Serine acetyltransferase (CysE) is a member of the left-handed β-helix family of acetyltransferases that catalyse the rate limiting step in de novo cysteine biosynthesis. There are two isoforms of CysE that differ in length, with the shorter isoform lacking approximately 76 amino acids at the N-terminus of the protein from the serine acetyltransferase (SATase) domain. Here, we analyse the distribution and diversity of CysE isoforms across the bacterial kingdom. The isoforms can be classified into two discrete groups, with the truncated isoform prevalent in Gram positive bacteria and the full-length isoform dominant in Proteobacteria. Moreover, we demonstrate that the truncation is discrete with the loss of four N-terminal α-helices conserved for the truncated isoform. Using predictive modelling we show that this truncation likely weakens the CysE trimer-trimer interface potentially resulting in a trimeric assembly instead of the canonical CysE hexamer. This expands our understanding of CysE enzymes and their distribution across bacterial species, an important consideration given the increasing interest in targeting CysE enzymes for developing potential antimicrobials.

DNA ligases catalyze bond formation in the backbone of nucleic acids via the formation of a phosphodiester bond between adjacent 5’ phosphates and 3’ hydroxyl groups on one strand of the duplex. While DNA ligases preferentially ligate single breaks in double stranded DNA (dsDNA), they are capable of ligating a multitude of other nucleic acids substrates like blunt-ended dsDNA, TA overhangs, short overhangs and various DNA-RNA hybrids. Here we report a novel DNA ligase from Chronobacter phage CR 9 (AZ R2D Ligase) with an unexpected DNA-to-RNA ligation activity. The R2D ligase shows excellent efficiency when ligating DNA to either end of RNA molecules using a DNA template. Furthermore, we show that DNA can be ligated simultaneously to both the 5’ and 3’ ends of microRNA-like molecules in a single reaction mixture. Abortive adenylated side product formation is suppressed at lower ATP concentrations and the ligase reaction reaches near completion when ligating RNA to DNA or DNA to RNA. The ligation of a DNA strand to the 5’-PO4 end of RNA is unique among the commercially available ligases and may facilitate novel workflows in microRNA analysis, RNA sequencing and in the preparation of chimeric guide DNA-RNA for gene editing applications.

The ATP-dependent DNA ligase Lig E is present as an accessory DNA ligase in numerous proteobacterial pathogen genomes, including many disease-causing species. Here we have constructed a genomic Lig E knock-out in the obligate human pathogen Neisseria gonorrhoeae and characterised its growth and infection characteristics. This demonstrates that N. gonorrhoeae Lig E is a non-essential gene and its deletion does not cause defects in replication or survival of DNA-damaging stressors. Knock-out strains were partially defective in biofilm formation on an artificial surface as well as adhesion to epithelial cells which coupled with the predicted extracellular/ periplasmic location of Lig E indicates a role in extracellular DNA joining. In addition to in vivo characterisation, we have recombinantly expressed and assayed N. gonorrhoeae Lig E and determined the crystal structure of the enzyme-adenylate engaged with DNA substrate in an open non-catalytic conformation, providing insight into the binding dynamics of these minimal DNA ligases.