Molecular analysis of the tagF gene, encoding CDP-Glycerol:Poly(glycerophosphate) glycerophosphotransferase of Staphylococcus epidermidis ATCC 14990

Staphylococcus epidermidis ATCC 14990 produces a wall-associated glycerol teichoic acid which is chemically identical to the major wall-associated teichoic acid of Bacillus subtilis 168. The S. epidermidis tagF gene was cloned from genomic DNA and sequenced. When introduced on a plasmid vector into B. subtilis 1A486 carrying the conditionally lethal temperature-sensitive mutation tagF1 (rodC1), it expressed an 85-kDa protein which allowed colonies to grow at the restrictive temperature. This showed that the cloned S. epidermidis gene encodes a functional CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase. An amino acid substitution at residue 616 in the recombinant TagF protein eliminated complementation. Unlike B. subtilis, where the tagF gene is part of the tagDEF operon, the tagF gene of S. epidermidis is not linked to any other tag genes. We attempted to disrupt the chromosomal tagF gene in S. epidermidis TU3298 by directed integration of a temperature-sensitive plasmid but this failed, whereas a control plasmid containing the 5' end of tagF on a similarly sized DNA fragment was able to integrate. This suggests that the tagF gene is essential and that the TagF and other enzymes involved in teichoic acid biosynthesis could be targets for new antistaphylococcal drugs.     

Purified,recombinant TagF protein from Bacillus subtilis 168 catalyzes the polymerization of glycerol phosphate onto a membrane acceptor in vitro

We report the first characterization of a recombinant protein involved in the polymerization of wall teichoic acid. Previously, a study of the teichoic acid polymerase activity associated with membranes from Bacillus subtilis 168 strains bearing thermosensitive mutations in tagB, tagD, and tagF implicated TagF as the poly(glycerol phosphate) polymerase (Pooley, H. M., Abellan, F. X., and Karamata, D. (1992) J. Bacteriol. 174, 646-649). In the work reported here, we have demonstrated an unequivocal role for tagF in the thermosensitivity of one such mutant (tagF1) by conditional complementation at the restrictive temperature with tagF under control of the xylose promoter at the amyE locus. We have overexpressed and purified recombinant B. subtilis TagF protein, and we provide direct biochemical evidence that this enzyme is responsible for polymerization of poly(glycerol phosphate) teichoic acid in B. subtilis 168. Recombinant hexahistidine-tagged TagF protein was purified from Escherichia coli and was used to develop a novel membrane pelleting assay to monitor poly(glycerol phosphate) polymerase activity. Purified TagF was shown to incorporate radioactivity from its substrate CDP-[(14)C]glycerol into a membrane fraction in vitro. This activity showed a saturable dependence on the concentration of CDP-glycerol (K(m) of 340 microm) and the membrane acceptor (half-maximal activity at 650 microg of protein/ml of purified B. subtilis membranes). High pressure liquid chromatography analysis confirmed the polymeric nature of the reaction product, approximately 35 glycerol phosphate units in length.     

Changes in wall teichoic acid during the rod-sphere transition of Bacillus subtilis 168.

Wall teichoic acid (WTA) is essential for the growth of Bacillus subtilis 168. To clarify the function of this polymer, the WTAs of strains 168, 104 rodB1, and 113 tagF1 (rodC1) grown at 32 and 42 degrees C were characterized. At the restrictive temperature, the rodB1 and tagF1 (rodC1) mutants undergo a rod-to-sphere transition that is correlated with changes in the WTA content of the cell wall. The amount of WTA decreased 33% in strain 104 rodB1 and 84% in strain 113 tagF1 (rodC1) when they were grown at the restrictive temperature. The extent of alpha-D-glucosylation (0.84) was not affected by growth at the higher temperature in these strains. The degree of D-alanylation decreased from 0.22 to 0.10 in the rodB1 mutant but remained constant (0.12) in the tagF1 (rodC1) mutant at both temperatures. Under these conditions, the degree of D-alanylation in the parent strain decreased from 0.27 to 0.21. The chain lengths of WTA in strains 168 and 104 rodB1 grown at both temperatures were approximately 53 residues, with a range of 45 to 60. In contrast, although the chain length of WTA from the tagF1 (rodC1) mutant at 32 degrees C was similar to that of strains 168 and 104 rodB1, it was approximately eight residues at the restrictive temperature. The results suggested that the rodB1 mutant is partially deficient in completed poly(glycerophosphate) chains. The precise biochemical defect in this mutant remains to be determined. The results for strain 113 tagF1(rodC1) are consistent with the temperature-sensitive defect in the CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase (H. M. Pooley, F.-X. Abellan, and D. Karamata, J. Bacteriol. 174:646-649, 1992).     

Genetic analysis of SCO2997, encoding a TagF homologue, indicates a role for wall teichoic acids in sporulation of Streptomyces coelicolor A3(2)

Streptomyces coelicolor contains two gene clusters putatively involved in wall teichoic acid biosynthesis. Inactivation of the tagF homologue SCO2997 or SCO2584, a component of the Streptomyces spore wall synthesizing complex, affected sporulation. The mutant phenotypes resembled those of mre mutants, suggesting a function of wall teichoic acids in the differentiation of Streptomyces.     

Teichoic acid is an essential polymer in Bacillus subtilis that is functionally distinct from teichuronic acid

Wall teichoic acids are anionic, phosphate-rich polymers linked to the peptidoglycan of gram-positive bacteria. In Bacillus subtilis, the predominant wall teichoic acid types are poly(glycerol phosphate) in strain 168 and poly(ribitol phosphate) in strain W23, and they are synthesized by the tag and tar gene products, respectively. Growing evidence suggests that wall teichoic acids are essential in B. subtilis; however, it is widely believed that teichoic acids are dispensable under phosphate-limiting conditions. In the work reported here, we carefully studied the dispensability of teichoic acid under phosphate-limiting conditions by constructing three new mutants. These strains, having precise deletions in tagB, tagF, and tarD, were dependent on xylose-inducible complementation from a distal locus (amyE) for growth. The tarD deletion interrupted poly(ribitol phosphate) synthesis in B. subtilis and represents a unique deletion of a tar gene. When teichoic acid biosynthetic proteins were depleted, the mutants showed a coccoid morphology and cell wall thickening. The new wall teichoic acid biogenesis mutants generated in this work and a previously reported tagD mutant were not viable under phosphate-limiting conditions in the absence of complementation. Cell wall analysis of B. subtilis grown under phosphate-limited conditions showed that teichoic acid contributed approximately one-third of the wall anionic content. These data suggest that wall teichoic acid has an essential function in B. subtilis that cannot be replaced by teichuronic acid.     

The atokous-epitokous border is determined before the onset of heteronereid development in Platynereis dumerilii (Annelida,Polychaeta)

Summary In most nereids sexual maturation is accompanied by a dramatic reorganization of the body that enables swarming of the formerly benthic worms. However, a border exists between unchanged anterior (atokous) and metamorphosed posterior (epitokous) segments. The site of this atokous-epitokous border (a/e border) is different in sexually mature males and females of Platynereis dumerilii. There is no correlation between the total number of setigerous segments of a specimen and the location of the a/e border. The location of the a/e border and sexual development are affected neither by cutting off caudal segments of juveniles (including the prospective a/e border) nor by transecting the ventral nerve cord. When parapodia are transplanted from prospective epitokous regions to prospective atokous regions and vice versa, they maintain their original character during metamorphosis. The results presented here suggest that prospective atokous as well as epitokous characters are determined at or only very shortly after formation of the respective segments. Thus the a/e border is established well in advance of the onset of epitokous metamorphosis.     

In the spiny lobster (Panulirus interruptus) the prophenoloxidase is located in plasma not in haemocytes

In the spiny lobster (Panulirus interruptus), unlike other crustaceans most of the prophenoloxidase (proPO) was detected in cell-free plasma (86.3%). In spite of its location, lobster proPO activating system has a similar activation mechanism to other crustacean proPO systems. Haemocyte lysate was able to activate the plasma proPO indicating location of the prophenoloxidase activating enzyme (PPAE) in haemocytes. Lobster haemocyte PPAE was isolated by affinity chromatography and its participation as activating enzyme was demonstrated. This enzyme is a serine-proteinase that transforms the inactive form (proPO) to an active one (phenoloxidase). The PPAE was also present in the cell-free supernatant of haemocytes previously incubated with Vibrio alginolyticus.     

The constant gene orf14.9, which belongs to the variable eps (exopolysaccharide) cluster, is involved in the cell growth of Streptococcus thermophilus

In Streptococcus thermophilus, the eps clusters involved in exopolysaccharide (EPS) biosynthesis are very polymorphic, nevertheless they all contain a highly conserved sequence corresponding to that of orf14.9. This open reading frame (ORF) is transcribed in a reverse direction with respect to eps genes. Amino acid sequence analysis showed a possible transmembrane location of the putative Orf14.9 protein but did not permit a proposed function. Insertional mutants of orf14.9 were obtained in strains NST2280 and A054 of S. thermophilus. EPS yields of these mutants are similar to those of their respective wild strains, suggesting that orf14.9 does not modify the quantity of produced EPS. Growth parameter determination for wild strains and their respective mutants showed that orf14.9 is involved in the cell growth of S. thermophilus.     

Waardenberg syndrome (WS) type I is caused by defects at multiple loci, one of which is near ALPP on chromosome 2: First report of the WS consortium

Previous studies have localized the gene for Waardenburg syndrome (WS) type I to the distal portion of chromosome 2q, near the ALPP locus. We pooled linkage data obtained from 41 WS type I and 3 WS type II families which were typed for six polymorphic loci on chromosome 2q in order to refine the location of the WS locus (WS1) and evaluate the extent of genetic heterogeneity. In the course of this work, we developed diagnostic criteria for genetic and phenotypic studies. Our findings, based on two-locus and multilocus analysis using a linkage map established from reference pedigrees, suggest that there are two or more mutations causing WS, one of which (i.e., WS1) is located on chromosome 2q, between the ALPP and FN1 loci, at distances of 7.8 cM and 11.2 cM for each marker, respectively. The results also indicate that WS1 is responsible for the illness in approximately 45% of all families in this sample. However, the odds favoring this position over a location between ALPP and SAG are only 2:1 when alternate assumptions about the proportion of linked families are considered. We conclude that a more saturated map of this region of chromosome 2q, including highly polymorphic markers, will be needed to accurately distinguish linked families and, ultimately, isolate the mutant gene.     

Two conserved histidine residues are critical to the function of the TagF-like family of enzymes

The TagF protein from Bacillus subtilis 168 is the poly(glycerol phosphate) polymerase responsible for the synthesis of wall teichoic acid and is the prototype member of a poorly understood family of similar teichoic acid synthetic enzymes. Here we describe in vitro and in vivo characterization of TagF, which localizes the active site to the carboxyl terminus of the protein and identifies residues that are critical for catalysis. We also establish the first mechanistic link among TagF and similar proteins by demonstrating that the identified residues are also critical in the function of TagB, a homologous enzyme implicated as the glycerophosphotransferase responsible for priming poly(glycerol phosphate) synthesis. We investigated the dependence of TagF activity on pH and showed that deprotonation of a residue with a pK(a) near neutral is critical for proper function. Alteration of histidine residues 474 and 612 by site-directed mutagenesis abolished TagF activity in vitro (5000-fold reduction in k(cat)/K(m)) while variants in four other conserved acidic residues showed minimal loss of activity. Complementation using H474A and H612A mutant alleles failed to suppress a lethal temperature-sensitive tagF defect in vivo despite confirmation of robust expression by Western blot. When corresponding mutations were made to the homologous tagB gene, these alleles were unable to suppress a tagB temperature-sensitive lethal phenotype. These results extend the mechanistic observations for TagF across a wider family of enzymes and provide the first biochemical evidence for the relatedness of these two enzymes.     

Morphological plasticity in the kelp Nereocystis luetkeana (Phaeophyceae) is sensitive to the magnitude,direction, and location of mechanical loading

Nereocystis luetkeana is a canopy-forming kelp that exhibits morphological plasticity across hydrodynamic gradients, producing broad, undulate blades in slow flow and narrow, flattened blades in fast flow, enabling thalli to reduce drag while optimizing photosynthesis. While the functional significance of this phenomenon has been well studied, the developmental and physiological mechanisms that facilitate the plasticity remain poorly understood. In this study, we conducted three experiments to characterize how the (1) magnitude, (2) direction, and (3) location of plasticity-inducing mechanical stimuli affect the morphology of Nereocystis blades. We found that applying a gradient of tensile force caused blades to grow progressively longer, narrower, less ruffled, and heavier in a linear fashion, suggesting that Nereocystis is equally well adapted for all conditions within its hydrodynamic niche. We also found that applying tension transversely across blades caused the growth response to rotate 90°, indicating that there is no substantial separation between the sites of stimulus perception and response and suggesting that a long-distance signaling mechanism, such as a hormone, is unlikely to mediate this phenomenon. Meristoderm cells showed morphological changes that paralleled those of their respective blades in this experiment, implying that tissue-level morphology is influenced by cell growth. Finally, we found that plasticity was only induced when tension was applied directly to the growing tissue, reinforcing that long-distance signaling is probably not involved and possibly indicating that the mechanism on display generally requires an intercalary meristem to facilitate mechanoperception.     

Urease in jack-bean (Canavalia ensiformis (L.) DC) seeds is a cytosolic protein

Urease (EC 3.5.1.5) is abundantly present in the seeds of many species of Leguminosae. There is at present conflicting information in the literature about its subcellular location and status as a glycoprotein. We have made a study of the subcellular location of urease in jack-bean cotyledons using an immunocytochemical approach; in addition, we studied the biosynthesis and glycoprotein nature of the enzyme using several biochemical approaches. All the results are in agreement with the interpretation that the seed urease is not a glycoprotein, is synthesized on free polysomes, and is present in the cytosol of the storage parenchyma cells.Abbreviations ConA Concanavalin A - ER endoplasmic reticulum - IgG immunoglobulin G - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis On leave from Laboratoire de Photobiologie (CNRS-UA 203), Faculté des Sciences de Rouen, F-76130 Mont Saint Aignan, France     

Activity-regulated, cytoskeleton-associated protein (Arc) is essential for visceral endoderm organization during early embryogenesis

Activity-regulated, cytoskeleton-associated protein (Arc) was first identified as an immediate-early gene regulated by synaptic activity. We have studied its functional role in vivo using a gene-targeting approach. We found that Arc is encoded by a single exon, and Arc mRNA is ubiquitously expressed in early mouse embryos. Homozygous Arc mutants are severely growth-retarded, fail to gastrulate and subsequently die before day 8.5 of embryogenesis. Further analysis revealed severe disorganization of visceral endoderm formation, and total separation and ectopic location of embryonic and extraembryonic structure. These findings demonstrate that Arc function is essential for early embryo development and patterning in mice, and support the hypothesis that signaling from visceral endoderm is essential for normal patterning of the extraembryonic and embryonic structure.     

Probe,VK5B,is located in the same interval as the autosomal dominant adult polycystic kidney disease locus,PKD1

Summary The polymorphic DNA probe VK5B (D16S94) was mapped by genetic linkage in families from the Centre d'Etude de Polymorphisme Humain (CEPH) as being in the same interval as the autosomal dominant adult polycystic kidney disease locus (PKD1). The maximum likelihood estimate of the genetic location of VK5B using multipoint linkage analysis was 9.6cM proximal to {ie286-01} (D16S85) and 5.4cM distal to CRI-0327 (D16S63), in males. The VK5B probe may be useful in PKD1 families for prenatal and presymptomatic diagnosis of the disease. Additional typing of PKD1 families is required to determine whether the location of VK5B is distal or proximal to (PKD1).     

Mate location and recognition in Glenea cantor (Fabr.) (Coleoptera: Cerambycidae: Lamiinae): roles of host plant health, female sex pheromone, and vision

Glenea cantor (Fabricius) is an important pest of kapok trees [Bombax ceiba L.=Gossampinus malabaricus (DC.) Merr.] in southern China and Vietnam, and its adults are diurnally active. We carried out both field and laboratory experiments to examine the mechanisms that brought G. cantor sexes together from a long distance and facilitated mate location and recognition in a close range. Long-range sex pheromones are not involved in mate location. Mutual attraction of sexes to weakened kapok trees where adult feeding, mating, and oviposition occur plays the key role in mate location from a long distance. In a close range, vision and a female sex pheromone that operates over a short distance (3-3.5 cm) and/or by contact are major cues males use for mate location and recognition. Males seem to use combined chemical and visual cues to achieve mating. Male antennae, particularly the terminal five segments, are critical for males to detect and recognize females. Removal of male palpi has no significant effect on mate location and recognition by males.     

An unusual genotype of Toxoplasma gondii is common in California sea otters (Enhydra lutris nereis) and is a cause of mortality

Toxoplasma gondii-associated meningoencephalitis is a significant disease of California sea otters (Enhydra lutris nereis), responsible for 16% of total mortality in fresh, beachcast carcasses. Toxoplasma gondii isolates were obtained from 35 California otters necropsied between 1998 and 2002. Based on multi-locus PCR-restriction fragment length polymorphism and DNA sequencing at conserved genes (18S rDNA, ITS-1) and polymorphic genes (B1, SAG1, SAG3 and GRA6), two distinct genotypes were identified: type II and a novel genotype, here called type x, that possessed distinct alleles at three of the four polymorphic loci sequenced. The majority (60%) of sea otter T. gondii infections were of genotype x, with the remaining 40% being of genotype II. No type I or III genotypes were identified. Epidemiological methods were used to examine the relationship between isolated T. gondii genotype(s) and spatial and demographic risk factors, such as otter stranding location and sex, as well as specific outcomes related to pathogenicity, such as severity of brain inflammation on histopathology and T. gondii-associated mortality. Differences were identified with respect to T. gondii genotype and sea otter sex and stranding location along the California coast. Localised spatial clustering was detected for both type II (centred within Monterey Bay) and x (centred near Morro Bay)-infected otters. The Morro Bay cluster of type x-infected otters overlaps previously reported high-risk areas for sea otter infection and mortality due to T. gondii. Nine of the 12 otters that had T. gondii-associated meningoencephalitis as a primary cause of death were infected with type x parasites.     

Breeding location choice in salmon: causes (habitat, competition, body size, energy stores) and consequences (life span, energy stores)

Breeding location choice provides a mechanism by which individuals can directly influence their reproductive success. Location choice should therefore reflect individual condition, habitat features, and the intensity of competition; with these factors then influencing reproductive success. To test whether such patterns were detectable in the wild, we tagged 705 sockeye salmon (Oncorhynchus nerka) in a natural population, and monitored them from when they started breeding until they died. We evaluated the role of individual condition (size, secondary sexual traits, energy stores) in the acquisition of breeding locations that differed in the intensity of competition (female density, sex ratio) and habitat features (water depth, water velocity). We then evaluated the influence of breeding location on reproductive life span and energy stores. At a coarse level (20‐m stream sections), females consistently settled in certain locations, and these locations sustained high densities and held larger females. At a fine scale (0.5‐m breeding sites), (1) larger fish occupied deeper water (males, r²=0.072; females, r²=0.199), (2) higher levels of competition reduced reproductive life span for males (r²=0.139) but not females, and (3) fish with shorter reproductive life spans died with more energy remaining in their muscle tissue (males, r²=0.414; females, r²=0.440). These patterns were nested within a tendency for late breeding fish to have shorter reproductive life spans. Energy stores and secondary sexual traits did not influence breeding location choice, and larger fish did not acquire locations of higher intrinsic quality (i.e., those sections settled first and sustaining higher competition). Our study provides evidence that some aspects of individual condition influence breeding location choice, which then influences components of reproductive success.     

The attached form of the endangered freshwater alga Aegagropila linnaei Kützing (Chlorophyta) is found in the Zuideindigerwiede, The Netherlands

The freshwater green macroalga Aegagropila linnaei used to occur in several locations in The Netherlands in unattached growths forms (including the enigmatic ‘lake balls’) more than 60 years ago. A recent survey of most of the historical collection sites of the species reported the attached growth form for the first time for The Netherlands, but only in miniscule quantities and from one location only. Here we report on the finding of the attached form from the Zuideindigerwiede, one of the two historical collection sites that had not been surveyed previously. Unattached growth forms do not seem to occur anymore in The Netherlands, and the species as a whole must be regarded as rare and endangered.     

Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias)

Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.