BACKGROUND
bacteria adapt to changing environments by regulating their gene expression through signal transduction systems.
two kinds of signal transduction systems exist in bacteria; the two component system  and serine/threonine kinases  and phosphatases  system  <cit> .
although both systems transduce signals by phosphorylation events, they have distinct ways of doing this.
while tcs uses a sensor histidine kinase and a regulator protein to transduce the signals, the stk /stp regulate gene expression by protein-protein interaction  <cit> .
however, it should be noted that not all kinases and phosphatases associated with serine or threonine residues in prokaryotes are stk/stp.
the stk/stp has special signature motifs  <cit>  and is restricted to selected species of bacteria.
it was once thought that bacteria have only tcs but not stk/stp.
however, evidence for the occurrence of stk/stp in bacteria continues to accumulate  <cit> .
also, it has been reported that bacterial tcs and stk/stp systems cross talk with each other  <cit> .
in addition to their role in the physiology, stk/stp plays a significant role in the virulence of some pathogenic bacteria, including bacteria relevant to public health such as yersinia and mycobacteria  <cit> .
for instance, ypka, an stk of yersinia pseudotuberculosis, is critical for the disruption of host cytoskeleton during infection  <cit> .
in mycobacterium tuberculosis, lack of stk pkng and pknh has been reported to show reduced viability of this bacterium and increased bacterial load, respectively, in mouse models  <cit> .
the significance of stk in the pathogenesis of staphylococcus aureus <cit> , streptococcus pneumoniae <cit> , s. pyogenes <cit> , pseudomonas aeruginosa <cit> , s. agalactiae <cit> , mycoplasma pneumoniae <cit>  and salmonella enterica <cit>  has also been reported.
with respect to stp, relatively few studies have been undertaken in understanding their role in bacterial virulence and most of them focus on pneumococcus  <cit> .
an stp  of s. pyogenes is required for the production of hemolysin and to cause apoptosis in the host cells  <cit> .
its homologue, stp <dig>  in group b streptococcus sp is also associated with the production of hemolysin and lack of this stp leads to less efficient systemic infection by this bacterium  <cit> .
very recently, an stp  of s. pneumoniae is found to have a role in the adherence of this species  <cit> .
besides, an stp of listeria monocytogenes is reported to be essential for the growth of this bacterium in murine model  <cit> .
mycoplasma genitalium is a bacterium that lacks a cell wall and is one of the smallest self-replicating organisms with a genome size of  <dig> kb  <cit> .
it is the etiological agent for the diseases non-gonococcal urethritis and cervicitis in men and women, respectively  <cit> .
in women, it is also implicated in diseases like endometritis, pelvic inflammatory syndrome and tubal infertility  <cit> .
additionally, m. genitailum coinfection in hiv patients has been reported to have increased shedding of hiv in urogenital mucosal regions of the female  <cit> .
although it was initially thought that m. genitalium primarily attaches with epithelial cells of the host to cause the disease, evidences indicate that it invades epithelial cells and is localized on the periphery of the nucleus of the infected cells  <cit> .
the intracellular m. genitailum is reported to persist within the infected cells for a long time  <cit> .
it should be noted that intracellular survival and persistence of this bacterium may require signal transduction mediated adaptation, as do other bacteria in similar circumstances  <cit> .
strikingly, however, m. genitalium and its close relative m. pneumoniae are lacking the classical bacterial tcs  <cit> , although a few mycoplasmas like m. penetrans and m. iowae do have tcs .
besides, both species have only a limited number of regulators controlling gene expression at the transcription level  <cit> , and this has been attributed to their small genomes due to reductive evolution.
nevertheless, these species have genes encoding stk and stp  <cit> .
in fact, the stk of m. pneumoniae has been demonstrated to have an effect on the adherence of this species  <cit> , although no such effect was noticed with an stp of this species   <cit> .
our long term objective is to determine the roles of stk and stp in m. genitalium pathogenesis and signal transduction.
ncbi database of m. genitalium genome sequence  <cit>  reveals that this bacterium possesses a gene encoding stk  and three genes encoding stp .
we initiated our studies first with mg_ <dig> because we had a mutant strain for this gene readily available from a transposon mutant library  <cit> .
here, we show that mg <dig> is an alkaline phosphatase and it dephosphorylates threonine phosphate.
we also report that m. genitalium lacking in mg <dig>  shows differentially phosphorylated proteins in two-dimensional gels.
in addition, we provide evidence that tim <dig> has reduced virulence as compared to wild type m. genitalium.
RESULTS
mg_ <dig> encodes a functional phosphatase
the gene mg_ <dig> is predicted to encode a serine/threonine phosphatase.
to verify this, we created the plasmid pmg207ex to overexpress mg <dig> protein in e. coli.
this plasmid was transformed into e. coli bl <dig>  strain and induced with iptg.
figure 1a shows the overexpression of his10mg <dig> protein by e. coli harboring the plasmid pmg207ex and its purification.
the purified his10mg <dig> protein separated onto sds-page and stained with coomassie blue exhibited a size of  <dig> kda .
this correlated with the predicted size of  <dig>  kda of mg_ <dig>
to determine if the purified his10mg <dig> protein was functional, we assayed the phosphatase activity of this protein using the substrate p-nitrophenyl phosphate .
the his10mg <dig> readily hydrolyzed pnpp in a dose dependent manner  in the alkaline ph of  <dig> .
to rule out the possibility that the observed phosphatase activity of his10mg <dig> was not due to e. coli host derived phosphatase, we used similarly overexpressed and purified his10ohr protein of m. genitalium as a control.
reactions with this protein  or reactions with heat inactivated his10mg <dig> or reactions with his10mg <dig> but without mg2+ in the reaction mixture showed no color formation with pnpp , indicating that the overexpressed protein was a functional phosphatase dependent upon mg2+ ion for its activity.
to further assess whether his10mg <dig> is a serine/threonine phosphatase, malachite green based phosphatase assay was performed using synthetic serine  or threonine  phosphopeptide.
no activity was noticed with either peptide in the presence of ni2+, a cation supplied with the assay kit .
however, substitution of ni2+ with mg2+ in the reaction mixture released the phosphate from threonine peptide , but this failed to release the phosphate from serine peptide.
we presume that the absence of activity with the serine phosphate peptide may be due to the requirement of appropriate conditions.
alternatively, it is possible that the serine phosphate in this particular peptide is un-accessible for the enzyme.
however, the fact that mg <dig> requires a metal  for its activity with pnpp or with threonine peptide suggests that it is a metal dependent phosphatase.
this observation is consistent with reports of other stps like stp of l. monocytogenes <cit> , phpp of s. pneumoniae <cit> , prpc of m. pneumoniae <cit>  and stp <dig> of s. agalactiae <cit> , all of which required divalent metal cofactor mn2+ for their activity.
in bacteria, stp belongs to two families, phosphoprotein phosphatases  and metal dependent phosphatases .
the major difference between these two groups appears to be their specificity for substrates.
while ppm specifically hydrolyzes serine or threonine phosphates, the ppp hydrolyzes, in addition to serine and threonine phosphates, histidine and tyrosine phosphates  <cit> .
although pp2c phosphatase, a member of the ppm family, has some catalytic similarities with ppp, this does not show any amino acid similarity with ppp  <cit> .
further, it appears that mg <dig> is only a closely related protein to pp2c phosphatase, because the cluster of orthologous groups  classification has placed this protein in a different group of bacterial phosphatase.
tim <dig> strain and its confirmation
to understand the role of mg <dig> in signal transduction and pathogenesis of m. genitalium, we sought to create a mutant strain through homologous recombination.
however, we were able to acquire a similar mutant strain from m. genitalium tn <dig> transposon mutant library generated by dr. john glass  <cit> .
the insertion of tn <dig> in the coding region of mg_ <dig> had already been determined by sequencing  <cit> .
in order to reconfirm this insertion and to check if this strain has any additional tn <dig> insertions due to sub-culturing, we probed the genomic dna of m. genitalium wild type g <dig> strain and tim <dig> cut with spei, in southern hybridization.
the membrane hybridized with radiolabeled dna of mg_ <dig> revealed strong signals around  <dig>  kb in the g <dig> strain and  <dig>  kb in the tim <dig>  in addition, a weak signal was also noticed in the tim <dig> strain around  <dig>  kb region .
the shift in hybridization signals for mg_ <dig> and also the presence of additional signals for mg_ <dig> in tim <dig> strain, as compared to g <dig> strain, reconfirmed that the gene was disrupted by tn <dig> insertion.
in addition, hybridization of the blot with gene fragment coding for gentamicin resistance, which is part of the transposon, revealed signals only at one position which corresponded to the signals of mg_ <dig> in the tim <dig> strain .
this indicated that this strain has no additional tn <dig> insertions in the chromosome and the mutant is stable.
further, to determine whether the transposon insertion indeed disrupted the expression of mg <dig> protein, we analyzed the proteins of g <dig> and tim <dig> strain in immunoblot with anti-mg <dig> antiserum.
this antiserum detected the mg <dig> protein only in the wild type g <dig> strain and not in the tim <dig> strain , indicating that the disruption of the gene affected the expression of the protein.
we do not expect that tn <dig> insertion in this strain  will have any polar effects on its downstream genes, because the transcription of the downstream genes is predicted to be in the opposite orientation .
this situation implies that complementation of the tim <dig> with a functional allele to assess the function of mg <dig> is of limited significance.
moreover, the only way by which the m. genitalium mutant strain can be complemented is through the use of a transposon which can insert a copy of the functional allele of the mutated gene in an unknown location of the chromosome.
it is very likely that the unknown location may be a functional gene and this will affect the interpretation of the complimented phenotype.
therefore, we have used a m. genitalium strain called tim <dig>  which bears the same transposon as in tim <dig>  inserted in the gene mg_ <dig>  as a control strain in some experiments.
the gene mg_ <dig> is predicted to code for a 3´-5´ exonuclease .
tim <dig> strain exhibits differentially phosphorylated proteins
as mg <dig> is a phosphatase presumed to be associated with signaling, it was predicted that absence of this protein might alter the phosphorylation status of some m. genitalium proteins.
to determine this, and also to identify some of the differentially phosphorylated proteins, we performed 2-d gel analysis of proteins from g <dig> and tim <dig> strains and stained them with pro-q diamond  and sypro ruby stains .
while the total proteins stained with sypro ruby showed similar profiles for g <dig> and tim <dig> strains, the phosphoproteins stained with pro-q diamond displayed different profiles for these strains.
these differences in phosphorylation appear not due to differences in the growth of the wild type  and mutant  strains as they showed no significant differences  in growth.
further, the differences do not appear due to variability in viability because both strains exhibited similar viability at the time of harvest .
the predominant difference was noticed to be at the high molecular weight  areas which are shown in large circles .
as can be seen, the gels from g <dig> showed relatively dense and larger stained areas as compared to gels from the tim <dig> strain, suggesting that some hmw proteins are less phosphorylated in tim <dig> strain.
however, these dense areas have shown no corresponding protein spots in sypro ruby stained gels, thus indicating that these areas do not represent real proteins but represent some artifacts.
therefore, we focused only on well separated and differentially phosphorylated proteins.
these included two proteins  which showed relatively dense staining in the gels of g <dig> strain but were weaker in the gels of tim <dig> strain, and three proteins  that showed stronger staining in the gels of tim <dig> strains but were weaker in the gels of g <dig>
to identify the differentially phosphorylated proteins, we subjected the protein spots 1– <dig> to mass spectrometry .
based on the number of peptide hits for each spots, the spots were identified as four different proteins  namely, pyruvate dehydrogenase complex e <dig> subunit α , a putative cytoskeleton protein , a conserved hypothetical protein  and thymidine phosphorylase .
interestingly, our study validates the findings of a previous study which identified pyruvate dehydrogenase e <dig> α chain and the protein encoded by mg_ <dig> as phosphoproteins of m. genitalium <cit> .
recently, both these proteins were found on the surface of m. genitalium <cit> , thus suggesting the possibility that they can play a role in m. genitalium-host interaction.
what is intriguing in this study, however, is the reduced phosphorylation of a protein  in a phosphatase  deficient mutant of m. genitalium.
theoretically, if proteins are dephosphorylated by a specific phosphatase, then the expectation, in the absence of the phosphatase, is no change in the phosphorylation levels or increased levels of phosphorylation of proteins.
in fact, the proteins identified in prpc mutant of m. pneumoniae <cit>  behave in this manner.
the differentially phosphorylated proteins identified in this mutant included rope, an adhesion related protein p <dig>  hmw <dig>  mpn <dig> and mpn <dig>  these proteins showed phosphorylation only in the prpc mutant but not in the wild type.
the contrasting situation in mg_ <dig> tends to speculate that additional protein with possible kinase property is affected in this mutant and this putative protein fails to phosphorylate some proteins in the mutant.
however, this is only a hypothesis and requires additional studies for confirmation.
nevertheless, differential phosphorylation of proteins has been shown in the stp mutant strains of l. monocytogenes and s. pneumoniae <cit> .
in addition to changes in protein phosphorylation, an stp  mutant of group b streptococcus has been shown to have altered expression of  <dig> genes which included stk <dig>  the gene encoding a major kinase  <cit> .
a refers protein spots marked in figure 3a and c.
ability of tim <dig> strain to adhere and invade eukaryotic cells
since tim <dig> strain showed differential phosphorylation of proteins, we speculated that this would have some impact on the adherence of this strain with eukaryotic cells.
consistent with this notion, we noticed that tim <dig> strain adhered only partially to culture flasks when grown in sp- <dig> medium.
the non-adherent mycoplasmas were seen as floating cell suspensions and this phenotype was highly reproducible.
however, growing of only the floating or adherent cells resulted into adherent and floating cells in subsequent cultures, indicating that the non-adherent phenotype to the culture flask is a transient phenomenon.
further, since surface attachment of m. genitalium to culture flasks often correlates with adherence to eukaryotic cells, we tested the tim <dig> strain for hemadsorption with sheep erythrocytes, a technique that we routinely use to assess the adherence of this species, and compared its phenotype with g <dig>  tim <dig> showed a hemadsorption positive phenotype similar to that of wild type g <dig> , suggesting that there is no difference between these two strains with regard to adherence to eukaryotic cells.
to evaluate this further, the ability of tim <dig> strain to adhere/invade epithelial cells was also assessed by infecting hela cells.
confocal microscopic analysis of the infected cells revealed that both g <dig> and tim <dig> strains exhibit similar levels of adherence/invasion, although the control strain tim <dig> was little different from these by showing relatively higher levels of adherence/invasion .
the reason for this difference is unknown at present.
however, the fact that both g <dig> and tim <dig> show more or less similar phenotype reiterates that the partial non-adherence to culture flasks by tim <dig> strain has no correlation with its adherence to or invasion of eukaryotic cells.
it has been shown  <cit>  that invasion of m. genitalium into cultured hela and em <dig> cervical epithelial cell lines occurs within  <dig> minutes after postinfection and the invaded bacteria are localized within nuclei.
interestingly, this study has also reported that only a subset of the bacteria  invades the cells.
this fact was confirmed by another group that used electron microscopy and they estimated that the invading bacteria would be around 50% of the total bacteria showing adherence  <cit> .
in this context, it will be of interest to know whether there exists any difference between the wild type and tim <dig> in the quantity of invading bacteria and this question will be addressed in our future studies.
nevertheless, the partial culture flask non-adherence phenotype that we observed with the tim <dig> strain is different from that of the completely culture flask non-adhering phenotype of m. genitalium strain reported earlier  <cit> .
feldner et al.
<cit>  reported that adherence of mycoplasma to culture flasks are based on electrostatic forces rather than adhesion mediated.
it is likely, therefore, that change in phosphorylation of some surface proteins, due to the absence of mg <dig>  leads to change in membrane potential which ultimately affects the electrostatic force.
in the case of m. pneumoniae, it is the stk, but not stp , mutant which failed to adhere with culture flasks  <cit> .
consistent with this negative adherence to culture flasks, this stk mutant strain  exhibits reduced levels of adherence related proteins, including p <dig>  in sds-page.
however, recent studies have demonstrated that deletion of stp in strains of s. pyogenes   <cit>  and s. pneumoniae  <cit>  leads to reduced adherence to pharyngeal cells.
it appears, therefore, that disruption of both stk and stp can lead to adherence negative phenotype but it varies from species to species.
however, the mechanism behind partial adherence of tim <dig> to cultures flask remains elusive and it requires further study.
tim <dig> strain is less cytotoxic to hela cells
further to understand whether the lack of mg <dig> has any effect on other pathogenic mechanisms of m. genitalium, we examined the ability of tim <dig> strain to cause cytotoxicity.
therefore, we infected hela cells with tim <dig> and other control strains.
figure  <dig> shows the confocal microscopy observation of hela cells infected with m. genitalium strains.
as can be seen, m. genitalium wild type strain g <dig> and a control strain tim <dig>  which hastn <dig> insertion in mg_ <dig> encoding 3´-5´ exonuclease, had severe cytotoxic effects on hela cells, while tim <dig> had no such effect and behaved similar to that of heat killed g <dig>  strain.
since cytotoxicity of mycoplasmas is due partly to the release of hydrogen peroxide by these species, we speculated that differences in cytotoxicity between the wild type and the mutant strains might be due to differences in the production of h2o <dig> by these strains.
to rule out this possibility, we determined the h2o <dig> levels in these strains by fox assay.
the results showed significantly reduced levels of h2o <dig> in tim <dig> strain as compared to g <dig> strain .
this indicated that deletion of mg_ <dig> had some direct or indirect effect on the synthesis of h2o <dig> by m. genitalium.
mycoplasmas produce h2o <dig> by oxidizing the glycerophosphate of the glycolytic pathway by glycerophosphate oxidase  <cit> .
it is likely that phosphorylation or dephosphorylation of some of the enzymes associated with this pathway leads to reduced production of h2o <dig> in tim <dig> strain.
besides, in m. pneumoniae reduced cytotoxicity and h2o <dig> production is linked to reduced ability to utilize glycerol  <cit> .
to understand if the reduced h2o <dig> production by tim <dig> has any correlation with glycerol utilization, we determined the growth of the tim <dig> strain in sp- <dig> medium containing glycerol instead of dextrose.
results presented in additional file 3: figure s <dig> reveal that this strain has a defect in the utilization of glycerol as compared to the wild type strain.
these results, taken together, reiterate that reduced cytotoxicity of tim <dig> is due partly to generation of relatively lower amount of h2o <dig> by this strain.
tim <dig> strain fails to differentiate thp- <dig> cells
thp- <dig> cell line is an undifferentiated monocyte cell line from human and its differentiation into macrophages requires incubation with  <dig> nm of phorbol-12-myristate-13-acetate  for  <dig> to  <dig> h. usually, differentiated thp- <dig> cells adhere to the surface of culture flask, while undifferentiated thp- <dig> cells only float in the culture medium.
we have recently discovered that m. genitalium can induce the differentiation of monocytic thp- <dig> cells into macrophages, similar to that of pma, and this ability of m. genitalium may be affected by the absence of protein like msra  <cit> .
to test whether absence of mg <dig> had any effect on the differentiation of thp- <dig> cells by m. genitalium, we labeled thp- <dig> cells with cfse and infected with tim <dig> strain and control strains of m. genitalium.
figure 7a shows confocal microscopy observation of thp- <dig> cells adhered to surface of the culture slides due to differentiation induced by m. genitalium strains.
although thp- <dig> cells infected with g <dig> and tim <dig> exhibited higher number of adhered cells, relatively less number of cells adhered with thp- <dig> cells infected with tim <dig> and heat killed bacteria of g <dig> strain .
this suggested that the product of mg_ <dig> plays an important role in the induction of differentiation of thp- <dig> cells by m. genitalium.
we presume that phosphorylation of some proteins associated with the differentiation of thp- <dig> cells is severely affected in this mutant which leads to reduced differentiation of thp- <dig> cells as compared to wild type.
it is unknown at present whether or not the surface proteins like pyruvate dehydrogenase e <dig> α chain and mg <dig>  which showed altered phosphorylation in this study, have any role in this process but such a possibility does exist.
nevertheless, since differentiation of monocytes is related to modulation of immune responses, the reduced ability of tim <dig> strain to differentiate these cells may suggest that this mutant will have only limited ability to alter the immune system to its favor.
this hypothesis is supported by the fact that an msra mutant  of m. genitalium, which differentiates thp- <dig> cells only moderately, could induce only limited amounts of proinflammatory cytokines il-1β and tnf-α as compared to wild type m. genitalium that has the full ability to differentiate thp- <dig> cells  <cit> .
it is our future goal to investigate whether absence of mg <dig> protein in m. genitalium has any relationship with induction of immune response in the host cells
CONCLUSIONS
in this study, we have shown that the product encoded by mg_ <dig> in m. genitalium is a phosphatase and its absence may affect the phosphorylation of some proteins.
we have also provided evidence that absence of mg <dig> leads to reduced virulence of this bacterium by affecting its ability to cause cytotoxicity and to differentiate monocytic cells.
however, the partial adherence phenotype to culture flasks that we observed with tim <dig> appears to be significant and what causes this transient phenotype remains a question.
similarly, the factors that led tim <dig> to cause reduced cytotoxicity and reduced induction of differentiation of thp- <dig> cells also remain indefinable at this point.
whether the differentially phosphorylated proteins like mg <dig>  mg <dig> and mg <dig> play any role in these processes needs additional investigation.

 bacteria adapt to changing environments by regulating their gene expression through signal transduction systems. two kinds of signal transduction systems exist in bacteria; the two component system and serine/threonine kinases and phosphatases system. although both systems transduce signals by phosphorylation events, they have distinct ways of doing this. while tcs uses a sensor histidine kinase and a regulator protein to transduce the signals, the stk /stp regulate gene expression by protein-protein interaction. however, it should be noted that not all kinases and phosphatases associated with serine or threonine residues in prokaryotes are stk/stp. the stk/stp has special signature motifs and is restricted to selected species of bacteria. it was once thought that bacteria have only tcs but not stk/stp. however, evidence for the occurrence of stk/stp in bacteria continues to accumulate. also, it has been reported that bacterial tcs and stk/stp systems cross talk with each other. in addition to their role in the physiology, stk/stp plays a significant role in the virulence of some pathogenic bacteria, including bacteria relevant to public health such as yersinia and mycobacteria. for instance, ypka, an stk of yersinia pseudotuberculosis, is critical for the disruption of host cytoskeleton during infection. in mycobacterium tuberculosis, lack of stk pkng and pknh has been reported to show reduced viability of this bacterium and increased bacterial load, respectively, in mouse models. the significance of stk in the pathogenesis of staphylococcus aureus, streptococcus pneumoniae, s. pyogenes, pseudomonas aeruginosa, s. agalactiae, mycoplasma pneumoniae and salmonella enterica has also been reported. with respect to stp, relatively few studies have been undertaken in understanding their role in bacterial virulence and most of them focus on pneumococcus. an stp of s. pyogenes is required for the production of hemolysin and to cause apoptosis in the host cells. its homologue, stp in group b streptococcus sp is also associated with the production of hemolysin and lack of this stp leads to less efficient systemic infection by this bacterium. very recently, an stp of s. pneumoniae is found to have a role in the adherence of this species. besides, an stp of listeria monocytogenes is reported to be essential for the growth of this bacterium in murine model. mycoplasma genitalium is a bacterium that lacks a cell wall and is one of the smallest self-replicating organisms with a genome size of kb. it is the etiological agent for the diseases non-gonococcal urethritis and cervicitis in men and women, respectively. in women, it is also implicated in diseases like endometritis, pelvic inflammatory syndrome and tubal infertility. additionally, m. genitailum coinfection in hiv patients has been reported to have increased shedding of hiv in urogenital mucosal regions of the female. although it was initially thought that m. genitalium primarily attaches with epithelial cells of the host to cause the disease, evidences indicate that it invades epithelial cells and is localized on the periphery of the nucleus of the infected cells. the intracellular m. genitailum is reported to persist within the infected cells for a long time. it should be noted that intracellular survival and persistence of this bacterium may require signal transduction mediated adaptation, as do other bacteria in similar circumstances. strikingly, however, m. genitalium and its close relative m. pneumoniae are lacking the classical bacterial tcs, although a few mycoplasmas like m. penetrans and m. iowae do have tcs. besides, both species have only a limited number of regulators controlling gene expression at the transcription level, and this has been attributed to their small genomes due to reductive evolution. nevertheless, these species have genes encoding stk and stp. in fact, the stk of m. pneumoniae has been demonstrated to have an effect on the adherence of this species, although no such effect was noticed with an stp of this species. our long term objective is to determine the roles of stk and stp in m. genitalium pathogenesis and signal transduction. ncbi database of m. genitalium genome sequence reveals that this bacterium possesses a gene encoding stk and three genes encoding stp. we initiated our studies first with mg_ because we had a mutant strain for this gene readily available from a transposon mutant library. here, we show that mg is an alkaline phosphatase and it dephosphorylates threonine phosphate. we also report that m. genitalium lacking in mg shows differentially phosphorylated proteins in two-dimensional gels. in addition, we provide evidence that tim has reduced virulence as compared to wild type m. genitalium.  mg_ encodes a functional phosphatase the gene mg_ is predicted to encode a serine/threonine phosphatase. to verify this, we created the plasmid pmg207ex to overexpress mg protein in e. coli. this plasmid was transformed into e. coli bl strain and induced with iptg. figure 1a shows the overexpression of his10mg protein by e. coli harboring the plasmid pmg207ex and its purification. the purified his10mg protein separated onto sds-page and stained with coomassie blue exhibited a size of kda. this correlated with the predicted size of kda of mg_ to determine if the purified his10mg protein was functional, we assayed the phosphatase activity of this protein using the substrate p-nitrophenyl phosphate. the his10mg readily hydrolyzed pnpp in a dose dependent manner in the alkaline ph of. to rule out the possibility that the observed phosphatase activity of his10mg was not due to e. coli host derived phosphatase, we used similarly overexpressed and purified his10ohr protein of m. genitalium as a control. reactions with this protein or reactions with heat inactivated his10mg or reactions with his10mg but without mg2+ in the reaction mixture showed no color formation with pnpp, indicating that the overexpressed protein was a functional phosphatase dependent upon mg2+ ion for its activity. to further assess whether his10mg is a serine/threonine phosphatase, malachite green based phosphatase assay was performed using synthetic serine or threonine phosphopeptide. no activity was noticed with either peptide in the presence of ni2+, a cation supplied with the assay kit. however, substitution of ni2+ with mg2+ in the reaction mixture released the phosphate from threonine peptide, but this failed to release the phosphate from serine peptide. we presume that the absence of activity with the serine phosphate peptide may be due to the requirement of appropriate conditions. alternatively, it is possible that the serine phosphate in this particular peptide is un-accessible for the enzyme. however, the fact that mg requires a metal for its activity with pnpp or with threonine peptide suggests that it is a metal dependent phosphatase. this observation is consistent with reports of other stps like stp of l. monocytogenes, phpp of s. pneumoniae, prpc of m. pneumoniae and stp of s. agalactiae, all of which required divalent metal cofactor mn2+ for their activity. in bacteria, stp belongs to two families, phosphoprotein phosphatases and metal dependent phosphatases. the major difference between these two groups appears to be their specificity for substrates. while ppm specifically hydrolyzes serine or threonine phosphates, the ppp hydrolyzes, in addition to serine and threonine phosphates, histidine and tyrosine phosphates. although pp2c phosphatase, a member of the ppm family, has some catalytic similarities with ppp, this does not show any amino acid similarity with ppp. further, it appears that mg is only a closely related protein to pp2c phosphatase, because the cluster of orthologous groups classification has placed this protein in a different group of bacterial phosphatase. tim strain and its confirmation to understand the role of mg in signal transduction and pathogenesis of m. genitalium, we sought to create a mutant strain through homologous recombination. however, we were able to acquire a similar mutant strain from m. genitalium tn transposon mutant library generated by dr. john glass. the insertion of tn in the coding region of mg_ had already been determined by sequencing. in order to reconfirm this insertion and to check if this strain has any additional tn insertions due to sub-culturing, we probed the genomic dna of m. genitalium wild type g strain and tim cut with spei, in southern hybridization. the membrane hybridized with radiolabeled dna of mg_ revealed strong signals around kb in the g strain and kb in the tim in addition, a weak signal was also noticed in the tim strain around kb region. the shift in hybridization signals for mg_ and also the presence of additional signals for mg_ in tim strain, as compared to g strain, reconfirmed that the gene was disrupted by tn insertion. in addition, hybridization of the blot with gene fragment coding for gentamicin resistance, which is part of the transposon, revealed signals only at one position which corresponded to the signals of mg_ in the tim strain. this indicated that this strain has no additional tn insertions in the chromosome and the mutant is stable. further, to determine whether the transposon insertion indeed disrupted the expression of mg protein, we analyzed the proteins of g and tim strain in immunoblot with anti-mg antiserum. this antiserum detected the mg protein only in the wild type g strain and not in the tim strain, indicating that the disruption of the gene affected the expression of the protein. we do not expect that tn insertion in this strain will have any polar effects on its downstream genes, because the transcription of the downstream genes is predicted to be in the opposite orientation. this situation implies that complementation of the tim with a functional allele to assess the function of mg is of limited significance. moreover, the only way by which the m. genitalium mutant strain can be complemented is through the use of a transposon which can insert a copy of the functional allele of the mutated gene in an unknown location of the chromosome. it is very likely that the unknown location may be a functional gene and this will affect the interpretation of the complimented phenotype. therefore, we have used a m. genitalium strain called tim which bears the same transposon as in tim inserted in the gene mg_ as a control strain in some experiments. the gene mg_ is predicted to code for a 3´-5´ exonuclease. tim strain exhibits differentially phosphorylated proteins as mg is a phosphatase presumed to be associated with signaling, it was predicted that absence of this protein might alter the phosphorylation status of some m. genitalium proteins. to determine this, and also to identify some of the differentially phosphorylated proteins, we performed 2-d gel analysis of proteins from g and tim strains and stained them with pro-q diamond and sypro ruby stains. while the total proteins stained with sypro ruby showed similar profiles for g and tim strains, the phosphoproteins stained with pro-q diamond displayed different profiles for these strains. these differences in phosphorylation appear not due to differences in the growth of the wild type and mutant strains as they showed no significant differences in growth. further, the differences do not appear due to variability in viability because both strains exhibited similar viability at the time of harvest. the predominant difference was noticed to be at the high molecular weight areas which are shown in large circles. as can be seen, the gels from g showed relatively dense and larger stained areas as compared to gels from the tim strain, suggesting that some hmw proteins are less phosphorylated in tim strain. however, these dense areas have shown no corresponding protein spots in sypro ruby stained gels, thus indicating that these areas do not represent real proteins but represent some artifacts. therefore, we focused only on well separated and differentially phosphorylated proteins. these included two proteins which showed relatively dense staining in the gels of g strain but were weaker in the gels of tim strain, and three proteins that showed stronger staining in the gels of tim strains but were weaker in the gels of g to identify the differentially phosphorylated proteins, we subjected the protein spots 1– to mass spectrometry. based on the number of peptide hits for each spots, the spots were identified as four different proteins namely, pyruvate dehydrogenase complex e subunit α, a putative cytoskeleton protein, a conserved hypothetical protein and thymidine phosphorylase. interestingly, our study validates the findings of a previous study which identified pyruvate dehydrogenase e α chain and the protein encoded by mg_ as phosphoproteins of m. genitalium. recently, both these proteins were found on the surface of m. genitalium, thus suggesting the possibility that they can play a role in m. genitalium-host interaction. what is intriguing in this study, however, is the reduced phosphorylation of a protein in a phosphatase deficient mutant of m. genitalium. theoretically, if proteins are dephosphorylated by a specific phosphatase, then the expectation, in the absence of the phosphatase, is no change in the phosphorylation levels or increased levels of phosphorylation of proteins. in fact, the proteins identified in prpc mutant of m. pneumoniae behave in this manner. the differentially phosphorylated proteins identified in this mutant included rope, an adhesion related protein p hmw mpn and mpn these proteins showed phosphorylation only in the prpc mutant but not in the wild type. the contrasting situation in mg_ tends to speculate that additional protein with possible kinase property is affected in this mutant and this putative protein fails to phosphorylate some proteins in the mutant. however, this is only a hypothesis and requires additional studies for confirmation. nevertheless, differential phosphorylation of proteins has been shown in the stp mutant strains of l. monocytogenes and s. pneumoniae. in addition to changes in protein phosphorylation, an stp mutant of group b streptococcus has been shown to have altered expression of genes which included stk the gene encoding a major kinase. a refers protein spots marked in figure 3a and c. ability of tim strain to adhere and invade eukaryotic cells since tim strain showed differential phosphorylation of proteins, we speculated that this would have some impact on the adherence of this strain with eukaryotic cells. consistent with this notion, we noticed that tim strain adhered only partially to culture flasks when grown in sp- medium. the non-adherent mycoplasmas were seen as floating cell suspensions and this phenotype was highly reproducible. however, growing of only the floating or adherent cells resulted into adherent and floating cells in subsequent cultures, indicating that the non-adherent phenotype to the culture flask is a transient phenomenon. further, since surface attachment of m. genitalium to culture flasks often correlates with adherence to eukaryotic cells, we tested the tim strain for hemadsorption with sheep erythrocytes, a technique that we routinely use to assess the adherence of this species, and compared its phenotype with g tim showed a hemadsorption positive phenotype similar to that of wild type g, suggesting that there is no difference between these two strains with regard to adherence to eukaryotic cells. to evaluate this further, the ability of tim strain to adhere/invade epithelial cells was also assessed by infecting hela cells. confocal microscopic analysis of the infected cells revealed that both g and tim strains exhibit similar levels of adherence/invasion, although the control strain tim was little different from these by showing relatively higher levels of adherence/invasion. the reason for this difference is unknown at present. however, the fact that both g and tim show more or less similar phenotype reiterates that the partial non-adherence to culture flasks by tim strain has no correlation with its adherence to or invasion of eukaryotic cells. it has been shown that invasion of m. genitalium into cultured hela and em cervical epithelial cell lines occurs within minutes after postinfection and the invaded bacteria are localized within nuclei. interestingly, this study has also reported that only a subset of the bacteria invades the cells. this fact was confirmed by another group that used electron microscopy and they estimated that the invading bacteria would be around 50% of the total bacteria showing adherence. in this context, it will be of interest to know whether there exists any difference between the wild type and tim in the quantity of invading bacteria and this question will be addressed in our future studies. nevertheless, the partial culture flask non-adherence phenotype that we observed with the tim strain is different from that of the completely culture flask non-adhering phenotype of m. genitalium strain reported earlier. feldner et al. reported that adherence of mycoplasma to culture flasks are based on electrostatic forces rather than adhesion mediated. it is likely, therefore, that change in phosphorylation of some surface proteins, due to the absence of mg leads to change in membrane potential which ultimately affects the electrostatic force. in the case of m. pneumoniae, it is the stk, but not stp, mutant which failed to adhere with culture flasks. consistent with this negative adherence to culture flasks, this stk mutant strain exhibits reduced levels of adherence related proteins, including p in sds-page. however, recent studies have demonstrated that deletion of stp in strains of s. pyogenes and s. pneumoniae leads to reduced adherence to pharyngeal cells. it appears, therefore, that disruption of both stk and stp can lead to adherence negative phenotype but it varies from species to species. however, the mechanism behind partial adherence of tim to cultures flask remains elusive and it requires further study. tim strain is less cytotoxic to hela cells further to understand whether the lack of mg has any effect on other pathogenic mechanisms of m. genitalium, we examined the ability of tim strain to cause cytotoxicity. therefore, we infected hela cells with tim and other control strains. figure shows the confocal microscopy observation of hela cells infected with m. genitalium strains. as can be seen, m. genitalium wild type strain g and a control strain tim which hastn insertion in mg_ encoding 3´-5´ exonuclease, had severe cytotoxic effects on hela cells, while tim had no such effect and behaved similar to that of heat killed g strain. since cytotoxicity of mycoplasmas is due partly to the release of hydrogen peroxide by these species, we speculated that differences in cytotoxicity between the wild type and the mutant strains might be due to differences in the production of h2o by these strains. to rule out this possibility, we determined the h2o levels in these strains by fox assay. the results showed significantly reduced levels of h2o in tim strain as compared to g strain. this indicated that deletion of mg_ had some direct or indirect effect on the synthesis of h2o by m. genitalium. mycoplasmas produce h2o by oxidizing the glycerophosphate of the glycolytic pathway by glycerophosphate oxidase. it is likely that phosphorylation or dephosphorylation of some of the enzymes associated with this pathway leads to reduced production of h2o in tim strain. besides, in m. pneumoniae reduced cytotoxicity and h2o production is linked to reduced ability to utilize glycerol. to understand if the reduced h2o production by tim has any correlation with glycerol utilization, we determined the growth of the tim strain in sp- medium containing glycerol instead of dextrose. results presented in additional file 3: figure s reveal that this strain has a defect in the utilization of glycerol as compared to the wild type strain. these results, taken together, reiterate that reduced cytotoxicity of tim is due partly to generation of relatively lower amount of h2o by this strain. tim strain fails to differentiate thp- cells thp- cell line is an undifferentiated monocyte cell line from human and its differentiation into macrophages requires incubation with nm of phorbol-12-myristate-13-acetate for to h. usually, differentiated thp- cells adhere to the surface of culture flask, while undifferentiated thp- cells only float in the culture medium. we have recently discovered that m. genitalium can induce the differentiation of monocytic thp- cells into macrophages, similar to that of pma, and this ability of m. genitalium may be affected by the absence of protein like msra. to test whether absence of mg had any effect on the differentiation of thp- cells by m. genitalium, we labeled thp- cells with cfse and infected with tim strain and control strains of m. genitalium. figure 7a shows confocal microscopy observation of thp- cells adhered to surface of the culture slides due to differentiation induced by m. genitalium strains. although thp- cells infected with g and tim exhibited higher number of adhered cells, relatively less number of cells adhered with thp- cells infected with tim and heat killed bacteria of g strain. this suggested that the product of mg_ plays an important role in the induction of differentiation of thp- cells by m. genitalium. we presume that phosphorylation of some proteins associated with the differentiation of thp- cells is severely affected in this mutant which leads to reduced differentiation of thp- cells as compared to wild type. it is unknown at present whether or not the surface proteins like pyruvate dehydrogenase e α chain and mg which showed altered phosphorylation in this study, have any role in this process but such a possibility does exist. nevertheless, since differentiation of monocytes is related to modulation of immune responses, the reduced ability of tim strain to differentiate these cells may suggest that this mutant will have only limited ability to alter the immune system to its favor. this hypothesis is supported by the fact that an msra mutant of m. genitalium, which differentiates thp- cells only moderately, could induce only limited amounts of proinflammatory cytokines il-1β and tnf-α as compared to wild type m. genitalium that has the full ability to differentiate thp- cells. it is our future goal to investigate whether absence of mg protein in m. genitalium has any relationship with induction of immune response in the host cells  in this study, we have shown that the product encoded by mg_ in m. genitalium is a phosphatase and its absence may affect the phosphorylation of some proteins. we have also provided evidence that absence of mg leads to reduced virulence of this bacterium by affecting its ability to cause cytotoxicity and to differentiate monocytic cells. however, the partial adherence phenotype to culture flasks that we observed with tim appears to be significant and what causes this transient phenotype remains a question. similarly, the factors that led tim to cause reduced cytotoxicity and reduced induction of differentiation of thp- cells also remain indefinable at this point. whether the differentially phosphorylated proteins like mg mg and mg play any role in these processes needs additional investigation. 
this recombinant protein readily hydrolyzed the substrate p-nitrophenyl phosphate in a dose-dependent manner. the results of the study suggest that mg is an important signaling protein of m. genitalium and its presence may be crucial for the virulence of this species. additional studies using synthetic peptides as substrates revealed that the recombinant protein was able to hydrolyze the threonine phosphate. further, a transposon insertion mutant strain of m. genitalium that lacks the protein mg showed differentially phosphorylated proteins when compared to the wild type g strain. this strain was also less efficient in inducing the differentiation of thp- cell line as compared to wild type m. genitalium. mycoplasma genitalium, a mollicute that causes the urogenital diseases urethritis and cervicitis in men and women, respectively, is a pathogen which lacks tcs but possesses stk/stp. mass spectrometry revealed that some of the key proteins differentially phosphorylated in tim strain were putative cytoskeletal protein encoded by the gene mg_ and pyruvate dehydrogenase e α chain encoded by the gene mg_ in addition, tim was noticed to be less cytotoxic to hela cells and this correlated with the production of less hydrogen peroxide by this strain. bacterial signal transduction systems like two component system and serine/threonine kinase and serine/threonine phosphatase play important roles in the virulence and pathogenesis of bacterial pathogens.
 bacterial signal transduction systems like two component system and serine/threonine kinase and serine/threonine phosphatase play important roles in the virulence and pathogenesis of bacterial pathogens. mycoplasma genitalium, a mollicute that causes the urogenital diseases urethritis and cervicitis in men and women, respectively, is a pathogen which lacks tcs but possesses stk/stp. in this study, we investigated the biochemical and virulence properties of an stp protein encoded by the gene mg_ of this species.  we overexpressed mg in escherichia coli overexpression system as a recombinant his10mg protein and purified it with affinity chromatography. this recombinant protein readily hydrolyzed the substrate p-nitrophenyl phosphate in a dose-dependent manner. additional studies using synthetic peptides as substrates revealed that the recombinant protein was able to hydrolyze the threonine phosphate. further, a transposon insertion mutant strain of m. genitalium that lacks the protein mg showed differentially phosphorylated proteins when compared to the wild type g strain. mass spectrometry revealed that some of the key proteins differentially phosphorylated in tim strain were putative cytoskeletal protein encoded by the gene mg_ and pyruvate dehydrogenase e α chain encoded by the gene mg_ in addition, tim was noticed to be less cytotoxic to hela cells and this correlated with the production of less hydrogen peroxide by this strain. this strain was also less efficient in inducing the differentiation of thp- cell line as compared to wild type m. genitalium.  the results of the study suggest that mg is an important signaling protein of m. genitalium and its presence may be crucial for the virulence of this species. serinethreonine kinasephosphatasemycoplasmavirulencepathogenesis 