BtuB-Dependent Infection of the T5-like Yersinia Phage ϕR2-01.

Yersinia enterocolitica is a food-borne Gram-negative pathogen responsible for several gastrointestinal disorders. Host-specific lytic bacteriophages have been increasingly used recently as an alternative or complementary treatment to combat bacterial infections, especially when antibiotics fail. Here, we describe the proteogenomic characterization and host receptor identification of the siphovirus vB_YenS_ϕR2-01 (in short, ϕR2-01) that infects strains of several Yersinia enterocolitica serotypes. The ϕR2-01 genome contains 154 predicted genes, 117 of which encode products that are homologous to those of Escherichia bacteriophage T5. The ϕR2-01 and T5 genomes are largely syntenic, with the major differences residing in areas encoding hypothetical ϕR2-01 proteins. Label-free mass-spectrometry-based proteomics confirmed the expression of 90 of the ϕR2-01 genes, with 88 of these being either phage particle structural or phage-particle-associated proteins. In vitro transposon-based host mutagenesis and ϕR2-01 adsorption experiments identified the outer membrane vitamin B12 receptor BtuB as the host receptor. This study provides a proteogenomic characterization of a T5-type bacteriophage and identifies specific Y. enterocolitica strains sensitive to infection with possible future applications of ϕR2-01 as a food biocontrol or phage therapy agent.

The B12 receptor BtuB alters the membrane integrity of Caulobacter crescentus.

Vitamin B12 is one of the most complex biomolecules in nature. Since few organisms can synthesize B12de novo, most bacteria utilize highly sensitive and specialized transporters to scavenge B12 and its precursors. In Gram-negative bacteria, BtuB is the outer membrane TonB-dependent receptor for B12. In the fresh water bacterium Caulobacter crescentus, btuB is among the most highly expressed genes. In this study, we characterized the function of BtuB in C. crescentus and unveiled a potential new function of this receptor involved in cellular fitness. Under standard minimal or rich growth conditions, we found that supplements of vitamin B12 to cultures of C. crescentus provided no significant advantage in growth rate. Using a B12 methionine auxotroph, we showed that BtuB in C. crescentus is capable of transporting B12 at low pico-molar range. A btuB knockout strain displayed higher sensitivity to detergents and to changes in osmotic pressure compared to the wild-type. Electron micrographs of this knockout strain revealed a morphology defect. The sensitivity observed in the btuB knockout strain was not due to changes in membrane permeability or altered S-layer levels. Our results demonstrate that btuB deletion mutants exhibit increased susceptibility to membrane stressors, suggesting a potential role of this receptor in membrane homeostasis. Because we only tested BtuB's function under laboratory conditions, we cannot eliminate the possibility that BtuB also plays a key role as a B12 scavenger in C. crescentus when growing in its highly variable and nutrient-limited natural environment.