Dynamics of Long-term Anhydrobiotic Survival of Lichen-dwelling Tardigrades

It is not rare to find in references that anhydrobiotic tardigrades can survive for more than a century. However, a closer look at the empirical evidence provides very little support that tardigrades are capable of surviving dried for such a long time. In order to resolve this discrepancy, we carried out a study to evaluate the long-term survival of naturally dried tardigrades. A large fragment of dry lichen (Xanthoria parietina) was collected in the field two days after a rainy day in 1999. The dry lichen was stored inside a paper bag in the laboratory at room temperature and humidity and under atmospheric oxygen exposure. Replicates of the dry lichen were re-hydrated after various time periods of storage, with all tardigrades extracted and the survivors enumerated. Five species of tardigrades were found, but two of them only occasionally. Ramazzottius oberhaeuseri, Echiniscus testudo and Echiniscus trisetosus were sufficiently represented for statistical analysis. At the beginning of the experiment 91.1% of R. oberhaeuseri and 71.7% of Echiniscus spp. were alive. R. oberhaeuseri survived up to 1604 days, while Echiniscus spp. lived up to 1085 days. Recovery after four years of anhydrobiosis has to be considered a very good long-term survival, which is important from an ecological and evolutionary point of view.     

Synthesis, characterization, X-ray molecular structure and catalase-like activity of a non-heme iron complex: Dichloro[N-propanoate-N,N-bis-(2-pyridylmethyl)amine]iron(III)

In this work we present the synthesis and characterization of the complex dichloro[N-propanoate-N,N-bis-(2-pyridylmethyl)amine]iron(III) [Fe^III(PBMPA)Cl₂]. The ligand LiPBMPA was synthesized through the Michael reaction of BMPA with methylacrylate, followed by alkaline hydrolysis. The complex [Fe^III(PBMPA)Cl₂] has been synthesized by the reaction of the ligand with FeCl₃ · H₂O and was mainly characterized by cyclic voltammetry, conductivimetry, and electronic, infrared and Mössbauer spectroscopies, and by X-ray structural analysis, which showed an iron center coordinated by one carboxylate oxygen in a monodentate way, one tertiary amine, two pyridine groups and two chloride ions. It has been proposed that in water the chloride ligands are shifted by the solvent molecules and the species [Fe^III(PBMPA)(H₂O)₂]Cl₂ is predominant. The catalase-like activity of the complex was tested in water, and it proved to be active in the hydrogen peroxide dismutation. Kinetics studies were conducted following the initial rates method. The reaction is first order in relation to both the complex and the hydrogen peroxide. Based on the presence of a lag phase that depends on the initial complex concentration, we propose that the active species that shows in situ catalase-like activity, is a binuclear complex.     

Phenological models for blooming of apple in a mountainous region

Six phenological series were available for ‘Golden Delicious’ apple blooming at six sites in Trentino, an alpine fruit-growing region. Several models were tested to predict flowering dates, all involving a “chilling and forcing” approach. In many cases, application of the models to different climatic conditions results in low accuracy of prediction of flowering date. The aim of this work is to develop a model with more general validity, starting from the six available series, and to test it against five other phenological series outside the original area of model development. A modified version of the “Utah” model was the approach that performed best. In fact, an algorithm using “chill units” for rest completion and a thermal sum for growing-degree-hours (GDH), whose efficiency changes over time depending on the fraction of forcing attained, yielded a very good prediction of flowering. Results were good even if hourly temperatures were reconstructed from daily minimum and maximum values. Errors resulting from prediction of flowering data were relatively small, and root mean square errors were in the range of 1–6 days, being <2 days for the longest phenological series. In the most general form of the model, the summation of GDH required for flowering is not a fixed value, but a function of topoclimatic variables for a particular site: slope, aspect and spring mean temperature. This approach allows extension of application of the model to sites with different climatic features outside the test area.     

Interactions between Streptomyces coelicolor and Bacillus subtilis: Role of surfactants in raising aerial structures

Using mixed-species cultures, we have undertaken a study of interactions between two common spore-forming soil bacteria, Bacillus subtilis and Streptomyces coelicolor. Our experiments demonstrate that the development of aerial hyphae and spores by S. coelicolor is inhibited by surfactin, a lipopeptide surfactant produced by B. subtilis. Current models of aerial development by sporulating bacteria and fungi postulate a role for surfactants in reducing surface tension at air-liquid interfaces, thereby removing the major barrier to aerial growth. S. coelicolor produces SapB, an amphipathic peptide that is surface active and required for aerial growth on certain media. Loss of aerial hyphae in developmental mutants can be rescued by addition of purified SapB. While a surfactant from a fungus can substitute for SapB in a mutant that lacks aerial hyphae, not all surfactants have this effect. We show that surfactin is required for formation of aerial structures on the surface of B. subtilis colonies. However, in contrast to this positive role, our experiments reveal that surfactin acts antagonistically by arresting S. coelicolor aerial development and causing altered expression of developmental genes. Our observations support the idea that surfactants function specifically for a given organism regardless of their shared ability to reduce surface tension. Production of surfactants with antagonistic activity could provide a powerful competitive advantage during surface colonization and in competition for resources.     

The Helicobacter pylori chemotaxis receptor TlpB (HP0103) is required for pH taxis and for colonization of the gastric mucosa

The location of Helicobacter pylori in the gastric mucosa of mammals is defined by natural pH gradients within the gastric mucus, which are more alkaline proximal to the mucosal epithelial cells and more acidic toward the lumen. We have used a microscope slide-based pH gradient assay and video data collection system to document pH-tactic behavior. In response to hydrochloric acid (HCl), H. pylori changes its swimming pattern from straight-line random swimming to arcing or circular patterns that move the motile population away from the strong acid. Bacteria in more-alkaline regions did not swim toward the acid, suggesting the pH taxis is a form of negative chemotaxis. To identify the chemoreceptor(s) responsible for the transduction of pH-tactic signals, a vector-free allelic replacement strategy was used to construct mutations in each of the four annotated chemoreceptor genes (tlpA, tlpB, tlpC, and tlpD) in H. pylori strain SS1 and a motile variant of strain KE26695. All deletion mutants were motile and displayed normal chemotaxis in brucella soft agar, but only tlpB mutants were defective for pH taxis. tlpD mutants exhibited more tumbling and arcing swimming, while tlpC mutants were hypermotile and responsive to acid. While tlpA, tlpC, and tlpD mutants colonized mice to near wild-type levels, tlpB mutants were defective for colonization of highly permissive C57BL/6 interleukin-12 (IL-12) (p40-/-)-deficient mice. Complementation of the tlpB mutant (tlpB expressed from the rdxA locus) restored pH taxis and infectivity for mice. pH taxis, like motility and urease activity, is essential for colonization and persistence in the gastric mucosa, and thus TlpB function might represent a novel target in the development of therapeutics that blind tactic behavior.     

A screen for genes of heme uptake identifies the FLC family required for import of FAD into the endoplasmic reticulum

Although Candida albicans and Saccharomyces cerevisiae express very similar systems of iron uptake, these species differ in their capacity to use heme as a nutritional iron source. Whereas C. albicans efficiently takes up heme, S. cerevisiae grows poorly on media containing heme as the sole source of iron. We identified a gene from C. albicans that would enhance heme uptake when expressed in S. cerevisiae. Overexpression of CaFLC1 (for flavin carrier 1) stimulated the growth of S. cerevisiae on media containing heme iron. In C. albicans, deletion of both alleles of CaFLC1 resulted in a decrease in heme uptake activity, whereas overexpression of CaFLC1 resulted in an increase in heme uptake. The S. cerevisiae genome contains three genes with homology to CaFLC1, and two of these, termed FLC1 and FLC2, also stimulated growth on heme when overexpressed in S. cerevisiae. The S. cerevisiae Flc proteins were detected in the endoplasmic reticulum and the FLC genes encoded an essential function, as strains deleted for either FLC1 or FLC2 were viable, but deletion of both FLC1 and FLC2 was synthetically lethal. FLC gene deletion resulted in pleiotropic phenotypes related to defects in cell wall integrity. High copy suppressors of this synthetic lethality included three mannosyltransferases, VAN1, KTR4, and HOC1. FLC deletion strains exhibited loss of cell wall mannose phosphates, defects in cell wall assembly, and delayed maturation of carboxypeptidase Y. Permeabilized cells lacking FLC proteins exhibited dramatic loss of FAD import activity. We propose that the FLC genes are required for import of FAD into the lumen of the endoplasmic reticulum, where it is required for disulfide bond formation.     

Disulfiram irreversibly aggregates betaine aldehyde dehydrogenase--a potential target for antimicrobial agents against Pseudomonas aeruginosa

In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacterium against the high-osmolality stress prevalent in the infected tissues. This tetrameric enzyme contains four cysteine residues per subunit and is a potential drug target. In our search for specific inhibitors, we mutated the catalytic Cys286 to alanine and chemically modified the recombinant wild-type and the four Cys-->Ala single mutants with thiol reagents. The small methyl-methanethiosulfonate inactivated the enzymes without affecting their stability while the bulkier dithionitrobenzoic acid (DTNB) and bis[diethylthiocarbamyl] disulfide (disulfiram) induced enzyme dissociation--at 23 degrees C--and irreversible aggregation--at 37 degrees C. Of the four Cys-->Ala mutants only C286A retained its tetrameric structure after DTNB or disulfiram treatments, suggesting that steric constraints arising upon the covalent attachment of a bulky group to C286 resulted in distortion of the backbone configuration in the active site region followed by a severe decrease in enzyme stability. Since neither NAD(P)H nor betaine aldehyde prevented disulfiram-induced PaBADH inactivation or aggregation, and reduced glutathione was unable to restore the activity of the modified enzyme, we propose that disulfiram could be a useful drug to combat infection by P. aeruginosa.     

Activity screening of carrier domains within nonribosomal peptide synthetases using complex substrate mixtures and large molecule mass spectrometry

For screening a pool of potential substrates that load carrier domains found in nonribosomal peptide synthetases, large molecule mass spectrometry is shown to be a new, unbiased assay. Combining the high resolving power of Fourier transform mass spectrometry with the ability of adenylation domains to select their own substrates, the mass change that takes place upon formation of a covalent intermediate thus identifies the substrate. This assay has an advantage over traditional radiochemical assays in that many substrates, the substrate pool, can be screened simultaneously. Using proteins on the nikkomycin, clorobiocin, coumermycin A1, yersiniabactin, pyochelin, and enterobactin biosynthetic pathways as proof of principle, preferred substrates are readily identified from substrate pools. Furthermore, this assay can be used to provide insight into the timing of tailoring events of biosynthetic pathways as demonstrated using the bromination reaction found on the jamaicamide biosynthetic pathway. Finally, this assay can provide insight into the role and function of orphan gene clusters for which the encoded natural product is unknown. This is demonstrated by identifying the substrates for two NRPS modules from the pksN and pksJ genes that are found on an orphan NRPS/PKS hybrid cluster from Bacillus subtilis. This new assay format is especially timely for activity screening in an era when new types of thiotemplate assembly lines that defy classification are being discovered at an accelerating rate.     

Morphology and lipid body and vacuole dynamics during secondary conidia formation in Colletotrichum acutatum: laser scanning confocal analysis

Colletotrichum acutatum may develop one or more secondary conidia after conidial germination and before mycelial growth. Secondary conidia formation and germination were influenced by conidia concentration. Concentrations greater than 1x105 conidia/mL were associated with germination decrease and with secondary conidia emergence. Secondary conidia can form either alone or simultaneously with germ tubes and appressoria. Confocal analysis showed numerous lipid bodies stored inside ungerminated conidia, which diminished during germ tube and appressoria formation, with or without secondary conidia formation. They were also reduced during secondary conidia formation alone. While there was a decrease inside germinated conidia, lipid bodies appeared inside secondary conidia since the initial stages. Intense vacuolization inside primary germinated conidia occurred at the same time as the decrease in lipid bodies, which were internalized and digested by vacuoles. During these events, small acidic vesicles inside secondary conidia were formed. Considering that the conidia were maintained in distilled water, with no exogenous nutrients, it is clear that ungerminated conidia contain enough stored lipids to form germ tubes, appressoria, and the additional secondary conidia replete with lipid reserves. These results suggested a very complex and well-balanced regulation that makes possible the catabolic and anabolic pathways of these lipid bodies.     

Preparation and biological evaluation of some 1,2-O-isopropylidene-D-hexofuranose esters

The synthesis and biological evaluation of some new glycose esters bearing the 1,2-O-isopropylidene-d-hexofuranose functionality and belonging to the 3-O-acyl-d-allose and 6-O-acyl-d-glucose series are reported. When the results concerning cell growth inhibition are compared, it appears that the 6-O-acyl-d-glucose derivatives are more active than the 3-O-acyl-d-allose compounds. Within both 6-O-acyl-d-glucose and 3-O-acyl-d-allose derivatives, butyric esters displayed the highest inhibitory effects. Inhibition of cell growth is not associated with high induction levels of erythroid differentiation, despite the fact that pivaloates induce erythroid differentiation to an extent similar to that exhibited by previously reported molecules [Bioorg. Med. Chem. Lett.1999, 9, 3153-3158].