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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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RT-PCR analysis of U. parvum. A. The positions of the different amplicons are shown below the schematic of the hit locus genes of U. parvum. The primers used (Table 2) and the lengths of the amplicons are indicated. B. The PCR products (A – D) for genomic DNA (g), cDNA (c) and RNA (r) were separated on a 0.6 % agarose gel and stained with ethidium bromide. Southern blot analysis was performed with digoxigenin (DIG)-labeled probes hybridizing to one of the four genes, (here shown for UU272 probing), and detected using chemiluminescence. M, Gene Ruler 1 kb DNA ladder (Fermentas).
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Figure 3: RT-PCR analysis of U. parvum. A. The positions of the different amplicons are shown below the schematic of the hit locus genes of U. parvum. The primers used (Table 2) and the lengths of the amplicons are indicated. B. The PCR products (A – D) for genomic DNA (g), cDNA (c) and RNA (r) were separated on a 0.6 % agarose gel and stained with ethidium bromide. Southern blot analysis was performed with digoxigenin (DIG)-labeled probes hybridizing to one of the four genes, (here shown for UU272 probing), and detected using chemiluminescence. M, Gene Ruler 1 kb DNA ladder (Fermentas).

Mentions: Next, we examined the genes flanking hitL 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 hitL (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.


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

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

RT-PCR analysis of U. parvum. A. The positions of the different amplicons are shown below the schematic of the hit locus genes of U. parvum. The primers used (Table 2) and the lengths of the amplicons are indicated. B. The PCR products (A – D) for genomic DNA (g), cDNA (c) and RNA (r) were separated on a 0.6 % agarose gel and stained with ethidium bromide. Southern blot analysis was performed with digoxigenin (DIG)-labeled probes hybridizing to one of the four genes, (here shown for UU272 probing), and detected using chemiluminescence. M, Gene Ruler 1 kb DNA ladder (Fermentas).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: RT-PCR analysis of U. parvum. A. The positions of the different amplicons are shown below the schematic of the hit locus genes of U. parvum. The primers used (Table 2) and the lengths of the amplicons are indicated. B. The PCR products (A – D) for genomic DNA (g), cDNA (c) and RNA (r) were separated on a 0.6 % agarose gel and stained with ethidium bromide. Southern blot analysis was performed with digoxigenin (DIG)-labeled probes hybridizing to one of the four genes, (here shown for UU272 probing), and detected using chemiluminescence. M, Gene Ruler 1 kb DNA ladder (Fermentas).
Mentions: Next, we examined the genes flanking hitL 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 hitL (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.

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