Mona Alqarni1, Silvia Spinelli2, Jennifer Mahony1, Christian Cambillau2 and Douwe van Sinderen1

Lactococcus lactis is the most widely employed starter bacterial species in dairy fermentations globally. Such starter bacteria are consistently threatened by (bacterio)phage predation and are, therefore, the subject of intensive and focused research efforts.. The P335 phage TP901-1 is classified as a Siphoviridae phage with the characteristic isometric head and long non-contractile tail. The overall objective of this study is to define the domains responsible for the varied role(s) of the tail tape measure protein (TMP) in the tail morphogenesis process using the prototype lactococcal bacteriophage TP901-1. It is known that the TMP forms the tail tube and is required for tail assembly as well as dictating the length of the tail. The TMP of TP901-1 is predicted to encode 29 repeats that are believed to be involved in tail length determination. The effect of deleting specific amino acids within one of the 29 repeats (comprised of 11 or 18 aa) of the TMP employing the ssDNA recombineering approach. 12 individual deletions in the TMP targeting certain amino acids were generated in order to assess the effect of the deletions on (1) the phage's infectivity on its host strain L. lactis subsp. cremoris 3107 and (2) the phage morphology. The deletion of certain amino acids was shown to reduce the level of infectivity of the phage as defined by plaque formation and lysogenic conversion assays; while electron microscopy revealed a slightly shorter tail compared to the wild type (wt) in the absence of repeat elements, thereby confirming the role of the TMP repeats in tail length determination. However, the mechanism by which the infectivity is reduced in some of the phage mutants with deletions in the TMP is as yet unknown.