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Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
Correspondence
Jaume Piñol
jaume.pinyol{at}uab.es
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ABSTRACT |
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A supplementary figure and table are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In particular, Mycoplasma genitalium has been linked to numerous genitourinary as well as extragenitourinary human pathologies (Baseman et al., 1988) and it is a leading cause of Chlamydia-negative, non-gonococcal urethritis (Taylor-Robinson & Horner, 2001; Tully et al., 1981). M. genitalium also has a unique and very attractive feature: with a genome size of 580 kb (Fraser et al., 1995), it is the smallest self-replicating cell known to be able to be grown in axenic culture. For this reason, this micro-organism has been considered to be one of the most suitable models for achieveing a complete understanding of the biology of a single replicating cell (Roberts, 2004).
M. genitalium cells display a differentiated tip structure, commonly known as the terminal organelle (TO), that gives them a flask-shaped appearance. The TO plays a key role in mycoplasma cytadherence and further parasitism of host target cells and it is defined by an electron-dense core (EDC) that is a component of the Triton X-100-insoluble fraction (Gobel et al., 1981; Meng & Pfister, 1980). The intriguing complexity and the multifunctional role of the TO have attracted the interest of many researchers. Most of the cytoskeletal proteins present in the complex network that constitutes the TO have been identified primarily as cyadhesins or cytadherence-associated proteins. In addition, the TO is the leading end as mycoplasma cells glide (Radestock & Bredt, 1977) and it constitutes the mycoplasma gliding motor (Hasselbring & Krause, 2007). Motility and cytadherence are closely related in mycoplasmas and many cytadherence-related proteins are also required for cell gliding (Burgos et al., 2007; Hasselbring et al., 2005; Hasselbring & Krause, 2007; Seto et al., 2005). However, the existence of a cytadherence-independent set of motility-related proteins has been disclosed recently for the slow gliding mycoplasmas M. genitalium (Pich et al., 2006a) and Mycoplasma pneumoniae (Hasselbring et al., 2006).
For several years the isolation and characterization of spontaneous as well as transposon-generated mutants involving different cytadherence-related genes has provided important information regarding the spatial and hierarchical development of the attachment organelle in M. pneumoniae (Krause & Balish, 2004). These studies have indicated that the cytoskeletal protein HMW2 is a major structural element of the EDC (Balish et al., 2003b). In addition, it has been suggested that polymers of HMW3 protein surround the core and the terminal button (TB) in a linear pattern, possibly serving to stabilize this structure (Krause, 1996). Unfortunately, the specific contribution of the different cytoskeletal proteins from M. genitalium to the TO organization has been much less studied (Burgos et al., 2006, 2007). Two independent factors have probably hindered the study of TO formation and the whole cytadherence scenario in the smallest mycoplasma. First, screening for haemadsorption-negative colonies has not allowed the isolation of transposon-generated mutants involving any of the cytadherence-accessory genes from M. genitalium (Reddy et al., 1996). Second, the high rates of spontaneous occurrence of class I and class II haemadsorption-negative mutants (Mernaugh et al., 1993), which probably reflect a subjacent cytadherence phase variation mechanism (Burgos et al., 2006), hampers the screening process and jeopardizes the characterization of the mutants isolated.
In the present study, we aimed to investigate the involvement of M. genitalium cytoskeletal proteins in the TO organization and to analyse their contribution to gliding mechanics. To achieve this goal, a methodology recently developed in our laboratory (Pich et al., 2006a) was modified in order to isolate transposon-generated gliding-deficient mutants also displaying surface adherence defects (SAD). Several mutants showing transposon insertions within the mg218 or mg317 genes, encoding the orthologues of M. pneumoniae HMW2 and HMW3 cytadherence-accessory proteins, respectively, were isolated using this procedure. The analysis of the transposon mutants obtained has revealed that the reduced gliding motility and cytadherence exhibited by these mutants is well correlated with the presence of defects in their respective EDC, revealing the particular roles of MG218 and MG317 proteins in the TO architecture of M. genitalium.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) at 37 °C under 5 % CO2 in tissue culture flasks (TPP). For standard colony development, mycoplasma cells were plated in SP-4 medium supplemented with 0.8 % agar (Difco). To check for gliding-deficient strains, cells were attached to cell culture dishes (Corning) and covered with SP-4 broth containing 0.5 % low-melting-point agarose (Iberlabo). SP-4 medium was supplemented with tetracycline (2 µg ml–1; Roche) to select for resistant strains. Transformation of M. genitalium by electroporation was performed as described previously (Pich et al., 2006b).
DNA manipulation.
Genomic DNA was isolated using the E.Z.N.A. Bacterial DNA kit (Omega Bio-tek). Sequencing of genomic DNA from the different transposon mutants obtained was performed with fluorescent dideoxynucleotides using the Big Dye 3.0 Terminator kit (Applied Biosystems) and Tc upstream and Tc downstream primers (Table 1), according to the recommendations of the manufacturer. Southern blot hybridization was performed using the Dig DNA labelling and detection kit (Roche).
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mg218.mg218, the flanking regions of mg218 were amplified by PCR. A 1 kb fragment corresponding to the upstream region of mg218 was amplified using primers URmg218-5' and URmg218-3' (Table 1
), cloned into an EcoRV-digested pBE plasmid (Pich et al., 2006b) and digested with PstI and EcoRI. Separately, a 0.8 kb fragment corresponding to the downstream region of mg218 was amplified using primers DRmg218-5' and DRmg218-3' (Table 1), cloned into an EcoRV-digested pBE plasmid, and digested with BamHI and ApaI. Both flanking regions and an EcoRI–BamHI-digested tetM438 marker were finally cloned into a PstI–ApaI-digested pBSK plasmid to obtain the pmg218 construct.
SDS-PAGE and MALDI-MS analysis.
The separation of total mycoplasma proteins by SDS-PAGE followed standard procedures. The identification of MG317 protein in the SDS-PAGE was achieved by peptide mass fingerprint analysis, as described previously (Pich et al., 2006a). MG317 protein was identified with a score of 136 and a protein sequence coverage of 38.9 %. Scores greater than 68 were considered significant (Perkins et al., 1999).
Western blotting and preparation of antisera.
Western blots were performed as described previously (Burgos et al., 2006), using MG218 monoclonal and polyclonal antibodies developed in this work, or rabbit antiserum to P41 (Krause et al., 1997), HMW1 (Stevens & Krause, 1991) and HMW3 proteins (Stevens & Krause, 1992). Monoclonal antibodies to MG218 protein were obtained by repeated immunization of BALB/c mice with a M. genitalium 0.1 % Triton X-100-insoluble fraction in sterile PBS. Polyclonal antibodies to the MG218 C-terminal region were obtained by repeated immunization of BALB/c mice with a purified, recombinant C-terminal MG218 polypeptide (amino acids 1627–1805). Previously, a 537 bp sequence corresponding to nucleotides 264055–264592 of the M. genitalium genome was amplified by PCR using primers mg218Exp5' and mg218Exp3' (Table 1), cloned into a pET21a vector (Novagen), expressed in Escherichia coli BL21(DE3) and purified following standard procedures.
Microcinematography.
Gliding motility data for the wild-type strain and the different mutants was obtained from microcinematographic studies (Pich et al., 2006a). The motility of 25 individual cells from each particular clone of mg317– or mg218– mutants was analysed. Data obtained from the different mg317– mutant clones or, separately, from the different mg218– mutant clones, were combined to obtain a single value. Similarly, a total of 300 cells from mg317– or mg218– mutants, including cells from different clones, were examined to calculate the respective gliding frequencies.
Haemadsorption activity.
For quantitative assessment of the haemadsorption activity, aliquots of 25 µl containing 
107c.f.u. ml–1 of the wild-type strain, mutants c9, c5, c12, c6, c4 and c16, and a representative clone of mg218 and mg191 mutants (Burgos et al., 2006), were incubated at 37 °C for 2 h with 475 µl diluted (1 : 50) human erythrocytes. The mixture was shaken gently throughout the incubation. Erythrocytes and bound mycoplasmas were pelleted by centrifugation at 300 g for 2 min. Different dilutions of the supernatant were plated onto solid SP-4 medium in order to estimate the mycoplasma concentration. Prior to the incubation with erythrocytes, different dilutions of the original samples were also plated onto solid SP-4 medium to determine the initial mycoplasma concentration. The haemadsorption activity values are derived from two independent experiments. In each experiment, data obtained from the two clones representing the same group of mutants were combined.
Scanning electron microscopy.
Scanning electron microscopy analyses were performed using Permanox chamber slides (Nunc). For this purpose, cells from the wild-type strain, all mg317– mutants, and mg218– mutants c12, c13, c11, c3 and c6 were grown for 24 h, while cells from mg218 mutants and mg218– mutants c4 and c16 were grown for 48 h. SP-4 medium was removed and cells were washed gently with PBS and fixed with 1 % glutaraldehyde for 1 h. Samples were washed again with PBS and dehydrated sequentially with 25, 50, 75 and 100 % ethanol concentrations for periods of 10 min each. Immediately, samples were critical-point dried (K850 Critical Point Drier, Emitech) and sputter coated with gold. Samples were observed using a Hitachi S-570 scanning electron microscope.
Transmission electron microscopy.
Cells from the wild-type strain, mg218– mutants c4, c16 and c6, mg317– mutant c5 and mg218 mutant c8 were grown to late-exponential phase, scraped, pelleted by centrifugation, and washed with PBS. Pellets were fixed with 2.5 % (v/v) glutaraldehyde and 2 % (v/v) paraformaldehyde (EM grade, TAAB) in 100 mM phosphate buffer (PB, pH 7.4) for 2 h and rinsed with 100 mM PB. Pellets were then post-fixed in 1 % (w/v) osmium tetroxide (TAAB) containing 0.8 % (w/v) potassium hexacyanoferrate (III) (Sigma) for 2 h and washed with 100 mM PB. All these steps were done at 4 °C. Samples were dehydrated through a graded ethanol series, infiltrated in Spurr's resin and polymerized for 48 h at 60 °C. Ultrathin sections were mounted on copper grids, contrasted with standard uranyl acetate and lead citrate double-staining, and observed in a Hitachi H-7000 transmission electron microscope at 75 kV. The length of the EDC in a particular strain was determined from measurements of 18–25 TO.
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RESULTS |
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). Transformed cells were dispensed into cell culture dishes, left to adsorb onto the plastic surface and covered with 0.5 % low-melting-point agarose SP-4 medium. In contrast to our previous report (Pich et al., 2006a), the adsorbed cells were not washed with PBS, in order to retain those mutants not strongly adhered to the plastic surface. The morphology of 12 000 colonies was examined and 40 colonies exhibiting a compact morphology, compatible with the existence of gliding deficiencies, were identified and isolated. Since gliding-deficient mutants without SAD are also isolated by this procedure, we first discarded those mutants showing transposon insertions in the mg200 and mg386 genes, analysed extensively in our previous report. Twenty-two transposon mutants lacking MG386 or MG200 polypeptides were identified by SDS-PAGE analysis and consequently were not examined. Separately, seven clones containing mixed populations were also discarded.
Identification of the transposon insertion points
Insertion points of MTnTetM438 in the eleven gliding-deficient mutants selected for further analysis (Table 2) were determined by sequencing the corresponding genomic DNA using the Tc upstream and Tc downstream primers (Table 1) present in the MTnTetM438 transposon. Seven transposon mutants showed MTnTetM438 insertions within the mg218 coding region (mg218– mutants; Fig. 1a). The remaining transposon mutants showed minitransposon insertions within the mg317 coding region (mg317– mutants; Fig. 1b).
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). Interestingly, cells from both clones did not attach avidly to the plastic surface of the cell culture flask. In contrast, under similar culture conditions, cells from the remaining mg218– mutant clones (Fig. 2b) and all the mg317– mutants (Fig. 2c) developed colonies with rough surfaces and a granulate appearance in the centre. The presence of some satellite microcolonies in these clones indicated that a certain degree of locomotion was still preserved. Cells from these nine transposon mutants attached quite well to the plastic surface of cell culture flasks. Hence, the two different classes of colony morphologies described here reflect the effect of the gene disruption on the surface-adherence capability of the mutant strain. This important divergence prompted us to consider the separation of the mg218– mutants into two groups: mg218– mutants with severe SAD (c4 and c16), and mg218– mutants with moderate SAD (c3, c6, c11, c12 and c13).
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mg218 mutantsmg218 was engineered to contain the tetM438 selectable marker (Pich et al., 2006b) and the flanking regions of mg218 (Fig. 3a). A double recombination event between plasmid pmg218 and the mycoplasma genome promotes the substitution of bases 43 to 5376 of mg218 (98.5 % of the mg218 coding region) by the tetM438 marker. Several tetracycline-resistant clones were isolated after electroporation of M. genitalium in the presence of pmg218. The presence of the expected deletion within mg218 in these strains was assessed by Southern blotting. Genomic DNA from the wild-type strain and eight pmg218 transformants was digested with Acc65I and probed with a 0.8 kb BamHI–ApaI fragment obtained from plasmid pmg218. A single band of 2.2 kb, clearly different from the 10.9 kb band expected for the wild-type strain, was detected for the eight pmg218 transformants analysed (Fig. 3b). This single band of 2.2 kb is compatible only with a double recombination event and confirms the expected deletion of mg218 in these strains. Hereinafter, we will refer to these strains as mg218 mutants.
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mg218 mutants was analysed quantitatively by microcinematography (Table 3). Motile cells from the wild-type strain represented 93.8 % of the cell population and exhibited a mean gliding speed of 0.152 µm s–1. However, attached cells from mg317– and mg218– mutants with moderate SAD displayed a mean gliding speed between four and fivefold below that of the wild-type strain. Motile cells from mg317– mutants represented 48.3 % of the cell population while the percentage of motile cells from mg218– mutants with moderate SAD was slightly lower (43.9 %). In contrast, mg218 and mg218– mutants with severe SAD were non-motile.
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). These filaments were often found connecting two different cells but were also present in isolated mycoplasmas. We observed that the filament linking two cells was broken as the cells became further apart (data not shown). When the resulting individual cells resumed gliding, the filament was clearly detected at the pole opposite to the direction of movement.
Protein profile
The protein profiles of the different mutants isolated in this study were analysed by SDS-PAGE (Fig. 4a) and Western blotting (Fig. 4b–f). As expected, a 220 kDa band corresponding to full-length (1805 amino acids) MG218 protein (Dhandayuthapani et al., 1999) was absent in both mg218 and mg218– mutants (Fig. 4a, b). Nevertheless, the use of MG218 monoclonal antibodies revealed the presence of N-terminal MG218 derivatives in all mg218– mutants with moderate SAD (Fig. 4b). Polyclonal antibodies raised against amino acids 1627 to 1805 of MG218 protein revealed also the presence of several C-terminal MG218 derivatives (bands of 118, 115 and 25 kDa, Fig. 4d), most of them visible both in the wild-type strain and in all the mg218– mutants isolated. As expected, no N-terminal and C-terminal MG218 derivatives were observed among the mg218 mutants.
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). However, the presence of truncated MG317 fragments was not detected in this case. mg218 and mg218– mutants c16 and c12 (Fig. 4a, c) as well as mutant c4 (data not shown) also exhibited reduced levels of MG317. Likewise, levels of MG312 protein, the orthologue of M. pneumoniae HMW1, were also reduced in mg218 and mg218– mutants c4, c16 and c12 (Fig. 4e). These observations strongly suggest that the stability of MG312 and MG317 depends on the expression of the MG218 N-terminal region, in particular of the short stretch located between amino acids 577 and 648 (the length of the N-terminal MG218 derivatives in mutants c12 and c13). In addition, all the mutants isolated in this study showed reduced levels of MG386, P140 and P110 proteins. The stability of MG386 protein seems to be directly linked to the expression of P140 and P110 proteins at wild-type levels (Burgos et al., 2006), suggesting a cooperative interaction between these proteins. Finally, the presence of MG218.1 protein, the orthologue of M. pneumoniae P41, was detected in all mg218 and mg218– mutants (Fig. 4f). This result rules out the existence of polar effects on the mg218.1 gene derived from disruption or deletion of mg218. Polar effects derived from the transposon insertion in mg317– mutants are not expected because the distance between mg316 and mg317 (40 bp) is too long to allow translational coupling of the corresponding polypeptides (Lluch-Senar et al., 2007).
Haemadsorption activity
Haemadsorption activity of the different mutants isolated in this study, the wild-type strain and the non-adherent mutant mg191 was analysed quantitatively. As expected, mg317– and mg218– mutants with moderate SAD retained around 50 % of the wild-type haemadsorption activity (Table 3). Accordingly, cells from these mutants grow mainly attached to the plastic surface of the cell culture flask in liquid cultures. In contrast, mg218 mutants displayed haemadsorption activities 20 % of that of the wild-type strain. Correspondingly, cells from mg218 mutants grow mainly in suspension in liquid cultures. The haemadsorption activity displayed by the two mg218– mutants with severe SAD was similar to the activity observed for mg218 mutants (data not shown). All data regarding mg218– mutants with severe SAD obtained in this study are similar to the data obtained for mg218 mutants and will not be addressed further in this manuscript.
Cell morphology
Scanning electron microscopy analyses revealed that cells from mg218– mutants with moderate SAD (Fig. 5b) and mg317– mutants (Fig. 5c) exhibit a similar morphology, characterized by the presence of a short TO. The thin neck that joins the cell body and the TB in the wild-type cells (Fig. 5a, arrows) is less apparent in these mutants. Interestingly, most of the cells from these mutants show long tails at the pole opposite to the TO. These long tails often join cells oriented in opposite directions. Among the population of mg218 mutants (Fig. 5d, e), we observed rounded cells, reminiscent of those described previously for mg191 and mg192 mutants (Burgos et al., 2006), as well as elongated cells. However, the most remarkable feature of mg218 mutants is the presence of rounded bodies, two or threefold larger than a single wild-type cell, suggesting the existence of strong deficiencies in cell division.
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). In agreement with previous work (Henderson & Jensen, 2006), some views revealed the origin of two independent rods at the proximal end of the EDC (Fig. 6a, numbers 1 and 2). In addition, a slightly electron-dense material that separates the cytoplasm from the proximal end of the EDC was observed (Fig. 6a, arrows). The distal end of the EDC showed the thicker region currently known as the TB. Notably, some transverse sections of the TB revealed the presence of several discrete highly electron-dense rounded particles (Fig. 6a, asterisk; see also Supplementary Fig. S1, available with the online version of this paper). Since the overall structure found in the M. genitalium TO seems similar to that observed in the M. pneumoniae TO, we hypothesize that these rounded particles correspond to the arched path of discrete globular proteins (Fig. 7b, component C) recently identified in M. pneumoniae by cryo-electron tomography (Henderson & Jensen, 2006).
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). TBs were still present and some sections retained the highly electron-dense rounded particles described for the wild-type strain. In contrast, these electron-dense particles were not observed in preparations from mg317– mutants. Most of the EDCs from mg317– mutants were devoid of a clear TB, and the distal end of the EDC often appeared unconnected to the cell membrane (Fig. 6c). Interestingly, we also observed electron-dense structures embedded in the cytoplasm, not associated with any membrane protrusion (Fig. 6c). These structures resemble bona fide EDCs that appear to be free in the cytoplasm, since the tip structure could not be identified in such cells. These observations suggest that the distal end of the EDC is not attached tightly to the cell membrane in these mutants. In contrast, EDCs could not be identified in cells from mg218 mutants (Fig. 6d). In accordance with the images obtained by scanning electron microscopy, cell bodies from mg218 mutants were larger than those observed for the wild-type strain. Finally, many cells from all the mutants analysed in this study displayed long tails with alternating electron-dense and electron-transparent regions, as could be seen for mg218 mutants (Fig. 6d). |
DISCUSSION |
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; Pich et al., 2006a; Seto et al., 2005), could also contribute to the reduced locomotion observed in the mg218– and mg317– mutant cells.
The two different phenotypes observed among mg218– mutants are well correlated with the presence of N-terminal MG218 truncated fragments. Supporting this information, we identified the presence of N-terminal MG218 derivatives in all mg218– mutants with moderate SAD. These MG218 truncated forms seem to be responsible for the EDC formation and the intermediate cytadherence activity displayed by this group of mg218– mutants. The stability of the N-terminal MG218 derivatives with lengths ranging from 577 to 799 amino acids is interesting, because it has been reported that N-terminal HMW2 derivatives smaller than 1040 amino acids are unstable in M. pneumoniae (Balish et al., 2003a). Moreover, in contrast to M. pneumoniae hmw2 transposon mutants (Hedreyda & Krause, 1995), N-terminal MG218 derivatives are not only stable but they are still functional in M. genitalium. Accordingly, the shorter EDCs observed in mg218– mutants with moderate SAD probably reflect the truncated nature of the N-terminal MG218 derivatives. Thereby, our results provide experimental evidence that the length of the EDC corresponds to the length of MG218 modelled as a fully extended 
-helix, as has been addressed previously for M. pneumoniae HMW2 (Balish et al., 2003b). Interestingly, we also identified the existence of multiple C-terminal MG218 derivatives in all mg218– mutants as well as in the wild-type strain. The presence of these internal products in mg218– mutants rules out the possibility that they could arise from the proteolytic processing of full-length MG218 protein and suggests the existence of additional transcriptional and translational start sites in the mg218 coding region. Two different ATG codons, encoding methionines 804 and 822 of MG218 protein, could also function as internal start codons, giving rise to polypeptides with a size compatible with the 118 and 115 kDa C-terminal MG218 derivatives identified in this work (see Fig. 4d and Supplementary Table S1, available with the online version of this paper). The function of these MG218 derivatives, not identified in M. pneumoniae, is still unknown. In contrast, the 25 kDa C-terminal MG218 derivative, which could arise from the translation of MG218 starting at either methionine 1574 or 1615 (Supplementary Table S1), is probably the orthologue of M. pneumoniae P28 protein (Krause et al., 1997). The presence of this small derivative in both mycoplasma species suggests that it plays a significant biological function that still remains unknown. Thus, further work is needed to unravel the significance of the different C-terminal derivatives found in M. genitalium. On the other hand, it is noteworthy that mg218 mutants, lacking such C-terminal MG218 derivatives, exhibit a phenotype similar to that of mg218– mutants with severe SAD. In addition, these C-terminal derivatives do not seem to be involved in the stabilization of MG312 and MG317 proteins, because MG312, and to a lesser extent MG317, are unstable in mg218– mutants with severe SAD. Thus, it seems that the stability of MG312 and MG317 depends on the expression of the MG218 N-terminal region, in particular of the short stretch located between amino acids 577 and 648 (the length of the N-terminal MG218 derivatives in mutants c12 and c13). Altogether, these observations indicate that the MG218 N-terminal region can fulfil many of the roles currently associated with the expression of a full-length MG218 protein, a result that is in acute contrast to that observed in M. pneumoniae (Balish et al., 2003a).
Electron microscopy analyses reveal that MG317 protein is required to build up a standard TB. Since many cells from mg317 transposon mutants show EDC free in the cytoplasm, the TB seems to be involved in the attachment of the distal end of the EDC to the inner surface of the plasma membrane. It is tempting to speculate that MG317 is involved in the attachment of the EDC to the intracellular domains of P140/P110 complexes present at the terminal tip membrane. To fulfil such a possible function, MG317 should be localized in the vicinity of the TB in M. genitalium, which is in agreement with the localization of HMW3 in M. pneumoniae revealed by immunoelectron microscopy (Stevens & Krause, 1992). In our micrographs, the chevron-like structures previously observed in a M. pneumoniae hmw3 mutant (Willby & Krause, 2002) could not be identified. Regardless of this, the intermediate cytadherence displayed by our mg317– mutants is in accordance with the phenotype described for the M. pneumoniae hmw3 mutant (Willby & Krause, 2002). Thus, the partial cytadherence activity displayed by mg317– and mg218– mutants provides an explanation for the previous failure to isolate M. genitalium haemadsorption-negative mutants showing transposon insertions in these two cytadherence-related genes (Reddy et al., 1996). Interestingly, a similar situation could be hampering the isolation of Mycoplasma gallisepticum haemadsorption-negative mutants bearing transposon insertion in genes different from gapA and crmA (Mudahi-Orenstein et al., 2003). Therefore, screening for colonies exhibiting a compact morphology could provide a direct way to isolate these mutants.
As stated previously, all the mutants isolated in this study show reduced levels of P140 and P110 proteins, which are involved in the maintenance of M. genitalium cytadherence (Burgos et al., 2006). This result indicates that beyond the mutual interdependence of P140 and P110, demonstrated previously in mg191 and mg192 mutants (Burgos et al., 2006), the levels of these two proteins seem to be also coordinately regulated in M. genitalium. In addition, the reduced levels of P140 and P110 also provide an explanation for the reduced haemadsorption activity exhibited by mg218– and mg317– mutants. However, additional factors are needed to explain the reduced cytadherence observed in mg218– mutants with severe SAD. Probably, the absence of a TO in these mutants is also hampering the correct localization of these two adhesins in the outer side of the cell membrane. Notably, a previous study in which M. genitalium mg218 was disrupted through homologous recombination allowed the isolation of some mutants (designated JB1 mutants) displaying a cytadherence-negative phenotype (Dhandayuthapani et al., 1999). JB1 mutants exhibited a dramatic instability of P140 and P110 that might also explain the complete loss of cytadherence of these mutants. In contrast, P140 and P110 are still found in all the mg218 mutants isolated in our study, although at reduced levels. At the present time, the origin of the different behaviour of P140 and P110 in JB1 and mg218 mutants is unclear.
Finally, most of the cells from the mg218– and mg317– mutants showed thin filaments similar to those described previously for mg386 and mg200 gliding-deficient mutants (Pich et al., 2006a). Scanning electron microscopy images from mg218– and mg317– mutants also revealed that cells coupled by these filaments are always oriented in opposite directions, a configuration characteristic of dividing mycoplasma cells. Altogether, these observations suggest that cytokinesis is impaired in M. genitalium mutants with gliding motility defects. In addition, locomotion clearly contributes to cell dispersion in adherent mycoplasmas, a fact that is supported by the formation of large cell aggregates in liquid cultures of mutants with gliding deficiencies (Pich et al., 2006a). The dramatic consequences for cell division, probably magnified by the complete loss of the TO, are particularly evident in mg218 mutants. However, the large cell bodies observed in mg218 mutants may reveal an additional role for MG218. Interestingly, further investigation of conserved domains within MG218 (Marchler-Bauer & Bryant, 2004) shows the presence of two independent but overlapping SMC domains (Structural Maintenance of Chromosomes; COG1196). Proteins that contain SMC-domains have been shown to participate in many aspects of higher-order chromosome organization. Like SMC proteins, MG218 is predicted to form large coiled-coils that are often involved in protein–DNA interactions (Akhmedov et al., 1999). Additional analyses are needed to determine whether DNA is present in the TO and whether MG218 protein is also involved in chromosome dynamics of M. genitalium.
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ACKNOWLEDGEMENTS |
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Edited by: C. Citti
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REFERENCES |
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Balish, M. F., Ross, S. M., Fisseha, M. & Krause, D. C. (2003a). Deletion analysis identifies key functional domains of the cytadherence-associated protein HMW2 of Mycoplasma pneumoniae. Mol Microbiol 50, 1507–1516.[CrossRef][Medline]
Balish, M. F., Santurri, R. T., Ricci, A. M., Lee, K. K. & Krause, D. C. (2003b). Localization of Mycoplasma pneumoniae cytadherence-associated protein HMW2 by fusion with green fluorescent protein: implications for attachment organelle structure. Mol Microbiol 47, 49–60.[CrossRef][Medline]
Baseman, J. B. & Tully, J. G. (1997). Mycoplasmas: sophisticated, reemerging, and burdened by their notoriety. Emerg Infect Dis 3, 21–32.[Medline]
Baseman, J. B., Dallo, S. F., Tully, J. G. & Rose, D. L. (1988). Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract. J Clin Microbiol 26, 2266–2269.
Burgos, R., Pich, O. Q., Ferrer-Navarro, M., Baseman, J. B., Querol, E. & Pinol, J. (2006). Mycoplasma genitalium P140 and P110 cytadhesins are reciprocally stabilized and required for cell adhesion and terminal-organelle development. J Bacteriol 188, 8627–8637.
Burgos, R., Pich, O. Q., Querol, E. & Pinol, J. (2007). Functional analysis of the Mycoplasma genitalium MG312 protein reveals a specific requirement of the MG312 N-terminal domain for gliding motility. J Bacteriol 189, 7014–7023.
Dhandayuthapani, S., Rasmussen, W. G. & Baseman, J. B. (1999). Disruption of gene mg218 of Mycoplasma genitalium through homologous recombination leads to an adherence-deficient phenotype. Proc Natl Acad Sci U S A 96, 5227–5232.
Fraser, C. M., Gocayne, J. D., White, O., Adams, M. D., Clayton, R. A., Fleischmann, R. D., Bult, C. J., Kerlavage, A. R., Sutton, G. & other authors (1995). The minimal gene complement of Mycoplasma genitalium. Science 270, 397–403.
Gobel, U., Speth, V. & Bredt, W. (1981). Filamentous structures in adherent Mycoplasma pneumoniae cells treated with nonionic detergents. J Cell Biol 91, 537–543.
Hasselbring, B. M. & Krause, D. C. (2007). Cytoskeletal protein P41 is required to anchor the terminal organelle of the wall-less prokaryote Mycoplasma pneumoniae. Mol Microbiol 63, 44–53.[CrossRef][Medline]
Hasselbring, B. M., Jordan, J. L. & Krause, D. C. (2005). Mutant analysis reveals a specific requirement for protein P30 in Mycoplasma pneumoniae gliding motility. J Bacteriol 187, 6281–6289.
Hasselbring, B. M., Page, C. A., Sheppard, E. S. & Krause, D. C. (2006). Transposon mutagenesis identifies genes associated with Mycoplasma pneumoniae gliding motility. J Bacteriol 188, 6335–6345.
Hedreyda, C. T. & Krause, D. C. (1995). Identification of a possible cytadherence regulatory locus in Mycoplasma pneumoniae. Infect Immun 63, 3479–3483.
Henderson, G. P. & Jensen, G. J. (2006). Three-dimensional structure of Mycoplasma pneumoniae's attachment organelle and a model for its role in gliding motility. Mol Microbiol 60, 376–385.[CrossRef][Medline]
Krause, D. C. (1996). Mycoplasma pneumoniae cytadherence: unravelling the tie that binds. Mol Microbiol 20, 247–253.[CrossRef][Medline]
Krause, D. C. & Balish, M. F. (2004). Cellular engineering in a minimal microbe: structure and assembly of the terminal organelle of Mycoplasma pneumoniae. Mol Microbiol 51, 917–924.[CrossRef][Medline]
Krause, D. C., Proft, T., Hedreyda, C. T., Hilbert, H., Plagens, H. & Herrmann, R. (1997). Transposon mutagenesis reinforces the correlation between Mycoplasma pneumoniae cytoskeletal protein HMW2 and cytadherence. J Bacteriol 179, 2668–2677.
Lluch-Senar, M., Vallmitjana, M., Querol, E. & Piñol, J. (2007). A new promoterless reporter vector reveals antisense transcription in Mycoplasma genitalium. Microbiology 153, 2743–2752.
Marchler-Bauer, A. & Bryant, S. H. (2004). CD-Search: protein domain annotations on the fly. Nucleic Acids Res 32, W327–W331.
Meng, K. E. & Pfister, R. M. (1980). Intracellular structures of Mycoplasma pneumoniae revealed after membrane removal. J Bacteriol 144, 390–399.
Mernaugh, G. R., Dallo, S. F., Holt, S. C. & Baseman, J. B. (1993). Properties of adhering and nonadhering populations of Mycoplasma genitalium. Clin Infect Dis 17 (Suppl. 1), S69–S78.[Medline]
Mudahi-Orenstein, S., Levisohn, S., Geary, S. J. & Yogev, D. (2003). Cytadherence deficient mutants of Mycoplasma gallisepticum generated by transposon mutagenesis. Infect Immun 71, 3812–3820.
Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. (1999). Probability based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567.[CrossRef][Medline]
Pich, O. Q., Burgos, R., Ferrer-Navarro, M., Querol, E. & Pinol, J. (2006a). Mycoplasma genitalium mg200 and mg386 genes are involved in gliding motility but not in cytadherence. Mol Microbiol 60, 1509–1519.[CrossRef][Medline]
Pich, O. Q., Burgos, R., Planell, R., Querol, E. & Pinol, J. (2006b). Comparative analysis of antibiotic resistance gene markers in Mycoplasma genitalium: application to studies of the minimal gene complement. Microbiology 152, 519–527.
Radestock, U. & Bredt, W. (1977). Motility of Mycoplasma pneumoniae. J Bacteriol 129, 1495–1501.
Reddy, S. P., Rasmussen, W. G. & Baseman, J. B. (1996). Isolation and characterization of transposon Tn4001-generated, cytadherence-deficient transformants of Mycoplasma pneumoniae and Mycoplasma genitalium. FEMS Immunol Med Microbiol 15, 199–211.[CrossRef][Medline]
Roberts, R. J. (2004). Identifying protein function – a call for community action. PLoS Biol 2, E42[CrossRef][Medline]
Seto, S., Kenri, T., Tomiyama, T. & Miyata, M. (2005). Involvement of P1 adhesin in gliding motility of Mycoplasma pneumoniae as revealed by the inhibitory effects of antibody under optimized gliding conditions. J Bacteriol 187, 1875–1877.
Stevens, M. K. & Krause, D. C. (1991). Localization of the Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW4 in the cytoskeleton-like Triton shell. J Bacteriol 173, 1041–1050.
Stevens, M. K. & Krause, D. C. (1992). Mycoplasma pneumoniae cytadherence phase-variable protein HMW3 is a component of the attachment organelle. J Bacteriol 174, 4265–4274.
Svenstrup, H. F., Jensen, J. S., Gevaert, K., Birkelund, S. & Christiansen, G. (2006). Identification and characterization of immunogenic proteins of Mycoplasma genitalium. Clin Vaccine Immunol 13, 913–922.
Taylor-Robinson, D. & Horner, P. J. (2001). The role of Mycoplasma genitalium in non-gonococcal urethritis. Sex Transm Infect 77, 229–231.
Tully, J. G., Rose, D. L., Whitcomb, R. F. & Wenzel, R. P. (1979). Enhanced isolation of Mycoplasma pneumoniae from throat washings with a newly-modified culture medium. J Infect Dis 139, 478–482.[Medline]
Tully, J. G., Taylor-Robinson, D., Cole, R. M. & Rose, D. L. (1981). A newly discovered mycoplasma in the human urogenital tract. Lancet 1, 1288–1291.[Medline]
Willby, M. J. & Krause, D. C. (2002). Characterization of a Mycoplasma pneumoniae hmw3 mutant: implications for attachment organelle assembly. J Bacteriol 184, 3061–3068.
Received 19 May 2008;
revised 18 June 2008;
accepted 18 June 2008.
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