Phylogeny of the Rhizobium–Allorhizobium–Agrobacterium clade supports the delineation of Neorhizobium gen. nov.

The genera Agrobacterium, Allorhizobium, and Rhizobium belong to the family Rhizobiaceae. However, the placement of a phytopathogenic group of bacteria, the genus Agrobacterium, among the nitrogen-fixing bacteria and the unclear position of Rhizobium galegae have caused controversy in previous taxonomic studies. To resolve uncertainties in the taxonomy and nomenclature within this family, the phylogenetic relationships of generic members of Rhizobiaceae were studied, but with particular emphasis on the taxa included in Agrobacterium and the “R. galegae complex” (R. galegae and related taxa), using multilocus sequence analysis (MLSA) of six protein-coding housekeeping genes among 114 rhizobial and agrobacterial taxa. The results showed that R. galegae, R. vignae, R. huautlense, and R. alkalisoli formed a separate clade that clearly represented a new genus, for which the name Neorhizobium is proposed. Agrobacterium was shown to represent a separate cluster of mainly pathogenic taxa of the family Rhizobiaceae. A. vitis grouped with Allorhizobium, distinct from Agrobacterium, and should be reclassified as Allorhizobium vitis, whereas Rhizobium rhizogenes was considered to be the proper name for former Agrobacterium rhizogenes. This phylogenetic study further indicated that the taxonomic status of several taxa could be resolved by the creation of more novel genera.     

Spiroplasma infection in Drosophila melanogaster: What is the advantage of killing males?

Male-killing bacteria are maternally inherited agents that cause death of sons of infected females. Their transmission rate is commonly high but imperfect and also sensitive to different environmental factors. Therefore, the proportion of infected females should be reduced in each generation. In order to explain male-killers spread and persistence in host population, a mechanism resulting in the relative increase of infected females must outweigh the losses caused by the imperfect transmission. The resource release hypothesis states that the males’ death results in increased resources available to sibling females which would otherwise be used by their male siblings. Infected females are then expected: to be larger than uninfected females in natural populations; or to have higher viability; or to have shorter development times; or any combination of these outcomes. Here, we tested the resource release hypothesis by measuring body size of infected and uninfected wild-caught Drosophila melanogaster females and carried out other fitness related measures in the laboratory. Wild-caught infected females produced more daughters than uninfected females in their first days in the laboratory. However, although no significant difference in viability was found in a controlled experiment with infected and uninfected flies from a standard laboratory strain, there was a decrease in development time probably mediated by reduced competition. Fitness effects conditioned by the host genetic background are pointed out as a possible explanation for this difference between wild and laboratory flies. Our findings are discussed in the context of the resource advantage hypothesis.     

Hormonal aspects of oögenesis in the flies Phormia regina and Sarcophaga bullata

The corpora allata (CA) and median neurosecretory cells (MNC) of Phormia regina and Sarcophaga bullata become active with increasing age of the fly, on a diet of sugar alone. To prevent or retard oögenesis the CA or MNCs must be removed shortly after emergence, with subsequent protein meals. Topical JH application partially compensates for CA or MNC removal. This shows that the MNC activate the CA, and not vice versa. The trauma of either operation slightly depresses egg development.Injection of ecdysone into both species in the stage of initial yolk deposition causes the primary oöcytes to degenerate. This leads to development of the penultimate oöcytes. Older and younger egg stages are not sensitive to ecdysone. In P. regina the application of JH to females with developing primary oöcytes stimulates yolk deposition in the penultimate oöcytes.     

Functional symmetry of the β-galactoside carrier in escherichia coli

Cytoplasmic membrane vesicles with either normal or inverted orientation were prepared from Escherichia coli. The lactose transport activity of these vesicle preparations was compared. The parameters measured were net efflux, counterflux, and K⁺/valinomycin-induced active uptake of lactose. With membrane vesicles derived from both wild-type and cytochrome-deficient strains the right-side-out and inverted membrane preparations showed similar rates of lactose flux in all assays. According to these criteria, the activity of the β-galactoside transport protein is inherently symmetrical.One major difference was observed between the native and inverted vesicle preparations: the inverted vesicles had approximately twice the specific activity of native vesicles in the counterflux and K⁺/valinomycin-induced uptake assays. This difference can be largely ascribed to the presence in the normal vesicle preparation of vesicles with a high passive permeability to lactose. Such vesicles are apparently absent from the inverted vesicle preparations.     

β-galactosidase activity in the germinating seeds of Vigna sinensis

The β-galactosidase activity in cotyledons of Vigna sinensis increases during seed germination and is inhibited by cycloheximide. The increasing activity may be due to the de novo synthesis of enzyme protein. The enzyme has been partially purified by gel filtration and characterized with respect to some biochemical parameters. The optimum pH and optimum temperature are 4.5 and 55°, respectively and the enzymes follows typical Michaelis kinetics with K_m and V_max of 4.5 x 10^?4 M and 2.0 x 10^?5 mol/hr respectively. K_i for galactose and lactose are 4.5 and 220 mM, respectively. The energy of activation of the enzyme for p-nitrophenyl β-D-galactoside is 9.83 kcal/mol. The apparent relative MW of the enzyme as determined by gel filtration was 56000.     

Determination of the CCR5∆32 frequency in Emiratis and Tunisians and the screening of the CCR5 gene for novel alleles in Emiratis

Background

The chemokine receptor components play crucial roles in the immune system and some of them serve as co-receptors for the HIV virus. Several studies have documented that variants in chemokine receptors are correlated with susceptibility and resistance to infection with HIV virus. For example, mutations in the chemokine receptor 5 gene (CCR5) resulting in loss-of-function (such as the homozygous CCR5?32) confer high degree of resistance to HIV infection. Heterozygotes for these variants exhibit slow progression to AIDS. The prevalence of CCR5 polymorphisms varies among ethnic and geographical groups. For example, the CCR5?32 variant is present in 10–15% of north Europeans but is rarely encountered among Africans. This study aims to identify the prevalence of some CCR5 variants in two geographically distant Arab populations (namely Emiratis and Tunisians).

Methodology

The prevalence of CCR5 gene variants including CCR5?32, FS299, C101X, A29S and C178R has been determined using PCR and direct DNA sequencing. A total of 403 unrelated healthy individuals (253 Emiratis and 150 Tunisians) were genotyped for the CCR5?32 variant using PCR amplification and gel electrophoresis. In addition, 200 Emiratis have been screened for other SNPs using Sanger DNA sequencing.

Results

Among Emiratis, the allele frequency of the CCR5?32 variant has been found to be 0.002. In addition, two variants L55Q and A159 were found at a frequency of 0.002. Moreover, the prevalence of the CCR5?32 variant in Tunisians was estimated to be 0.013 which is relatively higher than its frequency in Emiratis but lower than Europeans.

Conclusion

We conclude that the allele frequency of the most critical CCR5 polymorphism (?32) is extremely low among Emiratis compared to other Arabs and North Europeans. In addition, very low allele frequencies of other CCR5 polymorphisms have been detected among Emiratis.     

Environmental control of clonal growth in Carex nigra: What can be masked under the name Carex nigra subsp. juncella in the Czech Republic?

Carex nigra plants forming elevated dense tussocks are often named C. nigra subsp. juncella, as opposed to rhizomatous C. nigra subsp. nigra. It is uncertain, however, whether the cespitose growth form is a hereditary trait useful for definition of the distinct taxon or a site modification of little taxonomic value. We used vegetation analyses (phytosociological relevés) to reveal main patterns in ecological demands of the cespitose C. nigra plants in the Czech Republic, and three cultivation experiments to assess changes in clonal growth of C. nigra under various environmental conditions. In the field the cespitose C. nigra plants were typically found in abandoned wet meadows near open water, whereas the rhizomatous morphotypes frequently occurred also in regularly mown wet meadows and in peat bogs. The cespitose growth form disappeared in the cultivations, and the rhizome system responded plastically to immediate environmental stimuli. Number of rhizome branches and mean rhizome length decreased after defoliation of aboveground parts and denudation of belowground parts, whereas increased due to inundation. In the population from a cold site in high altitude (Modrava, Šumava Mts.), however, the originally cespitose plants repeatedly produced shorter and less numerous rhizome branches than the rhizomatous plants cultivated in the same conditions. This suggests ecotypic (genetic) differentiation in some populations of C. nigra, driven by environmental selection for more compact growth form in climatically severe sites. The cespitose C. nigra plants thus arise polytopically, by different mechanisms. The growth form itself therefore cannot serve as the character reliably delimiting C. nigra subsp. juncella as the distinct taxon.     

Darwin teleologist? Design in the Orchids

Focusing on the Orchids, this article aims at disentangling the concepts of teleology, design and natural theology. It refers to several contemporary critics of Darwin (Kölliker, Argyll, Royer, Candolle, Delpino) to challenge Huxley's interpretation that Darwin's system was “a deathblow” to teleology. The Orchids seem rather to be a “flank-movement” (Gray): it departs from the Romantic theories of transmutation and the “imaginary examples” of the Origin; it focuses on empirical data and on teleological structures. Although Darwin refers to natural selection, his readers mock him for his fascination for delicate morphological contrivances and co-adaptations – a sign that he was inescapably lured to finality. Some even suggested that his system was a “theodicy”. In the history of Darwinism, the Orchids reveal “another” quite unexpected and heterodox Darwin: freed from the hypothetical fancies of the Origin, and even suggesting a new kind of physico-theology.     

Variations of the lipid composition during the formation of cysts in the green alga Protosiphon botryoïdes

The formation of cysts in the alga Protosiphon botryoides, grown in a poor medium, is accompanied by important changes of the lipid composition of     

Rôle of phagostimulants of cotton leaves in the feeding behaviour of Spodoptera littoralis

Petroleum ether extract of cotton leaves was the most attractive to the hatched larvae of Spodoptera littoralis, and it remained so even after removal of the chlorophyll and other pigments. Steam distillation of this filtrate gave a volatile oil which also proved highly attractive to the larvae. In this fraction, 12 components were identified by thin-layer chromatography. Of these components, α-terpineol, citronellol and α-pinene were the most attractive, whereas humulene and linalool were less attractive. The remaining non-volatile lipid fraction was also attractive to the larva. In this fraction the unsaponifiable matter contained the attractive ingredients. This fraction proved to be a hydrocarbon. The infrared spectrum and nuclear magnetic resonance showed only typical hydrocarbon bonds.     

Observations on the histopathology of a systemic ciliate (Paranophrys sp.?) disease in the Dungeness crab, Cancer magister

Infections of the hemolymph of crabs by ciliates have been known for almost a century. Originally placed in the genus Anophrys, these crab-parasitizing ciliates have been recently transferred to the genus Paranophrys. Infections were long thought to be confined to the hemolymph in living crabs, with death caused by consumption of all hemocytes. Histological examination of heavily infected, but living, Dungeness crabs demonstrate that the ciliates actively invade and probably consume many tissues of the host prior to death rather than saprophytically feeding on the decomposing tissues subsequent to death as previously reported.     

Configuration of the carotenoid in the reaction centers of photosynthetic bacteria. Comparison of the resonance Raman spectrum of the reaction center of Rhodopseudomonas sphaeroides G1C with those of cis-trans isomers of β-carotene

The resonance Raman spectrum of the reaction center of Rhodopseudomonas sphaeroides G1C as well as those of the cis-trans isomers of β-carotene (all-trans, 9-cis, 13-cis, 15-cis and 9-cis, 13-cis- (or 9-cis, 13′-cis)) have been recorded at liquid N₂ temperature by use of the 457.9, 488.0 and 514.5 nm excitation lines. Comparison of the spectra indicated that the carotenoid in the reaction center takes the 15-cis configuration.     

Experimental analysis of the ‘Paarkultureffekt’ in the protandric polychaete, Ophryotrocha puerilis Clap. Mecz.

Female Ophyrotrocha puerilis Clap. Mecz. were coupled. Certain parts of the prostomium and of the pygidium in one or both partners of a couple were amputated in order to prove that they are responsible for the mutual influence which partners normally exert on their sexual differentiation. The results demonstrate that the palps and the ventrolateral prostomial cirri are the transmitters and the ciliated lateral pits on the prostomium the receivers of the mutual influence. The antennae and the pygidial cirri are not necessary as far as the ‘Paarkultureffekt’ is concerned. The nature of the stimulus is still unknown. At the present stage of these investigations, the favoured conception is of a pheromone transmitted during the contact of the partners.     

Chaperone-mediated native folding of a β-scorpion toxin in the periplasm of Escherichia coli

Background

Animal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria.

Methods

The β-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the Escherichia coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined.

Results

Western blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold.

Conclusions

The chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method.

General significance

We report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors.     

Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R_h) and reduced thermal stability in the mutant complex. Taken together, our data provide new structural insights into the β-hairpin’s role in stabilizing interactions between CyaA-ACD and N-CaM.     

Importance of spores and δ-endotoxin protein crystals of Bacillus thuringiensis in Galleria mellonella

Larvae of Galleria mellonella were fed on a honey-rich artificial food containing live spores or toxic crystals of Bacillus thuringiensis serotype V or various combinations of both. In this food; 1:1 combinations were 10 times more potent than live spores alone and about 10⁴ times more potent than crystals alone. Reduction in the proportion of spores, but not in that of crystals, decreased the slope of probit lines from 3.4 to 0.6. One or more factors in the spore are at least partly responsible for the potency of serotype V in G. mellonella. The results suggest than an observed gross loss of potency of this serotype in beehives is more likely to be due to death of spores than to deterioration of crystals. The reaction of G. mellonella to serotype V is nearest to that of a type 3 host species. Spores of serotype I are almost inactive in this host.