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Author(s): Vivek Srinivas [sup.1] , Hugo Lebrette [sup.1] , Daniel Lundin [sup.1] , Yuri Kutin [sup.2] , Margareta Sahlin [sup.1] , Michael Lerche [sup.1] , Jürgen Eirich [sup.3] , Rui [...], Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis.sup.1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target.sup.3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity.sup.5-7. Here we describe a group of RNR proteins in Mollicutes--including Mycoplasma pathogens--that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR--some of which are developing resistance to antibiotics--are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie.A subclass of ribonucleotide reductase in Mycoplasma pathogens contains a stable radical formed from a modified tyrosine residue, overturning the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase.

Mycoplasmas are thought to control gene expression through simple mechanisms. The switching mechanisms needed to regulate transcription during significant environmental shifts do not seem to be required for these host-adapted organisms. Mycoplasma hyopneumoniae, a swine respiratory pathogen, undergoes differential gene expression, but as for all mycoplasmas, the mechanisms involved are still unknown. Since mycoplasmas contain only a single sigma factor and few regulator-type proteins, it is likely that other mechanisms control gene regulation, possibly involving intergenic (IG) regions. To study this further, we investigated whether IG regions are transcribed in M. hyopneumoniae, and measured transcription levels across five specific regions. Microarrays were constructed with probes covering 343 IG regions of the M. hyopneumoniae genome, and RNA isolated from laboratory-grown cells was used to interrogate the arrays. Transcriptional signals were identified in 321 (93.6 %) of the IG regions. Five large (>500 bp) IG regions were chosen for further analysis by qRT-PCR by designing primer sets whose products reside in flanking ORFs, bridge flanking ORFs and the IG region, or reside solely within the IG region. The results indicate that no single transcriptional start site can account for transcriptional activity within IG regions. Transcription can end abruptly at the end of an ORF, but this does not seem to occur at high frequency. Rather, transcription continues past the end of the ORF, with RNA polymerase gradually releasing the template. Transcription can also be initiated within IG regions in the absence of accepted promoter-like sequences. DOI 10.1099/mic.0.038760-0

Lipoprotein T (LppT), a membrane-located 105-kDa lipoprotein of Mycoplasma conjunctivae, the etiological agent of infectious keratoconjunctivitis (IKC) of domestic sheep and wild Caprinae, was characterized. LppT was shown to promote cell attachment to LSM 192 primary lamb joint synovial cells. Adhesion of M. conjunctivae to LSM 192 cells is inhibited by antibodies directed against LppT. The RGD (Arg-Gly-Asp) motif of LppT was found to be a specific site for binding of M. conjunctivae to these eukaryotic host cells. Recombinant LppT fixed to polymethylmethacrylate slides binds LSM 192 cells, whereas LppT lacking the RGD site is deprived of binding capacity to LSM 192, and LppT containing RGE rather than RGD shows reduced binding. Synthetic nonapeptides derived from LppT containing RGD competitively inhibit binding of LSM 192 cells to LppT-coated slides, whereas nonapeptides containing RAD rather than RGD do not inhibit. RGD-containing, LppT-derived nonapeptides are able to directly inhibit binding of M. conjunctivae to LSM 192 cells by competitive inhibition, whereas the analogous nonapeptide containing RAD rather than RGD or the fibronectin-derived RGD hexapeptide has no inhibitory elect. These results reveal LppT as the first candidate of a RGD lectin in Mycoplasma species that is assumed to bind to [beta] integrins. doi: 10.1128/JB.00253-10

Mycoplasma gallisepticum (MG) is an economically important pathogen of poultry worldwide, causing chronic respiratory disease in chickens and turkeys. Differentiation of MG strains is critical, especially in countries where poultry flocks are vaccinated with live vaccines, In this study, oligonucleotide primers were designed based on a region preceding the trinucleotide repeat of a member of the vIhA gene family, and amplicons of 145-352 bp were generated from cultures of 10 different MG strains, including the ts-11, F and 6/85 vaccine strains. High-resolution melting (HRM) curve analysis of the resultant amplicons could differentiate all MG drains. Analysis of the nucleotide sequences of the amplicons from each strain revealed that each melting curve profile related to a unique DNA sequence. The HRM curve profiles (for ts-11) remained consistent after at least five passages under laboratory conditions. PCR-HRM curve analysis of 33 DNA extracts derived from respirator), swabs, or mycoplasma cultures grown from respiratory swabs, of ts-11-vaccinated commercial or specific pathogen-free chickens identified all these specimens, according to their sequences, as ts-11. The potential of the PCR-HRM curve analysis was also shown in the genotyping of 30 additional MG isolates from Europe, the USA and Israel. The results presented in this study indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of MG isolates/strains using both MG cultures and clinical swabs. DOI 10.1099/mic.0.031351-0

Mycoplasma mobile binds to solid surfaces and glides smoothly and continuously by a unique mechanism. A huge protein, Gli521 (521 kDa), is involved in the gliding machinery, and it is localized in the cell neck, the base of the membrane protrusion. This protein is thought to have the role of force transmission. In this study, the Gli521 protein was purified from M. mobile cells, and its molecular shape was studied. Gel filtration analysis showed that the isolated Gli521 protein forms mainly a monomer in Tween 80-containing buffer and oligomers in Triton X-100-containing buffer. Rotary shadowing electron microscopy showed that the Gli521 monomer consisted of three parts: an oval, a rod, and a hook. The oval was 15 nm long by 11 nm wide, and the filamentous part composed of the rod and the hook was 106 nm long and 3 nm in diameter. The Gli521 molecules form a trimer, producing a 'triskelion' reminiscent of eukaryotic clathrin, through association at the hook end. Image averaging of the central part of the triskelion suggested that there are stable and rigid structures. The binding site of a previously isolated monoclonal antibody on Gli521 images showed that the hook end and oval correspond to the C- and N-terminal regions, respectively. Partial digestion of Gli521 showed that the molecule could be divided into three domains, which we assigned to the oval, rod, and hook of the molecular image. The Gli521 molecule's role in the gliding mechanism is discussed. doi: 10.1128/JB.01143-09

Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits a very versatile surface architecture by switching multiple, related lipoproteins (Vpmas) on and off. In the type strain, PG2, Vpma phase variation is generated by a cluster of six vpma genes that undergo frequent DNA rearrangements via site-specific recombination. To further comprehend the degree of diversity that can be generated at the M. agalactiae surface, the vpma gene repertoire of a field strain, 5632, was analyzed and shown to contain an extended repertoire of 23 vpma genes distributed between two loci located 250 kbp apart. Loci I and II include 16 and 7 vpma genes, respectively, with all vpma genes of locus II being duplicated at locus I. Several Vpmas displayed a chimeric structure suggestive of homologous recombination, and a global proteomic analysis further indicated that at least 13 of the 16 Vpmas can be expressed by the 5632 strain. Because a single promoter is present in each vpma locus, concomitant Vpma expression can occur in a strain with duplicated loci. Consequently, the number of possible surface combinations is much higher for strain 5632 than for the type strain. Finally, our data suggested that insertion sequences are likely to be involved in 5632 vpma locus duplication at a remote chromosomal position. The role of such mobile genetic elements in chromosomal shuffling of genes encoding major surface components may have important evolutionary and epidemiological consequences for pathogens, such as mycoplasmas, that have a reduced genome and no cell wall.

Several mycoplasma species feature a membrane protrusion at a cell pole, and unknown mechanisms provide gliding motility in the direction of the pole defined by the protrusion. Mycoplasma gallisepticum, an avian pathogen, is known to form a membrane protrusion composed of bleb and infrableb and to glide. Here, we analyzed the gliding motility of M. gallisepticum cells in detail. They glided in the direction of the bleb at an average speed of 0.4 [micro]m/s and remained attached around the bleb to a glass surface, suggesting that the gliding mechanism is similar to that of a related species, Mycoplasma pneumoniae. Next, to elucidate the cytoskeletal structure of M. gallisepticum, we stripped the envelopes by treatment with Triton X-100 under various conditions and observed the remaining structure by negative-staining transmission electron microscopy. A unique cytoskeletal structure, about 300 nm long and 100 nm wide, was found in the bleb and infrableb. The structure, resembling an asymmetrical dumbbell, is composed of five major parts from the distal end: a cap, a small oval, a rod, a large oval, and a bowl. Sonication likely divided the asymmetrical dumbbell into a core and other structures. The cytoskeletal structures of M. gallisepticum were compared with those of M. pneumoniae in detail, and the possible protein components of these structures were considered.

The Mycoplasma hyorhinis protein p37 has been implicated in tumorigenic transformation for more than 20 years. Though there are many speculations as to its function, based solely on sequence homology, the issue has remained unresolved. Presented here is the 1.6-[Angstrom]-resolution refined crystal structure of M. hyorhinis p37, renamed the extracytoplasmic thiamine-binding lipoprotein (Cypl). The structure shows thiamine pyrophosphate (TPP) and two calcium ions are bound to Cypl and give the first insights into possible functions of the Cypl-like family of proteins. Sequence alignments of Cypl-like proteins between several different species of mycoplasma show that the thiamine-binding site is likely conserved and structural alignments reveal the similarity of Cypl to various binding proteins. While the experimentally determined function of Cypl remains unknown, the structure shows that the protein is a TPP-binding protein, opening up many avenues for future mechanistic studies and making Cypl a possible target for combating mycoplasma infections and tumorigenic transformation.

Mycoplasma penetrans has the ability to change its surface lipoprotein profiles frequently. The P35 family lipoproteins encoded by the mpl genes are key players in this profile variation. The M. penetrans HF-2 genome has 38 mpl genes that form three gene clusters. Most of these mpl genes have an invertible promoter sequence that is responsible for the ON/OFF switching of individual mpl gene expression. Here, we identified the recombinase that catalyses inversions of the mpl gene promoters. We focused on two open reading frames of the M. penetrans HF-2 genome, namely MYPE2900 and MYPE8180, which show significant homology to the tyrosine site-specific recombinase (Tsr) family proteins. Since genetic tools for M. penetrans are still not developed, we cloned the MYPE2900 and MYPE8180 genes and expressed them in Mycoplasma pneumoniae and Escherichia coll. The promoter regions of the mpl genes [p35 (MYPE6810) or p42 (MYPE6630) genes] were also introduced into M. pneumoniae and E. coil cells expressing MYPE2900 or MYPE8180. Inversion of these promoters occurred in the presence of the MYPE2900 gene but not in the presence of the MYPE8180 gene, indicating that the MYPE2900 gene product is the recombinase that catalyses mpl gene promoter inversions. We used a PCR-based method to detect mpl promoter inversion. This method also enabled us to detect inversions of 10 mpl gene promoters in M. penetrans HF-2 cells. All these promoter inversions occurred at the 12 bp inverted repeat (IR) sequences flanking the promoter sequence. The consensus sequence of these IRs was proposed as TAAYNNNDATTA (Y=C or T; D=A, G or T).

Mycoplasma mobile glides on solid surfaces by use of a unique mechanism that involves two large proteins, Gli349 and Gli521. Here we isolated and analyzed two antibodies and three mutants that modified mycoplasma gliding. Mapping of the target points of antibodies and mutations currently available suggested that a 301-amino-acid region on the whole 3,138-amino-acid sequence, a C-terminal region of Gli349, and an N-terminal region of Gli521 are directly involved in the movements of the gliding machinery.

Mycoplasma mycoides subspecies rnycoides small colony (SC) is the aetiologic agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease causing important losses in cattle production. The publication of the genome sequence of M. mycoides subsp, mycoides SC should facilitate the identification of putative virulence factors. However, real progress in the study of molecular mechanisms of pathogenicity also requires efficient molecular tools for gene inactivation. In the present study, we have developed a transposon-based approach for the random mutagenesis of M. mycoides subsp, mycoides SC. A PCR-based screening assay enabled the characterization of several mutants with knockouts of genes potentially involved in pathogenicity. The initial transposon was further improved by combining it with the transposon [gamma][delta] TnpR/res recombination system to allow the production of unmarked mutations. Using this approach, we isolated a mutant free of antibiotic-resistance genes, in which the gene encoding the main lipoprotein LppQ was disrupted. The mutant was found to express only residual amounts of the truncated N-terminal end of LppQ. This approach opens the way to study virulence factors and pathogen-host interactions of M. mycoides subsp, mycoides SC and to develop new, genetically defined vaccine strains.

The protein Mpn474 encoded by the mpn474 gene of the human-pathogenic Mycoplasma pneumoniae contains 1033 amino acids and has an isoelectric point of 4.79, which is caused by the large excess of glutamic acid residues (11%). Although the protein lacks recognizable export signals we showed by immuno-electron microscopy that Mpn474 is surface exposed, covering the cell completely. By combining cross-linking and careful treatment of the bacterial cells with Triton X-IO0, we found that this protein is weakly bound to the cell surface, while the true transmembrane protein Mpn141 (adhesin P1) is firmly attached under the same experimental conditions. A transposon mutant in the mpn474 gene, which has no obvious phenotype, served as negative control for the immunodetection.

As obligate parasites, Mycoplasma species are continuously exposed to oxidative damage due to hostgenerated peroxides and reactive oxygen species (ROS). In addition, the production of endogenous oxidants is believed to be a primary virulence mechanism of several Mollicute species, indicating that oxidative stress resistance is crucial to survival of these bacteria in the host milieu. Despite the abundance of oxidants at the site of infection, enzymes responsible for the detoxification of ROS have never been characterized in mycoplasmas. Here we characterize a homolog of the ohr (organic hydroperoxide resistance) family from Mycoplasma gallisepticum (encoding MGAl142). Unlike previously characterized ohr genes, the mga1142 gene is not upregulated in response to oxidative stress but displays a novel pattern of expression. Both organic and inorganic peroxides can act as substrates for MGA1142, but they are degraded with various efficiencies. Furthermore, cumene hydroperoxide, an aromatic peroxide metabolized with high efficiency by other Ohr proteins, was shown to rapidly inactivate MGA1142, accounting for the sensitivity of M. gallisepticum cells to this compound. Comparative modeling of the MGA1142 quaternary structure revealed that the active site of this molecule has a relatively wide conformation. These data indicate that the natural substrate for MGA1142 differs from that for previously characterized Ohr proteins. Triton X-114 partitioning demonstrated that MGA1142 is located in both cytosol and membrane fractions, suggesting that in vivo this molecule plays a role in the detoxification of both endogenous and exogenous peroxides. A model describing how MGA1142 is likely to be oriented in the cell membrane is presented.

Variable surface protein Vmm and five Vmm-type proteins from Mycoplasma mycoides subsp. mycoides SC were analysed to determine whether these proteins are expressed in vivo in animals affected by contagious bovine pleuropneumonia (CBPP) and in vitro. Recombinant versions of these proteins were constructed and expressed in Escherichia coli after mutation of the TGA Trp codons to TGG. These proteins were then analysed by dot and Western blotting with sera from CBPP-affected cattle. Furthermore, affinity-purified polyclonal antibodies to the recombinant proteins were used in Western and colony blotting to look for expression of the putative Vmm-type proteins in cultured M. mycoides SC. This study demonstrates that immunoglobulins in CBPP sera recognize all putative Vmm-type proteins tested, indicating that these proteins or their homologues are expressed by mycoplasmas during natural infections. Vmm and one of the putative Vmm-type proteins showed variable expression in vitro.

Mycoplasma mobile, a parasitic bacterium lacking a peptidoglycan layer, glides on solid surfaces in the direction of a membrane protrusion at a cell pole by a unique mechanism. Recently, we proposed a working model in which cells are propelled by leg proteins clustering at the protrusion's base. The legs repeatedly catch and release sialic acids on the solid surface, a motion that is driven by the force generated by ATP hydrolysis. Here, to clarify the subcellular structure supporting the gliding force and the cell shape, we stripped the membrane by Triton X-100 and identified a unique structure, designated the 'jellyfish' structure. In this structure, an oval solid 'bell' [approximately equal to] 235 wide and 155 nm long is filled with a 12-nm hexagonal lattice and connected to this structure are dozens of flexible 'tentacles' that are covered with particles of 20-nm diameter at intervals of [approximately equal to] 30 nm. The particles appear to have 180[degrees] rotational symmetry and a dimple at the center. The relation of this structure to the gliding mechanism was suggested by its cellular localization and by analyses of mutants lacking proteins essential for gliding. We identified 10 proteins as the components by mass spectrometry and found that these do not show sequence similarities with other proteins of bacterial cytoskeletons or the gliding proteins previously identified. Immunofluorescence and immunoelectron microscopy revealed that two components are localized at the bell and another that has the structure similar to the [F.sub.1]-ATPase [beta] subunit is localized at the tentacles. bacteria | electron microscopy | gliding motility | immunofluorescence | protein identification

Mycoplasma pneumoniae is a major cause of bronchitis and atypical pneumonia in humans. This cell wall-less bacterium has a complex terminal organelle that functions in cytadherence and gliding motility. The gliding mechanism is unknown but is coordinated with terminal- organelle development during cell division. Disruption of M. pneumoniae open reading frame MPN311 results in loss of protein P41 and downstream gene product P24. P41 localizes to the base of the terminal organelle and is required to anchor the terminal organelle to the cell body, but during cell division, MPN311 insertion mutants also fail to properly regulate nascent terminalorganelle development spatially or gliding activity temporally. We measured gliding velocity and frequency and used fluorescent protein fusions and time-lapse imaging to assess the roles of P41 and P24 individually in terminal-organelle development and gliding function. P41 was necessary, for normal gliding velocity and proper spatial positioning of new terminal organelles, while P24 was required for gliding frequency and new terminal-organelle formation at wild-type rates. However, P41 was essential for P24 function, and in the absence of P41, P24 exhibited a dynamic localization pattern. Finally, protein P28 requires P41 for stability, but analysis of a [P28.sup.-] mutant established that the MPN311 mutant phenotype was not a function of loss of P28.

The human pathogen Mycoplasma genitalium is known to mediate cell adhesion to target cells by the attachment organelle, a complex structure also implicated in gliding motility. The gliding mechanism of M. genitalium cells is completely unknown, but recent studies have begun to elucidate the components of the gliding machinery. We report the study of MG312, a cytadherence-related protein containing in the N terminus a box enriched in aromatic and glycine residues (EAGR), which is also exclusively found in MG200 and MG386 gliding motility proteins. Characterization of an MG_312 deletion mutant obtained by homologous recombination has revealed that the MG312 protein is required for the assembly of the M. genitalium terminal organelle. This finding is consistent with the intermediate-cytadherence phenotype and the complete absence of gliding motility exhibited by this mutant. Reintroduction of several MG_312 deletion derivatives into the MG_312 null mutant allowed us to identify two separate functional domains: an N-terminal domain implicated in gliding motility and a C-terminal domain involved in cytadherence and terminal organelle assembly functions. In addition, our results also provide evidence that the EAGR box has a specific contribution to mycoplasma cell motion. Finally, the presence of a conserved ATP binding site known as a Walker A box in the MG312 N-terminal region suggests that this structural protein could also play an active function in the gliding mechanism.

Mycoplasma genitalium is a human pathogen that mediates cell adhesion by a complex structure known as the attachment organelle. This structure is composed of cytadhesins and cytadherence-associated proteins, but few data are available about the specific role of these proteins in M. genitalium cytadherence. We have deleted by homologous recombination the mg191 and mg192 genes from the MgPa operon encoding the P140 and Pll0 cytadhesins. Molecular characterization of these mutants has revealed a reciprocal posttranslational stabilization between the two proteins. Loss of either P140 or P110 yields a hemadsorption-negative phenotype and correlates with decreased or increased levels of cytoskeleton-related proteins MG386 and DnaK, respectively. Scanning electron microscopy analysis reveals the absolute requirement of P140 and Pll0 for the proper development of the attachment organelle. The phenotype described for these mutants resembles that of the spontaneous class I and class II cytadherence-negative mutants [G. R. Mernaugh, S. F. Dallo, S. C. Holt, and J. B. Baseman, Clin. Infect. Dis. 17(Suppl. 1):S69-$78, 1993], whose genetic basis remained undetermined until now. Complementation assays and sequencing analysis demonstrate that class I and class II mutants are the consequence of large deletions affecting the mg192 and mg191-mg192 genes, respectively. These deletions originated from single-recombination events involving sequences of the MgPa operon and the MgPa island located immediately downstream. We also demonstrate the translocation of MgPa sequences to a particular MgPa island by double-crossover events. Based on these observations, we propose that in addition to being a source of antigenic variation, MgPa islands could be also involved in a general phase variation mechanism switching on and off, in a reversible or irreversible way, the adhesion properties of M. genitalium.

Mycoplasma can establish latent infections and are associated with arthritis, leukemia, and chronic lung disease. We developed an experimental model in which lung cells are deliberately infected with Mycoplasma fermentans. Human lung fibroblasts (HLF) were exposed to live M. fermentans and immune-modulating cytokine release was assessed with and without known inducers of cytokine production. M. fermentans increased IL-6, IL-8/CXCL8, MCP-1/CCL2, and Gro-[alpha]CXCL1 production. M. fermentans interacted with TNF-[beta] to release more IL-6, CXCL8, and CXCL1 than predicted by the responses to either stimulus alone. The effects of live infection were recapitulated by exposure to M. fermentans-derived macrophage-activating lipopeptide-2 (MALP-2), a Toll-like receptor-2- and receptor-6-specific ligand. The synergistic effect of combined stimuli was more pronounced with prolonged incubations. Preexposure to TNF-[beta] sensitized the cells to subsequent MALP-2 challenge, but preexposure to MALP-2 did not alter the IL-6 response to TNF-[beta]. Exposure to M. fermentans or MALP-2 did not enhance nuclear localization, DNA binding, or transcriptional activity of NF-[kappa]B and did not modulate early NF-[kappa]B activation in response to TNF-[beta]. Application of specific inhibitors of various MAPKs suggested that p38 and JNK/stress-activated protein kinase were involved in early IL-6 release after exposure to TNF-[beta] and M. fermentans, respectively. The combined response to M. fermentans and TNF-[beta], however, was uniquely sensitive to delayed application of SP-600125, suggesting that JNK/stress-activated protein kinase contributes to the amplification of IL-6 release. Thus M. fermentans interacts with stimuli such as TNF-[beta] to amplify lung cell production of immune-modulating cytokines. The mechanisms accounting for this interaction can now be dissected with the use of this in vitro model. interleukin 6; chemokines; host defense; innate immunity; Toll-like receptors

Mycoplasma mobile relies on an unknown mechanism to glide across solid surfaces including glass, animal cells, and plastics. To identify the direct binding target, we examined the factors that affect the binding of Mycoplasma pneumoniae to solid surfaces and concluded that N-acetylneuraminyllactose (sialyllactose) attached to a protein can mediate glass binding on the basis of the following four lines of evidence: (i) glass binding was inhibited by N-acetylneuraminidase, (ii) glass binding was inhibited by N-acetylneuraminyllactose in a structure-dependent manner, (iii) binding occurred on glass pretreated with bovine serum albumin attached to N-acetylneuraminyllactose, and (iv) gliding speed depended on the density of N-acetylneuraminyl-lactose on glass.

Unlike many bacterial pathogens, Mycoplasma pneumoniae is not known to produce classical toxins, and precisely how M. pneumoniae injures the respiratory epithelium has remained a mystery for >50 years. Here, we report the identification of a virulence factor (MPN372) possibly responsible for airway cellular damage and other sequelae associated with M. pneumoniae infections in humans. We show that M. pneumoniae MPN372 encodes a 68-kDa protein that possesses ADP-ribosyltransferase (ART) activity. Within its N terminus, MPN372 contains key amino acids associated with NAD binding and ADP-ribosylating activity, similar to pertussis toxin (PTX) $1 subunit (PTX-S1). Interestingly, MPN372 ADP ribosylates both identical and distinct mammalian proteins when compared with PTX-S1. Remarkably, MPN372 elicits extensive vacuolization and ultimate cell death of mammalian cells, including distinct and progressive patterns of cytopathology in tracheal rings in organ culture that had been previously ascribed to infection with WT virulent M. pneumoniae. We observed dramatic seroconversion to MPN372 in patients diagnosed with M. pneumoniae-associated pneumonia, indicating that this toxin is synthesized in vivo and possesses highly immunogenic epitopes. ADP ribosylation | community-acquired respiratory distress syndrome toxin | vacuolization

Mycoplasma pneumoniae lacks a cell wail but has internal cytoskeleton-like structures that are assumed to support the attachment organelle and asymmetric cell shape of this bacterium. To explore the fine details of the attachment organelle and the cytoskeleton-like structures, a fluorescent-protein tagging technique was applied to visualize the protein components of these structures. The focus was on the four proteins--P65, HMW2, P41, and P24--that are encoded in the crl operon (for 'cytadherence regulatory locus'), which is known to be essential for the adherence of M. pneumoniae to host cells. When the P65 and HMW2 proteins were fused to enhanced yellow fluorescent protein (EYFP), a variant of green fluorescent protein, the fused proteins became localized at the attachment organelle, enabling visualization of the organelles of living cells by fluorescence microscopy. The leading end of gliding M. pneumoniae cells, expressing the EYFP-P65 fusion, was observed as a focus of fluorescence. On the other hand, when the P41 and P24 proteins were labeled with EYFP, the fluorescence signals of these proteins were observed at the proximal end of the attachment organelle. Coexpression of the P65 protein labeled with enhanced cyan fluorescent protein clearly showed that the sites of localization of P41 and P24 did not overlap that of P65. The localization of P41 and P24 suggested that they are also cytoskeletal proteins that function in the formation of unknown structures at the proximal end of the attachment organelle. The fluorescent-protein fusion technique may serve as a powerful tool for identifying components of cytoskeleton-like structures and the attachment organelle. It can also be used to analyze their assembly.

Mycoplasma mobile glides on surfaces at up to 7 [micro]m/s by an unknown mechanism. We studied the energetics that power gliding by using a novel, growth medium-free system. We found that cells could glide in defined media if the glass substrate is preconditioned by exposure to horse serum. The active component that potentiates gliding is sensitive to proteinase K treatment. We used the defined medium system to test the effect of various inhibitors, ionophores, and poisons on motility of M. mobile. Valinomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), N,N'-dicyclohexylcarbodiimide, phenamil, amiloride, rifampin, and puromycin had no short-term effects on gliding. We also confirmed that we were able to modulate the membrane potential with valinomycin and FCCP by using a potential-sensitive dye. Shifting the pH likewise had no effect on motility. These results rule out the use of conventional ion motive forces to power gliding. Arsenate had a dramatic inhibitory effect on gliding, and both the speed and the fraction of cells moving tracked ATP levels. Sodium orthovanadate had a slight but significant inhibitory effect on gliding. Taken together, these results suggest that the motor system of M. mobile is likely an ATPase or is directly coupled to an ATPase.

Mycoplasma alligatoris causes lethal invasive disease of alligators and caimans. A homolog of the nagH gene, encoding a hyaluronidase secreted by Clostridium perfringens, and a C. perfringens hyalurunidase nagI or nagK pseudogene were discovered in the M. alligatoris genome. The nagH gene was detected by PCR in the closest relative of M. alligatoris, Mycoplasma crocodyli, but not in 40 other species representing the Mycoplasma hominis, Mycoplasma pneumoniae, and Spiroplasma phylogenetic clusters. The hyaluronidase activity in the cellular fraction of M. alligatoris and M. crocodyli SP4 broth cultures was equivalent to [10.sup.-16] U of Streptomyces hyalurolyticus hyaluronidase [CFU.sup.-1]. Negligible activity was present in the cell-free supernatant fraction. No chondruitinase activity was detected. There is also a novel homolog of the nanI gene, which encodes a sialidase secreted by C. perfringens, in the M. alligatoris genome. The signature YRIP and SXDXGXTW motifs and catalytic residues of the clostridial sialidase are conserved in the mycoplasmal gene, but the leader sequence necessary for its secretion by C. perfringens is absent. The gene was not detected by PCR in any other mycoplasma. Potent cell-associated sialidase activity was present in M. alligatoris colonies on agar but not in the cell-free superuatants of broth cultures or in M. crocodyli. The presence of hyaluronidase and sialidase in M. alligatoris is consistent with the rapid invasiveness and necrotizing effects of this organism, and the lack of sialidase in M. crocodyli is consistent with its comparatively attenuated virulence. This genetic and biochemical evidence suggests that the spreading factors hyaluronidase and sialidase, a combination unprecedented in mycoplasmas, are the basis of the virulence of M. alligatoris.

The complete genome of Mycoplasma gallisepticum strain [R.sub.low] has been sequenced. The genome is composed of 996 422 bp with an overall G + C content of 31 mol%. It contains 742 putative coding DNA sequences (CDSs), representing a 91% coding density. Function has been assigned to 469 of the CDSs, while 150 encode conserved hypothetical proteins and 123 remain as unique hypothetical proteins. The genome contains two copies of the rRNA genes and 33 tRNA genes. The origin of replication has been localized based on sequence analysis in the region of the dnaA gene. The vlhA family (previously termed pMGA) contains 43 genes distributed among five loci containing 8, 2, 9, 12 and 12 genes. This family of genes constitutes 10.4% (103 kb) of the total genome. Two CDSs were identified immediately downstream of gapA and crmA encoding proteins that share homology to cytadhesins GapA and CrmA. Based on motif analysis it is predicted that 80 genes encode lipoproteins and 149 proteins contain multiple transmembrane domains. The authors have identified 75 proteins putatively involved in transport of biomolecules, 12 transposases, and a number of potential virulence factors. The completion of this sequence has spawned multiple projects directed at defining the biological basis of M. gallisepticum.

Research has been conducted on Mycoplasma sp. bovine group 7 isolates from a clonal cluster recovered from various tissue sites during the outbreak lysed sheep red blood cells. The identification of the putative genes for glycerol transport and the flanking segment containing ORF6 and ORF5 on PG50 chromosome has been carried out in order to compare them with the homologues in M. mycoides subsp. mycoides small colony, and the results are presented.

Research has been conducted on serine-rich membrane protein LppS from Mycoplasma conjunctivae. The characterization of this protein has been carried out, and the results of gene cloning and sequence analysis of IppS gene have demonstrated that it encodes membrane protein precursor and that LppS is likely M. conjunctivae adhesin.

Research has been conducted on Mycoplasma genomes. The genetic element with unique features present in Mycoplasma fermentans PG18 chromosomes is described.

Research has been conducted on ruminant pathogen Mycoplasma agalactiae possessing expressed surface lipoproteins Vpmas. Genetic organization of the vpma multigene locus in one of M. agalactiae strains and molecular characterization of the mechanism which controls Vpma expression have been investigated, and the results are discussed.

The variation in the inhibition of the energy-generating pathway inhibitors on the growth of Mycoplasma species is tested using fluoride, iodoacetate, beta-fluoropyruvate, cibacron blue, L-citrulline, and carbonyl cyanide m-chlorophenylhydrazone. Data indicate fluoride selectively inhibits the growth of mycoplasmas, whereas iodoacetate inhibited organic acid-oxidizing species and L-citruline of arginine-hydrolyzing species.

Research has been conducted on HPr kinase/phosphatase, a regulatory protein encoded by Mycoplasma pneumoniae. Results demonstrate that this protein has both phosphatase and kinase activities.

Research has been conducted on the mycoplasma cytadherence of Mycoplasma genitalium. The identification of the new loci regulating this cytadherence has been carried out and the results indicate that msrA encodes the antioxidant repair enzyme peptide methionine sulfoxide reductase.

Results indicate that gliding feature of mycoplasma mutants is dependent on the cell-associated adherent components. Data further show that gliding activity also aids in the colony formationn in the mycoplasmas.

A study was undertaken to investigate the phospholipid degradation in Molt-3 lymphocytes infected by Mycoplasma penetrans and to identify the lipid intermediates produced. Mycoplasmas bind themselves to the host-cell surface preceded by internalization. Results indicated that infection of Molt-3 lymphocytes by M. penetrans led to the substantial production of fatty acids which indicated alterations in the lipid turnover of the host cells. The infection was related to the release of the lipid intermediates acting as signal transductors across the host-cell membrane.

Bacillus subtilis produces surfactin, a cyclic lipopeptide antibiotic and biosurfactant, that increases the proliferation rates and changes the morphology of mycoplasma-contaminated mammalian cells. The high level of membrane phospholipids in the Mycoplasma cells makes the membranes susceptible to surfactin. Surfactin causes the disintegration of the mycoplasma membranes due to its physicochemical interaction with the outer part of the lipid membrane bilayer. The interaction makes the membranes permeable. Surfactin is active against Mycoplasma hyorhinis and M. orale.

Mycoplasma arthritidis strains and substrains express different strain-variant antigens on the cell surface which are integral membrane proteins. These antigens are either non-lipid-modified proteins bound to the membrane hydrophobic domains or lipoproteins bound to the membrane fatty acid moieties. The variation is due to phase switching of the antigens. The antigens control interaction of the pathogen with the host cells and prevent damage by the host's immune system.

A variety of glycoconjugates was screened for the ability to serve as receptors for Mycoplasma hominis adherence. It was found that mycoplasmas bind strongly and specifically only to sulfatide in a temperature- and dose-dependent manner. The specificity and avidity of attachment suggest that the receptors to which M. hominis binds, particularly in the human genital tract, from which it is frequently isolated, are only sulfated glycolipids.

Mycoplasmas that are known to exhibit gliding motility possess a differentiated tip structure. This polar organelle mediates cytadherence and gliding motor activity and contains a cytoskeleton-like component that provides structural support. Here, we describe gliding motility and a unique cytoskeleton in Mycoplasma insons, which lacks any obviously differentiated tip structure.