Making Security Viral: Shifting Engineering Biology Culture and Publishing.

The ability to construct, synthesize, and edit genes and genomes at scale and with speed enables, in synergy with other tools of engineering biology, breakthrough applications with far-reaching implications for society. As SARS-CoV-2 spread around the world in early spring of 2020, researchers rapidly mobilized, using these tools in the development of diagnostics, therapeutics, and vaccines for COVID-19. The sharing of knowledge was crucial to making rapid progress. Several publications described the use of reverse genetics for the de novo construction of SARS-CoV-2 in the laboratory, one in the form of a protocol. Given the demonstrable harm caused by the virus, the unequal distribution of mitigating vaccines and therapeutics, their unknown efficacy against variants, and the interest in this research by laboratories unaccustomed to working with highly transmissible pandemic pathogens, there are risks associated with such publications, particularly as protocols. We describe considerations and offer suggestions for enhancing security in the publication of synthetic biology research and techniques. We recommend: (1) that protocol manuscripts for the de novo synthesis of certain pathogenic viruses undergo a mandatory safety and security review; (2) that if published, such papers include descriptions of the discussions or review processes that occurred regarding security considerations in the main text; and (3) the development of a governance framework for the inclusion of basic security screening during the publication process of engineering biology/synthetic biology manuscripts to build and support a safe and secure research enterprise that is able to maximize its positive impacts and minimize any negative outcomes.

Precision medicine: Beyond the inflection point.

A confluence of biological, physical, engineering, computer, and health sciences is setting the stage for a transformative leap toward data-driven, mechanism-based health and health care for each individual.

The promoter spacer influences transcription initiation via sigma70 region 1.1 of Escherichia coli RNA polymerase.

Transcription initiation is a dynamic process in which RNA polymerase (RNAP) and promoter DNA act as partners, changing in response to one another, to produce a polymerase/promoter open complex (RPo) competent for transcription. In Escherichia coli RNAP, region 1.1, the N-terminal 100 residues of sigma(70), is thought to occupy the channel that will hold the DNA downstream of the transcription start site; thus, region 1.1 must move from this channel as RPo is formed. Previous work has also shown that region 1.1 can modulate RPo formation depending on the promoter. For some promoters region 1.1 stimulates the formation of open complexes; at the P(minor) promoter, region 1.1 inhibits this formation. We demonstrate here that the AT-rich P(minor) spacer sequence, rather than promoter recognition elements or downstream DNA, determines the effect of region 1.1 on promoter activity. Using a P(minor) derivative that contains good sigma(70)-dependent DNA elements, we find that the presence of a more GC-rich spacer or a spacer with the complement of the P(minor) sequence results in a promoter that is no longer inhibited by region 1.1. Furthermore, the presence of the P(minor) spacer, the GC-rich spacer, or the complement spacer results in different mobilities of promoter DNA during gel electrophoresis, suggesting that the spacer regions impart differing conformations or curvatures to the DNA. We speculate that the spacer can influence the trajectory or flexibility of DNA as it enters the RNAP channel and that region 1.1 acts as a "gatekeeper" to monitor channel entry.

Identification of an AU-rich translational enhancer within the Escherichia coli fepB leader RNA.

The fepB gene encodes a periplasmic binding protein that is essential for the uptake of ferric enterobactin by Escherichia coli. Its transcription is regulated in response to iron levels by the Fur repressor. The fepB transcript includes a 217-nucleotide leader sequence with several features suggestive of posttranscriptional regulation. To investigate the fepB leader for its contribution to fepB expression, defined deletions and substitution mutations in the leader were characterized using fepB-phoA translational fusions. The fepB leader was found to be necessary for maximal fepB expression, primarily due to the influence of an AU-rich translational enhancer (TE) located 5' to the Shine-Dalgarno sequence. Deletions or substitutions within the TE sequence decreased fepB-phoA expression fivefold. RNase protection and in vitro transcription-translation assays demonstrated that the TE augmented translational efficiency, as well as RNA levels. Moreover, primer extension inhibition assays showed that the TE increases ribosome binding. In contrast to the enhancing effect of the TE, the natural fepB GUG start codon decreased ribosome binding and reduced fepB expression 2.5-fold compared with the results obtained with leaders bearing an AUG initiation codon. Thus, the TE-GUG organization in fepB results in an intermediate level of expression compared to the level with AUG, with or without the TE. Furthermore, we found that the TE-GUG sequence is conserved among the eight gram-negative strains examined that have fepB genes, suggesting that this organization may provide a selective advantage.

Transcription initiation by mix and match elements: flexibility for polymerase binding to bacterial promoters.

Bacterial RNA polymerase is composed of a core of subunits (beta, beta', alpha1, alpha2, omega), which have RNA synthesizing activity, and a specificity factor (sigma), which identifies the start of transcription by recognizing and binding to sequences elements within promoter DNA. Four core promoter consensus sequences, the -10 element, the extended -10 (TGn) element, the -35 element, and the UP elements, have been known for many years; the importance of a nontemplate G at position -5 has been recognized more recently. However, the functions of these elements are not the same. The AT-rich UP elements, the -35 elements ((-35)TTGACA(-30)), and the extended -10 ((-15)TGn(-13)) are recognized as double stranded binding elements, whereas the -5 nontemplate G is recognized in the context of single-stranded DNA at the transcription bubble. Furthermore, the -10 element ((-12)TATAAT(-7)) is recognized as both double strand DNA for the T:A bp at position -12 and as nontemplate, single-strand DNA from positions -11 to -7. The single-strand sequences at positions -11 to -7 as well as the -5 contribute to later steps in transcription initiation that involve isomerization of polymerase and separation of the promoter DNA around the transcription start site. Recent work has demonstrated that the double strand elements may be used in various combinations to yield an effective promoter. Thus, while some minimal number of contacts is required for promoter function, polymerase allows the elements to be mixed and matched. Interestingly, which particular elements are used does not appear to fundamentally alter the transcription bubble generated in the stable complex. In this review, we discuss the multiple steps involved in forming a transcriptionally competent polymerase/promoter complex, and we examine what is known about polymerase recognition of core promoter elements. We suggest that considering promoter elements according to their involvement in early (polymerase binding) or later (polymerase isomerization) steps in transcription initiation rather than simply from their match to conventional promoter consensus sequences is a more instructive form of promoter classification.

Escherichia coli RNA polymerase recognition of a sigma70-dependent promoter requiring a -35 DNA element and an extended -10 TGn motif.

Escherichia coli sigma70-dependent promoters have typically been characterized as either -10/-35 promoters, which have good matches to both the canonical -10 and the -35 sequences or as extended -10 promoters (TGn/-10 promoters), which have the TGn motif and an excellent match to the -10 consensus sequence. We report here an investigation of a promoter, P(minor), that has a nearly perfect match to the -35 sequence and has the TGn motif. However, P(minor) contains an extremely poor sigma70 -10 element. We demonstrate that P(minor) is active both in vivo and in vitro and that mutations in either the -35 or the TGn motif eliminate its activity. Mutation of the TGn motif can be compensated for by mutations that make the -10 element more canonical, thus converting the -35/TGn promoter to a -35/-10 promoter. Potassium permanganate footprinting on the nontemplate and template strands indicates that when polymerase is in a stable (open) complex with P(minor), the DNA is single stranded from positions -11 to +4. We also demonstrate that transcription from P(minor) incorporates nontemplated ribonucleoside triphosphates at the 5' end of the P(minor) transcript, which results in an anomalous assignment for the start site when primer extension analysis is used. P(minor) represents one of the few -35/TGn promoters that have been characterized and serves as a model for investigating functional differences between these promoters and the better-characterized -10/-35 and extended -10 promoters used by E. coli RNA polymerase.

Transcriptional takeover by sigma appropriation: remodelling of the sigma70 subunit of Escherichia coli RNA polymerase by the bacteriophage T4 activator MotA and co-activator AsiA.

Activation of bacteriophage T4 middle promoters, which occurs about 1 min after infection, uses two phage-encoded factors that change the promoter specificity of the host RNA polymerase. These phage factors, the MotA activator and the AsiA co-activator, interact with the sigma(70) specificity subunit of Escherichia coli RNA polymerase, which normally contacts the -10 and -35 regions of host promoter DNA. Like host promoters, T4 middle promoters have a good match to the canonical sigma(70) DNA element located in the -10 region. However, instead of the sigma(70) DNA recognition element in the promoter's -35 region, they have a 9 bp sequence (a MotA box) centred at -30, which is bound by MotA. Recent work has begun to provide information about the MotA/AsiA system at a detailed molecular level. Accumulated evidence suggests that the presence of MotA and AsiA reconfigures protein-DNA contacts in the upstream promoter sequences, without significantly affecting the contacts of sigma(70) with the -10 region. This type of activation, which is called 'sigma appropriation', is fundamentally different from other well-characterized models of prokaryotic activation in which an activator frequently serves to force sigma(70) to contact a less than ideal -35 DNA element. This review summarizes the interactions of AsiA and MotA with sigma(70), and discusses how these interactions accomplish the switch to T4 middle promoters by inhibiting the typical contacts of the C-terminal region of sigma(70), region 4, with the host -35 DNA element and with other subunits of polymerase.

Export of the siderophore enterobactin in Escherichia coli: involvement of a 43 kDa membrane exporter.

The enterobactin system for iron transport in Escherichia coli is well characterized with the exception of the mechanism of enterobactin secretion to the extracellular environment. Escherichia coli membrane protein P43, encoded by ybdA in the chromosomal region of genes involved in enterobactin synthesis, shows strong homology to the 12-transmembrane segment major facilitator superfamily of export pumps. A P43-null mutation was created and siderophore nutrition assays, performed with a panel of E. coli strains expressing one or more outer membrane receptors for enterobactin-related compounds, demonstrated that the P43 mutant was unable to secrete enterobactin efficiently. Products released from the mutant strain were identified with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), revealing that the P43 mutant secretes little, if any, enterobactin, but elevated levels of enterobactin breakdown products 2,3- dihydroxybenzoylserine (DHBS) monomer, dimer, and trimer. These data establish that P43 is a critical component of the E. coli enterobactin secretion machinery and provides a rationale for the designation of the previous genetic locus ybdA as entS to reflect its relevant biological function.