Analysis of a new phage, KZag1, infecting biofilm of Klebsiella pneumoniae: genome sequence and characterization.

BACKGROUND: This study investigates the effectiveness of the bacteriophage KZag1 against drug-resistant Klebsiella pneumoniae, aiming to assess its potential as a therapeutic agent. The novelty lies in the characterization of KZag1, a Myovirus with specific efficacy against multidrug-resistant K. pneumoniae strains. This highlights the significance of exploring alternative strategies, particularly phage therapy, in addressing biofilm-associated infections. METHODS: KZag1, characterized by a typical Myovirus structure with a 75 ± 5 nm diameter icosahedral head and a 15 ± 5 nm short tail, was evaluated in experimental trials against 15 strains of K. pneumoniae. The infection cycle duration was determined to be 50 min, resulting in an estimated burst size of approximately 83 plaque-forming units per colony-forming unit (PFU/CFU). Stability assessments were conducted within a pH range of 4 to 12 and temperatures ranging from 45°C to 60°C. Biofilm biomass reduction was observed, particularly at a multiplicity of infection (MOI) of 10. RESULTS: KZag1 demonstrated infection efficacy against 12 out of 15 tested K. pneumoniae strains. The phage exhibited stability across a broad pH range and at elevated temperatures. Notably, treatment with KZag1 significantly reduced K. pneumoniae biofilm biomass, emphasizing its potential in combating biofilm formation. Genomic analysis revealed a complete genome of 157,089 base pairs with a GC content of 46.38%, encompassing 203 open reading frames (ORFs) and a cysteine-specific tRNA sequence. Comparison with phage GP4 highlighted similarities, with KZag1 having a longer genome by approximately 4829 base pairs and a higher GC content by approximately 0.93%. Phylogenetic analysis classified KZag1 within the Myoviridae family. CONCLUSION: The efficacy of KZag1 against K. pneumoniae biofilm suggests its potential as a therapeutic candidate, especially for drug-resistant infections. Further clinical research is warranted to explore its synergy with other treatments, elucidate genomic traits, compare with Myoviridae phages, and understand its host interactions. These findings underscore the promising role of KZag1 in addressing drug-resistant bacterial infections.

Revealing the Viral Community in the Hadal Sediment of the New Britain Trench.

Marine viruses are widely distributed and influence matter and energy transformation in ecosystems by modulating hosts' metabolism. The hadal trenches represent the deepest marine habitat on Earth, for which the viral communities and related biogeochemical functions are least explored and poorly understood. Here, using the sediment samples (8720 m below sea level) collected from the New Britain Trench (NBT), we investigated the viral community, diversity, and genetic potentials in the hadal sediment habitat for the first time by deep shotgun metagenomic sequencing. We found the NBT sediment viral community was dominated by Siphoviridae, Myoviridae, Podoviridae, Mimiviridae, and Phycodnaviridae, which belong to the dsDNA viruses. However, the large majority of them remained uncharacterized. We found the hadal sediment virome had some common components by comparing the hadal sediment viruses with those of hadal aquatic habitats and those of bathypelagic and terrestrial habitats. It was also distinctive in community structure and had many novel viral clusters not associated with the other habitual virome included in our analyses. Further phylogenetic analysis on its Caudovirales showed novel diversities, including new clades specially evolved in the hadal sediment habitat. Annotation of the NBT sediment viruses indicated the viruses might influence microbial hydrocarbon biodegradation and carbon and sulfur cycling via metabolic augmentation through auxiliary metabolic genes (AMGs). Our study filled in the knowledge gaps on the virome of the hadal sediment habitats and provided insight into the evolution and the potential metabolic functions of the hadal sediment virome.

Phage vB_BveM-Goe7 represents a new genus in the subfamily Bastillevirinae.

Bacillus velezensis FZB42 is a Gram-positive, endospore-forming rhizobacterium that is associated with plant roots and promotes plant growth. It was used as host to isolate phage vB_BveM-Goe7 (Goe7). Goe7 exhibits a Myoviridae morphology with a contractile tail and an icosahedral head. Its genome is 158,674 bp in size and contains 5137-bp-long terminal repeats (LTRs). It also contains five tRNA-encoding genes and 251 coding DNA sequences (CDS), of which 65 were annotated. The adsorption constant of Goe7 is 6.1 ± 0.24 × 10-8 ml/min, with a latency period of 75 min and a burst size of 114 particles per burst. A BLASTn sequence comparison against the non-redundant nucleotide database of NCBI revealed that Goe7 is most similar to Bacillus subtilis phage vB_BsuM-Goe3.

Expanding the Diversity of Myoviridae Phages Infecting Lactobacillus plantarum-A Novel Lineage of Lactobacillus Phages Comprising Five New Members.

Lactobacillus plantarum is a bacterium with probiotic properties and promising applications in the food industry and agriculture. So far, bacteriophages of this bacterium have been moderately addressed. We examined the diversity of five new L. plantarum phages via whole genome shotgun sequencing and in silico protein predictions. Moreover, we looked into their phylogeny and their potential genomic similarities to other complete phage genome records through extensive nucleotide and protein comparisons. These analyses revealed a high degree of similarity among the five phages, which extended to the vast majority of predicted virion-associated proteins. Based on these, we selected one of the phages as a representative and performed transmission electron microscopy and structural protein sequencing tests. Overall, the results suggested that the five phages belong to the family Myoviridae, they have a long genome of 137,973-141,344 bp, a G/C content of 36.3-36.6% that is quite distinct from their host's, and surprisingly, 7 to 15 tRNAs. Only an average 41/174 of their predicted genes were assigned a function. The comparative analyses unraveled considerable genetic diversity for the five L. plantarum phages in this study. Hence, the new genus "Semelevirus" was proposed, comprising exclusively of the five phages. This novel lineage of Lactobacillus phages provides further insight into the genetic heterogeneity of phages infecting Lactobacillus sp. The five new Lactobacillus phages have potential value for the development of more robust starters through, for example, the selection of mutants insensitive to phage infections. The five phages could also form part of phage cocktails, which producers would apply in different stages of L. plantarum fermentations in order to create a range of organoleptic outputs.

Role of SH3b binding domain in a natural deletion mutant of Kayvirus endolysin LysF1 with a broad range of lytic activity.

The spontaneous host-range mutants 812F1 and K1/420 are derived from polyvalent phage 812 that is almost identical to phage K, belonging to family Myoviridae and genus Kayvirus. Phage K1/420 is used for the phage therapy of staphylococcal infections. Endolysin of these mutants designated LysF1, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase (CHAP) domain and C-terminal SH3b cell wall-binding domain, has deleted middle amidase domain compared to wild-type endolysin. In this work, LysF1 and both its domains were prepared as recombinant proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus species of the 43 tested. SH3b domain influenced antimicrobial activity of LysF1, since the lytic activity of the truncated variant containing the CHAP domain alone was decreased. The results of a co-sedimentation assay of SH3b domain showed that it was able to bind to three types of purified staphylococcal peptidoglycan 11.2, 11.3, and 11.8 that differ in their peptide bridge, but also to the peptidoglycan type 11.5 of Streptococcus uberis, and this capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy. Using several different approaches, including NMR, we have not confirmed the previously proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell wall. The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic spectrum, and therefore is an appropriate enzyme for practical use. The binding spectrum of SH3b domain covering all known staphylococcal peptidoglycan types is a promising feature for creating new chimeolysins by combining it with more effective catalytic domains.

Isolation and Characterization of Bacteriophages Against Pseudomonas syringae pv. actinidiae Causing Bacterial Canker Disease in Kiwifruit.

Pseudomonas syringae pv. actinidiae causes bacterial canker disease in kiwifruit. Owing to the prohibition of agricultural antibiotic use in major kiwifruit-cultivating countries, alternative methods need to be developed to manage this disease. Bacteriophages are viruses that specifically infect target bacteria and have recently been reconsidered as potential biological control agents for bacterial pathogens owing to their specificity in terms of host range. In this study, we isolated bacteriophages against P. syringae pv. actinidiae from soils collected from kiwifruit orchards in Korea and selected seven bacteriophages for further characterization based on restriction enzyme digestion patterns of genomic DNA. Among the studied bacteriophages, two belong to the Myoviridae family and three belong to the Podoviridae family, based on morphology observed by transmission electron microscopy. The host range of the selected bacteriophages was confirmed using 18 strains of P. syringae pv. actinidiae, including the Psa2 and Psa3 groups, and some were also effective against other P. syringae pathovars. Lytic activity of the selected bacteriophages was sustained in vitro until 80 h, and their activity remained stable up to 50°C, at pH 11, and under UV-B light. These results indicate that the isolated bacteriophages are specific to P. syringae species and are resistant to various environmental factors, implying their potential use in control of bacterial canker disease in kiwifruits.

Effects of actin-like proteins encoded by two Bacillus pumilus phages on unstable lysogeny, revealed by genomic analysis.

We characterized two newly isolated myoviruses, Bp8p-C and Bp8p-T, infecting the ginger rhizome rot disease pathogen Bacillus pumilus GR8. The plaque of Bp8p-T exhibited a clear center with a turbid rim, suggesting that Bp8p-T could transform into latent phage. Lysogeny assays showed that both the two phages could form latent states, while Bp8p-T could form latent phage at a higher frequency and stability than Bp8p-C. The genomes of Bp8p-C and Bp8p-T were 151,417 and 151,419 bp, respectively; both encoded 212 putative proteins, and only differed by three nucleotides. Moreover, owing to this difference, Bp8p-C encoded a truncated, putative actin-like plasmid segregation protein Gp27-C. Functional analysis of protein Gp27 showed that Gp27-T encoded by Bp8p-T exhibited higher ATPase activity and assembly ability than Gp27-C. The results indicate that the difference in Gp27 affected the phage lysogenic ability. Structural proteome analysis of Bp8p-C virion resulted in the identification of 14 structural proteins, among which a pectin lyase-like protein, a putative poly-gamma-glutamate hydrolase, and three proteins with unknown function, were firstly identified as components of the phage virion. Both phages exhibited specific lytic ability to the host strain GR8. Bp8p-C showed better control effect on the pathogen in ginger rhizome slices than Bp8p-T, suggesting that Bp8p-C has a potential application in bio-control of ginger rhizome rot disease.

The odd one out: Bacillus ACT bacteriophage CP-51 exhibits unusual properties compared to related Spounavirinae W.Ph. and Bastille.

The Bacillus ACT group includes three important pathogenic species of Bacillus: anthracis, cereus and thuringiensis. We characterized three virulent bacteriophages, Bastille, W.Ph. and CP-51, that infect various strains of these three species. We have determined the complete genome sequences of CP-51, W.Ph. and Bastille, and their physical genome structures. The CP-51 genome sequence could only be obtained using a combination of conventional and second and third next generation sequencing technologies - illustrating the problems associated with sequencing highly modified DNA. We present evidence that the generalized transduction facilitated by CP-51 is independent of a specific genome structure, but likely due to sporadic packaging errors of the terminase. There is clear correlation of the genetic and morphological features of these phages validating their placement in the Spounavirinae subfamily (SPO1-related phages) of the Myoviridae. This study also provides tools for the development of phage-based diagnostics/therapeutics for this group of pathogens.

Discovery and characterization of RecA protein of thermophilic bacterium Thermus thermophilus MAT72 phage Tt72 that increases specificity of a PCR-based DNA amplification.

The recA gene of newly discovered Thermus thermophilus MAT72 phage Tt72 (Myoviridae) was cloned and overexpressed in Escherichia coli. The 1020-bp gene codes for a 339-amino-acid polypeptide with an Mr of 38,155 which shows 38.7% positional identity to the E. coli RecA protein. When expressed in E. coli, the Tt72 recA gene did not confer the ability to complement the ultraviolet light (254nm) sensitivity of an E. coli recA mutant. Tt72 RecA protein has been purified with good yield to catalytic and electrophoretic homogeneity using a three-step chromatography procedure. Biochemical characterization indicated that the protein can pair and promote ATP-dependent strand exchange reaction resulting in formation of a heteroduplex DNA at 60°C under conditions otherwise optimal for E. coli RecA. When the Tt72 RecA protein was included in a standard PCR-based DNA amplification reaction, the specificity of the PCR assays was significantly improved by eliminating non-specific products.

Genome and proteome analysis of phage E3 infecting the soil-borne actinomycete Rhodococcus equi.

We report on the characterization and genomic analysis of bacteriophage E3 isolated from soil and propagating in Rhodococcus equi strains. Phage E3 has a circular genome of 142 563 bp and is the first Myoviridae reported for the genus Rhodococcus and for a non-mycobacterial actinomycete. Phylogenetic analyses placed E3 in a distinct Myoviridae clade together with Mycobacterium phages Bxz1 and Myrna. The highly syntenic genomes of this myoviridal group comprise vertically evolving core phage modules flanked by hyperplastic regions specific to each phage and rich in horizontally acquired DNA. The hyperplastic regions contain numerous tRNA genes in the mycobacteriophages which are absent in E3, possibly reflecting bacterial host-specific translation-related phage fitness constraints associated with rate-limiting tRNAs. A structural proteome analysis identified 28 E3 polypeptides, including 15 not previously known to be virion-associated proteins. The E3 genome and comparative analysis provide insight into short-term genome evolution and adaptive plasticity in tailed phages from the environmental microbiome.

Bacteriophages φMR299-2 and φNH-4 can eliminate Pseudomonas aeruginosa in the murine lung and on cystic fibrosis lung airway cells.

UNLABELLED: Pseudomonas aeruginosa is a common cause of infection in the lungs of patients with cystic fibrosis (CF). In addition, biofilm formation and antibiotic resistance of Pseudomonas are major problems that can complicate antibiotic therapy. We evaluated the efficacy of using bacteriophages to kill the pathogen in both biofilms and in the murine lung. We isolated and characterized two phages from a local wastewater treatment plant, a myovirus (φNH-4) and a podovirus (φMR299-2). Both phages were active against clinical isolates of P. aeruginosa. Together, the two phages killed all 9 clinical isolate strains tested, including both mucoid and nonmucoid strains. An equal mixture of the two phages was effective in killing P. aeruginosa NH57388A (mucoid) and P. aeruginosa MR299 (nonmucoid) strains when growing as a biofilm on a cystic fibrosis bronchial epithelial CFBE41o- cell line. Phage titers increased almost 100-fold over a 24-h period, confirming replication of the phage. Furthermore, the phage mix was also effective in killing the pathogen in murine lungs containing 1 × 10(7) to 2 × 10(7) P. aeruginosa. Pseudomonas was effectively cleared (reduced by a magnitude of at least 3 to 4 log units) from murine lungs in 6 h. Our study demonstrates the efficacy of these two phages in killing clinical Pseudomonas isolates in the murine lung or as a biofilm on a pulmonary cell line and supports the growing interest in using phage therapy for the control and treatment of multidrug-resistant Pseudomonas lung infections in CF patients. IMPORTANCE: Given the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment of Pseudomonas infection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can kill Pseudomonas growing on human lung cells and in an animal model of lung infection. The use of bacteriophages is particularly appropriate because the killing agent can replicate on the target cell, generating fresh copies of the bacteriophage. Thus, in the presence of a target, the killing agent multiplies. By using two bacteriophages we can reduce the risk of resistant colonies developing at the site of infection. Bacteriophage therapy is an exciting field, and this study represents an important demonstration of efficacy in validated infection models.

A novel bacteriophage Tail-Associated Muralytic Enzyme (TAME) from Phage K and its development into a potent antistaphylococcal protein.

BACKGROUND: Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. However, the rapid emergence of antibiotic resistance limits the choice of therapeutic options for treating infections caused by this organism. Muralytic enzymes from bacteriophages have recently gained attention for their potential as antibacterial agents against antibiotic-resistant gram-positive organisms. Phage K is a polyvalent virulent phage of the Myoviridae family that is active against many Staphylococcus species. RESULTS: We identified a phage K gene, designated orf56, as encoding the phage tail-associated muralytic enzyme (TAME). The gene product (ORF56) contains a C-terminal domain corresponding to cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), which demonstrated muralytic activity on a staphylococcal cell wall substrate and was lethal to S. aureus cells. We constructed N-terminal truncated forms of ORF56 and arrived at a 16-kDa protein (Lys16) that retained antistaphylococcal activity. We then generated a chimeric gene construct encoding Lys16 and a staphylococcal cell wall-binding SH3b domain. This chimeric protein (P128) showed potent antistaphylococcal activity on global clinical isolates of S. aureus including methicillin-resistant strains. In addition, P128 was effective in decolonizing rat nares of S. aureus USA300 in an experimental model. CONCLUSIONS: We identified a phage K gene that encodes a protein associated with the phage tail structure. The muralytic activity of the phage K TAME was localized to the C-terminal CHAP domain. This potent antistaphylococcal TAME was combined with an efficient Staphylococcus-specific cell-wall targeting domain SH3b, resulting in the chimeric protein P128. This protein shows bactericidal activity against globally prevalent antibiotic resistant clinical isolates of S. aureus and against the genus Staphylococcus in general. In vivo, P128 was efficacious against methicillin-resistant S. aureus in a rat nasal colonization model.

Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments.

T4-like myoviruses are ubiquitous, and their genes are among the most abundant documented in ocean systems. Here we compare 26 T4-like genomes, including 10 from non-cyanobacterial myoviruses, and 16 from marine cyanobacterial myoviruses (cyanophages) isolated on diverse Prochlorococcus or Synechococcus hosts. A core genome of 38 virion construction and DNA replication genes was observed in all 26 genomes, with 32 and 25 additional genes shared among the non-cyanophage and cyanophage subsets, respectively. These hierarchical cores are highly syntenic across the genomes, and sampled to saturation. The 25 cyanophage core genes include six previously described genes with putative functions (psbA, mazG, phoH, hsp20, hli03, cobS), a hypothetical protein with a potential phytanoyl-CoA dioxygenase domain, two virion structural genes, and 16 hypothetical genes. Beyond previously described cyanophage-encoded photosynthesis and phosphate stress genes, we observed core genes that may play a role in nitrogen metabolism during infection through modulation of 2-oxoglutarate. Patterns among non-core genes that may drive niche diversification revealed that phosphorus-related gene content reflects source waters rather than host strain used for isolation, and that carbon metabolism genes appear associated with putative mobile elements. As well, phages isolated on Synechococcus had higher genome-wide %G+C and often contained different gene subsets (e.g. petE, zwf, gnd, prnA, cpeT) than those isolated on Prochlorococcus. However, no clear diagnostic genes emerged to distinguish these phage groups, suggesting blurred boundaries possibly due to cross-infection. Finally, genome-wide comparisons of both diverse and closely related, co-isolated genomes provide a locus-to-locus variability metric that will prove valuable for interpreting metagenomic data sets.

[Physico-chemical properties of temperate bacteriophage ZF40 of Erwinia carotovora].

A method of efficient purification of the extremely instable virions of the temperate bacteriophage ZF40 of Erwinia carotovora was proposed in the work. The stage-by-stage centrifugation in mentrizamide and caesium chloride gradients permitted to obtain highly purified phage preparations which were used for studying the protein composition of the studied erwiniophage. It was established that the buoyant density of the phage ZF40 was 1.23 g/cm3, 1.49 g/cm3 and 1.27 g/cm3 in the gradients of metrizamide, caesium chloride and caesium sulphate, respectively. By the polypeptide composition of the virion the phage ZF40 has been included in the group of P2-like phages.

Studies on synonymous codon and amino acid usage biases in the broad-host range bacteriophage KVP40.

In this study, the relative synonymous codon and amino acid usage biases of the broad-host range phage, KVP40, were investigated in an attempt to understand the structure and function of its proteins/protein-coding genes, as well as the role of its tRNAs. Synonymous codons in KVP40 were determined to be ATrich at the third codon positions, and their variations are dictated principally by both mutational bias and translational selection. Further analysis revealed that the RSCU of KVP40 is distinct from that of its Vibrio hosts, V. cholerae and V. parahaemolyticus. Interestingly, the expression of the putative highly expressed genes of KVP40 appear to be preferentially influenced by the abundant host tRNA species, whereas the tRNAs expressed by KVP40 may be required for the efficient synthesis of all its proteins in a diverse array of hosts. The data generated in this study also revealed that KVP40 proteins are rich in low molecular weight amino acid residues, and that these variations are influenced primarily by hydropathy, mean molecular weight, aromaticity, and cysteine content.

Inversion within the haloalkaliphilic virus phi Ch1 DNA results in differential expression of structural proteins.

The sequence of phi Ch1 contains an open reading frame (int1) in the central part of its genome that belongs to the lambda integrase family of site-specific recombinases. Sequence similarities to known integrases include the highly conserved tetrad R-H-R-Y. The flanking sequences of int1 contain several direct repeats of 30 bp in length (IR-L and IR-R), which are orientated in an inverted direction. Here, we show that a recombination active region exists in the genome of phi Ch1: the number of those repeats, non-homologous regions within the repeat clusters IR-L and IR-R and the orientation of the int1 gene vary in a given virus population. Within this study, we identified circular intermediates, composed of the int1 gene and the inwards orientated repeat regions IR-L and IR-R, which could be involved in the recombination process itself. IR-L and IR-R are embedded within ORF34 and ORF36 respectively. As a consequence of the inversion within this region of phi Ch1, the C-terminal parts of the proteins encoded by ORF34 and 36 are exchanged. Both proteins, expressed in Escherichia coli, interact with specific antisera against whole virus particles, indicating that they could be parts of phi Ch1 virions. Expression of the protein(s) in Natrialba magadii could be detected 98 h after inoculation, which is similar to other structural proteins of phi Ch1. Taken together, the data show that the genome of phi Ch1 contains an invertible region that codes for a recombinase and structural proteins. Inversion of this segment results in a variation of these structural proteins.

The structural protein E of the archaeal virus phiCh1: evidence for processing in Natrialba magadii during virus maturation.

phiCh1 is a lysogenic virus for the haloalkalophilic archaeon Natrialba magadii. The virus morphology resembles other members of Myoviridae infecting Halobacterium species. The gene of the major capsid protein E of virus phiCh1 was cloned and the DNA sequence was determined. Gene E was mapped to a 3.2-kbp ClaI fragment, localized to the 5'-end of the phiCh1 genome. The complete nucleotide sequence of this region was determined and the identity of gene E was confirmed by comparing the experimentally determined N-terminal amino acid sequence of the purified protein to the translated DNA sequence of its open reading frame. We present evidence that the gene E product is proteolytically cleaved between Lys(16) and Asn(17) to yield the 305 residue polypeptides found in the mature viral capsid. Processing of the protein itself during virus development was determined by 2D gel electrophoresis using protein E-specific antibodies. Sequence similarity studies revealed an 80% identity to capsid protein Hp32 of phiH, infecting Halobacterium salinarum. RT-PCR analysis as well as Western blot studies revealed gene E as a late gene. Transcripts and proteins could be detected shortly before onset of lysis of the lysogenic strain N. magadii L11.

Resolution of the 1H-1H NOE spectrum of RNA into three dimensions using 15N-1H two-bond couplings.

The feasibility of using two-bond 15N-1H couplings to resolve the 1H-1H nuclear Overhauser effect spectrum of RNA into a third dimension was investigated, using the 36-nucleotide gene 32 messenger RNA pseudoknot of bacteriophage T2 as an example. The two-bond 15N-1H couplings present in adenosine and guanosine were found to be suitable for generating a three-dimensional 1H-1H-15N NOESY-HSQC spectrum with reasonably good sensitivity, as well as favorable chemical shift dispersion in the nitrogen dimension. The described NMR experiment provides a tool that can be used to complement other heteronuclear methods in the analysis of RNA structure.

Discovery of the tail tube gene of bacteriophage Mu and sequence analysis of the sheath and tube genes.

The nucleotide sequence was determined for 2.75 kbp of phage Mu DNA encoding the contractile tail sheath protein L. N-terminal sequence analysis of Mu tail tube and sheath proteins identified the open reading frame just downstream of gene L as the tube gene. This clustering and order of the sheath and tube genes appear to be common among the myoviridae. Database homology searches revealed high similarity between the Mu sheath and tube proteins and two proteins in a Haemophilus influenzae Mu-like prophage, suggesting that they are the sheath and tube proteins of that prophage.

Comparative studies of the phage T2 and T4 DNA (N6-adenine)methyltransferases: amino acid changes that affect catalytic activity.

The bacteriophage T2 and T4 dam genes code for a DNA (N6-adenine)methyltransferase (MTase). Nonglucosylated, hydroxymethylcytosine-containing T2gt- virion DNA has a higher level of methylation than T4gt- virion DNA does. To investigate the basis for this difference, we compared the intracellular enzyme levels following phage infection as well as the in vitro intrinsic methylation capabilities of purified T2 and T4 Dam MTases. Results from Western blotting (immunoblotting) showed that the same amounts of MTase protein were produced after infection with T2 and T4. Kinetic analyses with purified homogeneous enzymes showed that the two MTases had similar Km values for the methyl donor, S-adenosyl-L-methionine, and for substrate DNA. In contrast, they had different k(cat) values (twofold higher for T2 Dam MTase). We suggest that this difference can account for the ability of T2 Dam to methylate viral DNA in vivo to a higher level than does T4 Dam. Since the T2 and T4 MTases differ at only three amino acid residues (at positions 20 [T4, Ser; T2, Pro], 26 [T4, Asn; T2, Asp], and 188 [T4, Asp; T2, Glu]), we have produced hybrid proteins to determine which residue(s) is responsible for increased catalytic activity. The results of these analyses showed that the residues at positions 20 and 26 are responsible for the different k(cat) values of the two MTases for both canonical and noncanonical sites. Moreover, a single substitution of either residue 20 or 26 was sufficient to increase the k(cat) of T4 Dam.