A simple, robust, broadly applicable insertion mutagenesis method to create random fluorescent protein: target protein fusions.

A simple, broadly applicable method was developed using an in vitro transposition reaction followed by transformation into Escherichia coli and screening plates for fluorescent colonies. The transposition reaction catalyzes the random insertion of a fluorescent protein open reading frame into a target gene on a plasmid. The transposition reaction is employed directly in an E. coli transformation with no further procedures. Plating at high colony density yields fluorescent colonies. Plasmids purified from fluorescent colonies contain random, in-frame fusion proteins into the target gene. The plate screen also results in expressed, stable proteins. A large library of chimeric proteins was produced, which was useful for downstream research. The effect of using different fluorescent proteins was investigated as well as the dependence of the linker sequence between the target and fluorescent protein open reading frames. The utility and simplicity of the method were demonstrated by the fact that it has been employed in an undergraduate biology laboratory class without failure over dozens of class sections. This suggests that the method will be useful in high-impact research at small liberal arts colleges with limited resources. However, in-frame fusion proteins were obtained from 8 different targets suggesting that the method is broadly applicable in any research setting.

Systematic overexpression of genes encoded by mycobacteriophage Waterfoul reveals novel inhibitors of mycobacterial growth.

Bacteriophages represent an enormous reservoir of novel genes, many of which are unrelated to existing entries in public databases and cannot be assigned a predicted function. Characterization of these genes can provide important insights into the intricacies of phage-host interactions and may offer new strategies to manipulate bacterial growth and behavior. Overexpression is a useful tool in the study of gene-mediated effects, and we describe here the construction of a plasmid-based overexpression library of a complete set of genes for Waterfoul, a mycobacteriophage closely related to those infecting clinically important strains of Mycobacterium tuberculosis and/or Mycobacterium abscessus. The arrayed Waterfoul gene library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 32 Waterfoul gene products capable of inhibiting the growth of the host Mycobacterium smegmatis and providing a first look at the frequency and distribution of cytotoxic products encoded within a single mycobacteriophage genome. Several of these Waterfoul gene products were observed to confer potent anti-mycobacterial effects, making them interesting candidates for follow-up mechanistic studies.

A Bacterial Cell-Based Assay To Study SARS-CoV-2 Protein-Protein Interactions.

Methods for detecting and dissecting the interactions of virally encoded proteins are essential for probing basic viral biology and providing a foundation for therapeutic advances. The dearth of targeted therapeutics for the treatment of coronavirus disease 2019 (COVID-19), an ongoing global health crisis, underscores the importance of gaining a deeper understanding of the interactions of proteins encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we describe the use of a convenient bacterial cell-based two-hybrid (B2H) system to analyze the SARS-CoV-2 proteome. We identified 16 distinct intraviral protein-protein interactions (PPIs), involving 16 proteins. We found that many of the identified proteins interact with more than one partner. Further, our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified B2H system that permits the detection of disulfide bond-dependent PPIs in the normally reducing Escherichia coli cytoplasm, and we used this system to detect the interaction of the SARS-CoV-2 spike protein receptor-binding domain (RBD) with its cognate cell surface receptor ACE2. We then examined how the RBD-ACE2 interaction is perturbed by several RBD amino acid substitutions found in currently circulating SARS-CoV-2 variants. Our findings illustrate the utility of a genetically tractable bacterial system for probing the interactions of viral proteins and investigating the effects of emerging mutations. In principle, the system could also facilitate the identification of potential therapeutics that disrupt specific interactions of virally encoded proteins. More generally, our findings establish the feasibility of using a B2H system to detect and dissect disulfide bond-dependent interactions of eukaryotic proteins. IMPORTANCE Understanding how virally encoded proteins interact with one another is essential in elucidating basic viral biology, providing a foundation for therapeutic discovery. Here, we describe the use of a versatile bacterial cell-based system to investigate the interactions of the protein set encoded by SARS-CoV-2, the virus responsible for the current COVID-19 pandemic. We identified 16 distinct intraviral protein-protein interactions, involving 16 proteins, many of which interact with more than one partner. Our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified version of our bacterial cell-based system that permits detection of the interaction between the SARS-CoV-2 spike protein (specifically, its receptor-binding domain) and its cognate human cell surface receptor ACE2, and we investigated the effects of spike mutations found in currently circulating SARS-CoV-2 variants. Our findings illustrate the general utility of our system for probing the interactions of virally encoded proteins.

Control of a programmed cell death pathway in Pseudomonas aeruginosa by an antiterminator.

In Pseudomonas aeruginosa the alp system encodes a programmed cell death pathway that is switched on in a subset of cells in response to DNA damage and is linked to the virulence of the organism. Here we show that the central regulator of this pathway, AlpA, exerts its effects by acting as an antiterminator rather than a transcription activator. In particular, we present evidence that AlpA positively regulates the alpBCDE cell lysis genes, as well as genes in a second newly identified target locus, by recognizing specific DNA sites within the promoter, then binding RNA polymerase directly and allowing it to bypass intrinsic terminators positioned downstream. AlpA thus functions in a mechanistically unusual manner to control the expression of virulence genes in this opportunistic pathogen.

Diatoms: A novel cause of granulomatous inflammation of the head and neck.

OBJECTIVE: We report the first 4 cases of intraoral nonnecrotizing granulomatous foreign body reactions to diatoms, plausibly as a result of exogenous material introduced following iatrogenic or traumatic injury. STUDY DESIGN: Clinical and histopathologic findings of 4 intraoral cases of nonnecrotizing granulomatous foreign body reaction to diatoms, single-celled algae belonging to the taxonomic phylum Bacillariophyta, are reported. RESULTS: The lesions presented either in the jaws or in the soft tissue overlying the alveolar bone, in some instances mimicking an inflammatory lesion of odontogenic etiology. Microscopically, the lesions presented as nonnecrotizing granulomatous inflammation associated with either spherical and radially symmetric or rectangular and bilaterally symmetric diatomaceous foreign material. CONCLUSION: The diagnosis of a diatom-associated foreign body reaction necessitates familiarization with the histopathologic features of these organisms to accurately characterize the nature of such lesions.

Tombusvirus p19 Captures RNase III-Cleaved Double-Stranded RNAs Formed by Overlapping Sense and Antisense Transcripts in Escherichia coli.

Antisense transcription is widespread in bacteria. By base pairing with overlapping sense RNAs, antisense RNAs (asRNA) can form double-stranded RNAs (dsRNA), which are cleaved by RNase III, a dsRNA endoribonuclease. The ectopic expression of plant Tombusvirus p19 in Escherichia coli stabilizes ∼21-nucleotide (nt) dsRNA RNase III decay intermediates, which enabled us to characterize otherwise highly unstable asRNA by deep sequencing of p19-captured dsRNA. RNase III-produced small dsRNA were formed at most bacterial genes in the bacterial genome and in a plasmid. We classified the types of asRNA in genomic clusters producing the most abundant p19-captured dsRNA and confirmed RNase III regulation of asRNA and sense RNA decay at three type I toxin-antitoxin loci and at a coding gene, rsd Furthermore, we provide potential evidence for the RNase III-dependent regulation of CspD protein by asRNA. The analysis of p19-captured dsRNA revealed an RNase III sequence preference for AU-rich sequences 3 nucleotides on either side of the cleavage sites and for GC-rich sequences in the 2-nt overhangs. Unexpectedly, GC-rich sequences were enriched in the middle section of p19-captured dsRNA, suggesting some unexpected sequence bias in p19 protein binding. Nonetheless, the ectopic expression of p19 is a sensitive method for identifying antisense transcripts and RNase III cleavage sites in dsRNA formed by overlapping sense and antisense transcripts in bacteria.

A novel RNA polymerase-binding protein that interacts with a sigma-factor docking site.

Sporulation in Bacillus subtilis is governed by a cascade of alternative RNA polymerase sigma factors. We previously identified a small protein Fin that is produced under the control of the sporulation sigma factor σF to create a negative feedback loop that inhibits σF -directed gene transcription. Cells deleted for fin are defective for spore formation and exhibit increased levels of σF -directed gene transcription. Based on pull-down experiments, chemical crosslinking, bacterial two-hybrid experiments and nuclear magnetic resonance chemical shift analysis, we now report that Fin binds to RNA polymerase and specifically to the coiled-coil region of the ß' subunit. The coiled-coil is a docking site for sigma factors on RNA polymerase, and evidence is presented that the binding of Fin and σF to RNA polymerase is mutually exclusive. We propose that Fin functions by a mechanism distinct from that of classic sigma factor antagonists (anti-σ factors), which bind directly to a target sigma factor to prevent its association with RNA polymerase, and instead functions to inhibit σF by competing for binding to the ß' coiled-coil.

An Amino Acid Substitution in RNA Polymerase That Inhibits the Utilization of an Alternative Sigma Factor.

Sigma (σ) factors direct gene transcription by binding to and determining the promoter recognition specificity of RNA polymerase (RNAP) in bacteria. Genes transcribed under the control of alternative sigma factors allow cells to respond to stress and undergo developmental processes, such as sporulation in Bacillus subtilis, in which gene expression is controlled by a cascade of alternative sigma factors. Binding of sigma factors to RNA polymerase depends on the coiled-coil (or clamp helices) motif of the ß' subunit. We have identified an amino acid substitution (L257P) in the coiled coil that markedly inhibits the function of σH, the earliest-acting alternative sigma factor in the sporulation cascade. Cells with this mutant RNAP exhibited an early and severe block in sporulation but not in growth. The mutant was strongly impaired in σH-directed gene expression but not in the activity of the stress-response sigma factor σB Pulldown experiments showed that the mutant RNAP was defective in associating with σH but could still associate with σA and σB The differential effects of the L257P substitution on sigma factor binding to RNAP are likely due to a conformational change in the ß' coiled coil that is specifically detrimental for interaction with σH This is the first example, to our knowledge, of an amino acid substitution in RNAP that exhibits a strong differential effect on a particular alternative sigma factor.IMPORTANCE In bacteria, all transcription is mediated by a single multisubunit RNA polymerase (RNAP) enzyme. However, promoter-specific transcription initiation necessitates that RNAP associates with a σ factor. Bacteria contain a primary σ factor that directs transcription of housekeeping genes and alternative σ factors that direct transcription in response to environmental or developmental cues. We identified an amino acid substitution (L257P) in the B. subtilis ß' subunit whereby RNAPL257P associates with some σ factors (σA and σB) and enables vegetative cell growth but is defective in utilization of σH and is consequently blocked for sporulation. To our knowledge, this is the first identification of an amino acid substitution within the core enzyme that affects utilization of a specific sigma factor.

Nascent RNA length dictates opposing effects of NusA on antitermination.

The NusA protein is a universally conserved bacterial transcription elongation factor that binds RNA polymerase (RNAP). When functioning independently, NusA enhances intrinsic termination. Paradoxically, NusA stimulates the function of the N and Q antiterminator proteins of bacteriophage λ. The mechanistic basis for NusA's functional plasticity is poorly understood. Here we uncover an effect of nascent RNA length on the ability of NusA to collaborate with Q. Ordinarily, Q engages RNAP during early elongation when it is paused at a specific site just downstream of the phage late-gene promoter. NusA facilitates this engagement process and both proteins remain associated with the transcription elongation complex (TEC) as it escapes the pause and transcribes the late genes. We show that the λ-related phage 82 Q protein (82Q) can also engage RNAP that is paused at a promoter-distal position and thus contains a nascent RNA longer than that associated with the natively positioned TEC. However, the effect of NusA in this context is antagonistic rather than stimulatory. Moreover, cleaving the long RNA associated with the promoter-distal TEC restores NusA's stimulatory effect. Our findings reveal a critical role for nascent RNA in modulating NusA's effect on 82Q-mediated antitermination, with implications for understanding NusA's functional plasticity.

Mycobacterial mistranslation is necessary and sufficient for rifampicin phenotypic resistance.

Errors are inherent in all biological systems. Errors in protein translation are particularly frequent giving rise to a collection of protein quasi-species, the diversity of which will vary according to the error rate. As mistranslation rates rise, these new proteins could produce new phenotypes, although none have been identified to date. Here, we find that mycobacteria substitute glutamate for glutamine and aspartate for asparagine at high rates under specific growth conditions. Increasing the substitution rate results in remarkable phenotypic resistance to rifampicin, whereas decreasing mistranslation produces increased susceptibility to the antibiotic. These phenotypic changes are reflected in differential susceptibility of RNA polymerase to the drug. We propose that altering translational fidelity represents a unique form of environmental adaptation.

Phage-encoded inhibitor of Staphylococcus aureus transcription exerts context-dependent effects on promoter function in a modified Escherichia coli-based transcription system.

Promoter recognition in bacteria is mediated primarily by the σ subunit of RNA polymerase (RNAP), which makes sequence-specific contacts with the promoter -10 and -35 elements in the context of the RNAP holoenzyme. However, the RNAP α subunit can also contribute to promoter recognition by making sequence-specific contacts with upstream (UP) elements that are associated with a subset of promoters, including the rRNA promoters. In Escherichia coli, these interactions between the RNAP α subunit (its C-terminal domain [CTD], in particular) and UP element DNA result in significant stimulation of rRNA transcription. Among the many cellular and bacteriophage-encoded regulators of transcription initiation that have been functionally dissected, most exert their effects via a direct interaction with either the σ or the α subunit. An unusual example is provided by a phage-encoded inhibitor of RNA synthesis in Staphylococcus aureus. This protein, phage G1 gp67, which binds tightly to σ in the context of the S. aureus RNAP holoenzyme, has recently been shown to exert selective effects on transcription by inhibiting the function of the α subunit CTD (αCTD). Here we report the development of a gp67-responsive E. coli-based transcription system. We examine transcription in vitro from promoters that do or do not carry the UP element associated with a well-characterized E. coli rRNA promoter. Our findings indicate that the αCTD can increase promoter activity significantly even in the absence of an UP element. We also find that gp67 can exert αCTD-dependent or αCTD-independent effects on transcription depending on the particular promoter, indicating that the mechanism of gp67 action is context dependent.

Efficient and specific gene knockdown by small interfering RNAs produced in bacteria.

Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells and may be used for therapeutic purposes to knock down genes implicated in disease. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in Escherichia coli. This method relies on ectopic expression of p19, an siRNA-binding protein found in a plant RNA virus. When expressed in E. coli, p19 stabilizes an ∼21-nt siRNA-like species produced by bacterial RNase III. When mammalian cells are transfected by them, siRNAs that were generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene reproducibly knock down target gene expression by ∼90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes.

Crystal structure of the bacteriophage T4 late-transcription coactivator gp33 with the ß-subunit flap domain of Escherichia coli RNA polymerase.

Activated transcription of the bacteriophage T4 late genes, which is coupled to concurrent DNA replication, is accomplished by an initiation complex containing the host RNA polymerase associated with two phage-encoded proteins, gp55 (the basal promoter specificity factor) and gp33 (the coactivator), as well as the DNA-mounted sliding-clamp processivity factor of the phage T4 replisome (gp45, the activator). We have determined the 3.0 Å-resolution X-ray crystal structure of gp33 complexed with its RNA polymerase binding determinant, the ß-flap domain. Like domain 4 of the promoter specificity σ factor (σ(4)), gp33 interacts with RNA polymerase primarily by clamping onto the helix at the tip of the ß-flap domain. Nevertheless, gp33 and σ(4) are not structurally related. The gp33/ß-flap structure, combined with biochemical, biophysical, and structural information, allows us to generate a structural model of the T4 late promoter initiation complex. The model predicts protein/protein interactions within the complex that explain the presence of conserved patches of surface-exposed residues on gp33, and provides a structural framework for interpreting and designing future experiments to functionally characterize the complex.

Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex.

The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (α(2)ßß'ω) in complex with a σ factor that is essential for promoter recognition and transcription initiation. During early elongation, the stability of interactions between σ and the remainder of the transcription complex decreases. Nevertheless, there is no mechanistic requirement for release of σ upon the transition to elongation. Furthermore, σ can remain associated with RNAP during transcription elongation and influence regulatory events that occur during transcription elongation. Here we demonstrate that promoter-like DNA sequence elements within the initial transcribed region that are known to induce early elongation pausing through sequence-specific interactions with σ also function to increase the σ content of downstream elongation complexes. Our findings establish σ-dependent pausing as a mechanism by which initial transcribed region sequences can influence the composition and functional properties of the transcription elongation complex over distances of at least 700 base pairs.

A regulator from Chlamydia trachomatis modulates the activity of RNA polymerase through direct interaction with the beta subunit and the primary sigma subunit.

The obligate intracellular human pathogen Chlamydia trachomatis undergoes a complex developmental program involving transition between two forms: the infectious elementary body (EB), and the rapidly dividing reticulate body (RB). However, the regulators controlling this development have not been identified. To uncover potential regulators of transcription in C. trachomatis, we screened a C. trachomatis genomic library for sequences encoding proteins that interact with RNA polymerase (RNAP). We report the identification of one such protein, CT663, which interacts with the beta and sigma subunits of RNAP. Specifically, we show that CT663 interacts with the flap domain of the beta subunit (beta-flap) and conserved region 4 of the primary sigma subunit (sigma(66) in C. trachomatis). We find that CT663 inhibits sigma(66)-dependent (but not sigma(28)-dependent) transcription in vitro, and we present evidence that CT663 exerts this effect as a component of the RNAP holoenzyme. The analysis of C. trachomatis-infected cells reveals that CT663 begins to accumulate at the commencement of the RB-to-EB transition. Our findings suggest that CT663 functions as a negative regulator of sigma(66)-dependent transcription, facilitating a global change in gene expression. The strategy used here is generally applicable in cases where genetic tools are unavailable.

The bacteriophage lambda Q antiterminator protein contacts the beta-flap domain of RNA polymerase.

The multisubunit RNA polymerase (RNAP) in bacteria consists of a catalytically active core enzyme (alpha(2)beta beta'omega) complexed with a sigma factor that is required for promoter-specific transcription initiation. During early elongation the stability of interactions between sigma and core decreases, in part because of the nascent RNA-mediated destabilization of an interaction between region 4 of sigma and the flap domain of the beta-subunit (beta-flap). The nascent RNA-mediated destabilization of the sigma region 4/beta-flap interaction is required for the bacteriophage lambda Q antiterminator protein (lambdaQ) to engage the RNAP holoenzyme. Here, we provide an explanation for this requirement by showing that lambdaQ establishes direct contact with the beta-flap during the engagement process, thus competing with sigma(70) region 4 for access to the beta-flap. We also show that lambdaQ's affinity for the beta-flap is calibrated to ensure that lambdaQ activity is restricted to the lambda late promoter P(R'). Specifically, we find that strengthening the lambdaQ/beta-flap interaction allows lambdaQ to bypass the requirement for specific cis-acting sequence elements, a lambdaQ-DNA binding site and a RNAP pause-inducing element, that normally ensure lambdaQ is recruited exclusively to transcription complexes associated with P(R'). Our findings demonstrate that the beta-flap can serve as a direct target for regulators of elongation.

The bacteriophage lambdaQ anti-terminator protein regulates late gene expression as a stable component of the transcription elongation complex.

The Q protein of bacteriophage lambda (lambdaQ) is a transcription anti-terminator required for the expression of the phage's late genes under the control of promoter P(R'). To effect terminator read-through, lambdaQ must gain access to RNA polymerase (RNAP) via a promoter-restricted pathway. In particular, lambdaQ modifies RNAP by binding a specific DNA site embedded in P(R') and interacting with RNAP in the context of a specific paused early elongation complex. The resultant lambdaQ-modified transcription elongation complex is competent to read through downstream termination signals. Here we use a chromatin-immunoprecipitation assay to test the hypothesis that lambdaQ functions as a stable component of the transcription elongation complex. Our results indicate that, in vivo, the lambdaQ-modified transcription elongation complex contains Q as a stably associated subunit. Furthermore, we find that in the physiologically relevant context of an induced lambda lysogen, Q remains stably associated with RNAP as it transcribes at least 22 kb of the phage late operon. Thus, our findings suggest that the promoter-specific pathway leading to lambdaQ-mediated terminator read-through results in the formation of a highly stable lambdaQ-containing transcription elongation complex capable of traversing the entire late operon.

Conformational toggle triggers a modulator of RNA polymerase activity.

Members of a recently discovered class of transcription factor, which includes the Gre factors that stimulate transcript cleavage, function by directly modulating the catalytic properties of RNA polymerase (RNAP). Now, three research groups have determined crystal structures of a Gre homolog, Gfh1, which inhibits all RNAP catalytic activities. Strikingly, these structures reveal a puzzling discrepancy between the Gfh1 and GreA conformations, but the discovery that a pH-dependent conformational toggle alters Gfh1 activity suggests an elegant solution.

Characterization of the detachable Rho-dependent transcription terminator of the fimE gene in Escherichia coli K-12.

The fim genetic switch in the chromosome of Escherichia coli K-12 is an invertible DNA element that harbors the promoter for transcription of the downstream fim structural genes and a transcription terminator that acts on the upstream fimE regulatory gene. Switches oriented appropriately for structural gene transcription also allow fimE mRNA to read through, whereas those in the opposite orientation terminate the fimE message. We show here that termination is Rho dependent and is suppressed in a rho mutant or by bicyclomycin treatment when fimE mRNA is expressed by the fimE gene, either from a multicopy recombinant plasmid or in its native chromosomal location. Two cis-acting elements within the central portion of the 314-bp invertible DNA switch were identified as contributors to Rho-dependent termination and dissected. These fim sequence elements show similarities to well-characterized Rho utilization (rut) sites and consist of a boxA motif and a C-rich and G-poor region of approximately 40 bp. Deletion of the boxA motif alone had only a subtle negative effect on Rho function. However, when this element was deleted in combination with the C-rich, G-poor region, Rho function was considerably decreased. Altering the C-to-G ratio in favor of G in this portion of the switch also strongly attenuated transcription termination. The implications of the existence of a fimE-specific Rho-dependent terminator within the invertible switch are discussed in the context of the fim regulatory circuit.

An artificial activator that contacts a normally occluded surface of the RNA polymerase holoenzyme.

Many activators of transcription are sequence-specific DNA-binding proteins that stimulate transcription initiation through interaction with RNA polymerase (RNAP). Such activators can be constructed artificially by fusing a DNA-binding protein to a protein domain that can interact with an accessible surface of RNAP. In these cases, the artificial activator is directed to a target promoter bearing a recognition site for the DNA-binding protein. Here we describe an artificial activator that functions by contacting a normally occluded surface of promoter-bound RNAP holoenzyme. This artificial activator consists of a DNA-binding protein fused to the bacteriophage T4-encoded transcription regulator AsiA. On its own, AsiA inhibits transcription by Escherichia coli RNAP because it remodels the holoenzyme, disrupting an intersubunit interaction that is required for recognition of the major class of bacterial promoters. However, when tethered to the DNA via a DNA-binding protein, AsiA can exert a strong stimulatory effect on transcription by disrupting the same intersubunit interaction, contacting an otherwise occluded surface of the holoenzyme. We show that mutations that affect the intersubunit interaction targeted by AsiA modulate the stimulatory effect of this artificial activator. Our results thus demonstrate that changes in the accessibility of a normally occluded surface of the RNAP holoenzyme can modulate the activity of a gene-specific regulator of transcription.