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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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Quantitation of β-galactosidase activity. In yeast cells, co-transformed with pGADT7 (AD) and pGBKT7 (BD) constructs as indicated, β-galactosidase activity was quantified using 4-methylumbelliferyl-galactopyranoside (MUG) hydrolysis to the fluorescent molecule, 4-methylumbelliferone (4 MU). The fluorescence of 4 MU was excited at 360 nm and emission measured at 460 nm.
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Figure 6: Quantitation of β-galactosidase activity. In yeast cells, co-transformed with pGADT7 (AD) and pGBKT7 (BD) constructs as indicated, β-galactosidase activity was quantified using 4-methylumbelliferyl-galactopyranoside (MUG) hydrolysis to the fluorescent molecule, 4-methylumbelliferone (4 MU). The fluorescence of 4 MU was excited at 360 nm and emission measured at 460 nm.

Mentions: 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 proteins and their putative interaction partners in Mollicutes and Chlamydiaceae.

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

Quantitation of β-galactosidase activity. In yeast cells, co-transformed with pGADT7 (AD) and pGBKT7 (BD) constructs as indicated, β-galactosidase activity was quantified using 4-methylumbelliferyl-galactopyranoside (MUG) hydrolysis to the fluorescent molecule, 4-methylumbelliferone (4 MU). The fluorescence of 4 MU was excited at 360 nm and emission measured at 460 nm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Quantitation of β-galactosidase activity. In yeast cells, co-transformed with pGADT7 (AD) and pGBKT7 (BD) constructs as indicated, β-galactosidase activity was quantified using 4-methylumbelliferyl-galactopyranoside (MUG) hydrolysis to the fluorescent molecule, 4-methylumbelliferone (4 MU). The fluorescence of 4 MU was excited at 360 nm and emission measured at 460 nm.
Mentions: 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).

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