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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 M. pulmonis. A. The regions amplified by RT-PCR are shown below the genomic region encompassing MYPU_0060, MYPU_0070 and MYPU_0080. The primers used and the lengths of the amplicons are indicated. B. Amplicons were separated on a 0.8 % agarose gel and subjected to Southern blot analysis, here shown for the MYPU_0070 specific probe, using digoxigenin (DIG)-labeled probes hybridizing to each of the three genes, detected using chemiluminescence. Bands of lower length as expected are degradation products. M, DIG-labeled DNA molecular weight marker VII (Roche Biochemicals).
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Figure 2: RT-PCR analysis of M. pulmonis. A. The regions amplified by RT-PCR are shown below the genomic region encompassing MYPU_0060, MYPU_0070 and MYPU_0080. The primers used and the lengths of the amplicons are indicated. B. Amplicons were separated on a 0.8 % agarose gel and subjected to Southern blot analysis, here shown for the MYPU_0070 specific probe, using digoxigenin (DIG)-labeled probes hybridizing to each of the three genes, detected using chemiluminescence. Bands of lower length as expected are degradation products. M, DIG-labeled DNA molecular weight marker VII (Roche Biochemicals).

Mentions: 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/hitA gene and three primers downstream of the hitL 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 hitL 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 hitL (Figure 2B, lanes C1 and C2) suggesting that the mRNA terminates at the predicted hairpin 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 M. pulmonis. A. The regions amplified by RT-PCR are shown below the genomic region encompassing MYPU_0060, MYPU_0070 and MYPU_0080. The primers used and the lengths of the amplicons are indicated. B. Amplicons were separated on a 0.8 % agarose gel and subjected to Southern blot analysis, here shown for the MYPU_0070 specific probe, using digoxigenin (DIG)-labeled probes hybridizing to each of the three genes, detected using chemiluminescence. Bands of lower length as expected are degradation products. M, DIG-labeled DNA molecular weight marker VII (Roche Biochemicals).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: RT-PCR analysis of M. pulmonis. A. The regions amplified by RT-PCR are shown below the genomic region encompassing MYPU_0060, MYPU_0070 and MYPU_0080. The primers used and the lengths of the amplicons are indicated. B. Amplicons were separated on a 0.8 % agarose gel and subjected to Southern blot analysis, here shown for the MYPU_0070 specific probe, using digoxigenin (DIG)-labeled probes hybridizing to each of the three genes, detected using chemiluminescence. Bands of lower length as expected are degradation products. M, DIG-labeled DNA molecular weight marker VII (Roche Biochemicals).
Mentions: 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/hitA gene and three primers downstream of the hitL 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 hitL 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 hitL (Figure 2B, lanes C1 and C2) suggesting that the mRNA terminates at the predicted hairpin 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