# An N4-like Caulobacter phage requires host smooth lipopolysaccharide biosynthesis for infection

## TL;DR

This study identifies a new phage, Circe, that infects Caulobacter crescentus and depends on the host's smooth lipopolysaccharide for infection.

## Contribution

The study reveals that smooth lipopolysaccharide functions as a receptor for the N4-like phage Circe in Caulobacter.

## Key findings

- Circe variants CirceC and CirceH differ by a single amino acid substitution in Gp063.
- Loss-of-function mutations in S-LPS biosynthesis genes confer strong resistance to Circe infection.
- An S-LPS-defective mutant shows a strong defect in Circe adsorption.

## Abstract

Caulobacter species are Alphaproteobacteria that commonly inhabit plant-associated and aquatic microbial communities. Although Caulobacter is widespread and has long served as a model for the study of bacterial cell biology, our understanding of viruses that infect Caulobacter species remains limited. Here, we describe the discovery and characterization of Circe, a freshwater N4-like podophage belonging to the Schitoviridae family that infects Caulobacter crescentus. We isolated two variants, CirceC and CirceH, that differ by a single nucleotide, resulting in an F91I substitution in Gp063, an uncharacterized protein found in diverse bacteriophages and bacteria. While both Circe variants adsorb to C. crescentus with similar efficiency, they produce morphologically distinct plaques and display different infection dynamics. Through forward genetic selection and genome-wide transposon fitness profiling, we identified C. crescentus genes involved in cell envelope assembly, membrane sphingolipid biosynthesis, and envelope polysaccharide biosynthesis that influence susceptibility to Circe infection. Loss-of-function mutations in a predicted nucleoside diphosphate sugar epimerase and multiple genes required for smooth lipopolysaccharide (S-LPS) biosynthesis and export conferred strong resistance to infection. Furthermore, an in-frame deletion mutant defective in S-LPS production exhibited a strong defect in Circe adsorption. These results support a model in which S-LPS functions as a receptor for phage Circe. Our study expands the known repertoire of Caulobacter phages and adds to a growing understanding of the role of envelope polysaccharides in bacterial infection by N4-family phages.

Viruses that infect bacteria and archaea, known as phages, shape microbial communities through their effects on host survival and gene flow. Yet for many microbes, the phages that infect them and the host structures required for infection remain poorly characterized. Caulobacter species are ecologically important Alphaproteobacteria that can produce protein surface structures, such as pili, a flagellum, and an S-layer, which are established phage receptors in this genus. We discovered Caulobacter phage Circe and provided evidence that it relies on smooth lipopolysaccharide and other envelope carbohydrates to infect Caulobacter crescentus. This study broadens the understanding of phage–host interactions in Caulobacter and establishes Circe as a useful system for dissecting how phages engage with bacterial cells.