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 CBR-5884 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.