Organization of the hit-locus in Mollicutes

Hit loci with a genomic organization comparable to that of M. hominis [6], with the hit AB genes encoding two membrane proteins and hit L encoding the cytoplasmic HinT, were detected in nearly half of the mollicute genomes analyzed (Figure 1A). The highest similarity was found with the hit locus of M. pulmonis. The MYPU_0080 encoded protein had 44.3 % identity to the M. hominis HinT protein, and the predicted MYPU_0060 and MYPU_0070 proteins were 23.8 % and 26.6 % identical to the membrane proteins P80 and P60 of M. hominis, which were encoded by hit AB. MYPU_0060 had structural features similar to those of P80, with an amino-terminal signal sequence with a predicted signal peptidase I (SPase I) cleavage site and a predominantly alpha-helical structure [10]. MYPU_0070 began with an amino-terminal signal sequence of transmembrane helix (from AA 5 to AA 20) and a signal peptidase II recognition site with a lipoprotein attachment site at position 27. Thus, MYPU_0070 of M. pulmonis appears to encode a P60 homologue, a cysteine-anchored lipoprotein. The order of genes within the hit loci of M. mycoides subsp. mycoides SC (MSC), Mesoplasma florum (MF) and M. mobile (MMOB) was similar with two genes predicted to encode membrane proteins and a downstream hit L gene. The similarities between the predicted sequences of MSC_0500, MF_235 and MMOB_910, and P80 of M. hominis were quite low and the proteins would be significantly larger than P80. While MMOB_910 contained a signal peptidase I recognition site, like P80, MSC_0500 and MF_235 were pro-lipoproteins with an amino-terminal SPase II recognition sequence. Only MSC_500 was predicted to have a predominantly alpha-helical structure, MF_235 being predicted to have a secondary structure of alternate alpha helical and beta sheet regions and MMOB_910 to consist mainly of beta sheets (data not shown). The proteins encoded by the gene next to hit L had little similarity with P60 of M. hominis when the whole sequence was compared. However, when the P60 protein region from AA 164 to AA 177 (CS1; ELQKMLLAKLYLLK) was used, identities of 43 % (MMOB_900) to 57% (MYPU_0070) were detected and scrutiny of the sequence from AA 226 to AA 234 (CS2; LYLMKYLVE) revealed 60 % (MYPU_0070) to 70 % identity (MMOB_900). The corresponding proteins of Mesoplasma florum and M. mycoides subsp. mycoides SC did not contain these conserved sequences. The different HinT homologues had the highest identity with the HinT protein of M. hominis, ranging from 44.3 % (MF_233) to 55.7 % (MMOB_890).

Hit loci with a different genomic organization were identified in the mollicutes Ureaplasma parvum,M. pneumoniae and M. genitalium (Figure 1B). While UU271 of U. parvum, the gene immediately upstream of hit L encoded a putative P60 homologue possessing the P60 consensus sequences CS1 (35.7 % identity) and CS2 (30% identity), it differed from all other P60 homologues in possessing a SPase I recognition sequence and two other transmembrane helices (AA 355 to AA 375 and AA 407 to AA 427). The UU273 gene, immediately downstream of hit L, encoded a protein with two transmembrane spanning helices, but with no significant similarity to other known proteins. The deduced protein sequence of UU270 did not have any sequence similarity with P80. With six transmembrane segments, it appeared more likely to form a pore than to be surface exposed. The organization of the hit locus of U. parvum appeared intermediate between the hit loci described above and those of M. pneumoniae and M. genitalium. The sequence identity between the ureaplasma HinT and the HinT proteins of M. pneumoniae (42.7 %) and M. genitalium (47.4 %) was higher than that between HinT of U. parvum and M. hominis (39.2 %), and the genes immediately upstream of hit L encoded proteins with seven (MPN_274) or five (MG_133) transmembrane domains (Figure 1B). MPN_274 is predicted to encode a permease of an ABC transporter [11] suggesting a comparable function in M. genitalium and probably also in U. parvum. Thus, these hit loci of Mollicutes analyzed appear to have a hit L gene flanked by genes that are predicted to encode membrane-anchored proteins.

As in M. gallisepticum, the hit L gene is not flanked by other genes on the same strand, also exceptions of a polycistronic organization of the hit L gene seem to exist within the Mollicutes.

Organization of the hit loci in Chlamydiaceae

In the obligately intra cellular Chlamydiaceae the order of genes within the hit loci and the function of the encoded proteins appeared to be highly conserved, but distinctly different from that of the Mollicutes (Figure 1C). In all chlamydial species analyzed, the gene upstream of hit L encoded a putative cytoplasmic protein with the signature sequence of a metal-dependent protein hydrolase and a large number of metal binding residues (IPR003226/UPF0160), probably indicative of a phosphoesterase function, and an o ligonucleotide/oligosaccharide-b inding OB fold (IPR008994). The deduced protein sequence of the gene located downstream of hit L contained a 37–47 AA long tandemly repeated ARM repeat fold (IPR008938), which forms a right-handed superhelix and has been implicated in the mediation of protein-protein interactions [12]. Cp267 contained an RGD motif, which plays a role in cell adhesion [13]. However, the presence of RGD in a sequence alone is not sufficient to suggest a biological function for this motif [14]. The presence of two transmembrane helices (AA 314 to AA 334 and AA 428 to AA 448) suggested that it is more likely that Cp267 may interact with the bacterial cell membrane. In all other chlamydial species analyzed, the Cp267 homologues did not contain domains suggestive of membrane attachment.

Are the hit loci genes co-expressed?

In bacteria, overlapping genes on the same coding strand may indicate the polycistronic organization of these genes, and co-expressed proteins are often related in function.

As the hit locus of M. hominis is an operon containing three genes [6] the next step was to analyze whether the polycistronic organization of hit genes was conserved within the Mollicutes and whether this also occurred in Chlamydiaceae. Initially, we chose M. pulmonis, the species with the greatest similarity to M. hominis, for reverse transcription (RT) PCR analysis. To identify the likely boundaries of an mRNA containing MYPU_0060, MYPU_0070 and MYPU_0080, two different primers upstream of the MYPU_0060/hit A gene and three primers downstream of the hit L gene were used (Fig. 2A). Amplification occurred with primers binding just upstream of the predicted promoter regions 1 or 2 and just downstream of the TAA stop codon of the hit L gene (Fig. 2B, lanes A and B) indicating a polycistronic mRNA. Amplification was not seen with a primer hybridizing to the sequence downstream of a predicted hairpin loop structure after the TAA stop codon of hit L (Figure 2B, lanes C1 and C2) suggesting that the mRNA terminates at the predicted hairpin structure.

Next, we examined the genes flanking hit L in U. parvum. Amplicons which spanned the intergenic regions were obtained with primers hybridizing to the center of UU270 and to the 3'-end of UU272 (Fig. 3C), and with primers hybridizing to the 5'-end of UU271 and to the 3'-end of UU273 (Fig. 3D). No amplification occurred with a primer hybridizing to the 5'-end of UU270 and the 3'-end of hit L (Fig. 3A and 3B). These data suggest that HinT is expressed with the flanking genes UU271 and UU273. These findings are in accordance with the predicted termination of transcription by a hairpin loop with a stem energy of -9.1 kcal mole^-1 located 33 nt downstream of the UU273 gene [15]. Of the organisms analyzed so far U. parvum was the most demanding organism of the Mollicutes in terms of DNA-free, full-length total RNA preparation. Thus the detection of a common RNA from the center of UU270 up to UU273 (Fig. 3D) may be due to an unstable RNA or indicatory for UU270 not taking part in the operon structure.

To characterize the organization of hit loci genes within the Chlamydiaceae we analyzed the locus in Chlamydophila pneumoniae. The genes Cp266 (encoding HinT), Cp267 and Cp268, which is predicted to encode a solute symporter family protein [16], were predicted to comprise an operon. No amplification occurred when a primer pair that hybridized upstream of Cp265 and downstream of Cp267 was used (data not shown). Amplification was detected using primers binding within each gene (Figure 4A,B,C) and with primers binding within Cp265 and Cp266 (Fig. 4D) and within Cp266 and Cp267 (Fig. 4E). The predicted co-expression of Cp267 and Cp268 downstream of which several putative transcription terminators were detected, was not supported by RT-PCR analysis (Fig. 4F), with amplification being obtained when using genomic DNA as a template but not with cDNA.

Interactions between the proteins encoded by the hit locus of C. pneumoniae

As the stability of an RNA is increased in regions with pronounced secondary structures, such as transcriptional terminators, and is often decreased in regions lacking such protective structures [17], partial degradation of the chlamydial mRNA may have occurred. The results from RT-PCR analysis of the mRNA of CP265, Cp266 and Cp267 suggest an operon producing a transient mRNA thus suggesting a physical interaction between the products of these genes. Therefore we analyzed the interactions between Cp265, Cp266 and Cp267 using the yeast two-hybrid system [18]. The system is based on the modular organization of the transcription factor GAL4, which has a DNA-binding domain (DB) and an activation domain (AD). When GAL4 binds (via DB) to its cognate binding site, the activation domain AD is brought close to the promoter and activates transcription of the reporter genes lacZ and HIS3. In the yeast two-hybrid system these two domains, which alone are not able to activate transcription, are separately cloned into pGADT7 (AD) and pGBKT7 (BD) and expressed in fusion with the proteins under investigation. Growth of a histidine dependent yeast strain on histidine deficient media will only occur after transformation with both plasmids if the AD and DB domains are apposed by an interaction between the fusion proteins, which will result in transcription of the reporter genes. The plasmids pGADT7-T and pGBKT7-53, which encode the SV40 large T-antigen and the murine p53 protein, respectively served as a positive control (Fig. 5 K+; Fig. 6A).

HinT is known to dimerize [19, 20]. We introduced the coding region of M. hominis HinT into both pGADT7 and pGBKT7 and transformed the histidine dependent yeast strain AH109 with them. Quantification of transcriptional activation of the reporter gene was achieved using a liquid β-galactosidase assay measuring the hydrolysis of 4-methylumbelliferyl-β, D-galactopyranoside (MUG), yielding the fluorescent molecule 4-methylumbelliferone (4 MU). As expected, dimerization of the fused HinT peptides led to apposition of the AD and DB domains generating a functional transcription factor. The transcription of the reporter genes resulted in growth of the yeast on histidine-deficient agar plates (Fig. 5, top row, left plate) and production of β-galactosidase (Fig. 6C). Next we analyzed whether the proteins encoded by the hit locus of C. pneumoniae interacted. The coding regions of Cp265, Cp266 and Cp267 were introduced into pGADT7 and pGBKT7 and expressed in yeast in all possible combinations. As shown in Figure 5, large colonies, comparable in size to those of the positive control, were produced by yeast that expressed Cp265 fused to both the binding and the activation domain. This suggested a strong interaction between the molecules of the Cp265 protein. All other combinations of plasmids encoding Cp265, Cp266 and Cp267 only led to the growth of small colonies, a finding that was difficult to interpret. To differentiate between strong and weak interactions, the β-galactosidase assay was used. As shown in Fig. 6, the measurement of β-galactoside activity indicated an interaction between Cp267 and Cp265, Cp266 and Cp267 itself, independent of the fusion partner (the AD or DB domains of the GAL4 protein). Surprisingly, dimerization of Cp266 (HinT) was not detected, although Cp265 interacted strongly with itself. Cp267 did not activate transcription by itself (Fig. 6A. 267/BD and 267/AD) nor did it interact with the unrelated protein SV40 large T antigen (T/267) or murine tumor suppressor p53 (267/p53) to activate transcription, as in all these cases the β-galactosidase activity was at background levels (Fig. 6A).

Immune co-precipitation of Cp265, Cp266 and Cp267

To confirm the findings derived from the two-hybrid analyses we performed protein-binding assays in vitro. Protein C-tagged Cp265, Cp266 and Cp267 were expressed in E. coli and purified by affinity chromatography (Fig. 7A). Each protein was then incubated with equal amounts of [³⁵S]-labeled Cp265, Cp266 or Cp267 in vitro and precipitated with anti-Protein C antibodies. As shown in Fig. 7B, the supernatants of the different samples contained comparable amounts of the respective isotope labeled proteins. As expected the [³⁵S] labeled Cp proteins alone were neither precipitated by unrelated membrane proteins, such as Protein C-tagged OppA of M. hominis nor by the anti-Protein C-sepharose itself (Fig. 7C). In contrast, an interaction of Cp266 with Cp267 was suggested by co-precipitation of significant amounts of Cp266 [35S] with Cp267^C, and Cp267 [35S] with Cp266^C (Fig. 7C). The other findings from the two-hybrid analyses were also confirmed as both Cp265 and Cp267 were found to dimerize and there was no interaction between Cp265 and Cp266. Interestingly dimerization of Cp266 (HinT) which was not detected by the two-hybrid system, was demonstrated in this co-precipitation assay.

Thus, we can conclude that the chlamydial proteins Cp265, Cp266 (HinT) and Cp267 have a tendency to dimerize and Cp267 interacts with both Cp265 and Cp266.

DNA manipulations

All routine DNA manipulation techniques, including plasmid preparation, restriction endonuclease analysis, ligation and transformation of E. coli were performed as described by Sambrook et al. [31] or according to the manufacturers' recommendations (Qiagen, Hilden, Germany, and Roche Applied Science, Mannheim, Germany).

Bacterial strains and plasmids

The plasmids pXB (Roche Applied Science, Mannheim, Germany), pGADT7 and pGBKT7 (BD Bioscience Clontech, Palo Alto, USA) were used as expression vectors for the heterologous expression of Cp265, Cp266 and Cp267. The pXB plasmids were propagated in Escherichia coli DH5α F'(Life Technologies), pGADT7 and pGBKT7 in yeast AH109 cells (BD Biosciences Clontech, Palo Alto, CA USA).

Sequence analysis

Analysis of the DNA and protein sequences and the design of oligonucleotides were facilitated by the Lasergene software (DNA Star Inc., Madison Wisc.).

Computer-based prediction of transcription termination was performed using the GeneBee service (online http://www.genebee.msu.su/services/rna2_reduced.html). Distant relation-ships between HinT protein sequences of the analyzed Mollicutes were determined by using the BLAST method through Molligen 1.4 [32]. Conserved domains of proteins were predicted using the Conserved Domain Database (CDD) or the InterPro service (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, http://www.ebi.ac.uk/interpro/).

RT-PCR

RNA was prepared from exponential phase cultures of M. pulmonis and U. parvum as described by Chomczynski and Sacchi [33]. Chlamydophila pneumoniae respiratory isolate GiD was used for all experiments [34]. Chlamydial RNA was prepared from HEp-2 cells 2 days post-infection as described by Mölleken et al. (Mölleken et al., manuscript in preparartion). Before use as a template for RT-PCR, contaminating traces of DNA were digested with DNase I as described previously [35]. Overlapping regions of the Ureaplasma and Chlamydophila genes were amplified using the Expand Template PCR system (Roche Applied Science, Mannheim, Germany) by standard PCR conditions (initial cycle of 5 min at 95°C; 35 cycles of 1 min at 95°C, 1 min at 50°C (U. parvum) or 58°C (C. pneumoniae), 2.5 min at 68°C). The hit operon of M. pulmonis was amplified using the Expand Long Template PCR system by standard PCR conditions (initial cycle of 5 min at 95°C; 10 cycles of 1 min at 95°C, 1 min at 44°C, 4 min at 68°C; addition of 0.5 U polymerase; then 20 cycles of 1 min at 95°C, 30 sec at 44°C, 4 min (+ 20 sec per cycle) at 68 °C). Southern blot analysis was performed as described previously [6].

Construction of plasmids

For heterologous expression of the protein-C tagged proteins, CP265^C, Cp266^C and Cp267^C, in E. coli, the Cp inserts of the respective pGADT7 plasmids were digested with Sac I, the ends bluntened by incubation with 3 units S1 nuclease (Amersham Bioscience, Freiburg, Germany) for 30 min at 37°C and subsequently cut with Eco RI. The blunt-end/Eco RI digested Cp265, Cp266 and Cp267 fragments were cloned in-frame into the blunt-end/Eco RI digested plasmid pXB and propagated in E. coli Dh5α F'.

All plasmid constructs were confirmed by DNA sequencing (ABI 373 A machine) [36].

Expression and purification of Cp265^C, Cp266^C and Cp267^C

One liter of LB broth (Gibco BRL, Life Technologies Inc., Gaithersburg, USA) containing ampicillin (100 μg/ml) was inoculated with the respective E. coli DH5α F' clone and incubated for 16 h at 37°C with vigorous shaking. The cells were harvested by centrifugation (15,000 × g, 20 min, 4°C) and frozen at -70°C. After thawing on ice, the cells were resuspended in 40 ml buffer A (120 mM NaCl, 1 mM CaCl₂, 20 mM Tris-HCl pH 7.5) and disrupted by three repeated freeze-thaw cycles in liquid nitrogen, followed by three bursts of sonication on ice (5 min bursts at 95 W with a 1 min cooling period between each burst). Insoluble material was sedimented (15,000 × g, 20 min, 4°C) and the supernatant was transferred to an anti-Protein C affinity matrix (Roche Applied Science, Mannheim, Germany). Bound proteins were eluted from the anti-Protein C affinity matrix with 2 mM EDTA.

SDS-PAGE and immunostaining of proteins

Proteins were separated on 12 or 15 % polyacrylamide gels [37], transferred to nitrocellulose (Schleicher and Schüll, Dassel, Germany) using a semi-dry blotting apparatus (Phase, Mölln, Germany) [38], and immunostained using anti-Protein C peroxidase (Roche Applied Science, Mannheim, Germany), anti HA-antibody (pGADT7) or anti c-myc antibody (pGBKT7) (BD Bioscience Clontech, Palo Alto, USA).

Immune co-precipitation assay

The [³⁵S]-labeled Cp265, Cp266 and Cp267 proteins were generated from pGADT7 and pGBKT7 fusion vectors by in vitro translation using a T7 coupled transcription/translation system in the presence of [³⁵S]-methionine (Promega, Madison, USA). Two micrograms of purified Protein C-tagged protein were incubated with 10 μl of the [³⁵S]-labeled protein in buffer B (20 mM Tris/HCl (pH 7.5), 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl₂, 0.05 (v/v) % Igepal, 2 mM DTT, 10 (v/v) % glycerol and 2 mM CaCl₂) for 1 h [39]. Thereafter, a 5 μl aliquot of a slurry of the anti-Protein C matrix, equilibrated in buffer B containing 5 μg BSA, was added to each sample and the mixture further incubated for 1 h. The Protein C-matrix was washed eight times in buffer B, resuspended in 20 μl of SDS-PAGE sample buffer and heated at 95°C for 5 min. The proteins were then analyzed by 15 % SDS-PAGE and autoradiography using a phosphorimager (Fujifilm FLA 3000).

Yeast two-hybrid assays

The MATCHMAKER Gal4 two-hybrid system (BD Bioscience Clontech, Palo Alto, USA) was used for interaction assays. AH109 cells were co-transformed with the Cp265, Cp266 or Cp267 expressing pGADT-T7 and pGBKT7 plasmids, grown in small scale cultures and plated onto histidine deficient agar plates, followed by a β-galactosidase colony lift filter assay. All procedures were carried out as outlined in the BD Bioscience Clontech protocol. The β-galactosidase assay was performed after the inoculation of 7.5 ml YPD Medium with 0.5 ml overnight culture in selective medium and incubation for five hours at 30°C until the culture reached an OD₆₀₀ of 0.4 to 0.6. The cells were harvested by centrifugation at 14,000 × g for 1 min and re-suspended in 300 μl Buffer Z (Na₂HPO₄+ 7 × H₂O, NaH₂PO₄ + 4 × H₂O, KCl, MgSO₄ × 7 H₂O, pH7.0). Lysis of the cells was achieved with four cycles of freezing in liquid nitrogen and thawing at 37°C. Soluble and insoluble components were separated by centrifugation at 14000 × g for 2 min. The β-galactosidase activity was measured using a FluorAce™ β-galactosidase Reporter Assay kit (BioRad, Hercules, USA).