AtzC Is a New Member of the Amidohydrolase Protein Superfamily and Is Homologous to Other Atrazine-Metabolizing Enzymes

Pseudomonas sp. strain ADP metabolizes atrazine to cyanuric acid via three plasmid-encoded enzymes, AtzA, AtzB, and AtzC. The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hydroxyatrazine. The second enzyme, AtzB, catalyzes hydroxyatrazine deamidation, yielding N-isopropylammelide. In this study, the third gene in the atrazine catabolic pathway, atzC, was cloned from a Pseudomonas sp. strain ADP cosmid library as a 25-kb EcoRI DNA fragment in Escherichia coli. The atzC gene was further delimited by functional analysis following transposon Tn5 mutagenesis and subcloned as a 2.0-kb EcoRI-AvaI fragment. An E. coli strain containing this DNA fragment expressed N-isopropylammelide isopropylamino hydrolase activity, metabolizing N-isopropylammelide stoichiometrically to cyanuric acid and N-isopropylamine. The 2.0-kb DNA fragment was sequenced and found to contain a single open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids. AtzC showed modest sequence identity of 29 and 25%, respectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily. The sequence of AtzC was compared to that of E. coli cytosine deaminase in the regions containing the five ligands to the catalytically important metal for the protein. Pairwise comparison of the 35 amino acids showed 61% sequence identity and 85% sequence similarity. AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deaminase, and phosphotriester hydrolase. Similar sequence comparisons of the most highly conserved regions indicated that the AtzA and AtzB proteins also belong to the same amidohydrolase family. Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.     

A Putative Cell Surface Receptor for Anemia-Inducing Feline Leukemia Virus Subgroup C Is a Member of a Transporter Superfamily

Domestic cats infected with the horizontally transmitted feline leukemia virus subgroup A (FeLV-A) often produce mutants (termed FeLV-C) that bind to a distinct cell surface receptor and cause severe aplastic anemia in vivo and erythroblast destruction in bone marrow cultures. The major determinant for FeLV-C-induced anemia has been mapped to a small region of the surface envelope glycoprotein that is responsible for its receptor binding specificity. Thus, erythroblast destruction may directly or indirectly result from FeLV-C binding to its receptor. To address these issues, we functionally cloned a putative cell surface receptor for FeLV-C (FLVCR) by using a human T-lymphocyte cDNA library in a retroviral vector. Expression of the 2.0-kbp FLVCR cDNA in naturally resistant Swiss mouse fibroblasts and Chinese hamster ovary cells caused substantial susceptibility to FeLV-C but no change in susceptibilities to FeLV-B and other retroviruses. The predicted FLVCR protein contains 555 amino acids and 12 hydrophobic potential membrane-spanning sequences. Database searches indicated that FLVCR is a member of the major-facilitator superfamily of transporters and implied that it may transport an organic anion. RNA blot analyses showed that FLVCR mRNA is expressed in multiple hematopoietic lineages rather than specifically in erythroblasts. These results suggest that the targeted destruction of erythroblasts by FeLV-C may derive from their greater sensitivity to this virus rather than from a preferential susceptibility to infection.     

CgMFS1, a Major Facilitator Superfamily Transporter,Is Required for Sugar Transport,Oxidative Stress Resistance,and Pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis

Colletotrichum gloeosporioides is the main causal agent of anthracnose in various plant species. Determining the molecular mechanisms underlying the pathogenicity and fungicide resistance of C. gloeosporioides could help build new strategies for disease control. The major facilitator superfamily (MFS) has multiple roles in the transport of a diverse range of substrates. In the present study, an MFS protein CgMFS1 was characterized in C. gloeosporioides. This protein contains seven transmembrane domains, and its predicted 3D structure is highly similar to the reported hexose transporters. To investigate the biological functions of CgMFS1, the gene knock-out mutant ΔCgMFS1 was constructed. A colony growth assay showed that the mutant was remarkably decreased in vegetative growth in minimal medium supplemented with monosaccharides and oligosaccharides as the sole carbon sources, whereas it showed a similar growth rate and colony morphology as wild types when using soluble starch as the carbon source. A stress assay revealed that CgMFS1 is involved in oxidative stress but not in the fungicide resistance of C. gloeosporioides. Furthermore, its pathogenicity was significantly impaired in the mutant, although its appressorium formation was not affected. Our results demonstrate that CgMFS1 is required for sugar transport, resistance to oxidative stress, and the pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis.     

Molecular Cloning,Expression, and Characterization of a Ca2+-Dependent,Membrane-Associated Nuclease of Mycoplasma genitalium

In this study, we identified and characterized the enzymatic properties of MG_186, a calcium-dependent Mycoplasma genitalium nuclease. MG_186 displays the hallmarks of nucleases, as indicated by its amino acid sequence similarity to other nucleases. We cloned, UGA corrected, expressed, purified, and demonstrated that recombinant MG_186 (rMG_186) exhibits nuclease activity similar to that of typical sugar-nonspecific endonucleases and exonucleases. Biochemical characterization indicated that Ca²⁺ alone enhances its activity, which was inhibited by divalent cations, such as Zn²⁺ and Mn²⁺. Chelating agents EGTA and EDTA also inhibited nuclease activity. Mycoplasma membrane fractionation and Triton X-114 phase separation showed that MG_186 was a membrane-associated lipoprotein, and electron microscopy revealed its surface membrane location. Incubation of purified human endometrial cell nuclei with rMG_186 resulted in DNA degradation and morphological changes typical of apoptosis. Further, immunofluorescence analysis of rMG_186-treated nuclei indicated that morphological changes were linked to the disintegration of lamin and the internalization of rMG_186. Since M. genitalium has the capacity to invade eukaryotic cells and localize to the perinuclear and nuclear region of parasitized target cells, MG_186 has the potential to provide M. genitalium, which possesses the smallest genome of any self-replicating cell, with the ability to degrade host nucleic acids both as a source of nucleotide precursors for growth and for pathogenic purposes.Mycoplasma genitalium was first identified as a urogenital tract pathogen in men and subsequently implicated in a range of women pathologies, including pelvic inflammatory diseases, cervicitis, endometritis, salpingitis, and tubal factor infertility (5, 37, 40). In addition to its urogenital niche, M. genitalium has been detected in synovial and respiratory tract specimens (3, 39). M. genitalium DNA sequencing revealed a reduced genome size of 580 kb and a low GC content, along with 482 protein-encoding genes, of which 76 were categorized as hypothetical proteins (18). The streamlined genome of M. genitalium results in gene deficits that dramatically limit its biosynthetic capabilities, leading to a complete dependence on the host for metabolic precursors, such as nucleotides, amino acids, fatty acids, and sterols.Since M. genitalium, like most mollicutes, is unable to synthesize de novo purine and pyrimidine bases (27), it must scavenge nucleotides from the host in order to replicate and persist. Only Mycoplasma penetrans has an orotate-related pathway for converting carbamoyl-phosphate to uridine-5′-monophosphate (34). The importance of nucleases in the life cycle of mycoplasmas is reinforced by their detection in at least 20 Mycoplasma species (26). Purification of membrane-associated Ca²⁺/Mg²⁺-dependent M. penetrans and Mycoplasma hyorhinis nucleases and their relation to mycoplasma survival and pathogenesis have been reported (7, 8, 29, 30). Also, a membrane nuclease gene, mnuA, was identified and cloned from Mycoplasma pulmonis (20, 25). mnuA orthologous sequences were found in M. penetrans, Mycoplasma pneumoniae, Mycoplasma hyopneumoniae, Mycoplasma gallisepticum, and Ureaplasma urealyticum but not in M. genitalium. However, recent nuclease studies with M. hyopneumoniae (nuclease gene designated mhp379) revealed the existence of orthologous sequences in M. genitalium as well as in M. pneumoniae, M. pulmonis, M. gallisepticum, and Mycoplasma synoviae (35).M. genitalium was initially described as an extracellular pathogen. Subsequently, we reported that M. genitalium can be observed in the cytoplasmic and perinuclear regions of infected mammalian cells and can persist long-term within these compartments (4, 13, 24). The latter supports the contention that M. genitalium is capable of intracellular replication and survival. Furthermore, our recent evidence suggests that M. genitalium and its protein products are capable of intranuclear localization within infected endometrial cells (41). Therefore, understanding how M. genitalium overcomes its biosynthetic deficiencies and successfully parasitizes host tissues may provide insights into its biological uniqueness as the smallest pathogen capable of “independent” growth. In this report, we characterized a putative lipoprotein, MG_186, that retains the thermostable nuclease motif found in other bacterial nucleases. The gene encoding MG_186 was cloned and expressed in Escherichia coli, and the biochemical properties of purified recombinant MG_186 (rMG_186) nuclease protein were examined along with its impact on intact nuclei isolated from endometrial cells.     

Evidence that a Cerebellum-Enriched, Synaptic Junction Glycoprotein Is Related to Fodrin and Resists Extraction with Triton in a Calcium-Dependent Manner

Abstract: Subcellular fractions from rat cerebellum and other tissues were examined for the presence of a 240K glycoprotein, designated GP-A. Previous results have shown that GP-A is enriched in cerebellum synaptic junction (SJ) fractions when compared to parent synaptic plasma membrane (SPM) fractions and is not detected in forebrain SPM or SJ fractions. In the present studies, GP-A was not detected in myelin, mitochondria, purified nuclei, or cytosolic fractions from cerebellum, but was present in microsomal fractions. GP-A is partially soluble in the non-ionic detergent Triton X-100 and is completely soluble when cerebellum SPMs are treated with the ionic detergent N-lauryl sarcosinate. The solubilization of GP-A from cerebellum membranes was shown to be a function of bound calcium ions, e.g., pretreating SPMs with 100 μM-1mM Ca²⁺ decreased the solubility of GP-A in Triton by approximately threefold. GP-A is a major concanavalin A (Con A)-binding glycoprotein in cerebellum SJ fractions and migrates on sodium dodecyl sulfate (SDS) gels with a slower relative mobility than the 235K/ 230K fodrin doublet. Comparisons between purified fodrin and the 235K/230K doublet in cerebellum and fore-brain synaptic fractions by two-dimensional peptide mapping indicated that they were identical. The Con A-binding property of GP-A was exploited to purify it by affinity chromatography with agarose-Con A. Peptide mapping comparisons between affinity-purified GP-A and GP-A in SPM and SJ fractions indicated that GP-A in synaptic fractions is apparently homogeneous. Peptide map comparisons between GP-A and 235K fodrin polypeptide indicated that these two synaptic components are highly related (50% of their respective peptides are shared). The 235K fodrin polypeptide in SJs reacted with anti-fodrin antisera on Western blots; however, GP-A failed to cross-react. These observations, together with results from previous studies, indicate that GP-A is highly enriched in cerebellum compared to other neuronal and nonneural tissues. Moreover, GP-A is enriched in SJs relative to SPM fractions, is related to fodrin, and is most likely a cell-surface glycoprotein at asymmetric synapses in cerebellum. GP-A may be involved in neuronal recognition or synaptic transmission in the cerebellum. The important role of calcium in synaptic transmission, together with the decreased solubility of GP-A in Triton that results from micromolar concentrations of calcium, suggest that GP-A may play a role in stabilizing cerebellar synaptic junctions.     

Calcium Is a Major Determinant of Xylem Vulnerability to Cavitation

Xylem vulnerability to cavitation is a key parameter in the drought tolerance of trees, but little is known about the control mechanisms involved. Cavitation is thought to occur when an air bubble penetrates through a pit wall, and would hence be influenced by the wall''s porosity. We first tested the role of wall-bound calcium in vulnerability to cavitation in Fagus sylvatica. Stems perfused with solutions of oxalic acid, EGTA, or sodium phosphate (NaPO₄) were found to be more vulnerable to cavitation. The NaPO₄-induced increase in vulnerability to cavitation was linked to calcium removal from the wall. In contrast, xylem hydraulic conductance was unaffected by the chemical treatments, demonstrating that the mechanisms controlling vulnerability to cavitation and hydraulic resistance are uncoupled. The NaPO₄ solution was then perfused into stems from 13 tree species possessing highly contrasted vulnerability to cavitation. Calcium was found to be a major determinant of between-species differences in vulnerability to cavitation. This was evidenced in angiosperms as well as conifer species, thus supporting the hypothesis of a common mechanism in drought-induced cavitation.In plants, long-distance sap transport occurs under negative pressures in xylem conduits. Sap flows between adjoining conduits through pits that form pores in the walls, and that facilitate the flow of water while preventing the passage of air bubbles. Under water stress conditions, xylem tensions increase and the conduits become vulnerable to cavitation. Cavitation provokes an air embolism that leads to a loss of hydraulic conductance, thus exacerbating plant water deficit.Species resistance to cavitation has been intensively studied over the past two decades, and is now ranked among the traits with the highest functional and ecological significance. In woody species for instance, xylem vulnerability to cavitation correlates tightly with species-specific drought tolerance (Pockman and Sperry, 2000; Tyree et al., 2003; Maherali et al., 2004), with more xerophilous species proving less vulnerable to cavitation. Substantial variations have also been found between genotypes of a different species (Cochard et al., 2007; Dalla-Salda et al., 2009). This implies that this trait could potentially be used in breeding programs to identify more drought-tolerant species or genotypes. However, efforts in this direction are still strongly impeded by a lack of understanding of the molecular and genetic basis of cavitation resistance. Our work represents a first significant step toward resolving this challenging issue.Understanding the fine mechanism of cavitation formation is a pivotal step toward identifying the key structures and the key genes coding for these structures, yet we currently have only partial insights. According to a hypothesis first formulated by Zimmermann (1983), water stress-induced cavitation is thought to occur when a tiny air bubble penetrates through a pit membrane, and would consequently be strongly influenced by the porosity of the membrane (Tyree and Sperry, 1988; Cochard, 2006). There is also experimental evidence for a role of the mechanical properties of the pit membrane in this cavitation process (Choat et al., 2004; Sperry and Hacke, 2004). Clearly, the structural, physical, and chemical properties of pit membranes are central to the determinism of cavitation.Pit membranes are modified primary cell walls made of tightly interwoven cellulose microfibrils in a matrix of hydrated hemicelluloses and pectins. Pectins consist of a complex set of GalUA (GalA)-rich polysaccharides, and four pectic domains can be distinguished: homogalacturonan (HG), rhamnogalacturonan I, rhamnogalacturonan II, and xylogalacturonan (Willats et al., 2001). The high degree of structural complexity and heterogeneity across the pectin family is the result of both biosynthesis in the endomembrane system and the action of an array of wall-based pectin-modifying enzymes (Willats et al., 2001). HG units are synthesized in the Golgi apparatus and deposited in the cell wall in a form containing 70% to 80% methyl-esterified GalA residues (O''Neill et al., 1990; Mohnen, 1999). The removal of methyl ester groups by pectin methyl esterase (PME) within the cell wall matrix produces free carboxyl groups capable of being cross-linked by calcium cations in an “egg-box” structure (Grant et al., 1973; Pelloux et al., 2007). These calcium-dependent cross-linkages are dependent both on the degree and the distribution of methyl-esterified GalA units through the HG network (Willats et al., 2001). Calcium therefore plays a central role as it determines the supramolecular assembly of the pectic chains and the formation of a pectate gel.Pectins capable of Ca²⁺ cross-linking are particularly common in bordered pit membranes (Chaffey et al., 1997; Hafren et al., 2000). Moreover, pectin-bound calcium influences wall elasticity (Ezaki et al., 2005; Proseus and Boyer, 2006; Derbyshire et al., 2007), and could therefore influence the stretching properties of the pit membranes and, consequently, the mechanism of cavitation. We tested the hypothesis that calcium plays a major role in the determinism of cavitation. This hypothesis was formulated long ago (Sperry and Tyree, 1988) but has not yet been thoroughly tested. We designed a series of experiments to demonstrate the specific role of calcium in this mechanism, and analyzed a large number of woody species to establish the role of calcium cross-linkage in across- and within-species variation in cavitation resistance. The data strongly support our hypothesis.     

Evidence that a Chloroplast Surface Protein Is Associated with a Specific Binding Site for the Precursor to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase

Most chloroplast proteins are encoded by nuclear genes and synthesized in the cytoplasm as higher molecular weight precursors. These precursors are imported posttranslationally into the chloroplasts, where they are proteolytically processed, and sorted to their proper locations. The first step of this import process is thought to be the binding of precursors to putative receptors on the outer envelope membrane of chloroplasts. We have investigated the interaction of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with its putative receptor by using a heterobifunctional, photoactivatable cross-linker. The resulting cross-linked conjugate has a molecular weight of 86,000, and is present on the surface of chloroplasts as determined by its sensitivity to digestion with protease. Control experiments demonstrated that the label in the conjugate is derived from small subunit precursor and that the conjugate is formed only when modified precursor is reacted in the presence of chloroplasts. Based on these results, we postulate that a protein on the surface of chloroplasts is part of the receptor which interacts with the small subunit precursor.     

Identification and characterization of IS 1138, a transposable element from Mycoplasma pulmonis that belongs to the IS3 family

Insertion sequence (IS) elements are mobile genetic elements found in prokaryotes. We have identified a repetitive element from Mycoplasma pulmonis, a murine pathogen, that is similar to eubacterial IS elements. By subcloning a single strain of M. pulmonis, we isolated a variant clone in which the IS element had undergone an apparent transposition event. The nucleotide sequences of the element, designated IS 1138, and the target site into which it inserted were determined. IS1138 consists of 1288bp with 18bp perfect terminal inverted repeats. Sequence analysis of the target site before and after insertion of IS1138 identified a 3bp duplication of target DNA flanking the element. The predicted amino acids encoded by the major open reading frame of IS 1138 share significant similarity with the transposases of the IS3 family. Southern hybridization analysis indicates that repetitive sequences similar to IS 1138 are present in most, if not all, strains of M. pulmonis, but Is1138–like sequences were not detected in other mycoplasmal species.     

Occurrence,Plasticity, and Evolution of the vpma Gene Family,a Genetic System Devoted to High-Frequency Surface Variation in Mycoplasma agalactiae

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.Bacteria of the Mycoplasma genus belong to the class Mollicutes and represent a remarkable group of organisms that derived from the Firmicutes lineage by massive genome reduction (41, 51). Consequent to this regressive evolution, modern mycoplasmas have been left with small genomes (580 to 1,400 kb), a limited number of metabolic pathways, and no cell wall. Due to these particularities, members of the Mycoplasma genus have often been portrayed as “minimal self-replicating organisms.” Despite this apparent simplicity, a large number of mycoplasma species are successful pathogens of humans and a wide range of animals, in which they are known to cause diseases that are often chronic and debilitating (1, 33). The surface of their single membrane is considered a key interface in mediating adaptation and survival in the context of a complex, immunocompetent host (10, 13, 34, 40). Indeed, mycoplasmas possess a highly versatile surface architecture due to a number of sophisticated genetic systems that promote intraclonal variation in the expression and structure of abundant surface lipoproteins (9, 50). Usually, these systems combine a set of contingency genes with a molecular switch for turning expression on or off that is based on either (i) spontaneous mutation (slipped-strand mispairing), (ii) gene conversion, or (iii) specific DNA rearrangements (9). While high-frequency phenotypic variation using the two first mechanisms has been described thoroughly for other bacteria (47), switching of surface components by shuffling of silent genes at a particular single expression locus has been studied mainly in mycoplasmas (3, 8, 14, 16, 23, 39, 43).Mycoplasma agalactiae, an important pathogen responsible for contagious agalactia in small ruminants (listed by the World Organisation for Animal Health), possesses a family of lipoproteins encoded by the vpma genes for which phase variation in expression is driven by a “cut-and-paste” mechanism involving a tyrosine site-specific recombinase designated Xer1 (16). Data previously gathered with the PG2 type strain identified a single vpma cluster (42) composed of six vpma genes adjacent to one xer1 gene (Fig. ​(Fig.1A).1A). Based on fine genetic analyses, Xer1 was further shown to mediate frequent site-specific DNA rearrangements by targeting short DNA sequences located upstream of each vpma gene (8, 16). While some vpma rearrangements can be phenotypically silent, others result in Vpma on-off switching by linking a silent vpma gene sequence immediately downstream of the unique vpma promoter. Because site-specific recombination can be reciprocal, the initial vpma configuration can be restored without a loss of genetic information.Open in a separate windowFIG. 1.Comparison of M. agalactiae vpma loci between the PG2 type strain and strain 5632. Schematics represent the organization of the vpma loci in clonal variant 55.5 derived from PG2 (16, 42) (A) and in clonal variant c1 derived from strain 5632 (B). (C) Counterpart of locus II₅₆₃₂ in PG2 showing the absence of vpma genes in this region. (D) The presence of two distinct loci in 5632 was confirmed by PCR, using the primer pair xerF-phydR or xerF-agpR, and the resulting amplicons are shown. The locations of the primers are indicated by arrowheads in panels A, B, and C. Large white arrows labeled with letters represent Vpma CDSs. The positions of the promoters are represented by black arrowheads labeled “P.” The two non-Vpma-related CDSs (abiG_I and abiG_II) are indicated by large arrows filled with a dotted pattern. ISMag1 elements are indicated by hatched boxes. Recombination sites downstream of each vpma gene are indicated by black dots. An asterisk indicates that the corresponding vpma gene is present at two distinct loci. Schematics were drawn approximately to scale. HP, hypothetical protein; CHP, conserved hypothetical protein. Small letters and bars indicate the positions of short particular sequences mentioned in the text and in Fig. ​Fig.33 and ​and4.4. The pictures on the left side of panels A and B illustrate the variable surface expression of Vpma, as previously described (8, 17). These correspond to colony immunoblots using Vpma-specific polyclonal antibodies recognizing PG2 VpmaW (α W) and VpmaY (α Y) epitopes.The vsa family of the murine pathogen M. pulmonis (3, 39), the vsp family of the bovine pathogen M. bovis (2, 24), and the vpma family of M. agalactiae all generate intraclonal surface diversity by using very similar molecular switches (23), although their overall coding sequences seem to be specific to the Mycoplasma species. DNA rearrangements also govern phase variation of the 38 mpl genes of the human pathogen M. penetrans (27, 35, 38). However, in this mycoplasma species the molecular switch is slightly different, since each mpl gene possesses its own invertible promoter (19). In M. penetrans, the individual expression of each mpl gene can then be switched on and off in a combinatory manner, resulting in a large number of possible Mpl surface configurations. Since M. pulmonis, M. bovis, and M. agalactiae all belong to the Mycoplasma hominis phylogenetic cluster (48) and are relatively closely related, while M. penetrans belongs to the distinct Mycoplasma pneumoniae phylogenetic cluster (30, 48), it is tempting to speculate that the vsa, vsp, and vpma systems were all inherited from a common ancestor and that the bulk of their coding sequences evolved independently in their respective hosts while the molecular switch mechanism was retained.In so-called “minimal” bacteria, the occurrence of relatively large genomic portions dedicated to multigene families, with genes encoding phase-variable, related surface proteins, suggests that they serve an important function(s). Data accumulated over the years for several mycoplasma species tend to indicate that one general purpose of these systems is to provide the mycoplasma with a variable shield that modulates surface accessibility in order to escape the host response and to adapt to rapidly changing environments (10, 11, 13, 40, 50). On the other hand, the sequences of phase-variable proteins are relatively conserved within one species but divergent between species, suggesting a more specific role for these molecules.The role of the Vpma family of M. agalactiae has yet to be elucidated, but it was recently shown that Vpma switches in expression occur at a remarkably high rate in vitro (10⁻² to 10⁻³ per cell per generation) (8, 17). The vpma systems described for PG2 (16) and another M. agalactiae strain, isolated in Israel (in which Vpmas were designated Avg proteins [14]), both revealed a repertoire of six vpma genes and only one promoter, suggesting that in M. agalactiae the number of Vpma configurations is limited to six. This contrasts with the situation commonly found in other Mycoplasma variable systems, which can offer a larger mosaic of surface architecture because of the concomitant switches of several related surface proteins and/or because of a larger number of phase-variable genes.To further understand the degree of diversity that can be generated at the surface of M. agalactiae, we analyzed the vpma gene content of a field strain, 5632, whose genome was recently sequenced by our group (unpublished data). The present study shows that 5632 contains a total of 23 vpma genes distributed in two distinct loci that both contain a recombinase gene. Further genomic and proteomic analyses indicated that the capacity of 5632 to vary its Vpma surface architecture is far more complex than that described for the type strain. Unlike the case for PG2, both 5632 vpma loci are associated with several mobile genetic insertion elements (IS) that could play an evolutionary role in the dynamics of vpma repertoires, as suggested by data presented here. One 5632 vpma locus contains open reading frames (ORFs) that are highly conserved in both M. bovis, a closely related bovine mycoplasma, and the phylogenetically distant mycoplasmas of the M. mycoides cluster, which are also important ruminant pathogens. Whether these were acquired through evolution or through horizontal transfer is discussed. The present study reveals an additional degree of complexity for the Vpma system and further suggests that some field strains might have more dynamic genomes and a more variable surface than was first estimated (42).     

The Qrc Membrane Complex,Related to the Alternative Complex III,Is a Menaquinone Reductase Involved in Sulfate Respiration

Biological sulfate reduction is a process with high environmental significance due to its major contribution to the carbon and sulfur cycles in anaerobic environments. However, the respiratory chain of sulfate-reducing bacteria is still poorly understood. Here we describe a new respiratory complex that was isolated as a major protein present in the membranes of Desulfovibrio vulgaris Hildenborough. The complex, which was named Qrc, is the first representative of a new family of redox complexes. It has three subunits related to the complex iron-sulfur molybdoenzyme family and a multiheme cytochrome c and binds six hemes c, one [3Fe-4S]^+1/0 cluster, and several interacting [4Fe-4S]^2+/1+ clusters but no molybdenum. Qrc is related to the alternative complex III, and we show that it has the reverse catalytic activity, acting as a Type I cytochrome c₃:menaquinone oxidoreductase. The qrc genes are found in the genomes of deltaproteobacterial sulfate reducers, which have periplasmic hydrogenases and formate dehydrogenases that lack a membrane subunit for reduction of the quinone pool. In these organisms, Qrc acts as a menaquinone reductase with electrons from periplasmic hydrogen or formate oxidation. Binding of a menaquinone analogue affects the EPR spectrum of the [3Fe-4S]^+1/0 cluster, indicating the presence of a quinone-binding site close to the periplasmic subunits. Qrc is the first respiratory complex from sulfate reducers to have its physiological function clearly elucidated.     

Evidence That a Precursor Glycoprotein Is Cleaved to Yield the Major Glycoprotein of Avian Tumor Virus

A glycoprotein designated pr90, which is recognized by anti-gp85 serum, is present in lysates of pulse-labeled transformed cells. Under chase conditions, a reduction in the level of labeled pr90 is observed concomitant with the appearance of labeled, cell-associated viral glycoprotein.     

TcTASV-C,a Protein Family in Trypanosoma cruzi that Is Predominantly Trypomastigote-Stage Specific and Secreted to the Medium

Among the several multigene families codified by the genome of T. cruzi, the TcTASV family was the latest discovered. The TcTASV (Trypomastigote, Alanine, Serine, Valine) family is composed of ∼40 members, with conserved carboxi- and amino-termini but with a variable central core. According to the length and sequence of the central region the family is split into 3 subfamilies. The TcTASV family is conserved in the genomes of – at least – lineages TcI and TcVI and has no orthologues in other trypanosomatids. In the present work we focus on the study of the TcTASV-C subfamily, composed by 16 genes in the CL Brener strain. We determined that TcTASV-C is preferentially expressed in trypomastigotes, but it is not a major component of the parasite. Both immunoflourescence and flow cytometry experiments indicated that TcTASV-C has a clonal expression, i.e. it is not expressed by all the parasites of a certain population at the same time. We also determined that TcTASV-C is phosphorylated and glycosylated. TASV-C is attached to the parasite surface by a GPI anchor and is shed spontaneously into the medium. About 30% of sera from infected hosts reacted with TcTASV-C, confirming its exposition to the immune system. Its superficial localization and secretory nature suggest a possible role in host-parasite interactions.