Limits...
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.

Show MeSH

Related in: MedlinePlus

Schematic of the hit loci in Mollicutes and Chlamydiaceae. A schematic representation of genes within the hit loci of the following species: A) M. pulmonis, M. mycoides subsp. mycoides SC, Mesoplasma florum and M. mobile containing genes homologous to hitABL of M. hominis; B) U. parvum, M. pneumoniae and M. genitalium, each containing a gene upstream of hitL predicted to encode an integral pore-forming protein; and C) C. pneumoniae, C. trachomatis, C. muridarum and C. caviae containing hitL flanking genes predicted to encode, upstream, a protein with the signature sequence of a metal-dependent hydrolase (light blue region), with an OB nucleic acid binding fold (dark blue region) and, downstream, a protein with ARM repeats (red regions), which are known to mediate protein-protein interactions. Triangles indicate signal peptidase recognition sites of SPase I and SPase II. Transmembrane regions are depicted as striped regions. The position of the RGD tri-peptide is marked by a dotted region.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1173108&req=5

Figure 1: Schematic of the hit loci in Mollicutes and Chlamydiaceae. A schematic representation of genes within the hit loci of the following species: A) M. pulmonis, M. mycoides subsp. mycoides SC, Mesoplasma florum and M. mobile containing genes homologous to hitABL of M. hominis; B) U. parvum, M. pneumoniae and M. genitalium, each containing a gene upstream of hitL predicted to encode an integral pore-forming protein; and C) C. pneumoniae, C. trachomatis, C. muridarum and C. caviae containing hitL flanking genes predicted to encode, upstream, a protein with the signature sequence of a metal-dependent hydrolase (light blue region), with an OB nucleic acid binding fold (dark blue region) and, downstream, a protein with ARM repeats (red regions), which are known to mediate protein-protein interactions. Triangles indicate signal peptidase recognition sites of SPase I and SPase II. Transmembrane regions are depicted as striped regions. The position of the RGD tri-peptide is marked by a dotted region.

Mentions: Hit loci with a genomic organization comparable to that of M. hominis [6], with the hitAB genes encoding two membrane proteins and hitL encoding the cytoplasmic HinT, were detected in nearly half of the mollicute genomes analyzed (Figure 1A). The highest similarity was found with the hit locus of M. pulmonis. The MYPU_0080 encoded protein had 44.3 % identity to the M. hominis HinT protein, and the predicted MYPU_0060 and MYPU_0070 proteins were 23.8 % and 26.6 % identical to the membrane proteins P80 and P60 of M. hominis, which were encoded by hitAB. MYPU_0060 had structural features similar to those of P80, with an amino-terminal signal sequence with a predicted signal peptidase I (SPase I) cleavage site and a predominantly alpha-helical structure [10]. MYPU_0070 began with an amino-terminal signal sequence of transmembrane helix (from AA 5 to AA 20) and a signal peptidase II recognition site with a lipoprotein attachment site at position 27. Thus, MYPU_0070 of M. pulmonis appears to encode a P60 homologue, a cysteine-anchored lipoprotein. The order of genes within the hit loci of M. mycoides subsp. mycoides SC (MSC), Mesoplasma florum (MF) and M. mobile (MMOB) was similar with two genes predicted to encode membrane proteins and a downstream hitL gene. The similarities between the predicted sequences of MSC_0500, MF_235 and MMOB_910, and P80 of M. hominis were quite low and the proteins would be significantly larger than P80. While MMOB_910 contained a signal peptidase I recognition site, like P80, MSC_0500 and MF_235 were pro-lipoproteins with an amino-terminal SPase II recognition sequence. Only MSC_500 was predicted to have a predominantly alpha-helical structure, MF_235 being predicted to have a secondary structure of alternate alpha helical and beta sheet regions and MMOB_910 to consist mainly of beta sheets (data not shown). The proteins encoded by the gene next to hitL had little similarity with P60 of M. hominis when the whole sequence was compared. However, when the P60 protein region from AA 164 to AA 177 (CS1; ELQKMLLAKLYLLK) was used, identities of 43 % (MMOB_900) to 57% (MYPU_0070) were detected and scrutiny of the sequence from AA 226 to AA 234 (CS2; LYLMKYLVE) revealed 60 % (MYPU_0070) to 70 % identity (MMOB_900). The corresponding proteins of Mesoplasma florum and M. mycoides subsp. mycoides SC did not contain these conserved sequences. The different HinT homologues had the highest identity with the HinT protein of M. hominis, ranging from 44.3 % (MF_233) to 55.7 % (MMOB_890).


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

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

Schematic of the hit loci in Mollicutes and Chlamydiaceae. A schematic representation of genes within the hit loci of the following species: A) M. pulmonis, M. mycoides subsp. mycoides SC, Mesoplasma florum and M. mobile containing genes homologous to hitABL of M. hominis; B) U. parvum, M. pneumoniae and M. genitalium, each containing a gene upstream of hitL predicted to encode an integral pore-forming protein; and C) C. pneumoniae, C. trachomatis, C. muridarum and C. caviae containing hitL flanking genes predicted to encode, upstream, a protein with the signature sequence of a metal-dependent hydrolase (light blue region), with an OB nucleic acid binding fold (dark blue region) and, downstream, a protein with ARM repeats (red regions), which are known to mediate protein-protein interactions. Triangles indicate signal peptidase recognition sites of SPase I and SPase II. Transmembrane regions are depicted as striped regions. The position of the RGD tri-peptide is marked by a dotted region.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Schematic of the hit loci in Mollicutes and Chlamydiaceae. A schematic representation of genes within the hit loci of the following species: A) M. pulmonis, M. mycoides subsp. mycoides SC, Mesoplasma florum and M. mobile containing genes homologous to hitABL of M. hominis; B) U. parvum, M. pneumoniae and M. genitalium, each containing a gene upstream of hitL predicted to encode an integral pore-forming protein; and C) C. pneumoniae, C. trachomatis, C. muridarum and C. caviae containing hitL flanking genes predicted to encode, upstream, a protein with the signature sequence of a metal-dependent hydrolase (light blue region), with an OB nucleic acid binding fold (dark blue region) and, downstream, a protein with ARM repeats (red regions), which are known to mediate protein-protein interactions. Triangles indicate signal peptidase recognition sites of SPase I and SPase II. Transmembrane regions are depicted as striped regions. The position of the RGD tri-peptide is marked by a dotted region.
Mentions: Hit loci with a genomic organization comparable to that of M. hominis [6], with the hitAB genes encoding two membrane proteins and hitL encoding the cytoplasmic HinT, were detected in nearly half of the mollicute genomes analyzed (Figure 1A). The highest similarity was found with the hit locus of M. pulmonis. The MYPU_0080 encoded protein had 44.3 % identity to the M. hominis HinT protein, and the predicted MYPU_0060 and MYPU_0070 proteins were 23.8 % and 26.6 % identical to the membrane proteins P80 and P60 of M. hominis, which were encoded by hitAB. MYPU_0060 had structural features similar to those of P80, with an amino-terminal signal sequence with a predicted signal peptidase I (SPase I) cleavage site and a predominantly alpha-helical structure [10]. MYPU_0070 began with an amino-terminal signal sequence of transmembrane helix (from AA 5 to AA 20) and a signal peptidase II recognition site with a lipoprotein attachment site at position 27. Thus, MYPU_0070 of M. pulmonis appears to encode a P60 homologue, a cysteine-anchored lipoprotein. The order of genes within the hit loci of M. mycoides subsp. mycoides SC (MSC), Mesoplasma florum (MF) and M. mobile (MMOB) was similar with two genes predicted to encode membrane proteins and a downstream hitL gene. The similarities between the predicted sequences of MSC_0500, MF_235 and MMOB_910, and P80 of M. hominis were quite low and the proteins would be significantly larger than P80. While MMOB_910 contained a signal peptidase I recognition site, like P80, MSC_0500 and MF_235 were pro-lipoproteins with an amino-terminal SPase II recognition sequence. Only MSC_500 was predicted to have a predominantly alpha-helical structure, MF_235 being predicted to have a secondary structure of alternate alpha helical and beta sheet regions and MMOB_910 to consist mainly of beta sheets (data not shown). The proteins encoded by the gene next to hitL had little similarity with P60 of M. hominis when the whole sequence was compared. However, when the P60 protein region from AA 164 to AA 177 (CS1; ELQKMLLAKLYLLK) was used, identities of 43 % (MMOB_900) to 57% (MYPU_0070) were detected and scrutiny of the sequence from AA 226 to AA 234 (CS2; LYLMKYLVE) revealed 60 % (MYPU_0070) to 70 % identity (MMOB_900). The corresponding proteins of Mesoplasma florum and M. mycoides subsp. mycoides SC did not contain these conserved sequences. The different HinT homologues had the highest identity with the HinT protein of M. hominis, ranging from 44.3 % (MF_233) to 55.7 % (MMOB_890).

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