Characterization of Type II and III Restriction-Modification Systems from Bacillus cereus Strains ATCC 10987 and ATCC 14579

Xu, Shuang-yong; Nugent, Rebecca L.; Kasamkattil, Julie; Fomenkov, Alexey; Gupta, Y. K.; Aggarwal, Aneel K.; Wang, Xiaolong; Li, Zhi‐Ru; Zheng, Yu; Morgan, Richard D.

doi:10.1128/jb.06248-11

Cited by 26 publications

(34 citation statements)

References 46 publications

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“…cereus ATCC 10987 had previously been examined by Xu et al (25), who determined recognition sequences for four Type II and III REases and one orphan MTase by traditional methods. However, the sites of methylation for the Type II and III MTases were not determined and several other MTases were not examined including that in the Type I system (BCE_0839-BCE_0842) and a Type II MTase (BCE_0392) that was reported to be inactive (25). However, when we cloned this MTase and checked its activity, it was clearly a promiscuous m6 A MTase, which we have now named M.BceSVII (Supplementary Figure S9 and Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…It is important to note that while cloning the individual MTase genes showed five to be active only four seem to be active in the genome. M.BceSV, a multi-specific MTase characterized in the previous study by cloning and overexpression (25) is encoded on a prophage and does not show detectable activity in the native host genome. In addition to the m6 A and m4 C MTases mentioned earlier, our analysis indicated two more motifs that are likely modified by one or more of the predicted m5 C MTases in the B. cereus genome, as 179 of the 524 unassigned hits fell into two categories.…”

Section: Resultsmentioning

confidence: 99%

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The methylomes of six bacteria

Murray

Clark

Morgan

et al. 2012

Nucleic Acids Research

247

232

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Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and C. jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N6-methyladenine (m6A) and N4-methylcytosine (m4C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases, it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTase genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. Two of these proved active. No attempt was made to detect 5-methylcytosine (m5C) recognition motifs from the SMRT® sequencing data because this modification produces weaker signals using current methods. However, all predicted m6A and m4C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active but also revealing their recognition sequences.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

The methylomes of six bacteria

Murray

Clark

Morgan

et al. 2012

Nucleic Acids Research

247

232

View full text Add to dashboard Cite

show abstract

“…Therefore BCE_0392 might modify sequences that are not recognized by these REase, and new techniques like single-molecule DNA sequencing other than restriction analysis using commercialized REases should be useful in identifying the sequence specificity of BCE_0392 [29]. DNA nicking-associated concatenation activity was also detected for BCE_0392 in vivo [15], suggesting that this ParB-Methyltransferase might participate in phage DNA replication or phage packaging, since BCE_0392 was located in a prophage region in the chromosome of the B. cereus ATCC 10987 strain [11].…”

Section: Resultsmentioning

confidence: 99%

A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria

et al. 2012

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Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography–Mass Spectrometry (LC–MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate the functional genomics of the strains that are difficult to transform.

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“…Research on about half of these proteins has been successfully completed, and results for some have been published [4]–[8], while research on the other half is still in progress. For those results that have been reported, 65% of proteins (44 of 68) have been verified to have the predicted function described in the COMBREX grant proposal, while no activity was observed for the remaining 24 (Table S1).…”

Section: Combrex Grants: Experiments Fundedmentioning

confidence: 99%