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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 C. pneumoniae. A. Below the schematic of the hit locus of C. pneumoniae the positions of the PCR amplicons (A – F) are shown. Genomic DNA (g), cDNA (c) and RNA without RT reaction (r) are used as templates. B. The PCR products were separated on a 0.8 % agarose gel and subjected to Southern blot analysis with DIG-labeled probes hybridizing to Cp265 (A, D), Cp266 / hitL (B, E) Cp267 (C, F) with visualization using chemiluminescence. In E, the signals of lower length as 1.4 kb may be due to primer dimerization. M, DIG-labeled DNA molecular weight marker VII (Roche Biochemicals)
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Figure 4: RT-PCR analysis of C. pneumoniae. A. Below the schematic of the hit locus of C. pneumoniae the positions of the PCR amplicons (A – F) are shown. Genomic DNA (g), cDNA (c) and RNA without RT reaction (r) are used as templates. B. The PCR products were separated on a 0.8 % agarose gel and subjected to Southern blot analysis with DIG-labeled probes hybridizing to Cp265 (A, D), Cp266 / hitL (B, E) Cp267 (C, F) with visualization using chemiluminescence. In E, the signals of lower length as 1.4 kb may be due to primer dimerization. M, DIG-labeled DNA molecular weight marker VII (Roche Biochemicals)

Mentions: 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.


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

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

RT-PCR analysis of C. pneumoniae. A. Below the schematic of the hit locus of C. pneumoniae the positions of the PCR amplicons (A – F) are shown. Genomic DNA (g), cDNA (c) and RNA without RT reaction (r) are used as templates. B. The PCR products were separated on a 0.8 % agarose gel and subjected to Southern blot analysis with DIG-labeled probes hybridizing to Cp265 (A, D), Cp266 / hitL (B, E) Cp267 (C, F) with visualization using chemiluminescence. In E, the signals of lower length as 1.4 kb may be due to primer dimerization. M, DIG-labeled DNA molecular weight marker VII (Roche Biochemicals)
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Related In: Results  -  Collection

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

Figure 4: RT-PCR analysis of C. pneumoniae. A. Below the schematic of the hit locus of C. pneumoniae the positions of the PCR amplicons (A – F) are shown. Genomic DNA (g), cDNA (c) and RNA without RT reaction (r) are used as templates. B. The PCR products were separated on a 0.8 % agarose gel and subjected to Southern blot analysis with DIG-labeled probes hybridizing to Cp265 (A, D), Cp266 / hitL (B, E) Cp267 (C, F) with visualization using chemiluminescence. In E, the signals of lower length as 1.4 kb may be due to primer dimerization. M, DIG-labeled DNA molecular weight marker VII (Roche Biochemicals)
Mentions: 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.

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