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Journal of Microbiology & Biology... Apr 2024Many undergraduates struggle to interpret abstract concepts in molecular biology. Modeling can facilitate learning by making these abstract concepts tangible. Here, we...
Many undergraduates struggle to interpret abstract concepts in molecular biology. Modeling can facilitate learning by making these abstract concepts tangible. Here, we present an exercise based on the operon designed for undergraduate students using LEGO bricks. The operon is a classic example of transcriptional regulation taught in a variety of undergraduate biology courses and is fundamental to understanding the regulation of gene expression. This easy-to-implement active learning exercise demonstrates how the various components of the operon are oriented under a variety of nutritional conditions to control gene expression. In addition, higher-order concepts, such as the effect of mutation on operon expression, can be readily modeled. Overall, students not only found this exercise to be enjoyable but also helpful as a tool to engage with this course material.
PubMed: 38661411
DOI: 10.1128/jmbe.00034-24 -
Frontiers in Cell and Developmental... 2023Since Jacob and Monod's discovery of the lac operon ∼1960, the explanations offered for most metabolic adaptations have been genetic. The focus has been on the...
Since Jacob and Monod's discovery of the lac operon ∼1960, the explanations offered for most metabolic adaptations have been genetic. The focus has been on the adaptive changes in gene expression that occur, which are often referred to as "metabolic reprogramming." The contributions metabolism makes to adaptation have been largely ignored. Here we point out that metabolic adaptations, including the associated changes in gene expression, are highly dependent on the metabolic state of an organism prior to the environmental change to which it is adapting, and on the plasticity of that state. In support of this hypothesis, we examine the paradigmatic example of a genetically driven adaptation, the adaptation of to growth on lactose, and the paradigmatic example of a metabolic driven adaptation, the Crabtree effect in yeast. Using a framework based on metabolic control analysis, we have reevaluated what is known about both adaptations, and conclude that knowledge of the metabolic properties of these organisms prior to environmental change is critical for understanding not only how they survive long enough to adapt, but also how the ensuing changes in gene expression occur, and their phenotypes post-adaptation. It would be useful if future explanations for metabolic adaptations acknowledged the contributions made to them by metabolism, and described the complex interplay between metabolic systems and genetic systems that make these adaptations possible.
PubMed: 37377740
DOI: 10.3389/fcell.2023.1197226 -
International Journal of Molecular... Oct 2023Laccase genes produce laccase enzymes that play a crucial role in the production of lignin and oxidation reactions within plants. Lignin is a complex polymer that... (Review)
Review
Laccase genes produce laccase enzymes that play a crucial role in the production of lignin and oxidation reactions within plants. Lignin is a complex polymer that provides structure and toughness to the cell walls of numerous fruit plants. The genes that encode laccase enzymes play vital roles in plant physiology, including the synthesis of pigments like PA that contribute to the colors of fruits, and in defending against pathogens and environmental stresses. They are crucial for fruit development, ripening, structural maintenance in plants, and adaptation to various environmental factors. As such, these genes and enzymes are essential for plant growth and development, as well as for various biotechnological applications in environmental remediation and industrial processes. This review article emphasizes the significance of genes encoding laccase enzymes during fruit growth, specifically pertaining to the strengthening of the endocarp through lignification. This process is crucial for ensuring fruit defense and optimizing seed scattering. The information gathered in this article will aid breeders in producing future fruit-bearing plants that are resistant to disease, cost-effective, and nutrient-rich.
Topics: Fruit; Lignin; Laccase; Lac Operon; Seeds; Gene Expression Regulation, Plant
PubMed: 37958753
DOI: 10.3390/ijms242115768 -
Frontiers in Microbiology 2023[This corrects the article DOI: 10.3389/fmicb.2021.709259.].
[This corrects the article DOI: 10.3389/fmicb.2021.709259.].
PubMed: 38088962
DOI: 10.3389/fmicb.2023.1332365 -
MBio Apr 2024We previously demonstrated that mutation of in limits biofilm formation, cytotoxicity for osteoblasts and osteoclasts, and virulence in osteomyelitis, and that all of...
We previously demonstrated that mutation of in limits biofilm formation, cytotoxicity for osteoblasts and osteoclasts, and virulence in osteomyelitis, and that all of these phenotypes can be attributed to the increased production of extracellular proteases. Here we extend these studies to assess the individual importance of these proteases alone and in combination with each other using the methicillin-resistant USA300 strain LAC, the methicillin-susceptible USA200 strain UAMS-1, and isogenic mutants that were also unable to produce aureolysin (Aur), staphopain A (ScpA), staphylococcal serine protease A (subsp.), staphopain B (SspB), and the staphylococcal serine protease-like proteins A-F (SplA-F). Biofilm formation was restored in LAC and UAMS-1 mutants by subsequent mutation of and , while mutation of had the greatest impact on cytotoxicity to mammalian cells, particularly with conditioned medium (CM) from the more cytotoxic strain LAC. However, SDS-PAGE and western blot analysis of CM confirmed that mutation of was also required to mimic the phenotype of mutants unable to produce any extracellular proteases. Nevertheless, in a murine model of post-traumatic osteomyelitis, mutation of and had the greatest impact on restoring the virulence of LAC and UAMS-1 mutants, with concurrent mutation of and the operon having relatively little effect. These results demonstrate that the increased production of Aur and ScpA in combination with each other is a primary determinant of the reduced virulence of mutants in diverse clinical isolates including both methicillin-resistant and methicillin-susceptible strains.IMPORTANCEPrevious work established that SarA plays a primary role in limiting the production of extracellular proteases to prevent them from limiting the abundance of virulence factors. Eliminating the production of all 10 extracellular proteases in the methicillin-resistant strain LAC has also been shown to enhance virulence in a murine sepsis model, and this has been attributed to the specific proteases Aur and ScpA. The importance of this work lies in our demonstration that the increased production of these same proteases largely accounts for the decreased virulence of mutants in a murine model of post-traumatic osteomyelitis not only in LAC but also in the methicillin-susceptible human osteomyelitis isolate UAMS-1. This confirms that -mediated repression of Aur and ScpA production plays a critical role in the posttranslational regulation of virulence factors in diverse clinical isolates and diverse forms of infection.
Topics: Animals; Mice; Humans; Staphylococcus aureus; Virulence; Disease Models, Animal; Methicillin; Virulence Factors; Bacterial Proteins; Peptide Hydrolases; Osteomyelitis; Biofilms; Staphylococcal Infections; Gene Expression Regulation, Bacterial; Mammals; Metalloendopeptidases
PubMed: 38415646
DOI: 10.1128/mbio.03383-23 -
Microbial Biotechnology Mar 2024Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction...
Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction dynamic range upon the addition of effectors. In the P /LacI pair, the promoter of the lac operon in Escherichia coli is characterized by its leakiness, attributed to the moderate affinity of LacI for its operator target. In response to this limitation, the LacI regulatory protein underwent engineering to enhance its regulatory properties. The M7 mutant, carrying I79T and N246S mutations, resulted in the lac promoter displaying approximately 95% less leaky expression and a broader induction dynamic range compared to the wild-type LacI. An in-depth analysis of each mutation revealed distinct regulatory profiles. In contrast to the wild-type LacI, the M7 mutant exhibited a tighter binding to the operator sequence, as evidenced by surface plasmon resonance studies. Leveraging the capabilities of the M7 mutant, a high-value sugar biosensor was constructed. This biosensor facilitated the selection of mutant galactosidases with approximately a seven-fold improvement in specific activity for transgalactosylation. Consequently, this advancement enabled enhanced biosynthesis of galacto-oligosaccharides (GOS).
Topics: Lac Repressors; Escherichia coli; Escherichia coli Proteins; Mutation; Promoter Regions, Genetic; Bacterial Proteins
PubMed: 38465475
DOI: 10.1111/1751-7915.14427 -
Microbial Genomics Nov 2023Horizontal gene transfer by plasmids can confer metabolic capabilities that expand a host cell's niche. Yet, it is less understood whether the coalescence of specialized...
Horizontal gene transfer by plasmids can confer metabolic capabilities that expand a host cell's niche. Yet, it is less understood whether the coalescence of specialized catabolic functions, antibiotic resistances and metal resistances on plasmids provides synergistic benefits. In this study, we report whole-genome assembly and phenotypic analysis of five strains isolated in the 1980s from milk powder in Munich, Germany. All strains exhibited the unusual phenotype of lactose-fermentation and encoded either of two variants of the operon. Surprisingly, all strains encoded the mobilized colistin resistance gene 9 (), long before the first report of this gene in the literature. In two cases, the gene and the locus were linked within a large gene island that formed an IncHI2A-type plasmid in one strain but was chromosomally integrated in the other strain. In two other strains, the gene was found on a large IncHI1B/IncP-type plasmid, whereas the locus was encoded on a separate chromosomally integrated plasmidic island. The sequences were identical and genomic contexts could not explain the wide range of colistin resistances exhibited by the strains. Nucleotide variants did explain phenotypic differences in motility and exopolysaccharide production. The observed linkage of to lactose metabolism, an array of heavy-metal detoxification systems, and other antibiotic resistance genes may reflect a coalescence of specialized phenotypes that improve the spread of colistin resistance in dairy facilities, much earlier than previously suspected.
Topics: Colistin; Salmonella enterica; Lactose; Serogroup; Drug Resistance, Bacterial; Plasmids
PubMed: 38031909
DOI: 10.1099/mgen.0.001149 -
Archives of Toxicology Aug 2023Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide...
Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with > 80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.
Topics: Male; Mice; Animals; Procarbazine; Mutation Rate; Bone Marrow; Mutagens; Mutation; Mutagenicity Tests; Mice, Transgenic; Lac Operon
PubMed: 37341741
DOI: 10.1007/s00204-023-03527-y -
Physiological Reports Apr 2024HMG (high mobility group) proteins are a diverse family of nonhistone chromosomal proteins that interact with DNA and a wide range of transcriptional regulators to...
HMG (high mobility group) proteins are a diverse family of nonhistone chromosomal proteins that interact with DNA and a wide range of transcriptional regulators to regulate the structural architecture of DNA. HMGXB4 (also known as HMG2L1) is an HMG protein family member that contains a single HMG box domain. Our previous studies have demonstrated that HMGXB4 suppresses smooth muscle differentiation and exacerbates endotoxemia by promoting a systemic inflammatory response in mice. However, the expression of Hmgxb4 in vivo has not fully examined. Herein, we generated a mouse model that harbors a gene trap in the form of a lacZ gene insertion into the Hmgxb4 gene. This mouse enables the visualization of endogenous HMGXB4 expression in different tissues via staining for the β-galactosidase activity of LacZ which is under the control of the endogenous Hmgxb4 gene promoter. We found that HMGXB4 is widely expressed in mouse tissues and is a nuclear protein. Furthermore, the Hmgxb4 gene trap mice exhibit normal cardiac function and blood pressure. Measurement of β-galactosidase activity in the Hmgxb4 gene trap mice demonstrated that the arterial injury significantly induces Hmgxb4 expression. In summary, the Hmgxb4 gene trap reporter mouse described here provides a valuable tool to examine the expression level of endogenous Hmgxb4 in both physiological and pathological settings in vivo.
Topics: Animals; Male; Mice; beta-Galactosidase; High Mobility Group Proteins; Lac Operon; Mice, Inbred C57BL; Mice, Transgenic; Transcription Factors
PubMed: 38644513
DOI: 10.14814/phy2.16014