Cattle immunized against the pathogenic L-α-glycerol-3-phosphate oxidase of Mycoplasma mycoides subs. mycoides fail to generate neutralizing antibodies and succumb to disease on challenge.

The membrane-associated enzyme L-α-glycerol-3-phosphate oxidase (GlpO) of Mycoplasma mycoides subs. mycoides (Mmm), the causal agent of contagious bovine pleuropneumonia (CBPP) has been identified as a virulence factor responsible for the release of toxic by-products such as H2O2 that mediate host cell injury. Since CBPP pathogenesis is based on host inflammatory reactions, we have determined the capacity of recombinant GlpO to generate in vivo protective responses against challenge in immunized cattle. We also investigated whether sera raised against recombinant GlpO in cattle and mice inhibit production of H2O2 by Mmm. Immunization of cattle with recombinant GlpO did not protect against challenge with a virulent strain of Mmm. Further, although both murine and bovine antisera raised against recombinant GlpO detected recombinant and native forms of GlpO in immunoblot assays with similar titres, only murine antibodies could neutralize GlpO enzymatic function. The data raise the possibility that Mmm has adapted to evade potential detrimental antibody responses in its definitive host.

A Type III restriction-modification system in Mycoplasma mycoides subsp. capri.

The sequenced genome of Mycoplasma mycoides subsp. capri revealed the presence of a Type III restriction-modification system (MmyCI). The methyltransferase (modification) subunit of MmyCI (M.MmyCI) was shown to recognize the sequence 5'-TGAG-3' and methylate the adenine. The coding region of the methyltransferase gene contains 12 consecutive AG dinucleotide repeats that result in a translational termination at a TAA codon immediately beyond the repeat region. This strain does not have MmyCI activity. A clone was found with 10 AG repeats such that the gene is in frame, and this strain has MmyCI activity, suggesting that the expression of the MmyCI methyltransferase may be phase variable.

Specific evolution of F1-like ATPases in mycoplasmas.

F(1)F(0) ATPases have been identified in most bacteria, including mycoplasmas which have very small genomes associated with a host-dependent lifestyle. In addition to the typical operon of eight genes encoding genuine F(1)F(0) ATPase (Type 1), we identified related clusters of seven genes in many mycoplasma species. Four of the encoded proteins have predicted structures similar to the α, ß, γ and ε subunits of F(1) ATPases and could form an F(1)-like ATPase. The other three proteins display no similarity to any other known proteins. Two of these proteins are probably located in the membrane, as they have three and twelve predicted transmembrane helices. Phylogenomic studies identified two types of F(1)-like ATPase clusters, Type 2 and Type 3, characterized by a rapid evolution of sequences with the conservation of structural features. Clusters encoding Type 2 and Type 3 ATPases were assumed to originate from the Hominis group of mycoplasmas. We suggest that Type 3 ATPase clusters may spread to other phylogenetic groups by horizontal gene transfer between mycoplasmas in the same host, based on phylogeny and genomic context. Functional analyses in the ruminant pathogen Mycoplasma mycoides subsp. mycoides showed that the Type 3 cluster genes were organized into an operon. Proteomic analyses demonstrated that the seven encoded proteins were produced during growth in axenic media. Mutagenesis and complementation studies demonstrated an association of the Type 3 cluster with a major ATPase activity of membrane fractions. Thus, despite their tendency toward genome reduction, mycoplasmas have evolved and exchanged specific F(1)-like ATPases with no known equivalent in other bacteria. We propose a model, in which the F(1)-like structure is associated with a hypothetical X(0) sector located in the membrane of mycoplasma cells.

Thymidylate synthases of Mycoplasma mycoides subsp. mycoides SC and Ureaplasma parvum are flavin-dependent.

Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides subsp. mycoides SC) is the causative agent of contagious bovine pleuropneumonia (CBPP), one of the most serious bacterial diseases in cattle and buffalo. Ureaplasma parvum (U. parvum) colonizes the human urogenital tract, and has been associated with urethritis and premature birth. The de novo synthesis of thymidylate (dTMP) is essential and catalyzed by thymidylate synthase (TS), encoded by either the thyA or the thyX genes. No homologs to either thyA or thyX have been identified in the U. parvum and M. mycoides subsp. mycoides SC genomes. Here we report the identification, partial purification and characterization of M. mycoides subsp. mycoides and U. parvum TS. Our results showed that the M. mycoides subsp. mycoides SC and U. parvum TS apparently are flavin-dependent, having similar enzymatic activities but no sequence homology to other known ThyX proteins. Up to date there are 11 Mollicutes species lacking both thyA and thyX gene. Therefore, the finding described here most likely constitutes a new enzyme family specific for Mollicutes. These M. mycoides subsp. mycoides SC and U. parvum TS enzymes could be ideal targets for future development of agents against Myoplasma infections.

A PCR for the detection of mycoplasmas belonging to the Mycoplasma mycoides cluster: application to the diagnosis of contagious agalactia.

Contagious agalactia is a mycoplasmal infection caused by Mycoplasma agalactiae, Mycoplasma mycoides subsp. mycoides LC, M. mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens. Identification of the causative organisms is usually performed by isolation and classical biochemical and serological tests, though this is a lengthy and cumbersome process for mycoplasmas. Specific PCR assays have been developed for the identification of Mycoplasma agalactiae and M. putrefaciens. For members of the M. mycoides cluster existing PCR tests are based on the amplification of highly conserved genes coding for ribosomal proteins, hence a possibility of cross-reactions. The gene glk, coding for a glucokinase, that is found in this cluster is very distantly related to any other bacterial glucokinase described so far. It was therefore chosen as target to design a new PCR test. The validation was performed independently in three laboratories in France and India using over 100 mycoplasma strains of various geographical origins. All strains belonging to the M. mycoides cluster were detected by amplification of the expected PCR product (428 bp) while no amplification was obtained from M. agalactiae strains. Our results demonstrate the universality of this PCR in spite of the great heterogeneity found within this cluster. This new tool may be of great help for the implementation of control measures directed towards contagious agalactia.

Structure-function analysis of a bacterial deoxyadenosine kinase reveals the basis for substrate specificity.

Deoxyribonucleoside kinases (dNKs) catalyze the transfer of a phosphoryl group from ATP to a deoxyribonucleoside (dN), a key step in DNA precursor synthesis. Recently structural information concerning dNKs has been obtained, but no structure of a bacterial dCK/dGK enzyme is known. Here we report the structure of such an enzyme, represented by deoxyadenosine kinase from Mycoplasma mycoides subsp. mycoides small colony type (Mm-dAK). Superposition of Mm-dAK with its human counterpart's deoxyguanosine kinase (dGK) and deoxycytidine kinase (dCK) reveals that the overall structures are very similar with a few amino acid alterations in the proximity of the active site. To investigate the substrate specificity, Mm-dAK has been crystallized in complex with dATP and dCTP, as well as the products dCMP and dCDP. Both dATP and dCTP bind to the enzyme in a feedback-inhibitory manner with the dN part in the deoxyribonucleoside binding site and the triphosphates in the P-loop. Substrate specificity studies with clinically important nucleoside analogs as well as several phosphate donors were performed. Thus, in this study we combine structural and kinetic data to gain a better understanding of the substrate specificity of the dCK/dGK family of enzymes. The structure of Mm-dAK provides a starting point for making new anti bacterial agents against pathogenic bacteria.

Kinetic mechanism of deoxyadenosine kinase from Mycoplasma determined by surface plasmon resonance technology.

Surface plasmon resonance (SPR) detection technology was employed to investigate the kinetic mechanism of deoxyadenosine kinase from Mycoplasma mycoides ssp. mycoides SC. In our experimental approach, the enzyme was attached to the sensor surface, the reactants were injected in the mobile phase, and the product-enzyme complex formation was measured using the fact that the rate of product formation exceeds that of its dissociation. The pre-steady-state analysis of deoxyguanosine phosphorylation showed the presence of a burst phase, which is consistent with product dissociation being a rate-limiting step. High activity of the immobilized enzyme was demonstrated by analyzing the reaction mixture eluted from the chip and by determining the Michaelis-Menten constants for several phosphate acceptors (e.g., deoxyadenosine) and phosphate donors (e.g., ATP) using SPR detection. These values were in good agreement with those reported previously [Wang, L. et al. (2001) Mol. Microbiol. 42, 1065-1073]. The bisubstrate initial rate pattern obtained was characteristic of a sequential kinetic mechanism. Because in the method applied here it is the mass change on the surface that is monitored, a new mathematical approach to interpreting product inhibition experiments was proposed. According to that approach, product inhibition studies, supported by product binding experiments, indicated that the reaction mechanism was of Bi Bi sequential ordered type, involving the formation of a ternary complex, in which ATP and deoxyadenosine bound sequentially, followed by a transfer of the phosphate group, and an ordered release of products with ADP dissociating before dAMP.

Conformational variability of the GTPase domain of the signal recognition particle receptor FtsY.

The prokaryotic signal recognition particle Ffh and its receptor FtsY allow targeting of proteins into or across the plasma membrane. The targeting process is GTP dependent and the two proteins constitute a distinct GTPase family. The receptor FtsY is composed of A and NG domains where the NG's GTPase domain plays a critical role in the targeting process. In this study, we describe two X-ray structures determined independently of each other of the NG domain of FtsY from Mycoplasma mycoides (MmFtsY). The two structures are markedly different in three of the nucleotide-binding segments, GI (P-loop), GII, and GIII, making only one of the structures compatible with nucleotide binding. Interestingly, the two distinct conformations of the nucleotide-binding segments of MmFtsY are similar to the apo- and ADP-loaded forms of certain ATPases. The structure of the extended interface between the A and NG domains of MmFtsY provides new insights into the role of the A domain for phospholipid interaction.

A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony.

During evolution, pathogenic bacteria have developed complex interactions with their hosts. This has frequently involved the acquisition of virulence factors on pathogenicity islands, plasmids, transposons, or prophages, allowing them to colonize, survive, and replicate within the host. In contrast, Mycoplasma species, the smallest self-replicating organisms, have regressively evolved from gram-positive bacteria by reduction of the genome to a minimal size, with the consequence that they have economized their genetic resources. Hence, pathogenic Mycoplasma species lack typical primary virulence factors such as toxins, cytolysins, and invasins. Consequently, little is known how pathogenic Mycoplasma species cause host cell damage, inflammation, and disease. Here we identify a novel primary virulence determinant in Mycoplasma mycoides subsp. mycoides Small Colony (SC), which causes host cell injury. This virulence factor, released in significant amounts in the presence of glycerol in the growth medium, consists of toxic by-products such as H2O2 formed by l-alpha-glycerophosphate oxidase (GlpO), a membrane-located enzyme that is involved in the metabolism of glycerol. When embryonic calf nasal epithelial cells are infected with M. mycoides subsp. mycoides SC in the presence of physiological amounts of glycerol, H2O2 is released inside the cells prior to cell death. This process can be inhibited with monospecific anti-GlpO antibodies.

Variability of a glucose phosphotransferase system permease in Mycoplasma mycoides subsp. mycoides Small Colony.

Intraclonal antigenic variation in pathogenic mycoplasma species is considered an important feature of host-pathogen interaction. Such intraclonal protein variation was observed for the interaction of Mycoplasma mycoides subsp. mycoides Small Colony, the agent of contagious bovine pleuropneumonia, with mAb 3F3. Colony immunostaining allows the definition of 3F3 ON- and 3F3 OFF-type variants, which revert at low frequency. Targets of mAb 3F3 were shown to be surface located, and resided on multiple polypeptides in the 58-68 kDa size range. Phage display and a genomic database were combined to determine the gene encoding the proteins recognized by mAb 3F3. A gene encoding the putative permease of the glucose phosphotransferase system was identified. Genome sequence analysis of strain PG1 revealed two highly similar copies of this gene, resulting from duplication of the chromosomal region carrying the gene. Southern blot analysis demonstrated the presence of this duplication in almost every African strain tested, but not in European strains. DNA analysis revealed that ON/OFF switching is governed by a base substitution occurring upstream of the coding region for the 3F3 epitope. This event generates a stop codon that results in the premature termination of the PtsG protein.

Retrieving sequences of enzymes experimentally characterized but erroneously annotated : the case of the putrescine carbamoyltransferase.

BACKGROUND: Annotating genomes remains an hazardous task. Mistakes or gaps in such a complex process may occur when relevant knowledge is ignored, whether lost, forgotten or overlooked. This paper exemplifies an approach which could help to resuscitate such meaningful data. RESULTS: We show that a set of closely related sequences which have been annotated as ornithine carbamoyltransferases are actually putrescine carbamoyltransferases. This demonstration is based on the following points : (i) use of enzymatic data which had been overlooked, (ii) rediscovery of a short NH2-terminal sequence allowing to reannotate a wrongly annotated ornithine carbamoyltransferase as a putrescine carbamoyltransferase, (iii) identification of conserved motifs allowing to distinguish unambiguously between the two kinds of carbamoyltransferases, and (iv) comparative study of the gene context of these different sequences. CONCLUSIONS: We explain why this specific case of misannotation had not yet been described and draw attention to the fact that analogous instances must be rather frequent. We urge to be especially cautious when high sequence similarity is coupled with an apparent lack of biochemical information. Moreover, from the point of view of genome annotation, proteins which have been studied experimentally but are not correlated with sequence data in current databases qualify as "orphans", just as unassigned genomic open reading frames do. The strategy we used in this paper to bridge such gaps in knowledge could work whenever it is possible to collect a body of facts about experimental data, homology, unnoticed sequence data, and accurate informations about gene context.

Binding of GTP and GDP induces a significant conformational change in the GTPase domain of Ffh, a bacterial homologue of the SRP 54 kDa subunit.

The bacterial Ffh protein is homologous to the SRP54 subunit of the signal recognition particle. Ffh plays a key role in the targeting of proteins to the membrane and it is composed of a N-terminal domain (N), a middle GTPase (G) domain and a C-terminal M domain which has binding sites for SRP RNA and signal peptide. The GTP binding and hydrolysis of Ffh is critical to its function. We have used protease digestion to probe the conformation of the Mycoplasma mycoides Ffh N+G domain. In the absence of nucleotide the protein was comparatively sensitive to protease cleavage and we identified sites particularly prone to cleavage in a region near the C-terminus of the GTPase domain. However, in the presence of GTPgammaS or GDP this region is stabilized and the protein adopts a more ordered structure. The pattern of cleavage with GTPgammaS was indistinguishable from that when GDP was bound, indicating that the conformation of the nucleotide-free form is distinct from that when either GTPgammaS or GDP is bound to the protein. The possible functional role of this significant conformational change is discussed.

Lesiones anatomo e histopatologicas compatibles con neumoni enzootica porcina a nivel de matadero de Santa Cruz / Anatomic and histologic lesions consistent with endozootic pig neumonia in Santa Cruz

Se determino lesiones anatomo e histopatologicas compactibles con neumonia enzootica porcina (NEP) en cerdos faenados en los mataderos de Santa Cruz de la Sierra, en los meses de agosto y Septiembre de 1995. El muestreo se realizo dos veces por semana; de todos los animales faenados en el dia, se procedio al examen macroscopico de los pulmones en el momento de la evisceracion y se tomaron muestras a los que presentaban lesiones macroscopicas compatibles con NEP; estas fueron procesadas y se utilizo la tecnica de histopatologia de rutina. Los resultados obtenidos se sometieron a un analisis estadistico de comparacion de proporciones mediante la prueba de Chi Cuadrado. De 1000 pulmones inspeccionados 237 (23,7 por ciento) resultaron con lesiones macroscopicas

Cloning and characterization of the lipase operon from Mycoplasma mycoides subspecies mycoides LC.

Lipases, serine esterase enzymes, play an essential role in the mycoplasmal nutritional requirement for long-chain fatty acids. Although the lipase(s) activity in different mycoplasma species has been investigated, the molecular biology of the corresponding genes has not been studied. Using a single-primer PCR technique combined to more classical cloning systems, an operon containing three open reading frames (ORF), each of which could encode a lipase protein of 264, 264 or 269 amino acids (aa), was identified from Mycoplasma mycoides subsp. mycoides LC. Analysis of aa sequences of the encoded polypeptides showed that they display high aa similarity between each other (65-79%) and 28-31% identity to other prokaryotic lipases. Moreover, a lipase-esterase activity could be detected when the mycoplasmal lipase-encoding genes were expressed in a strong opal-suppressor-bearing Escherichia coli strain.

GTPase activity of a bacterial SRP-like complex.

We have recently identified a protein (SRPM54) in Mycoplasma mycoides homologous to SRP54, a subunit of the mammalian signal recognition particle (SRP). This protein forms a complex with a mycoplasma RNA related to the RNA component of SRP. We have now demonstrated that the protein has an intrinsic GTPase activity in vitro and kinetic parameters for the enzymatic reaction have been determined. The GTPase activity was not significantly affected by the presence of the mycoplasma SRP RNA. Different regions of the SRPM54 protein were expressed as recombinant proteins in E. coli and were purified to near homogeneity. On the basis of the properties of these SRPM54 fragments two different functional domains of the protein could be distinguished. An N-terminal part was found to contain the GTPase activity and this domain had approximately the same kinetic properties as the full-length protein. Another domain corresponding to a C-terminal fragment contained the RNA binding activity as shown using an assay based on the retention of RNA-protein complexes to nitrocellulose filters.

A mutant of Mycoplasma mycoides subsp. mycoides lacking the H2O2- producing enzyme L-alpha-glycerophosphate oxidase.

Cells of Mycoplasma mycoides subsp. mycoides grown without stirring or aeration in batch culture, and resuspended in a salts solution, oxidised a range of carbohydrates including glycerol. The rate of glycerol oxidation was not reduced when cells were passaged more than 20 times in batch culture. However, in cells grown in stirred and aerated chemostat culture for 100 generations the ability to oxidise glycerol, but not other carbohydrates, was lost or greatly reduced. A mutant strain isolated from chemostat even after several passages in batch culture. The growth rate and growth-yield of the mutant strain in batch culture were similar to those of the parent strain. The mutant possessed activity for glycerol kinase but had lost that for the hydrogen peroxide-producing enzyme, L-alpha-glycerophosphate oxidase. The selection pressure in favour of the mutant strain in chemostat culture may be a decreased production of hydrogen peroxide.

[Optimization of physico-chemical parameters of reaction media for determining the activity of RNA-polymerases of mollicutes]. / Optimizatsiia fiziko-khimicheskikh parametrov reaktsionnoi sredy dlia opredeleniia aktivnosti RNK-polimeraz mollikutov.

RNA-polymerases of mollicutes differ considerably from certain species of bacteria in the temperature optimum of the activity manifestation. The activity of mollicute enzymes is considerably higher in the presence of manganese ions than in the presence of magnesium ions. They differ from Escherichia coli transcriptase in this character but are similar to the RNA-polymerase of lactic bacteria, hypothetic ancestors of acholeplasm. Sensitivity of RNA-polymerases to metals may be one of arguments explaining phylogeny of mycoplasms. It is established that for studying mollicute transcriptase the reacting mixture for examining the enzymic activity besides the major components should have the following parameters: pH 8.0; the MnCl concentration--8-10 mM; for form II of the A. laidlawii subsp. granulum 118 enzyme--4.5 mM; ammonium sulphate concentration--40 mM; for form II of st. 118-20 mM; the reaction should be conducted at the temperature of 27 degrees C for 30 min.

Mollicutes DNA polymerases: characterization of a single enzyme from Mycoplasma mycoides and Ureaplasma urealyticum and of three enzymes from Acholeplasma laidlawii.

The DNA polymerase activity of different members of Mollicutes was studied. A single DNA polymerase was found in Mycoplasma mycoides and Ureaplasma urealyticum, type species of the genera Mycoplasma and Ureaplasma, and was compared with the previously described Mycoplasma orale enzyme. Most of their properties were comparable; an immunological relationship was demonstrated between M. orale and M. mycoides enzymes by immunoblotting. In contrast to these results, three different DNA polymerases were purified in Acholeplasma laidlawii, type species of the genus Acholeplasma which, in this aspect, resembles the genus Spiroplasma. A 3'-5' exonuclease activity was found in the different purified preparations. In M. mycoides, M. orale and one of the three A. laidlawii preparations, the 3'-5' exonuclease could be separated from the DNA polymerase by non-denaturing PAGE. The presence of a single DNA polymerase seems to be a typical feature of the Mycoplasmataceae, which include the genera Mycoplasma and Ureaplasma, in contrast to the occurrence of three enzymes within the Acholeplasmataceae and Spiroplasmataceae. These results are in agreement with the phylogenetic tree of Mollicutes proposed from their 5 S and 16 S rRNA sequence comparisons, in which the evolution of Acholeplasma and Spiroplasma branches led, by genome reductions, to Mycoplasma and Ureaplasma species.

Characterization of phospholipase A2 of mycoplasma species.

As phospholipases of mycoplasma species may play a role in the pathogenesis of respiratory tract and urogenital tract diseases Mycoplasma mycoides and Acholeplasma laidlawii were examined as to the production of phospholipase A2 (PLA) and attempts were made to purify and characterize it. Both species produced PLA. The purified enzyme was found to be heat-labile, active at alkaline pH, revealing a single band in polyacrylamide gel electrophoresis. Metal ions such as calcium and barium, increased its activity whereas solvents at high concentrations decreased it. It was resistant to surfactants.