Biology subjects

Butzin, N. C.

Publications and source records attributed to Butzin, N. C..

6 recordsLinked to original sources

bioRxiv2025

From Orphan Genes to Cryptogenic Gene Candidates: Reassessing Uniqueness

BackgroundOrphan genes lack recognizable homologues outside a given taxonomic unit; thus, they have elusive evolutionary origins. They have long been invoked to explain lineage specific traits in medicine and evolutionary biology. Here, we re-analyzed a 2023 repository of putative orphan genes compiled from over 80,000 bacterial species. Using iterative homology-based analyses, we reassessed each genes taxonomic distribution across a broader genomic landscape. ResultsThese results challenge the long-held assertion that species truly harbor large numbers of orphan genes and instead demonstrate that their prevalence has been overestimated. Many "orphan genes" from 2023 now match homologues in other bacterial taxa (2025), demonstrating that sparse sampling had inflated earlier orphan counts. To better reflect these findings, we propose the term Cryptogenic Gene Candidates (CGCs). This offers a more precise alternative to orphan genes, as it more accurately reflects the provisional and potentially temporary nature of these genes apparent uniqueness. This revised terminology acknowledges that many genes identified as having a unique origin may lose this status as more genomes are sequenced. Although our analyses substantially reduced the number of false-positive CGCs, it cannot determine whether a given CGC truly encodes a functional protein or is an artifact of bioinformatic analysis. To minimize annotation artifacts, we then applied additional computational filters to prioritize candidates most likely to encode bona fide proteins. ConclusionsThis work revises the understanding of orphan gene prevalence, establishes CGCs as a more accurate classification, and provides a prioritized set of candidates to guide future experimental studies.

bioinformatics↗

bioRxiv2024

Gram-positive bacteria are primed for surviving lethal doses of antibiotics and chemical stress

Antibiotic resistance kills millions worldwide yearly. However, a major contributor to recurrent infections lies in a small fraction of bacterial cells, known as persisters. These cells are not inherently antibiotic-resistant, yet they lead to increased antibiotic usage, raising the risk of developing resistant progenies. In a bacterial population, individual cells exhibit considerable fluctuations in their gene expression levels despite being cultivated under identical, stable conditions. This variability in cell-to-cell characteristics (phenotypic diversity) within an isogenic population enables persister cells to withstand antibiotic exposure by entering a non-dividing state. We recently showed the existence of "primed cells" in E. coli. Primed cells are dividing cells prepared for antibiotic stress before encountering it and are more prone to form persisters. They also pass their "prepared state" down for several generations through epigenetic memory. Here, we show that primed cells are common among distant bacterial lineages, allowing for survival against antibiotics and other chemical stress, and form in different growth phases. They are also responsible for increased persister levels in transition and stationary phases compared to the log phase. We tested and showed that the Gram-positive bacterium Bacillus megaterium, evolutionarily very distant from E. coli, forms primed cells and has a transient epigenetic memory that is maintained for 7 generations or more. We showed this using ciprofloxacin and the non-antibiotic chemical stress fluoride. It is well established that persister levels are higher in the stationary phase than in the log phase, and B. megaterium persisters levels are nearly identical from the early to late-log phase but are [~]2-fold and [~]4-fold higher in the transition and stationary phase, respectively. It was previously proposed that there are two distinct types of persisters: Type II forms in the log phase, while Type I forms in the stationary phase. However, we show that primed cells lead to increased persisters in the transition and stationary phase and found no evidence of Type I or II persisters with distant phenotypes. Overall, we have provided substantial evidence of the importance of primed cells and their transitory epigenetic memories to surviving stress.

microbiology↗

bioRxiv2023

An isogenic E. coli population gives rise to multiple persister phenotypes

Bacterial persisters are a subpopulation of multidrug-tolerant cells capable of surviving and resuming activity after exposure to bactericidal antibiotic concentrations, contributing to relapsing infections and the development of antibiotic resistance. We challenge the conventional view that persisters are metabolically dormant by providing compelling evidence that an isogenic population of Escherichia coli remains metabolically active in persistence. Our transcriptomic analysis, conducted at various time points following exposure to bactericidal concentrations of ampicillin (Amp), revealed a number of genes with differential expression over time. Some genes were consistently upregulated in Amp treated persisters compared to the untreated controls, a change that can only occur in metabolically active cells capable of increasing RNA levels. Some of these genes have been previously linked to persister cells, while others have not been associated with them before. If persister cells were metabolically dormant, we would expect minimal changes in the gene network across different time points of Amp treatment. However, network analysis revealed major shifts in gene network activity at various time points of antibiotic exposure. These findings reveal that persisters are metabolically active, non-dividing cells, thereby challenging the notion that they are dormant. Significance statementBacterial persisters are a subpopulation renowned for their multidrug tolerance and remarkable ability to survive bactericidal antibiotic treatments; understanding their formation and long-term survival presents significant challenges. These persisters play a critical role in driving antibiotic resistance, underscoring the urgency of deepening our knowledge about them as the threat of resistance continues to escalate. Our study challenges the long-held assumption that persisters are metabolically inactive and that persisters are not as dormant as previously thought.

microbiology↗

bioRxiv2022

Escherichia coli cells are primed for survival before lethal antibiotic stress

Non-genetic factors can cause significant fluctuations in gene expression levels. Regardless of growing in a stable environment, this fluctuation leads to cell-to-cell variability in an isogenic population. This phenotypic heterogeneity allows a tiny subset of bacterial cells in a population, referred to as persister cells, to tolerate long-term lethal antibiotic effects by entering into a non-dividing, metabolically altered state. One fundamental question is whether this heterogeneous persister population is due to a pre-existing genetic mutation or a result of a transiently-primed reversible cell state. To explore this, we tested clonal populations starting from a single cell using a modified Luria-Delbruck fluctuation test. Through we kept the conditions the same, the diversity in persistence level among clones was relatively consistent: varying from [~]60-100 and [~]40-70 fold for ampicillin (Amp) and apramycin (Apr), respectively. Then we divided and diluted each clone to observe whether the same clone had comparable persister levels for more than one generation. Replicates had similar persister levels even when clones were divided, diluted by 1:20, and allowed to grow for [~]5 generations. This result explicitly shows a cellular memory passed on for generations and eventually lost when cells are diluted to 1:100 and regrown (>7 generations). Our result demonstrates 1) the existence of a small population prepared for stress ("primed cells") resulting in higher persister numbers, 2) the primed memory state is reproducible and transient, passed down for generations but eventually lost, and 3) a heterogeneous persister population is a result of a transiently-primed reversible cell state and not due to a pre-existing genetic mutation.

synthetic biology↗

bioRxiv2020

Regulatory proteins direct a transcriptional network in response to antibiotic stress

Antibiotic treatment kills a large portion of a population, while a small, tolerant subpopulation survives. Tolerant bacteria disrupt antibiotic efficacy and increase the likelihood that a population gains antibiotic resistance, a growing health concern. We examined how E. coli transcriptional networks changed in response to lethal ampicillin concentrations. We are the first to apply transcriptional regulatory network (TRN) analysis to antibiotic tolerance by leveraging existing knowledge and our transcriptional data. TRN analysis shows that gene expression changes specific to ampicillin treatment are likely caused by specific sigma and transcription factors typically regulated by proteolysis. These results demonstrate that to survive lethal concentration of ampicillin specific regulatory proteins change activity and cause a coordinated transcriptional response that leverages multiple gene systems.

systems biology↗

bioRxiv2020

Antibiotic tolerance, persistence, and resistance of the evolved minimal cell, Mycoplasma mycoides JCVI-Syn3B.

Antibiotic resistance is a growing problem, but bacteria can evade antibiotic treatment via tolerance and persistence. Antibiotic persisters are a small subpopulation of bacteria that tolerate antibiotics due to a physiologically dormant state. Hence, persistence is considered a major contributor to the evolution of antibiotic-resistant and relapsing infections. Here, we used the synthetically developed minimal cell Mycoplasma mycoides JCVI-Syn3B to examine essential mechanisms of antibiotic survival. The minimal cell contains only 473 genes, and most genes are essential. Its reduced complexity helps to reveal hidden phenomenon and fundamental biological principles can be explored because of less redundancy and feedback between systems compared to natural cells. We found that Syn3B evolves antibiotic resistance to different types of antibiotics expeditiously. The minimal cell also tolerates and persists against multiple antibiotics. It contains a few already identified persister-related genes, although lacking many systems previously linked to persistence (e.g. toxin-antitoxin systems, ribosome hibernation genes, etc.).

synthetic biology↗