An electric alarm clock for spores Lombardino, Jonathan; Burton, Briana M.
Science (American Association for the Advancement of Science),
10/2022, Letnik:
378, Številka:
6615
Journal Article
Recenzirano
Inactive spores integrate stimuli over time through stored electrochemical potential
To survive among complex and transient environmental conditions, biological systems have evolved sensitive ...mechanisms to not only integrate informative stimuli but to mount a decisive response. Sensing the onset of harsh conditions, spore-forming organisms, including bacteria and fungi, undergo energetically costly developmental reprogramming to enter dormant states that persist through a multitude of biological insults including nutrient deprivation, extreme heat, and desiccation. Notably, spores formed by bacteria such as
Bacillus subtilis
can remain in their dormant, metabolically inactive state for extended periods of time, potentially even hundreds of years. How can a dormant entity respond in a dynamic way to its environment and thus commit to resume biological activity and growth at an appropriate time? On page 43 of this issue, Kikuchi
et al.
(
1
) reveal that exit of
B. subtilis
spores from dormancy may be explained by electrochemical-state switching, similar to that used by neurons.
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. First, the challenges inherent to DNA movement through the cell periphery will be discussed to provide context for DNA transport during natural competence. The following sections will trace the development of a comprehensive model for DNA translocation to the cytoplasmic membrane, highlighting the crucial studies performed over the last century that have contributed to building contemporary DNA import models. Finally, this review will conclude by reflecting on what is still unknown about the process and the possible solutions to overcome these limitations.
ABSTRACT
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. In strains that produce pilin monomers within tenfold of wild-type levels, the median length of detectable pili is 300 nm. These pili are retractile and associate with DNA. The analysis of pilus distribution at the cell surface reveals that they are predominantly located along the long axis of the cell. The distribution is consistent with localization of proteins associated with subsequent transformation steps, DNA binding, and DNA translocation in the cytosol. These data suggest a distributed model for
B. subtilis
transformation machinery, in which initial steps of DNA capture occur throughout the long axis of the cell and subsequent steps may also occur away from the cell poles.
IMPORTANCE
This work provides novel visual evidence for DNA translocation across the cell wall during
Bacillus subtilis
natural competence, an essential step in the natural transformation process. Our data demonstrate the existence of natural competence-associated retractile pili that can bind exogenous DNA. Furthermore, we show that pilus biogenesis occurs throughout the cell long axis. These data strongly support DNA translocation occurring all along the lateral cell wall during natural competence, wherein pili are produced, bind to free DNA in the extracellular space, and finally retract to pull the bound DNA through the gap in the cell wall created during pilus biogenesis.
This work provides novel visual evidence for DNA translocation across the cell wall during
Bacillus subtilis
natural competence, an essential step in the natural transformation process. Our data demonstrate the existence of natural competence-associated retractile pili that can bind exogenous DNA. Furthermore, we show that pilus biogenesis occurs throughout the cell long axis. These data strongly support DNA translocation occurring all along the lateral cell wall during natural competence, wherein pili are produced, bind to free DNA in the extracellular space, and finally retract to pull the bound DNA through the gap in the cell wall created during pilus biogenesis.
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. The DNA sensor strains can identify diverse bacterial species including major human pathogens with high specificity. Multiplexed detection of genomic DNA from different species in complex samples can be achieved by coupling the sensing mechanism to orthogonal fluorescent reporters. We also demonstrate that the DNA sensors can detect the presence of species in the complex samples without requiring DNA extraction. The modularity of the living cell-based DNA-sensing mechanism and simple detection procedure could enable programmable DNA sensing for a wide range of applications.
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. The donor is a key determinant of HGT efficiency as plasmids induce the SOS response, enter a multimerized state, and are released in high concentrations, enabling efficient HGT. However, HGT is reduced in response to high donor lysis rates. HGT is independent of the donor viability state as both live and dead cells transfer the plasmid with high efficiency. In sum, plasmid HGT via natural transformation depends on the interplay of plasmid properties, donor stress responses and lysis rates, and interspecies interactions.
Synopsis
Plasmid multimerization, donor stress response and donor lysis rate are major determinants of horizontal gene transfer via natural competence in a microbial co‐culture.
Horizontal gene transfer (HGT) via natural competence is highly efficient in a co‐culture of the recipient B. subtilis and donor E. coli and is modulated by initial species abundance.
The presence of RecA in the donor species substantially enhances HGT efficiency in the co‐culture by enhancing plasmid multimerization and eDNA release via the SOS response.
Efficient HGT can occur for both intact dead and live donor cells.
High donor cell lysis can reduce HGT efficiency in the co‐culture.
Plasmid multimerization, donor stress response and donor lysis rate are major determinants of horizontal gene transfer via natural competence in a microbial co‐culture.
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. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.
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. Using mixed dimers, we show that these motifs must form a composite secretion signal in which one motif is contributed by each subunit of the dimer. Finally, through targeted crosslinking we show that the dimer formed in the cell is likely secreted as a single unit. We discuss implications of this substrate recognition mechanism for the biogenesis and quality control of secretion substrates and describe its likely conservation across ESX systems.
Summary
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. Out of this body of work, a series of models have emerged and, ultimately, yielded two seemingly opposing models: the loading model and the target search model. We review these recent mechanistic insights into directional DNA movement and discuss the data that may serve to unite these suggested models, as well as propose future directions that may ultimately solve the debate.
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. In this study, we demonstrate that the B. subtilis yuk/yue locus codes for a nonessential ESX secretion system. We develop a functional secretion assay to demonstrate that each of the locus gene products is specifically required for secretion of the WXG100 virulence factor homolog, YukE. We then employ an unbiased approach to search for additional secreted substrates. By quantitative profiling of culture supernatants, we find that YukE may be the sole substrate that depends on the FtsK/SpoIIIE family ATPase for secretion. We discuss potential functional implications for secretion of a unique substrate.
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. Here, we show that SwrB likely acts substoichiometrically as it localizes as puncta at the membrane in numbers fewer than those of flagellar basal bodies. Moreover, the action of SwrB is likely transient as puncta of SwrB were not dependent on the presence of the basal bodies and rarely colocalized with flagellar hooks. Random mutagenesis of the SwrB sequence found that a histidine within the transmembrane segment was conditionally required for activity and punctate localization. Finally, three hydrophobic residues that precede a cytoplasmic domain of poor conservation abolished SwrB activity when mutated and caused aberrant migration during electrophoresis. Our data are consistent with a model in which SwrB interacts with the flagellum, changes conformation to activate type III secretion, and departs.
Type III secretion systems (T3SSs) are elaborate nanomachines that form the core of the bacterial flagellum and injectisome of pathogens. The machines not only secrete proteins like virulence factors but also secrete the structural components for their own assembly. Moreover, proper construction requires complex regulation to ensure that the parts are roughly secreted in the order in which they are assembled. Here, we explore a poorly understood activator of the flagellar T3SS activation in Bacillus subtilis called SwrB. To aid mechanistic understanding, we determine the rules for subcellular punctate localization, the topology with respect to the membrane, and critical residues required for SwrB function.
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