Metals in Motion: Understanding Labile Metal Pools in Bacteria.

Metal ions are essential for all life. In microbial cells, potassium (K) is the most abundant cation and plays a key role in maintaining osmotic balance. Magnesium (Mg) is the dominant divalent cation and is required for nucleic acid structure and as an enzyme cofactor.

Structural basis for transcription activation through cooperative recruitment of MntR.

The manganese transport regulator (MntR) from is a dual regulatory protein that responds to heightened Mn availability in the cell by both repressing the expression of uptake transporters and activating the expression of efflux proteins. Recent work indicates that, in its role as an activator, MntR binds several sites upstream of the genes encoding Mn exporters, leading to a cooperative response to manganese. Here, we use cryo-EM to explore the molecular basis of gene activation by MntR and report a structure of four MntR dimers bound to four 18-base pair sites across an 84-base pair regulatory region of the promoter.

Structural basis for transcription activation through cooperative recruitment of MntR.

The manganese transport regulator (MntR) from is a dual regulatory protein that responds to heightened Mn availability in the cell by both repressing the expression of uptake transporters and activating the expression of efflux proteins. Recent work indicates that, in its role as an activator, MntR binds several sites upstream of the genes encoding Mn exporters, leading to a cooperative response to manganese. Here, we use cryo-EM to explore the molecular basis of gene activation by MntR and report a structure of four MntR dimers bound to four 18-base pair sites across an 84-base pair regulatory region of the promoter.

Metalation of Extracytoplasmic Proteins and Bacterial Cell Envelope Homeostasis.

Cell physiology requires innumerable metalloenzymes supported by the selective import of metal ions. Within the crowded cytosol, most enzymes acquire their cognate cofactors from a buffered labile pool. Metalation of membrane-bound and secreted exoenzymes is more problematic since metal concentrations are highly variable outside the cell.

The operon protects magnesium-dependent enzymes by supporting manganese efflux.

Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux.

The operon protects magnesium-dependent enzymes by supporting manganese efflux.

Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux.

The cell envelope stress-inducible operon modulates membrane properties and contributes to bacitracin resistance.

Antibiotics that inhibit peptidoglycan synthesis trigger the activation of both specific and general protective responses. σ responds to diverse antibiotics that inhibit cell wall synthesis. Here, we demonstrate that cell wall-inhibiting drugs, such as bacitracin and cefuroxime, induce the σ-dependent operon.

TerC proteins function during protein secretion to metalate exoenzymes.

Cytosolic metalloenzymes acquire metals from buffered intracellular pools. How exported metalloenzymes are appropriately metalated is less clear. We provide evidence that TerC family proteins function in metalation of enzymes during export through the general secretion (Sec-dependent) pathway.

TerC Proteins Function During Protein Secretion to Metalate Exoenzymes.

Cytosolic metalloenzymes acquire metals from buffered intracellular pools. How exported metalloenzymes are appropriately metalated is less clear. We provide evidence that TerC family proteins function in metalation of enzymes during export through the general secretion (Sec-dependent) pathway.

TerC Proteins Function During Protein Secretion to Metalate Exoenzymes.

Cytosolic metalloenzymes acquire metals from buffered intracellular pools. How exported metalloenzymes are appropriately metalated is less clear. We provide evidence that TerC family proteins function in metalation of enzymes during export through the general secretion (Sec-dependent) pathway.

A Decrease in Fatty Acid Synthesis Rescues Cells with Limited Peptidoglycan Synthesis Capacity.

Proper synthesis and maintenance of a multilayered cell envelope are critical for bacterial fitness. However, whether mechanisms exist to coordinate synthesis of the membrane and peptidoglycan layers is unclear. In Bacillus subtilis, synthesis of peptidoglycan (PG) during cell elongation is mediated by an elongasome complex acting in concert with class A penicillin-binding proteins (aPBPs).

Meddling with Metal Sensors: Fur-Family Proteins as Signaling Hubs.

The ferric uptake regulator (Fur) protein is the founding member of the FUR superfamily of metalloregulatory proteins that control metal homeostasis in bacteria. FUR proteins regulate metal homeostasis in response to the binding of iron (Fur), zinc (Zur), manganese (Mur), or nickel (Nur). FUR family proteins are generally dimers in solution, but the DNA-bound complex can involve a single dimer, a dimer-of-dimers, or an extended array of bound protein.

A model industrial workhorse: Bacillus subtilis strain 168 and its genome after a quarter of a century.

The vast majority of genomic sequences are automatically annotated using various software programs. The accuracy of these annotations depends heavily on the very few manual annotation efforts that combine verified experimental data with genomic sequences from model organisms. Here, we summarize the updated functional annotation of Bacillus subtilis strain 168, a quarter century after its genome sequence was first made public.

The elements of life: A biocentric tour of the periodic table.

Living systems are built from a small subset of the atomic elements, including the bulk macronutrients (C,H,N,O,P,S) and ions (Mg,K,Na,Ca) together with a small but variable set of trace elements (micronutrients). Here, we provide a global survey of how chemical elements contribute to life. We define five classes of elements: those that are (i) essential for all life, (ii) essential for many organisms in all three domains of life, (iii) essential or beneficial for many organisms in at least one domain, (iv) beneficial to at least some species, and (v) of no known beneficial use.

Mutations in That Confer Rifampicin Resistance Can Alter Levels of Peptidoglycan Precursors and Affect β-Lactam Susceptibility.

Bacteria can adapt to stressful conditions through mutations affecting the RNA polymerase core subunits that lead to beneficial changes in transcription. In response to selection with rifampicin (RIF), mutations arise in the RIF resistance-determining region (RRDR) of that reduce antibiotic binding. These changes can also alter transcription and thereby have pleiotropic effects on bacterial fitness.

Iron homeostasis in relies on three differentially expressed efflux systems.

In iron homeostasis is maintained by the ferric uptake regulator (Fur) and manganese homeostasis relies on the manganese transport regulator (MntR). Both Fur and MntR function as bi-functional metalloregulators that repress import and activate metal ion efflux systems. The ferrous iron efflux ATPase, PfeT, is derepressed by hydrogen peroxide (HO) as sensed by PerR and induced by iron as sensed by Fur.

A Central Role for Magnesium Homeostasis during Adaptation to Osmotic Stress.

Osmotic stress is a significant physical challenge for free-living cells. Cells from all three domains of life maintain viability during osmotic stress by tightly regulating the major cellular osmolyte potassium (K) and by import or synthesis of compatible solutes. It has been widely established that in response to high salt stress, many bacteria transiently accumulate high levels of K, leading to bacteriostasis, with growth resuming only when compatible solutes accumulate and K levels are restored to biocompatible levels.

A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis.

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system from Streptococcus pyogenes has been widely deployed as a tool for bacterial strain construction. Conventional CRISPR-Cas9 editing strategies require design and molecular cloning of an appropriate guide RNA (gRNA) to target genome cleavage and a repair template for introduction of the desired site-specific genome modification. Here, we present a streamlined method that leverages the existing collection of nearly 4,000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin () resistance cassette.

Manganese impairs the QoxABCD terminal oxidase leading to respiration-associated toxicity.

Cell physiology relies on metalloenzymes and can be easily disrupted by imbalances in metal ion pools. Bacillus subtilis requires manganese for growth and has highly regulated mechanisms for import and efflux that help maintain homeostasis. Cells defective for manganese (Mn) efflux are highly sensitive to intoxication, but the processes impaired by Mn excess are often unknown.

Mini Review: Bacterial Membrane Composition and Its Modulation in Response to Stress.

Antibiotics and other agents that perturb the synthesis or integrity of the bacterial cell envelope trigger compensatory stress responses. Focusing on as a model system, this mini-review summarizes current views of membrane structure and insights into how cell envelope stress responses remodel and protect the membrane. Altering the composition and properties of the membrane and its associated proteome can protect cells against detergents, antimicrobial peptides, and pore-forming compounds while also, indirectly, contributing to resistance against compounds that affect cell wall synthesis.

The Bacillus subtilis monothiol bacilliredoxin BrxC (YtxJ) and the Bdr (YpdA) disulfide reductase reduce S-bacillithiolated proteins.

The bacterial cytosol is generally a reducing environment with protein cysteine residues maintained in their thiol form. The low molecular weight thiol bacillithiol (BSH) serves as a general thiol reductant, analogous to glutathione, in a wide range of bacterial species. Proteins modified by disulfide bond formation with BSH (S-bacillithiolation) are reduced by the action of bacilliredoxins, BrxA and BrxB.

Resource sharing between central metabolism and cell envelope synthesis.

Synthesis of the bacterial cell envelope requires a regulated partitioning of resources from central metabolism. Here, we consider the key metabolic junctions that provide the precursors needed to assemble the cell envelope. Peptidoglycan synthesis requires redirection of a glycolytic intermediate, fructose-6-phosphate, into aminosugar biosynthesis by the highly regulated branchpoint enzyme GlmS.

A regulatory pathway that selectively up-regulates elongasome function in the absence of class A PBPs.

Bacteria surround themselves with peptidoglycan, an adaptable enclosure that contributes to cell shape and stability. Peptidoglycan assembly relies on penicillin-binding proteins (PBPs) acting in concert with SEDS-family transglycosylases RodA and FtsW, which support cell elongation and division respectively. In , cells lacking all four PBPs with transglycosylase activity (aPBPs) are viable.

Group A Streptococcus AdcR Regulon Participates in Bacterial Defense against Host-Mediated Zinc Sequestration and Contributes to Virulence.

Colonization by pathogenic bacteria depends on their ability to overcome host nutritional defenses and acquire nutrients. The human pathogen group A streptococcus (GAS) encounters the host defense factor calprotectin (CP) during infection. CP inhibits GAS growth by imposing zinc (Zn) limitation.

Biphasic unbinding of a metalloregulator from DNA for transcription (de)repression in Live Bacteria.

Microorganisms use zinc-sensing regulators to alter gene expression in response to changes in the availability of zinc, an essential micronutrient. Under zinc-replete conditions, the Fur-family metalloregulator Zur binds to DNA tightly in its metallated repressor form to Zur box operator sites, repressing the transcription of zinc uptake transporters. Derepression comes from unbinding of the regulator, which, under zinc-starvation conditions, exists in its metal-deficient non-repressor forms having no significant affinity with Zur box.