Clostridioides difficile specific DNA adenine methyltransferase CamA squeezes and flips adenine out of DNA helix

Clostridioides difficile infections are an urgent medical problem. The newly discovered C.difficileadenine methyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. CamA is an orphan methyltransferase, unassociated with a restriction endonuclease. CamA-mediated methylation at CAAAAA is required for normal sporulation, biofilm formation, and intestinal colonization by C. difficile. We characterized CamA kinetic parameters, and determined its structure bound to DNA containing the recognition sequence. CamA contains an N-terminal domain for catalyzing methyl transfer, and a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These provide sufficient sequence discrimination to ensure high specificity. Finally, the surprisingly weak binding of the methyl donor S-adenosyl-l-methionine (SAM) might provide avenues for inhibiting CamA activity using SAM analogs.

The impact of DNA adenine methyltransferase knockout on the development of triclosan resistance and antibiotic cross-resistance in Escherichia coli

Background. DNA adenine methyltransferase (dam) has been well documented for its role in regulation of replication, mismatch repair and transposition. Recent studies have also suggested a role for dam in protection against antibiotic stress, although this is not yet fully defined. We therefore evaluated the role of dam in the development of antibiotic resistance and triclosan-associated cross-resistance. Results. A significant impact on growth rate was seen in the dam knockout compared to the parental strain. Known triclosan resistance-associated mutations in fabI were seen regardless of dam status, with an additional mutation in lrhA seen in the dam knockout. The expression of multiple antibiotic resistance-associated genes was significantly different between the parent and dam knockout post-resistance induction. Reversion rate assays showed that resistance mechanisms were stable. Conclusions. dam knockout had a significant effect on growth, but its role in the development of antibiotic resistance is likely confined to those antibiotics using acrAD-containing efflux pumps.

Identification of potential candidates for drug designing against Klebsiella Pneumoniae among hypothetical proteins of DNA Adenine Methyltransferase through subtractive genomics approach

ABSTRACTKlebsiella Pneumoniae is a gram-negative bacterium that is known for causing infection in nosocomial settings. As reported by WHO, this bacterial pathogen is classified as an urgent threat our most concern is that these bacterial pathogens acquired genetic traits that make them resistant towards antibiotics. The last class of antibiotics; carbapenems are not able to combat these bacterial pathogens allowing them to clonally expand their antibiotic-resistant strain. Most antibiotics target the essential pathways of the bacteria cell however these targets are no longer susceptible to the antibiotic. Hence in our study, we focus on Klebsiella Pneumoniae bacterial strains that contain DNA Adenine Methyltransferase domain which suggests a new potential site for a drug target. DNA methylation is seen to regulate the attenuation of bacterial virulence. In this study, all hypothetical proteins of Klebsiella Pneumoniae containing N6 DNA Adenine Methyltransferase domain were analysed for a potential drug target. About 32 hypothetical proteins were retrieved from Uniprot. 19 proteins were selected through a step-wise subtractive genomics approach like a selection of non-homologus proteins against the human host, selection of bacterial proteins contains an essential gene, broad-spectrum analysis, druggability analysis, Non-homology analysis against gut microbiota. Through drug target prioritization like sub-cellular analysis, drug property analysis, anti-target non-homology analysis, virulence factor analysis and protein-protein interaction analysis one drug target protein (Uniprot ID: A0A2U0NNR3) was prioritized. Identified drug target docked with potential inhibitors like are mahanine (PubChem ID: 375151), curcumin (PubChem ID: 969516), EGCG (PubChem ID: 65064), nanaomycin A (PubChem ID: 40586), parthenolide (PubChem ID: 7251185), quercetin (PubChem ID: 5280343) and trimethylaurintricarboxylic acid. Based on the moelcular docking analysis, mahanine has the highest binding affinity. In order to identify novel natural inhibitor based on mahanine fingerprint search is performed against NPASS (Natural Product Activity and Species Source databases) and Koenimbine was identified as a novel natural inhibitor based on virtual screening.

The transcriptional regulator CBX2 and ovarian function: A whole genome and whole transcriptome approach

The chromobox homolog 2 (CBX2) was found to be important for human testis development, but its role in the human ovary remains elusive. We conducted a genome-wide analysis based on DNA adenine methyltransferase identification (DamID) and RNA sequencing strategies to investigate CBX2 in the human granulosa cells. Functional analysis revealed that CBX2 was upstream of genes contributing to ovarian function like folliculogenesis and steroidogenesis (i.e. ESR1, NRG1, AKR1C1, PTGER2, BMP15, BMP2, FSHR and NTRK1/2). We identified CBX2 regulated genes associated with polycystic ovary syndrome (PCOS) such as TGFβ, MAP3K15 and DKK1, as well as genes implicated in premature ovarian failure (POF) (i.e. POF1B, BMP15 and HOXA13) and the pituitary deficiency (i.e. LHX4 and KISS1). Our study provided an excellent opportunity to identify genes surrounding CBX2 in the ovary and might contribute to the understanding of ovarian physiopathology causing infertility in women.

iDamIDseq and iDEAR: An improved method and a computational pipeline to profile chromatin-binding proteins of developing organisms

DNA adenine methyltransferase identification (DamID) has emerged as an alternative for profiling protein-DNA interactions, however critical issues in the method limit its applicability. Here we present iDamlDseq, a protocol that improves specificity and robustness making its use compatible with developing organisms. In addition, we present the analysis tool iDEAR (iDamlDseq Enrichment Analysis with R) to determine protein-DNA interactions genome wide. The combination of both allows establishing highly reliable transcription factor profiles, even in transient assays. For tissue specific expression we improved the Dam coding sequence to overcome predominant aberrant splicing of Dam fusions we discovered with the commonly used sequence.