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HinT proteins and their putative interaction partners in Mollicutes and Chlamydiaceae.

Hopfe M, Hegemann JH, Henrich B - BMC Microbiol. (2005)

Bottom Line: HinT proteins are found in prokaryotes and eukaryotes and belong to the superfamily of HIT proteins, which are characterized by an histidine-triad sequence motif.An cluster of hitABL genes, similar to that of M. hominis was found in M. pulmonis, M. mycoides subspecies mycoides SC, M. mobile and Mesoplasma florum.In the Mollicutes HinT proteins were shown to be linked with membrane proteins while in the Chlamydiaceae they were genetically and physically associated with cytoplasmic proteins, one of which is predicted to be a metal-dependent phosphoesterase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medical Microbiology, Moorenstrasse 5, 40225 Duesseldorf, Germany. Miriam.Hopfe@t-online.de

ABSTRACT

Background: HinT proteins are found in prokaryotes and eukaryotes and belong to the superfamily of HIT proteins, which are characterized by an histidine-triad sequence motif. While the eukaryotic variants hydrolyze AMP derivates and modulate transcription, the function of prokaryotic HinT proteins is less clearly defined. In Mycoplasma hominis, HinT is concomitantly expressed with the proteins P60 and P80, two domains of a surface exposed membrane complex, and in addition interacts with the P80 moiety.

Results: An cluster of hitABL genes, similar to that of M. hominis was found in M. pulmonis, M. mycoides subspecies mycoides SC, M. mobile and Mesoplasma florum. RT-PCR analyses provided evidence that the P80, P60 and HinT homologues of M. pulmonis were polycistronically organized, suggesting a genetic and physical interaction between the proteins encoded by these genes in these species. While the hit loci of M. pneumoniae and M. genitalium encoded, in addition to HinT, a protein with several transmembrane segments, the hit locus of Ureaplasma parvum encoded a pore-forming protein, UU270, a P60 homologue, UU271, HinT, UU272, and a membrane protein of unknown function, UU273. Although a full-length mRNA spanning the four genes was not detected, amplification of all intergenic regions from the center of UU270 to the end of UU273 by RT-PCR may be indicative of a common, but unstable mRNA. In Chlamydiaceae the hit gene is flanked upstream by a gene predicted to encode a metal dependent hydrolase and downstream by a gene putatively encoding a protein with ARM-repeats, which are known to be involved in protein-protein interactions. In RT-PCR analyses of C. pneumoniae, regions comprising only two genes, Cp265/Cp266 and Cp266/Cp267 were able to be amplified. In contrast to this in vivo interaction analysis using the yeast two-hybrid system and in vitro immune co-precipitation revealed an interaction between Cp267, which contains the ARM repeats, Cp265, the predicted hydrolase, and Cp266, the HinT protein.

Conclusion: In the Mollicutes HinT proteins were shown to be linked with membrane proteins while in the Chlamydiaceae they were genetically and physically associated with cytoplasmic proteins, one of which is predicted to be a metal-dependent phosphoesterase. Future work will elucidate whether these differing associations indicate that HinT proteins have evolved independently or are indeed two hotspots of a common sphere of action of bacterial HinT proteins.

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Immune co-precipitation. A. Affinity purified proteins, Cp265 (265C), Cp266 (266C), Cp267 (267C) and OppAC, were analyzed on 15 % SDS-polyacrylamide gels by Coomassie staining (C) or on Western blots by immunostaining with an anti-Protein C antibody (W). [35S]-labeled Cp265, Cp266 and Cp267 (Captives) were incubated at room temperature for 1 h with Cp265C, Cp266C, Cp267C and OppAC (Catcher). Immune complexes were precipitated with anti-Protein C-matrix, and the supernatants (B.) and precipitates (C.) analyzed on 15 % SDS-polyacrylamide gels by autoradiography using a phosphorimager. As a negative control, the [35S]-labeled proteins were subjected to the Protein C-matrix without a catcher (Sepha.αC). M, prestained low-molecular weight marker (Biorad)
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Figure 7: Immune co-precipitation. A. Affinity purified proteins, Cp265 (265C), Cp266 (266C), Cp267 (267C) and OppAC, were analyzed on 15 % SDS-polyacrylamide gels by Coomassie staining (C) or on Western blots by immunostaining with an anti-Protein C antibody (W). [35S]-labeled Cp265, Cp266 and Cp267 (Captives) were incubated at room temperature for 1 h with Cp265C, Cp266C, Cp267C and OppAC (Catcher). Immune complexes were precipitated with anti-Protein C-matrix, and the supernatants (B.) and precipitates (C.) analyzed on 15 % SDS-polyacrylamide gels by autoradiography using a phosphorimager. As a negative control, the [35S]-labeled proteins were subjected to the Protein C-matrix without a catcher (Sepha.αC). M, prestained low-molecular weight marker (Biorad)

Mentions: 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 Cp267C, and Cp267 [35S] with Cp266C (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.


HinT proteins and their putative interaction partners in Mollicutes and Chlamydiaceae.

Hopfe M, Hegemann JH, Henrich B - BMC Microbiol. (2005)

Immune co-precipitation. A. Affinity purified proteins, Cp265 (265C), Cp266 (266C), Cp267 (267C) and OppAC, were analyzed on 15 % SDS-polyacrylamide gels by Coomassie staining (C) or on Western blots by immunostaining with an anti-Protein C antibody (W). [35S]-labeled Cp265, Cp266 and Cp267 (Captives) were incubated at room temperature for 1 h with Cp265C, Cp266C, Cp267C and OppAC (Catcher). Immune complexes were precipitated with anti-Protein C-matrix, and the supernatants (B.) and precipitates (C.) analyzed on 15 % SDS-polyacrylamide gels by autoradiography using a phosphorimager. As a negative control, the [35S]-labeled proteins were subjected to the Protein C-matrix without a catcher (Sepha.αC). M, prestained low-molecular weight marker (Biorad)
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1173108&req=5

Figure 7: Immune co-precipitation. A. Affinity purified proteins, Cp265 (265C), Cp266 (266C), Cp267 (267C) and OppAC, were analyzed on 15 % SDS-polyacrylamide gels by Coomassie staining (C) or on Western blots by immunostaining with an anti-Protein C antibody (W). [35S]-labeled Cp265, Cp266 and Cp267 (Captives) were incubated at room temperature for 1 h with Cp265C, Cp266C, Cp267C and OppAC (Catcher). Immune complexes were precipitated with anti-Protein C-matrix, and the supernatants (B.) and precipitates (C.) analyzed on 15 % SDS-polyacrylamide gels by autoradiography using a phosphorimager. As a negative control, the [35S]-labeled proteins were subjected to the Protein C-matrix without a catcher (Sepha.αC). M, prestained low-molecular weight marker (Biorad)
Mentions: 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 Cp267C, and Cp267 [35S] with Cp266C (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.

Bottom Line: HinT proteins are found in prokaryotes and eukaryotes and belong to the superfamily of HIT proteins, which are characterized by an histidine-triad sequence motif.An cluster of hitABL genes, similar to that of M. hominis was found in M. pulmonis, M. mycoides subspecies mycoides SC, M. mobile and Mesoplasma florum.In the Mollicutes HinT proteins were shown to be linked with membrane proteins while in the Chlamydiaceae they were genetically and physically associated with cytoplasmic proteins, one of which is predicted to be a metal-dependent phosphoesterase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medical Microbiology, Moorenstrasse 5, 40225 Duesseldorf, Germany. Miriam.Hopfe@t-online.de

ABSTRACT

Background: HinT proteins are found in prokaryotes and eukaryotes and belong to the superfamily of HIT proteins, which are characterized by an histidine-triad sequence motif. While the eukaryotic variants hydrolyze AMP derivates and modulate transcription, the function of prokaryotic HinT proteins is less clearly defined. In Mycoplasma hominis, HinT is concomitantly expressed with the proteins P60 and P80, two domains of a surface exposed membrane complex, and in addition interacts with the P80 moiety.

Results: An cluster of hitABL genes, similar to that of M. hominis was found in M. pulmonis, M. mycoides subspecies mycoides SC, M. mobile and Mesoplasma florum. RT-PCR analyses provided evidence that the P80, P60 and HinT homologues of M. pulmonis were polycistronically organized, suggesting a genetic and physical interaction between the proteins encoded by these genes in these species. While the hit loci of M. pneumoniae and M. genitalium encoded, in addition to HinT, a protein with several transmembrane segments, the hit locus of Ureaplasma parvum encoded a pore-forming protein, UU270, a P60 homologue, UU271, HinT, UU272, and a membrane protein of unknown function, UU273. Although a full-length mRNA spanning the four genes was not detected, amplification of all intergenic regions from the center of UU270 to the end of UU273 by RT-PCR may be indicative of a common, but unstable mRNA. In Chlamydiaceae the hit gene is flanked upstream by a gene predicted to encode a metal dependent hydrolase and downstream by a gene putatively encoding a protein with ARM-repeats, which are known to be involved in protein-protein interactions. In RT-PCR analyses of C. pneumoniae, regions comprising only two genes, Cp265/Cp266 and Cp266/Cp267 were able to be amplified. In contrast to this in vivo interaction analysis using the yeast two-hybrid system and in vitro immune co-precipitation revealed an interaction between Cp267, which contains the ARM repeats, Cp265, the predicted hydrolase, and Cp266, the HinT protein.

Conclusion: In the Mollicutes HinT proteins were shown to be linked with membrane proteins while in the Chlamydiaceae they were genetically and physically associated with cytoplasmic proteins, one of which is predicted to be a metal-dependent phosphoesterase. Future work will elucidate whether these differing associations indicate that HinT proteins have evolved independently or are indeed two hotspots of a common sphere of action of bacterial HinT proteins.

Show MeSH
Related in: MedlinePlus