Selection for novel metabolic capabilities in Salmonella enterica

Bacteria are known to display extensive metabolic diversity and many studies have shown that they can use an extensive repertoire of small molecules as carbon‐ and energy sources. However, it is less clear to what extent a bacterium can expand its existing metabolic capabilities by acquiring mutations that, for example, rewire its metabolic pathways. To investigate this capability and potential for evolution of novel phenotypes, we sampled large populations of mutagenized Salmonella enterica to select very rare mutants that can grow on minimal media containing 124 low molecular weight compounds as sole carbon sources. We found mutants growing on 18 of these novel carbon sources, and identified the causal mutations that allowed growth for four of them. Mutations that relieve physiological constraints or increase expression of existing pathways were found to be important contributors to the novel phenotypes. For the remaining 14 novel phenotypes, whole genome sequencing of independent mutants and genetic analysis suggested that these novel metabolic phenotypes result from a combination of multiple mutations. This work, by virtue of identifying the genetic and mechanistic basis for new metabolic capabilities, sheds light on the properties of adaptive landscapes underlying the evolution of novel phenotypes.     

The complex barnacle perfume: identification of waterborne pheromone homologues in Balanus improvisus and their differential expression during settlement

A key question in barnacle biology is the nature of cues that induce gregarious settlement. One of the characterised cues is the waterborne settlement pheromone (WSP). This study aimed to identify WSP homologues in Balanus improvisus and to investigate their expression during settlement. Six WSP homologues were identified, all containing an N-terminal signal peptide, a conserved core region, and a variable C-terminus comprising several -GR- and -HDDH- motifs. The B. improvisus WSP homologues were expressed in all settlement stages but showed different expression patterns. The homologue most similar to the B. amphitrite WSP was the most abundant and was constantly expressed during settlement. In contrast, several of the other WSP homologues showed the greatest expression in the juvenile stage. The presence of several WSP homologues suggests the existence of a pheromone mix, where con-specificity might be determined by a combination of sequence characteristics and the concentration of the individual components.     

Sexual selection for bright females prevails under light pollution

The functions of sexually selected traits are particularly sensitive to changes in the environment because the traits have evolved to in-crease mating success u...     

Divide-and-conquer crystallographic approach towards an atomic structure of intermediate filaments

Intermediate filaments (IFs) represent an essential component of the cytoskeleton in higher eukaryotic cells. The elementary building block of the IF architecture is an elongated dimer with its dominant central part being a parallel double-stranded alpha-helical coiled coil. Filament formation proceeds via a specific multi-step association of the dimers into the unit-length filaments, which subsequently anneal longitudinally and finally radially compact into mature filaments. To tackle the challenge of a crystallographic structure determination, we have produced and characterised 17 overlapping soluble fragments of human IF protein vimentin. For six fragments ranging in length between 39 and 84 amino acid residues, conditions yielding macroscopic crystals could be established and X-ray diffraction data were collected to the highest resolution limit between 1.4 and 3 A. We expect that solving the crystal structures of these and further fragments will eventually allow us to patch together a molecular model for the full-length vimentin dimer. This divide-and-conquer approach will be subsequently extended to determining the crystal structures of a number of complexes formed by appropriate vimentin fragments, and will eventually allow us to establish the three- dimensional architecture of complete filaments at atomic resolution.     

A single nucleotide change in the 5' noncoding region of Sindbis virus confers neurovirulence in rats

Two pairs of Sindbis virus (SV) variants that differ in their neuroinvasive and neurovirulent traits in mice have been isolated. Recently, we mapped the genetic determinants responsible for neuroinvasiveness in weanling mice. Here, we extend this study to newborn and adult rats and to rat neuronal cultures. Remarkably, certain aspects of the pathogenesis of these strains in rats were found to be quite distinct from the mouse model. Suckling rats were susceptible to all four isolates, and replication in the brain was observed after both intraperitoneal and intracranial (i.c.) inoculation. None of the isolates was neuroinvasive in adult rats, although all replicated after i.c. inoculation. For the isolate pair that was highly neurovirulent in mice, SVN and SVNI, only SVNI caused death after i.c. inoculation of adult rats. Similarly, only SVNI was cytotoxic for primary cultures of mature neurons. The genetic determinants responsible for the pathogenic properties of SVNI were mapped to the E2 glycoprotein and the 5' noncoding region (5'NCR). Substitution of two amino acids in SVN E2 with the corresponding residues of SVNI (Met-190 and Lys-260) led to paralysis in 3- and 5-week-old rats. More dramatically, a single substitution in the 5'NCR of SVN (G at position 8) transformed the virus into a lethal pathogen for 3-week-old rats like SVNI. In 5-week-old rats, however, this recombinant was attenuated relative to SVNI by 2 orders of magnitude. Combination of the E2 and 5'NCR determinants resulted in a recombinant with virulence properties indistinguishable from those of SVNI. These data indicate that the 5'NCR and E2 play an instrumental role in determining the age-dependent pathogenic properties of SV in rats.     

A possible role for p190RhoGAP in PKCepsilon-induced morphological effects

We have previously shown that protein kinase C (PKC) epsilon induces neurite outgrowth via its regulatory domain. This is accompanied by PKC-induced stress fibre loss. Here, we show that the regulatory domain (RD) of PKCepsilon induces processes also in NIH-3T3 fibroblasts, similar to what has been observed with p190RhoGAP. This study also shows that p190RhoGAP induces neurite outgrowth in SK-N-BE(2) neuroblastoma cells. We therefore investigated whether p190RhoGAP may be downstream of PKCepsilon. We could detect a co-localization of p190RhoGAP and PKCepsilon at membrane ruffles and an increased association between the proteins in fibroblasts treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). The association is also seen in neuroblastoma cells, and nerve growth factor (NGF) treatment of SH-SYSY/TrkA cells decreases the interaction. However, overexpressed PKCepsilon did not coprecipitate overexpressed p190RhoGAP in CHO cells, indicating that the proteins do not interact directly. This raises the possibility that p190RhoGAP is involved in mediating the morphological effects of PKCepsilon.     

Lhca5 – an LHC-Type Protein Associated with Photosystem I

The light-harvesting antenna of higher plant photosystem (PS) I is known to be composed of four different types of light-harvesting complex (LHC) proteins (Lhca1-4). However, the genomic sequence of Arabidopsis thaliana contains open reading frames coding for two additional LHC type proteins (Lhca5-6) that are presumably associated with PSI. While Lhca6 might not be expressed at all, ESTs have been detected for the Lhca5 gene in Arabidopsis and a number of other plant species. Here we demonstrate the presence of the Lhca5 gene product in the thylakoid membrane of Arabidopsis as an additional type of Lhca-protein associated with PSI. Lhca5 seems to be regulated differently from the other LHC proteins since Lhca5 mRNA levels increase under high light conditions. Analyses reported here of Lhca5 in plants lacking individual Lhca1-4 proteins show that it is more abundant in plants lacking Lhca1/4, and suggest that it interacts in a direct physical fashion with Lhca2 or Lhca3. We propose that Lhca5 binds chlorophylls in a similar fashion to the other Lhca proteins and is associated with PSI only in sub-stoichiometric amounts.     

New fatty acid oxidation inhibitors with increased potency lacking adverse metabolic and electrophysiological properties

New inhibitors of palmitoylCoA oxidation were synthesized based on a structurally novel lead, CVT-3501 (1). Investigation of structure-activity relationships was conducted with respect to potency of inhibition of cardiac mitochondrial palmitoylCoA oxidation and metabolic stability. Potent and metabolically stable analogues 33, 42, and 43 were evaluated in vitro for cytochrome P450 inhibition and potentially adverse electrophysiological effects. Compound 33 was also found to have favorable pharmacokinetic properties in rat.     

CVT-4325: a potent fatty acid oxidation inhibitor with favorable oral bioavailability

New inhibitors of palmitoyl-CoA oxidation are based on the introduction of nitrogen heterocycles in the ‘Western Portion’ of the molecule. SAR studies led to the discovery of CVT-4325 (shown), a potent FOXi (IC₅₀ = 380 nM rat mitochondria) with favorable PK properties (F = 93%, t_1/2 = 13.6 h, dog).     

Single nucleotide polymorphisms in the apolipoprotein B and low density lipoprotein receptor genes affect response to antihypertensive treatment

Background

Dyslipidemia has been associated with hypertension. The present study explored if polymorphisms in genes encoding proteins in lipid metabolism could be used as predictors for the individual response to antihypertensive treatment.

Methods

Ten single nucleotide polymorphisms (SNP) in genes related to lipid metabolism were analysed by a microarray based minisequencing system in DNA samples from ninety-seven hypertensive subjects randomised to treatment with either 150 mg of the angiotensin II type 1 receptor blocker irbesartan or 50 mg of the β₁-adrenergic receptor blocker atenolol for twelve weeks.

Results

The reduction in blood pressure was similar in both treatment groups. The SNP C711T in the apolipoprotein B gene was associated with the blood pressure response to irbesartan with an average reduction of 19 mmHg in the individuals carrying the C-allele, but not to atenolol. The C16730T polymorphism in the low density lipoprotein receptor gene predicted the change in systolic blood pressure in the atenolol group with an average reduction of 14 mmHg in the individuals carrying the C-allele.

Conclusions

Polymorphisms in genes encoding proteins in the lipid metabolism are associated with the response to antihypertensive treatment in a drug specific pattern. These results highlight the potential use of pharmacogenetics as a guide for individualised antihypertensive treatment, and also the role of lipids in blood pressure control.