Biology subjects

McLellan, L. K.

Publications and source records attributed to McLellan, L. K..

4 recordsLinked to original sources

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↗

bioRxiv2022

TnSmu1 is a functional integrative and conjugative element in Streptococcus mutans that when expressed causes growth arrest of host bacteria

Integrative and conjugative elements (ICEs) are major drivers of horizontal gene transfer in bacteria. They mediate their own transfer from host cells (donors) to recipients and allow bacteria to acquire new phenotypes, including pathogenic and metabolic capabilities and drug resistances. Streptococcus mutans, a major causative agent of dental caries, contains a putative ICE, TnSmu1, integrated at the 3 end of a leucyl tRNA gene. We found that TnSmu1 is a functional ICE, containing all the genes necessary for ICE function. It excised from the chromosome and excision was stimulated by DNA damage. We identified the DNA junctions generated by excision of TnSmu1, defined the ends of the element, and detected the extrachromosomal circle. We found that TnSmu1 can transfer from S. mutans donors to recipients when co-cultured on solid medium. The presence of TnSmu1 in recipients inhibited successful acquisition of another copy and this inhibition was mediated, at least in part, by the likely transcriptional repressor encoded by the element. Using microscopy to track individual cells, we found that activation TnSmu1 caused an arrest of cell growth. Our results demonstrate that TnSmu1 is a functional ICE that affects the biology of its host cells.

microbiology↗

bioRxiv2022

Activation of the integrative and conjugative element Tn916 causes growth arrest and death of host bacteria

Integrative and conjugative elements (ICEs) serve as major drivers of bacterial evolution. These elements often confer some benefit to a host cell, including antibiotic resistance, metabolic capabilities, or pathogenic determinants. ICEs can also have negative impacts on their host cells. Here, we investigated the effects of the ICE (conjugative transposon) Tn916 on host cells. Because Tn916 is active in a relatively small subpopulation of host cells, we developed a fluorescent reporter system for monitoring activation of Tn916 in single cells. We found that when active in Bacillus subtilis and its natural host Enterococcus faecalis, Tn916 inhibited cell division and most cells died. We also observed these phenotypes on the population level in B. subtilis utilizing a modified version of Tn916 that can be activated in the majority of cells. We identified two genes (orf17 and orf16) in Tn916 that were sufficient to cause host growth defects and identified a single gene, yqaR, that is found in a defective phage (skin) in the B. subtilis chromosome that is required for this phenotype. However, these three genes are only partially responsible for the growth defect caused by Tn916, indicating that Tn916 possesses multiple mechanisms to affect growth and viability of host cells. These results highlight the complex relationships that conjugative elements have with their host cells and the interplay between mobile genetic elements.

microbiology↗

bioRxiv2021

Cooperativity mediates drug resistance and metabolism in Plasmodium falciparum malaria parasites

Efforts to control the global malaria health crisis are undermined by antimalarial resistance. Iden-tifying mechanisms of resistance will uncover the underlying biology of the Plasmodium falciparum malaria parasites that allow evasion of our most promising therapeutics and may reveal new drug targets. We utilized fosmidomycin (FSM) as a chemical inhibitor of plastidial isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway. We have thus identified an unusual metabolic regulation scheme in the malaria parasite through the essential glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Two parallel genetic screens converged on independent but functionally analogous resistance alleles in GAPDH. Metabolic profiling of FSM-resistant gapdh mutant parasites indicates that neither of these mutations disrupt overall glycolytic output. While FSM-resistant GAPDH variant proteins are catalytically active, they have reduced assembly into the homotetrameric state favored by wild-type GAPDH. Disrupted oligomerization of FSM-resistant GAPDH variant proteins is accompanied by altered enzymatic cooperativity and reduced susceptibility to inhibition by free heme. Together, our data identifies a new genetic biomarker of FSM-resistance and reveals the central role of GAPDH cooperativity in MEP pathway control and antimalarial sensitivity.

microbiology↗