Balancing redox cofactor generation and ATP synthesis: Key microaerobic responses in thermophilic fermentations

Geobacillus thermoglucosidasius is a Gram‐positive, thermophilic bacterium capable of ethanologenic fermentation of both C5 and C6 sugars and may have possible use for commercial bioethanol production [Tang et al., 2009; Taylor et al. (2009) Trends Biotechnol 27(7): 398–405]. Little is known about the physiological changes that accompany a switch from aerobic (high redox) to microaerobic/fermentative (low redox) conditions in thermophilic organisms. The changes in the central metabolic pathways in response to a switch in redox potential were analyzed using quantitative real‐time PCR and proteomics. During low redox (fermentative) states, results indicated that glycolysis was uniformly up‐regulated, the Krebs (tricarboxylic acid or TCA) cycle non‐uniformly down‐regulated and that there was little to no change in the pentose phosphate pathway. Acetate accumulation was accounted for by strong down‐regulation of the acetate CoA ligase gene (acs) in addition to up‐regulation of the pta and ackA genes (involved in acetate production), thus conserving ATP while reducing flux through the TCA cycle. Substitution of an NADH dehydrogenase (down‐regulated) by an up‐regulated NADH:FAD oxidoreductase and up‐regulation of an ATP synthase subunit, alongside the observed shifts in the TCA cycle, suggested that an oxygen‐scavenging electron transport chain likely remained active during low redox conditions. Together with the observed up‐regulation of a glyoxalase and down‐regulation of superoxide dismutase, thought to provide protection against the accumulation of toxic phosphorylated glycolytic intermediates and reactive oxygen species, respectively, the changes observed in G. thermoglucosidasius NCIMB 11955 under conditions of aerobic‐to‐microaerobic switching were consistent with responses to low pO₂ stress. Biotechnol. Bioeng. 2013; 110: 1057–1065. © 2012 Wiley Periodicals, Inc.     

Population changes of breeding waders on farmland in relation to agri-environment management

Capsule Population response of breeding waders to agri-environment management varied between management options and species; implementation has been on too small a scale to reverse national population declines.

Aims To test whether numbers of five breeding wader species have shown a more positive response between 1992 and 2005, at sites with appropriate agri-environment management, than at sites that have remained outside such schemes.

Methods Using data from 60 pairs of farmland study areas in Scotland first surveyed in 1992/93, before agri-environment scheme (AES) implementation, and again in 2005, after scheme implementation, we tested at both site and field scales whether changes in the abundance of five breeding wader species were associated with AES management options designed to benefit these species.

Results Changes in breeding wader abundance were more positive on sites in AES, especially for Northern Lapwings Vanellus vanellus and Common Redshanks Tringa totanus, even though management had not been targetted specially at breeding waders on those sites. However, AES management was associated only with modest population increase for Common Redshanks, and a reduction in the magnitude of decline for Northern Lapwings. At the field scale, there was evidence for Northern Lapwings, Common Redshanks and Common Snipe Gallinago gallinago that options which limited grazing and other agricultural activity were associated with more positive outcomes than those which also manipulated water levels.

Conclusions AES management for breeding waders slowed, and in some cases reversed, breeding wader decline at field and farm scales. These benefits were from options that limited grazing and agricultural operations during the breeding season, but not those that also aimed to raise water levels. A possible explanation is that when wetland options are applied to agriculturally marginal fields, grazing reduction or abandonment, and succession to rank vegetation cover then occur over the course of 5-year agreements, with detrimental effects for breeding waders. Verification arrangements need to be robust enough to guard against this. Levels of agri-environment provision in 2005 were too limited and too poorly targeted at remaining key areas for breeding waders to be able to halt or reverse national population declines.     

Habitat associations of Eurasian Skylarks Alauda arvensis breeding on Irish farmland and implications for agri-environment planning

Capsule Skylarks breeding in Ireland prefer extensive grassland habitats and almost completely avoid tillage habitats.

Aims To describe the distribution and habitat use of breeding Skylarks in Ireland, particularly in lowland agricultural habitats, and to use this information to inform conservation measures for this species.

Methods Countryside Bird Survey (CBS) and Farmland Bird Project (FBP) data were examined to determine large-scale (national) distribution and habitat selection, in addition to smaller-scale (farm- and field-level) habitat use. The CBS is a national breeding bird monitoring scheme involving 397 1-km squares. The FBP collected detailed bird and habitat data from 122 farms.

Results CBS and FBP data both showed significant regional differences in breeding Skylark densities, with the highest relative abundances in the northwest and west. Dry grassland/grass moor habitats supported the highest densities of breeding Skylarks in the CBS, which were significantly higher than in improved grassland or tillage. At the farm-level, Skylark numbers were positively related to wetland habitats but negatively associated with trees in field boundaries, dense ground vegetation and overall density of farm boundaries. At the field-scale, larger fields and unimproved grasslands were preferred.

Conclusion Agri-environment measures tailored to region-specific requirements and to the relatively local habitat preferences of target species are required if population declines of species of conservation concern, including Skylarks, are to be reversed.     

Tbx18 regulates development of the epicardium and coronary vessels

The epicardium and coronary vessels originate from progenitor cells in the proepicardium. Here we show that Tbx18, a T-box family member highly expressed in the proepicardium, controls critical early steps in coronary development. In Tbx18^−/− mouse embryos, both the epicardium and coronary vessels exhibit structural and functional defects. At E12.5, the Tbx18-deficient epicardium contains protrusions and cyst-like structures overlying a disorganized coronary vascular plexus that contains ectopic structures resembling blood islands. At E13.5, the left and right coronary stems form correctly in mutant hearts. However, analysis of PECAM-1 whole mount immunostaining, distribution of SM22α^lacZ/+ activity, and analysis of coronary vascular casts suggest that defective vascular plexus remodeling produces a compromised arterial network at birth consisting of fewer distributing conduit arteries with smaller lumens and a reduced capacity to conduct blood flow. Gene expression profiles of Tbx18^−/− hearts at E12.5 reveal altered expression of 79 genes that are associated with development of the vascular system including sonic hedgehog signaling components patched and smoothened, VEGF-A, angiopoietin-1, endoglin, and Wnt factors compared to wild type hearts. Thus, formation of coronary vasculature is responsive to Tbx18-dependent gene targets in the epicardium, and a poorly structured network of coronary conduit vessels is formed in Tbx18 null hearts due to defects in epicardial cell signaling and fate during heart development. Lastly, we demonstrate that Tbx18 possesses a SRF/CArG box dependent repressor activity capable of inhibiting progenitor cell differentiation into smooth muscle cells, suggesting a potential function of Tbx18 in maintaining the progenitor status of epicardial-derived cells.     

CONSUMER–RESOURCE INTERACTIONS AND THE EVOLUTION OF MIGRATION

Theoretical studies have demonstrated that selection will favor increased migration when fitnesses vary both temporally and spatially, but it is far from clear how pervasive those theoretical conditions are in nature. Although consumer–resource interactions are omnipresent in nature and can generate spatial and temporal variation, it is unknown even in theory whether these dynamics favor the evolution of migration. We develop a mathematical model to address whether and how migration evolves when variability in fitness is determined at least in part by consumer–resource coevolutionary interactions. Our analyses show that such interactions can drive the evolution of migration in the resource, consumer, or both species and thus supplies a general explanation for the pervasiveness of migration. Over short time scales, we show the direction of change in migration rate is determined primarily by the state of local adaptation of the species involved: rates increase when a species is locally maladapted and decrease when locally adapted. Our results reveal that long‐term evolutionary trends in migration rates can differ dramatically depending on the strength or weakness of interspecific interactions and suggest an explanation for the evolutionary divergence of migration rates among interacting species.     

STRONG SELECTION BARRIERS EXPLAIN MICROGEOGRAPHIC ADAPTATION IN WILD SALAMANDER POPULATIONS

Microgeographic adaptation occurs when populations evolve divergent fitness advantages across the spatial scales at which focal organisms regularly disperse. Although an increasing number of studies find evidence for microgeographic adaptation, the underlying causes often remain unknown. Adaptive divergence requires some combination of limited gene flow and strong divergent natural selection among populations. In this study, we estimated the relative influence of selection, gene flow, and the spatial arrangement of populations in shaping patterns of adaptive divergence in natural populations of the spotted salamander (Ambystoma maculatum). Within the study region, A. maculatum co‐occur with the predatory marbled salamander (Ambystoma opacum) in some ponds, and past studies have established a link between predation risk and adaptive trait variation in A. maculatum. Using 14 microsatellite loci, we found a significant pattern of genetic divergence among A. maculatum populations corresponding to levels of A. opacum predation risk. Additionally, A. maculatum foraging rate was strongly associated with predation risk, genetic divergence, and the spatial relationship of ponds on the landscape. Our results indicate the sorting of adaptive genotypes by selection regime and strongly suggest that substantial selective barriers operate against gene flow. This outcome suggests that microgeographic adaptation in A. maculatum is possible because strong antagonistic selection quickly eliminates maladapted phenotypes despite ongoing and substantial immigration. Increasing evidence for microgeographic adaptation suggests a strong role for selective barriers in counteracting the homogenizing influence of gene flow.     

THE IMPORTANCE OF MOSQUITO BEHAVIOURAL ADAPTATIONS TO MALARIA CONTROL IN AFRICA

Over the past decade the use of long‐lasting insecticidal nets (LLINs), in combination with improved drug therapies, indoor residual spraying (IRS), and better health infrastructure, has helped reduce malaria in many African countries for the first time in a generation. However, insecticide resistance in the vector is an evolving threat to these gains. We review emerging and historical data on behavioral resistance in response to LLINs and IRS. Overall the current literature suggests behavioral and species changes may be emerging, but the data are sparse and, at times unconvincing. However, preliminary modeling has demonstrated that behavioral resistance could have significant impacts on the effectiveness of malaria control. We propose seven recommendations to improve understanding of resistance in malaria vectors. Determining the public health impact of physiological and behavioral insecticide resistance is an urgent priority if we are to maintain the significant gains made in reducing malaria morbidity and mortality.     

VACCINATION AND REDUCED COHORT DURATION CAN DRIVE VIRULENCE EVOLUTION: MAREK’S DISEASE VIRUS AND INDUSTRIALIZED AGRICULTURE

Marek’s disease virus (MDV), a commercially important disease of poultry, has become substantially more virulent over the last 60 years. This evolution was presumably a consequence of changes in virus ecology associated with the intensification of the poultry industry. Here, we assess whether vaccination or reduced host life span could have generated natural selection, which favored more virulent strains. Using previously published experimental data, we estimated viral fitness under a range of cohort durations and vaccine treatments on broiler farms. We found that viral fitness maximized at intermediate virulence, as a result of a trade‐off between virulence and transmission previously reported. Our results suggest that vaccination, acting on this trade‐off, could have led to the evolution of increased virulence. By keeping the host alive, vaccination prolongs infectious periods of virulent strains. Improvements in host genetics and nutrition, which reduced broiler life spans below 50 days, could have also increased the virulence of the circulating MDV strains because shortened cohort duration reduces the impact of host death on viral fitness. These results illustrate the dramatic impact anthropogenic change can potentially have on pathogen virulence.     

Dodecyl Maltoside Protects Membrane Proteins In Vacuo

Molecular dynamics simulations have been used to characterize the effects of transfer from aqueous solution to a vacuum to inform our understanding of mass spectrometry of membrane-protein-detergent complexes. We compared two membrane protein architectures (an α-helical bundle versus a β-barrel) and two different detergent types (phosphocholines versus an alkyl sugar) with respect to protein stability and detergent packing. The β-barrel membrane protein remained stable as a protein-detergent complex in vacuum. Zwitterionic detergents formed conformationally destabilizing interactions with an α-helical membrane protein after detergent micelle inversion driven by dehydration in vacuum. In contrast, a nonionic alkyl sugar detergent resisted micelle inversion, maintaining the solution-phase conformation of the protein. This helps to explain the relative stability of membrane proteins in the presence of alkyl sugar detergents such as dodecyl maltoside.     

Nanoscale Distribution of Ryanodine Receptors and Caveolin-3 in Mouse Ventricular Myocytes: Dilation of T-Tubules near Junctions

We conducted super-resolution light microscopy (LM) imaging of the distribution of ryanodine receptors (RyRs) and caveolin-3 (CAV3) in mouse ventricular myocytes. Quantitative analysis of data at the surface sarcolemma showed that 4.8% of RyR labeling colocalized with CAV3 whereas 3.5% of CAV3 was in areas with RyR labeling. These values increased to 9.2 and 9.0%, respectively, in the interior of myocytes where CAV3 was widely expressed in the t-system but reduced in regions associated with junctional couplings. Electron microscopic (EM) tomography independently showed only few couplings with caveolae and little evidence for caveolar shapes on the t-system. Unexpectedly, both super-resolution LM and three-dimensional EM data (including serial block-face scanning EM) revealed significant increases in local t-system diameters in many regions associated with junctions. We suggest that this regional specialization helps reduce ionic accumulation and depletion in t-system lumen during excitation-contraction coupling to ensure effective local Ca²⁺ release. Our data demonstrate that super-resolution LM and volume EM techniques complementarily enhance information on subcellular structure at the nanoscale.The contraction of cardiac ventricular myocytes depends on the rapid cell-wide transient increase in intracellular [Ca²⁺] upon depolarization of the cell-membrane potential. The cardiac ryanodine receptor (RyR) (1), which is the intracellular Ca²⁺ release channel in the sarcoplasmic reticulum (SR), plays a central role in shaping Ca²⁺ transients. RyRs form clusters of various sizes (2,3) with the majority located within junctions between the SR and the surface membrane and its cytoplasmic extension, the transverse tubular (t-) system. It has been suggested that some RyR clusters are associated with caveolae, a specialized signaling microdomain of the surface membrane. Previous studies were complicated by the limited resolution of optical imaging methods of ∼250 nm, much larger than the nanometer scale of RyRs and caveolae. Accordingly, these studies report varying colocalization between RyRs and caveolin-3 (CAV3), a caveolar marker also expressed in the t-system (4,5).In this work, we investigated the relative distribution of CAV3 and RyRs in mouse ventricular myocytes both in the cytosol and near the cell surface with super-resolution fluorescence microscopy that achieves a resolution approaching 30 nm. Our data revealed unexpected local t-system swellings near junctional couplings, which was supported by two different three-dimensional electron microscopy (EM) modalities with <10-nm resolution: EM tomography and serial block-face scanning EM (SBFSEM).Super-resolution images of CAV3 and RyR labeling at the surface sarcolemma of mouse myocytes showed little overlap, suggesting that few RyRs were in couplings with caveolae (Fig. 1 A, for detailed methods, see the Supporting Material). Only ∼4.8% of RyR labeling was associated with CAV3 positive areas and ∼3.5% of CAV3 associated with RyR positive areas (n = 6 cells from three animals, Fig. 1 B, see also Table S1 in the Supporting Material), broadly consistent with previous data in rats (6). To support this finding, EM tomography was applied to mouse ventricular tissue that included a part of the surface sarcolemma, to our knowledge for the first time. Segmentation of peripheral couplings (containing RyR foot structures) and surface caveolae (∼60 nm in diameter and often interconnected) confirmed that the great majority of peripheral couplings were in regions devoid of caveolae (Fig. 1 C). A few junctional couplings containing feet were between caveolae and subsarcolemmal SR (Fig. 1 D, see also Fig. S1 and Movie S1 in the Supporting Material). We conducted a similar analysis in the cytosol where CAV3 expression occurs in the t-system (5) and RyRs are abundant in dyadic junctions between the t-system and SR terminal cisterns.Open in a separate windowFigure 1Colocalization of CAV3 and RyRs at the surface sarcolemma. (A) Super-resolution micrograph of the distribution of CAV3 (green) and RyRs (red) at the surface of a mouse cardiac myocyte. (B) Analysis of the association of CAV3 with RyRs. The fraction of RyR labeling within CAV3 positive areas was ∼4.8% (front data) whereas ∼3.5% of CAV3 was found in RyR-positive membrane areas. (C) Segmented EM tomogram containing a patch of surface sarcolemma (light blue) and associated caveolae (green) as well as peripheral couplings (red). (D) Detailed view of a region with abundant caveolae. (Arrows) Couplings with caveolae.As shown in Fig. 2 A, the spatial distribution of CAV3 and RyR clusters in super-resolution micrographs taken several microns below the surface sarcolemma is consistent with this view. The association of the two labels is slightly increased (as compared to the surface), according to distance analysis with 9% of CAV3 and 9.2% of RyR labeling associating with each other (Fig. 2 B, n = 6 cells from three animals). The similarity of manually traced t-system in EM tomograms (Fig. 2 C) and super-resolved CAV3 labeling suggested that CAV3 is widely distributed in the t-system except for regions where dyadic membrane junctions occur as CAV3 labeling was much weaker in regions with strong RyR labeling. It was notable that the t-system diameter appeared to increase at regions of strong RyR labeling (Fig. 2 D), broadly consistent with the behavior seen in tomograms (Fig. 2 C). This was confirmed by a quantitative analysis of t-tubule diameters in dyadic versus extradyadic regions on the basis of CAV3 and RyR labeling, with full-width at quarter-maximum mean diameters increasing from ∼150 nm distal to dyads, to ∼190 nm (using CAV3 signal only) or ∼280 nm (using CAV3 and RyR signal) near dyads (Fig. 2, G and H, see also Methods in the Supporting Material). The combined RyR and CAV3 signals seemed to be a better representation of the entire t-system lumen near junctions (see Fig. S2).Open in a separate windowFigure 2Distribution of CAV3 and RyRs in the cell interior. (A) Super-resolution micrograph of CAV3 (green) and RyR (red) distribution at t-system. (Arrow) Direction of longitudinal cell axis. (B) Distance analysis of the CAV3 and RyR association (N = 6 cells per group). (C) Segmented EM tomogram of a similar region with three-dimensional mesh models of t-system membrane (green) and dyadic couplings (red). (D) This image illustrates the tracing (white path) of t-tubules. The label distribution was extracted and linearized along the path (E) to calculate a mask that shows the full width at quarter-maximum diameter along tubules, CAV3 (green) and RyR (red) (F). (G) Histograms of local diameters extracted from traced t-tubules. (H) Mean diameters in junctional (dyad) and nonjunctional (ex-dyad) regions. See main text and the Supporting Material for details. ^**p < 0.01.Taken together, super-resolution imaging and EM tomography strongly support the presence of local t-system dilations in regions where the t-system opposes SR at dyads and such t-system bulges are connected by narrower tubule segments. Further support was provided by SBFSEM, another volume EM technique to study larger cell volumes (albeit at the expense of a slightly lower resolution). SBFSEM clearly showed local t-system dilations were regularly involved in the architecture of most (but not all) dyads (Fig. 3, see also Fig. S3 and Movie S2), as also observed in full three-dimensional super-resolution images (see Fig. S3 C).Open in a separate windowFigure 3Segmented SBFSEM data showing t-system dilations near dyadic junctions. (A) The overview shows t-system membranes (green) and jSR (red) in a mouse myocyte. (B, enlarged inset from panel A) Thin connecting tubules (arrows) and regular swellings in junctional regions at z-lines.Our data identify local dilations of the t-system associated with dyads in mouse cardiac myocytes. Frequent tubule distensions had been observed especially at the intersections of transverse and axial tubules (7), and constrictions were seen in rabbit myocytes although their relationship to dyads was unknown (8). The increased local t-system lumen near junctions may help reduce the predicted ionic accumulation/depletion during excitation-contraction coupling (9). Alternatively, it might simply be secondary to increasing local membrane area and allow the formation of large area junctions that harbor many RyRs. In connection with this point, it would be interesting to investigate the t-system near junctions in species that have larger average tubule diameters (e.g., human and rabbit (10)), or if this architecture changes in mouse heart failure models where t-tubule diameters are often increased.Most peripheral couplings were in regions void of surface caveolae, although a small number of RyR clusters were in junctional couplings between subsarcolemmal SR and caveolae as shown both by the low colocalization between CAV3 and RyRs as well as direct evidence from EM tomography. Similarly, a relatively small fraction of CAV3 colocalized with RyR clusters in the t-system although CAV3 was expressed widely in the t-system. A structural role of CAV3 in the t-system is still unclear—t-tubules in tomogram data did not reveal distinct caveolae shapes on the t-system membrane (see Fig. S4), although this might change in pathology (11). In any case, the t-system exhibits high curvature orthogonal to the tubule axis, which may be supported by CAV3 oligomerization. In addition, the presence of CAV3 in the t-system may be important for regulating other signaling systems (e.g., adrenergic signaling).Finally, our data demonstrate that complementary data from optical super-resolution and three-dimensional EM images assists data interpretation and reliability. We suggest that truly correlative optical and EM imaging approaches should provide further information and improve our knowledge of the basis of cardiac excitation-contraction coupling.