Development of Giant Bacteriophage ϕKZ Is Independent of the Host Transcription Apparatus

Abstract

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...

Identification of Essential Genes in the Salmonella Phage SPN3US Reveals Novel Insights into Giant Phage Head Structure and Assembly

“…Initial sequencing of a mixture of the candidates showed the presence of amber mutations in 33 SPN3US genes, 24 with homologs in ϕKZ. Numbers of these 24 genes would also be expected to be essential based on studies of other phages; for instance, the SbcC subunit of T4 was demonstrated to be essential using genetics, and the subunits of the ϕKZ multisubunit RNAPs would be expected to be essential based on the replication of the phage in the presence of rifampin (19). However, to confirm that any of the detected SPN3US amber mutations were actually in an essential gene, each mutation must be identified as the sole amber mutation in an individual genome.…”

“…Two multisubunit RNAPs are considered hallmark features of phages related to ϕKZ, with every member containing two sets of subunits (19), one set presumably arising from a gene duplication event(s) after becoming phage borne. This is quite remarkable considering that the two subunits (β and β′) themselves are hypothesized to have arisen from an ancient duplication event (50).…”

“…The SPN3US proteins gp241, gp218, gp240, and gp42 and their homologs in other giant phages that were previously identified as forming the vRNAP complex (19) account for all of the highly conserved regions in prokaryotic RNAP, with the exception of several hundred amino acids in the C-terminal region of β′ (approximately residues 1260 to 1524 of the RNAP β′ of Thermus thermophilus HB8). The C-terminal region of β′ was initially recognized as being conserved in both prokaryotic and eukaryotic RNAPs by Jokerst et al and designated region H (55).…”

“…The second RNAP, the nonvirion RNAP (nvRNAP), was recently purified from cells infected with ϕKZ and shown to be responsible for the transcription of late genes (18). There is great interest in the unusual RNAPs of these phages, as ϕKZ is able to infect its host in the presence of rifampin, indicating that phage transcription could be completely independent of the host transcriptional machinery (19). …”

Genomic and Transcriptional Mapping of PaMx41, Archetype of a New Lineage of Bacteriophages Infecting Pseudomonas aeruginosa

“…S2). Phage early mRNA shutdown has been reported in T7 (45) and in the phage PhiKZ; early mRNA shutdown is observed after the early stage of infection (46). Probes G, H, I, J, and K detected 11 middle mRNAs covering the leftward-oriented cluster in the right arm of the genome (Fig.…”