Potassium homeostasis is vital for all organisms, but is challenging in single-celled organisms like bacteria and yeast and immobile organisms like plants that constantly need to adapt to changing ...external conditions. KUP transporters facilitate potassium uptake by the co-transport of protons. Here, we uncover the molecular basis for transport in this widely distributed family. We identify the potassium importer KimA from Bacillus subtilis as a member of the KUP family, demonstrate that it functions as a K
/H
symporter and report a 3.7 Å cryo-EM structure of the KimA homodimer in an inward-occluded, trans-inhibited conformation. By introducing point mutations, we identify key residues for potassium and proton binding, which are conserved among other KUP proteins.
Aims
To demonstrate and characterize a portable lysis apparatus for rapid single‐step bacterial DNA extraction.
Methods and Results
Our portable lysis apparatus employed a novel design consisting of ...an annular piezo‐element with perforated diaphragm. Using Bacillus subtilis as target bacteria, our portable lysis apparatus was able to achieve a normalized percent lysis as high as 66% within 30 s. This is comparable to that by microprobe ultrasonication and almost 7 times higher than that by conventional bead beating. The effect from adding glass beads was predictable. However, the results from the addition of sodium dodecyl sulphate (SDS) were counter‐intuitive because a further increase from 0·5 to 1% concentration reduced the lysis performance. The portable lysis apparatus is also at least 1·5–5 times more power efficient than microprobe ultrasonication.
Conclusions
Our portable lysis apparatus is capable of rapidly extracting bacterial DNA and is more power efficient than microprobe ultrasonication. The addition of glass beads or SDS concentration (up to 0·5%) improves its performance.
Significance and Impact of the Study
The portable lysis apparatus provides a standalone, rapid, low cost and power efficient way of obtaining genomic constituents prior to a variety of bioassays used in the field of environmental, biomedical and other applied microbiology.
Bacillus subtilis is the most characterized gram-positive bacterium that has significant attributes, such as growing well on cheap carbon sources, possessing clear inherited backgrounds, having ...mature genetic manipulation methods, and exhibiting robustness in large-scale fermentations. Till date, B. subtilis has been identified as attractive hosts for the production of recombinant proteins and chemicals. By applying various systems and synthetic biology tools, the productivity features of B. subtilis can be thoroughly analyzed and further optimized via metabolic engineering. In the present review, we discussed why B. subtilis is the primary organisms used for metabolic engineering and industrial applications. Additionally, we summarized the recent advances in systems and synthetic biology, engineering strategies for improving cellular performances, and metabolic engineering applications of B. subtilis. In particular, we proposed emerging opportunities and essential strategies to enable the successful development of B. subtilis as microbial cell factories.
•The product portfolio of B. subtilis is expanding from proteins to bio-chemicals.•Reason of B. subtilis as a primary strain for metabolic engineering was discussed.•Recent advances in basic studies and applications of B. subtilis were summarized.•Essential strategies of developing B. subtilis to be cell factories were proposed.
Integrating the governing chemistry with the genomics and phenotypes of microbial colonies has been a “holy grail” in microbiology. This work describes a highly sensitive, broadly applicable, and ...cost-effective approach that allows metabolic profiling of live microbial colonies directly from a Petri dish without any sample preparation. Nanospray desorption electrospray ionization mass spectrometry (MS), combined with alignment of MS data and molecular networking, enabled monitoring of metabolite production from live microbial colonies from diverse bacterial genera, including Bacillus subtilis, Streptomyces coelicolor, Mycobacterium smegmatis , and Pseudomonas aeruginosa . This work demonstrates that, by using these tools to visualize small molecular changes within bacterial interactions, insights can be gained into bacterial developmental processes as a result of the improved organization of MS/MS data. To validate this experimental platform, metabolic profiling was performed on Pseudomonas sp. SH-C52, which protects sugar beet plants from infections by specific soil-borne fungi R. Mendes et al. (2011) Science 332:1097–1100. The antifungal effect of strain SH-C52 was attributed to thanamycin, a predicted lipopeptide encoded by a nonribosomal peptide synthetase gene cluster. Our technology, in combination with our recently developed peptidogenomics strategy, enabled the detection and partial characterization of thanamycin and showed that it is a monochlorinated lipopeptide that belongs to the syringomycin family of antifungal agents. In conclusion, the platform presented here provides a significant advancement in our ability to understand the spatiotemporal dynamics of metabolite production in live microbial colonies and communities.
The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned ...medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine and was reported to inhibit biofilm formation via the incorporation of these d-amino acids into the cell wall. Here, we show that l-amino acids were able to specifically reverse the inhibitory effects of their cognate d-amino acids. We also show that d-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding d-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of d-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of d-amino acids without losing the ability to incorporate at least one noncanonical d-amino acid, d-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of d-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis.
The WalR-WalK two component signaling system in Bacillus subtilis functions in the homeostatic control of the peptidoglycan (PG) hydrolases LytE and CwlO that are required for cell growth. When the ...activities of these enzymes are low, WalR activates transcription of
and
and represses transcription of
, a secreted inhibitor of LytE. Conversely, when PG hydrolase activity is too high, WalR-dependent expression of
and
is reduced and
is derepressed. In a screen for additional factors that regulate this signaling pathway, we discovered that overexpression of the membrane-anchored PG deacetylase PdaC increases WalR-dependent gene expression. We show that increased expression of PdaC, but not catalytic mutants, prevents cell wall cleavage by both LytE and CwlO, explaining the WalR activation. Importantly, the
gene, like
, is repressed by active WalR. We propose that derepression of
when PG hydrolase activity is too high results in modification of the membrane-proximal layers of the PG, protecting the wall from excessive cleavage by the membrane-tethered CwlO. Thus, the WalR-WalK system homeostatically controls the levels and activities of both elongation-specific cell wall hydrolases.
Bacterial growth and division requires a delicate balance between the synthesis and remodeling of the cell wall exoskeleton. How bacteria regulate the potentially autolytic enzymes that remodel the cell wall peptidoglycan remains incompletely understood. Here, we provide evidence that the broadly conserved WalR-WalK two-component signaling system homeostatically controls both the levels and activities of two cell wall hydrolases that are critical for cell growth.
Despite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall ...synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of treadmilling filaments. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling remains unclear. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.
Abstract
The Gram-positive bacterium Bacillus subtilis can initiate the process of sporulation under conditions of nutrient limitation. Here, we review some of the last 5 years of work in this area, ...with a particular focus on the decision to initiate sporulation, DNA translocation, cell–cell communication, protein localization and spore morphogenesis. The progress we describe has implications not only just for the study of sporulation but also for other biological systems where homologs of sporulation-specific proteins are involved in vegetative growth.
We describe progress over the past ~5 years in understanding the process of sporulation in Bacillus subtilis.
Based on technical advances in the sequencing and synthesis of genetic components as well as the genome, significant progress has recently been made in developing synthetic biology toolboxes and ...chassis for the model Gram-positive bacterium Bacillus subtilis. In this review, we discuss recently developed synthetic biology toolboxes, including gene expression toolsets and genome editing tools. Next, advances in the B. subtilis chassis and its applications are discussed in comparison to those of other model microorganisms. Finally, future directions for the integrative use of B. subtilis synthetic biology tools and the development of an advanced chassis for efficient biomanufacturing are discussed. These factors are expected to become a major driving force for facilitating biotechnological applications of B. subtilis.
Recent development of synthetic biology toolboxes for B. subtilis, including gene expression regulatory toolboxes and genome-wide editing tools, provides powerful tools for precise gene expression control and efficient genome editing.
Advances of B. subtilis chassis and their applications help to understand fundamental cellular processes and techniques for improving production of biomolecules or heterologous enzymes.
Comparing B. subtilis chassis development with E. coli and S. cerevisiae chassis may provide potential directions for B. subtilis chassis construction.
The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known ...and unknown defense systems are located in "defense islands" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria.
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