Pseudomonas aeruginosa bacteriophage KZ is the type representative of the giant phage genus, which is characterized by unusually large virions and genomes. By unraveling the transcriptional map of the ϳ280-kb KZ genome to single-nucleotide resolution, we combine 369 KZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the KZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to KZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the KZ genome is performed by the consecutive action of two KZ-encoded, noncanonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampin-resistant transcriptional machinery is conserved within the diverse giant phage genus. IMPORTANCEThe data presented in this paper offer, for the first time, insight into the complex transcriptional scheme of giant bacteriophages. We show that Pseudomonas aeruginosa giant phage KZ is able to infect and lyse its host cell and produce phage progeny in the absence of functional bacterial transcriptional machinery. This unique property can be attributed to two phage-encoded putative RNAP enzymes, which contain very distant homologues of bacterial ␤ and ␤=-like RNAP subunits. T ranscription is driven by DNA-dependent RNA polymerases (RNAPs), which synthesize RNA from DNA templates (1). RNAPs can be classified into two unrelated families: small singlesubunit enzymes (ssRNAPs), encoded by some bacteriophages and also found in mitochondria and chloroplasts, and large multisubunit cellular enzymes (msRNAPs), transcribing genes in bacterial, archaeal, and eukaryal genomes. The catalytic activity of enzymes from both families is accomplished through a common two-metal-ion mechanism. The canonical bacterial msRNAP is a 400-kDa complex consisting of five core subunits (␣ 2 ␤␤=) which are directed to specific promoter sequences by a variety of factors (2). The two largest RNAP subunits, ␤ and ␤=, contain conserved double-psi beta-barrel (DPBB) domains that together form the active center (3-5). Members of the ssRNAP family rely on different catalytic domains and amino acid motifs for catalysis of the RNA polymerization reaction and are related to DNA polymerases and reverse transcriptases (6, 7). Bacterial RNAPs are inactivated by the antibiotic rifampin (Rif), which acts by binding to the ␤-subunit pocket deep inside the active-site channel, preventing synthesis of RNA sequences longer than 3 to 4 nucleotides (nt) (8).Bacterial RNAPs play a key role during the infection of bacterial cells by bacteriophages. Most known phages do not encode their own RNAP but redirect the host transcription machinery to viral promoters by rel...

Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus. Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.

Acinetobacter baumannii is a gram-negative, non-fermenting aerobic bacterium which is often associated with hospital-acquired infections and known for its ability to develop resistance to antibiotics, form biofilms, and survive for long periods in hospital environments. In this study, we present two novel viruses, vB_AbaP_AS11 and vB_AbaP_AS12, specifically infecting and lysing distinct multidrug-resistant clinical A. baumannii strains with K19 and K27 capsular polysaccharide structures, respectively. Both phages demonstrate rapid adsorption, short latent periods, and high burst sizes in one-step growth experiments. The AS11 and AS12 linear double-stranded DNA genomes of 41,642 base pairs (bp) and 41,402 bp share 86% nucleotide sequence identity with the most variable regions falling in host receptor–recognition genes. These genes encode tail spikes possessing depolymerizing activities towards corresponding capsular polysaccharides which are the primary bacterial receptors. We described AS11 and AS12 genome organization and discuss the possible regulation of transcription. The overall genomic architecture and gene homology analyses showed that the phages are new representatives of the recently designated Fri1virus genus of the Autographivirinae subfamily within the Podoviridae family.

Escherichia coli phage phiEco32 encodes two proteins that bind to host RNA polymerase — gp79, a novel protein, and gp36, a distant homolog of σ70 family proteins. Here, we investigated the temporal pattern of phiEco32 and host gene expression during the infection. Host transcription shut-off and three distinct bacteriophage temporal gene classes – early, middle, and late – were revealed. A combination of bioinformatic and biochemical approaches allowed identification of phage promoters recognized by host RNA polymerase holoenzyme containing the σ70 factor. These promoters are located upstream of early phage genes. A combination of macroarray data, primer extension, and in vitro transcription analyses allowed identification of six promoters recognized by RNA polymerase holoenzyme containing gp36. These promoters are characterized by a single consensus element tAATGTAtA and are located upstream of the middle and late phage genes. Curiously, gp79, an inhibitor of host and early phage transcription by σ70-holoenzyme, activated transcription by the gp36-holoenzyme in vitro.

Type III CRISPR–Cas systems provide immunity to foreign DNA by targeting its transcripts. Target recognition activates RNases and DNases that may either destroy foreign DNA directly or elicit collateral damage inducing death of infected cells. While some Type III systems encode a reverse transcriptase to acquire spacers from foreign transcripts, most contain conventional spacer acquisition machinery found in DNA-targeting systems. We studied Type III spacer acquisition in phage-infected Thermus thermophilus, a bacterium that lacks either a standalone reverse transcriptase or its fusion to spacer integrase Cas1. Cells with spacers targeting a subset of phage transcripts survived the infection, indicating that Type III immunity does not operate through altruistic suicide. In the absence of selection spacers were acquired from both strands of phage DNA, indicating that no mechanism ensuring acquisition of RNA-targeting spacers exists. Spacers that protect the host from the phage demonstrate a very strong strand bias due to positive selection during infection. Phages that escaped Type III interference accumulated deletions of integral number of codons in an essential gene and much longer deletions in a non-essential gene. This and the fact that Type III immunity can be provided by plasmid-borne mini-arrays open ways for genomic manipulation of Thermus phages.

Bacteriophages and large dsDNA viruses encode sophisticated machinery to translocate their DNA into a preformed empty capsid. An essential part of this machine, the large terminase protein, processes viral DNA into constituent units utilizing its nuclease activity. Crystal structures of the large terminase nuclease from the thermophilic bacteriophage G20c show that it is most similar to the RuvC family of the RNase H-like endonucleases. Like RuvC proteins, the nuclease requires either Mn2+, Mg2+ or Co2+ ions for activity, but is inactive with Zn2+ and Ca2+. High resolution crystal structures of complexes with different metals reveal that in the absence of DNA, only one catalytic metal ion is accommodated in the active site. Binding of the second metal ion may be facilitated by conformational variability, which enables the two catalytic aspartic acids to be brought closer to each other. Structural comparison indicates that in common with the RuvC family, the location of the two catalytic metals differs from other members of the RNase H family. In contrast to a recently proposed mechanism, the available data do not support binding of the two metals at an ultra-short interatomic distance. Thus we postulate that viral terminases cleave DNA by the canonical RuvC-like mechanism.

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During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR–Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1–Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array.