Methylation-dependent DNA restriction in Bacillus anthracis

Sitaraman, Ramakrishnan; Leppla, Stephen H.

doi:10.1016/j.gene.2011.11.061

Cited by 16 publications

(16 citation statements)

References 28 publications

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“…Many bacterial strains have been found to restrict DNA with Dam and Dcm methylation. For instance, Bacillus anthracis was able to be transformed by DNA from dam, dcm-deficient E. coli strain but hardly by DNA from dam-proficient E. coli strain [37,38]; Dcm methylation was reported to be detrimental to plasmid transformation in Clostridium thermocellum [39]. In this study, the transformation efficiency of unmethylated pHT3101 was significantly increased by about 3001000-fold compared to methylated pHT3101 from E. coli DH5α (Tables 1 and 3), suggesting that B-9987 encodes restriction enzymes against Dam-methylated (containing m5A) or/and Dcm-methylated (containing m5C) DNA.…”

Section: Discussionmentioning

confidence: 99%

Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain

Liu

Zheng

Zhan

et al. 2014

Sci. China Life Sci.

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Marine-derived Bacillus strains have been proved to be a very promising source for natural product leads. However, transformation of environmental strains is much more difficult than that of domesticated strains. Here, we report the development of an efficient and robust electroporation-based transformation system for marine-derived Bacillus marinus B-9987, which is a macrolactin antibiotics producer and a very promising biological control agent against fungal plant diseases. The transformation efficiency was greatly enhanced 10 3 -fold by using unmethylated plasmid to bypass modification-restriction barrier, and using glycine betaine to protect cells from electrical damages during electroporation. Addition of HEPES and 2 mmol L 1 MgCl 2 further improved the efficiency by additional 2-fold, with a maximum value of 7.1×10 4 cfu/µg pHT3101. To demonstrate the feasibility and efficiency of the protocol, a green fluorescent protein reporter system was constructed; furthermore, phosphopantetheinyl transferase gene sfp, which is essential to the biosynthesis of polyketides and nonribosomal peptides, was overexpressed in B-9987, leading to increased production of macrolactin A by about 1.6-fold. In addition, this protocol is also applicable to marine-derived Bacillus licheniforms EI-34-6, indicating it could be a reference for other undomesticated Bacillus strains. To our knowledge, this is the first report regarding the transformation of marine-derived Bacillus strain. marine-derived, Bacillus marinus, electroporation, macrolactin, green fluorescent protein, phosphopantetheinyl transferase Citation:Liu Y, Zheng H, Zhan GH, Qin W, Tian L, Li WL. Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain.

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Section: Discussionmentioning

confidence: 99%

Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain

Liu

Zheng

Zhan

et al. 2014

Sci. China Life Sci.

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“…However, the plasmids for transformation into B. amyloliquefaciens still have to be extracted from E. coli JM110 ( dam − dcm − ). It was speculated that B. amyloliquefaciens LL3 might contain type IV R‐M systems, because the type IV, or methylation‐dependent restriction enzymes (MDREs), have a feature that is opposite with the other types, in which they cleave only when bases within the recognition sites are methylated (Sitaramana and Lepplab, ). Additionally, transformation experiments showed that only vectors prepared from E. coli JM110 ( dam − dcm − ) were capable to be transformed into LL3Δ upp , NK‐A0 or their derivatives.…”

Section: Discussionmentioning

confidence: 99%

“…S7). Besides, a BLAST homology search was performed between LL3Δ upp and the reported genes in no similar protein were found in Bacillus anthracis (Sitaramana and Lepplab, ). The possible explanations for the contrary results might be the unsuitable conditions in the enzymes extracts digestion experiments or that the enzymes belonging to type IV system were inactivated during the purification (Mulligan and Dunn, ).…”

Section: Discussionmentioning

confidence: 99%

Metabolic engineering ofBacillus amyloliquefaciensLL3 for enhanced poly‐γ‐glutamic acid synthesis

Gao

He²,

Zhang

et al. 2019

Microbial Biotechnology

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Summary Poly‐γ‐glutamic acid (γ‐PGA) is a biocompatible and biodegradable polypeptide with wide‐ranging applications in foods, cosmetics, medicine, agriculture and wastewater treatment. Bacillus amyloliquefaciens LL3 can produce γ‐PGA from sucrose that can be obtained easily from sugarcane and sugar beet. In our previous work, it was found that low intracellular glutamate concentration was the limiting factor for γ‐PGA production by LL3. In this study, the γ‐PGA synthesis by strain LL3 was enhanced by chromosomally engineering its glutamate metabolism‐relevant networks. First, the downstream metabolic pathways were partly blocked by deleting fadR, lysC, aspB, pckA, proAB, rocG and gudB. The resulting strain NK‐A6 synthesized 4.84 g l−1 γ‐PGA, with a 31.5% increase compared with strain LL3. Second, a strong promoter PC2up was inserted into the upstream of icd gene, to generate strain NK‐A7, which further led to a 33.5% improvement in the γ‐PGA titre, achieving 6.46 g l−1. The NADPH level was improved by regulating the expression of pgi and gndA. Third, metabolic evolution was carried out to generate strain NK‐A9E, which showed a comparable γ‐PGA titre with strain NK‐A7. Finally, the srf and itu operons were deleted respectively, from the original strains NK‐A7 and NK‐A9E. The resulting strain NK‐A11 exhibited the highest γ‐PGA titre (7.53 g l−1), with a 2.05‐fold improvement compared with LL3. The results demonstrated that the approaches described here efficiently enhanced γ‐PGA production in B. amyloliquefaciens fermentation.

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“…A restriction enzyme, McrBC (NEB) was used to analyze DNA methylation within the AR promoter. It specifically cleaves DNA containing methyl cytosine preceded by a purine nucleotide (A or G) [59,60]. The reaction consisted of 500ng of genomic DNA, 5 units of McrBC enzyme, 1x NEB buffer 2, 1x BSA, 1mM GTP and water in a total reaction volume of 50µl.…”

Section: Dna Methylation Analysis Using Methylation Dependent Restricmentioning

confidence: 99%

Activation of theAndrogen Receptorgene by BORIS/CTCFL in prostate cancer cells

Hari-Gupta

Kita

Farrar

et al. 2017

Preprint

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BORIS/CTCFL, a paralogue of the chromatin architectural protein CTCF, is a member of the cancer-testis antigen family, normally present in the testes. BORIS is expressed in various tumours, including prostate cancers, however the function of BORIS in cancer cells is not well defined. The androgen receptor (AR) plays a critical role in the normal development of a human prostate gland and pathogenesis of prostate cancer. In our previous study we described a positive correlation between elevated levels of BORIS and AR in prostate cancers, and activation of the AR gene by BORIS in prostate cancer cells. Elucidation of the mechanisms involved in the modulation of AR activity is important to understand prostate tumourigenesis and investigation of transcriptional regulation of the AR gene by BORIS may provide new insights into this issue. Here we report the ability of BORIS to not only positively regulate AR in androgen-dependent prostate cancer (ADPC) cells, but re-activate epigenetically silenced AR in androgen-independent prostate cancer (AIPC) cells leading to the production of biologically active AR protein. CTCF, on the other hand, had repressive effects on the AR. In both, ADPC and AIPC cells, introduction of ectopic BORIS was associated with the reduction in the AR promoter methylation, increase in active and decrease in repressive chromatin marks, and decrease in CTCF occupancies at the two main upstream BORIS/CTCF binding sites. We propose a model of epigenetic regulation of AR by BORIS in prostate cells whereby BORIS remodels the chromatin at the AR promoter leading to transcriptional activation.

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Methylation-dependent DNA restriction in Bacillus anthracis

Cited by 16 publications

References 28 publications

Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain

Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain

Metabolic engineering ofBacillus amyloliquefaciensLL3 for enhanced poly‐γ‐glutamic acid synthesis

Activation of theAndrogen Receptorgene by BORIS/CTCFL in prostate cancer cells

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