BACKGROUND
the contact of a pathogenic bacterium with its eukaryotic host provokes a multitude of reactions.
a prerequisite for successful infection with the host is the cytadhesion of the bacterium generally mediated by surface localized proteins  <cit> .
besides adhesion, pathogens like listeria, yersinia and even some of the mycoplasmas are able to invade the host cells  <cit> .
an intracellular localization is obviously a privileged niche, as the bacteria are well protected from the immune system.
moreover, bacteria not only remain concealed, but have evolved strategies for an attack on the eukaryotic cell.
in secreting virulence factors, such as antigenic or toxic proteins, bacteria can mislead the host immune response or damage the colonized tissue  <cit> .
the large majority of exported proteins possess an n-terminal signal sequence  <cit> .
most signal sequences are recognized by the sec-dependent protein translocation complex , which mediates membrane translocation of unfolded precursors  <cit> .
the signal sequences of proteins predicted to be recognized by type i signal peptidases are composed of a short, positively charged amino-terminal region , a central hydrophobic region  and a more polar carboxyl region  containing the cleavage site  <cit> .
the signal peptides present in pre-lipoproteins additionally contain a well-conserved lipobox with an invariant cysteine residue that is lipid-modified prior to precursor cleavage by signal peptidase ii  <cit> .
cleavage of the signal peptide is not required for translocation of the proteins through the membrane, but is generally the final step in processing  <cit> .
however, some precursors remain membrane bound because of an uncleaved hydrophobic signal peptide and diffuse laterally from the translocase  <cit> .
in the last few years, computer programs such as psort-ii, psort-b, exprot and signalp have been developed to facilitate the identification of putative secreted proteins  <cit> .
comparison of proteomes of gram-negative bacteria, gram-positive bacteria and archaea using exprot revealed that the fraction of putative secreted proteins ranged from 8% in the archaeal bacterium methanococcus jannaschii to 37% in the mollicute mycoplasma pneumoniae  <cit> .
analysis of the exported proteins of bacillus subtilis found that only 50% of the secreted proteins were detected by genomic prediction, indicating that proteomic analyses of secreted proteins  are necessary for a comprehensive definition of all secreted proteins  <cit> .
only a handful of mollicute genomes have been decoded, but no analyses of their secretomes have been conducted.
a secreted protein, probably processed by the classical mechanism described above, has been characterized in the swine pathogen m. hyopneumoniae.
p <dig> is encoded as a precursor protein carrying a type i signal sequence and is found exclusively in the extra-cellular milieu suggesting cleavage by signal peptidase i. the expression of secreted p <dig> is coupled to that of the surface-exposed cilium adhesin p <dig>  which seems to represent a new variant of processed surface antigens.
p <dig> is derived from a  <dig> kda precursor protein by cleavage at amino acid residue   <dig>  the cleaved  <dig> kda n-terminal fragment, which carries an uncleaved type i signal sequence is found embedded in the membrane, in the cytoplasm and in a soluble form in the supernatant, whereas the mature p <dig>  proposed to be membrane bound, is the target of complex proteolytic cleavage, which leads to the subsequent release of some fragments into the supernatant  <cit> .
the lipoprotein malp- <dig> of m. fermentans is a further example of a surface-localized protein undergoing proteolysis after reaching its target.
site-specific cleavage leads to the generation of the membrane bound immune-stimulatory lipopeptide malp- <dig> and the release of the remainder  in a soluble form into the supernatant  <cit> .
in m. hominis, which is mainly found as a commensal in the urogenital tract, but has also been associated with human urogenital tract infections  <cit> , two variants of a cell-surface protein exist, one a lipoprotein  and a homologous p120' without a lipid anchor, but containing an uncleaved n-terminal signal sequence for type i signal peptidases  <cit> .
recently we identified a gene locus coding for a surface-localized protein complex composed of a  <dig> kda lipoprotein  tightly bound to an  <dig> kda precursor protein  with an uncleaved type i signal sequence  <cit> .
we have proposed that the uncleaved n-terminus extends into the cytoplasm and thus mediates an interaction with the concomitantly expressed cytoplasmic hint protein.
hint is found in prokaryotes and eukaryotes and in the latter is known to function as an adenosine nucleotidyl-hydrolase  <cit> .
the data in this paper suggest that the signal peptidase i recognition site is protected in the membrane bound form of p <dig>  but is accessible as the first step in the release of the helical part of p <dig> into the cell culture supernatant, a process which is accompanied by a decrease in size of  <dig> kda.
RESULTS
to confirm our hypothesis that the p60/p <dig> membrane complex interacts with the cytoplasmic hint via the uncleaved n-terminus of p <dig> that extends into the cytoplasm, we set out to express different fragments of the p <dig> protein to map the domain of p <dig> that interacted with hint.
as mycoplasmas use tga as a tryptophan codon, rather than a stop codon as in e. coli, the tga codons were mutated to tgg to allow expression.
however, even though we mutated all tga codons to tgg in the hita gene, which encodes p <dig>  we were not able to express and purify p <dig> peptides with an intact n-terminus.
while p80- <dig>  the c-terminal region of p <dig> from aa  <dig> to aa  <dig>  was stably expressed in e. coli, p80-nt, the initial 21aa of the n-terminus, and p80- <dig>  the n-terminal portion of p <dig> from aa  <dig> to aa  <dig>  were degraded at the n-terminus during purification .
purification of the  <dig>  kda p80-nt fused to dihydrofolate reductase  led to complete loss of the p <dig> region.
as p <dig> nt did not contain the cleavage site of the signal peptidase i this proteolysis may be due to the presence of further processing signals within the first  <dig> aa of p <dig>  the isolation of p80- <dig> revealed rapid degradation of the  <dig>  kda peptide with loss of  <dig>   <dig> and  <dig> kda portions of the protein and loss of the poly his tag.
expression of the whole rp <dig> polypeptide chain  resulted in a protein about  <dig> kda smaller than expected, which lacked the poly his tag .
the conjecture that n-terminal degradation occurs was supported by western blot analyses with anti-tetra his antibodies in which the purified forms of p80- <dig> and rp <dig> were not detectable .
as conventional protease inhibitors had no effect on the p <dig> degradation  and as type i signal peptidases share the unusual feature of being resistant to general inhibitors of the four other peptidase classes  <cit>  we speculated that the degradation observed in the expressed rp <dig> may be in fact the result of physiological processing in e. coli.
analysis of the mechanism of rp <dig> processing
in order to test the hypothesis that the degradation of rp <dig> is mediated by e. coli signal peptidase i, we characterized different mutants of the recombinant p <dig> protein according to their processing in e. coli.
using pcr, we constructed clones expressing the mature p <dig> protein without the  <dig> aa signal peptide  and an  <dig> aa n-terminal truncated p <dig> protein, which corresponds to the proposed helical, surface-localized part of p <dig> .
as the absence of proline at the + <dig> position of the mature protein is consistent with the observation that the spase i of e.coli was inhibited by recombinant precursor proteins having a proline residue at this position  <cit> , we generated a p <dig> precursor with an asn to pro mutation  at the  position in the mature protein expecting a reduced or complete inhibition of signal peptide cleavage .
western-blot analysis of the heterologous expressed p <dig> variants in whole e. coli lysates revealed that a marked reduction of p <dig> processing was indeed observed for the  mutant, while the sizes of secreted p <dig> variants corresponded to that of the helical variant .
small amounts of rp <dig> precursor were only observed in the lysate after prolonged exposure in immunostaining with the p80-specific mab lf <dig> .
all variants, with the exception of the n/p mutant, were transported across the inner and the outer membranes and were of nearly the same size as the secreted  <dig> kda peptide of the p <dig> protein found in m. hominis .
in the case of p80- and p80-, the two variants lacking signal sequences as translocation signals, this transport may be mediated by an alternative, sec-independent export mechanism.
p <dig> secretion in mycoplasma hominis
the data suggest that the recombinant p <dig> processing in e. coli is signal peptidase i-mediated and therefore we investigated the protection of the signal peptidase i recognition site of p <dig> in mycoplasma hominis  <cit> .
therefore, we analyzed the cellular proteins and the proteins in the culture supernatant in a mycoplasma culture in early-, mid- and stationary growth phases.
to ensure that no viable cells were left in the supernatant we checked the separation efficiency by determining the titres of mycoplasmas in the supernatant and the original culture.
the titres in the supernatants were  <dig> % to  <dig> % of those of the original cultures, demonstrating that the western blot analysis would not be confounded by inclusion of cellular proteins in the supernatants .
as might be expected the stationary phase culture contained several proteins that might be released from lysed cells, including p <dig>  p <dig>  oppa and cytoplasmic proteins such as p <dig> .
however, the same membrane proteins were found in the mid-logarithmic phase suggesting release of these proteins from living cells into the supernatant.
indeed, the amount of the cytoplasmic p <dig> was reduced in this culture suggesting that in mid-logarithmic phase few proteins from lysed cells are present.
lipoproteins, such as p <dig> and p <dig> were also found in the supernatant of a mycoplasma culture at the beginning of logarithmic growth , whereas oppa was absent.
in general, immunostaining of p <dig> was weak in samples derived from the supernatant of a broth culture.
this might be due to a masking of p <dig> by albumin, which was present in high concentrations in the culture medium and of similar size as the secreted p <dig> protein.
interestingly, the p <dig> protein was the only one of the proteins analyzed that had a decreased size in the supernatant.
to exclude the possibility that the reduction in size was simply a result of the large amount of albumin in the culture medium, we purified p <dig> from the supernatant of a culture in mid logarithmic growth using affinity chromatography.
in addition we analyzed p <dig> as a control.
as shown in figure 3-b, the secreted p <dig> peptide was decreased in size by approximately  <dig> kda in comparison to the cellular form, whereas p <dig> remained unaltered.
a degree of processing of the p <dig> precursor was detectable, with several distinct p <dig> staining bands between  <dig> and  <dig> kda, running through distinct steps of degradation as shown for the recombinant p80- <dig> peptide .
as the activity of proteases often depends on the presence of divalent cations, we examined the secretion of mycoplasma proteins in the presence or absence of divalent cations.
mycoplasma cells were incubated for  <dig> h at 37°c in phosphate buffered saline  supplemented with up to  <dig> mm magnesium or calcium chloride, or chelating agents as edta and egta.
the addition of divalent cations did not increase the processing of p <dig> .
as the presence of  <dig> mm calcium ions resulted in a complete disappearence of p <dig> from the supernatant we examined the release of mycoplasma proteins from cells following stepwise addition of calcium ions at concentrations of  <dig> to  <dig> mm .
silver staining of the cell and supernatant preparations revealed that only a few proteins were released in the supernatant whereas most proteins remained cell-embedded .
increasing the calcium chloride concentration to more that  <dig>  mm led to a dramatic reduction in secretion.
all the antigens analyzed, with the exception of oppa, were found in the supernatant, even the cytoplasmic proteins p <dig> and elongation factor tu  .
the cytoplasmic proteins that were detected in the supernatant were not released from lysed cells as the titres of the cultures remained the same throughout the experiment .
these findings are in accordance with those of antelmann and coworkers, who found several cytoplasmic proteins, such as elongation factor g  and arginase  as to be components of the secretome of bacillus subtilis  <cit> .
increasing the concentration of calcium ions to more than  <dig>  mm cacl2inhibited a release of most mycoplasma proteins from the cells .
of the proteins analyzed, only ef-tu was found in the supernatant of a sample treated with  <dig>  mm cacl <dig> .
thus the surface-localized p <dig> protein, normally complexed with the p <dig> lipoprotein in the membrane, appears to be released into the extra-cellular environment as a  <dig> kda cleavage product.
our model of processing describes the initial cleavage of the  <dig>  kda signal peptide by a type i signal peptidase, followed by further degradation by unknown mechanisms, leading to a stable  <dig> kda peptide.
discussion
the data presented here indicate that the membrane protein p <dig> of mycoplasma hominis is a representative of a new group of proteins in the mollicutes.
p <dig> occurs as a precursor protein  anchored in the membrane and exposed on the surface, and is also released into the extra-cellular environment.
as p <dig> and p <dig> are concomitantly expressed and both complexed in the membrane fraction it is likely that the soluble p <dig> variant is derived from the membrane bound precursor.
interestingly, the findings from the heterologous expression of different p <dig> mutants in e. coli suggest that release of p <dig> is initiated by cleavage of the n-terminal type i signal sequence, followed by amino-terminal proteolysis by an unknown mechanism, leading to a stable p <dig> peptide with a predicted helical structure in the supernatant.
as the genome of m. genitalium, the smallest known self-sufficient organism, lacks a gene for the type i signal peptidase  <cit>  it was initially speculated that this enzyme may be absent in the mollicutes, despite the fact that several proteins with type i-signal sequences had been identified  <cit> .
however signal peptidase i genes have been found in the genomic sequences of mycoplasma gallisepticum  and m. pulmonis   <cit> .
the release of mycoplasmal proteins into the extra-cellular milieu may follow different pathways to those described previously for other bacteria.
while the exclusive detection of p <dig> of m. hyopneumoniae in the extra-cellular matrix suggests that the intrinsic n-terminal type i signal peptide is immediately cleaved after translocation of the precursor through the membrane, as would be expected from current knowledge of the classical protein secretion pathway, the precursor proteins p <dig> and p120' of m. hominis are attached to the membrane without being processed or released into the supernatant  <cit> .
the later release of a surface-localized peptide is described here for the first time.
davis and wise described site-specific proteolysis of the lipid-anchored malp- <dig> of m. fermentans that leads to the generation of the immune-stimulatory lipopeptide malp- <dig> and showed that the residual rf peptide was soluble and was released into the supernatant  <cit> .
processing of the p <dig> cilium adhesin precursor, p <dig>  of m. hyopneumoniae appears to be quite complex.
the precursor protein p <dig>  which carries a type i signal sequence with a cleavage site between aa  <dig> and aa  <dig>  is predominantly cleaved between aa  <dig> and aa  <dig>  suggesting that the sp-i signature may be essential only for the translocation of the whole precursor protein across the membrane.
the cleavage products p <dig> and p <dig> both remain closely associated with the membrane.
while p <dig> is generally subjected to further site-directed proteolysis, leading to the release of peptides into the extra-cellular milieu, further processing of p <dig> occurs to be strain dependent  <cit> .
release of proteins into the surroundings of a mycoplasma cell should lead to an immediate alteration of the cell surface architecture and, as most membrane proteins are targets for the host immune response  <cit> , may also interfere with the host effector response.
type ii secreted proteins appeared to be typically associated with non-invasive organisms colonizing mucosal surfaces, such as mycoplasma hominis, and are considered to be required for the establishment of an infection at these sites.
recently in legionella pneumophila a type ii protein secretion system was characterized as a virulence factor linked to an intracellular infection  <cit> .
p <dig> has significant similarity with two protein sequences, a hypothetical protein of m. pulmonis  the gene of which is followed by p <dig>  and hint  gene homologues, and a rhoptry protein of plasmodium yoelii yoelii .
rhoptry proteins are released during host cell invasion by apicomplexans  <cit> .
as p <dig> interacts with hint, a cytoplasmic protein found in all kingdoms, which in eukaryotes hydrolyses amp derivatives and in yeasts functions as a regulator of an rna polymerase ii domain  <cit> , a further scenario is imaginable: we do not know which factors promote the processing of the surface-exposed p <dig> protein.
however, release of the helical p <dig> peptide is accompanied by the retention of the signal peptide i in the membrane.
some signal peptides have been found to leak back into the cell where they bind to proteins, such as the ca2+-binding calmodulin  <cit> , or are presented to the immune system  <cit> , where they probably have a secondary signaling function distinct from their role in targeting  <cit> .
our results indicate that an increase in calcium ions prevents secretion of mycoplasma proteins.
thus, an extra- or intra- cellular stimulus – such as a local variation in the concentration of ca2+ ions – may activate the signal peptidase induced processing of the membrane bound p <dig> protein.
this would lead to a soluble p80-helix and the membrane anchored signal peptide.
after loss of the transmembrane spanning c-terminal amino acids or due to a leakage of the whole signal sequence  the signal peptide could reach the cytoplasm where an interaction with hint may be a further step in modulating or promoting cellular processes such as growth or activation of gene expression, a process we are currently investigating.
CONCLUSIONS
the data presented here clearly demonstrate that p <dig> is a secreted antigen of mycoplasma hominis.
this is, to our knowledge, the first description of secreted protein with a type i signal sequence that is stably embedded in the membrane as precursor protein and, as we propose as a model of secretion, is subsequently released from the membrane into the supernatant after signal peptidase i cleavage.
as in other mycoplasmas several proteins have been shown to either possess an uncleaved signal sequence or to be further processed after anchoring in the membrane, this may be a common phenomenon in mycoplasmas.
because of their minimal coding capacity, mycoplasmas may have evolved a strategy to use secretory proteins in a dual role, as surface localized proteins in the membrane for cell surface architecture, and, in response to a change in the environment, as a soluble protein released from the cell surface.

 the contact of a pathogenic bacterium with its eukaryotic host provokes a multitude of reactions. a prerequisite for successful infection with the host is the cytadhesion of the bacterium generally mediated by surface localized proteins. besides adhesion, pathogens like listeria, yersinia and even some of the mycoplasmas are able to invade the host cells. an intracellular localization is obviously a privileged niche, as the bacteria are well protected from the immune system. moreover, bacteria not only remain concealed, but have evolved strategies for an attack on the eukaryotic cell. in secreting virulence factors, such as antigenic or toxic proteins, bacteria can mislead the host immune response or damage the colonized tissue. the large majority of exported proteins possess an n-terminal signal sequence. most signal sequences are recognized by the sec-dependent protein translocation complex, which mediates membrane translocation of unfolded precursors. the signal sequences of proteins predicted to be recognized by type i signal peptidases are composed of a short, positively charged amino-terminal region, a central hydrophobic region and a more polar carboxyl region containing the cleavage site. the signal peptides present in pre-lipoproteins additionally contain a well-conserved lipobox with an invariant cysteine residue that is lipid-modified prior to precursor cleavage by signal peptidase ii. cleavage of the signal peptide is not required for translocation of the proteins through the membrane, but is generally the final step in processing. however, some precursors remain membrane bound because of an uncleaved hydrophobic signal peptide and diffuse laterally from the translocase. in the last few years, computer programs such as psort-ii, psort-b, exprot and signalp have been developed to facilitate the identification of putative secreted proteins. comparison of proteomes of gram-negative bacteria, gram-positive bacteria and archaea using exprot revealed that the fraction of putative secreted proteins ranged from 8% in the archaeal bacterium methanococcus jannaschii to 37% in the mollicute mycoplasma pneumoniae. analysis of the exported proteins of bacillus subtilis found that only 50% of the secreted proteins were detected by genomic prediction, indicating that proteomic analyses of secreted proteins are necessary for a comprehensive definition of all secreted proteins. only a handful of mollicute genomes have been decoded, but no analyses of their secretomes have been conducted. a secreted protein, probably processed by the classical mechanism described above, has been characterized in the swine pathogen m. hyopneumoniae. p is encoded as a precursor protein carrying a type i signal sequence and is found exclusively in the extra-cellular milieu suggesting cleavage by signal peptidase i. the expression of secreted p is coupled to that of the surface-exposed cilium adhesin p which seems to represent a new variant of processed surface antigens. p is derived from a kda precursor protein by cleavage at amino acid residue the cleaved kda n-terminal fragment, which carries an uncleaved type i signal sequence is found embedded in the membrane, in the cytoplasm and in a soluble form in the supernatant, whereas the mature p proposed to be membrane bound, is the target of complex proteolytic cleavage, which leads to the subsequent release of some fragments into the supernatant. the lipoprotein malp- of m. fermentans is a further example of a surface-localized protein undergoing proteolysis after reaching its target. site-specific cleavage leads to the generation of the membrane bound immune-stimulatory lipopeptide malp- and the release of the remainder in a soluble form into the supernatant. in m. hominis, which is mainly found as a commensal in the urogenital tract, but has also been associated with human urogenital tract infections, two variants of a cell-surface protein exist, one a lipoprotein and a homologous p120' without a lipid anchor, but containing an uncleaved n-terminal signal sequence for type i signal peptidases. recently we identified a gene locus coding for a surface-localized protein complex composed of a kda lipoprotein tightly bound to an kda precursor protein with an uncleaved type i signal sequence. we have proposed that the uncleaved n-terminus extends into the cytoplasm and thus mediates an interaction with the concomitantly expressed cytoplasmic hint protein. hint is found in prokaryotes and eukaryotes and in the latter is known to function as an adenosine nucleotidyl-hydrolase. the data in this paper suggest that the signal peptidase i recognition site is protected in the membrane bound form of p but is accessible as the first step in the release of the helical part of p into the cell culture supernatant, a process which is accompanied by a decrease in size of kda.  to confirm our hypothesis that the p60/p membrane complex interacts with the cytoplasmic hint via the uncleaved n-terminus of p that extends into the cytoplasm, we set out to express different fragments of the p protein to map the domain of p that interacted with hint. as mycoplasmas use tga as a tryptophan codon, rather than a stop codon as in e. coli, the tga codons were mutated to tgg to allow expression. however, even though we mutated all tga codons to tgg in the hita gene, which encodes p we were not able to express and purify p peptides with an intact n-terminus. while p80- the c-terminal region of p from aa to aa was stably expressed in e. coli, p80-nt, the initial 21aa of the n-terminus, and p80- the n-terminal portion of p from aa to aa were degraded at the n-terminus during purification. purification of the kda p80-nt fused to dihydrofolate reductase led to complete loss of the p region. as p nt did not contain the cleavage site of the signal peptidase i this proteolysis may be due to the presence of further processing signals within the first aa of p the isolation of p80- revealed rapid degradation of the kda peptide with loss of and kda portions of the protein and loss of the poly his tag. expression of the whole rp polypeptide chain resulted in a protein about kda smaller than expected, which lacked the poly his tag. the conjecture that n-terminal degradation occurs was supported by western blot analyses with anti-tetra his antibodies in which the purified forms of p80- and rp were not detectable. as conventional protease inhibitors had no effect on the p degradation and as type i signal peptidases share the unusual feature of being resistant to general inhibitors of the four other peptidase classes we speculated that the degradation observed in the expressed rp may be in fact the result of physiological processing in e. coli. analysis of the mechanism of rp processing in order to test the hypothesis that the degradation of rp is mediated by e. coli signal peptidase i, we characterized different mutants of the recombinant p protein according to their processing in e. coli. using pcr, we constructed clones expressing the mature p protein without the aa signal peptide and an aa n-terminal truncated p protein, which corresponds to the proposed helical, surface-localized part of p. as the absence of proline at the + position of the mature protein is consistent with the observation that the spase i of e.coli was inhibited by recombinant precursor proteins having a proline residue at this position, we generated a p precursor with an asn to pro mutation at the position in the mature protein expecting a reduced or complete inhibition of signal peptide cleavage. western-blot analysis of the heterologous expressed p variants in whole e. coli lysates revealed that a marked reduction of p processing was indeed observed for the mutant, while the sizes of secreted p variants corresponded to that of the helical variant. small amounts of rp precursor were only observed in the lysate after prolonged exposure in immunostaining with the p80-specific mab lf. all variants, with the exception of the n/p mutant, were transported across the inner and the outer membranes and were of nearly the same size as the secreted kda peptide of the p protein found in m. hominis. in the case of p80- and p80-, the two variants lacking signal sequences as translocation signals, this transport may be mediated by an alternative, sec-independent export mechanism. p secretion in mycoplasma hominis the data suggest that the recombinant p processing in e. coli is signal peptidase i-mediated and therefore we investigated the protection of the signal peptidase i recognition site of p in mycoplasma hominis. therefore, we analyzed the cellular proteins and the proteins in the culture supernatant in a mycoplasma culture in early-, mid- and stationary growth phases. to ensure that no viable cells were left in the supernatant we checked the separation efficiency by determining the titres of mycoplasmas in the supernatant and the original culture. the titres in the supernatants were % to % of those of the original cultures, demonstrating that the western blot analysis would not be confounded by inclusion of cellular proteins in the supernatants. as might be expected the stationary phase culture contained several proteins that might be released from lysed cells, including p p oppa and cytoplasmic proteins such as p. however, the same membrane proteins were found in the mid-logarithmic phase suggesting release of these proteins from living cells into the supernatant. indeed, the amount of the cytoplasmic p was reduced in this culture suggesting that in mid-logarithmic phase few proteins from lysed cells are present. lipoproteins, such as p and p were also found in the supernatant of a mycoplasma culture at the beginning of logarithmic growth, whereas oppa was absent. in general, immunostaining of p was weak in samples derived from the supernatant of a broth culture. this might be due to a masking of p by albumin, which was present in high concentrations in the culture medium and of similar size as the secreted p protein. interestingly, the p protein was the only one of the proteins analyzed that had a decreased size in the supernatant. to exclude the possibility that the reduction in size was simply a result of the large amount of albumin in the culture medium, we purified p from the supernatant of a culture in mid logarithmic growth using affinity chromatography. in addition we analyzed p as a control. as shown in figure 3-b, the secreted p peptide was decreased in size by approximately kda in comparison to the cellular form, whereas p remained unaltered. a degree of processing of the p precursor was detectable, with several distinct p staining bands between and kda, running through distinct steps of degradation as shown for the recombinant p80- peptide. as the activity of proteases often depends on the presence of divalent cations, we examined the secretion of mycoplasma proteins in the presence or absence of divalent cations. mycoplasma cells were incubated for h at 37°c in phosphate buffered saline supplemented with up to mm magnesium or calcium chloride, or chelating agents as edta and egta. the addition of divalent cations did not increase the processing of p. as the presence of mm calcium ions resulted in a complete disappearence of p from the supernatant we examined the release of mycoplasma proteins from cells following stepwise addition of calcium ions at concentrations of to mm. silver staining of the cell and supernatant preparations revealed that only a few proteins were released in the supernatant whereas most proteins remained cell-embedded. increasing the calcium chloride concentration to more that mm led to a dramatic reduction in secretion. all the antigens analyzed, with the exception of oppa, were found in the supernatant, even the cytoplasmic proteins p and elongation factor tu. the cytoplasmic proteins that were detected in the supernatant were not released from lysed cells as the titres of the cultures remained the same throughout the experiment. these findings are in accordance with those of antelmann and coworkers, who found several cytoplasmic proteins, such as elongation factor g and arginase as to be components of the secretome of bacillus subtilis. increasing the concentration of calcium ions to more than mm cacl2inhibited a release of most mycoplasma proteins from the cells. of the proteins analyzed, only ef-tu was found in the supernatant of a sample treated with mm cacl. thus the surface-localized p protein, normally complexed with the p lipoprotein in the membrane, appears to be released into the extra-cellular environment as a kda cleavage product. our model of processing describes the initial cleavage of the kda signal peptide by a type i signal peptidase, followed by further degradation by unknown mechanisms, leading to a stable kda peptide. discussion the data presented here indicate that the membrane protein p of mycoplasma hominis is a representative of a new group of proteins in the mollicutes. p occurs as a precursor protein anchored in the membrane and exposed on the surface, and is also released into the extra-cellular environment. as p and p are concomitantly expressed and both complexed in the membrane fraction it is likely that the soluble p variant is derived from the membrane bound precursor. interestingly, the findings from the heterologous expression of different p mutants in e. coli suggest that release of p is initiated by cleavage of the n-terminal type i signal sequence, followed by amino-terminal proteolysis by an unknown mechanism, leading to a stable p peptide with a predicted helical structure in the supernatant. as the genome of m. genitalium, the smallest known self-sufficient organism, lacks a gene for the type i signal peptidase it was initially speculated that this enzyme may be absent in the mollicutes, despite the fact that several proteins with type i-signal sequences had been identified. however signal peptidase i genes have been found in the genomic sequences of mycoplasma gallisepticum and m. pulmonis. the release of mycoplasmal proteins into the extra-cellular milieu may follow different pathways to those described previously for other bacteria. while the exclusive detection of p of m. hyopneumoniae in the extra-cellular matrix suggests that the intrinsic n-terminal type i signal peptide is immediately cleaved after translocation of the precursor through the membrane, as would be expected from current knowledge of the classical protein secretion pathway, the precursor proteins p and p120' of m. hominis are attached to the membrane without being processed or released into the supernatant. the later release of a surface-localized peptide is described here for the first time. davis and wise described site-specific proteolysis of the lipid-anchored malp- of m. fermentans that leads to the generation of the immune-stimulatory lipopeptide malp- and showed that the residual rf peptide was soluble and was released into the supernatant. processing of the p cilium adhesin precursor, p of m. hyopneumoniae appears to be quite complex. the precursor protein p which carries a type i signal sequence with a cleavage site between aa and aa is predominantly cleaved between aa and aa suggesting that the sp-i signature may be essential only for the translocation of the whole precursor protein across the membrane. the cleavage products p and p both remain closely associated with the membrane. while p is generally subjected to further site-directed proteolysis, leading to the release of peptides into the extra-cellular milieu, further processing of p occurs to be strain dependent. release of proteins into the surroundings of a mycoplasma cell should lead to an immediate alteration of the cell surface architecture and, as most membrane proteins are targets for the host immune response, may also interfere with the host effector response. type ii secreted proteins appeared to be typically associated with non-invasive organisms colonizing mucosal surfaces, such as mycoplasma hominis, and are considered to be required for the establishment of an infection at these sites. recently in legionella pneumophila a type ii protein secretion system was characterized as a virulence factor linked to an intracellular infection. p has significant similarity with two protein sequences, a hypothetical protein of m. pulmonis the gene of which is followed by p and hint gene homologues, and a rhoptry protein of plasmodium yoelii yoelii. rhoptry proteins are released during host cell invasion by apicomplexans. as p interacts with hint, a cytoplasmic protein found in all kingdoms, which in eukaryotes hydrolyses amp derivatives and in yeasts functions as a regulator of an rna polymerase ii domain, a further scenario is imaginable: we do not know which factors promote the processing of the surface-exposed p protein. however, release of the helical p peptide is accompanied by the retention of the signal peptide i in the membrane. some signal peptides have been found to leak back into the cell where they bind to proteins, such as the ca2+-binding calmodulin, or are presented to the immune system, where they probably have a secondary signaling function distinct from their role in targeting. our results indicate that an increase in calcium ions prevents secretion of mycoplasma proteins. thus, an extra- or intra- cellular stimulus – such as a local variation in the concentration of ca2+ ions – may activate the signal peptidase induced processing of the membrane bound p protein. this would lead to a soluble p80-helix and the membrane anchored signal peptide. after loss of the transmembrane spanning c-terminal amino acids or due to a leakage of the whole signal sequence the signal peptide could reach the cytoplasm where an interaction with hint may be a further step in modulating or promoting cellular processes such as growth or activation of gene expression, a process we are currently investigating.  the data presented here clearly demonstrate that p is a secreted antigen of mycoplasma hominis. this is, to our knowledge, the first description of secreted protein with a type i signal sequence that is stably embedded in the membrane as precursor protein and, as we propose as a model of secretion, is subsequently released from the membrane into the supernatant after signal peptidase i cleavage. as in other mycoplasmas several proteins have been shown to either possess an uncleaved signal sequence or to be further processed after anchoring in the membrane, this may be a common phenomenon in mycoplasmas. because of their minimal coding capacity, mycoplasmas may have evolved a strategy to use secretory proteins in a dual role, as surface localized proteins in the membrane for cell surface architecture, and, in response to a change in the environment, as a soluble protein released from the cell surface. 
all other p variants were released into the supernatant, in general as a kda protein encompassing the helical part of p incubation of m. hominis cells in phosphate buffered saline supplemented with divalent cations revealed that the release of mycoplasma proteins into the supernatant was inhibited by high concentrations of calciumions. in general the p protein is transported across the membrane and remains complexed to p surface-exposed and membrane anchored via the uncleaved signal sequence. in mycoplasma hominis, the genes encoding the surface-localized membrane complex p60/p are in an operon with a gene encoding a cytoplasmic, nucleotide-binding protein with a characteristic histidine triad motif. western blot analysis of recombinant p mutants expressed in e. coli and differing in the n-terminal region revealed that mutation of the + position of the mature protein which is important for signal peptidase i recognition resulted in reduced p secretion. as the membrane anchored p carries an n-terminal uncleaved signal peptide we have proposed that the n-terminus extends into the cytoplasm and interacts with the cytosolic hint. further characterization of p suggested that the kda signal peptide is protected from cleavage only in the membrane bound form. loss of the kda signal peptide seems to be a pre-requisite for p secretion, which is followed by a proteolytic process leading to a helical kda product.
 mycoplasmas are cell wall-less bacteria which encode a minimal set of proteins. in mycoplasma hominis, the genes encoding the surface-localized membrane complex p60/p are in an operon with a gene encoding a cytoplasmic, nucleotide-binding protein with a characteristic histidine triad motif. hint is found in both procaryotes and eukaryotes and known to hydrolyze adenosine nucleotides in eukaryotes. immuno-precipitation and biacore analysis revealed an interaction between hint and the p domain of the membrane complex. as the membrane anchored p carries an n-terminal uncleaved signal peptide we have proposed that the n-terminus extends into the cytoplasm and interacts with the cytosolic hint.  further characterization of p suggested that the kda signal peptide is protected from cleavage only in the membrane bound form. we found several proteins were released into the supernatant of a logarithmic phase mycoplasma culture, including p which was reduced in size by kda. western blot analysis of recombinant p mutants expressed in e. coli and differing in the n-terminal region revealed that mutation of the + position of the mature protein which is important for signal peptidase i recognition resulted in reduced p secretion. all other p variants were released into the supernatant, in general as a kda protein encompassing the helical part of p incubation of m. hominis cells in phosphate buffered saline supplemented with divalent cations revealed that the release of mycoplasma proteins into the supernatant was inhibited by high concentrations of calciumions.  our model for secretion of the p protein of m. hominis implies a two-step process. in general the p protein is transported across the membrane and remains complexed to p surface-exposed and membrane anchored via the uncleaved signal sequence. loss of the kda signal peptide seems to be a pre-requisite for p secretion, which is followed by a proteolytic process leading to a helical kda product. we propose that this novel form of two-step secretion is one of the solutions to a life with a reduced gene set. 