Structural and functional divergence of the newly identified GtrIc from its Gtr family of conserved Shigella flexneri serotype-converting glucosyltransferases

Abstract

Glucosyltransferases (Gtrs) and O-acetyltransferase (Oac) are integral membrane proteins embedded within the cytoplasmic membrane of Shigella flexneri. Gtrs and Oac are responsible for unidirectional host serotype conversion by altering the epitopic properties of the bacterial surface lipopolysaccharide (LPS) O-antigen. In this study, we present the membrane topology of a recently recognized Gtr, GtrIc, which is known to mediate S. flenxeri serotype switching from 1a to 1c. The GtrIc topology is shown to deviate from those typically seen in S. flexneri Gtrs. GtrIc has 11 hydrophilic loops, 10 transmembrane helices, a double intramembrane dipping loop 5, and a cytoplasmic N- and C-terminus. Along with a unique membrane topology, the identification of non-critical Gtr-conserved peptide motifs within large periplasmic loops (N-terminal D/ExD/E and C-terminal KK), which have previously been proven essential for the activity of other Gtrs, challenge current opinions of a similar mechanism for enzyme function between members of the S. flexneri Gtr family.     

Spermiogenesis and the spermatozoon ultrastructure of Robphildollfusium fractum (Digenea: Gyliauchenidae), an intestinal parasite of Sarpa salpa (Pisces: Teleostei)

Spermiogenesis in Robphildollfusium fractum begins with the formation of a differentiation zone containing: two centrioles, each bearing striated rootlets, nucleus, several mitochondria and an intercentriolar body constituted by seven electron-dense layers. The two centrioles originate two free flagella growing orthogonally to the median cytoplasmic process. Later, the free flagella rotate and undergo proximodistal fusion with the median cytoplasmic process. Nuclear and mitochondrial migrations occur before this proximodistal fusion. Finally, the young spermatozoon detaches from the residual cytoplasm after the constriction of the ring of arched membranes. The spermatozoon of R. fractum exhibits two axonemes of different length of the 9 + ‘1’ trepaxonematan pattern, nucleus, two mitochondria, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. Additionally, a shorter axoneme, which does not reach the nuclear region, the presence of an electron-dense material in the anterior spermatozoon extremity and the morphologies of both spermatozoon extremities characterize the mature sperm of R. fractum.     

A new species of the Rhinella margaritifera species group (Anura,Bufonidae) from the montane forest of the Selva Central,Peru

We describe a new species of the bufonid toad genus Rhinella from transition montane forest of the buffer zones of the Yanachaga-Chemillén National Park and the Pui Pui Protected Forest (eastern slopes of Andes, Selva Central, Peru). The new species belongs to the Rhinella margaritifera species group (confirmed by mtDNA data) and differs from all its members by the absence of tympanic membrane and tympanic annulus. It is characterized by medium size (SVL 57.5–65.5 mm, n = 5), moderately developed cranial crests, absence of neural crest of vertebrae, absence of bone protrusion at angle of jaw, presence of lateral rows of enlarged tubercles, and absence of subgular vocal sac and vocal slits in males. In addition, based on the molecular phylogenetic analyses of selected Rhinella species we propose the monophylum containing R. chavin, R. festae, R. macrorhina, R. manu, R. nesiotes, R. rostrata, and R. yanachaga as a new species group under the name Rhinella festae species group.     

PvD1 defensin,a plant antimicrobial peptide with inhibitory activity against Leishmania amazonensis

Plant defensins are small cysteine-rich peptides and exhibit antimicrobial activity against a variety of both plant and human pathogens. Despite the broad inhibitory activity that plant defensins exhibit against different micro-organisms, little is known about their activity against protozoa. In a previous study, we isolated a plant defensin named PvD₁ from Phaseolus vulgaris (cv. Pérola) seeds, which was seen to be deleterious against different yeast cells and filamentous fungi. It exerted its effects by causing an increase in the endogenous production of ROS (reactive oxygen species) and NO (nitric oxide), plasma membrane permeabilization and the inhibition of medium acidification. In the present study, we investigated whether PvD₁ could act against the protozoan Leishmania amazonensis. Our results show that, besides inhibiting the proliferation of L. amazonensis promastigotes, the PvD₁ defensin was able to cause cytoplasmic fragmentation, formation of multiple cytoplasmic vacuoles and membrane permeabilization in the cells of this organism. Furthermore, we show, for the first time, that PvD₁ defensin was located within the L. amazonensis cells, suggesting the existence of a possible intracellular target.     

Distribution of PLD and FagA,B, C and D genes in Corynebacterium pseudotuberculosis isolates from sheep and goats with caseus lymphadenitis

Caseous lymphadenits (CL) is a chronic and subclinical disease that affects goats and sheep and, consequently, causes economic losses, especially to small producers. The purpose of this study, through use of Polymerase Chain Reaction (PCR), was to verify the presence of virulence genes of phospholipase D (PLD), integral membrane protein (FagA), iron enterobactin transporter (FagB), ATP binding cytoplasmic membrane protein (FagC) and iron siderophore binding protein (FagD) in 168 isolates of C. pseudotuberculosis obtained from cases of caseous lymphadenitis in goats and sheep. FagA, FagB and PLD genes were detected in all 145 strains isolated from abscesses in superficial lymph nodes and in 23 strains isolated from viscera. The FagC gene was positive in 167 (99.40%) isolates. The FagD gene was detected in 160 (95.23%) isolates. All virulence factors analyzed were found more frequently among isolates collected in the viscera of animals with CL, indicating a multifactorial nature, as well as variations, in the invasive potential of C. pseudotuberculosis strains.     

Identification of an endocytosis motif in an intracellular loop of Wntless protein, essential for its recycling and the control of Wnt protein signaling

The secretion of Wnt signaling proteins is dependent upon the transmembrane sorting receptor, Wntless (Wls), which recycles between the trans-Golgi network and the cell surface. Loss of Wls results in impairment of Wnt secretion and defects in development and homeostasis in Drosophila, Caenorhabditis elegans, and the mouse. The sorting signals for the internalization and trafficking of Wls have not been defined. Here, we demonstrate that Wls internalization requires clathrin and dynamin I, components of the clathrin-mediated endocytosis pathway. Moreover, we have identified a conserved YXXφ endocytosis motif in the third intracellular loop of the multipass membrane protein Wls. Mutation of the tyrosine-based motif YEGL to AEGL (Y425A) resulted in the accumulation of human mutant Wls on the cell surface of transfected HeLa cells. The cell surface accumulation of Wls^AEGL was rescued by the insertion of a classical YXXφ motif in the cytoplasmic tail. Significantly, a Drosophila Wls^AEGL mutant displayed a wing notch phenotype, with reduced Wnt secretion and signaling. These findings demonstrate that YXXφ endocytosis motifs can occur in the intracellular loops of multipass membrane proteins and, moreover, provide direct evidence that the trafficking of Wls is required for efficient secretion of Wnt signaling proteins.     

Crystal structure of FtsA from Staphylococcus aureus

The bacterial cell-division protein FtsA anchors FtsZ to the cytoplasmic membrane. But how FtsA and FtsZ interact during membrane division remains obscure. We have solved 2.2 Å resolution crystal structure for FtsA from Staphylococcus aureus. In the crystals, SaFtsA molecules within the dimer units are twisted, in contrast to the straight filament of FtsA from Thermotoga maritima, and the half of S12–S13 hairpin regions are disordered. We confirmed that SaFtsZ and SaFtsA associate in vitro, and found that SaFtsZ GTPase activity is enhanced by interaction with SaFtsA.     

Management of Rotylenchulus reniformis in Pineapple,Ananas comosus,by Intercycle Cover Crops

The effects of intercycle cover crops on Rotylenchulus reniformis population densities in pineapple were evaluated in one greenhouse and two field experiments. In the greenhouse, Crotalaria juncea, Brassica napus, and Tagetes erecta were planted for 3 months and then incorporated. These treatments were compared to weedy fallow with or without 1,3-dichloropropene (1,3-D) in three soils (Makawao fallow, Wahiawa fallow, and Wahiawa pineapple) naturally infested with R. reniformis. All cover crop incorporation suppressed R. reniformis numbers in cowpea more than did the weedy treatment in the Makawao (P < 0.05) but not in the Wahiawa soils. Crotalaria juncea treatment increased bacterivorous nematodes and nematode-trapping fungal population densities more than the other treatments in Makawao fallow and Wahiawa pineapple-planted soils. The field trials included the same plants as well as Sinapis alba. Treatments with Crotalaria juncea and 1,3-D maintained lower R. reniformis population densities on pineapple longer than other cover crops or weedy fallow treatments. Crotalaria juncea could have suppressed R. reniformis because it is a poor host and because it enhances nematode-trapping fungi when incorporated into soil. Treatment with 1,3-D reduced microbial activities but produced the greatest pineapple yield.     

Cellular pattern formation, establishment of polarity and segregation of colored cytoplasm in embryos of the nematode Romanomermis culicivorax

We have begun to analyze the early embryogenesis of Romanomermis culicivorax, an insect-parasitic nematode phylogenetically distant to Caenorhabditis elegans. Development of R. culicivorax differs from C. elegans in many aspects including establishment of polarity, formation of embryonic axes and the pattern of asymmetric cleavages. Here, a polarity reversal in the germline takes place already in P₁ rather than P₂, the dorsal-ventral axis appears to be inverted and gut fate is derived from the AB rather than from the EMS blastomere. So far unique for nematodes is the presence of colored cytoplasm and its segregation into one specific founder cell. Normal development observed after experimentally induced abnormal partitioning of pigment indicates that it is not involved in cell specification. Another typical feature is prominent midbodies (MB). We investigated the role of the MB region in the establishment of asymmetry. After its irradiation the potential for unequal cleavage in somatic and germline cells as well as differential distribution of pigment are lost. This indicates a crucial involvement of this region for spindle orientation, positioning, and cytoplasmic segregation. A scenario is sketched suggesting why and how during evolution the observed differences between R. culicivorax and C. elegans may have evolved.     

Effect of Mutations of Arginine 94 on Proton Pumping,Electron Transfer,and Superoxide Anion Generation in Cytochrome b of the bc1 Complex from Rhodobacter sphaeroides

Proton transfer involving internal water molecules that provide hydrogen bonds and facilitate proton diffusion has been identified in some membrane proteins. Arg-94 in cytochrome b of the Rhodobacter sphaeroides bc₁ complex is fully conserved and is hydrogen-bonded to the heme propionate and a chain of water molecules. To further elucidate the role of Arg-94, we generated the mutations R94A, R94D, and R94N. The wild-type and mutant bc₁ complexes were purified and then characterized. The results show that substitution of Arg-94 decreased electron transfer activity and proton pumping capability and increased O₂^˙̄ production, suggesting the importance of Arg-94 in the catalytic mechanism of the bc₁ complex in R. sphaeroides. This also suggests that the transport of H⁺, O₂, and O₂^˙̄ in the bc₁ complex may occur by the same pathway.     

Homodimeric intrinsic membrane proteins. Identification and modulation of interactions between mitochondrial transporter (carrier) subunits

Transporter (carrier) proteins of the inner mitochondrial membrane link metabolic pathways within the matrix and the cytosol with transport/exchange of metabolites and inorganic ions. Their strict control of these fluxes is required for oxidative phosphorylation. Understanding the ternary complex transport mechanism with which most of these transporters function requires an accounting of the number and interactions of their subunits. The phosphate transporter (PTP, Mir1p) subunit readily forms homodimers with intersubunit affinities changeable by mutations. Cys28, likely at the subunit interface, is a site for mutations yielding transport inhibition or a channel-like transport mode. Such mutations yield a small increase or decrease in affinity between the subunits. The PTP inhibitor N-ethylmaleimide decreases subunit affinity by a small amount. PTP mutations that yield the highest (40%) and the lowest (2%) liposome incorporation efficiencies (LIE) are clustered near Cys28. Such mutant subunits show the lowest and highest subunit affinities respectively. The oxaloacetate transporter (Oac1p) subunit has an almost twofold lower affinity than the PTP subunit. The Oac1p, dicarboxylate (Dic1p) and PTP transporter subunits form heterodimers with even lower affinities. These results form a firm basis for detailed studies to establish the effect of subunit affinities on transport mode and activity and for the identification of the mechanism that prevents formation of heterodimers that surely will negatively impact oxidative phosphorylation and ATP levels with serious consequences for the cell.     

A review of Rheocricotopus (Psilocricotopus) chalybeatus species group from China,with the description of three new species (Diptera,Chironomidae)

The Rheocricotopus (Psilocricotopus) chalybeatus species group from China is reviewed. Three new species, R. (P.) brochus sp. n., R. (P.) rotundus sp. n. and R. (P.) serratus sp. n. are described as adult males. R. (P.) imperfectus Makarchenko & Makarchenko, 2005, R. (P.) robacki (Beck & Beck, 1964) and R. (P.) valgus Chaudhuri & Sinharay, 1983 are recorded from China for the first time and annotated. The diagnosis for the species group is emended and a key to adult males of the species group in China is presented.     

Mode of action of iturin A, an antibiotic isolated from Bacillus subtilis, on Micrococcus luteus.

Iturin A has an antibacterial activity on M. luteus which is strongly reduced in presence of MgCl₂. Iturin A lyses M. luteus protoplast, this lysis is enhanced by EDTA and inhibited by MgCl₂. These results suggest an action of iturin A on cytoplasmic membrane with interactions of both lipophilic and polypeptidic moieties of the antibiotic, respectively with membrane lipids and membrane polar components. Polar interactions involve the participation of mineral ions as magnesium ions have a strong inhibition effect on the activity of iturin A. The effect of iturin A on the incorporation of radio-active thymidine, uracil, isoleucine and alanine seems unspecific and is probably a consequence of the primary action on cytoplasmic membrane.     

Characterization of a New Burrowing Nematode Population,Radopholus citrophilus,from Hawaii

Karyotype, host preference, isozyzme patterns, morphometrics, and mating behavior of two burrowing nematode populations from Hawaii, one infecting Anthurium sp. and the second infecting Musa sp., were compared with Radopholus similis and R. citrophilus populations from Florida. The population from Anthurium sp. had five chromosomes (n = 5), and that from Musa sp. had four (n = 4). Neither of the Hawaiian nematode populations persisted in roots of Citrus limon or C. aurantium. Anthurium clarinerivum and A. hookeri were hosts of the burrowing nematode population from anthurium in Hawaii and of R. citrophilus from Florida, whereas the two anthurium species were poor hosts of the population from Musa sp. in Hawaii and R. similis from Florida. The isozyme pattern of the population isolated from anthurium was identical to that of R. citrophigus, whereas the pattern of the population from banana in Hawaii was identical to that of R. similis. Mating behavior between the burrowing nematode population isolated from Anthurium sp. and a Florida population of R. citrophilus supports their close taxonomic relationship. Mating was observed between the population from Anthurium sp. and the Florida population of R. citrophilus but not between the Hawaiian burrowing nematode population isolated from Musa sp. and a Florida population of R. citrophilus. These findings indicate that a previously unidentified population of R. citrophilus which does not parasitize citrus occurs in Hawaii.     

Biosynthesis of a 42-kD Polypeptide in the Cytoplasmic Membrane of the Cyanobacterium Anacystis nidulans Strain R2 during Adaptation to Low CO(2) Concentration

When cells of Anacystis nidulans strain R2 grown under high CO₂ conditions (3%) were transferred to low CO₂ conditions (0.05%), their ability to accumulate inorganic carbon (C_i) increased up to 8 times. Cytoplasmic membranes (plasmalemma) isolated at various stages of low CO₂ adaptation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase of a 42-kilodalton polypeptide in the cytoplasmic membrane during adaptation; a linear relationship existed between the amount of this polypeptide and the C_i-accumulating capability of the cells. No significant changes were observed during this process in the amount of other polypeptides in the cytoplasmic membranes or in the polypeptide profiles of the thylakoid membranes, cell walls, and soluble fractions. Spectinomycin, an inhibitor of protein biosynthesis, inhibited both the increase of the 42-kilodalton polypeptide and the induction of high C_i-accumulating capability. The incorporation of [³⁵S]sulfate into membrane proteins was greatly reduced during low CO₂ adaptation. Radioautograms of the ³⁵S-labeled membrane proteins revealed that synthesis of the 42-kilodalton polypeptide in the cytoplasmic membrane was specifically activated during the adaptation, while that of most other proteins was greatly suppressed. These results suggested that the 42-kilodalton polypeptide in the cytoplasmic membrane is involved in the active C_i transport by A. nidulans strain R2 and its synthesis under low CO₂ conditions leads to high C_i-transporting activity.     

The ultrastructure of Chlorobaculum tepidum revealed by cryo-electron tomography

Chlorobaculum (Cba) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. As other anoxygenic green photosynthetic bacteria, Cba tepidum synthesizes bacteriochlorophylls for the assembly of a large light-harvesting antenna structure, the chlorosome. Chlorosomes are sac-like structures that are connected to the reaction centers in the cytoplasmic membrane through the BChl α-containing Fenna–Matthews–Olson protein. Most components of the photosynthetic machinery are known on a biophysical level, however, the structural integration of light harvesting with charge separation is still not fully understood. Despite over two decades of research, gaps in our understanding of cellular architecture exist. Here we present an in-depth analysis of the cellular architecture of the thermophilic photosynthetic green sulfur bacterium of Cba tepidum by cryo-electron tomography. We examined whole hydrated cells grown under different electron donor conditions. Our results reveal the distribution of chlorosomes in 3D in an unperturbed cell, connecting elements between chlorosomes and the cytoplasmic membrane and the distribution of reaction centers in the cytoplasmic membrane.     

The Dynamin-related GTPase,Dnm1p,Controls Mitochondrial Morphology in Yeast

The Saccharomyces cerevisiae Dnm1 protein is structurally related to dynamin, a GTPase required for membrane scission during endocytosis. Here we show that Dnm1p is essential for the maintenance of mitochondrial morphology. Disruption of the DNM1 gene causes the wild-type network of tubular mitochondrial membranes to collapse to one side of the cell but does not affect the morphology or distribution of other cytoplasmic organelles. Dnm1 proteins containing point mutations in the predicted GTP-binding domain or completely lacking the GTP-binding domain fail to rescue mitochondrial morphology defects in a dnm1 mutant and induce dominant mitochondrial morphology defects in wild-type cells. Indirect immunofluorescence reveals that Dnm1p is distributed in punctate structures at the cell cortex that colocalize with the mitochondrial compartment. These Dnm1p-containing structures remain associated with the spherical mitochondria found in an mdm10 mutant strain. In addition, a portion of Dnm1p cofractionates with mitochondrial membranes during differential sedimentation and sucrose gradient fractionation of wild-type cells. Our results demonstrate that Dnm1p is required for the cortical distribution of the mitochondrial network in yeast, a novel function for a dynamin-related protein.     

Escherichia coli lacking the AcrAB multidrug efflux pump also lacks nonproteinaceous, PHB-polyphosphate Ca channels in the membrane

PHB(polyP) complexes bind calcium and form calcium channels in the cytoplasmic membrane in Escherichia coli and are likely to be important in Ca²⁺ homeostasis in this organism. E. coli N43, which lacks the AcrA component of a major multidrug resistance pump, was shown to be defective in calcium handling, with an inability to maintain submicromolar levels of free Ca²⁺ in the cytoplasm. Therefore, using an N-phenyl-1-napthylamine (NPN)-dependent fluorescence assay, we measured temperature-dependent phase transitions in the membranes of intact cells. These transitions specifically depend on the presence of PHB(Ca²⁺polyP) complexes. PHB(Ca²⁺polyP) channel complexes, particularly in stationary phase cultures, were detected in wild-type strains; however, in contrast, isogenic acrA⁻ strains had greatly reduced amounts of the complexes. This indicates that the AcrAB transporter may have a novel, hitherto undetected physiological role, either directly in the membrane assembly of the PHB complexes or the transport of a component of the membrane, which is essential for assembly of the complexes into the membrane. In other experiments, we showed that the particular defective calcium handling detected in N43 was not due to the absence of AcrA but to other unknown factors in this strain.