Les crocodiliens fossiles du Jurassique moyen et supérieur de Bourgogne

The middle and upper Jurassic limestones of Côte-d'Or (France) contain some crocodilian remains referred to the genera Teleidosaurus, Steneosaurus, and Metriorhynchus. Their preservation allows a determination at a specific level only for Teleidosaurus gaudryi (Lower Bathonian), Steneosaurus larteti (Upper Bathonian), S. heberti and S. cf. intermedius (Middle Oxfordian). Sedimentological evidence shows that Steneosaurus and Teleidosaurus are found either in very pure limestones sedimented on an epicontinental platform, in a very calm and sheltered environment, in the intertidal or supratidal zone, often associated with subrecifal facies; or in the infratidal zone, more distinctly subjected to open-sea influences (deposits, currents, reworkings). Specialised Metriorhynchidae such as Metriorhynchus are completely absent in the former type of sediment but are present in the later. One may infer, considering anatomical evidence (no dermal armour, caudal fin…) that Metriorhynchus was better adapted to swimming and rather lived in the open sea. The Teleosauridae preferred very shallow or temporarily emerged places, where they could move around thanks to their limbs which were still capable of walking on land; however, this did not prevent them from going out to sea, where the habitats of both families probably largely overlapped.     

The fine structure of Ameson pulvis (Microspora,Microsporida) and its implications regarding classification and chromosome cycle

Nosema pulvisPerez, 1905, Ameson pulvis (Perez) Sprague, 1977, in muscles of the crabs Carcinus maenas and C. mediterraneus from the coast of France, was observed with the electron microscope. It was found to be structurally similar to the type species A. michaelis (Sprague, 1970). Sprague, 1977, having moniliform sporogonial plasmodia, unikaryotic sporoblasts, and hirsute sporulation stages. It is treated as distinct from A. michaelis because it has slightly smaller spores (by comparison with syntype material of A. michaelis) and appears to have fewer coils in the polar filament. The results require the removal of the genus Ameson from the family Nosematidae Labbé, 1899, where Sprague (1977) had placed it under the erroneous supposition that its sporoblasts are diplokaryotic. Ameson is transferred to family Unikaryonidae Sprague, 1977. Ameson is distinguished from PereziaLéger and Duboscq, 1909, shown by Ormieres et al. to have a similar developmental pattern, by presence of appendages on its sporulation stage. A. nelsoni (Sprague, 1950), the third, and only other species of Ameson, lacks the appendages and is transferred to genus Perezia.     

Asp1, a Conserved 1/3 Inositol Polyphosphate Kinase,Regulates the Dimorphic Switch in Schizosaccharomyces pombe

The ability to undergo dramatic morphological changes in response to extrinsic cues is conserved in fungi. We have used the model yeast Schizosaccharomyces pombe to determine which intracellular signal regulates the dimorphic switch from the single-cell yeast form to the filamentous invasive growth form. The S. pombe Asp1 protein, a member of the conserved Vip1 1/3 inositol polyphosphate kinase family, is a key regulator of the morphological switch via the cAMP protein kinase A (PKA) pathway. Lack of a functional Asp1 kinase domain abolishes invasive growth which is monopolar, while an increase in Asp1-generated inositol pyrophosphates (PP) increases the cellular response. Remarkably, the Asp1 kinase activity encoded by the N-terminal part of the protein is regulated negatively by the C-terminal domain of Asp1, which has homology to acid histidine phosphatases. Thus, the fine tuning of the cellular response to environmental cues is modulated by the same protein. As the Saccharomyces cerevisiae Asp1 ortholog is also required for the dimorphic switch in this yeast, we propose that Vip1 family members have a general role in regulating fungal dimorphism.Eucaryotic cells are able to define and maintain a particular cellular organization and thus cellular morphology by executing programs modulated by internal and external signals. For example, signals generated within a cell are required for the selection of the growth zone after cytokinesis in the fission yeast Schizosaccharomyces pombe or the emergence of the bud in Saccharomyces cerevisiae (37, 44, 81). Cellular morphogenesis is also subject to regulation by a wide variety of external signals, such as growth factors, temperature, hormones, nutrient limitation, and cell-cell or cell-substrate contact (13, 34, 66, 75, 81). Both types of signals will lead to the selection of growth zones accompanied by the reorganization of the cytoskeleton.The ability to alter the growth form in response to environmental conditions is an important virulence-associated trait of pathogenic fungi which helps the pathogen to spread in and survive the host''s defense system (7, 32). Alteration of the growth form in response to extrinsic signals is not limited to pathogenic fungi but is also found in the model yeasts S. cerevisiae and S. pombe, in which it appears to represent a foraging response (1, 24).The regulation of polarized growth and the definition of growth zones have been studied extensively with the fission yeast S. pombe. In this cylindrically shaped organism, cell wall biosynthesis is restricted to one or both cell ends in a cell cycle-regulated manner and to the septum during cytokinesis (38). This mode of growth requires the actin cytoskeleton to direct growth and the microtubule cytoskeleton to define the growth sites (60). In interphase cells, microtubules are organized in antiparallel bundles that are aligned along the long axis of the cell and grow from their plus ends toward the cell tips. Upon contact with the cell end, microtubule growth will first pause and then undergo a catastrophic event and microtubule shrinkage (21). This dynamic behavior of the microtubule plus end is regulated by a disparate, conserved, microtubule plus end group of proteins, called the +TIPs. The +TIP complex containing the EB1 family member Mal3 is required for the delivery of the Tea1-Tea4 complex to the cell tip (6, 11, 27, 45, 77). The latter complex docks at the cell end and recruits proteins required for actin nucleation (46, 76). Thus, the intricate cross talk between the actin and the microtubule cytoskeleton at specific intracellular locations is necessary for cell cycle-dependent polarized growth of the fission yeast cell.The intense analysis of polarized growth control in single-celled S. pombe makes this yeast an attractive organism for the identification of key regulatory components of the dimorphic switch. S. pombe multicellular invasive growth has been observed for specific strains under specific conditions, such as nitrogen and ammonium limitation and the presence of excess iron (1, 19, 50, 61).Here, we have identified an evolutionarily conserved key regulator of the S. pombe dimorphic switch, the Asp1 protein. Asp1 belongs to the highly conserved family of Vip1 1/3 inositol polyphosphate kinases, which is one of two families that can generate inositol pyrophosphates (PP) (17, 23, 42, 54). The inositol polyphosphate kinase IP6K family, of which the S. cerevisiae Kcs1 protein is a member, is the “classical” family that can phosphorylate inositol hexakisphosphate (IP₆) (70, 71). These enzymes generate a specific PP-IP₅ (IP₇), which has the pyrophosphate at position 5 of the inositol ring (20, 54). The Vip1 family kinase activity was unmasked in an S. cerevisiae strain with KCS1 and DDP1 deleted (54, 83). The latter gene encodes a nudix hydrolase (14, 68). The mammalian and S. cerevisiae Vip1 proteins phosphorylate the 1/3 position of the inositol ring, generating 1/3 diphosphoinositol pentakisphosphate (42). Both enzyme families collaborate to generate IP₈ (17, 23, 42, 54, 57).Two modes of action have been described for the high-energy moiety containing inositol pyrophosphates. First, these molecules can phosphorylate proteins by a nonenzymatic transfer of a phosphate group to specific prephosphorylated serine residues (2, 8, 69). Second, inositol pyrophosphates can regulate protein function by reversible binding to the S. cerevisiae Pho80-Pho85-Pho81 complex (39, 40). This cyclin-cyclin-dependent kinase complex is inactivated by inositol pyrophosphates generated by Vip1 when cells are starved of inorganic phosphate (39, 41, 42).Regulation of phosphate metabolism in S. cerevisiae is one of the few roles specifically attributed to a Vip1 kinase. Further information about the cellular function of this family came from the identification of the S. pombe Vip1 family member Asp1 as a regulator of the actin nucleator Arp2/3 complex (22). The 106-kDa Asp1 cytoplasmic protein, which probably exists as a dimer in vivo, acts as a multicopy suppressor of arp3-c1 mutants (22). Loss of Asp1 results in abnormal cell morphology, defects in polarized growth, and aberrant cortical actin cytoskeleton organization (22).The Vip1 family proteins have a dual domain structure which consists of an N-terminal “rimK”/ATP-grasp superfamily domain found in certain inositol signaling kinases and a C-terminal part with homology to histidine acid phosphatases present in phytase enzymes (28, 53, 54). The N-terminal domain is required and sufficient for Vip1 family kinase activity, and an Asp1 variant with a mutation in a catalytic residue of the kinase domain is unable to suppress mutants of the Arp2/3 complex (17, 23, 54). To date, no function has been described for the C-terminal phosphatase domain, and this domain appears to be catalytically inactive (17, 23, 54).Here we describe a new and conserved role for Vip1 kinases in regulating the dimorphic switch in yeasts. Asp1 kinase activity is essential for cell-cell and cell-substrate adhesion and the ability of S. pombe cells to grow invasively. Interestingly, Asp1 kinase activity is counteracted by the putative phosphatase domain of this protein, a finding that allows us to describe for the first time a function for the C-terminal part of Vip1 proteins.     

Bacteria,Archaea, and Crenarchaeota in the Epilimnion and Hypolimnion of a Deep Holo-Oligomictic Lake

In a deep, subalpine holo-oligomictic lake, the relative abundance of Archaea and Crenarchaeota, but not that of Bacteria, increases significantly with depth and varies seasonally. Cell-specific prokaryotic productivity is homogeneous along the water column. The concept of active Archaea observed in the deep ocean can therefore be extended to a deep oxic lake.The abundance, activity, and community composition of epilimnetic and hypolimnetic prokaryotes have been less thoroughly investigated in deep lakes than in oceans. Strong evidence that the depth gradient plays a role in modulating the balance between the domains of Bacteria and Archaea has been found in various marine systems (8, 12, 13, 20). It has been shown that the percentage of Bacteria in the deep marine hypolimnion decreases (up to 5,000 m) while, conversely, the percentage of Archaea increases. The percentage of Crenarchaeota is also higher in the mesopelagic zone than in surface waters (10).Although Archaea have been found in a variety of freshwater habitats (3), little has thus far been published on differentiating between Bacteria, Archaea, and Crenarchaeota in the hypolimnion of deep lakes. An exception is a study of the high-altitude ultraoligotrophic Crater Lake (21, 22), where group I marine Crenarchaeota were observed in deep-water populations (22). This study and another study of various lakes from three continents (9) are based on summer sampling, making it impossible to ascertain the effects of temporal variability on the vertical distribution of Archaea and Crenarchaeota, as has been done for marine systems and shallow lakes (for examples, see references 8 and 11).Our primary objective was to follow variations in the relative abundance of Bacteria, Archaea, and Crenarchaeota found in the hypolimnetic waters of a deep holo-oligomictic lake with a permanent oxic hypolimnion and compare them with those in the epilimnetic assemblages. We used the catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) technique and compared the data thus obtained with prokaryotic productivity.     

Envelope Proteins of the CsmB/CsmF and CsmC/CsmD Motif Families Influence the Size,Shape, and Composition of Chlorosomes in Chlorobaculum tepidum

The chlorosome envelope of Chlorobaculum tepidum contains 10 proteins that belong to four structural motif families. A previous mutational study (N.-U. Frigaard, H. Li, K. J. Milks, and D. A. Bryant, J. Bacteriol. 186:646-653, 2004) suggested that some of these proteins might have redundant functions. Six multilocus mutants were constructed to test the effects of eliminating the proteins of the CsmC/CsmD and CsmB/CsmF motif families, and the resulting strains were characterized physiologically and biochemically. Mutants lacking all proteins of either motif family still assembled functional chlorosomes, and as measured by growth rates of the mutant strains, light harvesting was affected only at the lowest light intensities tested (9 and 32 μmol photons m⁻² s⁻¹). The size, composition, and biogenesis of the mutant chlorosomes differed from those of wild-type chlorosomes. Mutants lacking proteins of the CsmC/CsmD motif family produced smaller chlorosomes than did the wild type, and the Q_y absorbance maximum for the bacteriochlorophyll c aggregates in these chlorosomes was strongly blueshifted. Conversely, the chlorosomes of mutants lacking proteins of the CsmB/CsmF motif family were larger than wild-type chlorosomes, and the Q_y absorption for their bacteriochlorophyll c aggregates was redshifted. When CsmH was eliminated in addition to other proteins of either motif family, chlorosomes had smaller diameters. These data show that the chlorosome envelope proteins of the CsmB/CsmF and CsmC/CsmD families play important roles in determining chlorosome size as well as the assembly and supramolecular organization of the bacteriochlorophyll c aggregates within the chlorosome.Green sulfur bacteria (GSB; phylum Chlorobi) are obligate photolithoautotrophs that utilize chlorosomes for light harvesting (2, 13). Chlorosomes additionally occur in some green-nonsulfur bacteria, also known as filamentous anoxygenic phototrophs (phylum Chloroflexi), and in a recently discovered chlorophototrophic member of the phylum Acidobacteria, “Candidatus Chloracidobacterium thermophilum” (2, 3). Chlorosomes are the largest known light-harvesting organelles and can contain up to 250,000 bacteriochlorophyll (BChl) molecules (13, 29, 30, 39). They do not have a fixed stoichiometric ratio of the major pigment, which may be BChl c, d, or e, to any protein component, and as a result they are highly variable in size, shape, and composition. In spite of this structural heterogeneity (34), the detailed molecular and supramolecular structures of the BChls in chlorosomes of Chlorobaculum tepidum were recently solved by combining systems biology, solid-state nuclear magnetic resonance (NMR), cryo-electron microscopy, and molecular modeling (22). The fundamental structural units were found to be syn-anti monomer stacks that form coaxial nanotubes, which have a 2.1-nm spacing between the adjacent BChl layers. In addition to the major BChl species, chlorosomes contain carotenoids, isoprenoid quinones, wax esters, and a small quantity of BChl a. BChl a is known to be associated with CsmA, the most highly conserved protein in chlorosomes (13).Although the structural organization of the BChl molecules in all chlorosomes may be similar (4, 22, 25, 37, 38), with the exception of CsmA, the composition and sequences of the envelope proteins of chlorosomes of the phyla Chlorobi, Chloroflexi, and Acidobacteria are not well conserved. Blankenship (1) suggested that lateral gene transfer might have been responsible for the presence of the genes for chlorosome biogenesis among some of these three groups of bacteria. However, because chlorosomes are found in each of three, early-diverging bacterial lineages that contain chlorophototrophs, two of which additionally contain homodimeric type 1 reaction centers (2, 3), it is possible that chlorosomes represent one of the earliest types of photosynthetic antennae and were present in a common ancestor of these phyla.A protein-stabilized, glycolipid envelope surrounds the chlorosome BChls, and this membrane can be considered to be an asymmetric bilayer membrane in which glycolipids form the outer leaflet and the hydrophobic tails of BChls form the inner leaflet (13, 24, 50, 53). In C. tepidum, a genetically tractable model GSB, this envelope contains 10 proteins, which are designated CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX (6-8, 14, 47, 50). The structural organization of these proteins has been studied by cross-linking and immunoblotting, which led to a model for the organization of these proteins in the chlorosome envelope (28, 50, 53). CsmA, the only protein for which any detailed structural information is available, probably binds both BChl a and carotenoids (13, 23, 31, 35, 40) and forms a large, paracrystalline array known as the “baseplate” (8, 23, 28, 35, 36, 42). The structure for apo-CsmA in an organic solvent was recently determined by NMR spectroscopy, and a model for the structural organization of CsmA in the chlorosome baseplate of C. tepidum was proposed (35, 36).Sequence comparisons suggest that the chlorosome envelope proteins can be assigned to four motif families: 1, CsmA/CsmE; 2, CsmB/CsmF (CsmH); 3, CsmC/CsmD (CsmH); and 4, CsmI/CsmJ/CsmX (48, 50). CsmA and CsmE are 49% identical and are both synthesized as precursors, which are proteolytically processed by the removal of ∼20 amino acids at their carboxy termini to generate the mature polypeptides (7, 8). CsmB and CsmF are 29% identical and 63% similar in sequence (6, 50). Moreover, the amino-terminal domain of CsmH is related in sequence to these two proteins (50). The CsmC and CsmD proteins are 26% identical and 45% similar in sequence, and these two proteins additionally share sequence similarity to the carboxyl-terminal region of CsmH. The other three chlorosome proteins (CsmI, CsmJ, and CsmX) share some sequence similarities to the precursor forms of CsmA and CsmE in their carboxyl-terminal regions, while their amino-terminal domains are obviously related to adrenodoxin-type [2Fe-2S] ferredoxins (47-50). These sequence relationships strongly imply that gene duplication and divergence have occurred among a small number of ancestral gene types, and these observations additionally suggest that some of these proteins might be functionally redundant (47, 50). This view was supported by mutational studies that showed that only CsmA was essential for the viability of C. tepidum. Mutants lacking any other single chlorosome protein still assembled functional chlorosomes that were similar in pigment composition and functionality to those of the wild type (14).Because of the possible functional redundancy of chlorosome proteins of the different motif classes, double, triple, and quadruple mutants were constructed to study the roles of the CsmC/CsmD/CsmH and CsmB/CsmF/CsmH protein motif families in chlorosome biogenesis and structure. Mutants lacking CsmI, CsmJ, and CsmX, which form the Fe/S motif family of envelope proteins, were also constructed, and these mutants will be described in detail elsewhere (27; H. Li, N.-U. Frigaard, and D. A. Bryant, unpublished data). The results presented here show that functional chlorosomes assemble in the complete absence of proteins of the CsmC/CsmD or CsmB/CsmF motif families, but the size, shape, and composition of the resulting chlorosomes are altered. The results suggest that the chlorosome envelope proteins may also influence the structural organization of the BChls in chlorosomes and thus help to define chlorosome assembly and shape.     

Genome-wide identification,in silico characterization and expression analysis of ZIP-like genes from <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> in response to Zinc and Iron

Trace elements such as Zinc and Iron are essential components of metalloproteins and serve as cofactors or structural elements for enzymes involved in several important biological processes in almost all organisms. Because either excess or insufficient levels of Zn and Fe can be harmful for the cells, the homeostatic levels of these trace minerals must be tightly regulated. The Zinc regulated transporter, Iron regulated transporter-like Proteins (ZIP) comprise a diverse family, with several paralogues in diverse organisms and are considered essential for the Zn and Fe uptake and homeostasis. Zn and Fe has been shown to regulate expression of proteins involved in metabolism and pathogenicity mechanisms in the protozoan pathogen Trichomonas vaginalis, in contrast high concentrations of these elements were also found to be toxic for T. vaginalis trophozoites. Nevertheless, Zn and Fe uptake and homeostasis mechanisms is not yet clear in this parasite. We performed a genome-wide analysis and localized the 8 members of the ZIP gene family in T. vaginalis (TvZIP1-8). The bioinformatic programs predicted that the TvZIP proteins are highly conserved and show similar properties to the reported in other ZIP orthologues. The expression patterns of TvZIP1, 3, 5 and 7 were diminished in presence of Zinc, while the rest of the TvZIP genes showed an unchanged profile in this condition. In addition, TvZIP2 and TvZIP4 showed a differential expression pattern in trophozoites growth under different Iron conditions. These results suggest that TvZIP genes encode membrane transporters that may be responsible for the Zn and Fe acquisition in T. vaginalis.     

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).     

Identification,evolution and expression analyses of Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene family in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) serves as a plentiful leaf protein which functions in both eukaryote and prokaryote photosynthesis. The small subunits of Rubisco (RBCS) exist as a multigene family which regulates the catalytic efficiency of holoenzyme. Here, 20 RBCS family genes were identified in Triticum aestivum genome, and were clustered into 4 clades according to phylogenetic analysis. On the basis of the identified 9 and 8 RBCSs in Triticum urartu and Aegilops tauschii, homology analysis revealed some TaRBCS genes were orthologous to TuRBCSs and AetRBCSs, and the number of in-paralog pairs between RBCSs in wheat were much more than that in T. urartu or A. tauschii. Gene structure, protein motif and cis-acting element analysis exhibited that TaRBCSs in each clade shared some identity. The in silico expression of RBCS genes showed that RBCSs mainly expressed in leaf, flower and caryopsis. Quantitative real-time PCR analysis showed that TaRBCSs were remarkably responsive to drought, salt, ABA and darkness stresses. The work comprehensively studies the RBCS family genes in wheat, and lays the foundation for subsequent functional research of TaRBCSs.     

ICEEc2, a New Integrative and Conjugative Element Belonging to the pKLC102/PAGI-2 Family,Identified in Escherichia coli Strain BEN374

The diversity of the Escherichia coli species is in part due to the large number of mobile genetic elements that are exchanged between strains. We report here the identification of a new integrative and conjugative element (ICE) of the pKLC102/PAGI-2 family located downstream of the tRNA gene pheU in the E. coli strain BEN374. Indeed, this new region, which we called ICEEc2, can be transferred by conjugation from strain BEN374 to the E. coli strain C600. We were also able to transfer this region into a Salmonella enterica serovar Typhimurium strain and into a Yersinia pseudotuberculosis strain. This transfer was then followed by the integration of ICEEc2 into the host chromosome downstream of a phe tRNA gene. Our data indicated that this transfer involved a set of three genes encoding DNA mobility enzymes and a type IV pilus encoded by genes present on ICEEc2. Given the wide distribution of members of this family, these mobile genetic elements are likely to play an important role in the diversification of bacteria.The fantastic diversity of the Escherichia coli species has been known for a long time. With modern sequencing strategies, the molecular bases of this diversity are now being unraveled (49). Analyzing the genome of 20 E. coli strains, Touchon et al. recently showed that only a minority of genes, approximately 1,900 genes, were shared by all E. coli strains and constituted the core genome of the E. coli species (50). Additionally, the total number of genes found in all E. coli strains, the pan-genome, is an order of magnitude larger than this core genome (50). The non-core genome of a strain, also called flexible gene pool, is therefore made of a wide diversity of genes. This genetic diversity of the E. coli species translates into a diversity of phenotypic properties. While most E. coli strains are commensal of the gastrointestinal tract of humans and warm-blooded animals, a significant number are responsible for different diseases in humans and animals (22), including extraintestinal infections in chickens; strains isolated from such cases are designated by the term APEC for avian pathogenic E. coli (10).This diversity arises from frequent horizontal gene transfers of mobile genetic elements such as transposons, plasmids, phages, genomic islands, or integrative and conjugative elements (ICEs) (11, 21, 34). Among these mobile genetic elements, ICEs have a particular place as they share properties with both plasmids, genomic islands, and transposons; they can be defined as elements that encode all the necessary machineries that allow their excision from the chromosome, their transfer to a recipient strain, and their integration into the recipient strain''s genome (5, 6, 46, 54). Well-known representatives of this class of genetic elements include Tn916 discovered in Enterococcus faecalis, the conjugative transposon CTnDOT in Bacteroides thetaiotaomicron, ICEKp1 in Klebsiella pneumoniae, SXT/R391-related elements, PFGI-1 in Pseudomonas fluorescens, and the clc element in Pseudomonas sp. strain B13 as well as ICEBs1 in Bacillus subtilis and ICEEc1 in the E. coli strain ECOR31 (1, 39, 44, 46, 54). Typically, ICEs contain at least three modules that are required for key steps in the ICE''s life cycle: an excision/integration module, a transfer module, and a regulation module (54). Besides these, ICEs often contain cargo regions that confer on their host a diverse array of properties, such as virulence properties (ICEEc1), antibiotic resistance (SXT), or degradation of chemical compounds (clc). Because of their self-transfer abilities and their diverse accessory gene repertoires, ICEs are very likely to play a major role in bacteria evolution (46).A new family of ICEs has recently gained interest and was named the pKLC102/PAGI-2 family. The first element of this family, the clc element, was discovered in Pseudomonas sp. strain B13 and confers on the bacteria the possibility to degrade aromatic compounds (42). The transfer of this element was discovered long before its complete sequence was characterized (16). Other members of this family include several elements present in Pseudomonas strains such as PAGI-1 and PAGI-2 as well as the pKLC102 element first considered to be a plasmid but later on shown to be an ICE because of its ability to integrate into the chromosome of its host (23, 52). pKLC102/PAGI-2 elements share a set of core genes (33) and, like most ICEs and genomic islands, are all integrated downstream of tRNA genes (26, 52). The transfer between strains has been demonstrated, albeit with different frequencies, for only a few members, such as the clc element, Pseudomonas aeruginosa pathogenicity island 1 (PAPI-1), and ICEHin1056 from Haemophilus influenzae (20, 37, 41); this transfer involves the type IV pilus (20), the integrase (40), and in some cases the formation of a circular intermediate of the excised ICE (24).In order to identify new accessory genes of APEC strains, we previously described tRNA loci in the E. coli genome that could represent potential insertion sites for new genomic islands (18). We had already used this strategy to characterize the AGI-3 region that is involved in the virulence of an avian pathogenic E. coli strain and that confers the ability to grow on fructooligosaccharides (7, 43). During this tRNA screening, we showed that genomic islands might potentially be present downstream of the tRNA genes argW, leuX, pheU, pheV, selC, serU, and thrW in several APEC strains.In this report, we describe the identification of a new genomic island located downstream of pheU in the APEC strain BEN374. This region, which we named ICEEc2, was fully sequenced, and its properties were analyzed in detail; ICEEc2 is a new ICE found in E. coli and belongs to the pKLC102/PAGI-2 family described above.     

Molecular Basis of ChvE Function in Sugar Binding,Sugar Utilization,and Virulence in Agrobacterium tumefaciens

ChvE is a chromosomally encoded protein in Agrobacterium tumefaciens that mediates a sugar-induced increase in virulence (vir) gene expression through the activities of the VirA/VirG two-component system and has also been suggested to be involved in sugar utilization. The ChvE protein has homology to several bacterial periplasmic sugar-binding proteins, such as the ribose-binding protein and the galactose/glucose-binding protein of Escherichia coli. In this study, we provide direct evidence that ChvE specifically binds the vir gene-inducing sugar d-glucose with high affinity. Furthermore, ChvE mutations resulting in altered vir gene expression phenotypes have been isolated and characterized. Three distinct categories of mutants have been identified. Strains expressing the first class are defective in both virulence and d-glucose utilization as a result of mutations to residues lining the sugar-binding cleft. Strains expressing a second class of mutants are not adversely affected in sugar binding but are defective in virulence, presumably due to impaired interactions with the sensor kinase VirA. A subset of this second class of mutants includes variants of ChvE that also result in defective sugar utilization. We propose that these mutations affect not only interactions with VirA but also interactions with a sugar transport system. Examination of a homology model of ChvE shows that the mutated residues associated with the latter two phenotypes lie in two overlapping solvent-exposed sites adjacent to the sugar-binding cleft where conformational changes associated with the binding of sugar might have a maximal effect on ChvE''s interactions with its distinct protein partners.Virulent strains of Agrobacterium tumefaciens contain the tumor-inducing (Ti) plasmid that carries virulence (vir) operons. Products of vir operons are involved in infecting wound sites of dicotyledonous plants and initiating tumor formation. The expression of vir genes in A. tumefaciens is activated by plant-released signals, namely, phenolic derivatives, acidic pH, and monosaccharides (for a review, see reference 6), via the combined activities of the periplasmic protein ChvE and the VirA/VirG two-component regulatory system. Upon perception of these plant signals, autophosphorylated VirA, a transmembrane histidine kinase, transfers a phosphoryl group to VirG, a response regulator, and then the phosphorylated VirG activates the expression of vir genes by binding vir boxes in their promoters (8, 19, 24, 31, 52).Perception and transduction of the sugar signals is crucial to the virulence of A. tumefaciens: strains lacking ChvE, a chromosomally encoded putative sugar-binding protein, are significantly less virulent than wild-type strains (17, 18). Previous studies have shown that, in fact, sugar signaling is neither sufficient for nor absolutely required for vir gene expression. Rather, sugars vastly increase both the sensitivity of VirA to phenol derivatives, such as acetosyringone (AS), and the maximal levels of vir gene expression observed at saturating levels of such compounds (for a review, see reference 26). The periplasmic domain of VirA is required for transduction of the sugar and pH signals (7, 8, 16, 41), whereas the so-called “linker” domain, located in the cytoplasm between the second transmembrane domain and the kinase domain, is required for perception and transduction of the phenolic signals (8, 46, 47).A working model for the ChvE/sugar/VirA signaling pathway suggests that monosaccharide-bound ChvE interacts with the periplasmic domain of VirA to relieve periplasmic repression, resulting in maximal sensitivity of VirA to phenolic signals (7, 11, 32, 41). However, limited evidence has been presented to reveal how ChvE recognizes monosaccharides and how it interacts with the periplasmic domain of VirA. Shimoda et al. (41) identified a mutant chvE allele [chvE(T211M)] that is able to suppress a sugar-insensitive virA allele [virA(E210V)], thereby restoring the sugar-sensing ability. The suppressing effect of chvE(T211M) was then proposed to be the result of the specific restoration of the capacity of VirA^E210V to bind ChvE^T211M. However, ChvE^T211M also activated wild-type VirA in the absence of sugars (32), suggesting that this mutant may not be a site-specific suppressor of VirA^E210V. Based on a homology model of ChvE, a recent study (16) does predict, though, that the residue T211 is located on the surface of the ChvE protein, consistent with the model that T211 is in a position to interact with the periplasmic domain of VirA.Based on sequence similarity, ChvE is a member of the periplasmic sugar-binding protein (PSBP) family. The structures of some PSBPs, including two ChvE homologues in Escherichia coli, ribose-binding protein (RBP) and glucose/galactose-binding protein (GBP), have been solved. The family of PSBPs shares very similar structural features, and each of them contains two similar but distinct globular domains connected by a flexible hinge (38). A sugar-binding site is located at the cleft between the two domains. PSBPs play an important role in active sugar transport, and some of them also serve as an initial receptor for sugar chemotaxis (45). A wealth of evidence has demonstrated that some specialized regions located on the surfaces of PSBPs are important for transport and chemotactic functions. In the case of RBP, four distinct regions spanning the N-terminal and C-terminal domains are involved in interaction with its permease (a transport partner), its chemotransducer (a chemotactic partner), or both (5, 15). In GBP, one residue was identified as being specifically involved in chemotaxis but not transport (36, 49). For maltose-binding protein (MBP), which is also a member of the PSBP family, two well-defined regions located on each domain of the protein are involved in interaction with its chemotransducer (54). These regions partially overlap with the regions involved in interaction with its permease (25, 54). Structural analysis indicates that both domains of MBP have direct interactions with its transport partners (35).ChvE also appears to be a highly versatile protein: not only does it play an important role in virulence, but as in the case of the PSPBs described above, it has been indicated to be a primary receptor for transport of and chemotaxis toward some sugars (7). This raises important biological/biochemical questions. How can ChvE interact with three presumably different periplasmic components of systems that are respectively involved in virulence, sugar utilization, and chemotaxis? How are the interactions of ChvE with these periplasmic components structurally segregated: do the interactions occur on the same or different regions of ChvE? To address these issues, we employed genetic and biophysical approaches to identify the residues of ChvE involved in sugar utilization versus the residues involved in virulence. The residues of both groups were mapped onto a homology model of ChvE based on a high-resolution crystal structure of E. coli GBP (PDB ID, 2ipn). Our results identify an extended surface spanning both the N-terminal and C-terminal domains of ChvE that is essential for interacting with VirA and that partially overlaps the surface responsible for the interaction of ChvE with a putative ABC sugar transport protein.     

<Emphasis Type="Italic">Larkinella terrae</Emphasis> sp. nov., isolated from soil on Jeju Island,South Korea

A Gram-stain negative, non-motile, rod-shaped, aerobic bacterium, designated 15J8-8^T, was isolated from a soil sample collected on Jeju Island, South Korea, and characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain 15J8-8^T belongs to the family Cytophagaceae and is related to Larkinella bovis M2TB15^T (95.0%), ‘Larkinella harenae’ 15J9-9 (94.5%), Larkinella arboricola Z0532^T (93.2%), and Larkinella insperata LMG 22510^T (93.0%). The DNA G+C content of strain 15J8-8^T was 50.5 mol%. The detection of phosphatidylethanolamine and two unidentified polar lipids as major polar lipids; menaquinone-7 as the predominant quinone; and C_16:1 ω5c, C_16:0 N alcohol, and iso-C_15:0 as the major fatty acids also supported the affiliation of the isolate to the genus Larkinella. Based on its phenotypic properties and phylogenetic distinctiveness, strain 15J8-8^T should be classified in the genus Larkinella as representative of a novel species, for which the name Larkinella terrae sp. nov. is proposed. The type strain is 15J8-8^T (= KCTC 52001^T = JCM 31990^T).