Search

The distinct conjugation machineries encoded by plasmids pNP40 and pUC11B represent the most prevalent plasmid transfer systems among lactococcal strains. In the current study, we identified genetic determinants that underpin pNP40‐ and pUC11B‐mediated, high‐frequency mobilisation of other, non‐conjugative plasmids. The mobilisation frequencies of the smaller, non‐conjugative plasmids and the minimal sequences required for their mobilisation were determined, owing to the determination of the oriT sequences of both pNP40 and pUC11B, which allowed the identification of similar sequences in some of the non‐conjugative plasmids that were shown to promote their mobilisation. Furthermore, the auxiliary gene mobC, two distinct functional homologues of which are present in several plasmids harboured by the pNP40‐ and pUC11B‐carrying host strains, was observed to confer a high‐frequency mobilisation phenotype. These findings provide mechanistic insights into how lactococcal conjugative plasmids achieve conjugation and promote mobilisation of non‐conjugative plasmids. Ultimately, these insights would be harnessed to optimise conjugation and mobilisation strategies for the rapid and predictable development of robust and technologically improved strains. [ABSTRACT FROM AUTHOR]

Summary: Available 3D structures of bacteriophage modules combined with predictive bioinformatic algorithms enabled the identification of adhesion modules in 57 siphophages infecting Streptococcus thermophilus (St). We identified several carbohydrate‐binding modules (CBMs) in so‐called evolved distal tail (Dit) and tail‐associated lysozyme (Tal) proteins of St phage baseplates. We examined the open reading frame (ORF) downstream of the Tal‐encoding ORF and uncovered the presence of a putative p2‐like receptor‐binding protein (RBP). A 21 Å resolution electron microscopy structure of the baseplate of cos‐phage STP1 revealed the presence of six elongated electron densities, surrounding the core of the baseplate, that harbour the p2‐like RBPs at their tip. To verify the functionality of these modules, we expressed GFP‐ or mCherry‐coupled Tal and putative RBP CBMs and observed by fluorescence microscopy that both modules bind to their corresponding St host, the putative RBP CBM with higher affinity than the Tal‐associated one. The large number of CBM functional domains in St phages suggests that they play a contributory role in the infection process, a feature that we previously described in lactococcal phages and beyond, possibly representing a universal feature of the siphophage host‐recognition apparatus. [ABSTRACT FROM AUTHOR]

Streptococcus thermophilus strain ST64987 was exposed to a member of a recently discovered group of S. thermophilus phages (the 987 phage group), generating phage‐insensitive mutants, which were then characterized phenotypically and genomically. Decreased phage adsorption was observed in selected bacteriophage‐insensitive mutants, and was partnered with a sedimenting phenotype and increased cell chain length or aggregation. Whole genome sequencing of several bacteriophage‐insensitive mutants identified mutations located in a gene cluster presumed to be responsible for cell wall polysaccharide production in this strain. Analysis of cell surface‐associated glycans by methylation and NMR spectroscopy revealed a complex branched rhamno‐polysaccharide in both ST64987 and phage‐insensitive mutant BIM3. In addition, a second cell wall‐associated polysaccharide of ST64987, composed of hexasaccharide branched repeating units containing galactose and glucose, was absent in the cell wall of mutant BIM3. Genetic complementation of three phage‐resistant mutants was shown to restore the carbohydrate and phage resistance profiles of the wild‐type strain, establishing the role of this gene cluster in cell wall polysaccharide production and phage adsorption and, thus, infection. [ABSTRACT FROM AUTHOR]