Differential expression of the operon in a biofilm.

Type VII protein secretion systems play an important role in the survival and virulence of pathogens and in the competition among some microbes. Potential polymorphic toxin substrates of the type VII secretion system (T7SS) in are important for competition in the context of biofilm communities. Within a biofilm, there is significant physiological heterogeneity as cells within the population take on differential cell fates.

ComI inhibits transformation in by selectively killing competent cells.

Unlabelled: Many bacteria build elaborate molecular machines to import DNA via natural competence, yet this activity is often not identified until strains have been handled and domesticated in laboratory settings. For example, one of the best studied Gram-positive model organisms, has a poorly transformable ancestor. Transformation in the ancestral strain is inhibited by a transmembrane peptide, ComI, which is encoded on an extrachromosomal plasmid.

From isotopically labeled DNA to fluorescently labeled dynamic pili: building a mechanistic model of DNA transport to the cytoplasmic membrane.

SUMMARYNatural competence, the physiological state wherein bacteria produce proteins that mediate extracellular DNA transport into the cytosol and the subsequent recombination of DNA into the genome, is conserved across the bacterial domain. DNA must successfully translocate across formidable permeability barriers during import, including the cell membrane(s) and the cell wall, that are normally impermeable to large DNA polymers. This review will examine the mechanisms underlying DNA transport from the extracellular space to the cytoplasmic membrane.

Visualizing dynamic competence pili and DNA capture throughout the long axis of .

The first step in the process of bacterial natural transformation is DNA capture. Although long hypothesized based on genetics and functional experiments, the pilus structure responsible for initial DNA binding had not yet been visualized for . Here, we visualize functional competence pili in using fluorophore-conjugated maleimide labeling in conjunction with epifluorescence microscopy.

Visualizing dynamic competence pili and DNA capture throughout the long axis of .

The first step in the process of bacterial natural transformation is DNA capture. Although long-hypothesized based on genetics and functional experiments, the pilus structure responsible for initial DNA-binding had not yet been visualized for Bacillus subtilis. Here, we visualize functional competence pili in Bacillus subtilis using fluorophore-conjugated maleimide labeling in conjunction with epifluorescence microscopy.

Programming bacteria for multiplexed DNA detection.

DNA is a universal and programmable signal of living organisms. Here we develop cell-based DNA sensors by engineering the naturally competent bacterium Bacillus subtilis (B. subtilis) to detect specific DNA sequences in the environment.

Efficient plasmid transfer via natural competence in a microbial co-culture.

The molecular and ecological factors shaping horizontal gene transfer (HGT) via natural transformation in microbial communities are largely unknown, which is critical for understanding the emergence of antibiotic-resistant pathogens. We investigate key factors shaping HGT in a microbial co-culture by quantifying extracellular DNA release, species growth, and HGT efficiency over time. In the co-culture, plasmid release and HGT efficiency are significantly enhanced than in the respective monocultures.

An electric alarm clock for spores.

Inactive spores integrate stimuli over time through stored electrochemical potential.

Natural Transformation Protein ComFA Exhibits Single-Stranded DNA Translocase Activity.

Natural transformation is one of the major mechanisms of horizontal gene transfer in bacterial populations and has been demonstrated in numerous species of bacteria. Despite the prevalence of natural transformation, much of the molecular mechanism remains unexplored. One major outstanding question is how the cell powers DNA import, which is rapid and highly processive.

Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights.

Bacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, here we report broad and efficient genetic code expansion in B.

Molecular and Cell Biological Analysis of SwrB in Bacillus subtilis.

Swarming motility is flagellum-mediated movement over a solid surface, and Bacillus subtilis cells require an increase in flagellar density to swarm. SwrB is a protein of unknown function required for swarming that is necessary to increase the number of flagellar hooks but not basal bodies. Previous work suggested that SwrB activates flagellar type III secretion, but the mechanism by which it might perform this function is unknown.

Pervasive prophage recombination occurs during evolution of spore-forming Bacilli.

Phages are the main source of within-species bacterial diversity and drivers of horizontal gene transfer, but we know little about the mechanisms that drive genetic diversity of these mobile genetic elements (MGEs). Recently, we showed that a sporulation selection regime promotes evolutionary changes within SPβ prophage of Bacillus subtilis, leading to direct antagonistic interactions within the population. Herein, we reveal that under a sporulation selection regime, SPβ recombines with low copy number phi3Ts phage DNA present within the B.

DNA-Membrane Anchor Facilitates Efficient Chromosome Translocation at a Distance in Bacillus subtilis.

Chromosome segregation in sporulating involves the tethering of sister chromosomes at opposite cell poles. RacA is known to mediate chromosome tethering by interacting with both centromere-like elements in the DNA and with DivIVA, a membrane protein which localizes to the cell poles. RacA has a secondary function in which it assists in nucleoid condensation.

Quantitative Transformation Efficiency Assay for .

is a model Gram-positive organism used to study a variety of physiological states and stress responses, one of which is the development of competence. Competence refers to the physiological state of a cell which allows it to be transformed naturally. Through induction of competence, the efficiency of natural transformation can be quantified by plating colony forming units (CFU) and transforming units (TFU).

Bacterial transformation: ComFA is a DNA-dependent ATPase that forms complexes with ComFC and DprA.

Pneumococcal natural transformation contributes to genomic plasticity, antibiotic resistance development and vaccine escape. Streptococcus pneumoniae, like many other naturally transformable species, has evolved sophisticated protein machinery for the binding and uptake of DNA. Two proteins encoded by the comF operon, ComFA and ComFC, are involved in transformation but their exact molecular roles remain unknown.

A Conserved Metal Binding Motif in the Bacillus subtilis Competence Protein ComFA Enhances Transformation.

Genetic competence is a process in which cells are able to take up DNA from their environment, resulting in horizontal gene transfer, a major mechanism for generating diversity in bacteria. Many bacteria carry homologs of the central DNA uptake machinery that has been well characterized in It has been postulated that the competence helicase ComFA belongs to the DEAD box family of helicases/translocases. Here, we made a series of mutants to analyze conserved amino acid motifs in several regions of ComFA.

Missense Mutations Allow a Sequence-Blind Mutant of SpoIIIE to Successfully Translocate Chromosomes during Sporulation.

SpoIIIE directionally pumps DNA across membranes during Bacillus subtilis sporulation and vegetative growth. The sequence-reading domain (γ domain) is required for directional DNA transport, and its deletion severely impairs sporulation. We selected suppressors of the spoIIIEΔγ sporulation defect.

Structural characterization of the late competence protein ComFB from Bacillus subtilis.

Many bacteria take up DNA from their environment as part of the process of natural transformation. DNA uptake allows microorganisms to gain genetic diversity and can lead to the spread of antibiotic resistance or virulence genes within a microbial population. Development of genetic competence (Com) in Bacillus subtilis is a highly regulated process that culminates in expression of several late competence genes and formation of the DNA uptake apparatus.

Do the same traffic rules apply? Directional chromosome segregation by SpoIIIE and FtsK.

Over a decade of studies have tackled the question of how FtsK/SpoIIIE translocases establish and maintain directional DNA translocation during chromosome segregation in bacteria. FtsK/SpoIIIE translocases move DNA in a highly processive, directional manner, where directionality is facilitated by sequences on the substrate DNA molecules that are being transported. In recent years, structural, biochemical, single-molecule and high-resolution microscopic studies have provided new insight into the mechanistic details of directional DNA segregation.

Linked domain architectures allow for specialization of function in the FtsK/SpoIIIE ATPases of ESX secretion systems.

Among protein secretion systems, there are specialized ATPases that serve different functions such as substrate recognition, substrate unfolding, and assembly of the secretory machinery. ESX (early secretory antigen target 6 kDa secretion) protein secretion systems require FtsK/SpoIIIE family ATPases but the specific function of these ATPases is poorly understood. The ATPases of ESX secretion systems have a unique domain architecture among proteins of the FtsK/SpoIIIE family.

Dimer recognition and secretion by the ESX secretion system in Bacillus subtilis.

Protein secretion typically involves translocation of unfolded polypeptides or transport of monomeric folded proteins. Here we provide, to our knowledge, the first experimental evidence for secretion of an intact multimeric complex requiring a signal formed by both members of the complex. Using systematic mutagenesis of a substrate involved in early secretory antigen 6 kDa (ESX) secretion in Bacillus subtilis, we demonstrate that export of the substrate requires two independent motifs.

The ESX system in Bacillus subtilis mediates protein secretion.

Esat-6 protein secretion systems (ESX or Ess) are required for the virulence of several human pathogens, most notably Mycobacterium tuberculosis and Staphylococcus aureus. These secretion systems are defined by a conserved FtsK/SpoIIIE family ATPase and one or more WXG100 family secreted substrates. Gene clusters coding for ESX systems have been identified amongst many organisms including the highly tractable model system, Bacillus subtilis.

Mechanistic study of classical translocation-dead SpoIIIE36 reveals the functional importance of the hinge within the SpoIIIE motor.

SpoIIIE/FtsK ATPases are central players in bacterial chromosome segregation. It remains unclear how these DNA translocases harness chemical energy (ATP turnover) to perform mechanical work (DNA movement). Bacillus subtilis sporulation provides a dramatic example of intercompartmental DNA transport, in which SpoIIIE moves 70% of the chromosome across the division plane.

SpoIIIE protein achieves directional DNA translocation through allosteric regulation of ATPase activity by an accessory domain.

Bacterial chromosome segregation utilizes highly conserved directional translocases of the SpoIIIE/FtsK family. These proteins employ an accessory DNA-binding domain (γ) to dictate directionality of DNA transport. It remains unclear how the interaction of γ with specific recognition sequences coordinates directional DNA translocation.