Amino acid starvation in Escherichia coli results a spectrum of changes gene expression, including inhibition rRNA and tRNA promoters activation certain for amino biosynthesis transport. The unusual nucleotide ppGpp plays an important role both negative positive regulation. Previously, we others suggested that effects might be indirect, resulting from the transcription and, thus, liberation RNA polymerase binding to other promoters. Recently, showed DksA binds greatly enhances direct on...

ABSTRACT The ability of Escherichia coli and Bacillus subtilis to regulate their cytoplasmic pH is well studied in cell suspensions but poorly understood individual adherent cells biofilms. We observed the using ratiometric pHluorin. A standard curve equating fluorescence ratio with was obtained by perfusion at a range external 5.0 9.0, uncouplers that collapse transmembrane difference. Adherent were acid stressed switching medium from 7.5 5.5. E. fell value varied among (range 6.2 6.8),...

DNA replication occurs at discrete sites in the cell. To gain insight into spatial and temporal organization of Bacillus subtilis cycle, we simultaneously visualized origins machinery (replisomes) inside live cells. We found that origin is positioned near midcell prior to replication. After initiation, replisome colocalizes with origin, confirming initiates midcell. The remains after duplicated separate. Artificially mispositioning region leads mislocalization indicating location time...

In many bacteria, there is a strong bias for genes to be encoded on the leading strand of DNA, resulting in coorientation replication and transcription. Bacillus subtilis, transcription majority (75%) cooriented with replication. By using genome-wide profiling DNA microarrays, we found that this reduces adverse effects We wild-type cells, did not appear affect rate elongation. However, mutants reversed an extended region chromosome, elongation was slower. This reduced depended limited which...

Horizontal gene transfer contributes to evolution and the acquisition of new traits. In bacteria, horizontal is often mediated by conjugative genetic elements that directly from cell cell. Integrative (ICEs; also known as transposons) are mobile reside within a host genome but can excise form circle conjugation recipient cells. ICEs contribute spread genes involved in pathogenesis, symbiosis, metabolism, antibiotic resistance. Despite its importance, little about mechanisms Gram-positive...

Abstract Biology and biochemistry students must learn to visualize comprehend the complex three‐dimensional (3D) structures of macromolecules such as proteins or DNA. However, most tools available for teaching biomolecular typically operate in two dimensions. Here, we present protocols pedagogical approaches using immersive augmented reality (AR) visors, specifically Microsoft HoloLens, reinforce learning with large scale 3D holographic structures. We developed a novel workflow render...

Background Bacillus subtilis encounters a wide range of environmental pH. The bacteria maintain cytoplasmic pH within narrow range. Response to acid stress is poorly understood function external and permeant acids that conduct protons into the cytoplasm. Methods Principal Findings Cytoplasmic acidification benzoate transcriptome were observed in subtilis. was measured with 4-s time resolution using GFPmut3b fluorimetry. Rapid (pH 7.5 6.0) acidified B. cytoplasm, followed by partial recovery....

ABSTRACT ICE Bs1 is an integrative and conjugative element found in the chromosome of Bacillus subtilis . encodes functions needed for its excision transfer to recipient cells. We that gene conE (formerly yddE ) required conjugation requires a conserved ATPase motif ConE. ConE belongs HerA/FtsK superfamily ATPases, which includes well-characterized proteins FtsK, SpoIIIE, VirB4, VirD4. ConE-GFP (green fluorescent protein) fusion associated with membrane predominantly at cell poles donor At...

Regions of bacterial chromosomes occupy characteristic locations within the cell. In Bacillus subtilis, origin replication, oriC, is located at 0 degrees /360 on circular chromosome. After duplication, sister regions rapidly move to and then reside near cell quarters. It has been hypothesized that function or oriC sequences contribute positioning movement region. We found position a given chromosomal region does not depend initiation replication from an mutant strain replicates heterologous...

Conjugation, or mating, plays a profound role in bacterial evolution by spreading genes that allow bacteria to adapt and colonize new niches. ICEBs1, an integrative conjugative element of Bacillus subtilis, can transfer itself mobilize resident plasmids. DNA is mediated type IV secretion system (T4SS). Characterized components the ICEBs1 T4SS include conserved VirB4-like ATPase ConE, bifunctional cell wall hydrolase CwlT, presumed VirD4-like coupling protein ConQ. A fusion ConE green...

Abstract Biochemistry and molecular biology students are asked to understand analyze the structures of small molecules complex three‐dimensional (3D) macromolecules. However, most tools help learn visualization skills limited two‐dimensional (2D) images on screens in textbooks. The virtual reality (VR) App Nanome, designed for collaborative drug discovery, allows users visualize, build, modify, interact with 3D. Here, we describe our pedagogical approach using Nanome teach biomolecular...

Students in the fields of biochemistry and molecular biology face challenges understanding interpreting structures large macromolecules, such as proteins, RNA DNA. While these complex are three-dimensional (3D) nature, most existing tools for teaching biomolecular visualization, screens textbooks, operate two dimensions. After introducing students to visualization using conventional tools, we used Virtual Reality (VR) App Nanome (www.nanome.ai) on immersive Meta Quest headsets reinforce...

Students are engaged and improve their critical thinking skills in laboratory courses when they have the opportunity to design conduct inquiry-based experiments that generate novel results. A discovery-driven project for a microbiology, genetics, or multidisciplinary research course was developed familiarize students with scientific method. In this multi-lab module, determine whether chosen stress conditions induce conjugation and/or cell death of model BSL-1 Gram-positive bacterium Bacillus...

Fumarate hydratase deficiency (FHD) is a rare metabolic disease caused by two defective copies of the FH gene, which encodes Krebs cycle enzyme fumarase. FHD associated with brain and developmental abnormalities, seizures, high childhood mortality. We describe symptoms treatment patient FHD. While infantile spasms are common in FHD, presented epileptic later childhood. Also unexpectedly, responded excellently to lacosamide for her non-convulsive status epilepticus after three first-line...

Bacterial conjugation, also known as mating, is a major form of horizontal gene transfer. During donor bacterium passes genetic material through translocation channel to recipient cell. This process allows for the evolution bacterial populations and spread antibiotic resistance genes. Our research focuses on studying integrative conjugative element ICEBs1 Bacillus subtilis, Gram-positive bacterium. We studied membrane topology ConC VirB6-like protein ConG, which are two multi-pass proteins...

Students are routinely asked to understand the three-dimensional nature of macromolecules and their interactions. However, resources provided for this learning typically include two dimensional images on screens. Building simple molecules with physical models is an effective way learn about three concepts such as chirality but these often limited in scope do not easily allow docking into more complex proteins. We trained students use virtual reality app Nanome along Meta Quest headsets build...

Students are engaged and improve their critical thinking skills in laboratory courses when they have the opportunity to design conduct inquiry‐based experiments that generate novel results. A discovery‐driven project for a microbiology, genetics, or multidisciplinary research course was developed familiarize students with scientific method. In this multi‐lab module, determine whether chosen stress conditions induce conjugation and/or cell death of model BSL‐1 Gram‐positive bacterium Bacillus...

Fumarase, or fumarate hydratase, is an enzyme that participates in the citric acid cycle and catalyzes reversible hydration of to malate. Homozygous germline mutations FH , human hydratase gene, are linked deficiency (FHD). Clinical features this inborn error metabolism include encephalitis, seizures, brain abnormalities, developmental delay. Fumarase protein sequences evolutionarily conserved, with Escherichia coli FumC sharing a stunning 59% amino sequence identity. Crystallographic...

Conjugation is the transfer of DNA from a donor cell to recipient through Type IV Secretion System (T4SS). The T4SS encoded by conjugative element and assembled in membrane. T4SSs can also mediate mobilization resident plasmids that do not encode their own conjugation machinery recipient. In addition, some cases, have been shown retro‐transfer, or reverse mobilization, which containing receives cell. Conjugation, are all important mechanisms horizontal gene contribute bacterial evolution...