SUMMARY Bacteria are central to human health and disease, but existing tools to edit microbial consortia are limited. For example, broad-spectrum antibiotics are unable to accurately manipulate bacterial communities. Bacteriophages can provide highly specific targeting of bacteria, but assembling well-defined phage cocktails solely with natural phages can be a time-, labor- and cost-intensive process. Here, we present a synthetic-biology strategy to modulate phage host ranges by engineering phage genomes in Saccharomyces cerevisiae. We used this technology to redirect Escherichia coli phage scaffolds to target pathogenic Yersinia and Klebsiella bacteria, and conversely, Klebsiella phage scaffolds to target E. coli by modular swapping of phage tail components. The synthetic phages achieved efficient killing of their new target bacteria and were used to selectively remove bacteria from multi-species bacterial communities with cocktails based on common viral scaffolds. We envision that this approach will accelerate phage-biology studies and enable new technologies for bacterial population editing.

The rapid emergence of antibiotic-resistant infections is prompting increased interest in phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the successful development of phage therapies. Through natural evolution and structural modeling, we identified host-range-determining regions (HRDRs) in the T3 phage tail fiber protein and developed a high-throughput strategy to genetically engineer these regions through site-directed mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional diversity while minimizing disruptions to the overall tail fiber structure, resulting in synthetic ''phagebodies.'' We showed that mutating HRDRs yields phagebodies with altered host-ranges, and select phagebodies enable long-term suppression of bacterial growth in vitro, by preventing resistance appearance, and are functional in vivo using a murine model. We anticipate that this approach may facilitate the creation of next-generation antimicrobials that slow resistance development and could be extended to other viral scaffolds for a broad range of applications.

RNase LS was originally identified as a potential antagonist of bacteriophage T4 infection. When T4 dmd is defective, RNase LS activity rapidly increases after T4 infection and cleaves T4 mRNAs to antagonize T4 reproduction. Here we show that rnlA, a structural gene of RNase LS, encodes a novel toxin, and that rnlB (formally yfjO), located immediately downstream of rnlA, encodes an antitoxin against RnlA. Ectopic expression of RnlA caused inhibition of cell growth and rapid degradation of mRNAs in DrnlAB cells. On the other hand, RnlB neutralized these RnlA effects. Furthermore, overexpression of RnlB in wild-type cells could completely suppress the growth defect of a T4 dmd mutant, that is, excess RnlB inhibited RNase LS activity. Pull-down analysis showed a specific interaction between RnlA and RnlB. Compared to RnlA, RnlB was extremely unstable, being degraded by ClpXP and Lon proteases, and this instability may increase RNase LS activity after T4 infection. All of these results suggested that rnlA-rnlB define a new toxin-antitoxin (TA) system. B ACTERIAL toxin-antitoxin (TA) systems are composed of a stable toxin and an unstable antitoxin (reviewed in Engelberg-Kulka and Glaser 1999). There are two different types of TA systems depending on the nature of antitoxin. In the type I systems, antitoxin is a small regulatory RNA that blocks the translation of toxin (Gerdes and Wagner 2007). In the type II systems, both toxin and antitoxin are proteins and antitoxin neutralizes toxin by direct interaction (Zhang et al. 2003a). When expression from type II TA loci is impaired by various kinds of stresses, such as amino acid starvation or translational inhibition by antibiotics (Christensen et al. 2001;Sat et al. 2001), antitoxin is rapidly decreased and consequently the level of toxin unbound (UB) with antitoxin is increased, leading to the activation of toxin (reviewed in Gerdes et al. 2005).RNase LS contributes to mRNA turnover in Escherichia coli, although its effect seems modest in comparison to that of a major RNase, RNase E (Otsuka and Yonesaki 2005). Recently we found one important role for this RNase in the physiology of E. coli cells: it targets cyaA mRNA (encoding adenylate cyclase) to reduce its expression (Iwamoto et al. 2008). Interestingly, the activity of RNase LS becomes much stronger after T4 infection ( We surveyed the E. coli DNA sequence in the vicinity of rnlA and found a promoter-like sequence, the open reading frame (ORF) of rnlA, the ORF of the downstream gene rnlB (formerly yfjO), and a terminator-like sequence consistently aligned in this order, suggesting that rnlA and rnlB form an operon. In addition, the terminal region in the rnlA ORF and the start region of the rnlB ORF overlap by 7 bp, implying an intimate coupling in their expression. These features prompted us to inquire whether rnlB is involved in RNase LS activity. In this study, we demonstrate that RnlB suppresses RNase LS activity. We also demonstrated that expression of RnlA in the absence of RnlB degrades E. coli bulk ...

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Many species of bacteria harbor multiple prophages in their genomes. Prophages often carry genes that confer a selective advantage to the bacterium, typically during host colonization. Prophages can convert to infectious viruses through a process known as induction, which is relevant to the spread of bacterial virulence genes. The paradigm of prophage induction, as set by the phage Lambda model, sees the process initiated by the RecA-stimulated self-proteolysis of the phage repressor. Here we show that a large family of lambdoid prophages found in Salmonella genomes employs an alternative induction strategy. The repressors of these phages are not cleaved upon induction; rather, they are inactivated by the binding of small antirepressor proteins. Formation of the complex causes the repressor to dissociate from DNA. The antirepressor genes lie outside the immunity region and are under direct control of the LexA repressor, thus plugging prophage induction directly into the SOS response. GfoA and GfhA, the antirepressors of Salmonella prophages Gifsy-1 and Gifsy-3, each target both of these phages' repressors, GfoR and GfhR, even though the latter proteins recognize different operator sites and the two phages are heteroimmune. In contrast, the Gifsy-2 phage repressor, GtgR, is insensitive to GfoA and GfhA, but is inactivated by an antirepressor from the unrelated Fels-1 prophage (FsoA). This response is all the more surprising as FsoA is under the control of the Fels-1 repressor, not LexA, and plays no apparent role in Fels-1 induction, which occurs via a Lambda CI-like repressor cleavage mechanism. The ability of antirepressors to recognize non-cognate repressors allows coordination of induction of multiple prophages in polylysogenic strains. Identification of non-cleavable gfoR/gtgR homologues in a large variety of bacterial genomes (including most Escherichia coli genomes in the DNA database) suggests that antirepression-mediated induction is far more common than previously recognized.

15Bacteria are central to human health and disease, but the tools available for modulating and 16 editing bacterial communities are limited. New technologies for tuning microbial populations 17 would facilitate the targeted manipulation of the human microbiome and treatment of bacterial 18 infections. For example, antibiotics are often broad spectrum in nature and cannot be used to 19 accurately manipulate bacterial communities. Bacteriophages can provide highly specific 20 targeting of bacteria, but relying solely on natural phage isolation strategies to assemble well-21 defined and uniform phage cocktails that are amenable to engineering can be a time-consuming 22 and labor-intensive process. Here, we present a synthetic-biology strategy to modulate phage 23 host ranges by manipulating phage genomes in Saccharomyces cerevisiae. We used this 24 technology to swap multiple modular phage tail components and demonstrated that Escherichia 25 coli phage scaffolds can be redirected to target pathogenic Yersinia and Klebsiella bacteria, and 26 conversely, Klebsiella phage scaffolds can be redirected to target E. coli. The synthetic phages 27 achieved multiple orders-of-magnitude killing of their new target bacteria and were used to 28 selectively remove specific bacteria from multi-species bacterial communities. We envision that 29 this approach will accelerate the study of phage biology, facilitate the tuning of phage host 30 ranges, and enable new tools for microbiome engineering and the treatment of infectious diseases. 31 32 65 their manipulation within bacterial hosts. Finally, all existing approaches are limited in the 66 number of mutations that can be introduced simultaneously. Multiple rounds of mutations are 67 therefore often required, making the process inefficient. Here, we demonstrate a high-throughput 68 phage-engineering platform that leverages the tools of synthetic biology to overcome these 69 challenges and use this platform to engineer model phages with tunable host ranges. 70 71 RESULTS 72Yeast platform for bacteriophage genome engineering. 73 We used an efficient yeast-based platform (Jaschke et al., 2012; Lu et al., 2013) to create phages 74 with novel host ranges based on common viral scaffolds. Inspired by gap-repair cloning in yeast 75 (Ma et al., 1987) and the pioneering work of Gibson and co-workers (Gibson, 2012; Gibson et al., 76 2008; Gibson et al., 2009), we captured phage genomes into Saccharomyces cerevisiae, thus 77 enabling facile genetic manipulation of modified genomes that can be subsequently re-activated 78 or "rebooted" into functional phages after transformation of genomic DNA into bacteria (Figure 79 1A). The workflow is split into two parts. In the first part, the entirety of the viral genome to be 80 assembled in yeast is amplified by PCR in such a way that each adjacent fragment has homology 81 over at least 30 bp. The first and last fragments of the phage genome are amplified with primers 82 that carry "arms" that have homology with a yeast artificial chromosome (YA...

Bacteriophage research has been instrumental to advancing many fields of biology, such as genetics, molecular biology, and synthetic biology. Many phage-derived technologies have been adapted for building gene circuits to program biological systems. Phages also exhibit significant medical potential as antibacterial agents and bacterial diagnostics due to their extreme specificity for their host, and our growing ability to engineer them further enhances this potential. Phages have also been used as scaffolds for genetically programmable biomaterials that have highly tunable properties. Furthermore, phages are central to powerful directed evolution platforms, which are being leveraged to enhance existing biological functions and even produce new ones. In this review, we discuss recent examples of how phage research is influencing these next-generation biotechnologies.

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