Subtilis Chromosome Research Articles

The Construction of an Environmentally Friendly Super-Secreting Strain of Bacillus subtilis through Systematic Modulation of Its Secretory Pathway Using the CRISPR-Cas9 System.

Achieving commercially significant yields of recombinant proteins in Bacillus subtilis requires the optimization of its protein production pathway, including transcription, translation, folding, and secretion. Therefore, in this study, our aim was to maximize the secretion of a reporter α-amylase by overcoming potential bottlenecks within the secretion process one by one, using a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system. The strength of single and tandem promoters was evaluated by measuring the relative α-amylase activity of AmyQ integrated into the B. subtilis chromosome. Once a suitable promoter was selected, the expression levels of amyQ were upregulated through the iterative integration of up to six gene copies, thus boosting the α-amylase activity 20.9-fold in comparison with the strain harboring a single amyQ gene copy. Next, α-amylase secretion was further improved to a 26.4-fold increase through the overexpression of the extracellular chaperone PrsA and the signal peptide peptidase SppA. When the final expression strain was cultivated in a 3 L fermentor for 90 h, the AmyQ production was enhanced 57.9-fold. The proposed strategy allows for the development of robust marker-free plasmid-less super-secreting B. subtilis strains with industrial relevance.

A historical sequence deletion in a commonly used Bacillus subtilis chromosome integration vector generates undetected loss-of-function mutations.

Since the 1980s, chromosome-integration vectors have been used as a core method of engineering Bacillus subtilis. One of the most frequently used vector backbones contains chromosomally derived regions that direct homologous recombination into the amyE locus. Here, we report a gap in the homology region inherited from the original amyE integration vector, leading to erroneous recombination in a subset of transformants and a loss-of-function mutation in the downstream gene. Internal to the homology arm that spans the 3' portion of amyE and the downstream gene ldh, an unintentional 227 bp deletion generates two crossover events. The major event yields the intended genotype, but the minor event, occurring in ~10 % of colonies, results in a truncation of ldh, which encodes lactate dehydrogenase. Although both types of colonies test positive for amyE disruption by starch plating, the potential defect in fermentative metabolism may be left undetected and confound the results of subsequent experiments.

Rok from B. subtilis: Bridging genome structure and transcription regulation.

Bacterial genomes are folded and organized into compact yet dynamic structures, called nucleoids. Nucleoid orchestration involves many factors at multiple length scales, such as nucleoid-associated proteins and liquid-liquid phase separation, and has to be compatible with replication and transcription. Possibly, genome organization plays an intrinsic role in transcription regulation, in addition to classical transcription factors. In this review, we provide arguments supporting this view using the Gram-positive bacterium Bacillus subtilis as a model. Proteins BsSMC, HBsu and Rok all impact the structure of the B. subtilis chromosome. Particularly for Rok, there is compelling evidence that it combines its structural function with a role as global gene regulator. Many studies describe either function of Rok, but rarely both are addressed at the same time. Here, we review both sides of the coin and integrate them into one model. Rok forms unusually stable DNA-DNA bridges and this ability likely underlies its repressive effect on transcription by either preventing RNA polymerase from binding to DNA or trapping it inside DNA loops. Partner proteins are needed to change or relieve Rok-mediated gene repression. Lastly, we investigate which features characterize H-NS-like proteins, a family that, at present, lacks a clear definition.

The DNA replication initiation protein DnaD recognises a specific strand of the Bacillus subtilis chromosome origin.

Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. However, the molecular mechanisms underpinning helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC, the DnaD Recognition Element (DRE). Considered with single particle cryo-electron microscopy (cryo-EM) imaging of DnaD, we propose that the location of the DRE within oriC orchestrates strand-specific recruitment of helicase during DNA replication initiation. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin.

Target search by an imported conjugative DNA element for a unique integration site along a bacterial chromosome during horizontal gene transfer.

Integrative and conjugative elements (ICEs) are mobile genetic elements that can transfer by conjugation to recipient cells. Some ICEs integrate into a unique site in the genome of their hosts. We studied quantitatively the process by which an ICE searches for its unique integration site in the Bacillus subtilis chromosome. We followed the motion of both ICEBs1 and the chromosomal integration site in real time within individual cells. ICEBs1 exhibited a wide spectrum of dynamical behaviors, ranging from rapid sub-diffusive displacements crisscrossing the cell, to kinetically trapped states. The chromosomal integration site moved sub-diffusively and exhibited pronounced dynamical asymmetry between longitudinal and transversal motions, highlighting the role of chromosomal structure and the heterogeneity of the bacterial interior in the search. The successful search for and subsequent recombination into the integration site is a key step in the acquisition of integrating mobile genetic elements. Our findings provide new insights into intracellular transport processes involving large DNA molecules.

Barriers to simultaneous multilocus integration in Bacillus subtilis tumble down: development of a straightforward screening method for the colorimetric detection of one-step multiple gene insertion using the CRISPR-Cas9 system

BackgroundDespite recent advances in genetic engineering tools for effectively regulating and manipulating genes, efficient simultaneous multigene insertion methods have not been established in Bacillus subtilis. To date, multilocus integration systems in B. subtilis, which is one of the main industrial enzyme producers and a GRAS (generally regarded as safe) microbial host, rely on iterative rounds of plasmid construction for sequential insertions of genes into the B. subtilis chromosome, which is tedious and time consuming.ResultsIn this study, we present development and proof-of-concept of a novel CRISPR-Cas9-based genome-editing strategy for the colorimetric detection of one-step multiple gene insertion in B. subtilis. First, up to three copies of the crtMN operon from Staphylococcus aureus, encoding a yellow pigment, were incorporated at three ectopic sites within the B. subtilis chromosome, rendering engineered strains able to form yellow colonies. Second, a single CRISPR-Cas9-based plasmid carrying a highly specific single guide RNA (sgRNA) targeting crtMN operon and a changeable editing template was constructed to facilitate simultaneous insertion of multiple gene-copies through homology-directed repair (HDR). Upon transformation of engineered strains with engineered plasmids, strains harboring up to three gene copies integrated into the chromosome formed white colonies because of the removal of the crtMN operon, clearly distinguishable from yellow colonies harboring undesired genetic modifications. As a result, construction of a plasmid-less, marker-free, high-expression stable producer B. subtilis strain can be completed in only seven days, demonstrating the potential that the implementation of this technology may bring for biotechnology purposes.ConclusionsThe novel technology expands the genome-editing toolset for B. subtilis and means a substantial improvement over current methodology, offering new application possibilities that we envision should significantly boost the development of B. subtilis as a chassis in the field of synthetic biology.

Profiling a single-stranded DNA region within an rDNA segment that affects the loading of bacterial condensin.

Bacterial condensin preferentially loads onto single-stranded DNA (ssDNA) invitro and onto rDNA invivo to support proper chromosome compaction. Thus, the actively transcribing rDNA would provide the ssDNA region for the loading of bacterial condensin. We attempted to detect the ssDNA region in the rrnI gene in situ. Non-denaturing sodium bisulfite treatment catalyzed the conversion of cytosines to thymines (CT-conversion) at the melted DNA of a genome. Using next-generation sequencing, we generated an average of 11,000 reads covering each cytosine on the rDNA segment. In principle, the CT-conversion rate is an accurate guide to detect ssDNA segment. We detected multiple ssDNA segments throughout the rDNA. The deletion mutations of the rDNA hindered the ssDNA formation at the 100-500bp segment downstream of the promoter. These data support the idea that the ssDNA segment plays a crucial role as the condensin-loading site and suggest the mechanism of condensin loading onto rDNA.

Activation of the integrative and conjugative element Tn916 causes growth arrest and death of host bacteria.

Integrative and conjugative elements (ICEs) serve as major drivers of bacterial evolution. These elements often confer some benefit to host cells, including antibiotic resistance, metabolic capabilities, or pathogenic determinants. ICEs can also have negative effects on host cells. Here, we investigated the effects of the ICE (conjugative transposon) Tn916 on host cells. Because Tn916 is active in a relatively small subpopulation of host cells, we developed a fluorescent reporter system for monitoring activation of Tn916 in single cells. Using this reporter, we found that cell division was arrested in cells of Bacillus subtilis and Enterococcus faecalis (a natural host for Tn916) that contained an activated (excised) Tn916. Furthermore, most of the cells with the activated Tn916 subsequently died. We also observed these phenotypes on the population level in B. subtilis utilizing a modified version of Tn916 that can be activated in the majority of cells. We identified two genes (orf17 and orf16) in Tn916 that were sufficient to cause growth defects in B. subtilis and identified a single gene, yqaR, that is in a defective phage (skin) in the B. subtilis chromosome that was required for this phenotype. These three genes were only partially responsible for the growth defect caused by Tn916, indicating that Tn916 possesses multiple mechanisms to affect growth and viability of host cells. These results highlight the complex relationships that conjugative elements have with their host cells and the interplay between mobile genetic elements.

Molecular Analysis of pSK1 par: A Novel Plasmid Partitioning System Encoded by Staphylococcal Multiresistance Plasmids

The segregation of prokaryotic plasmids typically requires a centromere-like site and two proteins, a centromere-binding protein (CBP) and an NTPase. By contrast, a single 245 residue Par protein mediates partition of the prototypical staphylococcal multiresistance plasmid pSK1 in the absence of an identifiable NTPase component. To gain insight into centromere binding by pSK1 Par and its segregation function we performed structural, biochemical and in vivo studies. Here we show that pSK1 Par binds a centromere consisting of seven repeat elements. We demonstrate this Par-centromere interaction also mediates Par autoregulation. To elucidate the Par centromere binding mechanism, we obtained a structure of the Par N-terminal DNA-binding domain bound to centromere DNA to 2.25 Å. The pSK1 Par structure, which harbors a winged-helix-turn-helix (wHTH), is distinct from other plasmid CBP structures but shows homology to the B. subtilis chromosome segregation protein, RacA. Biochemical studies suggest the region C-terminal to the Par wHTH forms coiled coils and mediates oligomerization. Fluorescence microscopy analyses show that pSK1 Par enhances the separation of plasmids from clusters, driving effective segregation upon cell division. Combined the data provide insight into the molecular properties of a single protein partition system.

A novel method for transforming Geobacillus kaustophilus with a chromosomal segment of Bacillus subtilis transferred via pLS20-dependent conjugation

BackgroundGeobacillus kaustophilus is a thermophilic Gram-positive bacterium. Methods for its transformation are still under development. Earlier studies have demonstrated that pLS20catΔoriT mobilized the resident mobile plasmids from Bacillus subtilis to G. kaustophilus and transferred long segments of chromosome from one cell to another between B. subtilis.ResultsIn this study, we applied mobilization of the B. subtilis chromosome mediated by pLS20catΔoriT to transform G. kaustophilus. We constructed a gene cassette to be integrated into G. kaustophilus and designed it within the B. subtilis chromosome. The pLS20catΔoriT-mediated conjugation successfully transferred the gene cassette from the B. subtilis chromosome into the G. kaustophilus allowing for the desired genetic transformation.ConclusionsThis transformation approach described here will provide a new tool to facilitate the flexible genetic manipulation of G. kaustophilus.

Galactolipids from <i>Arabidopsis thaliana</i> can replace the function of gluco lipids in <i>Bacillus subtilis</i>.

Arabidopsis thaliana monogalactosyldiacylglycerol synthase 1 (AtMGD1) and digalactosyldiacylglycerol synthase 2 (AtDGD2) genes introduced into a Bacillus subtilis chromosome with disrupted galE, which encodes UDP-glucose 4-epi merase, enabled the mutant to produce monogalactosyldiacylglycerol. When galE mutant cells are cultivated in galactose containing medium they show ab normal morphology. This phenotype is correlated with a decrease in the amount of glucolipids. Nucleoids of the ugtP and galE mutants were stained by propidium iodide, which does not permeate intact cell membranes, whereas nucleoids of wild type and of a pssA mutant we examined were not stained. Expression of the AtMGD1 gene in a ugtP galE double mutant restored cell membrane integrity. Expression of galactolipid synthase genes from a multi-copy plasmid, pDGHisN4, allowed higher production of galactolipids. Activation of the extracytoplasmic function sigma factors SigM, SigV, and SigX, in the ugtP mutant was decreased by expression of AtMGD1, and SigX activity was strongly repressed when both AtMGD1 and AtDGD2 genes were expressed in the mutant. We conclude that the number of sugars that bind to diacylglycerol - rather than the exact sugar species - is important for glycolipid function in B. subtilis.

DNA-loop-extruding SMC complexes can traverse one another in vivo.

Chromosome organization mediated by structural maintenance of chromosomes (SMC) complexes is vital in many organisms. SMC complexes act as motors that extrude DNA loops, but it remains unclear what happens when multiple complexes encounter one another on the same DNA in living cells and how these interactions may help to organize an active genome. We therefore created a crash-course track system to study SMC complex encounters in vivo by engineering defined SMC loading sites in the Bacillus subtilis chromosome. Chromosome conformation capture (Hi-C) analyses of over 20 engineered strains show an amazing variety of chromosome folding patterns. Through three-dimensional polymer simulations and theory, we determine that these patterns require SMC complexes to bypass each other in vivo, as recently seen in an in vitro study. We posit that the bypassing activity enables SMC complexes to avoid traffic jams while spatially organizing the genome.

SR7 – a dual-function antisense RNA from Bacillus subtilis

ABSTRACT Here, we describe SR7, a dual-function antisense RNA encoded on the Bacillus subtilis chromosome. This RNA was earlier described as SigB-dependent regulatory RNA S1136 and reported to reduce the amount of the small ribosomal subunit under ethanol stress. We found that the 5ʹ portion of SR7 encodes a small protein composed of 39 amino acids which we designated SR7P. It is translated from a 185 nt SigB-dependent mRNA under five different stress conditions and a longer SigB-independent RNA constitutively. About three-fold higher amounts of SR7P were detected in B. subtilis cells exposed to salt, ethanol, acid or heat stress. Co-elution experiments with SR7PC-FLAG and Far-Western blotting demonstrated that SR7P interacts with the glycolytic enzyme enolase. Enolase is a scaffolding component of the B. subtilis degradosome where it interacts with RNase Y and phosphofructokinase PfkA. We found that SR7P increases the amount of RNase Y bound to enolase without affecting PfkA. RNA does not bridge the SR7P-enolase-RNase Y interaction. In vitro-degradation assays with the known RNase Y substrates yitJ and rpsO mRNA revealed enhanced enzymatic activity of enolase-bound RNase Y in the presence of SR7P. Northern blots showed a major effect of enolase and a minor effect of SR7P on the half-life of rpsO mRNA indicating a fine-tuning role of SR7P in RNA degradation.

Multiple integration of the gene ganA into the Bacillus subtilis chromosome for enhanced \u03b2-galactosidase production using the CRISPR/Cas9 system

The ganA gene from Bacillus subtilis encoding a β-galactosidase for degradation of the galactomannan was integrated in different loci of the B. subtilis chromosome employing the CRISPR/Cas9 system. Hereby a total of five copies of ganA cassettes in which the ganA gene was fused with the glucitol-promoter were inserted in the recipient chromosome wherein hypothetical, sporulation and protease genes were deleted. The strain with five copies of ganA expression cassette showed a β-galactosidase activity similar to the one with the same gene on a pUB110 derived multi-copy plasmid and under the same regulatory control of the glucitol promoter and GutR activator. The production of β-galactosidase in the strain with the multi-copy plasmid decreased rapidly when growth was performed under induced conditions and without antibiotic selection. In contrast, the strain with the five copies of ganA in the chromosome produced β-galactosidase for at least 40 generations. This demonstrates that the CRISPR/Cas9 system is a valuable and easy tool for constructing stable producer strains. The bigger efforts that are needed for the multiple target gene integration into the chromosome compared to cloning in expression vectors were justified by the higher stability of the target genes and the lack of antibiotic resistance genes.

Identification of a basal system for unwinding a bacterial chromosome origin

Genome duplication is essential for cell proliferation, and DNA synthesis is generally initiated by dedicated replication proteins at specific loci termed origins. In bacteria, the master initiator DnaA binds the chromosome origin (oriC) and unwinds the DNA duplex to permit helicase loading. However, despite decades of research it remained unclear how the information encoded within oriC guides DnaA‐dependent strand separation. To address this fundamental question, we took a systematic genetic approach in vivo and identified the core set of essential sequence elements within the Bacillus subtilis chromosome origin unwinding region. Using this information, we then show in vitro that the minimal replication origin sequence elements are necessary and sufficient to promote the mechanical functions of DNA duplex unwinding by DnaA. Because the basal DNA unwinding system characterized here appears to be conserved throughout the bacterial domain, this discovery provides a framework for understanding oriC architecture, activity, regulation and diversity.

Mutational Analysis of DnaA‐trio Motifs in E. coli OriC Reveals Multiple Modes for Bacterial Replication Origin Activation

The chromosomal replication origin (oriC) in bacteria is a unique region that directs assembly of the orisome, a nucleoprotein complex that unwinds origin DNA and assists with the loading of the replicative helicase. All orisome functions are mediated by the initiator protein, DnaA, which binds to specific sites in oriC, and, after DNA unwinding, binds to and stabilizes the unwound region. Orisome assembly is well characterized in E. coli, but bacterial origins are not conserved with regard to the number and spacing of DnaA recognition sites, and so it is difficult to determine whether there are common sequence patterns that are essential for origin activity. However, one promising feature, the “DnaA‐trio” motif, can be found in the origins of most bacterial types, with multiple copies residing near the AT‐rich region where DNA strand separation takes place. DnaA‐trios are essential for B. subtilis chromosome replication initiation, where they facilitate delivery of DnaA‐ATP oligomeric filaments to the AT‐rich region. Three DnaA‐trio motifs also reside in E. coli oriC, but their role is unclear, since we recently reported that E. coli oriC could be activated without formation of DnaA‐ATP oligomers. Therefore, to test their essentiality in E. coli, we used site‐directed mutagenesis to inactivate the trios, and then used recombineering to replace wild type chromosomal oriC with the mutated versions. Eliminating trio sites had no effect on E. coli oriC activity or initiation timing. Based on these results, we hypothesized that E. coli might use an alternative mechanism to deliver DnaA to the AT‐rich region, one that does not depend on trios or DnaA‐ATP filaments. We hypothesized that the sharp DNA bend formed late in E. coli orisome assembly and stabilized by the DNA bending protein IHF, might eliminate the need for trio sites by positioning the AT‐rich region for cross‐strand DnaA donations provided by the DnaA bound to oriC's left half. To test this, we removed the bend region from oriC using site‐directed mutagenesis, and even though the mutated origin (oriCno bend) contained all three DnaA trios, it was unable to function as the sole chromosomal origin of replication. In vitro unwinding studies suggested that the defect could be caused by a failure to deliver DnaA to the AT‐rich region, so we tried to rescue oriCno bend function by activating a trio‐dependent mode of DnaA delivery. Successful rescue was achieved by inserting a DnaA binding site between R1 and the trio sites, and origin activity became completely dependent on the DnaA‐trio elements. However, rescued cells displayed perturbed initiation timing, suggesting that the bend mode of DnaA delivery plays both mechanical and regulatory roles in E. coli. We propose that there are at least two possible modes of bacterial origin activation; one that depends on DnaA‐trio elements, and one that uses DNA bending and cross‐strand DnaA binding to regulate late stage orisome assembly.Support or Funding InformationNational Institutes of Health (GM54042 to A.L.) and Florida Institute of Technology Holzer Lequear Fund for Molecular Genetics (to J.G.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High-Throughput Allelic Replacement Screening in Bacillus subtilis.

Site-directed mutagenesis is a key tool in the analysis of biological mechanisms. We have established an efficient and systematic gene targeting strategy for Bacillus subtilis based on the Golden Gate cloning methodology. Our approach permits the introduction of single or multiple point mutations or of heavily engineered alleles into the endogenous gene locus in a single step using a 96-well microtiter plate format. We have successfully applied this system for high-throughput functional screening of resized variants of the Structural Maintenance of Chromosome (Smc) protein and for exhaustive cysteine cross-linking mutagenesis. Here we describe, in detail, the experimental setup for high-throughput introduction of modifications into the B. subtilis chromosome. With minor modifications, the approach should be applicable to other bacteria and yeast.

Bacillus subtilis Type I antitoxin SR6 Promotes Degradation of Toxin yonT mRNA and Is Required to Prevent Toxic yoyJ Overexpression.

yonT/SR6 is the second type I toxin-antitoxin (TA) system encoded on prophage SPβ in the B. subtilis chromosome. The yonT ORF specifying a 58 aa toxin is transcribed on a polycistronic mRNA under control of the yonT promoter. The antitoxin SR6 is a 100 nt antisense RNA that overlaps yonT at its 3′ end and the downstream gene yoyJ encoding a second, much weaker, toxin at its 5′ end. SR6 displays a half-life of >60 min, whereas yonT mRNA is less stable with a half-life of ≈8 min. SR6 is in significant excess over yonT mRNA except in minimal medium with glucose. It interacts with the 3′ UTR of yonT mRNA, thereby promoting its degradation by RNase III. By contrast, SR6 does not affect the amount or half-life of yoyJ mRNA. However, in its absence, a yoyJ overexpression plasmid could not be established in Bacillus subtilis suggesting that SR6 inhibits yoyJ translation by directly binding to its ribosome-binding site. While the amounts of both yonT RNA and SR6 were affected by vancomycin, manganese, heat-shock and ethanol stress as well as iron limitation, oxygen stress decreased only the amount of SR6.

Overproduction of Rummeliibacillus pycnus arginase with multi-copy insertion of the arg R.pyc cassette into the Bacillus subtilis chromosome.

A plasmid-less and marker-less strain with multi-copy integration of the arginase gene from Rummeliibacillus pycnus was constructed using Bacillus subtilis 168 as a host. A total of nine copies of the arg R.pyc cassettes, in which the R. pycnus arginase gene was fused with the strong promoter P43, were inserted into the recipient chromosome. These multiple insertions were completed via step-by-step integrations into designed (2 copies) and random (9 copies) sites, respectively. A strategy for random site integration was developed based on the construction of the arg R.pyc cassette sandwiched between "front" and "back" homologous arms which were randomly restricted from chromosomal DNA. An antibiotic resistance marker was applied in transformant selection and was eliminated via the Cre/lox system. Performance showed that the highest enzyme activity (14.5U/mL) was obtained after culture in flasks, and this segregation stable strain could efficiently hydrolyze L-arginine with a 97.2% molar yield, showing potential application in the food industry.

Transposition of insertion sequence IS256Bsu1 in Bacillus subtilis 168 is strictly dependent on recA.

We developed an insertion sequence transposition detection system called the "jumping cat assay" and applied it to the Bacillus subtilis chromosome using IS256Bsu1 derived from B. subtilis natto. The high frequency of transposition enabled us to explore host factors; combining the assay and genetic analyses revealed that recA is essential for the transposition of IS256Bsu1. Detailed analyses using various domain mutants of recA demonstrated that this essentiality is not related to the function of recA in homologous recombination. Instead, the ATP binding and hydrolysis function seemed to be crucial for IS transposition. To elucidate the role of recA, we focused on the muB gene of the enterobacteriophage Mu. Based on information from the NCBI Conserved Domain Database, both MuB and RecA belong to the P-loop dNTPase superfamily. Further experiments revealed that muB complements the transposition-defective phenotype of a recA deletant, although it could not rescue UV sensitivity. These results suggest that recA shares a common function with muB that helps the transposition of IS256Bsu1 in B. subtilis.