• 2018-07
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  • br Introduction Acinetobacter baumannii is a gram negative


    Introduction Acinetobacter baumannii is a gram-negative opportunistic pathogen that causes nosocomial infections including pneumonia and bloodstream infections which is associated with an increased mortality and multi-drug resistance [[1], [2], [3]]. A. baumannii was rated as one of the critical priority 1 pathogens for the development of new antibiotics by the WHO in 2017 [4]. Despite the name “Acinetobacter” meaning non-motile bacteria and despite the lack of flagella, members of the genus are able to move [5,6]. At least two forms of motility are known for Acinetobacter species. The so called “twitching motility” depends on retraction of type IV pili [[7], [8], [9]]. Another form of movement, the surface-associated motility, occurs at the surface of semi-dry media and is independent of type IV pili [10]. Almost all tested clinical isolates can move along surfaces [11] and a number of genes required for this form of motility have been identified [10,12,13]. Motility is known to be affected by epigenetic regulation in various bacteria [14]. Epigenetics deals with heritable changes in gene expression without any changes in the DNA sequence. In bacteria the most studied epigenetic mechanism is DNA methylation [15] that is performed by DNA methyltransferases [16]. These enzymes transfer methyl groups from S-adenosyl-l-methionine (SAM) to Glycopyrrolate molecular or cytosine bases. This process protects DNA against digestion from restriction endonucleases and is important for the regulation of various physiological processes such as mismatch repair and transcription [17,18]. Most DNA methyltransferases are part of a restriction-modification system (R-M system). In this case the host DNA gets methylated by the DNA methyltransferase that protects the DNA against digestion by the corresponding endonuclease [19]. In addition, “orphan” methyltransferases are known that act without any associated endonuclease [17]. The most studied orphan DNA adenine methyltransferase called Dam was found in E. coli and was shown to methylate adenine bases at GATC sites [20,21]. Salmonella enterica dam mutants exhibit a reduced motility [22] and dam overexpression in Yersinia enterocolitica resulted in an increased motility [23]. The Acinetobacter baumannii genome encodes a putative DNA-(adenine N6)-methyltransferase, designated A1S_0222 in strain ATCC 17978 that seems to act without a corresponding endonuclease. We hypothesized that the putative DNA adenine methyltransferase A1S_0222 does impose epigenetic control in Acinetobacter baumannii and since little is known about orphan methyltransferases we approached its characterization.
    Materials and methods Transformation was performed by electroporation [24]. The EZ-Tn5™ transposon mutants in A. baumannii ATCC 17978 were generated by using the EZ-Tn5™ insertion kit (Epicentre Biotechnologies) as previously described [11]. Surface-associated motility. Motility plates were composed of 0.5% agarose (w/v), 5 g/L of tryptone, and 2.5 g/L of NaCl as previously described [11]. A single colony from a nutrient agar plate (Oxoid) or selective agar plates (supplemented with 50 μg/mL of kanamycin for the A1S_0222 mutant) of either wild-type or A. baumannii ATCC 17978 mutant was taken with the pipette tip and then the surface of a motility plate was touched. Pictures were taken after incubating the plates for 16 h at 37 °C. Construction of protein expression plasmids. The a1s_0222 gene of A. baumannii ATCC 17978 was amplified by PCR using the oligonucleotides 0222-pGEX-6P-3-for: 5′-ATTAGGATCCAATTCAGAGCCTTCGGTATACCAC-3’ (BamHI restriction site underlined) and 0222-pGEX-6P-3-rev: 5′-ATTAGCGGCCGCTTACCAAAGTGCGAGCTGTGTAC-3’ (NotI restriction site underlined). The amplified a1s_0222 gene was inserted into pGEX-6P-3 expression vector (GE Healthcare) after digestion of insert and vector with BamHI and NotI restriction enzymes. The pGEX-6P-3 expression vector carries a glutathione S-transferase (GST-tag) gene, a PreScission protease restriction site, an ampicillin resistance (AmpR) cassette, encodes a tac promotor and is inducible with isopropyl β-d-1-thiogalactopyranoside (IPTG). The plasmid (pGEX-6P-3-A1S_0222) was confirmed by DNA sequencing and transformed into E. coli BL21 (DE3) pLysS expression strain.