Pseudomonas fluorescens' view of the periodic table

Growth in a biofilm modulates microbial metal susceptibility, sometimes increasing the ability of microorganisms to withstand toxic metal species by several orders of magnitude. In this study, a high-throughput metal toxicity screen was initiated with the aim of correlating biological toxicity data in planktonic and biofilm cells to the physiochemical properties of metal ions. To this end, Pseudomonas fluorescens ATCC 13525 was grown in the Calgary Biofilm Device (CBD) and biofilms and planktonic cells of this microorganism were exposed to gradient arrays of different metal ions. These arrays included 44 different metals with representative compounds that spanned every group of the periodic table (except for the halogens and noble gases). The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentration (MBEC) values were obtained after exposing the biofilms to metal ions for 4 h. Using these values, metal ion toxicity was correlated to the following ion-specific physicochemical parameters: standard reduction-oxidation potential, electronegativity, the solubility product of the corresponding metal–sulfide complex, the Pearson softness index, electron density and the covalent index. When the ions were grouped according to outer shell electron structure, we found that heavy metal ions gave the strongest correlations to these parameters and were more toxic on average than the other classes of the ions. Correlations were different for biofilms than for planktonic cells, indicating that chemical mechanisms of metal ion toxicity differ between the two modes of growth. We suggest that biofilms can specifically counter the toxic effects of certain physicochemical parameters, which may contribute to the increased ability of biofilms to withstand metal toxicity.     

Prostate Tumor-Derived Exosomes Down-Regulate NKG2D Expression on Natural Killer Cells and CD8+ T Cells: Mechanism of Immune Evasion

Tumor-derived exosomes, which are nanometer-sized extracellular vesicles of endosomal origin, have emerged as promoters of tumor immune evasion but their role in prostate cancer (PC) progression is poorly understood. In this study, we investigated the ability of prostate tumor-derived exosomes to downregulate NKG2D expression on natural killer (NK) and CD8⁺ T cells. NKG2D is an activating cytotoxicity receptor whose aberrant loss in cancer plays an important role in immune suppression. Using flow cytometry, we found that exosomes produced by human PC cells express ligands for NKG2D on their surface. The NKG2D ligand-expressing prostate tumor-derived exosomes selectively induced downregulation of NKG2D on NK and CD8⁺ T cells in a dose-dependent manner, leading to impaired cytotoxic function in vitro. Consistent with these findings, patients with castration-resistant PC (CRPC) showed a significant decrease in surface NKG2D expression on circulating NK and CD8⁺ T cells compared to healthy individuals. Tumor-derived exosomes are likely involved in this NKG2D downregulation, since incubation of healthy lymphocytes with exosomes isolated from serum or plasma of CRPC patients triggered downregulation of NKG2D expression in effector lymphocytes. These data suggest prostate tumor-derived exosomes as down-regulators of the NKG2D-mediated cytotoxic response in PC patients, thus promoting immune suppression and tumor escape.     

Myosin-X provides a motor-based link between integrins and the cytoskeleton

Unconventional myosins are actin-based motors with a growing number of attributed functions. Interestingly, it has been proposed that integrins are transported by unidentified myosins to facilitate cellular remodelling. Here we present an interaction between the unconventional myosin-X (Myo10) FERM (band 4.1/ezrin/radixin/moesin) domain and an NPXY motif within beta-integrin cytoplasmic domains. Importantly, knock-down of Myo10 by short interfering RNA impaired integrin function in cell adhesion, whereas overexpression of Myo10 stimulated the formation and elongation of filopodia in an integrin-dependent manner and relocalized integrins together with Myo10 to the tips of filopodia. This integrin relocalization and filopodia elongation did not occur with Myo10 mutants deficient in integrin binding or with a beta(1)-integrin point mutant deficient in Myo10 binding. Taken together, these results indicate that Myo10-mediated relocalization of integrins might serve to form adhesive structures and thereby promote filopodial extension.     

Copulatory courtship signals male genetic quality in cucumber beetles

In the spotted cucumber beetle, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae), males court females during copulation by stroking them with their antennae. Stroking occurs exclusively during the first stages of copulation, after a male has penetrated a female's vaginal duct but before he is allowed access to her bursa copulatrix. Females accept the spermatophore of fast-stroking males and reject those of slow-stroking males by relaxing or constricting muscles distorting the vaginal duct. Here, we measure the repeatability of stroking behaviour within males, examine the effect of losing one antenna on male attractiveness and test whether such female control results in direct phenotypic benefits for the discriminating female or indirect genetic benefits that appear in her offspring. We also use a half-sibling design to quantify the variance and heritability of stroking speed and endurance. Female beetles were paired with a male that was known to stroke either quickly or slowly. No difference was found in the resulting fecundity or egg-hatching rate of the females, or in the survivorship, development rate, size, age at first reproduction or fecundity of their offspring indicating that no direct benefits are gained by discriminating among males on the basis of stroking speed. There were, however, good-genes benefits for the mates of fast-stroking males. Offspring of fast-stroking fathers were also fast strokers and were more likely to be accepted as mates than offspring of slow-stroking fathers. There was substantial variance among sires in stroking speed and endurance and the heritability of each trait was high. The antennal stroking rate was highly repeatable in successive mating attempts and males with only one antenna were not accepted as mates. The repeatability within males, variability between males and heritability between generations of copulatory stroking combine to provide females with a reliable and honest signal of the genetic quality of courting males.     

The J-domain of Hsp40 couples ATP hydrolysis to substrate capture in Hsp70

The Escherichia coli Hsp40 DnaJ uses its J-domain to target substrate polypeptides for binding to the Hsp70 DnaK, but the mechanism of J-domain function has been obscured by a substrate-like interaction between DnaJ and DnaK. ATP hydrolysis in DnaK is associated with a conformational change that captures the substrate, and both DnaJ and substrate can stimulate ATP hydrolysis. However, substrates cannot trigger capture by DnaK in the presence of ATP, and substrates stimulate a DnaK conformational change that is uncoupled from ATP hydrolysis. The role of the J-domain was examined using the fluorescent derivative of a fusion protein composed of the J-domain and a DnaK-binding peptide. In the absence of ATP, DnaK-binding affinity of the fusion protein is similar to that of the unfused peptide. However, in the presence of ATP, the affinity of the fusion protein is dramatically increased, which is opposite to the decrease in DnaK affinity typically exhibited by peptides. Binding of a fusion protein that contains a defective J-domain is insensitive to ATP. According to results from isothermal titration calorimetry, the J-domain binds to the DnaK ATPase domain with weak affinity (K(D) = 23 microM at 20 degrees C). The interaction is characterized by a positive enthalpy, small heat capacity change (DeltaC(p)= -33 kcal mol(-1)), and increasing binding affinity for increasing temperatures in the physiological range. In conditions that support binding of the J-domain to the ATPase domain, the J-domain accelerates ATP hydrolysis and a simultaneous conformational change in DnaK that is associated with peptide capture. The defective J-domain is inactive, despite the fact that it binds to the DnaK ATPase domain with higher than wild-type affinity. The results are most consistent with an allosteric mechanism of J-domain action in which the J-domain couples ATP hydrolysis to peptide capture by accelerating ATP hydrolysis and delaying DnaK closure until ATP is hydrolyzed.     

Aquatic biodiversity and saline lakes: Lake Bogoria National Reserve,Kenya

Lake Bogoria, in the Rift Valley of Kenya is an extreme saline lake (conductivity 40–80 mS cm^–1, alkalinity 1500 m equ l^–1). It is hydrologically more stable than the other, endorheic lakes in Kenya, because it is deep – maximum depth at present just over 10 m in an area of 3000 ha – and so does not have periods when it is dry. It is ecologically simple, with only one species dominating the phytoplankton – the cyanobacterium `spirulina', Arthrospira fusiformis. Its biomass and productivity were very high – biomass between 38 and 365 g l<sup>–1</sup> chlorophyll `a' and 3.4–21 × 10³ coils ml^–1 and net production between 0.24 and 1 gm C m³ h, the latter in a narrow zone of less than a metre. There were no macro-zooplankton in the plankton and the only grazer of A. fusiformis was the lesser flamingo, Phoeniconaias minor,which occurred irregularly in very high concentrations (in excess of 1 × 10⁶). Detritivory in the benthos was effected by a single chironomid species, Paratendipes sp., at a maximum density of 4 × 10⁴ m^–2. The mean daily emergence of adult chironomids was estimated to be 1 × 10³ m^–2, the maximum 3. There was no littoral plant community within the lake but 44 dicotyledonous and 31 monocotyledonous plant species in the drawn-down zone and adjacent to it. A diverse draw-down terrestrial invertebrate fauna, only superficially described here, processed the flamingo feathers and carcasses, with other detritus such as chironomid pupal exuviae and decaying A. fusiformis scum. About 50 bird species depended upon the chironomids, either as they emerged through the water column as flying adults or later on the shoreline as floating pupal exuvia and dead adults. The lake has high conservation value because of three bird species in particular – lesser flamingo, Cape teal and black-necked grebe. The former provides real economic value in a region otherwise impoverished, because of the spectacle of tens of thousands of flamingos set against the landscape of hot springs and fumaroles at the lake edge, which draws 15000 visitors per annum. P. minor has experienced three periods during the past ten years when major mortalities have occurred, the last of which killed 700 birds day^–1. This could have involved as many as 200000 birds (about 1/5th of the maximum population at this lake) if mortality was at a constant rate for the nine months it was observed. Causes of mortality have been suggested as avian tuberculosis, poisoning from cyanobacterial toxins or from heavy metal contamination at Lake Nakuru, but it is still not yet clear what contribution each makes to the problem.     

In vivo assay for low-activity mutant forms of Escherichia coli ribonucleotide reductase

Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells. In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic Escherichia coli cells. The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR. This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays. E. coli RNR is composed of two homodimeric proteins, R1 and R2. The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site. The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues. Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity. In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.