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.     

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.     

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.     

Past and current gene flow in the selfing, wind-dispersed species Mycelis muralis in western Europe

The distribution of genetic diversity in Mycelis muralis, or wall lettuce, was investigated at a European scale using 12 microsatellite markers to infer historical and contemporary forces from genetic patterns. Mycelis muralis has the potential for long-distance seed dispersal by wind, is mainly self-pollinated, and has patchily distributed populations, some of which may show metapopulation dynamics. A total of 359 individuals were sampled from 17 populations located in three regions, designated southern Europe (Spain and France), the Netherlands, and Sweden. At this within-region scale, contemporary evolutionary forces (selfing and metapopulation dynamics) are responsible for high differentiation between populations (0.34 < F(ST) < 0.60) but, contrary to expectation, levels of within-population diversity, estimated by Nei's unbiased expected heterozygosity (H(E)) (0.24 < H(E) < 0.68) or analyses of molecular variance (50% of the variation found within-populations), were not low. We suggest that the latter results, which are unusual in selfing species, arise from efficient seed dispersal that counteracts population turnover and thus maintains genetic diversity within populations. At the European scale, northern regions showed lower allelic richness (A = 2.38) than populations from southern Europe (A = 3.34). In light of postglacial colonization hypotheses, these results suggest that rare alleles may have been lost during recolonization northwards. Our results further suggest that mutation has contributed to genetic differentiation between southern and northern Europe, and that Sweden may have been colonized by dispersers originating from at least two different refugia.     

Arabidopsis thaliana GLN2-encoded glutamine synthetase is dual targeted to leaf mitochondria and chloroplasts

In higher plants, photorespiratory Gly oxidation in leaf mitochondria yields ammonium in large amounts. Mitochondrial ammonium must somehow be recovered as glutamate in chloroplasts. As the first step in that recovery, we report glutamine synthetase (GS) activity in highly purified Arabidopsis thaliana mitochondria isolated from light-adapted leaf tissue. Leaf mitochondrial GS activity is further induced in response to either physiological CO(2) limitation or transient darkness. Historically, whether mitochondria are fully competent for oxidative phosphorylation in actively photorespiring leaves has remained uncertain. Here, we report that light-adapted, intact, leaf mitochondria supplied with Gly as sole energy source are fully competent for oxidative phosphorylation. Purified intact mitochondria efficiently use Gly oxidation (as sole energy, NH(3), and CO(2) source) to drive conversion of l-Orn to l-citrulline, an ATP-dependent process. An A. thaliana genome-wide search for nuclear gene(s) encoding mitochondrial GS activity yielded a single candidate, GLN2. Stably transgenic A. thaliana ecotype Columbia plants expressing a p35S::GLN2::green fluorescent protein (GFP) chimeric reporter were constructed. When observed by laser scanning confocal microscopy, leaf mesophyll and epidermal tissue of transgenic plants showed punctate GFP fluorescence that colocalized with mitochondria. In immunoblot experiments, a 41-kD chimeric GLN2::GFP protein was present in both leaf mitochondria and chloroplasts of these stably transgenic plants. Therefore, the GLN2 gene product, heretofore labeled plastidic GS-2, functions in both leaf mitochondria and chloroplasts to faciliate ammonium recovery during photorespiration.     

Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA

Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in a microarray format. Forty-five SNPs were genotyped and the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of genotype discrimination, yield and imbalanced amplification of alleles in the MDA product. Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA. For PEP products the power of genotype discrimination was lower than for MDA due to a 2-fold lower signal-to-noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied. To obtain faithful representation of the SNP alleles at least 0.3 ng DNA should be used per MDA reaction. We conclude that the use of WGA, and MDA in particular, is a highly promising procedure for producing DNA in sufficient amounts even for genome wide SNP mapping studies.     

Substrate reduction therapy in mouse models of the glycosphingolipidoses

Substrate reduction therapy uses small molecules to slow the rate of glycolipid biosynthesis. One of these drugs, N-butyldeoxynojirimycin (NB-DNJ), shows efficacy in mouse models of Tay-Sachs, Sandhoff and Fabry diseases. This offers the prospect that NB-DNJ may be of therapeutic benefit, at least in the juvenile and adult onset variants of these disorders. The infantile onset variants will require an additional enzyme-augmenting modality if the pathology is to be significantly improved. A second drug, N-butyldeoxyglactonojirimycin, looks very promising for treating storage diseases with neurological involvement as high systemic dosing is achievable without any side-effects.