Isolation and Characterization of Phages That Bypass the Requirement for RNA-Mediated Antitermination.

Introduction: The rpoCY75N mutation in the zinc-binding domain of the ß' subunit of Escherichia coli RNA polymerase blocks the RNA-based mechanism of transcription antitermination utilized by bacteriophage HK022. Materials and Methods: Mutant phages that overcome the block imposed by the rpoCY75N mutation are described. These phages, designated "orc" (overcomes rpoC), carry mutations that create new promoters. Promoter activity was assessed by cloning the respective regions from the wild-type and orc phages into a promoterless lacZ reporter vector. Results: Reporter assays showed that the sequence originating from orc phages had significant promoter activity when compared with the equivalent sequence cloned from the parental phage. Conclusions: The newly created promoters facilitate the expression of phage genes that are essential for growth on the rpoCY75N strain by bypassing transcription terminators. The small plaque phenotype of orc phages, when grown on the mutant host, suggests that suppression of the rpoCY75N mutation is incomplete.

Structural basis of transcriptional regulation by a nascent RNA element, HK022 putRNA.

Transcription, in which RNA polymerases (RNAPs) produce RNA from DNA, is the first step of gene expression. As such, it is highly regulated either by trans-elements like protein factors and/or by cis-elements like specific sequences on the DNA. Lambdoid phage HK022 contains a cis-element, put, which suppresses pausing and termination during transcription of the early phage genes. The putRNA transcript solely performs the anti-pausing/termination activities by interacting directly with the E.coli RNAP elongation complex (EC) by an unknown structural mechanism. In this study, we reconstituted putRNA-associated ECs and determined the structures using cryo-electron microscopy. The determined structures of putRNA-associated EC, putRNA-absent EC, and σ70-bound EC suggest that the putRNA interaction with the EC counteracts swiveling, a conformational change previously identified to promote pausing and σ70 might modulate putRNA folding via σ70-dependent pausing during elongation.

Whole genome sequencing identifies an allele responsible for clear vs. turbid plaque morphology in a Mycobacteriophage.

BACKGROUND: Whole genome sequencing promises to revolutionize our ability to link genotypic and phenotypic variation in a wide range of model and non-model species. RESULTS: Here we describe the isolation and characterization of a novel mycobacteriophage named BGlluviae that grows on Mycobacterium smegmatis mc2155. BGlluviae normally produces turbid plaques but a spontaneous clear plaque was also recovered. The genomic DNA from pure populations of the BGlluviae phage and the clear plaque mutant were sequenced. A single substitution, at amino acid 54 (I to T), in the immunity repressor protein resulted in a clear plaque phenotype. CONCLUSIONS: This substitution is predicted to be located at the subunit interaction interface of the repressor protein, and thus prevents the establishment of lysogeny.

Sequence Analysis of a Hybrid HK022/λ Bacteriophage and the Precise Identification of the λb515 and λb519 Deletion Endpoints.

Bacteriophage O276 is a laboratory-generated hybrid that carries the immunity region of bacteriophage HK022 and all remaining genes from phage λ. Its construction was instrumental in the discovery of RNA-mediated antitermination, an intriguing alternative to the protein-mediated mechanism of transcription antitermination found in most lambdoid phages.

Complete Genome Sequences of 44 Arthrobacter Phages.

We report here the complete genome sequences of 44 phages infecting Arthrobacter sp. strain ATCC 21022. These phages have double-stranded DNA genomes with sizes ranging from 15,680 to 70,707 bp and G+C contents from 45.1% to 68.5%. All three tail types (belonging to the families Siphoviridae, Myoviridae, and Podoviridae) are represented.

Genome Sequences of Subcluster K5 Mycobacteriophages AlleyCat, Edugator, and Guillsminger.

Bacteriophages AlleyCat, Edugator, and Guillsminger were isolated on Mycobacterium smegmatis mc2155 from enriched soil samples. All are members of mycobacteriophage subcluster K5, with genomes of 62,112 to 63,344 bp. Each genome contains 92 to 99 predicted protein-coding genes and one tRNA. Guillsminger is the first mycobacteriophage to carry an IS1380 family transposon.

Comparative genomics of Cluster O mycobacteriophages.

Mycobacteriophages--viruses of mycobacterial hosts--are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages--Corndog, Catdawg, Dylan, Firecracker, and YungJamal--designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8-9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange.

A broadly implementable research course in phage discovery and genomics for first-year undergraduate students.

UNLABELLED: Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students' interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training. IMPORTANCE: Engagement of undergraduate students in scientific research at early stages in their careers presents an opportunity to excite students about science, technology, engineering, and mathematics (STEM) disciplines and promote continued interests in these areas. Many excellent course-based undergraduate research experiences have been developed, but scaling these to a broader impact with larger numbers of students is challenging. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunting Advancing Genomics and Evolutionary Science (SEA-PHAGES) program takes advantage of the huge size and diversity of the bacteriophage population to engage students in discovery of new viruses, genome annotation, and comparative genomics, with strong impacts on bacteriophage research, increased persistence in STEM fields, and student self-identification with learning gains, motivation, attitude, and career aspirations.

Newly discovered antiterminator RNAs in bacteriophage.

Antiterminator RNA directly modifies the transcription elongation complex so that it terminates less efficiently at intrinsic and factor-dependent terminators. These unusual RNAs were first discovered in bacteriophage HK022, where the nascent transcripts of the phage put sites promote full expression of phage genes during lytic infection. The activity of antiterminator RNA depends on specific structural elements that form as the transcript exits RNA polymerase. To further our understanding of the critical sequence features that permit RNA to serve as a transcriptional antiterminator, we have identified eight antiterminator RNA sequences in bacteriophages or prophages. There is strong sequence conservation among most of the put sequences, but sequence divergence is tolerated if critical structural elements are preserved. The most diverged antiterminator RNA is found in bacteriophage HK639. The HK639 putL transcript is an efficient antiterminator, and it has a novel structural feature that is critical for its activity. HK639 also displays a unique pattern of sensitivity to amino acid substitutions in the ß' subunit zinc binding domain of RNA polymerase, adding to existing evidence that this domain interacts specifically with antiterminator RNA.

Inhibition of a transcriptional pause by RNA anchoring to RNA polymerase.

We describe a mechanism by which nascent RNA inhibits transcriptional pausing. PutL RNA of bacteriophage HK022 suppresses transcription termination at downstream terminators and pausing within a nearby U-rich sequence. In vitro transcription and footprinting assays reveal that this pausing results from backtracking of RNA polymerase and that binding of nascent putL RNA to polymerase limits backtracking by restricting re-entry of the transcript into the RNA exit channel. The restriction is local and relaxes as the transcript elongates. Our results suggest that putL RNA binds to the surface of polymerase close to the RNA exit channel, a region that includes amino acid residues important for antitermination. Although binding is essential for antipausing and antitermination, these two activities of put differ: antipausing is limited to the immediate vicinity of the putL site, but antitermination is not. We propose that RNA anchoring to the elongation complex is a widespread mechanism of pause regulation.

Characterization of Salmonella bacteriophages isolated from swine lagoon effluent.

Four Salmonella bacteriophages that had been originally isolated from swine manure lagoons were characterized and compared to each other and to well-known Salmonella phages P22 and Felix 01. Host ranges of the lagoon phages were similar to each other in spot tests on reference strains of Salmonella, but differed slightly from each other on a panel of Salmonella lagoon strains. In single-step growth at 35 degrees C the lagoon phages had latent periods of 15 to 20 min and burst sizes from 100 to 230. The lagoon phages and P22 were purified by cesium chloride (CsCl) gradient centrifugation and used to produce specific antisera and DNA. The lagoon phages were indistinguishable from each other but distinct from P22 and Felix 01 in immunodiffusion and infectivity neutralization tests and in restriction digest analysis.

Protection of antiterminator RNA by the transcript elongation complex.

Nascent transcripts encoded by the putL and putR sites of phage HK022 bind the transcript elongation complex and suppress termination at downstream transcription terminators. We report here that the chemical stability of putL RNA is considerably greater than that of the typical Escherichia coli message because the elongation complex protects this RNA from degradation. When binding to the elongation complex was prevented by mutation of either putL or RNA polymerase, RNA stability decreased more than 50-fold. The functional modification conferred by putL RNA on the elongation complex is also long-lived: the efficiency of terminator suppression remained high for at least 10 kb from the putL site. We find that RNase III rapidly and efficiently cleaved the transcript just downstream of the putL sequences, but such cleavage changed neither the stability of putL RNA nor the efficiency of antitermination. These results argue that the continuity of the RNA that connects put sequences to the growing point is not required for persistence of the antiterminating modification in vivo.

A conserved zinc binding domain in the largest subunit of DNA-dependent RNA polymerase modulates intrinsic transcription termination and antitermination but does not stabilize the elongation complex.

An evolutionarily conserved zinc-binding motif is found close to the amino terminus of the largest subunits of DNA-dependent RNA polymerases from bacteria, archaea, and eukaryotes. In bacterial RNA polymerase, this motif, the zinc binding domain, has been implicated in protein-DNA interactions that stabilize the transcription elongation complex and that occur downstream of the catalytic center. Here, we show that this view is incorrect, and instead, the zinc binding domain interacts with product RNA located upstream of the catalytic center and the RNA-DNA hybrid, a view consistent with structural studies of the elongation complex. We engineered mutations that alter or remove the zinc binding domain of Escherichia coli RNA polymerase. Several mutants, including one that lacked all four zinc ligands and another that lacked the entire domain, produced enzymes that were active in vitro and formed stable elongation complexes. However, they were defective in two functions that require interaction of polymerase with product RNA. First, they terminated less efficiently than the wild-type at intrinsic transcription terminators. Second, enzymes lacking the tip of the zinc binding domain or the zinc ligands did not antiterminate in response to an intrinsic antiterminator encoded by the put site of phage HK022. Termination, but not antitermination, was restored by the bacterial termination factor NusA. Surprisingly, a mutant that lacks the entire zinc binding domain regained a partial response to put. To account for this we suggest that put RNA interacts with an additional site in the elongation complex to mediate antitermination, and that this site is occluded by the wild-type zinc binding domain.

Suppression of factor-dependent transcription termination by antiterminator RNA.

Nascent transcripts of the phage HK022 put sites modify the transcription elongation complex so that it terminates less efficiently at intrinsic transcription terminators and accelerates through pause sites. We show here that the modification also suppresses termination in vivo at two factor-dependent terminators, one that depends on the bacterial Rho protein and a second that depends on the HK022-encoded Nun protein. Suppression was efficient when the termination factors were present at physiological levels, but an increase in the intracellular concentration of Nun increased termination both in the presence and absence of put. put-mediated antitermination thus shows no apparent terminator specificity, suggesting that put inhibits a step that is common to termination at the different types of terminator.

Sequence-specific interaction of nascent antiterminator RNA with the zinc-finger motif of Escherichia coli RNA polymerase.

The N-terminal Zn-finger motif of the beta' subunit of RNA polymerase contains two pairs of invariant cysteines flanking a moderately well-conserved segment of 13 amino acids that is rich in basic residues. Previous work showed that replacement of certain Zn-finger residues prevented transcription antitermination in response to phage HK022 put sites. Nascent put RNA binds to and modifies transcribing polymerase, so that it becomes resistant to termination. To characterize the Zn finger further, we replaced each of the basic residues with alanine and determined the effects of the substitutions on termination, antitermination and cell viability. All the mutants were defective in put-mediated antitermination. The severity of the defect depended on the mutant and on the sequence of the upstream stem-loop of put RNA. Some, but not all, mutants distinguished between put variants that differed in this region. This suggests that the Zn-finger motif interacts directly and specifically with put RNA. All the mutants in the basic residues complemented a temperature-sensitive beta' mutant for cell growth at a non-permissive temperature, and those mutant enzymes that were tested transcribed and terminated normally in vitro on a template that lacked a put site.