Growth of hyperthermophilic archaeon Pyrococcus furiosus on chitin involves two family 18 chitinases

Pyrococcus furiosus was found to grow on chitin, adding this polysacharide to the inventory of carbohydrates utilized by this hyperthermophilic archaeon. Accordingly, two open reading frames (chiA [Pf1234] and chiB [Pf1233]) were identified in the genome of P. furiosus, which encodes chitinases with sequence similarity to proteins from the glycosyl hydrolase family 18 in less-thermophilic organisms. Both enzymes contain multiple domains that consist of at least one binding domain and one catalytic domain. ChiA (ca. 39 kDa) contains a putative signal peptide, as well as a binding domain (ChiA(BD)), that is related to binding domains associated with several previously studied bacterial chitinases. chiB, separated by 37 nucleotides from chiA and in the same orientation, encodes a polypeptide with two different proline-threonine-rich linker regions (6 and 3 kDa) flanking a chitin-binding domain (ChiB(BD) [11 kDa]), followed by a catalytic domain (ChiB(cat) [35 kDa]). No apparent signal peptide is encoded within chiB. The two chitinases share little sequence homology to each other, except in the catalytic region, where both have the catalytic glutamic acid residue that is conserved in all family 18 bacterial chitinases. The genes encoding ChiA, without its signal peptide, and ChiB were cloned and expressed in Escherichia coli. ChiA exhibited no detectable activity toward chitooligomers smaller than chitotetraose, indicating that the enzyme is an endochitinase. Kinetic studies showed that ChiB followed Michaelis-Menten kinetics toward chitotriose, although substrate inhibition was observed for larger chitooligomers. Hydrolysis patterns on chitooligosaccharides indicated that ChiB is a chitobiosidase, processively cleaving off chitobiose from the nonreducing end of chitin or other chitooligomers. Synergistic activity was noted for the two chitinases on colloidal chitin, indicating that these two enzymes work together to recruit chitin-based substrates for P. furiosus growth. This was supported by the observed growth on chitin as the sole carbohydrate source in sulfur-free media.     

Distribution of individual members of the mosquito Anopheles maculipennis complex in Germany identified by newly developed real‐time PCR assays

Owing to their role as vectors of malaria parasites, species of the Anopheles maculipennis complex (Diptera: Culicidae) Meigen were intensively studied in the past, but with the disappearance of malaria in Germany in the middle of the last century, the interest in this field of research declined. A comprehensive ecological analysis of the current species distribution for Germany is lacking. Between 2010 and 2013, a total of 1445 mosquitoes of the An. maculipennis complex were collected at 72 different sites in Germany. The samples comprise 722 single individuals as well as 723 individuals in 90 pools of up to 25 mosquitoes. All samples were analysed with newly developed species‐specific qPCR assays for the identification of the four German species using nucleotide differences within the internal transcribed spacer 2 (ITS2) ribosomal DNA. All gathered data were used for species distribution modelling. The overall prevalence of An. messeae s.l. was highest with 98.89% of all pools; An. daciae with 6.93% of all individuals and An. messeae s.s. with 69.53%. The prevalence of the other two species was relatively low: An. maculipennis s.s. with 13.30% of all individuals (6.67% of all pools) and An. atroparvus with 1.80% of all individuals (1.11% of all pools).     

Screening of different algae for green synthesis of gold nanoparticles

The cyanobacteria Phormidium valderianum, P. tenue and Microcoleus chthonoplastes and the green algae Rhizoclonium fontinale, Ulva intestinalis, Chara zeylanica and Pithophora oedogoniana were exposed to hydrogen tetrachloroaurate solution and were screened for their suitability for producing nano‐gold. All three cyanobacteria genera and two of the green algae (Rhizoclonium fontinale and Ulva intestinalis) produced gold nanoparticles intracellularly, confirmed by purple colouration of the thallus within 72?h of treatment at 20°C. Extracted nanoparticle solutions were examined by UV‐vis spectroscopy, transmission electron microscopy (TEM) and X‐ray diffractometry (XRD). XRD confirmed the reduction of Au (III) to Au (0). UV‐vis spectroscopy and TEM studies indicated the production of nanoparticles having different shapes and sizes. Phormidium valderianum synthesized mostly spherical nanoparticles, along with hexagonal and triangular nanoparticles, at basic and neutral pHs (pH 9 and pH 7, respectively). Medicinally important gold nanorods were synthesized (together with gold nanospheres) only by P. valderianum at acidic pH (pH 5); this was initially determined by two surface plasmon bands in UV‐vis spectroscopy and later confirmed by TEM. Spherical to somewhat irregular particles were produced by P. tenue and Ulva intestinalis (TEM studies). The UV‐vis spectroscopy of the supernatant of other algal extracts indicated the formation of mostly spherical particles. Production of gold nanoparticles by algae is more ecofriendly than purely chemical synthesis. However, the choice of algae is important: Chara zeylanica and Pithophora oedogoniana were found to be unable to produce nanoparticles.     

Cuticular Membrane Fine Structure of Nicotiana tabacum L. Leaves

The cuticular membrane (CM) ofNicotiana tabacumL., includingthe cellin wall (CW), was examined to gain more informationabout the nature and chemical constitution of its fine structurefor possible inclusion in a model system, as recent literaturequestions its function as a major water permeability barrier.Different preparation techniques were used and the results evaluatedto select a method for future studies on tobacco leaf cuticles.Fixation with OsO₄included in the primary fixative, either asa vapour or in combination with other agents, followed by OsO₄aspost-fixative, gave good contrast of the CM. The lamellar structureof the tobacco cuticle proper (CP) was revealed by contrastingwith uranyl acetate and lead citrate. The fine lamellar structureof the CP was very clearly contrasted when KMnO₄was includedin the primary fixative. This was interpreted as indicatingthe tobacco CP to be polar. The reticulate fibrillar patternof the tobacco cuticular layer (CL) containing polysaccharideswas well contrasted when either OsO₄or paraformaldehyde wereincluded in the primary fixative. Cold fixation with glutaraldehydeand dimethyl sulphoxide and post-fixation with OsO₄revealedelectron-opaque material in the outer cutinized, irregularlyoutlined, region of the CW. These ultrahistochemical reactionsare discussed in relation to the known chemical compositionand possible water permeability of the CM. Cuticular fine structure; cuticular transpiration; Nicotiana tabacumL.