Mutations and genomic islands can explain the strain dependency of sugar utilization in 21 strains of Propionibacterium freudenreichii

Background

Propionibacterium freudenreichii (PF) is an actinobacterium used in cheese technology and for its probiotic properties. PF is also extremely adaptable to several ecological niches and can grow on a variety of carbon and nitrogen sources. The aim of this work was to discover the genetic basis for strain-dependent traits related to its ability to use specific carbon sources. High-throughput sequencing technologies were ideal for this purpose as they have the potential to decipher genomic diversity at a moderate cost.

Results

21 strains of PF were sequenced and the genomes were assembled de novo. Scaffolds were ordered by comparison with the complete reference genome CIRM-BIA1, obtained previously using traditional Sanger sequencing. Automatic functional annotation and manual curation were performed. Each gene was attributed to either the core genome or an accessory genome. The ability of the 21 strains to degrade 50 different sugars was evaluated. Thirty-three sugars were degraded by none of the sequenced strains whereas eight sugars were degraded by all of them. The corresponding genes were present in the core genome. Lactose, melibiose and xylitol were only used by some strains. In this case, the presence/absence of genes responsible for carbon uptake and degradation correlated well with the phenotypes, with the exception of xylitol. Furthermore, the simultaneous presence of these genes was in line the metabolic pathways described previously in other species. We also considered the genetic origin (transduction, rearrangement) of the corresponding genomic islands. Ribose and gluconate were degraded to a greater or lesser extent (quantitative phenotype) by some strains. For these sugars, the phenotypes could not be explained by the presence/absence of a gene but correlated with the premature appearance of a stop codon interrupting protein synthesis and preventing the catabolism of corresponding carbon sources.

Conclusion

These results illustrate (i) the power of correlation studies to discover the genetic basis of binary strain-dependent traits, and (ii) the plasticity of PF chromosomes, probably resulting from horizontal transfers, duplications, transpositions and an accumulation of mutations. Knowledge of the genetic basis of nitrogen and sugar degradation opens up new strategies for the screening of PF strain collections to enable optimum cheese starter, probiotic and white biotechnology applications.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1467-7) contains supplementary material, which is available to authorized users.     

Aberrant post‐translational modifications compromise human myosin motor function in old age

Novel experimental methods, including a modified single fiber in vitro motility assay, X‐ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging‐related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force‐generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X‐ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age‐specific myosin post‐translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.     

RPA prevents G‐rich structure formation at lagging‐strand telomeres to allow maintenance of chromosome ends

Replication protein A (RPA) is a highly conserved heterotrimeric single‐stranded DNA‐binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1‐D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging‐strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G‐quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G‐quadruplex. We propose that RPA prevents the formation of G‐quadruplex structures at lagging‐strand telomeres to promote shelterin association and facilitate telomerase action at telomeres.     

Native mass spectrometry and ion mobility characterization of trastuzumab emtansine,a lysine-linked antibody drug conjugate

Antibody–drug conjugates (ADCs) are biochemotherapeutics consisting of a cytotoxic chemical drug linked covalently to a monoclonal antibody. Two main classes of ADCs, namely cysteine and lysine conjugates, are currently available on the market or involved in clinical trials. The complex structure and heterogeneity of ADCs makes their biophysical characterization challenging. For cysteine conjugates, hydrophobic interaction chromatography is the gold standard technique for studying drug distribution, the naked antibody content, and the average drug to antibody ratio (DAR). For lysine ADC conjugates on the other hand, which are not amenable to hydrophobic interaction chromatography because of their higher heterogeneity, denaturing mass spectrometry (MS) and UV/Vis spectroscopy are the most powerful approaches. We report here the use of native MS and ion mobility (IM-MS) for the characterization of trastuzumab emtansine (T-DM1, Kadcyla®). This lysine conjugate is currently being considered for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer, and combines the anti-HER2 antibody trastuzumab (Herceptin®), with the cytotoxic microtubule-inhibiting maytansine derivative, DM1. We show that native MS combined with high-resolution measurements and/or charge reduction is beneficial in terms of the accurate values it provides of the average DAR and the drug load profiles. The use of spectral deconvolution is discussed in detail. We report furthermore the use of native IM-MS to directly determine DAR distribution profiles and average DAR values, as well as a molecular modeling investigation of positional isomers in T-DM1.     

Tree cover at fine and coarse spatial grains interacts with shade tolerance to shape plant species distributions across the Alps

The role of competition for light among plants has long been recognised at local scales, but its importance for plant species distributions at larger spatial scales has generally been ignored. Tree cover modifies the local abiotic conditions below the canopy, notably by reducing light availability, and thus, also the performance of species that are not adapted to low‐light conditions. However, this local effect may propagate to coarser spatial grains, by affecting colonisation probabilities and local extinction risks of herbs and shrubs. To assess the effect of tree cover at both the plot‐ and landscape‐grain sizes (approximately 10‐m and 1‐km), we fit generalised linear models (GLMs) for the plot‐level distributions of 960 species of herbs and shrubs using 6935 vegetation plots across the European Alps. We ran four models with different combinations of variables (climate, soil and tree cover) at both spatial grains for each species. We used partial regressions to evaluate the independent effects of plot‐ and landscape‐grain tree cover on plot‐level plant communities. Finally, the effects on species‐specific elevational range limits were assessed by simulating a removal experiment comparing the species distributions under high and low tree cover. Accounting for tree cover improved the model performance, with the probability of the presence of shade‐tolerant species increasing with increasing tree cover, whereas shade‐intolerant species showed the opposite pattern. The tree cover effect occurred consistently at both the plot and landscape spatial grains, albeit most strongly at the former. Importantly, tree cover at the two grain sizes had partially independent effects on plot‐level plant communities. With high tree cover, shade‐intolerant species exhibited narrower elevational ranges than with low tree cover whereas shade‐tolerant species showed wider elevational ranges. These findings suggest that forecasts of climate‐related range shifts for herb and shrub species may be modified by tree cover dynamics.     

Coping with fast climate change in northern ecosystems: mechanisms underlying the population‐level response of a specialist avian predator

Northern ecosystems are facing unprecedented climate modifications, which pose a major threat for arctic species, especially the specialist predator guild. However, the mechanisms underlying responses of predators to climate change remain poorly understood. Climate can influence fitness parameters of predators either through reduced reproduction or survival following adverse weather conditions, or via changes in the population dynamics of their main prey. Here, we combined three overlapping long‐term datasets on the breeding density and parameters of a rodent‐specialist predator, the rough‐legged buzzard Buteo lagopus, its main prey population dynamics and climate variables, collected in subarctic areas of Finland and Norway, to assess the impact of changing climate on the predator reproductive response. Rough‐legged buzzards responded to ongoing climate change by advancing their laying date (0.1 d yr^?1 over the 21 yr of the study period), as a consequence of earlier snowmelt. However, we documented for the same period a decrease in breeding success, which principally resulted from an indirect effect of changes in the dynamics of their main prey, i.e. grey‐sided voles Microtus oeconomus, and not from the expected negative effect of unfavorable weather conditions during the brood‐rearing period on nestling survival. Additionally, we showed the striking impact of autumn and winter weather conditions on vole population growth rates in subarctic ecosystems, with a strong positive correlation between mean snow depth in autumn and winter and both winter and summer population growth rates. Our results highlighted that, in northern ecosystems, ongoing climate change has the potential to impact specialist predator species through two mechanistic linkages, which may in the long‐run, threaten the viability of their populations, and lead to potential severe cascading trophic effects at the ecosystem level.     

Multispectral fluorescence lifetime imaging system for intravascular diagnostics with ultrasound guidance: in vivo validation in swine arteries

Fluorescence lifetime technique has demonstrated potential for analysis of atherosclerotic lesions and for complementing existing intravascular imaging modalities such as intravascular ultrasound (IVUS) in identifying lesions at high risk of rupture. This study presents a multimodal catheter system integrating a 40 MHz commercial IVUS and fluorescence lifetime imaging (FLIm) using fast helical motion scanning (400 rpm, 0.75 mm/s), able to acquire in vivo in pulsatile blood flow the autofluorescence emission of arterial vessels with high precision (5.08 ± 0.26 ns mean average lifetime over 13 scans). Co‐registered FLIm and IVUS data allowed 3D visualization of both biochemical and morphological vessel properties. Current study supports the development of clinically compatible intravascular diagnostic system integrating FLIm and demonstrates, to our knowledge, the first in vivo intravascular application of a fluorescence lifetime imaging technique. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Targeted Exon Capture and Sequencing in Sporadic Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that results in progressive degeneration of motor neurons, ultimately leading to paralysis and death. Approximately 10% of ALS cases are familial, with the remaining 90% of cases being sporadic. Genetic studies in familial cases of ALS have been extremely informative in determining the causative mutations behind ALS, especially as the same mutations identified in familial ALS can also cause sporadic disease. However, the cause of ALS in approximately 30% of familial cases and in the majority of sporadic cases remains unknown. Sporadic ALS cases represent an underutilized resource for genetic information about ALS; therefore, we undertook a targeted sequencing approach of 169 known and candidate ALS disease genes in 242 sporadic ALS cases and 129 matched controls to try to identify novel variants linked to ALS. We found a significant enrichment in novel and rare variants in cases versus controls, indicating that we are likely identifying disease associated mutations. This study highlights the utility of next generation sequencing techniques combined with functional studies and rare variant analysis tools to provide insight into the genetic etiology of a heterogeneous sporadic disease.