Biology subjects

Wolfe, A. J.

Publications and source records attributed to Wolfe, A. J..

16 recordsLinked to original sources

bioRxiv2026

An expanded urine culturing workflow to cultivate and characterize diverse urobiome isolates

Despite increased recognition of the diverse resident microbiome of the urinary tract (i.e., the urobiome) in postmenopausal women, the roles and functions of these microbes remain largely unknown. Further empirical research is needed to understand the physiology, interactions, and antibiotic resistance evolution of urobiome members with pathogenic potential. However, experimental work relies on viable, culturable isolates. Standard urine culturing practices are designed for identifying a narrow set of known urinary microbes, and are thus poorly suited for cultivating taxa from the resident urobiome. Here we expand the urine culturing toolkit to reliably recover diverse urobiome taxa for downstream empirical research. Urine samples collected from postmenopausal women with recurrent urinary tract infections were shipped at ambient temperature to a central point for culturing. Microbial viability was maintained using boric acid preservative tubes during multi-day transport of sample aliquots. Selective media incubated under specialized conditions were used to promote recovery of diverse urobiome members, including fastidious taxa. Under 5% CO2 -enriched atmospheric conditions and with longer incubation times, we leveraged a chromogenic agar (UTIC) to further differentiate isolates based on colony color and morphology. We evaluated the workflow for its ability to isolate and characterize urobiome taxa, as determined by morphological differentiation and taxonomic identification. Across 108 urine samples, 6.3 {+/-} 3.2 distinct isolates were recovered, with no detectable relationship between sample shipment duration and isolate richness. On chromogenic agar, colony growth and color intensity was improved with CO2 -enriched atmospheric conditions and extended incubation times. We identified diverse taxa that are typically underrepresented in standard diagnostic culture and provide novel morphological characterizations for members of the genera Actinotignum, Aerococcus, Facklamia, Lactobacillus, Latilactobacillus, Limosilactobacillus, and Streptococcus species, which have not been previously described on UTIC chromogenic agar. Using this novel workflow, we recovered a diverse collection of urobiome isolates from urine samples shipped over multiple days. We also demonstrated the utility of a chromogenic agar for the visual differentiation of key urobiome taxa. While sequencing approaches have enhanced our understanding of urobiome composition, culturing is needed to investigate microbial interactions, virulence mechanisms, and antimicrobial susceptibility. This protocol adds to the growing toolkit for the cultivation of diverse urobiome isolates needed to support downstream empirical studies and advance urinary tract infection research.

microbiology↗

bioRxiv2025

SimUrine: A Novel, Fully Defined Artificial Urinary Media for Enhanced Microbiological Research of Urinary Bacteria

Urinary tract infections represent one of the most prevalent bacterial diseases, yet current diagnostic and research methodologies are hampered by inadequate culture media that fail to replicate the bladder biochemical environment. Conventional artificial urine formulations contain undefined components, lack essential nutrients, or inadequately support urinary microbiome (urobiome) growth. To address these limitations, we developed SimUrine, a fully defined synthetic urine medium that aims to replicate human bladder chemistry while supporting diverse microbial growth requirements. SimUrine was systematically developed through iterative optimization of multi-purpose artificial urine, incorporating defined concentrations of carbon sources, vitamins, trace elements, and amino acids within physiologically relevant ranges. The modular design enables component substitution without complete reformulation, facilitating customization for culturomics, antimicrobial susceptibility testing, and microbial ecology studies, while reducing batch-to-batch variability associated with authentic urine. Performance evaluation demonstrated SimUrines capability to support growth of fastidious urobiome members, including Lactobacillus species, Aerococcus urinae, and Corynebacterium riegelii, which fail to proliferate in conventional minimal media. Physicochemical characterization confirmed that SimUrine formulation exhibits properties within normal human urine ranges for density, conductivity, osmolarity, and viscosity, ensuring physiological relevance. Clinical applications revealed reduced antibiotic susceptibility compared to standard media, suggesting more accurate representation of in vivo conditions. Co-culture experiments using Escherichia coli and Enterococcus faecalis demonstrated previously unobserved microbial interactions, highlighting SimUrines utility for investigating urobiome dynamics. SimUrine represents a significant advancement in urobiome research methodology, providing a standardized, reproducible platform for investigating urobiome under physiologically relevant conditions, potentially improving fundamental understanding and clinical diagnostic approaches. IMPORTANCEUrinary tract infections affect millions globally, yet current research and diagnostic methods rely on inadequate culture media that fail to replicate the bladders unique biochemical environment. This fundamental limitation has hindered accurate UTI research and potentially compromised clinical treatment decisions. SimUrine addresses this critical gap as the first fully defined synthetic urine medium that mimics human bladder chemistry while supporting growth of diverse urinary microbes. The breakthrough enables cultivation of urobiome organisms in a minimal medium that resembles natural conditions, revealing novel microbial interactions that influence urinary health. Crucially, SimUrine demonstrates different antimicrobial susceptibility patterns compared to standard clinical media, suggesting current testing protocols may inaccurately predict treatment outcomes. This standardized, reproducible platform eliminates the variability of authentic urine samples while maintaining physiological relevance, potentially transforming urobiome research methodology and improving clinical diagnostic accuracy for urinary tract infections worldwide.

microbiology↗

bioRxiv2025

Complex carbohydrates catabolism capacity of bladder microbiota inhabiting healthy and overactive female bladders

Overactive bladder syndrome (OAB) is a poorly understood symptom complex that affects 40% of females over the age of 40, with clinical features including urinary urgency and incontinence. In addition to inflammation, oxidative stress, nerve damage and reduced blood flow, alterations in the urinary microbiome (urobiome), specifically in bladder bacterial diversity, have been reported to be associated with OAB. Bladder bacteria are members of the urobiome along with viruses, archaea, fungi, and protozoans. The urobiome metabolism, particularly in relationship to host complex sugars (glycans), has been investigated recently in terms of glycosaminoglycan (GAG) utilization. Nevertheless, other urinary free oligosaccharides (FOS) have not yet been explored in both OAB and urobiome contexts. Similarly, a comprehensive search of microbial genes involved in host glycan metabolism in the bladder of adult females with or without OAB has not yet been reported. In this study, we investigated urinary FOS by mass spectrometry in women without OAB (asymptomatic controls), with OAB without incontinence (dry OAB), or with OAB with incontinence (wet OAB or urgency urinary incontinence, UUI). We also questioned the ability of commensal bladder bacteria to digest these FOS and other glycans, using bioinformatic tools to query publicly available bladder genomes isolated from affected and unaffected adult females to identify genes that encode polysaccharide lyases (PL) and glycoside hydrolases (GH). Our results show that FOS are present in a similar level in affected and unaffected controls with a few exceptions: ten FOS were found to differ between the OAB dry groups and either the control (four) or UUI (six) groups. Our results indicate that bladder microbiota from adult females both with and without OAB have the genetic capacity to digest host glycans and dietary sugars with subtle differences. Bladder bacteria isolated from females with OAB possess more GH/PL genes for host mucins, whereas bladder bacteria from controls possess more GH/PL genes for GAG digestion. In the control group, specifically, the genus Streptococcus possessed genes for the PL8 and GH88 enzymes, known to be involved in host GAG digestion. These novel bioinformatic data can enable future biochemical exploration of the urobiomes metabolism toward specific host glycans, such as GAGs, mucins O-glycans and N-glycans.

biochemistry↗

bioRxiv2025

Genomic reconstruction of Bacillus anthracis from complex environmental samples enables high throughput identification and lineage assignment in Pakistan

Bacillus anthracis, the causative agent of anthrax, is a highly virulent zoonotic pathogen primarily affecting domesticated and wild herbivores. Human exposure to B. anthracis is primarily through contact with infected animals or contaminated animal products. In Pakistan, where livestock vaccines are largely unavailable and infected carcasses are often disposed of improperly, the risk to humans, wildlife and livestock is significant. Currently, diagnosis of anthrax infections and outbreak tracing necessitates the isolation and culturing of B. anthracis, a process that requires BSL-3 facilities. In this study, we show that positive identification, genome reconstruction and lineage assignment can be accomplished using bioinformatic analysis of DNA extracted directly from environmental samples that would otherwise provide the starting material for isolation and culturing. This approach does not require laboratory target enrichment as is necessary for other pathogens, due in part to the extremely high bacterial load in the bloodstream in the deceased animals. Using these methods, we greatly expand the knowledge of endemic B. anthracis in Pakistan. We provide the first reference B. anthracis genomes from Pakistan since the 1970s and identify A.Br.014 Aust94 as a minor circulating sublineage alongside dominant A.Br.047 Vollum. Future work will focus on limits of detection and will determine if this bioinformatic method can be expanded more broadly for B. anthracis or other pathogens to replace typical culture-based methods.

bioinformatics↗

bioRxiv2024

Urinary Metabolomic Profile is Minimally Impacted by Common Storage Conditions and Additives

Metabolomics reflects the molecular communications within a biological system. Urine can be obtained non-invasively and is rich in metabolites that are potential biomarkers for human health and disease. The optimal urine storage conditions for metabolomic analysis are unknown. We measured the impact of common storage conditions and the presence of a DNA-stabilizer, AssayAssure(R) (Thermo Scientific), on metabolite content of voided human urine. Urinary metabolite composition was not altered across storage conditions. Although addition of AssayAssure(R) significantly alters metabolic profile by adduct addition, this does not preclude the identification of parent metabolites or overshadow biological differences. These data suggest biobanked urine stored in any of the tested conditions for metabolomic studies may be considered for metabolomic analysis.

molecular biology↗

bioRxiv2022

A Generalizable Nanopore Sensor for Highly Specific Protein Detection at Single-Molecule Precision

Protein detection and biomarker profiling have wide-ranging implications in many areas of basic research and molecular diagnostics. Substantial progress has been made in protein analytics using nanopores and the resistive-pulse technique. Yet, a long-standing challenge is implementing specific binding interfaces for detecting proteins without the steric hindrance of the pore interior. To overcome this technological difficulty, we formulate a new class of sensing elements made of a programmable antibody-mimetic binder fused to a monomeric protein nanopore. This way, such a modular design significantly expands the utility of nanopore sensors to numerous proteins while preserving their architecture, specificity, and sensitivity. We prove the power of this approach by developing and validating nanopore sensors for protein analytes that drastically vary in size, charge, and structural complexity. These analytes produce unique electrical signatures that depend on their identity and quantity and the binder-analyte assembly at the nanopore tip. From a practical point of view, our sensors unambiguously probe protein recognition events without the necessity of using any additional exogenous tag. The outcomes of this work will impact biomedical diagnostics by providing a fundamental basis and tools for protein biomarker detection in biofluids.

biophysics↗

bioRxiv2022

Regulation of translation by lysine acetylation in Escherichia coli

N{varepsilon}-lysine acetylation is a common post-translational modification observed in diverse species of bacteria. Aside from a few central metabolic enzymes and transcription factors, little is known about how this post-translational modification regulates protein activity. In this work, we investigated how lysine acetylation affects translation in Escherichia coli. In multiple species of bacteria, ribosomal proteins are highly acetylated at conserved lysine residues, suggesting that this modification may regulate translation. In support of this hypothesis, we found that the addition of the acetyl donors, acetyl phosphate or acetyl-Coenzyme A, inhibits translation but not transcription using an E. coli cell-free system. Further investigations using in vivo assays revealed that acetylation does not appear to alter the rate of translation elongation but rather increases the proportion of dissociated 30S and 50S ribosomes, based on polysome profiles of mutants or growth conditions known to promote lysine acetylation. Furthermore, ribosomal proteins are more acetylated in the disassociated 30S and 50S ribosomal subunit than in the fully assembled 70S complex. The effect of acetylation is also growth rate dependent, with disassociation of the subunits most pronounced during late exponential and early stationary phase growth - the same growth phase where protein acetylation is greatest. Collectively, our data demonstrate that lysine acetylation inhibits translation, most likely by interfering with subunit association. These results have also uncovered a new mechanism for coupling translation to the metabolic state of the cell. IMPORTANCENumerous cellular processes are regulated in response to the metabolic state of the cell. One such regulatory mechanism involves lysine acetylation, a covalent modification involving the transfer of an acetyl group from the central metabolites acetyl coenzyme A or acetyl phosphate to a lysine residue in a protein. This post-translational modification is known to regulate some central metabolic enzymes and transcription factors in bacteria, though a comprehensive understanding of its effect on cellular physiology is still lacking. In the present study, lysine acetylation was also found to inhibit translation in Escherichia coli by impeding ribosome association, most likely by disrupting salt-bridges along the binding interface of the 30S and 50S ribosomal subunits. These results further our understanding of lysine acetylation by uncovering a new target of regulation, protein synthesis, and aid in the design of bacteria for biotechnology applications where the growth conditions are known to promote lysine acetylation.

microbiology↗

bioRxiv2022

Urinary F plasmids reduce permissivity to coliphage infection

The microbial community of the urinary tract (urinary microbiota or urobiota) has been associated with human health. Bacteriophages (phages) and plasmids present in the urinary tract, like in other niches, may shape urinary bacteria dynamics. While urinary E. coli, often associated with urinary tract infection (UTI), and their phages have been catalogued for the urobiome, the dynamics of their interactions have yet to be explored. In this study, we characterized urinary E. coli F plasmids and their ability to decrease permissivity to E. coli phage (coliphage) infection. Putative F plasmids were present in 47 of 67 urinary E. coli isolates, and most of these plasmids carry genes that encode for toxin-antitoxin (TA) modules, antibiotic resistance, and/or virulence. Two urinary E. coli F plasmids, from urinary microbiota (UMB) 0928 and 1284, were conjugated into E. coli K-12 strains; plasmids included genes for antibiotic resistance and virulence. These plasmids, pU0928 and pU1284, decreased permissivity to coliphage infection by the laboratory phage P1vir and the urinary phages Greed and Lust. Furthermore, pU0928 could be maintained in E. coli K-12 for up to ten days in the absence of antibiotic resistance selection; this included maintenance of the antibiotic resistance phenotype and decreased permissivity to phage. Finally, we discuss how F plasmids present in urinary E. coli could play a role in coliphage dynamics and maintenance of antibiotic resistance in urinary E. coli. ImportanceThe microbial community of the urinary tract (urinary microbiota or urobiota) has been associated with human health. Bacteriophages (phages) and plasmids present in the urinary tract, like in other niches, may shape urinary bacteria dynamics. While urinary E. coli, often associated with urinary tract infection, and their phages have been catalogued for the urobiome, the dynamics of their interactions have yet to be explored. In this study, we characterized urinary E. coli F plasmids and their ability to decrease permissivity to E. coli phage (coliphage) infection. Two urinary E. coli F plasmids, each encoding antibiotic resistance and transferred by conjugation into naive laboratory E. coli K-12 strains decreased permissivity to coliphage infection. We propose a model by which F plasmids present in urinary E. coli could help to maintain antibiotic resistance of urinary E. coli.

microbiology↗

bioRxiv2022

The bladder microbiome, metabolome, cytokines, and phenotypes in patients with systemic lupus erythematosus

Background and aimsEmerging studies reveal a unique bacterial community in the human bladder, with alteration of composition associated to disease states. Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is characterized by frequent impairment of the kidney. Here, we explored the bladder microbiome, metabolome, and cytokine profiles in SLE patients, as well as correlations between microbiome and metabolome, cytokines, and disease profiles. Methods and materialsWe recruited a cohort of 50 SLE patients and 50 individually matched asymptomatic controls. We used transurethral catheterization to collect urine samples, 16S rRNA gene sequencing to profile bladder microbiomes, and LC-MS/MS to perform untargeted metabolomic profiling. ResultsCompared to controls, SLE patients possessed a unique bladder microbial community and increased alpha diversity. These differences were accompanied by differences in urinary metabolomes, cytokines, and patients disease profiles. The SLE-enriched genera, including Bacteroides, were positively correlated with several SLE-enriched metabolites, including olopatadine. The SLE-depleted genera, such as Pseudomonas, were negatively correlated to SLE-depleted cytokines, including IL-8. Alteration of the bladder microbiome was associated with disease profile. For example, the genera Megamonas and Phocaeicola were negatively correlated with serum complement C3, and Streptococcus was positively correlated with IgG. ConclusionsOur present study reveals associations between the bladder microbiome and the urinary metabolome, cytokines, and disease phenotypes. Our results could help identify biomarkers for SLE.

microbiology↗

bioRxiv2020

Species level resolution of female bladder microbiota from marker gene surveys

The human bladder contains bacteria in the absence of infection. Interest in studying these bacteria and their association with bladder conditions is increasing. However, the chosen experimental method can limit the resolution of the taxonomy that can be assigned to the bacteria found in the bladder. 16S rRNA amplicon sequencing is commonly used to identify bacteria in urinary specimens, but is typically restricted to genus-level identification. Our primary aim was to determine if accurate species-level identification of bladder bacteria is possible using 16S rRNA amplicon sequencing. We evaluated the ability of different classification schemes, each consisting of combinations of a reference database, a 16S rRNA gene variable region and a taxonomic classification algorithm to correctly classify bladder bacteria. We show that species-level identification is possible, and that the reference database chosen is the most important component, followed by the 16S variable region sequenced. ImportanceSpecies-level information may deepen our understanding of associations between bladder microbiota and bladder conditions, such as lower urinary tract symptoms and urinary tract infections. The capability to identify bacterial species depends on large databases of sequences, algorithms that leverage statistics and available computer hardware, and knowledge of bacterial genetics and classification. Taken together, this is a daunting body of knowledge to become familiar with before the simple question of bacterial identity can be answered. Our results show the choice of taxonomic database and variable region of the 16S rRNA gene sequence makes species level identification possible. We also show this improvement can be achieved through the more careful application of existing methods and use of existing resources.

bioinformatics↗

bioRxiv2020

Association of common genetic variants in the CPSF7 and SDHAF2 genes with Canine Idiopathic Pulmonary Fibrosis in the West Highland White Terrier

Canine Idiopathic Pulmonary Fibrosis (CIPF) is a chronic fibrotic lung disease that is observed at a higher frequency in the West Highland White Terrier dog breed (WHWT) and may have molecular pathological overlap with human lung fibrotic disease. We conducted a Genome-Wide Association Study (GWAS) in the WHWT using Whole Genome Sequencing (WGS) to discover genetic variants associated with CIPF. Saliva-derived DNA samples were sequenced using the Riptide DNA library prep kit. After quality controls, 28 affected, 44 unaffected and 1,843,695 informative Single Nucleotide Polymorphisms (SNPs) were included in the GWAS. Data were analyzed both at the single SNP and gene levels using the GEMMA and GATES methods, respectively. We detected significant signals at the gene level in both the CPSF7 and SDHAF2 genes (adjusted p = 0.016 and p = 0.025, respectively), two overlapping genes located on chromosome 18. The top SNP for both genes was rs22669389, however it did not reach genome-wide significance in the GWAS (adjusted p = 0.078). Our studies provide, for the first time, candidate loci for CIPF in the WHWT. CPSF7 was recently associated with lung adenocarcinoma further highlighting the potential relevance of our results since IPF and lung cancer share several pathological mechanisms.

genomics↗

bioRxiv2019

Female lower urinary tract microbiota do not correspond to IC/PBS symptoms: a case-controlled study

ABSTRACTO_ST_ABSOBJECTIVEC_ST_ABSCurrent etiology of interstitial cystitis/painful bladder syndrome (IC/PBS) is poorly understood and multifactorial. Recent studies suggest the female urinary microbiota (FUM) contribute to IC/PBS symptoms. This study was designed to determine if the FUM, analyzed using mid-stream voided urine samples, differs between IC/PBS patients and controls.\n\nMATERIALS AND METHODSThis prospective case-controlled study compared the voided FUM of women with symptoms of urinary frequency, urgency, and bladder pain for greater than 6 months to the voided FUM of healthy female controls without pain. Bacterial identification was performed using 16S rRNA gene sequencing and EQUC, a validated enhanced urine culture approach. Urotype was defined by a genus present at >50% relative abundance. If no genus was present above this threshold, the urotype was classified as mixe. Chi-square and Fishers exact tests were performed. P-values <0.05 were considered significant.\n\nRESULTSA mid-stream voided specimen was collected from 21 IC/PBS patients and 20 asymptomatic controls. Both groups had similar demographics. Urotypes did not differ between cohorts as assessed by either EQUC or 16S rRNA gene sequencing. We detected no significant differences between cohorts in terms of alpha-diversity. Cohorts also were not distinct using Principle Component Analysis or hierarchical clustering. Detection by EQUC of bacterial species considered uropathogenic was high in both cohorts, but detection of these uropathogenic species did not differ between groups (p=0.10).\n\nCONCLUSIONSEnhanced culture and modern DNA sequencing methods provide evidence that IC/PBS symptoms may not be related to differences in the FUM, at least not its bacterial components. Future larger studies are needed to confirm this preliminary finding.

microbiology↗

bioRxiv2018

Global lysine acetylation in Escherichia coli results from growth conditions that favor acetate fermentation

Lysine acetylation is thought to provide a mechanism for regulating metabolism in diverse bacteria. Indeed, many studies have shown that the majority of enzymes involved in central metabolism are acetylated and that acetylation can alter enzyme activity. However, the details regarding this regulatory mechanism are still unclear, specifically with regards to the signals that induce lysine acetylation. To better understand this global regulatory mechanism, we profiled changes in lysine acetylation during growth of Escherichia coli on the hexose glucose or the pentose xylose at both high and low sugar concentrations using label-free mass spectrometry. The goal was to see whether lysine acetylation differed during growth on these two different sugars. No significant differences, however, were observed. Rather, the initial sugar concentration was the principal factor governing changes in lysine acetylation, with higher sugar concentrations causing more acetylation. These results suggest that acetylation does not target specific metabolic pathways but rather simply targets accessible lysines, which may or may not alter enzyme activity. They further suggest that lysine acetylation principally results from conditions that favor accumulation of acetyl phosphate, the principal acetate donor in E. coli.\n\nIMPORTANCEBacteria alter their metabolism in response to nutrient availability, growth conditions, and environmental stresses. This process is best understood at the level of transcriptional regulation, where many metabolic genes are conditionally expressed in response to diverse cues. However, additional modes of regulations are known to exist. One is lysine acetylation, a post-translational modification known to target many metabolic enzymes. However, unlike transcriptional regulation, little is known about this regulatory mode. We investigated the factors inducing changes in lysine acetylation by comparing growth on glucose and xylose. We found that the specific sugar used for growth did not alter the pattern of acetylation; rather, the principal factor was the amount of sugar, with more sugar yielding more acetylation. These results imply lysine acetylation is a global regulatory mechanism that is not responsive to the specific carbon source per se but rather the accumulation of downstream metabolites.

microbiology↗

bioRxiv2018

Identification of novel protein lysine acetyltransferasesin Escherichia coli

Post-translational modifications, such as N{varepsilon}-lysine acetylation, regulate protein function. N{varepsilon}-lysine acetylation can occur either non-enzymatically or enzymatically. The non-enzymatic mechanism uses acetyl phosphate (AcP) or acetyl coenzyme A (AcCoA) as acetyl donors to modify an N{varepsilon}-lysine residue of a protein. The enzymatic mechanism uses N{varepsilon}-lysine acetyltransferases (KATs) to specifically transfer an acetyl group from AcCoA to N{varepsilon}-lysine residues on proteins. To date, only one KAT (YfiQ, also known as Pka and PatZ) has been identified in E. coli. Here, we demonstrate the existence of 4 additional E. coli KATs: RimI, YiaC, YjaB, and PhnO. In a genetic background devoid of all known acetylation mechanisms (most notably AcP and YfiQ) and one deacetylase (CobB), overexpression of these putative KATs elicited unique patterns of protein acetylation. We mutated key active site residues and found that most of them eliminated enzymatic acetylation activity. We used mass spectrometry to identify and quantify the specificity of YfiQ and the four novel KATs. Surprisingly, our analysis revealed a high degree of substrate specificity. The overlap between KAT-dependent and AcP-dependent acetylation was extremely limited, supporting the hypothesis that these two acetylation mechanisms play distinct roles in the post-translational modification of bacterial proteins. We further showed that these novel KATs are conserved across broad swaths of bacterial phylogeny. Finally, we determined that one of the novel KATs (YiaC) and the known KAT (YfiQ) can negatively regulate bacterial migration. Together, these results emphasize distinct and specific non-enzymatic and enzymatic protein acetylation mechanisms present in bacteria.\n\nImportanceN{varepsilon}-lysine acetylation is one of the most abundant and important post-translational modifications across all domains of life. One of the best-studied effects of acetylation occurs in eukaryotes, where acetylation of histone tails activates gene transcription. Although bacteria do not have true histones, N{varepsilon}-lysine acetylation is prevalent; however, the role of these modifications is mostly unknown. We constructed an E. coli strain that lacked both known acetylation mechanisms to identify four new N{varepsilon}-lysine acetyltransferases (RimI, YiaC, YjaB, and PhnO). We used mass spectrometry to determine the substrate specificity of these acetyltransferases. Structural analysis of selected substrate proteins revealed site-specific preferences for enzymatic acetylation that had little overlap with the preferences of the previously reported acetyl-phosphate non-enzymatic acetylation mechanism. Finally, YiaC and YfiQ appear to regulate flagellar-based motility, a phenotype critical for pathogenesis of many organisms. These acetyltransferases are highly conserved and reveal deeper and more complex roles for bacterial post-translational modification.

microbiology↗

bioRxiv2018

Controlling for contaminants in low biomass 16S rRNA gene sequencing experiments

BackgroundMicrobial communities are commonly studied using culture-independent methods such as 16S rRNA gene sequencing. However, one challenge in accurately characterizing microbial communities is exogenous bacterial DNA contamination. This is particularly problematic for sites of low microbial biomass such as the urinary tract, placenta, and lower airway. Computational approaches have been proposed as a post-processing step to identify and remove potential contaminants, but their performance has not been independently evaluated.\n\nTo identify the impact of decreasing microbial biomass on polymicrobial 16S rRNA gene sequencing experiments, we used a serial dilution of a mock microbial community. We evaluated two computational approaches to identify and remove contaminants: 1) identifying sequences that have an inverse correlation with DNA concentration implemented in Decontam and 2) predicting the proportion of experimental sample arising from defined contaminant sources implemented in SourceTracker.\n\nResultsAs expected, the proportion of contaminant bacterial DNA increased with decreasing starting microbial biomass, with 79.12% of the most dilute sample arising from contaminant sequences. Inclusion of contaminant sequences in analyses leads to overinflated diversity estimates (up to 12 times greater than the expected values) and distorts microbiome composition. SourceTracker successfully removed over 98% of contaminants when the experimental environments are well defined. However, SourceTracker performed poorly when the experimental environment is unknown, failing to remove the majority of contaminants. Decontam successfully removed 74-91% of contaminants regardless of prior knowledge of the experimental environment.\n\nConclusionsOur study indicates that computational methods can reduce the amount of contaminants in 16S rRNA gene sequencing experiments. The appropriate computational approach for removing contaminant sequences from an experiment depends on the prior knowledge about the microbial environment under investigation and can be evaluated with a dilution series of a mock microbial community.

bioinformatics↗

bioRxiv2016

A lottery simulation model for predicting cancer risk based on the strength and abundance of individual mutations

Models for predicting an individuals risk of developing cancer are complicated by a lack of well-conserved mutations present in most types of disease. To address this challenge we developed a simple lottery simulation of cancer development based solely on the abundance and relative survival advantage of mutations present within individual stem or progenitor cells. In simulations requiring few mutations or that involve mutations exhibiting a strong survival advantage cancers develop in our simulation in a progressive manner amenable to oncogene-based risk prediction. In contrast, in simulations involving numerous mutations that lack a strong survival advantage, a stochastic process of neutral drift and punctuated equilibrium determines eventual tumour formation. In these situations, the development of cancer is largely attributable to the rate of mutational variability amongst progenitor cells rather than the identity and relative survival advantage of the mutations present at any single time point. These results suggest that measuring rates of stem cell mutational variability represents an important component of predicting an individuals risk of developing cancer.

cancer biology↗