Identification of mutants with increased daptomycin resistance.

Unlabelled: Daptomycin is a cyclic lipopeptide antibiotic used to treat infections caused by some Gram-positive bacteria. Daptomycin disrupts synthesis of the peptidoglycan (PG) cell wall by inserting into the cytoplasmic membrane and binding multiple forms of the undecaprenyl carrier lipid required for PG synthesis. Membrane insertion requires phosphatidylglycerol, so studies of daptomycin can provide insight into assembly and maintenance of the cytoplasmic membrane.

From regulation to ruin: a rogue sigma factor causes cell death in .

A rogue, plasmid-encoded sigma factor that kills is the focus of a new study by A. T. Burton, D.

Identification of DraRS in , a Two-Component Regulatory System That Responds to Lipid II-Interacting Antibiotics.

is a Gram-positive opportunistic pathogen that results in 220,000 infections, 12,000 deaths, and upwards of $1 billion in medical costs in the United States each year. is highly resistant to a variety of antibiotics, but we have a poor understanding of how senses and responds to antibiotic stress and how such sensory systems affect clinical outcomes. We have identified a spontaneous mutant that displays increased daptomycin resistance.

HexSDF Is Required for Synthesis of a Novel Glycolipid That Mediates Daptomycin and Bacitracin Resistance in C. difficile.

Clostridioides difficile is a Gram-positive opportunistic pathogen responsible for 250,000 hospital-associated infections, 12,000 hospital-associated deaths, and $1 billion in medical costs in the United States each year. There has been recent interest in using a daptomycin analog, surotomycin, to treat C. difficile infections.

The WalRK Two-Component System Is Essential for Proper Cell Envelope Biogenesis in Clostridioides difficile.

The WalR-WalK two-component regulatory system (TCS) is found in all , in which it regulates the expression of multiple genes required for remodeling the cell envelope during growth and division. Unlike most TCSs, WalRK is essential for viability, so it has attracted interest as a potential antibiotic target. In this study, we used overexpression of WalR and CRISPR interference to investigate the Wal system of Clostridioides difficile, a major cause of hospital-associated diarrhea in high-income countries.

Activation of the Extracytoplasmic Function σ Factor σ in Clostridioides difficile Requires Regulated Intramembrane Proteolysis of the Anti-σ Factor RsiV.

Clostridioides (Clostridium) difficile is one of the leading causes of nosocomial diarrhea. Lysozyme is a common host defense against many pathogenic bacteria. C.

Signal Peptidase-Mediated Cleavage of the Anti-σ Factor RsiP at Site 1 Controls σ Activation and β-Lactam Resistance in Bacillus thuringiensis.

In Bacillus thuringiensis, β-lactam antibiotic resistance is controlled by the extracytoplasmic function (ECF) σ factor σ. σ activity is inhibited by the anti-σ factor RsiP. In the presence of β-lactam antibiotics, RsiP is degraded and σ is activated.

Identification of the Extracytoplasmic Function σ Factor σ Regulon in Bacillus thuringiensis.

Bacillus thuringiensis and other members of the Bacillus cereus family are resistant to many β-lactams. Resistance is dependent upon the extracytoplasmic function sigma factor σ. We used label-free quantitative proteomics to identify proteins whose expression was dependent upon σ.

Activation of the extracytoplasmic function σ factor σ by lysozyme in Clostridioides difficile.

Clostridioides difficile is naturally resistant to high levels of lysozyme an important component of the innate immune defense system. C. difficile encodes both constitutive as well as inducible lysozyme resistance genes.

The Penicillin-Binding Protein PbpP Is a Sensor of β-Lactams and Is Required for Activation of the Extracytoplasmic Function σ Factor σ in Bacillus thuringiensis.

β-Lactams are a class of antibiotics that target the synthesis of peptidoglycan, an essential component of the cell wall. β-Lactams inhibit the function of penicillin-binding proteins (PBPs), which form the cross-links between strands of peptidoglycan. Resistance to β-lactams complicates the treatment of bacterial infections.

Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases.

() is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall.

Activation of the Extracytoplasmic Function σ Factor σ by β-Lactams in Bacillus thuringiensis Requires the Site-2 Protease RasP.

Bacteria can utilize alternative σ factors to regulate sets of genes in response to changes in the environment. The largest and most diverse group of alternative σ factors are the extracytoplasmic function (ECF) σ factors. σ is an ECF σ factor found in , , and Previous work showed that σ is induced by ampicillin, a β-lactam antibiotic, and required for resistance to ampicillin.

Activation of the extracytoplasmic function σ factor σ by lysozyme.

σ is an extracytoplasmic function (ECF) σ factor that is found exclusively in Firmicutes including Bacillus subtilis and the opportunistic pathogens Clostridioides difficile and Enterococcus faecalis. σ is activated by lysozyme and is required for lysozyme resistance. The activity of σ is normally inhibited by the anti-σ factor RsiV, a transmembrane protein.

25 Annual Midwest Microbial Pathogenesis Conference.

The 25th annual Midwest Microbial Pathogenesis Conference (MMPC) was held at the University of Iowa from 28 to 30 September 2018. The conference has a long-standing tradition of providing scientists from the Midwest with a forum to present and discuss cutting-edge advances in microbial pathogenesis with particular focus on bacterial interactions with the environment, host, and other microbes. This review summarizes the genesis of the MMPC, topics presented at the conference, and articles found in the special MMPC sections of this issue of the .

A Xylose-Inducible Expression System and a CRISPR Interference Plasmid for Targeted Knockdown of Gene Expression in Clostridioides difficile.

Here we introduce plasmids for xylose-regulated expression and repression of genes in The xylose-inducible expression vector allows for ∼100-fold induction of an reporter gene. Induction is titratable and uniform from cell to cell. The gene repression plasmid is a CRISPR interference (CRISPRi) system based on a nuclease-defective, codon-optimized allele of the Cas9 protein () that is targeted to a gene of interest by a constitutively expressed single guide RNA (sgRNA).

Glycerol Monolaurate (GML) and a Nonaqueous Five-Percent GML Gel Kill and Spores.

Glycerol monolaurate is a broadly antimicrobial fatty acid monoester, killing bacteria, fungi, and enveloped viruses. The compound kills stationary-phase cultures of , suggesting that the molecule may kill spores. In this study, we examined the ability of glycerol monolaurate alone or solubilized in a nonaqueous gel to kill vegetative cells and spores of aerobic , , and and anaerobic and () Glycerol monolaurate alone was bactericidal for all five organisms tested.

Multiple factors contribute to bimodal toxin gene expression in Clostridioides (Clostridium) difficile.

Clostridioides (formerly Clostridium) difficile produces two major toxins, TcdA and TcdB, upon entry into stationary phase. Transcription of tcdA and tcdB requires the specialized sigma factor, σ , which also directs RNA Polymerase to transcribe tcdR itself. We fused a gene for a red fluorescent protein to the tcdA promoter to study toxin gene expression at the level of individual C.

Bacterial sensing: A putative amphipathic helix in RsiV is the switch for activating σV in response to lysozyme.

Extra Cytoplasmic Function (ECF) σ factors are a diverse group of alternate σ factors bacteria use to respond to changes in the environment. The Bacillus subtilis ECF σ factor σV responds to lysozyme. In the absence of lysozyme, σV is held inactive by the anti-σ factor, RsiV.

Signal Peptidase Is Necessary and Sufficient for Site 1 Cleavage of RsiV in Bacillus subtilis in Response to Lysozyme.

Extracytoplasmic function (ECF) σ factors are a diverse family of alternative σ factors that allow bacteria to sense and respond to changes in the environment. σ is an ECF σ factor found primarily in low-GC Gram-positive bacteria and is required for lysozyme resistance in several opportunistic pathogens. In the absence of lysozyme, σ is inhibited by the anti-σ factor RsiV.

The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for σV Activation.

σ factors provide RNA polymerase with promoter specificity in bacteria. Some σ factors require activation in order to interact with RNA polymerase and transcribe target genes. The Extra-Cytoplasmic Function (ECF) σ factor, σV, is encoded by several Gram-positive bacteria and is specifically activated by lysozyme.

Use of mCherryOpt Fluorescent Protein in Clostridium difficile.

Here we describe protocols for using the red fluorescent protein mCherryOpt in Clostridium difficile. The protocols can be readily adapted to similar fluorescent proteins (FPs), such as green fluorescent protein (GFP) and cyan fluorescent protein (CFP). There are three critical considerations for using FPs in C.