Bacteriophage F1 Research Articles

Atomic Structure of Bacteriophage Sf6 Tail Needle Knob

Podoviridae are double-stranded DNA bacteriophages that use short, non-contractile tails to adsorb to the host cell surface. Within the tail apparatus of P22-like phages, a dedicated fiber known as the "tail needle" likely functions as a cell envelope-penetrating device to promote ejection of viral DNA inside the host. In Sf6, a P22-like phage that infects Shigella flexneri, the tail needle presents a C-terminal globular knob. This knob, absent in phage P22 but shared in other members of the P22-like genus, represents the outermost exposed tip of the virion that contacts the host cell surface. Here, we report a crystal structure of the Sf6 tail needle knob determined at 1.0 Å resolution. The structure reveals a trimeric globular domain of the TNF fold structurally superimposable with that of the tail-less phage PRD1 spike protein P5 and the adenovirus knob, domains that in both viruses function in receptor binding. However, P22-like phages are not known to utilize a protein receptor and are thought to directly penetrate the host surface. At 1.0 Å resolution, we identified three equivalents of l-glutamic acid (l-Glu) bound to each subunit interface. Although intimately bound to the protein, l-Glu does not increase the structural stability of the trimer nor it affects its ability to self-trimerize in vitro. In analogy to P22 gp26, we suggest the tail needle of phage Sf6 is ejected through the bacterial cell envelope during infection and its C-terminal knob is threaded through peptidoglycan pores formed by glycan strands.

Preliminary Studies of Bacteriophage Sf6 Virions: Icosahedral, Asymmetric, and Tomographic Reconstructions

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

EXPERIMENTAL EVOLUTION OF RNA VERSUS DNA VIRUSES

Based on their extremely high mutation rates, RNA viruses have been traditionally considered as the fastest evolving entities in nature. However, recent work has revealed that, despite their greater replication fidelity, single-stranded (ss) DNA viruses can evolve fast in a similar way. To further investigate this issue, we have compared the rates of adaptation and molecular evolution of ssRNA and ssDNA viruses under highly controlled laboratory conditions using the bacteriophages ΦX174, G4, f1, Qβ, SP, and MS2 as model systems. Our results indicate that ssRNA phages evolve faster than ssDNA phages under strong selective pressure, and that their extremely high mutation rates appear to be optimal for this kind of scenario. However, their performance becomes similar to that of ssDNA phages over the longer term or when the population is moderately well-adapted. Interestingly, the roughly 100-fold difference between the mutation rates of ssRNA and ssDNA phages yields less than a fivefold difference in adaptation and nucleotide substitution rates. The results are therefore consistent with the observation that, despite their lower mutation rates, ssDNA viruses can sometimes match the evolvability of RNA viruses.

Specificity of Interactions among the DNA-packaging Machine Components of T4-related Bacteriophages

Tailed bacteriophages use powerful molecular motors to package the viral genome into a preformed capsid. Packaging at a rate of up to ∼2000 bp/s and generating a power density twice that of an automobile engine, the phage T4 motor is the fastest and most powerful reported to date. Central to DNA packaging are dynamic interactions among the packaging components, capsid (gp23), portal (gp20), motor (gp17, large "terminase"), and regulator (gp16, small terminase), leading to precise orchestration of the packaging process, but the mechanisms are poorly understood. Here we analyzed the interactions between small and large terminases of T4-related phages. Our results show that the gp17 packaging ATPase is maximally stimulated by homologous, but not heterologous, gp16. Multiple interaction sites are identified in both gp16 and gp17. The specificity determinants in gp16 are clustered in the diverged N- and C-terminal domains (regions I-III). Swapping of diverged region(s), such as replacing C-terminal RB49 region III with that of T4, switched ATPase stimulation specificity. Two specificity regions, amino acids 37-52 and 290-315, are identified in or near the gp17-ATPase "transmission" subdomain II. gp16 binding at these sites might cause a conformational change positioning the ATPase-coupling residues into the catalytic pocket, triggering ATP hydrolysis. These results lead to a model in which multiple weak interactions between motor and regulator allow dynamic assembly and disassembly of various packaging complexes, depending on the functional state of the packaging machine. This might be a general mechanism for regulation of the phage packaging machine and other complex molecular machines.

The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion

Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379–394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus may be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.

Exposure of conjugative plasmid carrying Escherichia coli biofilms to male-specific bacteriophages

Escherichia coli carrying a natural conjugative F-plasmid generates F-pili mating pairs, which is important for early biofilm formation. In this study, we investigated the effect of male-specific filamentous single stranded DNA bacteriophage (f1) and RNA bacteriophage (MS2) on the formation of biofilms by E. coli carrying a natural conjugative F-plasmid. We showed that the early biofilm formation was completely inhibited by addition of the f1 phage, but not the MS2 phage. This suggests that the tip of F-pili is the specific attachment site for mating pairs formation and the side of F-pili has a non-obligatory role during biofilm formation. The inhibitory effect of the f1 phage was dependent on the time of addition during the biofilm formation. No inhibitory effect was observed when the f1 phages were added to the mature biofilms. This resistant mechanism of the mature biofilms could be attributed to the biofilm-specific phenotypes representing that the F-pili mating pairs were already formed and then the curli production commenced during the biofilm maturation. The pre-formed mating pairs seemed to resist the f1 phages. Altogether, our results indicate a close relationship between the presence of conjugative plasmid and male-specific bacteriophages within sessile biofilm communities, as well as the possibility of using the male-specific bacteriophages to control biofilm formation.

The K5 Lyase KflA Combines a Viral Tail Spike Structure with a Bacterial Polysaccharide Lyase Mechanism

K5 lyase A (KflA) is a tail spike protein (TSP) encoded by a K5A coliphage, which cleaves K5 capsular polysaccharide, a glycosaminoglycan with the repeat unit [-4)-betaGlcA-(1,4)- alphaGlcNAc(1-], displayed on the surface of Escherichia coli K5 strains. The crystal structure of KflA reveals a trimeric arrangement, with each monomer containing a right-handed, single-stranded parallel beta-helix domain. Stable trimer formation by the intertwining of strands in the C-terminal domain, followed by proteolytic maturation, is likely to be catalyzed by an autochaperone as described for K1F endosialidase. The structure of KflA represents the first bacteriophage tail spike protein combining polysaccharide lyase activity with a single-stranded parallel beta-helix fold. We propose a catalytic site and mechanism representing convergence with the syn-beta-elimination site of heparinase II from Pedobacter heparinus.

Distribution of Fitness Effects Caused by Single-Nucleotide Substitutions in Bacteriophage f1

Empirical knowledge of the fitness effects of mutations is important for understanding many evolutionary processes, yet this knowledge is often hampered by several sources of measurement error and bias. Most of these problems can be solved using site-directed mutagenesis to engineer single mutations, an approach particularly suited for viruses due to their small genomes. Here, we used this technique to measure the fitness effect of 100 single-nucleotide substitutions in the bacteriophage f1, a filamentous single-strand DNA virus. We found that approximately one-fifth of all mutations are lethal. Viable ones reduced fitness by 11% on average and were accurately described by a log-normal distribution. More than 90% of synonymous substitutions were selectively neutral, while those affecting intergenic regions reduced fitness by 14% on average. Mutations leading to amino acid substitutions had an overall mean deleterious effect of 37%, which increased to 45% for those changing the amino acid polarity. Interestingly, mutations affecting early steps of the infection cycle tended to be more deleterious than those affecting late steps. Finally, we observed at least two beneficial mutations. Our results confirm that high mutational sensitivity is a general property of viruses with small genomes, including RNA and single-strand DNA viruses infecting animals, plants, and bacteria.

Determining the filtration efficiency of half-face medical protection mask (N99) against viral aerosol.

Hospital-based outbreaks of severe acute respiratory syndrome (SARS) have once again highlighted the vulnerability of healthcare workers (HCWs). Use of personal respiratory protective equipment was the main method used by HCWs to avoid nosocomial transmission. This paper describes the technology used to evaluate the filtration efficiency of the half-face medical protection mask (N99), manufactured by Firmshield Biotechnology, against viral aerosol. Viral aerosol was generated and then sampled simultaneously with and without the test mask. This enables a percentage efficiency value to be calculated against test phage f2 aerosols (surrogates of viral pathogen aerosols). At the same time the mask filtration efficiency against NaCl particle aerosol was determined by use of TSI8130 equipment and face-fit factor was tested by use of TSI8020 equipment. The half-face medical protection mask (N99) evaluated by use of the viral aerosol had a filtration efficiency >99%. The mask filtration efficiency against NaCl particle aerosol was 99.634 ± 0.024% and it had a good face-fit factor. This half-face medical protection mask (N99) can protect the wearer from viral aerosol disease transmission. The test method can be used to assess filtration efficacy against viral aerosol of masks used for respiratory protection.

Identification of the gate regions in the primary structure of the secretin pIV

Secretins are a family of large bacterial outer membrane channels that serve as exit ports for folded proteins, filamentous phage and surface structures. Despite the large size of their substrates, secretins do not compromise the barrier function of the outer membrane, implying a gating mechanism. The region in the primary structure that forms the putative gate has not previously been determined for any secretin. To identify residues involved in gating the pIV secretin of filamentous bacteriophage f1, we used random mutagenesis of the gene followed by positive selection for mutants with compromised barrier function ('leaky' mutants). We identified mutations in 34 residues, 30 of which were clustered into two regions located in the centre of the conserved C-terminal secretin family domain: GATE1 (that spanned 39 residues) and GATE2 (that spanned 14 residues). An internal deletion constructed in the GATE2 region resulted in a severely leaky phenotype. Three of the four remaining mutations are located in the region that encodes the N-terminal, periplasmic portion of pIV and could be involved in triggering gate opening. Two missense mutations in the 24-residue region that separates GATE1 and GATE2 were also constructed. These mutant proteins were unstable, defective in multimerization and non-functional.

Effects of different sampling solutions on the survival of bacteriophages in bubbling aeration.

The present study focuses on the effects of three different sampling solutions, namely distilled water, phosphatic buffer solution, and suspension medium (SM), adding antifoam or not, on the survival probability of several different bacteriophages (EcP1,PhiX174,SM702, and F2) as surrogates for the mammalian viruses in the bubbling process. AGI-10 impinger was used as the representative for all the impingers which would bubble during operation. It was found that the survival probability of the same bacteriophage bubbling with different sampling solutions was different except that there was no significant difference observed for the bacteriophage F2. The use of SM as the collection fluid was relative to a high survival probability for the four bacteriophages. And the endurance or resistance of different kinds of bacteriophages in the same sampling solution was different. We conclude that SM is a promising sampling solution for liquid impingers in the process of sampling phages.

Modeling the functional consequences of single residue replacements in bacteriophage f1 gene V protein

A computational mutagenesis methodology utilizing a four-body, knowledge-based, statistical contact potential is applied toward globally quantifying relative environmental perturbations (residual scores) in bacteriophage f1 gene V protein (GVP) due to single amino acid substitutions. We show that residual scores correlate well with experimentally measured relative changes in protein function upon mutation. Residual scores also distinguish between GVP amino acid positions grouped according to protein structural or functional roles or based on similarities in physicochemical characteristics. For each mutant, the in silico mutagenesis additionally yields local measures of environmental change (EC scores) occurring at every residue position (residual profile) relative to the native protein. Implementation of the random forest (RF) algorithm, utilizing experimental GVP mutants whose feature vector components include EC scores at the mutated position and at six structurally nearest neighbors, correctly classifies mutants based on function with up to 77% cross-validation accuracy while achieving 0.82 area under the receiver operating characteristic curve. A control experiment highlights the effectiveness of mutant feature vector signals, and a variety of learning curves are generated to analyze the impact of GVP mutant data set size on performance measures. An optimally trained RF model is subsequently used for inferring function for all the remaining unexplored GVP mutants.

Brucella inopinata sp. nov., isolated from a breast implant infection

A Gram-negative, non-motile, non-spore-forming coccoid bacterium (strain BO1(T)) was isolated recently from a breast implant infection of a 71-year-old female patient with clinical signs of brucellosis. Affiliation of strain BO1(T) to the genus Brucella was confirmed by means of polyamine pattern, polar lipid profile, fatty acid profile, quinone system, DNA-DNA hybridization studies and by insertion sequence 711 (IS711)-specific PCR. Strain BO1(T) harboured four to five copies of the Brucella-specific insertion element IS 711, displaying a unique banding pattern, and exhibited a unique 16S rRNA gene sequence and also grouped separately in multilocus sequence typing analysis. Strain BO1(T) reacted with Brucella M-monospecific antiserum. Incomplete lysis was detected with bacteriophages Tb (Tbilisi), F1 and F25. Biochemical profiling revealed a high degree of enzymic activity and metabolic capabilities. In multilocus VNTR (variable-number tandem-repeat) analysis, strain BO1(T) showed a very distinctive profile and clustered with the other 'exotic' Brucella strains, including strains isolated from marine mammals, and Brucella microti, Brucella suis biovar 5 and Brucella neotomae. Comparative omp2a and omp2b gene sequence analysis revealed the most divergent omp2 sequences identified to date for a Brucella strain. The recA gene sequence of strain BO1(T) differed in seven nucleotides from the Brucella recA consensus sequence. Using the Brucella species-specific multiplex PCR assay, strain BO1(T) displayed a unique banding pattern not observed in other Brucella species. From the phenotypic and molecular analysis it became evident that strain BO1( T) was clearly different from all other Brucella species, and therefore represents a novel species within the genus Brucella. Because of its unexpected isolation, the name Brucella inopinata with the type strain BO1(T) (=BCCN 09-01(T)=CPAM 6436(T)) is proposed.

A methodological study on testing and evaluating of filtration efficiency of canister against microbial aerosol

To establish a testing and evaluating method for filtration efficiency of the canister against microbial aerosol. Serratia marcescens aerosol served as model of bacterial aerosol, Bacillus subtilis var niger aerosol as model of spores aerosol, bacteriophage f(2) aerosol as model of viral aerosol. Employing the microbial aerosol testing platform was established in lab, models of microbial aerosol generated artificially were sampled quantitatively by air samplers before and after filtrating by canisters, respectively. Filtration efficiency was determined by the concentration of microbial aerosol in the air sample before and after filtrating. The four canisters of 1-1, 1-2, 1-3, 1-4 were tested for the filtration efficiency against Serratia marcescens, Bacillus subtilis var niger and phage f(2) aerosol. The two canisters of 543 and 544 canisters equipped with active carbon were tested for the filtration efficiencies against Serratia marcescens aerosol. The filtration efficiency of 1-1, 1-2, 1-3 canisters against Serratia marcescens, Bacillus subtilis var niger and phage f(2) aerosol was 100.000%. The filtration efficiency of 1-4 canister filtration efficiency against Bacillus subtilis var niger spores aerosol was 99.997% and efficiency of the other two aerosol was 100.000%. The filtration efficiency of the two canisters of 543 and 544 to those attached with active carbon against Serratia marcescens aerosol was 100.000%. The testing method might be used to evaluate the protective performance of the canister against microbiological aerosol. The effect of the canisters (including those equipped with active carbon) against microbiological aerosol should be reliable.

Phage Tailspike Proteins with β‐Solenoid Fold as Thermostable Carbohydrate Binding Materials

We have investigated the stability of three tailspike proteins (TSPs) from bacteriophages Sf6, P22, and HK620. Tailspikes are rod-like homotrimers with comparable beta-solenoid folds and similarly high kinetic stability in spite of different amino acid sequences. As tailspikes bind polysaccharides to recognize the bacterial host cell, their stability is required for maintenance of bacteriophage infectivity under harsh extracellular conditions. They resist denaturation by SDS at ambient temperature and their unfolding is slow even in 6 M guanidinium hydrochloride (GdmHCl). This makes them interesting candidates for very stable carbohydrate binding protein materials.

Polyaluminum chloride-enhanced concentration efficiency of poliovirus and f2phage from sewage water

To enhance the recovery of f(2) bacteriophage and poliovirus by an established method based on the adsorption to and elution from positively-charged Al(OH)(3)-treated silica gel. Polyaluminum Chloride (PAC) was added to water samples to neutralize the negatively charged materials, which can reduce virus recovery by providing a competing adsorption mode on the media surface. Using this improved process (PAC 30 mg l(-1), pH 6.5, temperature 20 approximately 30 degrees C), the recoveries of Poliovirus I and f(2) from small-volume sewage (100 ml) were 110.76 +/- 36.0% and 92.06 +/- 8.65%, respectively (P < 0.05 vs. traditional methods). Recovery from a 20-L volume of sewage averaged 85.65 +/- 4.43% for f(2) and 88.73 +/- 9.76% for poliovirus, significantly higher than the recoveries in the traditional methods (P < 0.05). PAC could enhance concentration efficiency of poliovirus and f(2) phage from sewage water. This method should significantly improve the recovery of viruses from sewage.

Polyaluminum chloride enhanced concentration efficiency of poliovirus and f2 phage from sewage water

Polyaluminum chloride enhanced concentration efficiency of poliovirus and f2 phage from sewage water

Crystal structure of Escherichia coli phage HK620 tailspike: podoviral tailspike endoglycosidase modules are evolutionarily related

Bacteriophage HK620 infects Escherichia coli H and is closely related to Shigella phage Sf6 and Salmonella phage P22. All three Podoviridae recognize and cleave their respective host cell receptor polysaccharide by homotrimeric tailspike proteins. The three proteins exhibit high sequence identity in the 110 residues of their N-terminal particle-binding domains, but no apparent sequence similarity in their major, receptor-binding parts. We have biochemically characterized the receptor-binding part of HK620 tailspike and determined its crystal structure to 1.38 A resolution. Its major domain is a right-handed parallel beta-helix, as in Sf6 and P22 tailspikes. HK620 tailspike has endo-N-acetylglucosaminidase activity and produces hexasaccharides of an O18A1-type O-antigen. As indicated by the structure of a hexasaccharide complex determined at 1.6 A resolution, the endoglycosidase-active sites are located intramolecularly, as in P22, and not between subunits, as in Sf6 tailspike. In contrast, the extreme C-terminal domain of HK620 tailspike forms a beta-sandwich, as in Sf6 and unlike P22 tailspike. Despite the different folds, structure-based sequence alignments of the C-termini reveal motifs conserved between the three proteins. We propose that the tailspike genes of P22, Sf6 and HK620 have a common precursor and are not mosaics of unrelated gene fragments.

An Intersubunit Active Site between Supercoiled Parallel β Helices in the Trimeric Tailspike Endorhamnosidase of Shigella flexneri Phage Sf6

Sf6 belongs to the Podoviridae family of temperate bacteriophages that infect gram-negative bacteria by insertion of their double-stranded DNA. They attach to their hosts specifically via their tailspike proteins. The 1.25 A crystal structure of Shigella phage Sf6 tailspike protein (Sf6 TSP) reveals a conserved architecture with a central, right-handed beta helix. In the trimer of Sf6 TSP, the parallel beta helices form a left-handed, coiled-beta coil with a pitch of 340 A. The C-terminal domain consists of a beta sandwich reminiscent of viral capsid proteins. Further crystallographic and biochemical analyses show a Shigella cell wall O-antigen fragment to bind to an endorhamnosidase active site located between two beta-helix subunits each anchoring one catalytic carboxylate. The functionally and structurally related bacteriophage, P22 TSP, lacks sequence identity with Sf6 TSP and has its active sites on single subunits. Sf6 TSP may serve as an example for the evolution of different host specificities on a similar general architecture.

Brucella microti sp. nov., isolated from the common vole Microtus arvalis

Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915(T) and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA-DNA hybridization studies. Both strains reacted equally with Brucella M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other Brucella species. The omp2a and omp2b genes of isolates CCM 4915(T) and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915(T) and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915(T) and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus Brucella, for which the name Brucella microti sp. nov. with the type strain CCM 4915(T) (=BCCN 07-01(T)=CAPM 6434(T)) is proposed.