Engineering of Ion Sensing by the Cystathionine ��-Synthase Module of the ABC Transporter OpuA

We have previously shown that the C-terminal cystathionine β-synthase (CBS) domains of the nucleotide-binding domains of the ABC transporter OpuA, in conjunction with an anionic membrane surface function, act as sensor of internal ionic strength (I_in). Here, we show that a surface-exposed cationic region in the CBS module domain is critical for ion sensing. The consecutive substitution of up to five cationic residues led to a gradual decrease of the ionic strength dependence of transport. In fact, a 5-fold mutant was essentially independent of salt in the range from 0 to 250 mm KCl (or NaCl), supplemented to medium of 30 mm potassium phosphate. Importantly, the threshold temperature for transport was lowered by 5–7 °C and the temperature coefficient Q₁₀ was lowered from 8 to ∼1.5 in the 5-fold mutant, indicating that large conformational changes are accompanying the CBS-mediated regulation of transport. Furthermore, by replacing the anionic C-terminal tail residues that extend the CBS module with histidines, the transport of OpuA became pH-dependent, presumably by additional charge interactions of the histidine residues with the membrane. The pH dependence was not observed at high ionic strength. Altogether the analyses of the CBS mutants support the notion that the osmotic regulation of OpuA involves a simple biophysical switching mechanism, in which nonspecific electrostatic interactions of a protein module with the membrane are sufficient to lock the transporter in the inactive state.In their natural habitats microorganisms are often exposed to changes in the concentration of solutes in the environment (1). A sudden increase in the medium osmolality results in loss of water from the cell, loss of turgor, a decrease in cell volume, and an increase in intracellular osmolyte concentration. Osmoregulatory transporters such as OpuA in Lactococcus lactis, ProP in Escherichia coli, and BetP in Corynebacterium glutamicum diminish the consequences of the osmotic stress by mediating the uptake of compatible solutes upon an increase in extracellular osmolality (2–4). For the ATP-binding cassette (ABC)⁵ transporter OpuA, it has been shown that the system, reconstituted in proteoliposomes, is activated by increased concentrations of lumenal ions (increased internal ionic strength) (2, 5, 6). This activation is instantaneous both in vivo and in vitro and only requires threshold levels of ionic osmolytes. Moreover, the ionic threshold for activation is highly dependent of the ionic lipid content (charge density) of the membrane and requires the presence of so-called cystathionine β-synthase (CBS) domains, suggesting that the ionic signal is transduced to the transporter via critical interactions of the protein with membrane lipids.The ABC transporter OpuA consists of two identical nucleotide-binding domains (NBD) fused to CBS domains and two identical substrate-binding domains fused to transmembrane domains. The NBD-CBS and substrate-binding domain-transmembrane domain subunits are named OpuAA and OpuABC, respectively. Two tandem CBS domains are linked to the C-terminal end of the NBD; each domain (CBS1 and CBS2) has a β-α-β-β-α secondary structure (5) (Fig. 1A). The CBS domains are widely distributed in most if not all species of life but their function is largely unknown. Most of the CBS domains are found as tandem repeats but data base searches have also revealed tetra-repeat units (5). The crystal structures of several tandem CBS domains have been elucidated (7–9, 32), and in a number of cases it has been shown that two tandem CBS domains form dimeric structures with a total of four CBS domains per structural module (hereafter referred to as CBS module). The crystal structures of the full-length MgtE Mg²⁺ transporter confirm the dimeric configuration and show that the CBS domains undergo large conformational changes upon Mg²⁺ binding or release (10, 11). In general, ABC transporters are functional as dimers, which implies that two tandem CBS domains are present in the OpuA complex. Preliminary experiments with disulfides engineered at the interface of two tandem CBS domains in OpuA suggest that large structural rearrangements (association-dissociation of the interfaces) play a determining role in the ionic strength-regulated transport. Finally, a subset of CBS-containing proteins has a C-terminal extension, which in OpuA is highly anionic (sequence: ADIPDEDEVEEIEKEEENK) and modulates the ion sensing activity (6).Open in a separate windowFIGURE 1.Domain structure of CBS module of OpuA. A, sequence of tandem CBS domains. The predicted secondary structure is indicated above the sequence. The residues modified in this study are underlined. The amino acid sequence end-points of OpuAΔ61 and OpuAΔ119 are indicated by vertical arrows. B, homology model of tandem CBS domain of OpuA. The CBS domains were individually modeled on the crystal structure of the tandem CBS protein Ta0289 from T. acidophilum (PDB entry 1PVM), using Phyre. Ta0289 was used for the initial modeling, because its primary sequence was more similar to the CBS domains of OpuA than those of the other crystallized CBS proteins. The individual domain models were then assembled with reference to the atomic coordinates of the tandem CBS domains of IMPDH from Streptococcus pyogenes (PDB entry 1ZFJ) to form the tandem CBS pair, using PyMOL (DeLano). The positions of the (substituted) cationic residues are indicated.In this study, we have engineered the surface-exposed cationic residues of the CBS module and the C-terminal anionic tail of OpuA (Fig. 1B). The ionic strength and lipid dependence of the OpuA mutants were determined in vivo and in vitro. We show that substitution of five cationic residues for neutral amino acids is sufficient to inactivate the ionic strength sensor and convert OpuA into a constitutively active transporter. Moreover, by substituting six anionic plus four neutral residues of the C-terminal anionic tail for histidines, the transport reaction becomes strongly pH-dependent.     

Intracellular β-Carbonic Anhydrase of the Unicellular Green Alga Coccomyxa : Cloning of the cDNA and Characterization of the Functional Enzyme Overexpressed in Escherichia coli

Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO₂, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular β-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to β-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO₂-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic.     

Validity of the bereavement exclusion to major depression: does the empirical evidence support the proposal to eliminate the exclusion in DSM-5?

The DSM-IV major depression "bereavement exclusion" (BE), which recognizes that depressive symptoms are sometimes normal in recently bereaved individuals, is proposed for elimination in DSM-5. Evidence cited for the BE's invalidity comes from two 2007 reviews purporting to show that bereavement-related depression is similar to other depression across various validators, and a 2010 review of subsequent research. We examined whether the 2007 and 2010 reviews and subsequent relevant literature support the BE's invalidity. Findings were: a) studies included in the 2007 reviews sampled bereavement-related depression groups most of whom were not BE-excluded, making them irrelevant for evaluating BE validity; b) three subsequent studies cited by the 2010 review as supporting BE elimination did examine BE-excluded cases but were in fact inconclusive; and c) two more recent articles comparing recurrence of BE-excluded and other major depressive disorder cases both support the BE's validity. We conclude that the claimed evidence for the BE's invalidity does not exist. The evidence in fact supports the BE's validity and its retention in DSM-5 to prevent false positive diagnoses. We suggest some improvements to increase validity and mitigate risk of false negatives.     

Inhibition of the Water Oxidizing Complex of Photosystem II and the Reoxidation of the Quinone Acceptor QA ? by Pb2+

The action of the environmental toxic Pb²⁺ on photosynthetic electron transport was studied in thylakoid membranes isolated from spinach leaves. Fluorescence and thermoluminescence techniques were performed in order to determine the mode of Pb²⁺ action in photosystem II (PSII). The invariance of fluorescence characteristics of chlorophyll a (Chl a) and magnesium tetraphenylporphyrin (MgTPP), a molecule structurally analogous to Chl a, in the presence of Pb²⁺ confirms that Pb cation does not interact directly with chlorophyll molecules in PSII. The results show that Pb interacts with the water oxidation complex thus perturbing charge recombination between the quinone acceptors of PSII and the S₂ state of the Mn₄Ca cluster. Electron transfer between the quinone acceptors Q_A and Q_B is also greatly retarded in the presence of Pb²⁺. This is proposed to be owing to a transmembrane modification of the acceptor side of the photosystem.     

Analyzing the Interaction of RseA and RseB, the Two Negative Regulators of the ��E Envelope Stress Response, Using a Combined Bioinformatic and Experimental Strategy

The Escherichia coli envelope stress response is controlled by the alternative sigma factor, σ^E, and is induced when unfolded outer membrane proteins accumulate in the periplasm. The response is initiated by sequential cleavage of the membrane-spanning antisigma factor, RseA. RseB is an important negative regulator of envelope stress response that exerts its negative effects onσ^E activity through its binding to RseA. In this study, we analyze the interaction between RseA and RseB. We found that tight binding of RseB to RseA required intact RseB. Using programs that performed global and local sequence alignment of RseB and RseA, we found regions of high similarity and performed alanine substitution mutagenesis to test the hypothesis that these regions were functionally important. This protocol is based on the hypothesis that functionally dependent regions of two proteins co-evolve and therefore are likely to be sequentially conserved. This procedure allowed us to identify both an N-terminal and C-terminal region in RseB important for binding to RseA. We extensively analyzed the C-terminal region, which aligns with a region of RseA coincident with the major RseB binding determinant in RseA. Both allele-specific suppression analysis and cysteine-mediated disulfide bond formation indicated that this C-terminal region of similarity of RseA and RseB identifies a contact site between the two proteins. We suggest a similar protocol can be successfully applied to pairs of non-homologous but functionally linked proteins to find specific regions of the protein sequences that are important for establishing functional linkage.The Escherichia coli σ^E-mediated envelope stress response is the major pathway to ensure homeostasis in the envelope compartment of the cell (1-3). σ^E regulon members encode periplasmic chaperones and proteases, the machinery for inserting β-barrel proteins into the outer membrane and components controlling the synthesis and assembly of LPS (4-6). This pathway is highly conserved among γ-proteobacteria (6).The σ^E response is initiated when periplasmic protein folding and assembly is compromised (7-9). During steady state growth, σ^E is inhibited by its antisigma factor, RseA, a membrane-spanning protein whose cytoplasmic domain binds to σ^E with picomolar affinity (10-13). Accumulation of unassembled porin monomers serves as a signal to activate the DegS protease to cleave RseA in its periplasmic domain (14, 15). This initiates a proteolytic cascade in which RseP cleaves periplasmically truncated RseA near or within the cytoplasmic membrane to release the RseA_cytoplasmic-σ^E complex, and cytoplasmic ATP-dependent proteases complete the degradation of RseA thereby releasing active σ^E (16-19).RseB, a second negative regulator of the envelope stress response (11, 20, 21), binds to the periplasmic domain of RseA with nanomolar affinity. RseB is an important regulator of the response (2, 22, 23). It prevents RseP from degrading intact RseA, thereby ensuring that proteolysis is initiated only when the DegS protease is activated by a stress signal (21). Additionally, RseB prevents activated DegS from cleaving RseA, suggesting that interaction of RseB with RseA must be altered before the signal transduction cascade is activated (23).The goal of the present studies was to explore how RseB binds to RseA. The interaction partner of RseB is the unstructured periplasmic domain of RseA (RseA-peri). Within RseA-peri, amino acids ∼169-186 constitute a major binding determinant to RseB (23, 24). This peptide alone binds RseB with 6 μm affinity, and deleting this region abrogates binding to RseB (23). Additional regions of RseA-peri also contribute to RseB binding, as intact RseA-peri binds with 20 nm affinity to RseB (23). Much less is known about the regions of RseB required for interaction with RseA. RseB is homodimeric two-domain protein, whose large N-terminal domain shares structural homology with LolA, a protein that transports lipoproteins to outer membrane (24, 25). The smaller C-terminal domain is connected to the N-terminal domain by a linker, and the two domains share a large interface, which may facilitate interdomain signaling. Glutaraldehyde cross-linking studies indicate that the C-terminal domain interacts with RseA, but the regions of interaction were not identified (25).In the present report, we study the interaction of RseB and RseA. We establish that both domains of RseB interact with RseA-peri. Using a global sequence alignment, we discovered several regions in RseA and RseB that had high sequence similarity, despite the low overall sequence similarity between these two proteins, a finding that was independently confirmed by a local sequence similarity algorithm. This suggested that these regions were functionally dependent, and we performed a set of mutagenesis experiments designed to test this idea. Our studies of the binding properties of these mutants revealed that regions in both the N terminus and C terminus of RseB modulate interaction with RseA. Moreover, genetic suppression analysis and cysteine-mediated disulfide bond formation suggest that the region of RseA/B with highest similarity (RseA residues 165-191 (major binding determinant in RseA) and RseB residues 233-258) are interacting partners.     

Catalytic Role of the Metal Ion in the Metallo-��-lactamase GOB

Metallo-β-lactamases (MβLs) stand as one of the main mechanisms of bacterial resistance toward carbapenems. The rational design of an inhibitor for MβLs has been limited by an incomplete knowledge of their catalytic mechanism and by the structural diversity of their active sites. Here we show that the MβL GOB from Elizabethkingia meningoseptica is active as a monometallic enzyme by using different divalent transition metal ions as surrogates of the native Zn(II) ion. Of the metal derivatives in which Zn(II) is replaced, Co(II) and Cd(II) give rise to the most active enzymes and are shown to occupy the same binding site as the native ion. However, Zn(II) is the only metal ion capable of stabilizing an anionic intermediate that accumulates during nitrocefin hydrolysis, in which the C–N bond has already been cleaved. This finding demonstrates that the catalytic role of the metal ion in GOB is to stabilize the formation of this intermediate prior to nitrogen protonation. This role may be general to all MβLs, whereas nucleophile activation by a Zn(II) ion is not a conserved mechanistic feature.     

The First Bite— Profiling the Predatosome in the Bacterial Pathogen Bdellovibrio

Bdellovibrio bacteriovorus is a Gram-negative bacterium that is a pathogen of other Gram-negative bacteria, including many bacteria which are pathogens of humans, animals and plants. As such Bdellovibrio has potential as a biocontrol agent, or living antibiotic. B. bacteriovorus HD100 has a large genome and it is not yet known which of it encodes the molecular machinery and genetic control of predatory processes. We have tried to fill this knowledge-gap using mixtures of predator and prey mRNAs to monitor changes in Bdellovibrio gene expression at a timepoint of early-stage prey infection and prey killing in comparison to control cultures of predator and prey alone and also in comparison to Bdellovibrio growing axenically (in a prey-or host independent “HI” manner) on artificial media containing peptone and tryptone. From this we have highlighted genes of the early predatosome with predicted roles in prey killing and digestion and have gained insights into possible regulatory mechanisms as Bdellovibrio enter and establish within the prey bdelloplast. Approximately seven percent of all Bdellovibrio genes were significantly up-regulated at 30 minutes of infection- but not in HI growth- implicating the role of these genes in prey digestion. Five percent were down-regulated significantly, implicating their role in free-swimming, attack-phase physiology. This study gives the first post- genomic insight into the predatory process and reveals some of the important genes that Bdellovibrio expresses inside the prey bacterium during the initial attack.     

α-Synemin localizes to the M-band of the sarcomere through interaction with the M10 region of titin

α-Synemin contains a unique 312 amino acid insert near the end of its C-terminal tail. Therefore we set out to determine if the insert is a site of protein–protein interaction that regulates the sub-cellular localization of this large isoform of synemin. Yeast-two hybrid analysis indicated that this region is a binding site for the M10 region of titin. This was confirmed with GST pull-down assays. Co-immunoprecipitation of endogenous proteins indicated close association of the two proteins in vivo and immunostaining of cardiomyocytes demonstrated co-localization of the proteins at the M-band of the sarcomere.     

Different contributions of the indirect effects of γ-rays on the cytotoxicity in M10 and XRCC4 transfected M10 cells

The protective effects of dimethyl sulfoxide (DMSO) against cell killing by ¹³⁷Cs γ-rays were investigated in XRCC4-deficient cell line M10, XRCC4-complemented M10 and the parental mouse leukemia cell line L5178Y. Cell survival was determined by the colony-forming ability. M10 cells were more sensitive to γ-ray-induced cell death than L5178Y and complemented M10 cells. Cell survival was increased in both M10 and L5178Y in the presence of DMSO. However, estimation of the DMSO-protectable fraction revealed a smaller protectable fraction for M10 cells than for L5178Y cells, indicating that indirect effects contributed in a smaller extent to the cytotoxicity in M10 than that in L5178Y. This effect is due to XRCC4 deficiency, since transfection of XRCC4 cDNA into M10 cells restored the radioprotective effects of DMSO to the level seen in L5178Y. In M10 cells, the killing effects of high LET radiation (Auger electrons from ¹²⁵I-antipyrine, carbon ions with an LET of 166 keV μm⁻¹) were similar to those of low LET radiation (¹³⁷Cs γ-rays, characteristic X-rays from ¹²⁵I-bovine serum albumin). We discuss that lethal lesions produced by indirect actions in L5178Y and XRCC4-complemented M10 cells may differ, at least in part, from DNA double-strand breaks repairable by non-homologous end joining.     

Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4 Gene

cDNA corresponding to the GA4 gene of Arabidopsis thaliana L. (Heynh.) was expressed in Escherichia coli, from which cell lysates converted [¹⁴C]gibberellin (GA)₉ and [¹⁴C]GA₂₀ to radiolabeled GA₄ and GA₁, respectively, thereby confirming that GA4 encodes a GA 3β-hydroxylase. GA₉ was the preferred substrate, with a Michaelis value of 1 μm compared with 15 μm for GA₂₀. Hydroxylation of these GAs was regiospecific, with no indication of 2β-hydroxylation or 2,3-desaturation. The capacity of the recombinant enzyme to hydroxylate a range of other GA substrates was investigated. In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs. Therefore, no activity was detected using GA₁₂-aldehyde, GA₁₂, GA₁₉, GA₂₅, GA₅₃, or GA₄₄ as the open lactone (20-hydroxy-GA₅₃), whereas GA₁₅, GA₂₄, and GA₄₄ were hydroxylated to GA₃₇, GA₃₆, and GA₃₈, respectively. The open lactone of GA₁₅ (20-hydroxy-GA₁₂) was hydroxylated but less efficiently than GA₁₅. In contrast to the free acid, GA₂₅ 19,20-anhydride was 3β-hydroxylated to give GA₁₃. 2,3-Didehydro-GA₉ and GA₅ were converted by recombinant GA4 to the corresponding epoxides 2,3-oxido-GA₉ and GA₆.Dwarf mutants with reduced biosynthesis of the GA plant hormones have been valuable tools in studies of the function of these compounds (Ross, 1994). In Arabidopsis thaliana, mutations at six loci (GA1-GA6) that result in reduced GA biosynthesis have been identified (Koorneef and van der Veen, 1980; Sponsel et al., 1997), and three of these loci have recently been cloned. The GA1 locus was isolated by genomic subtraction (Sun et al., 1992) and shown by heterologous expression in Escherichia coli to encode the enzyme that cyclizes geranylgeranyl diphosphate to copalyl diphosphate (Sun and Kamiya, 1994). This enzyme was formerly referred to as ent-kaurene synthase A but has been renamed copalyl diphosphate synthase (Hedden and Kamiya, 1997; MacMillan, 1997). The GA5 locus was shown to correspond to one of the GA 20-oxidase genes (Xu et al., 1995), the products of which catalyze the conversion of GA₁₂ to GA₉ and GA₅₃ to GA₂₀ (Phillips et al., 1995; Xu et al., 1995). GA 20-oxidases are 2-oxoglutarate-dependent dioxygenases that are encoded by small multigene families, members of which are differentially expressed in plant tissues (Phillips et al., 1995; Garcia-Martinez et al., 1997).The GA4 locus was isolated by T-DNA tagging and, on the basis of the derived amino acid sequence, was also shown to encode a dioxygenase (Chiang et al., 1995). Several lines of evidence indicate that the GA4 gene encodes a GA 3β-hydroxylase. Shoots of a ga4 mutant, all alleles of which are semidwarf, contained reduced concentrations of the 3β-hydroxy GAs GA₁, GA₄, and GA₈ compared with the Landsberg erecta wild type, whereas levels of immediate precursors to these GAs were elevated (Talon et al., 1990). Furthermore, metabolism of [¹³C]GA₂₀ to [¹³C]GA₁ was substantially less in the mutant than in the wild type (Kobayashi et al., 1994). In the present paper we confirm by functional expression of its cDNA in E. coli that GA4 encodes a GA 3β-hydroxylase. In addition, we determine the substrate specificity of recombinant GA4 using a number of C₂₀- and C₁₉-GAs and show by kinetic analysis that the enzyme has a higher affinity for GA₉ than for GA₂₀, which is consistent with the non-13-hydroxylation pathway predominating in Arabidopsis (Talon et al., 1990).     

Probing the Conformation of the Fibronectin III1�C2 Domain by Fluorescence Resonance Energy Transfer

Fibronectin (FN) matrix is crucial for cell and tissue functions during embryonic development, wound healing, and oncogenesis. Assembly of FN matrix fibrils requires FN domains that mediate interactions with integrin receptors and with other FN molecules. In addition, regulation of FN matrix assembly depends on the first two FN type III modules, III₁ and III₂, which harbor FN-binding sites. We propose that interactions between these two modules sequester FN-binding sites in soluble FN and that these sites become exposed by FN conformational changes during assembly. To test the idea that III_1–2 has a compact conformation, we constructed CIIIY, a conformational sensor of III_1–2 based on fluorescent resonance energy transfer between cyan and yellow fluorescent proteins conjugated at its N and C termini. We demonstrate energy transfer in CIIIY and show that fluorescent resonance energy transfer was eliminated by proteolysis and by treatment with mild denaturants that disrupted intramolecular interactions between the two modules. We also show that mutations of key charged residues resulted in conformational changes that exposed binding sites for the N-terminal 70-kDa FN fragment. Collectively, these results support a conformation-dependent mechanism for the regulation of FN matrix assembly by III_1–2.Fibronectin (FN)³ is a 500-kDa modular dimeric protein and a major component of the extracellular matrix. It exists in the blood and other body fluids as a soluble compact molecule and undergoes cell-mediated assembly to form an insoluble three-dimensional fibrillar matrix (reviewed in Ref. 1). The process of FN matrix assembly has been implicated in embryonic development, wound healing, and cancer (2–4). FN is composed of type I–III modules, and sets of these modules comprise binding domains for cells and for other extracellular matrix components (see Fig. 1A). Three of these binding domains are essential for matrix assembly (1). Integrin receptor interactions with the cell-binding domain tether disulfide-bonded FN dimers to the cell surface, where FN-FN interactions involving the N-terminal assembly domain form dimers into fibrils. In addition to these essential domains, other FN-binding sites have been implicated in assembly. In particular, the III_1–2 FN-binding domain plays a regulatory role in matrix assembly. Within this domain reside a cryptic FN-binding site in III₁ and a site available for FN binding in the native form of III₂ (5–8). Recombinant FN lacking III₁ is assembled into a matrix at wild-type levels, but that lacking the III_1–2 domain results in short immature FN fibrils (8). Peptides derived from the III_1–2 domain or antibodies against III_1–2 block matrix assembly by cultured cells (9–11). Furthermore, FN binding to this region is enhanced when FN is mechanically stretched (12). Taken together, these results suggest that conformational changes in the III_1–2 domain may control its interactions during FN assembly.Open in a separate windowFIGURE 1.The FN III_1–2 FRET conformational sensor. A, representation of the domain structure of FN and major interaction sites. FN is composed of repeating modules that form binding domains for other FN molecules, cell receptors, and other extracellular matrix components as indicated. The first two type III modules III₁ and III₂ (black), have FN-binding sites and regulate FN matrix assembly. The N-terminal 70-kDa region contains a matrix assembly domain with FN-binding activity. The cell-binding domain (cell), the heparin-binding domain (heparin), the dimerization site (SS), and the alternatively spliced type IIIA (A), IIIB (B), and variable regions (V) are indicated. 70kD, N-terminal 70-kDa FN fragment. B, schematic of proposed model of III_1–2 domain conformation. Panel i, in solution, the FN-binding sites in III₁ and III₂ (hatched areas) are sequestered through domain orientations that are facilitated by the linker between modules (thin line). Panel ii, binding sites are exposed through conformational changes resulting from cell-mediated extension of FN (arrows). The length of the linker and the height and width of the modules are drawn to scale for a linear peptide and published data on FN type III modules, respectively. C, ribbon diagram representation of CIIIY, a FRET sensor of the model in B (panel i), oriented with N and C termini 50 Å apart. CIIIY consists of the III_1–2 domain with CFP at the N terminus and YFP at the C terminus.To more fully understand the roles of native and cryptic FN-binding sites in matrix assembly, the conformational dynamics of III_1–2 must be characterized. One approach to this problem is to tag III_1–2 with fluorescent probes, which, in conjunction with fluorescent resonance energy transfer (FRET), create a molecular conformational sensor. FRET involves the radiationless transfer of energy from an excited donor fluorophore to an acceptor fluorophore, a process that is very sensitive to the distance between the two fluorophores (13–15). Two fluorescent protein variants, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), are highly related to green fluorescent protein (GFP). Because the emission spectrum of CFP is well matched to the excitation spectrum of YFP, these two fluorophores have been widely used as a donor-acceptor pair in FRET studies (13–15).In this study, we describe a FRET conformational sensor designed to test the idea that intramolecular interactions between III₁ and III₂ sequester key FN-binding and assembly sites. We show that III_1–2 with CFP and YFP fused to the N and C termini, respectively, displays a clear FRET signal, indicating that the attached fluorescent proteins and thus the ends of III_1–2 are in close proximity. FRET data from III_1–2 mutants support the presence of a stabilizing intermodule salt bridge that regulates FN-binding activity.     

Reaching the Ball or Missing the Flight? Collective Dispersal in the Two-Spotted Spider Mite Tetranychus urticae

The two-spotted spider mite is a worldwide phytophagous pest displaying a peculiar dispersal. At high density, when plants are exhausted, individuals gather at the plant apex to form a collective silk-ball. This structure can be dispersed by wind or phoresy. Individuals initiating the ball are enclosed in the centre and have a high risk to die. For the first time, the ultimate and proximate mechanisms leading to this group dispersal are examined. To explore if a particular mite genotype was involved in the ball formation, plants were infested with individuals of different genetic background. After the silk-ball formation, the mites in the ball and those remaining on the plant were collected and genotyped. The balls were harvested after 4h and 24h to determine the role of timing between the formation and dispersal on the mortality of mites. Mites do not segregate according to their degree of relatedness, stage, or sex. Mites parallel humans using public transportation: they climb up in the ball whatever their genetic background. Silk-balls composed of unrelated individuals may help avoiding inbreeding when colonizing a new plant. Our results also emphasize the importance of an adequate timing for efficient dispersal between the time spent between ball formation and dispersal.     

Identification of the Gene Encoding the Tryptophan Synthase β-Subunit from Chlamydomonas reinhardtii

We report the isolation of a Chlamydomonas reinhardtii cDNA that encodes the β-subunit of tryptophan synthase (TSB). This cDNA was cloned by functional complementation of a trp-operon-deleted strain of Escherichia coli. Hybridization analysis indicated that the gene exists in a single copy. The predicted amino acid sequence showed the greatest identity to TSB polypeptides from other photosynthetic organisms. With the goal of identifying mutations in the gene encoding this enzyme, we isolated 11 recessive and 1 dominant single-gene mutation that conferred resistance to 5-fluoroindole. These mutations fell into three complementation groups, MAA2, MAA7, and TAR1. In vitro assays showed that mutations at each of these loci affected TSB activity. Restriction fragment-length polymorphism analysis suggested that MAA7 encodes TSB. MAA2 and TAR1 may act to regulate the activity of MAA7 or its protein product.     

How unique is the tiger beetle fauna (Coleoptera,Cicindelidae) of the Balkan Peninsula?

The tiger beetle fauna of the Balkan Peninsula is one of the richest in Europe and includes 19 species or 41% of the European tiger beetle fauna. Assembled by their biogeographical origins, the Balkan tiger beetle species fall into 14 different groups that include, Mediterranean, Middle Oriental, Central Asiatic, Euro-Siberian, South and East European, Pannonian-Sarmatian, West Palaearctic, Turano-European and Afrotropico Indo-Mediterranean species. The Mediterranean Sclerophyl and the Pontian Steppe are the Balkan biogeographical provinces with the highest species richness, while the Balkan Highlands has the lowest Cicindelidae diversity. Most species are restricted to single habitat types in lowland areas of the Balkan Peninsula and only Calomera aulica aulica and Calomera littoralis nemoralis occur in respectively 3 and 4 different types of habitat. About 60% of all Balkan Cicindelidae species are found in habitats potentially endangered by human activity.     

Interleukin–10 promoter haplotypes are differently distributed in the Brazilian versus the Dutch population

The frequency of five different single nucleotide polymorphisms of the promoter interleukin-10 (IL-10) gene (-3575, -2849, 2763, -1082, -819) was compared between two healthy populations, one originating from the Netherlands and one from Rio de Janeiro, Brazil. A total of 321 Caucasian Dutch individuals and 293 Brazilians, grouped as Afro-Brazilians and Euro-Brazilians, were genotyped using PCR-RFLP. The frequencies of the genotypes in the Brazilian population were different (P<0.05) from the frequencies in the Dutch population in all but one (-2763) genotype. The comparison of genotype frequencies between Afro- and Euro-Brazilians did not demonstrate any differences. The haplotype combination of the most-distant three polymorphisms showed strong linkage disequilibrium. All eight possible combinations were observed in Brazilians, but only seven in Dutch Caucasians. The haplotype frequencies were also significantly different between Brazilians when compared with Dutch and also between Euro-Brazilians and Dutch. No differences were observed in haplotype frequencies between Afro-Brazilians and Euro-Brazilians. The -3575T/-2849G/-2763C is more frequent, while the AAA haplotype was much less represented in the Brazilian than in the Dutch population. The haplotype TAC, which was described in African-Americans, was observed only in Brazilians, almost exclusively among those of European origin. The results corroborate the data indicating that the Brazilian population exhibits a genetic admixture of Africans, Europeans, and Amerindians, and the data may serve as a background for clinical and immunological studies involving the IL-10 locus.