Biology subjects

Schiro, G.

Publications and source records attributed to Schiro, G..

11 recordsLinked to original sources

bioRxiv2026

Conserved patterns of mammal-microbe codiversification and interspecific bacterial sharing

What determines whether a gut microbial lineage has fidelity to one host species or is shared freely across multiple species? Factors that predict microbial community composition, including host species, diet, and environmental variation, have been well documented in wild animals. However, the eco-evolutionary processes maintaining host species- or population-specific microbial lineages remain unresolved. Here, we analyze 519 gut microbiome samples collected from 15 species of wild rodents across two mountains in the Madrean Sky Islands of southeastern Arizona using full-length 16S rRNA sequencing. This montane system provides a natural experiment in which adjacent replicated elevational gradients bring different combinations of rodent species into sympatry, allowing host evolutionary history to be disentangled from shared environmental effects. We find that host phylogeny, rather than environment, is the primary driver of microbial strain sharing across host species. We recover replicated signatures of codiversification on both mountains, indicating shared evolutionary history between hosts and their microbes, despite ample opportunities for cross-species environmental transmission. This evolutionary coupling is associated with predicted differences in bacterial oxygen tolerance and optimal growth temperature: codiversifying lineages are predominantly obligate anaerobes with optimal growth near host body temperature, suggesting more limited out-of-host persistence than non-codiversifying taxa. This pattern mirrors findings from the human gut microbiome, suggesting conserved constraints on microbial dispersal across mammalian hosts. The cost-effectiveness of long-read amplicon sequencing for phylogenetic inference within bacterial species opens new opportunities for large-scale evolutionary and ecological studies of host-microbe interactions in wildlife.

ecology↗

bioRxiv2026

Maternal effects shape scale-dependent convergence in gut microbial responses to environmental change

Animals can experience environmental variability across multiple temporal scales, from predictable within-year seasonal fluctuations to rarer, irregular resource pulses that occur across generations. While gut microbiome responses to seasonal changes are well documented, far less is understood about how host-associated microbiomes respond to episodic environmental resource pulses, and whether such responses can persist across generations. Here, we investigate how gut microbial communities of wild North American red squirrels (Tamiasciurus hudsonicus) respond to intra-annual seasonal variation and episodic resource pulses happening years apart (specifically, seed masting). We then test the hypothesis that these responses are shared by mothers and their offspring. In line with prior work across mammals, we found that seasonal variation strongly structured gut microbiomes, driving widespread and convergent shifts in composition across the year. However, episodic resource pulses predicted microbial scatter such that gut microbiomes became more dissimilar across individuals in resource pulse years compared to typical years. Despite this population-level divergence, mothers and their adult offspring exhibited microbial convergence when encountering resource pulses occurring years apart, whereas seasonal responses were shared across the population and not unique to mother-offspring pairs. Overall, our results highlight the mammalian gut microbiome as a flexible component of the host phenotype, capable of adjusting to both predictable seasonal variation and large-scale, episodic resource pulses. Patterns of mother-offspring similarity across resource pulse years further provide evidence that some host-microbial responses to environmental change may be transmitted across generations.

ecology↗

bioRxiv2026

Beyond distance-decay: The Mississippi River shapes gut microbiome communities in the Peromyscus maniculatus species complex

Biogeographic barriers are fundamental in shaping the distribution of animals and plants, yet the role of discrete landscape features in constraining microbial dispersal remains poorly understood. Identifying the barriers that partition gut microbial communities is essential for modeling the distribution of hosts and their symbionts across physical space and evolutionary time. The deer mouse (Peromyscus maniculatus species complex), which inhabits nearly all terrestrial environments in North America, provides an ideal model for testing these biogeographic drivers of the mammalian gut microbiome. Using NSF NEON biorepository samples, we characterized the gut microbiomes of 13 populations across the contiguous United States using full-length 16S rRNA long-read sequencing. While we observed a consistent distance-decay relationship across the continent, our results reveal that the Mississippi River acts as a major biogeographic break, significantly increasing microbial dissimilarity beyond the levels predicted by geographic distance alone. This "river effect" suggests that large fluvial systems impose a discrete barrier to microbial transmission, likely due to restricted host dispersal. Furthermore, we identified specific microbial lineages that exhibit differential sensitivity to this barrier, suggesting a gradient in microbial acquisition patterns ranging from the environment to host-host transmission. Together, these findings demonstrate that the gut microbiome may act as a sensitive bio-indicator of landscape-level ecological connectivity, revealing that large-scale landscape barriers disrupt microbial transmission even among closely related host populations that lack reproductive barriers.

microbiology↗

bioRxiv2026

Structural basis of the two-photon photoactivation mechanism of orange carotenoid protein

Cyanobacteria have produced Earths oxygen for 2.4 billion years by adapting to fluctuating irradiance. This adaptation relies on orange carotenoid protein (OCP), which mediates light-intensity- dependent photoprotective energy dissipation using a unique two-photon absorption mechanism. Photon absorption by ground-state OCP (OCPO) generates a metastable intermediate (OCP1h{nu}) that either relaxes thermally or, upon absorption of a second photon within [~]1 s, converts to the active photoprotective state (OCPR). By integrating static and time-resolved crystallography, cryo-EM, computation, spectroscopy and biochemistry, we assign the structure of OCP1h{nu}, establish its functional relevance and capture structural snapshots along the OCPO[->]OCP1h{nu} and OCP1h{nu}[->]OCPR photochemical pathways. We elucidate the molecular mechanism of OCP, which serves as a unique biological circuit breaker protecting the photosynthetic machinery from high light flux.

biochemistry↗

bioRxiv2025

Dynamics and fermentation patterns of stool microbiota on simple carbon sources in in vitro batch cultures reveal dysbiosis in Crohn's disease.

Crohns disease (CD)-associated dysbiosis is characterized by reduced microbial diversity, depletion of short-chain fatty acid (SCFA) producers, especially butyrate-forming taxa, and altered metabolic profiles. This study examined whether CD-related dysbiosis is reflected in the fermentation behavior of stool-derived microbial communities cultured in vitro on simple carbon substrates (glucose or acetate+lactate). Shotgun metagenomics and metabolomics revealed that CD-derived communities produced lower levels of butyrate (mainly via lactate and acetate), valerate, caproate, and propionate, and higher levels of ethanol and certain amino acids compared to healthy controls. These metabolic alterations aligned with compositional shifts, including a loss of beneficial commensals (e.g., Coprococcus catus, Ruminococcus torques, Eubacterium rectale, Fusicatenibacter saccharivorans, and Faecalibacterium prausnitzii) and an overrepresentation of CD-associated taxa, particularly Escherichia coli. Metabolic potential analysis revealed an enrichment of genes linked to ethanol and amino acid synthesis in CD-associated microbiotas, underscoring the metabolic adaptability of E. coli. Notably, acetate+lactate substrates supported the growth of healthy microbiota-associated bacteria, whereas glucose favored CD-associated taxa, highlighting disease-specific metabolic imbalances. These findings suggest that in vitro fermentation profiling may help distinguish CD-associated dysbiosis from a healthy microbiome and might support the development of microbiome-informed diagnostic or therapeutic approaches for CD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/656803v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@809890org.highwire.dtl.DTLVardef@a04b89org.highwire.dtl.DTLVardef@1bf39c0org.highwire.dtl.DTLVardef@1687974_HPS_FORMAT_FIGEXP M_FIG C_FIG A short abstract: CD-related dysbiosis alters stool microbial fermentation, reducing butyrate, propionate, valerate, caproate, and increasing ethanol/amino acids in vitro. Fermentation tests may help distinguish CD-associated patterns, suggesting potential for future microbiome-based diagnostics.

microbiology↗

bioRxiv2024

Crystal structure of a bacterial photoactivated adenylate cyclase determined at room temperature by serial femtosecond crystallography

OaPAC is a recently discovered blue-light using flavin adenosine dinucleotide (BLUF) photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata that uses adenosine triphosphate and translates the light signal into the production of cyclic adenosine monophosphate. Here, we report the crystal structures of the enzyme in the absence of its natural substrate determined from room temperature serial crystallography data collected at both an X-ray free electron laser and a synchrotron and we compare them with the cryo macromolecular crystallography structures obtained at a synchrotron by us and others. These results reveal slight differences in the structure of the enzyme due to data collection at different temperatures and X-ray sources. We further investigate the effect of the Y6 mutation in the blue-light using flavin adenosine dinucleotide domain, a mutation which results in a rearrangement of the hydrogen-bond network around the flavin and a notable rotation of the side-chain of the critical Q48 residue. These studies pave the way for ps - ms time-resolved serial crystallography experiments at X-ray free electron lasers and synchrotrons in order to determine the early structural intermediates and correlate them with the well-studied ps - ms spectroscopic intermediates. SynopsisStructures of the dark-adapted state of a photoactivated adenylate cyclase are determined from serial crystallography (SX) data collected at room temperature at an X-ray free electron laser (XFEL) and a synchrotron and are compared with cryo macromolecular crystallography (MX) synchrotron structures obtained by us and others. These structures of the wild-type enzyme in combination with the cryo MX synchrotron structure of a light-sensor domain mutant provide insight into the hydrogen bond network rearrangement upon blue-light illumination and pave the way for the determination of structural intermediates of the enzyme by time-resolved SX.

biophysics↗

bioRxiv2022

Influence of pump laser fluence on ultrafast structural changes in myoglobin

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump probe experiments for investigating electronic and nuclear changes during light-induced reactions. On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1. However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore2-14. As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern15 whether this experimental approach16 allows valid inferences to be drawn vis-a-vis biologically relevant single-photon-induced reactions17. Here we describe ultrafast pump-probe SFX experiments on photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics15) are seen to depend strongly on pump laser energy. Our results confirm both the feasibility and necessity of performing TR-SFX pump probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing design and interpretation of ultrafast TR-SFX pump probe experiments16 such that biologically relevant insight emerges.

biophysics↗

bioRxiv2022

Oligomerization processes limit photoactivation and recovery of the Orange Carotenoid Protein.

The Orange Carotenoid Protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection, by quenching of the excess of light harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which protein structural changes take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy quenching activity. Here, we probed photo-induced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in regulation of the OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation, a numbed-state forms, which remains in a non-photoexcitable structural state for at least [~]0.5 {micro}s after absorption of a first photon. Significance StatementThe orange carotenoid protein (OCP) is a photoactivatable protein involved in cyanobacterial photoprotection. Upon photoactivation, OCP becomes able to quench the excess of energy uptaken by the light-harvesting antennae, thereby evading damage to the cells. It remains unclear, however, what is the exact OCP photoactivation mechanism, and whether or not oligomerization partakes in the regulation of the OCP function. Here, we investigated these issues by combining static and time-resolved (TR) X-ray scattering and optical spectroscopy. Our results show that OCP oligomerizes in both the dark-adapted inactive and light-adapted active states, suggesting a functional role for oligomerization. TR scattering data furthermore reveal that the first large-scale conformational changes associated with OCP photoactivation take place on the {micro}s time scale.

biophysics↗

bioRxiv2021

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

molecular biology↗

bioRxiv2021

Rational control of structural off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, a key parameter that largely dictates the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

biophysics↗

bioRxiv2020

Zinc Determines Dynamical Properties and Aggregation Kinetics of Human Insulin

Protein aggregation is a widespread process leading to deleterious consequences in the organism, with amyloid aggregates being important not only in biology but also for drug design and biomaterial production. Insulin is a protein largely used in diabetes treatment and its amyloid aggregation is at the basis of the so-called insulin-derived amyloidosis. Here we uncover the major role of zinc in both insulin dynamics and aggregation kinetics at low pH, where the formation of different amyloid superstructures (fibrils and spherulites) can be thermally induced. Amyloid aggregation is accompanied by zinc release and the suppression of water-sustained insulin dynamics, as shown by particle-induced X-ray emission and X-ray absorption spectroscopy and by neutron spectroscopy, respectively. Our study shows that zinc binding stabilizes the native form of insulin by facilitating hydration of this hydrophobic protein and suggests that introducing new binding sites for zinc can improve insulin stability and tune its aggregation propensity.Statement of Significance Localized amyloidosis occurs at insulin injection sites for diabetes treatment, leading to deleterious inflammations known as insulin-derived amyloidosis. Amyloid superstructures are also promising candidates in the field of biomaterials. Here we revealed that zinc, coordinated to insulin in the native form, is released upon amyloid aggregation, when insulin forms superstructures known as fibrils and spherulites. Zinc release leads to a full suppression of functionally essential protein dynamics through a modification of the protein’s hydration properties and completely modifies insulin amyloid kinetics. The results suggest that changes in protein hydration upon zinc binding/release modifies both stability and dynamics of insulin and might then be a general strategy to control protein stability and tune protein aggregation into amorphous and ordered superstructures.View Full Text

biophysics↗