Control of lysogeny and antiphage defense by a prophage-encoded kinase-phosphatase module.

The filamentous 'Pf' bacteriophages of Pseudomonas aeruginosa play roles in biofilm formation and virulence, but mechanisms governing Pf prophage activation in biofilms are unclear. Here, we identify a prophage regulatory module, KKP (kinase-kinase-phosphatase), that controls virion production of co-resident Pf prophages and mediates host defense against diverse lytic phages. KKP consists of Ser/Thr kinases PfkA and PfkB, and phosphatase PfpC. The kinases have multiple host targets, one of which is MvaU, a host nucleoid-binding protein and known prophage-silencing factor. Characterization of KKP deletion and overexpression strains with transcriptional, protein-level and prophage-based approaches indicates that shifts in the balance between kinase and phosphatase activities regulate phage production by controlling MvaU phosphorylation. In addition, KKP acts as a tripartite toxin-antitoxin system that provides defense against some lytic phages. A conserved lytic phage replication protein inhibits the KKP phosphatase PfpC, stimulating toxic kinase activity and blocking lytic phage production. Thus, KKP represents a phosphorylation-based mechanism for prophage regulation and antiphage defense. The conservation of KKP gene clusters in >1000 diverse temperate prophages suggests that integrated control of temperate and lytic phage infection by KKP-like regulatory modules may play a widespread role in shaping host cell physiology.

A phage tail-like bacteriocin suppresses competitors in metapopulations of pathogenic bacteria.

Bacteria can repurpose their own bacteriophage viruses (phage) to kill competing bacteria. Phage-derived elements are frequently strain specific in their killing activity, although there is limited evidence that this specificity drives bacterial population dynamics. Here, we identified intact phage and their derived elements in a metapopulation of wild plant-associated Pseudomonas genomes. We discovered that the most abundant viral cluster encodes a phage remnant resembling a phage tail called a tailocin, which bacteria have co-opted to kill bacterial competitors. Each pathogenic Pseudomonas strain carries one of a few distinct tailocin variants that target the variable polysaccharides in the outer membrane of co-occurring pathogenic Pseudomonas strains. Analysis of herbarium samples from the past 170 years revealed that the same tailocin and bacterial receptor variants have persisted in Pseudomonas populations. These results suggest that tailocin genetic diversity can be mined to develop targeted "tailocin cocktails" for microbial control.

Characterization of a lipid-based jumbo phage compartment as a hub for early phage infection.

Viral genomes are most vulnerable to cellular defenses at the start of the infection. A family of jumbo phages related to phage ΦKZ, which infects Pseudomonas aeruginosa, assembles a protein-based phage nucleus to protect replicating phage DNA, but how it is protected prior to phage nucleus assembly is unclear. We find that host proteins related to membrane and lipid biology interact with injected phage protein, clustering in an early phage infection (EPI) vesicle. The injected virion RNA polymerase (vRNAP) executes early gene expression until phage genome separation from the vRNAP and the EPI vesicle, moving into the nascent proteinaceous phage nucleus. Enzymes involved in DNA replication and CRISPR/restriction immune nucleases are excluded by the EPI vesicle. We propose that the EPI vesicle is rapidly constructed with injected phage proteins, phage DNA, host lipids, and host membrane proteins to enable genome protection, early transcription, localized translation, and to ensure faithful genome transfer to the proteinaceous nucleus.

Lytic bacteriophages induce the secretion of antiviral and proinflammatory cytokines from human respiratory epithelial cells.

Phage therapy is a therapeutic approach to treat multidrug-resistant (MDR) infections that employs lytic bacteriophages (phages) to eliminate bacteria. Despite the abundant evidence for its success as an antimicrobial in Eastern Europe, there is scarce data regarding its effects on the human host. Here, we aimed to understand how lytic phages interact with cells of the airway epithelium, the tissue site that is colonized by bacterial biofilms in numerous chronic respiratory disorders. Using a panel of Pseudomonas aeruginosa phages and human airway epithelial cells (AECs) derived from a person with cystic fibrosis (CF), we determined that interactions between phages and epithelial cells depend on specific phage properties as well as physiochemical features of the microenvironment. Although poor at internalizing phages, the airway epithelium responds to phage exposure by changing its transcriptional profile and secreting antiviral and proinflammatory cytokines that correlate with specific phage families. Overall, our findings indicate that mammalian responses to phages are heterogenous and could potentially alter the way that respiratory local defenses aid in bacterial clearance during phage therapy. Thus, besides phage receptor specificity in a particular bacterial isolate, the criteria to select lytic phages for therapy should be expanded to include mammalian cell responses.

Development of an enzyme-linked phage receptor-binding protein assay (ELPRA) based on a novel biorecognition molecule- receptor-binding protein Gp130 of Pseudomonas aeruginosa bacteriophage Henu5.

Pseudomonas aeruginosa is a Gram-negative bacterium associated with life-threatening healthcare-associated infections (HAIs), including burn wound infections, pneumonia and sepsis. Moreover, P. aeruginosa has been considered a pathogen of global concern due to its rising antibiotic resistance. Efficient identification of P. aeruginosa would significantly benefit the containment of bacterial infections, prevent pathogen transmission, and provide orientated treatment options. The accuracy and specificity of bacterial detection are primarily dictated by the biorecognition molecules employed. Lytic bacteriophages (or phages) could specifically attach to and lyse host bacterial cells. Phages' host specificity is typically determined by their receptor-binding proteins (RBPs), which recognize and adsorb phages to particular bacterial host receptors. This makes RBPs promising biorecognition molecules in bacterial detection. This study identified a novel RBP (Gp130) from the P. aeruginosa phage Henu5. A modified enzyme-linked phage receptor-binding protein assay (ELPRA) was developed for P. aeruginosa detection employing Gp130 as biorecognition molecules. Optimized conditions provided a calibration curve for P. aeruginosa with a range from 1.0 × 103 to 1.0 × 107 CFU/mL, with a limit of detection as low as 10 CFU/mL in phosphate-buffered saline (PBS). With VITEKⓇ 2 Compact system identification (40 positives and 21 negatives) as the gold standard, the sensitivity of ELPRA was 0.950 (0.818-0.991), and the specificity was 0.905 (0.682-0.983) within a 95 %confidence interval. Moreover, the recovery test in spiked mouse serum showed recovery rates ranging from 82.79 %to 98.17%, demonstrating the prospect of the proposed ELPRA for detecting P. aeruginosa in biological samples.

The Dynamics of Synthesis and Localization of Jumbo Phage RNA Polymerases inside Infected Cells.

A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early gene products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection were studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.

Bacterial cytological profiling reveals interactions between jumbo phage φKZ infection and cell wall active antibiotics in Pseudomonas aeruginosa.

The emergence of antibiotic resistance in bacteria has led to the investigation of alternative treatments, such as phage therapy. In this study, we examined the interactions between the nucleus-forming jumbo phage ФKZ and antibiotic treatment against Pseudomonas aeruginosa. Using the fluorescence microscopy technique of bacterial cytological profiling, we identified mechanism-of-action-specific interactions between antibiotics that target different biosynthetic pathways and ФKZ infection. We found that certain classes of antibiotics strongly inhibited phage replication, while others had no effect or only mildly affected progression through the lytic cycle. Antibiotics that caused an increase in host cell length, such as the cell wall active antibiotic ceftazidime, prevented proper centering of the ФKZ nucleus via the PhuZ spindle at midcell, leading us to hypothesize that the kinetic parameters of the PhuZ spindle evolved to match the average length of the host cell. To test this, we developed a computational model explaining how the dynamic properties of the PhuZ spindle contribute to phage nucleus centering and why some antibiotics affect nucleus positioning while others do not. These findings provide an understanding of the molecular mechanisms underlying the interactions between antibiotics and jumbo phage replication.

Structural basis for anti-CRISPR repression mediated by bacterial operon proteins Aca1 and Aca2.

It has been shown that phages have evolved anti-CRISPR (Acr) proteins to inhibit host CRISPR-Cas systems. Most acr genes are located upstream of anti-CRISPR-associated (aca) genes, which is instrumental for identifying these acr genes. Thus far, eight Aca families (Aca1-Aca8) have been identified, all proteins of which share low sequence homology and bind to different target DNA sequences. Recently, Aca1 and Aca2 proteins were discovered to function as repressors by binding to acr-aca promoters, thus implying a potential anti-anti-CRISPR mechanism. However, the structural basis for the repression roles of Aca proteins is still unknown. Here, we elucidated apo-structures of Aca1 and Aca2 proteins and their complex structures with their cognate operator DNA in two model systems, the Pseudomonas phage JBD30 and the Pectobacterium carotovorum template phage ZF40. In combination with biochemical and cellular assays, our study unveils dimerization and DNA-recognition mechanisms of Aca1 and Aca2 family proteins, thus revealing the molecular basis for Aca1-and Aca2-mediated anti-CRISPR repression. Our results also shed light on understanding the repression roles of other Aca family proteins and autoregulation roles of acr-aca operons.

The In Vitro Anti-Pseudomonal Activity of Cu2+, Strawberry Furanone, Gentamicin, and Lytic Phages Alone and in Combination: Pros and Cons.

In this study, we investigated the anti-pseudomonal activity of cupric ions (Cu2+), strawberry furanone (HDMF), gentamicin (GE), and three lytic Pseudomonas aeruginosa bacteriophages (KT28, KTN4, LUZ19), separately and in combination. HDMF showed an anti-virulent effect but only when applied with Cu2+ or GE. GE, at a sub-minimal inhibitory concentration, slowed down phage progeny production due to protein synthesis inhibition. Cu2+ significantly reduced both the bacterial cell count and the number of infective phage particles, likely due to its genotoxicity or protein inactivation and cell membrane disruption effects. Furthermore, Cu2+'s probable sequestration by phage particles led to the reduction of free toxic metal ions available in the solution. An additive antibacterial effect was only observed for the combination of GE and Cu2+, potentially due to enhanced ROS production or to outer membrane permeabilization. This study indicates that possible interference between antibacterial agents needs to be carefully investigated for the preparation of effective therapeutic cocktails.

The bacteriophage LUZ24 "Igy" peptide inhibits the Pseudomonas DNA gyrase.

The bacterial DNA gyrase complex (GyrA/GyrB) plays a crucial role during DNA replication and serves as a target for multiple antibiotics, including the fluoroquinolones. Despite it being a valuable antibiotics target, resistance emergence by pathogens including Pseudomonas aeruginosa are proving problematic. Here, we describe Igy, a peptide inhibitor of gyrase, encoded by Pseudomonas bacteriophage LUZ24 and other members of the Bruynoghevirus genus. Igy (5.6 kDa) inhibits in vitro gyrase activity and interacts with the P. aeruginosa GyrB subunit, possibly by DNA mimicry, as indicated by a de novo model of the peptide and mutagenesis. In vivo, overproduction of Igy blocks DNA replication and leads to cell death also in fluoroquinolone-resistant bacterial isolates. These data highlight the potential of discovering phage-inspired leads for antibiotics development, supported by co-evolution, as Igy may serve as a scaffold for small molecule mimicry to target the DNA gyrase complex, without cross-resistance to existing molecules.

The Repressor C Protein, Pf4r, Controls Superinfection of Pseudomonas aeruginosa PAO1 by the Pf4 Filamentous Phage and Regulates Host Gene Expression.

It has been shown that the filamentous phage, Pf4, plays an important role in biofilm development, stress tolerance, genetic variant formation and virulence in Pseudomonas aeruginosa PAO1. These behaviours are linked to the appearance of superinfective phage variants. Here, we have investigated the molecular mechanism of superinfection as well as how the Pf4 phage can control host gene expression to modulate host behaviours. Pf4 exists as a prophage in PAO1 and encodes a homologue of the P2 phage repressor C and was recently named Pf4r. Through a combination of molecular techniques, ChIPseq and transcriptomic analyses, we show a critical site in repressor C (Pf4r) where a mutation in the site, 788799A>G (Ser4Pro), causes Pf4r to lose its function as the immunity factor against reinfection by Pf4. X-ray crystal structure analysis shows that Pf4r forms symmetric homo-dimers homologous to the E.coli bacteriophage P2 RepC protein. A mutation, Pf4r*, associated with the superinfective Pf4r variant, found at the dimer interface, suggests dimer formation may be disrupted, which derepresses phage replication. This is supported by multi-angle light scattering (MALS) analysis, where the Pf4r* protein only forms monomers. The loss of dimerisation also explains the loss of Pf4r's immunity function. Phenotypic assays showed that Pf4r increased LasB activity and was also associated with a slight increase in the percentage of morphotypic variants. ChIPseq and transcriptomic analyses suggest that Pf4r also likely functions as a transcriptional regulator for other host genes. Collectively, these data suggest the mechanism by which filamentous phages play such an important role in P. aeruginosa biofilm development.

Introducing differential RNA-seq mapping to track the early infection phase for Pseudomonas phage ɸKZ.

As part of the ongoing renaissance of phage biology, more phage genomes are becoming available through DNA sequencing. However, our understanding of the transcriptome architecture that allows these genomes to be expressed during host infection is generally poor. Transcription start sites (TSSs) and operons have been mapped for very few phages, and an annotated global RNA map of a phage - alone or together with its infected host - is not available at all. Here, we applied differential RNA-seq (dRNA-seq) to study the early, host takeover phase of infection by assessing the transcriptome structure of Pseudomonas aeruginosa jumbo phage ɸKZ, a model phage for viral genetics and structural research. This map substantially expands the number of early expressed viral genes, defining TSSs that are active ten minutes after ɸKZ infection. Simultaneously, we record gene expression changes in the host transcriptome during this critical metabolism conversion. In addition to previously reported upregulation of genes associated with amino acid metabolism, we observe strong activation of genes with functions in biofilm formation (cdrAB) and iron storage (bfrB), as well as an activation of the antitoxin ParD. Conversely, ɸKZ infection rapidly down-regulates complexes IV and V of oxidative phosphorylation (atpCDGHF and cyoABCDE). Taken together, our data provide new insights into the transcriptional organization and infection process of the giant bacteriophage ɸKZ and adds a framework for the genome-wide transcriptomic analysis of phage-host interactions.

Genomic Characterisation of Mushroom Pathogenic Pseudomonads and Their Interaction with Bacteriophages.

Bacterial diseases of the edible white button mushroom Agaricus bisporus caused by Pseudomonas species cause a reduction in crop yield, resulting in considerable economic loss. We examined bacterial pathogens of mushrooms and bacteriophages that target them to understand the disease and opportunities for control. The Pseudomonastolaasii genome encoded a single type III protein secretion system (T3SS), but contained the largest number of non-ribosomal peptide synthase (NRPS) genes, multimodular enzymes that can play a role in pathogenicity, including a putative tolaasin-producing gene cluster, a toxin causing blotch disease symptom. However, Pseudomonasagarici encoded the lowest number of NRPS and three putative T3SS while non-pathogenic Pseudomonas sp. NS1 had intermediate numbers. Potential bacteriophage resistance mechanisms were identified in all three strains, but only P. agarici NCPPB 2472 was observed to have a single Type I-F CRISPR/Cas system predicted to be involved in phage resistance. Three novel bacteriophages, NV1, ϕNV3, and NV6, were isolated from environmental samples. Bacteriophage NV1 and ϕNV3 had a narrow host range for specific mushroom pathogens, whereas phage NV6 was able to infect both mushroom pathogens. ϕNV3 and NV6 genomes were almost identical and differentiated within their T7-like tail fiber protein, indicating this is likely the major host specificity determinant. Our findings provide the foundations for future comparative analyses to study mushroom disease and phage resistance.

The Concerted Action of Two B3-Like Prophage Genes Excludes Superinfecting Bacteriophages by Blocking DNA Entry into Pseudomonas aeruginosa.

In this study, we describe seven vegetative phage genomes homologous to the historic phage B3 that infect Pseudomonas aeruginosa Like other phage groups, the B3-like group contains conserved (core) and variable (accessory) open reading frames (ORFs) grouped at fixed regions in their genomes; however, in either case, many ORFs remain without assigned functions. We constructed lysogens of the seven B3-like phages in strain Ps33 of P. aeruginosa, a novel clinical isolate, and assayed the exclusion phenotype against a variety of temperate and virulent superinfecting phages. In addition to the classic exclusion conferred by the phage immunity repressor, the phenotype observed in B3-like lysogens suggested the presence of other exclusion genes. We set out to identify the genes responsible for this exclusion phenotype. Phage Ps56 was chosen as the study subject since it excluded numerous temperate and virulent phages. Restriction of the Ps56 genome, cloning of several fragments, and resection of the fragments that retained the exclusion phenotype allowed us to identify two core ORFs, so far without any assigned function, as responsible for a type of exclusion. Neither gene expressed separately from plasmids showed activity, but the concurrent expression of both ORFs is needed for exclusion. Our data suggest that phage adsorption occurs but that phage genome translocation to the host's cytoplasm is defective. To our knowledge, this is the first report on this type of exclusion mediated by a prophage in P. aeruginosaIMPORTANCEPseudomonas aeruginosa is a Gram-negative bacterium frequently isolated from infected immunocompromised patients, and the strains are resistant to a broad spectrum of antibiotics. Recently, the use of phages has been proposed as an alternative therapy against multidrug-resistant bacteria. However, this approach may present various hurdles. This work addresses the problem that pathogenic bacteria may be lysogenized by phages carrying genes encoding resistance against secondary infections, such as those used in phage therapy. Discovering phage genes that exclude superinfecting phages not only assigns novel functions to orphan genes in databases but also provides insight into selection of the proper phages for use in phage therapy.

Structure and conformational cycle of a bacteriophage-encoded chaperonin.

Chaperonins are ubiquitous molecular chaperones found in all domains of life. They form ring-shaped complexes that assist in the folding of substrate proteins in an ATP-dependent reaction cycle. Key to the folding cycle is the transient encapsulation of substrate proteins by the chaperonin. Here we present a structural and functional characterization of the chaperonin gp146 (ɸEL) from the phage EL of Pseudomonas aeruginosa. ɸEL, an evolutionarily distant homolog of bacterial GroEL, is active in ATP hydrolysis and prevents the aggregation of denatured protein in a nucleotide-dependent manner. However, ɸEL failed to refold the encapsulation-dependent model substrate rhodanese and did not interact with E. coli GroES, the lid-shaped co-chaperone of GroEL. ɸEL forms tetradecameric double-ring complexes, which dissociate into single rings in the presence of ATP. Crystal structures of ɸEL (at 3.54 and 4.03 Å) in presence of ATP•BeFx revealed two distinct single-ring conformational states, both with open access to the ring cavity. One state showed uniform ATP-bound subunit conformations (symmetric state), whereas the second combined distinct ATP- and ADP-bound subunit conformations (asymmetric state). Cryo-electron microscopy of apo-ɸEL revealed a double-ring structure composed of rings in the asymmetric state (3.45 Å resolution). We propose that the phage chaperonin undergoes nucleotide-dependent conformational switching between double- and single rings and functions in aggregation prevention without substrate protein encapsulation. Thus, ɸEL may represent an evolutionarily more ancient chaperonin prior to acquisition of the encapsulation mechanism.

Isolation and characterization of bacteriophage to control multidrug-resistant Pseudomonas aeruginosa planktonic cells and biofilm.

Pseudomonas aeruginosa is Gram-negative bacterium, one of the leading cause of drug-resistant nosocomial infections in developing countries. This bacterium possesses chromosomally encoded efflux pumps, poor permeability of outer-membrane and high tendency for biofilm formation which are tools to confer resistance. Bacteriophages are regarded as feasible treatment option for control of resistant P. aeruginosa. The aim of the current study was isolate and characterized a bacteriophage against P. aeruginosa with MDR and biofilm ability. A bacteriophage MA-1 with moderate host range was isolated from waste water. The phage was considerable heat and pH stable. Electron microscopy revealed that phage MA-1 belongs to Myoviridae family. Its genome was dsDNA (≈50 kb), coding for eighteen different proteins (ranging from 12 to 250 KDa). P. aeruginosa-2949 log growth phase was significantly reduced by phage MA-1 (2.5 × 103 CFU/ml) as compared to control (without phage). Phage MA-1 also showed significant reductions of 2.0, 2.5 and 3.2 folds in 24, 48, and 74 h old biofilms after 6 h treatment with phage respectively as compared to control. It was concluded from this study that phage MA-1 has capability of killing P. aeruginosa planktonic cells and biofilm, but for complete eradication cocktail will more effective to avoid resistance.

'Drc', a structurally novel ssDNA-binding transcription regulator of N4-related bacterial viruses.

Bacterial viruses encode a vast number of ORFan genes that lack similarity to any other known proteins. Here, we present a 2.20 Å crystal structure of N4-related Pseudomonas virus LUZ7 ORFan gp14, and elucidate its function. We demonstrate that gp14, termed here as Drc (ssDNA-binding RNA Polymerase Cofactor), preferentially binds single-stranded DNA, yet contains a structural fold distinct from other ssDNA-binding proteins (SSBs). By comparison with other SSB folds and creation of truncation and amino acid substitution mutants, we provide the first evidence for the binding mechanism of this unique fold. From a biological perspective, Drc interacts with the phage-encoded RNA Polymerase complex (RNAPII), implying a functional role as an SSB required for the transition from early to middle gene transcription during phage infection. Similar to the coliphage N4 gp2 protein, Drc likely binds locally unwound middle promoters and recruits the phage RNA polymerase. However, unlike gp2, Drc does not seem to need an additional cofactor for promoter melting. A comparison among N4-related phage genera highlights the evolutionary diversity of SSB proteins in an otherwise conserved transcription regulation mechanism.

Viral Capsid Trafficking along Treadmilling Tubulin Filaments in Bacteria.

Cargo trafficking along microtubules is exploited by eukaryotic viruses, but no such examples have been reported in bacteria. Several large Pseudomonas phages assemble a dynamic, tubulin-based (PhuZ) spindle that centers replicating phage DNA sequestered within a nucleus-like structure. Here, we show that capsids assemble on the membrane and then move rapidly along PhuZ filaments toward the phage nucleus for DNA packaging. The spindle rotates the phage nucleus, distributing capsids around its surface. PhuZ filaments treadmill toward the nucleus at a constant rate similar to the rate of capsid movement and the linear velocity of nucleus rotation. Capsids become trapped along mutant static PhuZ filaments that are defective in GTP hydrolysis. Our results suggest a transport and distribution mechanism in which capsids attached to the sides of filaments are trafficked to the nucleus by PhuZ polymerization at the poles, demonstrating that the phage cytoskeleton evolved cargo-trafficking capabilities in bacteria.

Meet the Anti-CRISPRs: Widespread Protein Inhibitors of CRISPR-Cas Systems.

The constant selective pressure exerted by phages, the viruses that infect bacteria, has led to the evolution of a wide range of anti-phage defenses. One of these defense mechanisms, CRISPR-Cas, provides an adaptive immune system to battle phage infection and inhibit horizontal gene transfer by plasmids, transposons, and other mobile genetic elements. Although CRISPR-Cas systems are widespread in bacteria and archaea, they appear to have minimal long-term evolutionary effects with respect to limiting horizontal gene transfer. One factor that may contribute to this may be the presence of potent inhibitors of CRISPR-Cas systems, known as anti-CRISPR proteins. Forty unique families of anti-CRISPR proteins have been described to date. These inhibitors, which are active against both Class 1 and 2 CRISPR-Cas systems, have a wide range of mechanisms of activity. Studies of these proteins have provided important insight into the evolutionary arms race between bacteria and phages, and have contributed to the development of biotechnological tools that can be harnessed for control of CRISPR-Cas genome editing.

The study of the phiKZ phage non-canonical non-virion RNA polymerase.

Non-canonical multisubunit DNA-dependent RNA-polymerases (RNAP) form a new group of the main transcription enzymes, which have only distinct homology to the catalytic subunits of canonical RNAPs of bacteria, archaea and eukaryotes. One of the rare non-canonical RNAP, which was partially biochemically characterized, is non-virion RNAP (nvRNAP) encoded by Pseudomonas phage phiKZ. PhiKZ nvRNAP consists of five subunits, four of which are homologs of ß and ß' subunit of bacterial RNAP, and the fifth subunits with unknown function. To understand the role of the fifth subunit in phiKZ nvRNAP, we created co-expression system allowing to get recombinant full five-subunit (5s) and four-subunit (4s) complexes and performed their comparison. The 5s recombinant complex is active on phage promoters in vitro as the native nvRNAP. The 4s complex cannot extend RNA, so 4s complex is not a catalytically active core of phiKZ nvRNAP. Thus, the phiKZ fifth subunit is not only a promoter-recognition subunit, but it plays an important role in the formation of active phiKZ nvRNAP.