Eco-Evolutionary Processes Generating Diversity Among Bottlenose Dolphin, <Emphasis Type="Italic">Tursiops truncatus</Emphasis>, Populations off Baja California,Mexico

For highly mobile species that nevertheless show fine-scale patterns of population genetic structure, the relevant evolutionary mechanisms determining structure remain poorly understood. The bottlenose dolphin (Tursiops truncatus) is one such species, exhibiting complex patterns of genetic structure associated with local habitat dependence in various geographic regions. Here we studied bottlenose dolphin populations in the Gulf of California and Pacific Ocean off Baja California where habitat is highly structured to test associations between ecology, habitat dependence and genetic differentiation. We investigated population structure at a fine geographic scale using both stable isotope analysis (to assess feeding ecology) and molecular genetic markers (to assess population structure). Our results show that there are at least two factors affecting population structure for both genetics and feeding ecology (as indicated by stable isotope profiles). On the one hand there is a signal for the differentiation of individuals by ecotype, one foraging more offshore than the other. At the same time, there is differentiation between the Gulf of California and the west coast of Baja California, meaning that for example, nearshore ecotypes were both genetically and isotopically differentiated either side of the peninsula. We discuss these data in the context of similar studies showing fine-scale population structure for delphinid species in coastal waters, and consider possible evolutionary mechanisms.     

Acyl Enzyme Intermediates in Sortase-Catalyzed Pilus Morphogenesis in Gram-Positive Bacteria

In gram-positive bacteria, covalently linked pilus polymers are assembled by a specific transpeptidase enzyme called pilus-specific sortase. This sortase is postulated to cleave the LPXTG motif of a pilin precursor between threonine and glycine and to form an acyl enzyme intermediate with the substrate. Pilus polymerization is believed to occur through the resolution of this intermediate upon specific nucleophilic attack by the conserved lysine located within the pilin motif of another pilin monomer, which joins two pilins with an isopeptide bond formed between threonine and lysine. Here, we present evidence for sortase reaction intermediates in Corynebacterium diphtheriae. We show that truncated SrtA mutants that are loosely bound to the cytoplasmic membrane form high-molecular-weight complexes with SpaA polymers secreted into the extracellular milieu. These complexes are not formed with SpaA pilin mutants that have alanine substitutions in place of threonine in the LPXTG motif or lysine in the pilin motif. The same phenotype is observed with alanine substitutions of either the conserved cysteine or histidine residue of SrtA known to be required for catalysis. Remarkably, the assembly of SpaA pili, or the formation of intermediates, is abolished with a SrtA mutant missing the membrane-anchoring domain. We infer that pilus polymerization involves the formation of covalent pilin-sortase intermediates, which occurs within a molecular platform on the exoplasmic face of the cytoplasmic membrane that brings together both sortase and its cognate substrates in close proximity to each other, likely surrounding a secretion apparatus. We present electron microscopic data in support of this picture.Adherence to specific host tissue is a key step in bacterial colonization and the establishment of a successful infection by bacterial pathogens. Bacteria express a variety of adhesive cell surface molecules to bind host cells or other substrates in their natural habitat. The proteinaceous filaments known as pili or fimbriae are a clinically important class of these molecules. Both gram-negative and gram-positive bacteria express pili (6, 8). The gram-positive bacterial pili are unique in three respects (12, 25, 31). First, they represent heterodimeric or heterotrimeric protein polymers in which individual pilin subunits are covalently joined to each other (2, 9, 32). Second, the polymer itself is covalently attached to the cell wall (3, 31). Third, unlike pilus assembly in gram-negative bacteria, many of which require chaperones (26), the polymerization of the gram-positive pili and their cell wall attachment require specific transpeptidase enzymes called sortases (31).Mazmanian and colleagues discovered the sortase SrtA as an enzyme that linked the surface protein A of Staphylococcus aureus to its cell wall (15). Genome sequences revealed that sortases are ubiquitously expressed in gram-positive bacteria, including significant pathogens, such as Actinomyces naeslundii, Bacillus cereus, Corynebacterium diphtheriae, Enterococcus faecalis, Streptococcus agalactiae, and Streptococcus pneumoniae (4, 5, 28). Sortases are classified according to their functions and phylogenic relationships (4, 5). The class that closely matches SrtA of S. aureus in structure and function is now called a housekeeping sortase. Its function is to attach numerous surface proteins to the cell wall (16). Common to each of these cell surface proteins is a cell wall sorting signal with an LPXTG motif that is absolutely necessary for cell wall anchoring (18). Elegant genetic, biochemical, and structural work by the Schneewind laboratory illuminated the universal reaction mechanism of protein sorting in the gram-positive cell wall (14). Cell wall anchoring of surface proteins is catalyzed in two steps. In the first step, SrtA cleaves the TG peptide bond of the LPXTG motif of protein A and forms an acyl enzyme intermediate involving the threonine of protein A and the catalytic cysteine of sortase (22, 27, 29). In the second step, the cleaved protein A is transferred to the cell wall when a nucleophile amine from the lipid II precursor attacks and resolves the acyl enzyme intermediate (20, 21, 30). This seminal work formed the basis of our current model of pilus assembly catalyzed by pilus-specific sortases (12).We have used C. diphtheriae as a model for studies of the mechanism of pilus biogenesis. The corynebacterial genome encodes six different sortases (32). We now know that while five of these sortases (SrtA to -E) are devoted to pilus assembly, even the housekeeping sortase, SrtF, is required for efficient attachment of pili to the cell wall (23). Corynebacteria produce three distinct types of heterotrimeric pili, which are encoded by three pilus islands, each encoding three pilins (namely, SpaABC, SpaDEF, and SpaGHI) plus one or two cognate sortases essential for the assembly of the respective pilus (7, 24, 32). In each case, the prototype pilus represents a shaft structure made of a specific major pilin (namely, SpaA, SpaD, and SpaH) (12). Each type of pilus also contains a minor pilin at the tip (SpaC, SpaF, and SpaG) and another minor pilin dispersed along the shaft, as well as at the base of the pilus (SpaB, SpaE, and SpaI) (12). How are these polymers assembled, and how are they attached to the cell wall? All pilin proteins are predicted to contain in their amino termini a hydrophobic signal sequence necessary for export to the exoplasm by the Sec machinery. In addition, like the cell wall-anchored protein A of S. aureus, a cell wall sorting signal including the LPXTG motif is also present at the carboxy terminus of each of the Spa proteins of corynebacteria and other pilus proteins found in different gram-positive organisms (17). It is thus logical to imagine that the pilus-specific sortase utilizes the LPXTG motif for pilus polymerization, its cell wall anchoring, or both. Substantial genetic, biochemical, and ultrastructural analyses have proved this prediction. Consequently, Ton-That and Schneewind proposed a model of pilus assembly which posited that the basic mechanism of catalysis is conserved between cell wall sorting of surface proteins and the assembly of the pilus (31).According to our current working model (Fig. ​(Fig.1A),1A), the prototype SpaA pilus is assembled as follows. SrtA, which is essential and also specific for SpaA pilus formation, captures and cleaves cognate pilins at the LPXTG motif and forms an acyl enzyme intermediate. To form a dimer of SpaA and SpaC, the proposed tip entity, a conserved lysine in the SpaA pilin motif attacks the Cys-Thr bond of the SpaC-SrtA acyl enzyme intermediate. Shaft formation ensues by the cyclic addition of SpaA to the SpaC-SpaA dimer and the SpaC-SpaA_n oligomer formed in the preceding reaction. When a SpaB is attached to the growing pilus terminus by a similar mechanism involving a critical lysine of SpaB, it acts as a switch, terminating pilus polymerization in favor of cell wall anchoring (11). This happens by the classic resolution reaction mentioned above, which involves the lipid II precursor (28), followed by its linkage to the cell wall (11). Alternatively, the SpaB-containing pilus can elongate further by adding a SpaA subunit to SpaB (11). This model explains all the available genetic and biochemical data we have obtained so far in the corynebacterial system, as well as other systems reported by various investigators.Open in a separate windowFIG. 1.(A) Working model of pilus assembly in C. diphtheriae. Spa pilins are synthesized in the cytoplasm and transported across the cytoplasmic membrane by the Sec machinery. The membrane-bound pilus-specific sortase SrtA cleaves the Spa pilins at the LPXTG motif and forms an acyl enzyme intermediate with the substrates. Pilus polymerization occurs when this intermediate is resolved by a nucleophilic attack by the lysine residue within the pilin motif of an adjacent intermediate. Cell wall anchoring terminates pilus polymerization when SpaB is incorporated into the pilus base by the housekeeping sortase, SrtF (see the text for details). (B) Membrane localization of the pilus-specific sortase SrtA. Corynebacteria grown to mid-log phase were separated from the culture medium (M) by centrifugation. The cell wall (W) was removed from its protoplast by muramidase treatment of the cells. The protoplasts were lysed, and membrane (P*) and cytoplasmic (C) compartments were obtained by ultracentrifugation. Protein samples were separated on 4 to 12% Tris-glycine gradient gels and detected by immunoblotting them with the specific antisera α-SrtA, α-SecA, and α-SpaA. The positions of molecular mass markers (kDa) are indicated. WT, wild type.Significantly, there has been no report demonstrating the proposed intermediates of pilus assembly, to our knowledge. The present study was initiated to explore this key element of our model of pilus assembly, as well as the localization of the sortase in the membrane and its organization in the exoplasmic membrane in relation to the cognate pilins and the general secretion machinery.     

Adaptive radiation of venomous marine snail lineages and the accelerated evolution of venom peptide genes

An impressive biodiversity (>10,000 species) of marine snails (suborder Toxoglossa or superfamily Conoidea) have complex venoms, each containing approximately 100 biologically active, disulfide-rich peptides. In the genus Conus, the most intensively investigated toxoglossan lineage (～500 species), a small set of venom gene superfamilies undergo rapid sequence hyperdiversification within their mature toxin regions. Each major lineage of Toxoglossa has its own distinct set of venom gene superfamilies. Two recently identified venom gene superfamilies are expressed in the large Turridae clade, but not in Conus. Thus, as major venomous molluscan clades expand, a small set of lineage-specific venom gene superfamilies undergo accelerated evolution. The juxtaposition of extremely conserved signal sequences with hypervariable mature peptide regions is unprecedented and raises the possibility that in these gene superfamilies, the signal sequences are conserved as a result of an essential role they play in enabling rapid sequence evolution of the region of the gene that encodes the active toxin.     

Phytomonas: transport of amino acids, hexoses and polyamines

Phytomonas cells (Phytomonas Jma) isolated from the latex of Jatropha macrantha were assayed for amino acid, hexose and polyamine transport. Results showed high transport rates for glucose and fructose (193 and 128 pmol min(-1) 10(-7) cells, respectively) and lower, but significant rates, for proline, arginine, cysteine and glutamate (between 1.7 and 5.8 pmol min(-1) 10(-7) cells). Minor transport activities were observed for serine, glycine and aspartate (<1 pmol min(-1) 10(-7) cells). Amino acid transport processes do not seem to be regulated by starvation or during the growth phases. Polyamine transport was also evaluated showing a clear preference for spermidine over putrescine (3.4 and 0.4 pmol min(-1) 10(-7) cells, respectively). This work represents the first report on metabolite transport in phytomonads.     

Mutations in Interaction Surfaces Differentially Impact E. coli Hfq Association with Small RNAs and Their mRNA Targets

The RNA chaperone protein Hfq is required for the function of all small RNAs (sRNAs) that regulate mRNA stability or translation by limited base pairing in Escherichia coli. While there have been numerous in vitro studies to characterize Hfq activity and the importance of specific residues, there has been only limited characterization of Hfq mutants in vivo. Here, we use a set of reporters as well as co-immunoprecipitation to examine 14 Hfq mutants expressed from the E. coli chromosome. The majority of the proximal face residues, as expected, were important for the function of sRNAs. The failure of sRNAs to regulate target mRNAs in these mutants can be explained by reduced sRNA accumulation. Two of the proximal mutants, D9A and F39A, acted differently from the others in that they had mixed effects on different sRNA/mRNA pairs and, in the case of F39A, showed differential sRNA accumulation. Mutations of charged residues at the rim of Hfq interfered with positive regulation and gave mixed effects for negative regulation. Some, but not all, sRNAs accumulated to lower levels in rim mutants, suggesting qualitative differences in how individual sRNAs are affected by Hfq. The distal face mutants were expected to disrupt binding of ARN motifs found in mRNAs. They were more defective for positive regulation than negative regulation at low mRNA expression, but the defects could be suppressed by higher levels of mRNA expression. We discuss the implications of these observations for Hfq binding to RNA and mechanisms of action.     

Bacillus anthracis IsdG, a heme-degrading monooxygenase

Bacillus anthracis, the causative agent of anthrax, utilizes hemin and hemoglobin for growth in culture, suggesting that these host molecules serve as sources for the nutrient iron during bacterial infection. Bioinformatic analyses of the B. anthracis genome revealed genes with similarity to the iron-regulated surface determinant (isd) system responsible for heme uptake in Staphylococcus aureus. We show that the protein product of one of these genes, isdG, binds hemin in a manner resembling the heme binding of known heme oxygenases. Formation of IsdG:hemin complexes in the presence of a suitable electron donor, e.g., ascorbate or cytochrome P450 reductase, promotes catalytic degradation of hemin to biliverdin with concomitant release of iron. IsdG is required for B. anthracis utilization of hemin as a sole iron source, and it is also necessary for bacterial protection against heme-mediated toxicity. These data suggest that IsdG functions as a heme-degrading monooxygenase in B. anthracis.     

Expression of the maize ZmGF14-6 gene in rice confers tolerance to drought stress while enhancing susceptibility to pathogen infection

14-3-3 proteins are found in all eukaryotes where they act as regulators of diverse signalling pathways associated with a wide range of biological processes. In this study the functional characterization of the ZmGF14-6 gene encoding a maize 14-3-3 protein is reported. Gene expression analyses indicated that ZmGF14-6 is up-regulated by fungal infection and salt treatment in maize plants, whereas its expression is down-regulated by drought stress. It is reported that rice plants constitutively expressing ZmGF14-6 displayed enhanced tolerance to drought stress which was accompanied by a stronger induction of drought-associated rice genes. However, rice plants expressing ZmGF14-6 either in a constitutive or under a pathogen-inducible regime showed a higher susceptibility to infection by the fungal pathogens Fusarium verticillioides and Magnaporthe oryzae. Under infection conditions, a lower intensity in the expression of defence-related genes occurred in ZmGF14-6 rice plants. These findings support that ZmGF14-6 positively regulates drought tolerance in transgenic rice while negatively modulating the plant defence response to pathogen infection. Transient expression assays of fluorescently labelled ZmGF14-6 protein in onion epidermal cells revealed a widespread distribution of ZmGF14-6 in the cytoplasm and nucleus. Additionally, colocalization experiments of fluorescently labelled ZmGF14-6 with organelle markers, in combination with cell labelling with the endocytic tracer FM4-64, revealed a subcellular localization of ZmGF14-6 in the early endosomes. Taken together, these results improve our understanding of the role of ZmGF14-6 in stress signalling pathways, while indicating that ZmGF14-6 inversely regulates the plant response to biotic and abiotic stresses.     

Incomplete chromatin condensation in enlarged rat myelocytic leukemia cells

The distinguishable morphologic features of nuclei of acute myelogenous leukemia cells with enlarged size and finely distributed nuclear chromatin indicate incomplete chromosome condensation that can be related to elevated gene expression. To confirm this, interphase chromosome structures were studied in exponentially growing rat myelomonocytic leukemia 1 cells isolated at the University of Debrecen (My1/De cells). This cell line was established from primary rat leukemia chemically induced by 7,12-dimethylbenz[a]anthracene treatment. The enlarged nuclei of My1/De cells allowed improved fluorescent visualization of chromosomal structures. Increased resolution revealed major interphase intermediates consisting of (1) veil-like chromatin, (2) chromatin ribbon, (3) chromatin funnel, (4) chromatin bodies, (5) elongated prechromosomes, (6) seal-ring, spiral shaped, and circular chromosomal subunits, (7) elongated, bent, u- and v-shaped prechromosomes, and (8) metaphase chromosomes. Results confirmed the existence of the chromatin funnel, the first visible interphase chromosome generated by the supercoiling of the chromatin ribbon. Other intermediates not seen previously included the spiral subunits that are involved in the chromonemic folding of metaphase chromosomes. The existence of spiral subunits favors the helical coil model of chromosome condensation. Incomplete chromatin condensation in leukemia cells throughout the cell cycle is an indication of euchromatization contributing to enhanced gene expression and is regarded as a leukemic factor.