A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis

Sachla, Ankita J.; Alfonso, Alexander J; Helmann, John D.

doi:10.1128/spectrum.00754-21

Cited by 12 publications

(6 citation statements)

References 35 publications

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“…In these strains, the coding region is replaced by an erythromycin cassette that can be removed using plasmid pDR244 expressing the Cre-Lox recombinase (19). In addition, we used a CRISPR-based mutagenesis approach (20) to delete just the S936 element, the S936- sodA region, or the yqgC-sodA operon in its entirety (τι yqgC -S936- sodA; YS). The yqgC , sodA , and S936- sodA deletion strains all grew well in LB medium both with and without amendment with 150 μM Mn (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The repair template had compatible SfiI flanking the 5’ and 3’ ends. Upon digestion with SfiI at 50 °C for 2 hr, a similar restriction digest was performed for pAJS23 plasmid containing guide RNA against erm cassette as described previously (20). The ligation of the digested repair template into pre-digested pAJS23 was performed and was then transformed into E. coli DH5α selected on Luria-Bertani (LB) medium (Affymetrix) supplemented with 30 μg/ml of kanamycin.…”

Section: Methodsmentioning

confidence: 99%

“…The repair template had compatible SfiI flanking the 5' and 3' ends. Upon digestion with SfiI at 50 °C for 2 hr, a similar restriction digest was performed for pAJS23 plasmid containing guide RNA against erm cassette as described previously (20).…”

Section: Genetic Manipulations and Strain Construction A) Crispr Editingmentioning

confidence: 99%

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TheBacillus subtilis yqgC-sodAoperon protects magnesium-dependent enzymes by supporting manganese efflux

Sachla,

Soni,

Piñeros

et al. 2024

Preprint

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Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux. Here, we demonstrate that deletion of theBacillus subtilis yqgC-sodA(YS) complex operon, but not deletion of the individual genes, causes hypersensitivity to manganese (Mn). YqgC is an integral membrane protein of unknown function and SodA is a Mn-dependent superoxide dismutase (MnSOD). The YS strain has reduced expression of two Mn efflux proteins, MneP and MneS, consistent with the observed Mn sensitivity. The YS strain accumulated high levels of Mn, had increased reactive radical species (RRS), and had broad metabolic alterations that can be partially explained by the inhibition of Mg-dependent enzymes. Although the YS operon deletion strain and an efflux-deficientmneP mneSdouble mutant both accumulate Mn and have similar metabolic perturbations they also display phenotypic differences. Several mutations that suppressed Mn intoxication of themneP mneSefflux mutant did not benefit the YS mutant. Further, Mn intoxication in the YS mutant, but not themneP mneSstrain, was alleviated by expression of Mg-dependent, chorismate-utilizing enzymes of the menaquinone, siderophore, and tryptophan (MST) family. Therefore, despite their phenotypic similarities, the Mn sensitivity in themneP mneSand theyqgC-sodAdeletion mutants results from distinct enzymatic vulnerabilities.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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TheBacillus subtilis yqgC-sodAoperon protects magnesium-dependent enzymes by supporting manganese efflux

Sachla,

Soni,

Piñeros

et al. 2024

Preprint

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“…To this end, we used long-flanking homology (LFH) PCR to generate allelic variants of mntR with sufficient upstream and downstream homology. These repair template PCR fragments were cloned into pAJS23 plasmid, which expresses an erm -directed guide RNA 36 . The recombinant plasmids were transformed into mntR :: erm B. subtilis strains at 30°C and transformants were selected onto LB (Lennox L Broth, RPI) containing kanamycin (15mg/mL) and 0.2% mannose.…”

Section: Methodsmentioning

confidence: 99%

“…The erythromycin and kanamycin negative strains were selected for Sanger sequencing for further confirmation. The components of minimal media were the same as previously described 36 . Whenever applicable, we used MLS (erythromycin (1μg/mL) plus lincomycin (25μg/mL)).…”

Section: B Subtilis Bacterial Strain Construction and Growth Conditionsmentioning

confidence: 99%

Structural basis for transcription activation through cooperative recruitment of MntR

Shi,

Fu,

Kodyte

et al. 2024

Preprint

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The manganese transport regulator (MntR) fromB. subtilisis a dual regulatory protein that responds to heightened Mn2+availability in the cell by both repressing the expression of uptake transporters and activating the expression of efflux proteins. Recent work indicates that, in its role as an activator, MntR binds several sites upstream of the genes encoding Mn2+exporters, leading to a cooperative response to manganese. Here, we use cryo-EM to explore the molecular basis of gene activation by MntR and report a structure of four MntR dimers bound to four 18-base pair sites across an 84-base pair regulatory region of themnePpromoter. Our structures, along with solution studies including mass photometry andin vivotranscription assays, reveal that MntR dimers employ polar and non-polar contacts to bind cooperatively to an array of low-affinity DNA-binding sites. These results reveal the molecular basis for cooperativity in the activation of manganese efflux.

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Recent advances of the biological and biomedical applications of CRISPR/Cas systems

Wang

Zhao

2022

Mol Biol Rep

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It has also been reported that about 40% of spacers from lactic acid bacteria, namely Streptococcus thermophiles, have a homologue corresponding to either phage or plasmid DNA sequences among the isolated and sequenced phage's genome [3]. Thus, the spacers are proposed to be derived from foreign DNA including phage and plasmids. Bacteria generally acquire new spacers once they are exposed to new viruses in order to confer resistance to the cognate virus. Therefore, spacers present in a certain bacterium typically reveal the historical record of phages that the bacterium has been exposed to [4]. The leader sequence is commonly located 5' to the CRISPR locus and is rich in AT nucleotides. However, it has been demonstrated that the leader sequence is incapable of encoding RNAs or proteins, and that it is located next to the short repeats. Apart from the leader sequence, the genes encoding Cas proteins are found to be localized either upstream or downstream of the CRISPR locus.The CRISPR/Cas system is postulated to be formed in three stages. In the adaption stage, bacterial cells acquire sequences derived from foreign DNA such as viral DNA, and then incorporate them into the bacterial CRISPR array. During the expression stage, precursor CRISPR RNA (crRNA)

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A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis

Cited by 12 publications

References 35 publications

TheBacillus subtilis yqgC-sodAoperon protects magnesium-dependent enzymes by supporting manganese efflux

TheBacillus subtilis yqgC-sodAoperon protects magnesium-dependent enzymes by supporting manganese efflux

Structural basis for transcription activation through cooperative recruitment of MntR

Recent advances of the biological and biomedical applications of CRISPR/Cas systems

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