Enterobacteriaceae are gram negative rods
Enterobacteriaceae are gram negative rods that include the Escherichia coli and Shigella. There are several DNA ligases posted on the GenBank belong mostly to Enterobacteria phages, such as the T4 bacteriophage . There are several protein markers used to differentiate between T4 phage superfamily, including the capsid proteins gp20 and gp23 . These markers however are not specific enough in differentiating between T4 and T-like 2-Amino-ATP synthesis . DNA ligases are highly conserved among these strains with identity score over 98%, but still can show variations that can be used as tools in distant bacteriophage classification. In this study, a DNA ligase was cloned from a bacteriophage that was able to host on a commercially designed strain of E. coli and its sequence was compared with related DNA ligases. The sequence supports the idea that ligases can be used as markers to classify bacteriophages.
Materials and methods
Results and disussion
Acknowledgments The authors would like to thank Dr. Yaqoub Ashhab for advice and Dr. Rami Arafeh for carefully reading the manuscript, Mrs. Amal Abu Rayan for carrying out the sequencing, and Mrs. Asma Tamimi for technical support. The project was support by an IIRG Grant from Palestine Polytechnic University.
Introduction DNA ligases are enzymes which catalyze the formation of a phosphodiester bond between adjacent 5′ PO4 and 3′ OH ends in double-stranded DNA, and are essential for sealing breaks during DNA replication and repair . DNA ligases can be divided into two types based on the nucleotide cofactor they use as an AMP donor: ATP-dependent DNA ligases (EC 126.96.36.199) which are found in eukaryotes and archaea, and NAD-dependent DNA ligases (EC 188.8.131.52) which are found exclusively in bacteria . In addition to their house-keeping NAD-dependent enzymes, many bacteria have one or more ATP-dependent DNA ligases, the evolutionary origin and cellular function of which have not been entirely determined . A number of these accessory enzymes have been biochemically characterized and some are postulated to play a role in DNA repair , , ,  while others are suggested to be involved in competence and DNA uptake , . The genome of the pathogenic psychrophile Aliivibrio salmonicida encodes one such putative ATP-dependent DNA ligase . In order to study its structure and activity in vitro we have undertaken the recombinant production and purification of this DNA ligase from an Escherichia coli based expression system. Expression and purification of this protein presented two significant challenges. First, the gene product was moderately toxic to E. coli cells, second, crude lysates contained a large amount of bacterial DNA which needed to be removed prior to purification without contaminating the ligase protein preparation. Numerous publications have focused on the utility of large fusion partners in increasing protein solubility and expression levels (for example see , ), and comprehensive protocols for the production of MBP fusion constructs are available . However the application of large fusion partners to overcome toxic effects of intracellularly-expressed proteins on the host cells has not been systematically reported to the same extent. In the case of the two Vib-Lig variants described here, the decreased host-cell growth rate with smaller tags presented a significant loss of efficiency during protein production, even before solubility issues were taken into consideration. As the A. salmonicida gene encodes a predicted leader peptide, both the full-length and mature forms of the protein were produced, and ATP-dependent DNA ligation activity was verified for both constructs. This work represents the first instance of successful production, purification and preliminary characterization of active A. salmonicida ATP-dependent DNA ligase.
Results and discussion