Genome-wide screens reveal Escherichia coli genes required for growth of T1-like phage LL5 and V5-like phage LL12

Abstract

The host factor requirements of phages and mechanisms of mutational phage insensitivity must be characterized for rational design of phage cocktails. To characterize host dependencies of two novel Escherichia coli phages, the T1-like siphophage LL5 and the V5-like myophage LL12, forward genetic screens were conducted against the Keio collection, a library of single non-essential gene deletions in E. coli str. BW25113. These screens and subsequent experiments identified genes required by phages LL5 and LL12. E. coli mutants deficient in heptose II and the phosphoryl substituent of heptose I of the inner core lipopolysaccharide (LPS) were unable to propagate phage LL5, as were mutants deficient in the outer membrane protein TolC. Mutants lacking glucose I of the LPS outer core failed to propagate LL12. Two additional genes encoding cytoplasmic chaperones, PpiB and SecB, were found to be required for efficient propagation of phage LL5, but not LL12. This screening approach may be useful for identifying host factors dependencies of phages, which would provide valuable information for their potential use as therapeutics and for phage engineering. Escherichia coli is a Gram-negative facultative anaerobic bacterium which is commonly found as a member of the commensal gut flora in mammals 1. While commensal E. coli strains generally do not cause disease in humans, multiple strains of E. coli have acquired virulence factors and may cause disease with symptoms ranging from mild discomfort to life-threatening bacteremia. These strains have been categorized into several pathotypes, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) 2. Enterotoxigenic E. coli strains can be distinguished from other E. coli pathotypes by the presence of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (HT) 2. These enterotoxins induce traveler's diarrhea (TD), characterized by mild to severe watery diarrhea 2 , which may be accompanied by nausea, vomiting, abdominal pain, fever or blood in stool 3. TD is one of the most common illnesses contracted by people from developed countries during international travel, with ETEC being the major causative agent of TD in Latin America, Africa, South Asia and the Middle East 4. TD typically self-resolves or may be successfully treated with antibiotics, but the global increase in the emergence of antibiotic resistance warrants evaluation of alternative treatment approaches 4. Bacteriophages (phages) are the natural viral predators of bacteria. Due to the ongoing emergence of multidrug-resistant bacteria and limited development of new antibiotics, there has been a renewed interest in the use of phages as antimicrobials 5-9. However, knowledge of the basic biology of phages outside of a number of well-studied model organisms is limited. Phages are highly diverse, and they are constantly co-evolving with their bacterial hosts 10,11. In the ...

High-throughput mapping of the phage resistance landscape in E. coli

“…There have been few attempts to use genetic approaches for studying genome-wide host factors essential in phage infection. These loss-of-function (LOF) genetic screens broadly use bacterial saturation mutagenesis [ 49 , 54 , 58 – 61 ] or an arrayed library of single-gene deletion strains for studying phage-host interactions [ 50 , 51 , 53 , 62 , 63 ]. Consequently, these studies have generally involved laborious experiments on relatively few phages and their hosts, and scaling the approach to characterize hundreds of phages is challenging.…”

Systematic exploration ofEscherichia coliphage-host interactions with the BASEL phage collection

“…Just like the much larger Markadamsvirinae siphoviruses (see below), Drexlerviridae phages use a small set of outer membrane porins as their secondary / final receptors for irreversible adsorption and DNA injection. To the best of our knowledge, previous work had only identified the receptors of T1 and IME18 (FhuA), IME347 (YncD), IME253 (FepA), and of LL5 as well as TLS (TolC) while for an additional phage, RTP, no protein receptor could be identified [40,[52][53][54]. Using available single-gene mutants, we readily determined the terminal receptor of eleven out of our thirteen Drexlerviridae phages as FhuA, BtuB, YncD, and TolC (Figs 4B and 4C) but failed for two others, AugustePiccard (Bas01) and JeanPiccard (Bas02).…”

“…Three different, co-expressed lateral tail fibers have been directly visualized in the cryogenic 371 electron microscopy structure of phi92, and large orthologous loci are found in all Vequintavirinae and 372 relatives [23,70,71]. The considerable repertoire of glycan-hydrolyzing protein domains in these tail fiber proteins has been compared to a "nanosized Swiss army knife" and is probably responsible for the exceptionally broad host range of these phages and their ability to infect even diverse capsulated strains of enterobacteria [23,52,70,71]. However, it is far from clear which genes code for which components of the different lateral tail fibers.…”

“…The terminal receptor of Vequintavirinae has been unraveled genetically for phage LL12, a close relative of rV5 ( Fig 9B), and seems to be at the first, heptose-linked glucose of the LPS outer core which is shared by all E. coli core LPS types [52,72] (Fig 9A). Consistently, we found that all Vequintavirinae in the BASEL collection are completely unable to infect the waaC and waaG mutants with core LPS defects that result in the absence of this sugar ( Fig 9C, see also Fig 3A).…”

Complete Genome Sequence of Escherichia coli Bacteriophage U136B

“…A whole-genome BLASTn search (18) indicated that phage U136B is in the Tlsvirus phage group, which includes E. coli bacteriophage TLS (GenBank accession number AY308796.1) (93% identity) and E. coli bacteriophage LL5 (NCBI reference sequence NC_047985.1) (94% identity), both of which are also reliant on the E. coli TolC efflux protein (23,24) and have siphophage morphology with flexible tails (23,25). Host range analysis of phage U136B indicates a limited host range of some, but not all, E. coli hosts (2).…”