Biology subjects

Shields, R. C.

Publications and source records attributed to Shields, R. C..

10 recordsLinked to original sources

bioRxiv2026

Deciphering the Streptococcus mutans essentialome: multi-omic resolution of hypothetical genes and identification of a functional RocS equivalent

Genome-wide viability catalogs produced by transposon sequencing (Tn-seq) and CRISPR interference (CRISPRi) have successfully mapped the essential genome of Streptococcus mutans. In this study, we combined predictive bioinformatics, conditional CRISPRi transcriptional silencing, transmission electron microscopy, transcriptomics, and genetic suppressor screens to investigate nine poorly characterized essential genes in S. mutans. From this screen, phenotypic and genetic analyses identified SMU_393 as a functional homolog of the pneumococcal chromosome segregation factor, RocS. Depletion of SMU_393 resulted in abnormal cell widening, hypersensitivity to DNA damage, and a significant subpopulation of anucleate cells. These phenotypes were bypassed by a spontaneous surface-exposed missense mutation (dnaAQ197E) within the AAA+ ATPase domain of the replication initiator. Together, this study refines annotations within the S. mutans essential genome and provides genetic insights into streptococcal chromosome segregation and cell cycle control.

microbiology↗

bioRxiv2026

Alternative management strategy reshapes litter microbiome dynamics in a commercial broiler rearing system

Pre-harvest litter management is a key determinant of broiler production conditions, influencing NH generation, pathogen exposure, nutrient retention, and microbial reservoirs that accumulate across production cycles. Conventional chemical and physical management strategies can support flock health, but their effects on pathogen-associated bacterial populations are often transient and may not account for the microbial interactions that govern persistence, exclusion, and community succession. Here, we evaluated an alternative litter management strategy combining IndigoLT(R) pre-/postbiotic with reduced-rate NaHSO across two broiler growouts, with litter sampled at the end of each flock to determine impact on prokaryotic microbiome structure, physicochemistry, and Enterococcus abundance. Alternative management influenced observed richness, phylogenetic diversity, community composition, and co-occurrence network structure while reducing the relative abundance of Enterococcus, including E. cecorum and E. hirae. Digital PCR corroborated sequencing-based Enterococcus abundance patterns, although 16S-based treatment effects were not always reflected as lower absolute copy number at terminal sampling, consistent with reduced proportional dominance rather than sustained absolute suppression. Complementary biofilm- and growth-inhibition assays performed with IndigoLT(R) demonstrated context-dependent antibiofilm and bacteriostatic activity against reference and poultry-derived Enterococcus isolates, with stronger responses for E. cecorum than E. hirae and bactericidal-level reductions in viable recovery at higher exposure levels. These findings demonstrate that biologic-based litter management can alter microbiome structure and pathogen-associated taxa under commercial production conditions, providing a basis for microbiome-informed amendment strategies aimed at reducing pathogen load and supporting broiler health.

microbiology↗

bioRxiv2026

Genomic instability and biofilm determinants in Streptococcus mutans: insights from a sequence-defined arrayed transposon library

Streptococcus mutans is a primary architect of dental caries, utilizing complex genetic networks to build resilient, acid-producing biofilms. While pooled screens (Tn-seq) have identified important fitness factors, they often fail to capture extracellular or moderate-effect determinants due to community-level masking. Therefore, to study biofilm phenotypes, we constructed a comprehensive arrayed library of 9,216 mutants and used Cartesian Pooling-Coordinate Sequencing (CP-CSeq) to establish a sequence-defined resource covering 51% of non-essential genes. By screening the entire collection in isolation, we identified several novel biofilm determinants, including the putative metal transporter SMU_635 and the glycosylation-associated protein SMU_2160. However, systematic whole-genome sequencing (WGS) of our hits revealed an interesting level of genomic instability: 25% of biofilm-defective mutants had undergone spontaneous recombination at the gtfBC locus, while 7% had lost the TnSmu1 element, an excision rate 1,000-fold higher than previously reported. While targeted mutagenesis confirmed that TnSmu1 loss does not impact biofilm integrity, the gtfBC deletions directly accounted for the most severe phenotypes, highlighting a systemic risk of misattributing gene functions to primary transposon insertions. Our findings provide a powerful new genetic resource for the S. mutans community while establishing a critical new standard: an arrayed library is only as defined as its underlying genome, making systematic genomic verification an essential prerequisite for accurate functional genomics. ImportanceStreptococcus mutans is a major human pathogen responsible for dental caries, a global public health challenge driven in part by the organisms ability to form resilient, acidogenic biofilms. While traditional pooled genetic screens have identified many fitness factors, they often fail to capture extracellular or moderate-effect determinants because neighboring healthy bacteria can mask these defects. This work provides the scientific community with a sequence-defined arrayed mutant library, an essential resource for dissecting the individual contributions of genes to biofilm integrity in isolation. Beyond identifying well-known machinery, this study uncovers novel determinants, including the putative metal transporter SMU_635 and the putative glycosylation-associated protein SMU_2160. Crucially, the discovery of pervasive genomic instability within the library, specifically at the gtfBC and TnSmu1 loci, reveals a systemic risk in functional genomics: the potential to misattribute phenotypes to primary mutations when the underlying background has undergone large-scale rearrangements. By establishing systematic whole-genome verification as a necessary standard, this research ensures that the identification of future therapeutic targets is built upon a verified genetic foundation.

microbiology↗

bioRxiv2025

Post-translational modifications via serine/threonine phosphorylation and GpsB in Streptococcus mutans

Post-translational modifications (PTMs), such as protein phosphorylation, are critical regulators of bacterial physiology. Here, we present the first comprehensive phosphoproteomic analysis of Streptococcus mutans, revealing extensive O-phosphorylation under non-stressed conditions. Using tandem mass tag (TMT)-based mass spectrometry and phosphopeptide enrichment, we identified 231 high-confidence phosphosites on 131 proteins, representing approximately 6.7% of the detected proteome. These phosphorylated proteins were enriched in pathways related to translation, carbohydrate metabolism, and the cell cycle, suggesting a broad role for O-phosphorylation in core cellular functions. To define the functional roles of the sole serine/threonine protein kinase (PknB) and phosphatase (PppL) encoded by S. mutans, we analyzed phosphoproteomic and proteomic changes in {Delta}pknB and {Delta}pppL mutants. These mutants exhibited widespread alterations in protein abundance and phosphorylation, revealing overlapping but distinct sets of putative kinase and phosphatase substrates, including DivIVA, MapZ, MltG, and ribosomal proteins. Notably, we discovered that repression of gpsB, a predicted PknB binding partner, causes lethal defects that can be rescued by a suppressor mutation (G98R) in pppL. This mutation restores phosphorylation of DivIVA, suggesting that GpsB regulates the PknB/PppL signaling axis to maintain appropriate phosphorylation of essential targets. This work highlights conserved and unique features of bacterial phospho-signaling and provides a foundation for future studies on PTM-mediated regulation in S. mutans.

microbiology↗

bioRxiv2022

Investigating CRISPR spacer targets and their impact on genomic diversification of Streptococcus mutans

CRISPR-Cas is a bacterial immune system that restricts the acquisition of mobile DNA elements. These systems provide immunity against foreign DNA by encoding CRISPR spacers that help target DNA if it re-enters the cell. In this way, CRISPR spacers are a type of molecular tape recorder of foreign DNA encountered by the host microorganism. Here, we extracted [~]8,000 CRISPR spacers from a collection of over three hundred Streptococcus mutans genomes. Phage DNA is a major target of S. mutans spacers. Strains have also generated immunity against mobile DNA elements such as plasmids and integrative and conjugative elements. There may also be considerable immunity generated against bacterial DNA, although the relative contribution of self-targeting versus bona fide intra- or inter-species targeting needs to be investigated further. While there was clear evidence that these systems have acquired immunity against foreign DNA, there appeared to be minimal impact on horizontal gene transfer (HGT) constraints on a species-level. There was little or no impact on genome size, GC content and openness of the pangenome when comparing between S. mutans strains with low or high CRISPR spacer loads. In summary, while there is evidence of CRISPR spacer acquisition against self and foreign DNA, CRISPR-Cas does not act as a barrier on the expansion of the S. mutans accessory genome. Impact StatementCRISPR-Cas is a widespread bacterial immune system that has been repurposed as a molecular biology tool. This study investigates the role of these systems in the biology and evolution of the dental caries pathogen Streptococcus mutans. CRISPR spacers, that encode immunity against foreign DNA, were extracted from over three hundred S. mutans isolates. Sequence analysis showed that the CRISPR spacers match against phage, mobile element, and bacterial DNA. This shows that S. mutans is actively acquiring immunity against horizontally acquired DNA. However, additional analysis revealed little to no impact of CRISPR-Cas systems on diversification of the S. mutans genome. This suggests that while these systems are actively acquiring CRISPR spacers to defend against foreign DNA, the overall impact on the S. mutans genome might be small. Data SummarySupporting data provided on the Github platform: https://github.com/theshieldslab/Streptococcus-mutans-CRISPR-Spacers-Analysis The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

genomics↗

bioRxiv2019

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function

A recent genome-wide screen identified [~]300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3 end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as the Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

microbiology↗

bioRxiv2019

Carbohydrate and PepO control bimodality in competence development by Streptococcus mutans

In Streptococcus mutans, the alternative sigma factor ComX controls entry into genetic competence. Competence signaling peptide (CSP) induces bimodal expression of comX, with only a fraction of cells in the population becoming transformable. Curiously, bimodal comX activation in response to CSP is affected by peptides in the growth medium and by carbohydrate source. CSP elicits bimodal expression of comX in media rich in small peptides, but in defined media lacking small peptides CSP induces no response in comX. In addition, growth on certain sugars other than glucose increases the proportion of the population that activates comX in response to CSP, relative to growth on glucose. By investigating the connection between media and bimodal comX expression, we find evidence for two mechanisms that modulate transcriptional positive feedback in the ComRS system, which is the origin of comX bimodality. We find that the endopeptidase PepO suppresses the ComRS feedback loop, most likely by degrading the intracellular XIP/ComS signal. Deletion of pepO eliminates bimodality in comX, leading to a unimodal comX response to CSP in defined and complex media. We also find that CSP upregulates comR in a carbohydrate source-dependent fashion, providing an additional stimulus to the ComRS feedback system. Our data provide mechanistic insight into how CSP regulates the bistable competence circuit and explain the puzzle of growth medium-dependence in S. mutans competence regulation.

biophysics↗

bioRxiv2019

Long-term fish assemblages of the Ohio River: altered trophic and life history strategies with hydrologic alterations and landuse modifications

Long-term monitoring of species assemblages provides a unique opportunity to test hypotheses regarding environmentally-induced directional trajectories of freshwater species assemblages. We used 57 years of lockchamber fish rotenone and boat electrofishing survey data (1957-2014) collected by the Ohio River Valley Water Sanitation Commission (ORSANCO) to test for directional trajectories in taxonomy, trophic classifications, and life history strategies of freshwater fish assemblages in the Ohio River Basin. We found significant changes in taxonomic and trophic composition of freshwater fishes in the Ohio River Basin. Annual species richness varied from 31 to 90 species and generally increased with year. Temporal trajectories were present for taxonomic and trophic assemblages. Assemblage structure based on taxonomy was correlated with land use change (decrease in agriculture and increase in forest). Taxonomic assemblage structure was also correlated with altered hydrology variables of increased minimum discharge, decreased fall rate, and increased rise rate. Trophic composition of fish catch correlated with land use change (decrease in agriculture and increase in forest) and altered hydrology. Altered hydrology of increased minimum discharge, increased fall discharge, decreased base flows, and increased number of high pulse events was correlated with increased counts of herbivore-detritivores and decreased counts of piscivores and planktivores. We did not find directional changes in life history composition. We hypothesized a shift occurred from benthic to phytoplankton production throughout the basin that may have decreased secondary production of benthic invertebrates. This may also be responsible for lower trophic position of invertivore and piscivore fishes observed in other studies.

ecology↗

bioRxiv2018

Intracellular signaling through the comRS system in Streptococcus mutans genetic competence

AbstractEntry into genetic competence in streptococci is controlled by ComX, an alternative sigma factor for genes that enable the import of exogenous DNA. In Streptococcus mutans, the immediate activator of comX is the ComRS signaling system, which consists of the cytosolic receptor ComR and the 7-residue signal peptide XIP, which is derived from ComS. Extracellular XIP imported by an oligopeptide permease interacts with ComR to form a transcriptional activator for both comX and comS. Therefore, extracellular XIP can function as an exogenous signal to trigger S. mutans competence. However, the mechanisms that process ComS and export it as XIP are not fully known in S. mutans. The observation that comX is expressed bimodally under some environmental conditions suggests that ComR may also interact with endogenously produced XIP or ComS, creating an intracellular positive feedback loop in comS transcription. Here we use single cell and microfluidic methods to compare the effects of the native comS gene and extracellular XIP on comX expression. We find that deletion of comS reduces the response of comX to extracellular XIP. We also find that comS-overexpressing cells autoactivate their comX even when their growth medium is rapidly exchanged, although this autoactivation requires an intact copy of comS under control of its own promoter. However comS-overexpressing cells do not activate comS-deficient mutants growing in coculture. These data show that individual cells can activate comX without exporting or importing the XIP or ComS signal, and that endogenously and exogenously produced ComS/XIP have inequivalent effects on comX behavior. These data are fully consistent with a model in which intracellular positive feedback in comS transcription plays a role in ComRS signaling, and is responsible for the bimodal expression of comX.\n\nAuthor SummaryHeterogeneous gene expression in genetically identical populations plays an important role in bacterial persistence and survival under changing environmental conditions. In the oral pathogen Streptococcus mutans, the physiological state of genetic competence can exhibit bimodality, with only some cells becoming competent. S. mutans controls its entry into competence by using the ComRS signaling system to activate comX, a gene encoding the master competence regulator ComX. The ComRS system is understood as a quorum sensing system, in which the extracellular accumulation of the small signal peptide XIP, derived from ComS, induces comX expression. We coupled observation of bacteria that fluoresce when comX is active with mathematical analysis and chemical binding assays to show that activation of comX does not necessarily require extracellular XIP or ComS, and that comX-active cells do not necessarily export XIP. Our experiments and mathematical modeling indicate that a positive feedback loop in comS transcription allows a cell to activate comX in response to its own XIP or ComS in the absence of extracellular XIP, or to amplify its comX response to extracellular XIP if present. Such positive feedback loops are often the cause of bimodal gene expression like that seen in S. mutans competence.

microbiology↗

bioRxiv2017

Intercellular communication via the comX-Inducing Peptide (XIP) of Streptococcus mutans

Gram-positive bacteria utilize exported peptides to coordinate genetic and physiological processes required for biofilm formation, stress responses and ecological competitiveness. One example is activation of natural genetic competence by ComR and the comX-inducing peptide (XIP) in Streptococcus mutans. Although the competence pathway can be activated by addition of synthetic XIP in defined medium, the hypothesis that XIP is able to function as an intercellular signal molecule has not been rigorously tested. Co-culture model systems were developed that included a \"sender\" strain that overexpressed the XIP precursor (ComS) and a \"responder\" strain harboring a GFP reporter fusion to a ComR-activated gene (comX) promoter. The ability of the sender strain to provide a signal to activate GFP expression was monitored at the individual cell and population levels using i) planktonic culture systems, ii) cells suspended in an agarose matrix or iii) cells growing in biofilms. XIP was shown to be freely diffusible and XIP signaling between the S. mutans sender and responder strains did not require cell-to-cell contact. The presence of a sucrose-derived exopolysaccharide matrix diminished the efficiency of XIP signaling in biofilms, possibly by affecting spatial distribution of XIP senders and potential responders. Intercellular signaling was greatly impaired in a strain lacking the primary autolysin, AtlA, and was substantially greater when the sender strain underwent lysis. Collectively, these data provide evidence that S. mutans XIP can indeed function as a peptide signal between cells and highlight the importance of studying signaling with endogenously-produced peptide(s) in populations in various environments and physiologic states.\n\nIMPORTANCEThe comX-inducing peptide (XIP) of Streptococcus mutans is a key regulatory element in the activation of genetic competence, which allows cells to take up extracellular DNA. XIP has been found in cell culture fluids and addition of synthetic XIP to physiologically receptive cells can robustly induce competence gene expression. However, there is a lack of consensus as to whether XIP can function as an intercellular communication signal. Here, we show that XIP indeed signals between cells in S. mutans, but that cell lysis may be a critical factor, as opposed to a dedicated secretion/processing system, in allowing for release of XIP into the environment. The results have important implications in the context of the ecology, virulence and evolution of a ubiquitous human pathogen and related organisms.

microbiology↗