14 research outputs found
Mm19, a Mycoplasma meleagridis Major Surface Nuclease that Is Related to the RE_AlwI Superfamily of Endonucleases.
No full textMycoplasma meleagridis infection is widespread in turkeys, causing poor growth and feathering, airsacculitis, osteodystrophy, and reduction in hatchability. Like most mycoplasma species, M. meleagridis is characterized by its inability to synthesize purine and pyrimidine nucleotides de novo. Consistent with this intrinsic deficiency, we here report the cloning, expression, and characterization of a M. meleagridis 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_AlwI 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 M. meleagridis cells, the E. coli-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 M. meleagridis intact cells and fully neutralized the surface-bound nuclease activity. Collectively, the results show that M. meleagridis expresses a strong surface-bound nuclease activity, which is the product of a single gene sequence that is related to the RE_AlwI superfamily of endonucleases
Virulence Is More than Adhesion and Invasion Ability, an In Vitro Cell Infection Assay of Bovine Mycoplasma spp.
No full textMycoplasma bovis is the most common mycoplasma associated with cattle diseases worldwide. However, other seemingly less virulent Mycoplasma spp. such as M. bovigenitalium and M. bovirhinis have also been associated with mycoplasmosis. The study objective was to compare the adhesion and cellular invasion characteristics of these bovine Mycoplasma spp. using Madin–Darby Bovine Kidney (MDBK) epithelial cells. MDBK cells were separately infected with 12 M. bovis strains and one strain each of M. bovigenitalium and M. bovirhinis. Following infection, a gentamicin protection assay was performed and the cells lysed at 6 and 54 h post-infection. The MDBK cell lysates were cultured for Mycoplasma spp. and qPCR was used to estimate the average number of Mycoplasma bacterial cells that infected each MDBK cell (Myc/Cell ratio). Confocal and electron microscopy studies using M. bovis mNeonGreen strain were also performed. All 14 Mycoplasma strains multiplied within the MDBK cells, a finding confirmed by microscopy studies of the M. bovis mNeonGreen strain. Unexpectedly, the M. bovis strains, obtained from diseased and asymptomatic cattle and bison, had lower Myc/Cell ratios than M. bovirhinis and M. bovigenitalium strains. These findings suggest that the ability for mycoplasmas to invade and replicate within host cells does not account for the differences in virulence between species
Nuclease activity of <i>Mycoplasma meleagridis</i>.
No full text(A) Nuclease activity associated with intact M. meleagridis cell suspensions against plasmid pCR2.1 DNA. Lane MW, 1 kb DNA ladder (Amersham); lane C, untreated plasmid DNA control; lanes 1–13, plasmid pCR2.1 DNA incubated with two-fold serial dilutions of M. meleagridis cells, containing 1.25 μg to 0.0003 μg of total cell protein. (B) Nuclease activity of M. meleagridis against various nucleic acid substrates. The three panels from the left to the right show the effect of M. meleagridis cells on Vero cell double-stranded DNA, M13 phage single-stranded DNA, and E. coli strain BL21 total RNA. Molecular weight markers (1 kb DNA ladder, Amersham) are shown on the left of each panel. Lane C was loaded with undigested nucleic acid (incubated with buffer alone). M. meleagridis cell suspensions (containing 1.25 μg total cell protein) were incubated with nucleic acid substrates at 37°C for different period times (indicated in minutes above each lane).</p
<i>In silico</i> characterization of Mm19 nuclease.
No full text(A) Schematic illustration of RE_AlwI conserved domain as determined by BLASTP analysis of Mm19 amino acid sequence. The deduced 300-residue sequence of the RE_AlwI superfamily domain mapped to the C-terminal region of Mm19, from residues 307 to 607. (B) Genomic location of M. meleagridis Mm19 nuclease ORF and methyltransferase sequences. The two genes encoding these enzymes are located next to each other with an intergenic region of 130 bp and opposing orientations; Mm19 is on the positive strand, while the methyltransferase 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 Mm19 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
Neutralization of <i>M</i>. <i>meleagridis</i> membrane-associated endonuclease activity.
No full textNuclease 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 M. meleagridis cells; Lane C3, plasmid pCR2.1 DNA incubated with intact M. meleagridis cells that had been treated with rabbit antiserum against GST. Lanes 1–5, plasmid pCR2.1 DNA incubated with M. meleagridis 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
Hypothetical and experimental evidence of Mm19 surface exposure.
No full text(A) 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) Surface expression of M. meleagridis Mm19 as revealed by colony blotting. M. meleagridis 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-M. meleagridis (panel 2) sera were used as negative and positive controls, respectively. The reactivity of anti-GST-Mm19 serum against non- and trypsin-treated M. meleagridis colonies is shown in panels 3 and 4, respectively. Colored imprints of colonies were observed under a microscope at 100 x magnification.</p
Nuclease activity of GST-Mm19 fusion protein.
No full text(A) 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) 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 E. coli 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
Genome-Wide Association Study of Nucleotide Variants Associated with Resistance to Nine Antimicrobials in Mycoplasma bovis
No full textAntimicrobial resistance (AMR) studies of Mycoplasma bovis have generally focused on specific loci versus using a genome-wide association study (GWAS) approach. A GWAS approach, using two different models, was applied to 194 Mycoplasma bovis genomes. Both a fixed effects linear model (FEM) and a linear mixed model (LMM) identified associations between nucleotide variants (NVs) and antimicrobial susceptibility testing (AST) phenotypes. The AMR phenotypes represented fluoroquinolones, tetracyclines, phenicols, and macrolides. Both models identified known and novel NVs associated (Bonferroni adjusted p < 0.05) with AMR. Fluoroquinolone resistance was associated with multiple NVs, including previously identified mutations in gyrA and parC. NVs in the 30S ribosomal protein 16S were associated with tetracycline resistance, whereas NVs in 5S rRNA, 23S rRNA, and 50S ribosomal proteins were associated with phenicol and macrolide resistance. For all antimicrobial classes, resistance was associated with NVs in genes coding for ABC transporters and other membrane proteins, tRNA-ligases, peptidases, and transposases, suggesting a NV-based multifactorial model of AMR in M. bovis. This study was the largest collection of North American M. bovis isolates used with a GWAS for the sole purpose of identifying novel and non-antimicrobial-target NVs associated with AMR
Genome Sequence of Mycoplasma meleagridis Type Strain 17529
No full textInternational audienceMycoplasma meleagridis is a prominent turkey bacterial pathogen associated with airsacculitis and reproductive disorders. Notwithstanding the economic losses caused by M. meleagridis, its genome has still not been sequenced. For a better understanding of its genetic background and pathogenicity mechanisms, we sequenced the genome of M. meleagridis type strain ATCC 25294
