Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

wrwyrggrywrw is a single-chain functional analog of the Holliday junction-binding homodimer, (wrwycr)2.

DNA repair pathways in bacteria that use homologous recombination involve the formation and subsequent resolution of Holliday junction (HJ) intermediates. We have previously identified several hexameric peptides that bind to HJs and interfere with HJ processing enzymes in vitro. The peptide WRWYCR and its D-amino acid stereoisomer wrwycr, are potent antibacterial agents. These hexapeptides must form homodimers in order to interact stably with HJs, and inhibit bacterial growth, and this represents a potential limitation. Herein we describe a disulfide bond-independent inhibitor, WRWYRGGRYWRW and its D-stereoisomer wrwyrggrywrw. We have characterized these single-chain, linear analogs of the hexapeptides, and show that in addition to effectively binding to HJs, and inhibiting the activity of DNA repair enzymes that process HJs, they have equal or greater potency against Gram-positive and Gram-negative bacterial growth. The analogs were also shown to cause DNA damage in bacteria, and disrupt the integrity of the bacterial cytoplasmic membrane. Finally, we found that they have little toxicity toward several eukaryotic cell types at concentrations needed to inhibit bacterial growth.

Similarities between exogenously- and endogenously-induced envelope stress: the effects of a new antibacterial molecule, TPI1609-10.

Antibiotics with novel and/or multiple targets are highly desirable in the face of the steady rise of clinical antibiotic resistance. We have screened and identified small molecules, typified by the compound TPI1609-10 (aka SM10), with antibiotic activity against both gram-positive and gram-negative bacteria. SM10 was screened in vitro to bind branched Holliday junction intermediates of homologous recombination and tyrosine recombinase-mediated recombination; thus, the cellular targets of the small molecules were expected to include the RuvABC Holliday junction resolvasome and the XerCD complex involved in proper segregation of replicated chromosomes to daughter cells. SM10 indeed induces DNA damage and filamentation in E. coli. However, SM10 also induces envelope stress and causes increased production of intracellular reactive oxygen species. In addition, SM10 has similar effects to endogenously-induced envelope stress via overproducing outer membrane proteins (OmpC and OmpF), which also induces the SOS response, chromosome fragmentation, and production of reactive oxygen species. The synergy between SM10, and cerulenin, a fatty acid synthesis inhibitor, together with the SM10 hypersensitivity of cpx and rpoE mutants, further support that SM10's mode of action damages membrane damage. The lethality of SM10 treatment and of OmpC overproduction are observed in both aerobically- and anaerobically-grown cells, and is accompanied by substantial DNA damage even anaerobically. Thus, only some DNA damage is due to reactive oxygen. We propose that membrane depolarization and the potential reduction in intracellular pH, leading to abasic site formation, cause a substantial amount of the DNA damage associated with both SM10 treatment and endogenous envelope stress. While it is difficult to completely exclude effects related to envelope damage as the sources of DNA damage, trapping intermediates associated with DNA repair and chromosome segregation pathways remains very likely. Thus SM10 may have distinct but synergistic modes of action.

Escherichia coli enterobactin synthesis and uptake mutants are hypersensitive to an antimicrobial peptide that limits the availability of iron in addition to blocking Holliday junction resolution.

The peptide wrwycr inhibits Holliday junction resolution and is a potent antimicrobial. To study the physiological effects of wrwycr treatment on Escherichia coli cells, we partially screened the Keio collection of knockout mutants for those with increased sensitivity to wrwycr. Strains lacking part of the ferric-enterobactin (iron-bound siderophore) uptake and utilization system, parts of the enterobactin synthesis pathway, TolC (an outer-membrane channel protein) or Fur (an iron-responsive regulator) were hypersensitive to wrwycr. We provide evidence that the ΔtolC mutant was hypersensitive to wrwycr due to its reduced ability to efflux wrwycr from the cell rather than due to its export of newly synthesized enterobactin. Deleting ryhB, which encodes a small RNA involved in iron regulation, mostly relieved the wrwycr hypersensitivity of the fur and ferric-enterobactin uptake mutants, indicating that the altered regulation of a RyhB-controlled gene was at least partly responsible for the hypersensitivity of these strains. Chelatable iron in the cell, measured by electron paramagnetic resonance spectroscopy, increased dramatically following wrwycr treatment, as did expression of Fur-repressed genes and, to some extent, mutation frequency. These incongruous results suggest that while wrwycr treatment caused accumulation of chelatable iron in the cell, iron was not available to bind to Fur. This is corroborated by the observed induction of the suf system, which assembles iron-sulfur clusters in low-iron conditions. Disruption of iron metabolism by wrwycr, in addition to its effects on DNA repair, may make it a particularly effective antimicrobial in the context of the low-iron environment of a mammalian host.