An Improved Method for Eliminating or Creating Intragenic Bacterial Promoters.

Recent advances in genomic refactoring have been hindered by the ever-present complication of internal or cryptic transcriptional regulation. Typical approaches to these features have been to randomize or perform mass alterations to the gene sequences thought to contain the regulatory motifs; however, this approach can cause problems by altering translational speeds, introducing long distance DNA-DNA interaction effects, and inducing RNA toxicity. Previously, we developed a rational design approach named COdon Restrained Promoter SilEncing (CORPSE) which takes externally identified promoter sequences and uses position-specific scoring matrices as proxy promoter strengths to make minimal changes to promoter sequences to disable their activity. Additionally, through inverting our system we were also able to modify weak internal promoters to increase their activity. In this chapter, we augment our previous process with the biophysical model Promoter Calculator v1.0 developed by LaFleur et al. to combine promoter identification and activity prediction, with our algorithm to silently modify promoter sequences, to provide more robust promoter elimination and creation.

IbpAB small heat shock proteins are not host factors for bacteriophage ϕX174 replication.

Bacteriophage ϕX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ϕX174 infections of Escherichia coli, the most highly upregulated host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against ϕX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on ϕX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for ϕX174 replication; moreover, their absence has no discernible effect on ϕX174 fecundity. These results suggest IbpA/B upregulation is a response to ϕX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors.

Discovery and characterisation of new phage targeting uropathogenic Escherichia coli.

Antimicrobial resistance is an escalating threat with few new therapeutic options in the pipeline. Urinary tract infections (UTIs) are one of the most prevalent bacterial infections globally and are prone to becoming recurrent and antibiotic resistant. We discovered and characterized six novel Autographiviridae and Guernseyvirinae bacterial viruses (phage) against uropathogenic Escherichia coli (UPEC), a leading cause of UTIs. The phage genomes were between 39,471 bp - 45,233 bp, with 45.0%-51.0% GC%, and 57-84 predicted coding sequences per genome. We show that tail fiber domain structure, predicted host capsule type, and host antiphage repertoire correlate with phage host range. In vitro characterisation of phage cocktails showed synergistic improvement against a mixed UPEC strain population and when sequentially dosed. Together, these phage are a new set extending available treatments for UTI from UPEC, and phage vM_EcoM_SHAK9454 represents a promising candidate for further improvement through engineering.

Creating De Novo Overlapped Genes.

Future applications of synthetic biology will rely on deploying engineered cells outside of lab environments for long periods of time. Currently, a significant roadblock to this application is the potential for deactivating mutations in engineered genes. A recently developed method to protect engineered coding sequences from mutation is called Constraining Adaptive Mutations using Engineered Overlapping Sequences (CAMEOS). In this chapter we provide a workflow for utilizing CAMEOS to create synthetic overlaps between two genes, one essential (infA) and one non-essential (aroB), to protect the non-essential gene from mutation and loss of protein function. In this workflow we detail the methods to collect large numbers of related protein sequences, produce multiple sequence alignments (MSAs), use the MSAs to generate hidden Markov models and Markov random field models, and finally generate a library of overlapping coding sequences through CAMEOS scripts. To assist practitioners with basic coding skills to try out the CAMEOS method, we have created a virtual machine containing all the required packages already installed that can be downloaded and run locally.

Codon-Restrained Method for Both Eliminating and Creating Intragenic Bacterial Promoters.

Future applications of synthetic biology will require refactored genetic sequences devoid of internal regulatory elements within coding sequences. These regulatory elements include cryptic and intragenic promoters, which may constitute up to a third of the predicted Escherichia coli promoters. The promoter activity is dependent on the structural interaction of core bases with a σ factor. Rational engineering can be used to alter key promoter element nucleotides interacting with σ factors and eliminate downstream transcriptional activity. In this paper, we present codon-restrained promoter silencing (CORPSE), a system for removing intragenic promoters. CORPSE exploits the DNA-σ factor structural relationship to disrupt σ70 promoters embedded within gene coding sequences with a minimum of synonymous codon changes. Additionally, we present an inverted CORPSE system, iCORPSE, which can create highly active promoters within a gene sequence while not perturbing the function of the modified gene.

Proteomic and Transcriptomic Analysis of Microviridae φX174 Infection Reveals Broad Upregulation of Host Escherichia coli Membrane Damage and Heat Shock Responses.

Measuring host-bacteriophage dynamics is an important approach to understanding bacterial survival functions and responses to infection. The model Microviridae bacteriophage φX174 is endemic to the human gut and has been studied for over 70 years, but the host response to infection has never been investigated in detail. To address this gap in our understanding of this important interaction within our microbiome, we have measured host Escherichia coli C proteomic and transcriptomic response to φX174 infection. We used mass spectrometry and RNA sequencing (RNA-seq) to identify and quantify all 11 φX174 proteins and over 1,700 E. coli proteins, enabling us to comprehensively map host pathways involved in φX174 infection. Most notably, we see significant host responses centered on membrane damage and remodeling, cellular chaperone and translocon activity, and lipoprotein processing, which we speculate is due to the peptidoglycan-disruptive effects of the φX174 lysis protein E on MraY activity. We also observe the massive upregulation of small heat shock proteins IbpA/B, along with other heat shock pathway chaperones, and speculate on how the specific characteristics of holdase protein activity may be beneficial for viral infections. Together, this study enables us to begin to understand the proteomic and transcriptomic host responses of E. coli to Microviridae infections and contributes insights to the activities of this important model host-phage interaction.IMPORTANCE A major part of the healthy human gut microbiome is the Microviridae bacteriophage, exemplified by the model φX174 phage, and their E. coli hosts. Although much has been learned from studying φX174 over the last half-century, until this work, the E. coli host response to infection has never been investigated in detail. We reveal the proteomic and transcriptomic pathways differentially regulated during the φX174 infection cycle and uncover the details of a coordinated cellular response to membrane damage that results in increased lipoprotein processing and membrane trafficking, likely due to the phage antibiotic-like lysis protein. We also reveal that small heat shock proteins IbpA/B are massively upregulated during infection and that these holdase chaperones are highly conserved across the domains of life, indicating that reliance on them is likely widespread across viruses.

A high-resolution map of bacteriophage ϕX174 transcription.

Bacteriophage ϕX174 is a model virus for studies across the fields of structural biology, genetics, gut microbiomics, and synthetic biology, but did not have a high-resolution transcriptome until this work. In this study we used next-generation sequencing to measure the RNA produced from ϕX174 while infecting its host E. coli C. We broadly confirm the past transcriptome model while revealing several interesting deviations from previous knowledge. Additionally, we measure the strength of canonical ϕX174 promoters and terminators and discover both a putative new promoter that may be activated by heat shock sigma factors, as well as rediscover a controversial Rho-dependent terminator. We also provide evidence for the first antisense transcription observed in the Microviridae, identify two promoters that may be involved in generating this transcriptional activity, and discuss possible reasons why this RNA may be produced.