Directionality of auditory nerve fiber responses to pure tone stimuli in the grassfrog, Rana temporaria. II. Spike timing

We studied the directionality of spike timing in the responses of single auditory nerve fibers of the grass frog, Rana temporaria, to tone burst stimulation. Both the latency of the first spike after stimulus onset and the preferred firing phase during the stimulus were studied. In addition, the directionality of the phase of eardrum vibrations was measured. The response latency showed systematic and statistically significant changes with sound direction at both low and high frequencies. The latency changes were correlated with response strength (spike rate) changes and were probably the result of directional changes in effective stimulus intensity. Systematic changes in the preferred firing phase were seen in all fibers that showed phaselocking (i.e., at frequencies below 500–700 Hz). The mean phase lead for stimulation from the contralateral side was approximately 140° at 200 Hz and decreased to approximately 100° at 700 Hz. These phaseshifts correspond to differences in spike timing of approximately 2 ms and 0.4 ms respectively. The phaseshifts were nearly independent of stimulus intensity. The phase directionality of eardrum vibrations was smaller than that of the nerve fibers. Hence, the strong directional phaseshifts shown by the nerve fibers probably reflect the directional characteristics of extratympanic pathways. Accepted: 23 November 1996     

High Organic Carbon Export Precludes Eutrophication Responses in Experimental Rocky Shore Communities

We studied the effect of nutrient inputs on the carbon (C) budget of rocky shore communities using a set of eight large experimental mesocosms. The mesocosms received a range of inorganic nitrogen (N) and phosphorus (P) additions, at an N:P ratio of 16. These additions were designed to elevate the background concentration, relative to that in eutrophic Oslofjord (Norway) waters, by 1, 2, 4, 8, 16, 32 μmol dissolved inorganic nitrogen (DIN)l⁻¹ (and the corresponding P increase). Two unamended mesocosms were used as controls. The nutrients were added continuously for 27 months before gross primary production (GPP), respiration (R), net community production (NCP), and dissolved organic carbon (DOC) production were assessed for the dominant algal species (Fucus serratus) and for the whole experimental ecosystem. Inputs and outputs of DOC and particulate organic carbon (POC) from the mesocosms were also quantified. The F. serratus communities were generally autotrophic (average P/R ratio = 1.33 ± 0.12), with the GPP independent of the nutrient inputs to the mesocosms, and maintained a high net DOC production during both day (0.026 ± 0.008 g C m⁻² h⁻¹) and night (0.015 ± 0.004 g C m⁻² h⁻¹). All the experimental rocky shore ecosystems were autotrophic (P/R ratio = 2.04 ± 0.28), and neither macroalgal biomass nor production varied significantly with increasing nutrient inputs. Most of the excess production from these autotrophic ecosystems was exported from the systems as DOC, which accounted for 69% and 58% of the NCP of the dominant community and the experimental ecosystem, respectively, the rest being lost as POC. High DOC release and subsequent export from the highly energetic environments occupied by rocky shore communities may prevent the development of eutrophication symptoms and render these communities resistant to eutrophication. Received 10 October 2001; accepted 18 July 2002.     

A Combined Zinc/Cadmium Sensor and Zinc/Cadmium Export Regulator in a Heavy Metal Pump

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn²⁺ nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn²⁺ and Cd²⁺ chelator with capacity to bind 10 Zn²⁺ ions per C terminus. When AtHMA4 is expressed in a Zn²⁺-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn²⁺ and Cd²⁺ tolerance and lowered Zn²⁺ and Cd²⁺ content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn²⁺ and Cd²⁺ chelator (sensor) and as a regulator of the efficiency of Zn²⁺ and Cd²⁺ export. The identification of a post-translational handle on Zn²⁺ and Cd²⁺ transport efficiency opens new perspectives for regulation of Zn²⁺ nutrition and tolerance in eukaryotes.     

In silico prediction of human pathogenicity in the γ-proteobacteria

Background

Although the majority of bacteria are innocuous or even beneficial for their host, others are highly infectious pathogens that can cause widespread and deadly diseases. When investigating the relationships between bacteria and other living organisms, it is therefore essential to be able to separate pathogenic organisms from non-pathogenic ones. Using traditional experimental methods for this purpose can be very costly and time-consuming, and also uncertain since animal models are not always good predictors for pathogenicity in humans. Bioinformatics-based methods are therefore strongly needed to mine the fast growing number of genome sequences and assess in a rapid and reliable way the pathogenicity of novel bacteria.

Methodology/Principal Findings

We describe a new in silico method for the prediction of bacterial pathogenicity, based on the identification in microbial genomes of features that appear to correlate with virulence. The method does not rely on identifying genes known to be involved in pathogenicity (for instance virulence factors), but rather it inherently builds families of proteins that, irrespective of their function, are consistently present in only one of the two kinds of organisms, pathogens or non-pathogens. Whether a new bacterium carries proteins contained in these families determines its prediction as pathogenic or non-pathogenic. The application of the method on a set of known genomes correctly classified the virulence potential of 86% of the organisms tested. An additional validation on an independent test-set assigned correctly 22 out of 24 bacteria.

Conclusions

The proposed approach was demonstrated to go beyond the species bias imposed by evolutionary relatedness, and performs better than predictors based solely on taxonomy or sequence similarity. A set of protein families that differentiate pathogenic and non-pathogenic strains were identified, including families of yet uncharacterized proteins that are suggested to be involved in bacterial pathogenicity.     

The Abi Proteins and Their Involvement in Bacteriocin Self-Immunity

The Abi protein family consists of putative membrane-bound metalloproteases. While they are involved in membrane anchoring of proteins in eukaryotes, little is known about their function in prokaryotes. In some known bacteriocin loci, Abi genes have been found downstream of bacteriocin structural genes (e.g., pln locus from Lactobacillus plantarum and sag locus from Streptococcus pyogenes), where they probably are involved in self-immunity. By modifying the profile hidden Markov model used to select Abi proteins in the Pfam protein family database, we show that this family is larger than presently recognized. Using bacteriocin-associated Abi genes as a means to search for novel bacteriocins in sequenced genomes, seven new bacteriocin-like loci were identified in Gram-positive bacteria. One such locus, from Lactobacillus sakei 23K, was selected for further experimental study, and it was confirmed that the bacteriocin-like genes (skkAB) exhibited antimicrobial activity when expressed in a heterologous host and that the associated Abi gene (skkI) conferred immunity against the cognate bacteriocin. Similar investigation of the Abi gene plnI and the Abi-like gene plnL from L. plantarum also confirmed their involvement in immunity to their cognate bacteriocins (PlnEF and PlnJK, respectively). Interestingly, the immunity genes from these three systems conferred a high degree of cross-immunity against each other''s bacteriocins, suggesting the recognition of a common receptor. Site-directed mutagenesis demonstrated that the conserved motifs constituting the putative proteolytic active site of the Abi proteins are essential for the immunity function of SkkI, and to our knowledge, this represents a new concept in self-immunity.Bacteriocins are ribosomally synthesized antimicrobial peptides and proteins produced by a wide variety of bacterial genera. The majority of bacteriocins from Gram-positive bacteria are classified into two groups: the class I lantibiotics, containing posttranslationally modified peptides with ring-forming lanthionine or methyllanthionine residues, and the nonmodified class II peptide bacteriocins (8, 33, 34). Class II bacteriocins are further subdivided into pediocin-like bacteriocins (class IIa), two-peptide bacteriocins (class IIb), and nonpediocin one-peptide bacteriocins (class IIc) (33). Bacteriocin-producing bacteria normally possess a mechanism of immunity to protect themselves from their own bacteriocins, and such self-immunity is often mediated by a dedicated protein (32). For a few bacteriocin systems, the mechanisms by which these proteins confer immunity have been elucidated. For instance, immunity to the lantibiotic nisin (class I) involves a combined action which includes (i) sequestering of bacteriocins on the bacterial cell membrane by a protein called NisI and (ii) removal of the bacteriocins from cells by a dedicated ABC transporter (NisFEG) (39, 44). On the other hand, proteins conferring immunity to pediocin-like bacteriocins (class IIa) as well as lactococcins A and B (class IIc) have been shown to bind directly to the bacteriocin receptor and thereby inhibit pore formation (13). Hitherto, no immunity mechanism is known for any class IIb two-peptide bacteriocins.Recently, we reported that several bacteriocin loci encode proteins belonging to the Pfam Abi protein family (Pfam accession no. PF02517) (14). These loci include the plantaricin (pln) locus of Lactobacillus plantarum, encoding two two-peptide bacteriocins (12), the multibacteriocin pnc locus of Streptococcus pneumoniae (25), and the streptolysin S (sag) locus found in group A streptococci (35) (Fig. ​(Fig.1A).1A). Some of the Abi proteins encoded in these loci (PlnI in L. plantarum, PncO in S. pneumoniae, and SagE in Streptococcus pyogenes) are probable bacteriocin self-immunity proteins on the basis of gene knockout studies (10, 25) and genetic organization (i.e., being closely associated with bacteriocin structural genes), while others (e.g., PlnP and PlnTUVW in L. plantarum and PncP in S. pneumoniae) have completely unknown functions.Open in a separate windowFIG. 1.Abi-associated bacteriocin and bacteriocin-like loci. (A) Three known bacteriocin loci containing Abi genes, including the pln locus of L. plantarum (14), the pnc locus of S. pneumoniae (25), and the sag locus of S. pyogenes (10). (B) Seven potential new bacteriocin loci identified by genome mining for bacteriocin-associated Abi genes. Abi genes are shown as black arrows, bacteriocin structural genes are shown in red, ABC transporter genes are shown in blue, regulatory genes are shown in green, and other genes are shown in white. Gene names or locus tags are shown below the arrows. The genes are drawn approximately to scale. The boxed arrows in the L. sakei 23K genome represent a disrupted histidine protein kinase gene.The Abi family, also known as the CAAX amino-terminal protease family, consists of putative membrane-bound metalloproteases from both eukaryotes and prokaryotes (38), with the majority being bacterial proteins (90%). The Abi family is recognized by three highly conserved motifs (38): motif 1, consisting of two neighboring and invariant glutamate residues and a conserved arginine separated by three residues (EEXXXR, where X denotes any amino acid); motif 2, consisting of a conserved phenylalanine and a conserved histidine separated by three residues (FXXXH); and motif 3, with an invariant histidine. The three conserved motifs are thought to constitute the active site of the Abi protease, and their importance in proteolytic activity has been demonstrated by mutational analysis of the yeast Abi protease RCE1 (15). In eukaryotic cells, Abi family proteins are involved in membrane targeting of proteins harboring the C-terminal sequence CAAX (C, cysteine; A, aliphatic amino acid; and X, any amino acid) via a process known as prenylation, which consists of the following three sequential reactions (23): (i) a geranylgeranyltransferase/farnesyltransferase attaches a prenyl group (lipid anchor) to the cysteine in the fourth-to-last position, (ii) a CAAX protease of the Abi family cleaves off the three C-terminal amino acids (-AAX), and (iii) an isoprenylcysteine carboxyl methyltransferase then attaches a methyl group to the C-terminal cysteine.Despite being widespread in prokaryotic genomes, the function of Abi proteins has not been investigated much in bacteria, with the exception of gene knockout experiments on the Abi genes sagE and pncO, which implies their involvement in bacteriocin self-immunity in streptococci (10, 25). Another Abi-like gene, prsW in Bacillus subtilis, has been studied to some extent (16). PrsW does not belong to any Pfam family but contains the same three motifs as the Abi proteins, with the exception of the conserved histidine in motif 2, which has been replaced by a glutamate in this protein. PrsW is a protease involved in response to antimicrobial peptides via a process known as regulated intracellular proteolysis (16). In this process, PrsW together with another protease (YluC) cleaves an anti-σ factor (RsiW) to release σ^W, which subsequently regulates gene expression in a manner that protects the cells from antimicrobial peptides. However, the PrsW target protein RsiW in B. subtilis does not harbor the C-terminal consensus sequence CAAX found for Abi target proteins in eukaryotes.Pfam (http://pfam.sanger.ac.uk/) is a comprehensive collection of protein families and domains. For each protein family in Pfam, a profile hidden Markov model (profile HMM) is built from an alignment of sequences from nonredundant representatives of the family (seed sequences), and this profile HMM is then used in an iterative fashion to find new members of the protein family (18, 43). The Abi family in Pfam contains a large number of sequences (1,966 by September 2009). However, several proteins containing Abi or Abi-like motifs are somehow not detected by the current search tool. Examples of this include PrsW from B. subtilis and PlnL from L. plantarum, which apparently contain all three motifs but somehow do not fit into Pfam''s Abi family. These observations suggest that the profile HMM for Abi may be based upon a slightly skewed sample of seed sequences, resulting in a low sensitivity. We provide here an updated profile HMM to detect Abi-like proteins in prokaryotes that are omitted in the present protein family. Furthermore, searches for novel Abi-associated bacteriocin loci were also performed in silico. Several potentially novel bacteriocin loci were identified, and one of them was assessed further by experimentation. The role of bacteriocin-associated Abi genes in self-immunity was also addressed by heterologous expression and site-directed mutagenesis.     

Modeling Mechanisms of Cell Secretion

Secretion is a fundamental cellular process involving the regulated release of intracellular products from cells. Physiological functions such as neurotransmission, or the release of hormones and digestive enzymes, are all governed by cell secretion. Anomalies in the processes involved in secretion contribute to the development and progression of diseases such as diabetes and other hormonal disorders. To unravel the mechanisms that govern such diseases, it is essential to understand how hormones, growth factors and neurotransmitters are synthesized and processed, and how their signals are recognized, amplified and transmitted by intracellular signaling pathways in the target cells. Here, we discuss diverse aspects of the detailed mechanisms involved in secretion based on mathematical models. The models range from stochastic ones describing the trafficking of secretory vesicles to deterministic ones investigating the regulation of cellular processes that underlie hormonal secretion. In all cases, the models are closely related to experimental results and suggest theoretical predictions for the secretion mechanisms.     

Spatio-temporal distribution patterns of the invasive macroalga Sargassum muticum within a Danish Sargassum-bed

Sargassum muticum was first observed in Scandinavia in Limfjorden (Denmark) in 1984, where it is now the most abundant and conspicuous macroalga. Despite the ecological importance of Sargassum, few studies have described seasonal patterns within Scandinavian Sargassum beds. We quantified the dynamics of macroalgae among years and seasons along a depth transect through a typical Sargassum bed in Limfjorden. The annual investigations (summer transects 1989–1999) showed a gradual increase in the dominance of Sargassum, especially at the 2–4-m depth interval. Significant seasonal dynamics in macroalgal abundance and assemblage structure were observed in this depth interval; the mean cover of Sargassum varied from ca. 5% (autumn and winter) to 25% (mid-summer). In comparison, encrusting algae had high and relatively stable covers throughout the year (ca. 20%). Other perennial macroalgae had low mean covers (<2%) characterized by a few patches of higher abundances. Except from a spring bloom, filamentous algae had low covers throughout the year. Within this relatively uniform bed, Sargassum abundance was positively related to boulders >10 cm in diameter and species richness was negatively correlated to depth and stones <10 cm in diameter, and non-correlated to other algal form-groups or grazer densities. Thus, in Limfjorden, the distribution of Sargassum is determined by large- (>6 m) and small-scale (<1 m) depth differences where low light limits Sargassum at depth, physical disturbance and sediment stress limits Sargasum in shallow waters, and the presence of stable boulder substratum facilitate Sargassum. Competition for space from other macroalgae and herbivory are probably of minor importance.     

High-pressure studies of the reaction mechanism of nitric-oxide synthase

Nitric-oxide synthase (NOS) generates nitric oxide from l-arginine in two reaction cycles with N(omega)-hydroxy-l-arginine as an obligate intermediate. Although much progress has been made in recent years in the elucidation of the reaction mechanism of NOS, many questions remain to be answered. The use of low temperature has been instrumental in the revelation of the mechanism of NO synthesis, particularly regarding the role of the cofactor 5,6,7,8-tetrahydrobopterin (BH4). High-pressure studies may be expected to be similarly useful, but have been very few so far. In this short review, we depict the present state of knowledge about the reaction mechanism of NO synthesis, and the role(s) BH4 plays in it. This exposition is followed by a summary of the results obtained thus far in high-pressure studies and of the conclusions that can be drawn from them.     

Recombinant human PPAR-beta/delta ligand-binding domain is locked in an activated conformation by endogenous fatty acids

High-resolution crystallographic structures of recombinant human peroxisome proliferator-activated receptor ligand-binding domain (isotype beta/delta) reveal a fatty acid in the binding site. Mass spectrometry confirmed the presence of C16:0, C16:1, C18:0 and C18:1 in a ratio of approximately 3:2:1:4 with 11, Z-octadecenoic acid (cis-vaccenic acid) identified as the predominant species. These are endogenous fatty acids acquired from the bacterial expression system, and serve to lock the ligand-binding domain into the activated conformation. A requirement for crystal growth, the additive n-heptyl-beta-d-glucopyranoside, binds near the activation function helix where recognition of co-activator proteins occurs. Our observations suggest potential physiological ligands for human PPAR-beta/delta and highlight that reported binding studies must be treated with caution unless endogenous fatty acids have been removed from the sample prior to analysis.     

Evaluation of the ‘maximum price paid’ as an index of motivational strength for farmed silver foxes (Vulpes vulpes)

To measure farmed foxes’ motivations for full, naturalistic social contact, we constructed an apparatus where they could perform an operant to access stimuli, but then leave freely and thence determine their own bout lengths. Motivational measures based on demand curves can be invalid in such set-ups, and we therefore sought to validate the measure ‘maximum price paid’. This was achieved by measuring six silver fox males’ maximum operant responding for access or proximity to three resources differing in biological significance: food, vixens in oestrus and males. We predicted that if valid, maximum price paid would be highest for food and vixens. Maximum price were 970 ± 399 (S.E.) for food, 677 ± 173 (S.E.) for vixens and 389 ± 101 (S.E.) for other males (P < 0.05). In contrast, our complementary measures of motivation – price elasticity, expenditure and consumer surplus – did not differentiate between the resources, and ranked them in different orders (albeit not significantly). This was because the foxes rescheduled their behaviour with increasing costs, decreasing bout number while increasing bout length, to different extents with the three resources. Additional findings showed that all subjects ‘overpaid’, performing the operant response more than was required. This increased as the costs increased, perhaps due to increasing ‘time outs’ on the time-restricted schedule (DRH) as the task got harder. However, the overpayment was also highest when males were the resource, suggesting that operant responding was slowest and least efficient when working for less-valued resources. The resources present also affected how the foxes used the rest of the apparatus and influenced their behaviour; subjects staying more in the operant compartment when the resource was social (especially a female), but retreating to a distant compartment when it was food. While proximity to oestrous vixens elicited higher levels of tail wagging and only low levels of pacing, indicating a positive motivation, proximity to males elicited relatively high levels of pacing plus agonistic gaping, suggesting that the motives for seeking contact with males related to aggression. Thus, although our operant set-up reveals a drive to approach other males, the possible aggressive motives suggest that this sort of social contact would not necessarily improve their welfare in a traditional housing system. Overall, these results help improve the design and interpretation of preference tests, and confirm maximum price paid as a useful motivational measure for farmed foxes.