants remain phenotypically stable when overproducing HslU and HslV proteases, denoting a probable substrate overlapping among these proteins under certain physiological conditions (Wu et al., 1999). This suggests that the lack of the HslUV system in mycoplasmas could be surpassed by the presence of Lon. FtsH is an endopeptidase, dependent on ATP and Zn2+, that degrades abnormally-folded proteins and the proteolysis products that otherwise cause cellular abnormalities. The absence of the HslUV system and the presence of Lon and FtsH appear to be conserved among mycoplasmas, except for M. florum that does not possess FtsH. The same applies to the absence of ClpPX, observed in Mollicute genomes except the Onion Yellow Phytoplasma, which possesses ClpX (Table 2).

Protease secretion in order to obtain peptides from the milieu is a common feature of bacteria (Morales et al., 2001). Most subtilisin-like and other serine proteases are secreted endopeptidases with little specificity for their substrates. The subtilisin-like serine proteases in the mycoplasma genomes belong to the subfamily S8A, which are endopeptidases. It is important to note that the gram-negative bacterium Dichelobacter has one subtilisin-like serine protease directly implicated in pathogenesis. Microbial pathogens often utilize secreted proteases as virulence factors, which may contribute largely to their pathogenicity. These proteases participate in tissue destruction, inactivation of host defense molecules, activation of key regulatory proteins or peptides and in nutrient acquisition. Some bacterial proteases can also activate bacterial toxins, thus triggering toxigenic pathogenesis. These proteases are also capable of degrading immunoglobulins and components of the complement system assisting infection propagation. Microbial proteases are very critical in enhancing pathogenesis of many severe diseases. However, only three out of the nine mycoplasmas analyzed here possess putative genes coding for serine proteases (Table 2).

Intracellular peptidases were also found in the present genome survey (Table 2). Bacterial leucyl aminopeptidases are involved in processing and maintaining a regular turnover of intracellular proteins and peptide breakdown products generated by intracellular proteases (Jenal and Hengge-Aronis, 2003). Methionyl aminopeptidase (Map) degrades "Ala/Ser-Pro" dipeptides, avoiding their accumulation, which could become toxic to the cell. Furthermore, oligopeptidase F (functions) acts in the degradation of intracellular oligopeptides generated from cell protease activity and can also cleave signal peptides. The main role of Map is to remove the initial methionine of many proteins during translation. The Xaa-Pro aminopeptidases, that hydrolyse "Xaa-Pro" dipeptides, and the prolyl dipeptidases, that release N-terminal residues from peptides, preferably (but not exclusively) a proline , were annotated in mycoplasma genomes (Table 2). The o-sialoglycoprotein endopeptidases cleave heavily o-sialoglycosylated proteins. These enzymes do not possess identifiable signal peptides and, therefore, are probably not secreted, establishing themselves as intracellular proteins. The involvement of these enzymes in pathogenicity remains to be demonstrated.

Protein secretion

The first description of secretion systems in mycoplasmas came with the genomes of Mycoplasma genitalium (Fraser et al., 1995) and M. pneumoniae (Himmelreich et al., 1996). These authors referred to the incomplete Sec translocation machinery in mycoplasmas compared to Haemophilus influenzae and E. coli, as a result of the lower complexity of the mycoplasma cell membrane. Now, the characterization of secretion systems in phylogenetically closer bacteria (Tjalsma et al., 2000; van Wely et al., 2001) and the availability of several Mollicute genomes allow a more accurate comparison. A survey of mycoplasma genomes, based on similarity searches using conserved domains of the Sec translocation proteins, yielded near-complete secretion systems in all individuals (Table 3). The absence of identifiable SecB protein is in agreement to the Sec translocation machinery of B. subtilis, in which other chaperone-like protein(s) would function as SecB-like protein (Tjalsma et al., 2000). The most intriguing fact is that some mycoplasmas are devoid of annotated pore-forming transmembrane proteins SecG or SecE.

Signal peptidases, such as signal peptidase I (Spase I) and signal peptidase II (Spase II), are responsible for cleaving off the hydrophobic N-terminal signal peptide regions of proteins to be exported or held in specific parts of the cell, such as the cytoplasmic membrane. Several types of Spase I from gram-negative and gram-positive bacteria have clear differences concerning gene size, gene copy number and substrate specificity, although there are substantial sequence similarities, as indicated by six different regions with conserved amino acids. Besides that, Spases type I are essential for cell life. The distribution of type I and type II Spases was found to be quite different in the mycoplasmas analyzed here (Table 2). Signal peptidases are thought to be fundamental in the pathogenicity of mycoplasmas, since their activities were found to be involved in the processing of a cilium adhesin from M. hyopneumoniae (Djordjevic et al., 2004) and M. pneumoniae and hence its their role in pathogenicity.


C.C.S, J.B. and L.B. are recipients of CAPES pre-doctoral fellowships. This work was supported by MCT/ CNPq and FAPERGS. The authors thank the two anonymous referees for their useful suggestions to improve the manuscript.

Received: April 4, 2006; Accepted: October 5, 2006.

Associate Editor: Ana Tereza Vasconcelos

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  • Send correspondence to

    Augusto Schrank
    Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul
    Caixa Postal 15005, 91501-970 Porto Alegre, RS, Brazil
  • Publication Dates

    • Publication in this collection
      14 May 2007
    • Date of issue


    • Accepted
      05 Oct 2006
    • Received
      04 Apr 2006
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