Building Radical Listening and Empathy through an Implementation Lab in an Undergraduate Microbiology Course.

With rapid advances in science and technology, individuals are faced with the challenging process of making decisions based on sound and accurate information. As a result, to promote scientific literacy, scientists must be able to engage with a wide range of audiences in an inclusive and engaging manner. In addition to a solid knowledge of facts and data, effective scientific communication requires an empathetic approach that comes from a place of understanding and values the knowledge and experience of the intended audience.

Poly-Gamma-Glutamic Acid Secretion Protects Bacillus subtilis from Zinc and Copper Intoxication.

Zinc and copper are essential micronutrients that serve as a cofactors for numerous enzymes. However, when present at elevated concentrations, zinc and copper are highly toxic to bacteria. To combat the effects of zinc and copper excess, bacteria have evolved a wide array of defense mechanisms.

Metal sensing and regulation of adaptive responses to manganese limitation by MtsR is critical for group A streptococcus virulence.

Pathogenic bacteria encounter host-imposed manganese (Mn) limitation during infection. Herein we report that in the human pathogen Streptococcus pyogenes, the adaptive response to Mn limitation is controlled by a DtxR family metalloregulator, MtsR. Genes upregulated by MtsR during Mn limitation include Mn (mtsABC) and Fe acquisition systems (sia operon), and a metal-independent DNA synthesis enzyme (nrdFEI.

Bacillus subtilis FolE is sustained by the ZagA zinc metallochaperone and the alarmone ZTP under conditions of zinc deficiency.

Bacteria tightly regulate intracellular zinc levels to ensure sufficient zinc to support essential functions, while preventing toxicity. The bacterial response to zinc limitation includes the expression of putative zinc metallochaperones belonging to subfamily 1 of the COG0523 family of G3E GTPases. However, the client proteins and the metabolic processes served by these chaperones are unclear.

A metabolic checkpoint protein GlmR is important for diverting carbon into peptidoglycan biosynthesis in Bacillus subtilis.

The Bacillus subtilis GlmR (formerly YvcK) protein is essential for growth on gluconeogenic carbon sources. Mutants lacking GlmR display a variety of phenotypes suggestive of impaired cell wall synthesis including antibiotic sensitivity, aberrant cell morphology and lysis. To define the role of GlmR, we selected suppressor mutations that ameliorate the sensitivity of a glmR null mutant to the beta-lactam antibiotic cefuroxime or restore growth on gluconeogenic carbon sources.

Antagonism of Two Plant-Growth Promoting Bacillus velezensis Isolates Against Ralstonia solanacearum and Fusarium oxysporum.

Plant growth promoting rhizobacteria (PGPR) provide an effective and environmentally sustainable method to protect crops against pathogens. The spore-forming Bacilli are attractive PGPR due to their ease of storage and application. Here, we characterized two rhizosphere-associated Bacillus velezensis isolates (Y6 and F7) that possess strong antagonistic activity against Ralstonia solanacearum and Fusarium oxysporum under both laboratory and greenhouse conditions.

Modulation of extracytoplasmic function (ECF) sigma factor promoter selectivity by spacer region sequence.

The ability of bacteria to adapt to stress depends on the conditional expression of specific sets of genes. Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma (σ) factors that regulate functions important for survival under conditions eliciting cell envelope stress. Of these, four have been studied in detail: σM, σW, σX and σV.

A Critical Role of Zinc Importer AdcABC in Group A Streptococcus-Host Interactions During Infection and Its Implications for Vaccine Development.

Bacterial pathogens must overcome host immune mechanisms to acquire micronutrients for successful replication and infection. Streptococcus pyogenes, also known as group A streptococcus (GAS), is a human pathogen that causes a variety of clinical manifestations, and disease prevention is hampered by lack of a human GAS vaccine. Herein, we report that the mammalian host recruits calprotectin (CP) to GAS infection sites and retards bacterial growth by zinc limitation.

Metal homeostasis and resistance in bacteria.

Metal ions are essential for many reactions, but excess metals can be toxic. In bacteria, metal limitation activates pathways that are involved in the import and mobilization of metals, whereas excess metals induce efflux and storage. In this Review, we highlight recent insights into metal homeostasis, including protein-based and RNA-based sensors that interact directly with metals or metal-containing cofactors.

The Role of Bacillithiol in Gram-Positive Firmicutes.

Significance: Since the discovery and structural characterization of bacillithiol (BSH), the biochemical functions of BSH-biosynthesis enzymes (BshA/B/C) and BSH-dependent detoxification enzymes (FosB, Bst, GlxA/B) have been explored in Bacillus and Staphylococcus species. It was shown that BSH plays an important role in detoxification of reactive oxygen and electrophilic species, alkylating agents, toxins, and antibiotics. Recent Advances: More recently, new functions of BSH were discovered in metal homeostasis (Zn buffering, Fe-sulfur cluster, and copper homeostasis) and virulence control in Staphylococcus aureus.

Lack of formylated methionyl-tRNA has pleiotropic effects on Bacillus subtilis.

Bacteria initiate translation using a modified amino acid, N-formylmethionine (fMet), adapted specifically for this function. Most proteins are processed co-translationally by peptide deformylase (PDF) to remove this modification. Although PDF activity is essential in WT cells and is the target of the antibiotic actinonin, bypass mutations in the fmt gene that eliminate the formylation of Met-tRNAMet render PDF dispensable.

Intracellular Zn(II) Intoxication Leads to Dysregulation of the PerR Regulon Resulting in Heme Toxicity in Bacillus subtilis.

Transition metal ions (Zn(II), Cu(II)/(I), Fe(III)/(II), Mn(II)) are essential for life and participate in a wide range of biological functions. Cellular Zn(II) levels must be high enough to ensure that it can perform its essential roles. Yet, since Zn(II) binds to ligands with high avidity, excess Zn(II) can lead to protein mismetallation.

Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters.

Most Escherichia coli promoters initiate transcription with a purine 7 or 8 nt downstream from the -10 hexamer, but some promoters, including the ribosomal RNA promoter rrnB P1, start 9 nt from the -10 element. We identified promoter and RNA polymerase determinants of this noncanonical rrnB P1 start site using biochemical and genetic approaches including mutational analysis of the promoter, Fe(2+) cleavage assays to monitor template strand positions near the active-site, and Bpa cross-linking to map the path of open complex DNA at amino acid and nucleotide resolution. We find that mutations in several promoter regions affect transcription start site (TSS) selection.

Bacillithiol is a major buffer of the labile zinc pool in Bacillus subtilis.

Intracellular zinc levels are tightly regulated since zinc is an essential cofactor for numerous enzymes, yet can be toxic when present in excess. The majority of intracellular zinc is tightly associated with proteins and is incorporated during synthesis from a poorly defined pool of kinetically labile zinc. In Bacillus subtilis, this labile pool is sensed by equilibration with the metalloregulator Zur, as an indication of zinc sufficiency, and by CzrA, as an indication of zinc excess.

A Small-Group Activity Introducing the Use and Interpretation of BLAST.

As biological sequence data are generated at an ever increasing rate, the role of bioinformatics in biological research also grows. Students must be trained to complete and interpret bioinformatic searches to enable them to effectively utilize the trove of sequence data available. A key bioinformatic tool for sequence comparison and genome database searching is BLAST (Basic Local Alignment Search Tool).

Methylglyoxal resistance in Bacillus subtilis: contributions of bacillithiol-dependent and independent pathways.

Methylglyoxal (MG) is a toxic by-product of glycolysis that damages DNA and proteins ultimately leading to cell death. Protection from MG is often conferred by a glutathione-dependent glyoxalase pathway. However, glutathione is absent from the low-GC Gram-positive Firmicutes, such as Bacillus subtilis.

Suppression of a dnaKJ deletion by multicopy dksA results from non-feedback-regulated transcripts that originate upstream of the major dksA promoter.

DksA is an RNA polymerase (RNAP) binding transcription factor that controls expression of a large number of genes in concert with the small-molecule "alarmone" ppGpp. DksA also aids in the resolution of conflicts between RNAP and DNA polymerase (DNAP) during genome replication. DksA was originally identified as a multicopy suppressor of the temperature sensitivity caused by deletion of the genes coding for the DnaKJ chaperone system.

The dksA promoter is negatively feedback regulated by DksA and ppGpp.

Escherichia coli DksA is an RNA polymerase (RNAP)-binding protein required for the regulation of a number of promoters, including those for the biosynthesis of rRNA, many ribosomal proteins, components of the flagellum, and several amino acids. Previous work demonstrated that DksA protein levels do not vary greatly in different growth conditions. We show here that DksA is a stable protein whose levels are kept constant by a negative feedback loop in which transcription from the dksA promoter is inhibited by DksA protein in conjunction with its cofactor ppGpp.

6S RNA regulation of relA alters ppGpp levels in early stationary phase.

6S RNA is a small, non-coding RNA that interacts directly with σ(70)-RNA polymerase and regulates transcription at many σ(70)-dependent promoters. Here, we demonstrate that 6S RNA regulates transcription of relA, which encodes a ppGpp synthase. The 6S RNA-dependent regulation of relA expression results in increased ppGpp levels during early stationary phase in cells lacking 6S RNA.

The cell-penetrating peptide, Pep-1, has activity against intracellular chlamydial growth but not extracellular forms of Chlamydia trachomatis.

Objectives: In the course of studies to identify novel treatment strategies against the pathogenic bacterium, Chlamydia, we tested the carrier peptide, Pep-1, for activity against an intracellular infection.

Methods: Using a cell culture model of Chlamydia trachomatis infection, the effect of Pep-1 was measured by incubating the peptide with extracellular chlamydiae prior to infection, or by adding Pep-1 to the medium at varying times after infection, and assaying for inhibition of inclusion formation.

Results: Pep-1 had a concentration-dependent effect on chlamydial growth with 100% inhibition of inclusion formation at 8 mg/L peptide.