Nuclease activity of GST-Mm19 fusion protein.

<p><b>(A)</b> Effect of divalent cations on nuclease activity of purified GST-Mm19 against pasmid pCR2.1 DNA. Nuclease activity was analyzed in the absence or presence of 2 mM of different divalent cations (indicated under each panel). Molecular weight markers, 1 kb DNA Plus Ladder (Thermoscientific), are shown on the left margin of each panel. Lanes C1 and C2 in each panel are plasmid DNA untreated or treated with GST protein, respectively. Purified GST-Mm19 fusion protein was incubated with pasmid pCR2.1 DNA. Incubation times in minutes are indicated above each lane. All reactions were performed at 37°C. <b>(B)</b> Substrate specificity of nuclease activity of purified GST-Mm19. Nuclease activity of GST-Mm19 was assessed against Vero cell double-stranded DNA (left panel), M13 phage single-stranded DNA (middle panel), and <i>E</i>. <i>coli</i> strain BL21 total RNA (right panel). The positions of 1 kb DNA ladder markers are indicated as MW on the left of each panel. Lanes C1 and C2 in each panel indicate end point reactions of the undigested nucleic acid controls after incubation in the nuclease buffer alone or supplemented with GST protein, respectively. Reactions were stopped at the times indicated above each lane.</p

<i>In silico</i> characterization of Mm19 nuclease.

<p><b>(A)</b> Schematic illustration of RE_<i>Alw</i>I conserved domain as determined by BLASTP analysis of Mm19 amino acid sequence. The deduced 300-residue sequence of the RE_<i>Alw</i>I superfamily domain mapped to the C-terminal region of Mm19, from residues 307 to 607. <b>(B)</b> Genomic location of <i>M</i>. <i>meleagridis Mm19</i> nuclease ORF and <i>methyltransferase</i> sequences. The two genes encoding these enzymes are located next to each other with an intergenic region of 130 bp and opposing orientations; <i>Mm19</i> is on the positive strand, while the <i>methyltransferase</i> gene is encoded on the negative strand. A “TATAAT box” (in red) was detected upstream of Mm19 ORF, at position -77. In the same orientation and approximately 10 kb upstream of the <i>Mm19</i> gene sequence, genes coding for an ABC transporter ATP-binding protein and a transport system permease protein were detected. Genomic coordinates of coding sequences are indicated. All genomic data were extracted from the MolliGen database.</p

Mm19, a <i>Mycoplasma meleagridis</i> Major Surface Nuclease that Is Related to the RE_<i>Alw</i>I Superfamily of Endonucleases

<div><p><i>Mycoplasma meleagridis</i> infection is widespread in turkeys, causing poor growth and feathering, airsacculitis, osteodystrophy, and reduction in hatchability. Like most mycoplasma species, <i>M</i>. <i>meleagridis</i> is characterized by its inability to synthesize purine and pyrimidine nucleotides <i>de novo</i>. Consistent with this intrinsic deficiency, we here report the cloning, expression, and characterization of a <i>M</i>. <i>meleagridis</i> gene sequence encoding a major surface nuclease, referred to as Mm19. Mm19 consists of a 1941- bp ORF encoding a 646-amino-acid polypeptide with a predicted molecular mass of 74,825 kDa. BLASTP analysis revealed a significant match with the catalytic/dimerization domain of type II restriction enzymes of the RE_<i>Alw</i>I superfamily. This finding is consistent with the genomic location of Mm19 sequence, which dispalys characteristics of a typical type II restriction-modification locus. Like intact <i>M</i>. <i>meleagridis</i> cells, the <i>E</i>. <i>coli-</i>expressed Mm19 fusion product was found to exhibit a nuclease activity against plasmid DNA, double-stranded DNA, single-stranded DNA, and RNA. The Mm19-associated nuclease activity was consistently enhanced with Mg²⁺ divalent cations, a hallmark of type II restriction enzymes. A rabbit hyperimmune antiserum raised against the bacterially expressed Mm19 strongly reacted with <i>M</i>. <i>meleagridis</i> intact cells and fully neutralized the surface-bound nuclease activity. Collectively, the results show that <i>M</i>. <i>meleagridis</i> expresses a strong surface-bound nuclease activity, which is the product of a single gene sequence that is related to the RE_<i>Alw</i>I superfamily of endonucleases.</p></div

Hypothetical and experimental evidence of Mm19 surface exposure.

<p><b>(A)</b> Schematic representation of the secondary structure of Mm19 protein as predicted by PSIPRED based on PSI-BLAST. Using MEMSAT-SVM, a segment of 464 amino acids, including the N-terminal region of the Mm19 nuclease protein, was predicted to be located in the cytoplasm. The C-terminal 167 residues were predicted to be exposed to the extracellular milieu, while 15 amino acid residues were embedded within the membrane. A similar topology was predicted by MEMSAT3 but with a few differences in the segments lengths (cytoplasmic segment, 581 aa; transmembrane segment, 18 aa; extracellular segment, 47 aa). <b>(B)</b> Surface expression of <i>M</i>. <i>meleagridis</i> Mm19 as revealed by colony blotting. <i>M</i>. <i>meleagridis</i> colonies growing on agar plates were left untreated or subjected to trypsin digestion then imprinted onto nitrocellulose discs. The anti-GST (panel 1) and the specific polyclonal anti-<i>M</i>. <i>meleagridis</i> (panel 2) sera were used as negative and positive controls, respectively. The reactivity of anti-GST-Mm19 serum against non- and trypsin-treated <i>M</i>. <i>meleagridis</i> colonies is shown in panels 3 and 4, respectively. Colored imprints of colonies were observed under a microscope at 100 x magnification.</p

Neutralization of <i>M</i>. <i>meleagridis</i> membrane-associated endonuclease activity.

<p>Nuclease activity neutralization was tested with three controls (lanes C1, C2, and C3). Lane C1, plasmid pCR2.1 DNA incubated in reaction buffer only; Lane C2, plasmid pCR2.1 DNA incubated with untreated <i>M</i>. <i>meleagridis</i> cells; Lane C3, plasmid pCR2.1 DNA incubated with intact <i>M</i>. <i>meleagridis</i> cells that had been treated with rabbit antiserum against GST. Lanes 1–5, plasmid pCR2.1 DNA incubated with <i>M</i>. <i>meleagridis</i> cells treated with rabbit anti-GST-Mm19 antiserum diluted 1/10, 1/100, 1/1000, 1/2000, or 1/4000, respectively. Lane MW, 1 kb DNA ladder (Amersham).</p

Representative illustration of PCR amplification of the 510-bp fragment of

<p><b>Copyright information:</b></p><p>Taken from "Genetic variability of the P120' surface protein gene of isolates recovered from Tunisian patients with uro-genital and infertility disorders"</p><p>http://www.biomedcentral.com/1471-2334/7/142</p><p>BMC Infectious Diseases 2007;7():142-142.</p><p>Published online 5 Dec 2007</p><p>PMCID:PMC2225410.</p><p></p> M: 100 bp molecular size ladder, lanes 1 and 7: PG21 strain (positive control), lane 2: No DNA with Mhp120' primers (negative control), lanes 3–6: Mhp120' primers with DNA from ATCC 33530 (lane 3), ATCC 19989 (lane 4), ATCC 15531 (lane 5), and UuipT3 (lane 6), lanes 8–19: isolates

Multiple alignment of the deduced amino acid sequences of the P120'surface exposed region of isolates (Mh1 to Mh27) relative to the reference strain PG21

<p><b>Copyright information:</b></p><p>Taken from "Genetic variability of the P120' surface protein gene of isolates recovered from Tunisian patients with uro-genital and infertility disorders"</p><p>http://www.biomedcentral.com/1471-2334/7/142</p><p>BMC Infectious Diseases 2007;7():142-142.</p><p>Published online 5 Dec 2007</p><p>PMCID:PMC2225410.</p><p></p> Dots indicate identical residues. The 3 regions where amino acid changes which tend to occur are underlined