Biology subjects

Whitaker, R.

Publications and source records attributed to Whitaker, R..

14 recordsLinked to original sources

bioRxiv2026

Resistome, Virulome, Mobilome, And Biosynthetic Gene Clusters Adaptations of Acinetobacter Baumannii Mexican Strains Across the Pre- and of the COVID-19 Period: Insights from Whole-Genome Sequencing

BackgroundAcinetobacter baumannii is a critical multidrug-resistant pathogen whose genomic landscape in Mexico has been reshaped by the COVID-19 pandemic. While global studies have highlighted distinctive sequence type distributions, systematic analyses in Mexico remain limited. MethodsWe analyzed 194 genomes, including 47 newly sequenced post-COVID isolates (MIQ), alongside 147 publicly available genomes (HPG). Whole-genome sequencing was combined with phylogenetic reconstruction, resistome and virulome profiling based on gene presence and absence, mobilome analysis, and biosynthetic gene cluster (BGC) characterization. ResultsTwo major clades dominated by Oxford STs 758, 208, 417, and 369 were identified. Resistome profiling uncovered 128 distinct resistome profiles (combinations of genes) and 44 emerging antimicrobial resistance genes (ARGs), with an increased number of resistance genes in the strains obtained during the pandemic. Virulome analysis revealed enrichment of metabolic adaptation genes (argG, carA, ilvC) in MIQ strains. Mobilome profiling demonstrated enrichment of ISAbA1 and ISAbA3 elements, known to mobilize carbapenemase genes. BGC analysis showed conserved siderophores involved in virulence, alongside diversification of the secondary metabolite repertoires in MIQ genomes. Additional observations included geographic mixing of clades across Jalisco, Aguascalientes, and Mexico City and referral bias toward carbapenemase-positive isolates. ConclusionThe genomic landscape of A. baumannii in Mexico has diversified post-COVID, with evidence of inter-regional transmission, referral bias, virulome expansion, mobilome-driven ARG dissemination, and metabolic adaptation. These findings underscore the urgent need for coordinated genomic surveillance, functional and clinical validation of adaptation signals, and regionally integrated infection control strategies to mitigate resistance trajectories. Graphical abstractIntegrative genomic profiling of A. baumannii isolates from Mexico. Workflow summarizing the analysis of 194 genomes (147 historic public genomes, 47 novel MIQ strains). Clinical isolates were identified by MALDI Biotyper and tested with BD Phoenix M50. Whole genome sequencing enabled phylogenetic and MLST analyses, resistome and virulome profiling, mobilome characterization, and BGC identification using BIGSCAPE and antiSMASH. Outputs include phylogenetic clustering, resistance/virulence gene distributions, and biosynthetic potential across Mexican regions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/712163v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@74c87borg.highwire.dtl.DTLVardef@1160aa7org.highwire.dtl.DTLVardef@1564c1org.highwire.dtl.DTLVardef@89e91b_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Analyses of Genome-Wide Recombination in Sulfolobus islandicus

Recombination is a fundamental process that both maintains genome integrity and dramatically shapes evolutionary dynamics in populations from all domains of life. Here we cross two genetically distinct but highly syntenic sympatric strains of Sulfolobus (Saccharolobus) islandicus and identify the pattern of recombination across the archaeal chromosome. We quantify recombination breakpoints occurring at neutral chromosomal markers through high-throughput short read Illumina sequencing of selected recombinant strains. We focus on three datasets that provide different, but corroborating information: i) individual colony sequences, ii) large colony pools of strains sorted by background genotype, and iii) a large colony pool of combined background genotypes. For each dataset we apply a newly designed and optimized computational tool called MARBL (Multiply Anchored Recombination Breakpoint Locator) for quantifying breakpoints and testing their statistical significance relative to sources of error introduced by sequencing technology. By combining the results of each dataset, we determine the short tract recombination preference in this organism and discuss its implications for mechanisms of recombination in archaea. We demonstrate uniform rates of recombination throughout the chromosome, which differ from previous studies of natural variation and discuss this in light of previous data suggesting speciation occurs through selection against inter-species hybrids. Importance StatementRecombination is a critical process in all domains of life that facilitates the essential repair of chromosomes and exchanges variation between organisms. Here we investigate genome-wide patterns of recombination in the model archaeaon Saccharolobus islandicus to better understand mechanistic aspects of their DNA exchange and recombination processes. Our observations of recombination in a controlled laboratory setting identified patterns of recombination that support the ability to incorporate short pieces of DNA in a piecemeal fashion from larger transferred segments. By comparing these observations to inferred recombination from natural populations, we can gain insight into evolutionary parameters including natural selection that would be otherwise difficult to determine from genomic evidence alone.

microbiology↗

bioRxiv2025

A continuum of ecology and evolution contributes to mutualism breakdown between legumes and rhizobia

Ecology and evolution jointly shape community responses to environmental perturbations, yet these processes are often examined separately, even in microorganisms where both occur over short time-scales. For example, during mutualism breakdown, shifts in diverse microbial communities might coincide with evolutionary changes in mutualistic traits within key symbionts. Here we study the legume-rhizobium mutualism after 33 years of nitrogen fertilization. Pairing a manipulative inoculation study with full-length 16S rRNA amplicon sequencing provides incomparable resolution across biological scales: whole bacterial community, genus Rhizobium, Rhizobium ASVs, and symbiosis plasmids. Clover host plant growth is first limited by symbiont availability; clovers preferred partner declines in N-addition soils, while a diverse, largely uncharacterized Rhizobium community increases. This ecological change is compounded by a concurrent evolutionary decline in the quality of the preferred symbiont through changing frequencies of symbiotic plasmids. These shifts make mutualists both rarer and inferior, reducing the benefits hosts receive from mutualism.

microbiology↗

bioRxiv2025

Discovery, characterization, and application of chromosomal integration sites in the hyperthermophilic crenarchaeon Sulfolobus islandicus

Sulfolobus islandicus, an emerging crenarchaeal model organism, offers unique advantages for metabolic engineering and synthetic biology applications owing to its ability to thrive in extreme environments. Although several genetic tools have been established for this organism, the lack of well-characterized chromosomal integration sites has considerably limited its potential as a cellular factory. Here, we systematically identified and characterized 13 artificial CRISPR RNAs (crRNAs) targeting eight chromosomal integration sites in S. islandicus using the CRISPR-COPIES pipeline and a multi-omics-informed computational workflow. By leveraging the endogenous CRISPR-Cas systems, we integrated the reporter gene lacS into these sites and validated heterologous gene expression through a {beta}-galactosidase reporter assay, which revealed significant positional effects on expression levels. As a proof of concept, we utilized these characterized sites to genetically manipulate lipid ether composition by overexpressing GDGT (glycerol dibiphytanyl glycerol tetraether) ring synthase B (GrsB) in S. islandicus, a key enzyme in GDGT biosynthesis. This study expands the genetic toolbox for S. islandicus and highlights a concept that could be widely applicable to other Sulfolobales, advancing their potential as robust platforms for archaeal synthetic biology and industrial biotechnology.

synthetic biology↗

bioRxiv2025

Host Competitive Asymmetries Accelerate Viral Evolution in a Microbe-Virus Coevolutionary System

Microbial host populations evolve traits conferring specific resistance to viral predators via various defense mechanisms, while viruses reciprocally evolve traits to evade these defenses. Such co-evolutionary dynamics often involve diversification promoted by negative frequency-dependent selection. However, microbial traits conferring competitive asymmetries can induce directional selection, opposing diversification. Despite extensive research on microbe-virus co-evolution, the combined effect of both host trait types and associated selection remains unclear. Using a CRISPR-mediated co-evolutionary system, we examine how the co-occurrence of both trait types impacts viral evolution and persistence, previously shown to be transient and non-stationary in computational models. A stochastic model incorporating host competitive asymmetries via variation of intrinsic growth rates reveals that competitively-advantaged host clades generate the majority of immune diversity. Greater asymmetries extend viral extinction times, accelerate viral adaptation locally in time, and augment long-term local adaptation. These findings align with previous experiments, and provide further insights into long-term co-evolutionary dynamics.

evolutionary biology↗

bioRxiv2024

Effects of injury size on local and systemic immune cell dynamics in volumetric muscle loss

We took a systems approach to the analysis of macrophage phenotype in regenerative and fibrotic volumetric muscle loss outcomes in mice together with analysis of systemic inflammation and of other leukocytes in the muscle, spleen, and bone marrow. Macrophage dysfunction in the fibrotic group occurred as early as day 1, persisted to at least day 28, and was associated with increased numbers of leukocytes in the muscle and bone marrow, increased pro-inflammatory marker expression in splenic macrophages, and changes in the levels of pro-inflammatory cytokines in the blood. The most prominent differences were in muscle neutrophils, which were much more abundant in fibrotic outcomes compared to regenerative outcomes at day 1 after injury. However, neutrophil depletion had little to no effect on macrophage phenotype or on muscle repair outcomes. Together, these results suggest that the entire system of immune cell interactions must be considered to improve muscle repair outcomes.

bioengineering↗

bioRxiv2023

Heparan sulfate modifications of betaglycan promote TIMP3-dependent ectodomain shedding to fine-tune TGF-β signaling.

In pathologies such as cancer, aberrant Transforming Growth Factor-{beta} (TGF-{beta}) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target this pivotal pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-{beta} responses. Betaglycan/type III TGF-{beta} receptor (T{beta}RIII), is an established co-receptor for the TGF-{beta} superfamily known to bind directly to TGF-{beta}s 1-3 and inhibin A/B. While betaglycan can be membrane-bound, it can also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. The extracellular domain of betaglycan undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. Here we report the unexpected discovery that the heparan sulfate modifications are critical for the ectodomain shedding of betaglycan. In the absence of such modifications, betaglycan is not shed. Such shedding is indispensable for the ability of betaglycan to suppress TGF-{beta} signaling and the cells responses to exogenous TGF-{beta} ligands. Using unbiased transcriptomics, we identified TIMP3 as a key regulator of betaglycan shedding and thereby TGF-{beta} signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-{beta} signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan modifications of betaglycan for shedding and influence on TGF-{beta} signaling responses. Dysregulated shedding of TGF-{beta} receptors plays a vital role in determining the response and availability of TGF-{beta}s, which is crucial for prognostic predictions and understanding of TGF-{beta} signaling dynamics.

biochemistry↗

bioRxiv2023

Ca2+/Calmodulin Dependent Protein Kinase Kinase-2 (CaMKK2) promotes Protein Kinase G (PKG)-dependent actin cytoskeletal assembly to increase tumor metastasis

Triple-negative breast cancers (TNBCs) tend to become highly invasive early during cancer development. Despite some successes in the initial treatment of patients diagnosed with early-stage localized TNBC, the rate of metastatic recurrence remains high with poor long-term survival outcomes. Here we show that elevated expression of the serine/threonine-kinase, Calcium/Calmodulin (CaM)-dependent protein kinase kinase-2 (CaMKK2), is highly correlated with tumor invasiveness. We determined that genetic disruption of CaMKK2 expression, or inhibition of its activity, disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor-prognosis ovarian cancer subtype, shares many genetic features with TNBC, and importantly, CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Probing the mechanistic links between CaMKK2 and metastasis we defined the elements of a new signaling pathway that impacts actin cytoskeletal dynamics in a manner which increases cell migration/invasion and metastasis. Notably, CaMKK2 increases the expression of the phosphodiesterase PDE1A which decreases the cGMP-dependent activity of protein kinase G1 (PKG1). This inhibition of PKG1 results in decreased phosphorylation of Vasodilator-Stimulated Phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate contraction/cell movement. Together, these data establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis. Further, it credentials CaMKK2 as a therapeutic target that can be exploited in the discovery of agents for use in the neoadjuvant/adjuvant setting to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC.

cancer biology↗

bioRxiv2023

Horizontal gene transfer and CRISPR targeting drive phage-bacterial host interactions and co-evolution in pink berry marine microbial aggregates

Bacteriophages (phages), viruses that infect bacteria, are the most abundant components of microbial communities and play roles in community dynamics and host evolution. The study of phage-host interactions, however, is made difficult by a paucity of model systems from natural environments and known and cultivable phage-host pairs. Here, we investigate phage-host interactions in the "pink berry" consortia, naturally-occurring, low-diversity, macroscopic aggregates of bacteria found in the Sippewissett Salt Marsh (Falmouth, MA, USA). We leverage metagenomic sequence data and a comparative genomics approach to identify eight compete phage genomes, infer their bacterial hosts from host-encoded clustered regularly interspaced short palindromic repeats (CRISPR), and observe the potential evolutionary consequences of these interactions. Seven of the eight phages identified infect the known pink berry symbionts Desulfofustis sp. PB-SRB1, Thiohalocapsa sp. PB-PSB1, and Rhodobacteraceae sp. A2, and belong to entirely novel viral taxa, except for one genome which represents the second member of the Knuthellervirus genus. We further observed increased nucleotide variation over a region of a conserved phage capsid gene that is commonly targeted by host CRISPR systems, suggesting that CRISPRs may drive phage evolution in pink berries. Finally, we identified a predicted phage lysin gene that was horizontally transferred to its bacterial host, potentially via a transposon intermediary, emphasizing the role of phages in bacterial evolution in pink berries. Taken together, our results demonstrate that pink berry consortia contain diverse and variable phages, and provide evidence for phage-host co-evolution via multiple mechanisms in a natural microbial system. IMPORTANCEPhages (viruses that infect bacteria) are important components of all microbial systems, where they drive the turnover of organic matter by lysing host cells, facilitate horizontal gene transfer (HGT), and co-evolve with their bacterial hosts. Bacteria resist phage infection, which is often costly or lethal, through a diversity of mechanisms. One of these mechanisms are CRISPR systems, which encode arrays of phage-derived sequences from past infections to block subsequent infection with related phages. Here, we investigate bacteria and phage populations from a simple marine microbial community known as "pink berries" found in salt marshes of Falmouth, Massachusetts, as a model of phage-host co-evolution. We identify eight novel phages, and characterize a case of putative CRISPR-driven phage evolution and an instance of HGT between phage and host, together suggesting that phages have large evolutionary impacts in a naturally-occuring microbial community.

microbiology↗

bioRxiv2022

G6PD Inhibition Sensitizes Ovarian Cancer Cells to Oxidative Stress in the Metastatic Omental Microenvironment

Ovarian cancer (OC) is the most lethal gynecological malignancy, with aggressive metastatic disease responsible for the majority of ovarian cancer related deaths. In particular, OC tumors preferentially metastasize to and proliferate rapidly in the omentum. Here, we show metastatic OC cells experience increased oxidative stress in the omental microenvironment. Metabolic reprogramming, including upregulation of the pentose phosphate pathway (PPP), a key cellular redox homeostasis mechanism, allows OC cells to compensate for this challenge. Inhibition of G6PD, the rate-limiting enzyme of the PPP, reduces tumor burden in pre-clinical models of OC, suggesting this adaptive metabolic dependency is important for OC omental metastasis. HighlightsO_LIThe omental microenvironment poses a high oxidative stress metastatic niche for ovarian cancer cells. C_LIO_LIG6PD, a key enzyme involved in redox homeostasis and the rate-limiting enzyme of the pentose phosphate pathway (PPP) is upregulated in omental metastasis. C_LIO_LIInhibition of G6PD increases oxidative stress and cytotoxicity in the omental microenvironment. C_LIO_LIPharmacological G6PD inhibition reduces omental metastases in vivo. C_LI

cancer biology↗

bioRxiv2022

Reciprocal epigenetic Sox2 regulation by SMAD1-SMAD3 is critical for anoikis resistance and metastasis in cancer

Growth factors in the tumor environment are key regulators of cell survival and metastasis. Here we reveal, dichotomy between TGF-{beta} superfamily growth factors BMP and TGF-{beta}/activin, and their downstream SMAD effectors. Gene expression profiling uncovered Sox2 as a key signaling node regulated in an opposing manner by anoikis-promoting BMP2, 4 and 9, and anoikis-suppressing TGF-{beta} and activin A. We find that Sox2 repression by BMPs robustly inhibits intraperitoneal tumor burden and increases survival in multiple ovarian cancer models. Repression of Sox2 is driven by SMAD1 dependent histone H3K27me3 recruitment and DNA methylation at SOX2s promoter. Conversely, TGF-{beta} and activin A promote Sox2 expression, and anoikis resistance by SMAD3 mediated histone H3K4me3 recruitment. We find that balancing Sox2 levels is critical for anoikis, as transcriptomics reveals regulation of key cell death pathways. Moreover, BMP-driven SMAD1 signaling can override TGF-{beta} and activins effect on Sox2, which has clinical significance due to the high levels of TGF-{beta} we find in ovarian cancer patients. Together, our findings identify Sox2 as a contextual and contrastingly regulated key node, downstream of TGF-{beta} superfamily members controlling anoikis and metastasis in ovarian cancers. HighlightsO_LISox2 is a key node for anoikis resistance in cancer C_LIO_LISox2 is differentially regulated by TGF-{beta}/activin and BMPs in broad cancers C_LIO_LIBMP9 is a robust metastasis suppressor by lowering Sox2 C_LIO_LISox2 regulation is contextual, epigenetic and at the transcriptional level C_LI

cancer biology↗

bioRxiv2021

A short prokaryotic argonaute cooperates with membrane effector to confer antiviral defense

Argonaute (Ago) proteins are widespread nucleic acid-guided enzymes that recognize targets through complementary base pairing. While in eukaryotes Agos are involved in RNA silencing, the functions of prokaryotic Agos (pAgos) remain largely unknown. In particular, a clade of truncated and catalytically inactive pAgos (short pAgos) lacks characterization. Here, we reveal that a short pAgo protein in Sulfolobus islandicus, together with its two genetically associated proteins, Aga1 and Aga2, provide robust antiviral protection via abortive infection. Aga2 is a membrane-associated toxic effector that binds anionic phospholipids via a basic pocket, which is essential for its cell killing ability. Ago and Aga1 form a stable complex that exhibits RNA-directed nucleic acid recognition ability and directly interacts with Aga2, pointing to an immune sensing mechanism. Together, our results highlight the cooperation between pAgos and their widespread associated proteins, suggesting an uncharted diversity of pAgo-derived immune systems that await to be discovered.

microbiology↗

bioRxiv2021

Interrogation of the integrated mobile genetic elements in gut-associated Bacteroidaceae with a consensus prediction approach

Exploration of mobile genetic element (MGE) diversity and relatedness is vital to understanding microbial communities, especially the gut microbiome, where the mobilization of antibiotic resistance and pathogenicity genes has important clinical consequences. Current MGE prediction tools are biased toward elements similar to previously-identified MGEs, especially tailed phages of proteobacterial hosts. Further, there is a need for methods to examine relatedness and gene sharing among MGEs. We present VICSIN, a consensus approach for MGE prediction and clustering of predictions to provide classification. Testing of VICSIN on datasets of Pseudomonas aeruginosa and Bacteroides fragilis genomes suggests VICSIN is the optimal approach to predict integrated MGEs from poorly-explored host taxa, because of its increased sensitivity and accuracy. We applied VICSIN to a dataset of gut-associated Bacteroidaceae genomes, identifying 816 integrated MGEs falling into 95 clusters, most of which are novel. VICSINs fast and simple network-building scheme revealed a high degree of gene sharing within and between related MGE clusters. Shared gene functions across MGEs include core mobilization functions and accessory gene content, such as type VI secretion systems and antibiotic resistance genes. The MGEs identified here encode a large portion of unknown gene content, emphasizing the fact that the full diversity of MGEs and the factors they encode remain very poorly understood. Together, this work motivates more exploration of the gut mobilome, which is likely one of the most potent drivers of microbial evolution in the human microbiome. IMPORTANCEMobile genetic elements (MGEs), including phages and integrative and conjugative elements (ICEs), drive the diversity and function of microbial communities through horizontal gene transfer. Current tools to predict MGEs in genomic sequence data are highly focused on phages, and are biased against the discovery of novel MGEs. We present VICSIN, a consensus approach to MGE prediction that is able to find a diversity of MGEs, particularly in poorly-understood bacterial taxa. By applying VICSIN to a large database of diverse Bacteroidaceae genomes, we have been able to get a distinct view of the gut mobilome, extending beyond the phageome. These novel MGEs belong to related groups, sharing a significant amount of functional gene content within and between groups, supporting a mosaic model of evolution for ICEs. Understanding how phages evolve in Bacteroidaceae hosts, however, remains elusive and highlights the need for more experimental research.

microbiology↗

bioRxiv2020

Infection with novel Bacteroides phage BV01 alters host transcriptome and bile acid metabolism in a common human gut microbe

The bacterial genus Bacteroides is among the most abundant and common taxa in the human gut, yet little is known about the phages infecting the group. Bacteroides phage BV01 (BV01) was identified as a prophage integrated on the chromosome of its host, Bacteroides vulgatus ATCC 8482. Phage BV01 is actively produced, and infects susceptible B. vulgatus hosts in the mouse gut. Infection with BV01 causes a generalized repression of the B. vulgatus transcriptome, downregulating 103 transcripts and upregulating only 12. Integration of BV01 disrupts the promoter sequence of a downstream gene encoding a putative tryptophan-rich sensory protein (tspO). Deletion of tspO and subsequent RNAseq analysis revealed that more than half of the differentially-regulated transcripts are shared with the BV01 lysogen, suggesting the transcriptomic response to BV01 is linked to tspO. Among these differentially-regulated transcripts are two encoding bile salt hydrolases. Bile acid deconjugation assays show that BV01 represses its hosts ability to hydrolyze bile acids in a tspO-dependent manner. Analysis of 256 published healthy human gut metagenomes suggests that phage integration adjacent to B. vulgatus-like tspO genes is rare within an individual, but common among humans. Finally, this work proposes a novel phage family that includes BV01, the Salyersviridae, whose host range spans the Bacteroides and is detectable in human-associated samples. Together, these findings highlight the importance of phage-host interactions to our understanding of how gut microbes sense and interact with their environment. IMPORTANCEThe links between human disease and the gut microbiome are numerous. Most mechanisms by which most gut microbes and their activities change and impact human health remain elusive. Phages, viruses that infect bacteria, are hypothesized to play a central role in modulating both community dynamics and functional activities. Here we have characterized an active prophage, BV01, which infects a pervasive and abundant human gut-associated species. BV01 infection alters its hosts transcriptional profile including its metabolism of bile acids, molecules implicated in mediating health and disease states in the gut. This highlights that prophages and other components of the variable genome should not be overlooked in bacterial genomes because they may dramatically alter host phenotypes. Furthermore, BV01 represents a new family of phages infecting human gut symbionts, providing a foundation for future investigations of phage-host interactions in these clinically-relevant but underexplored hosts.

microbiology↗