HinT proteins and their putative interaction partners in Mollicutes and Chlamydiaceae

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.

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.

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).

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).

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)

Figure 5

The yeast-two hybrid assay. AH109 were co-transformed with the pGADT7 (AD) and pGBKT7 (BD) constructs containing hitL of M. hominis, Cp265, Cp266 or Cp267 from C. pneumoniae, pGADT7-T, expressing the SV40 large T-antigen and pGBKT7-53, expressing the murine p53 protein (K+), or pGADT7 and pGBKT7 without fusion partners (K-). Yeast cultures were incubated at 30°C on histidine-deficient agar plates for 4 days.

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.

Figure 7

Immune co-precipitation. A. Affinity purified proteins, Cp265 (265^C), Cp266 (266^C), Cp267 (267^C) and OppA^C, were analyzed on 15 % SDS-polyacrylamide gels by Coomassie staining (C) or on Western blots by immunostaining with an anti-Protein C antibody (W). [³⁵S]-labeled Cp265, Cp266 and Cp267 (Captives) were incubated at room temperature for 1 h with Cp265^C, Cp266^C, Cp267^C and OppA^C (Catcher). Immune complexes were precipitated with anti-Protein C-matrix, and the supernatants (B.) and precipitates (C.) analyzed on 15 % SDS-polyacrylamide gels by autoradiography using a phosphorimager. As a negative control, the [³⁵S]-labeled proteins were subjected to the Protein C-matrix without a catcher (Sepha.^αC). M, prestained low-molecular weight marker (Biorad)