Detection of Bacillus anthracis spores from environmental water using bioluminescent reporter phage.

AIMS: We investigated the ability of a temperate Bacillus anthracis reporter phage (Wß::luxAB-2), which transduces bioluminescence to infected cells, to detect viable spores from deliberately contaminated environmental water samples. METHODS AND RESULTS: Environmental water was inoculated with spores and assayed with Wß::luxAB-2. Bioluminescent signals directly correlated with input phage and spore concentrations. A limit of detection of 101 and 102 CFU per ml within 8 h was achieved from pond and lake water, respectively. Detection was greatly simplified by minimizing sample processing steps without spore extraction. The complex endogenous microbial flora and salt content of brackish water challenged the assay, extending the detection time to 12 h for a sensitivity of 102 CFU per ml. Phage-mediated bioluminescence was strictly dependent on bacterial physiology, being significantly reduced in mid/late log phase cells. This was shown to be due to an inability of the phage to adsorb. CONCLUSIONS: The reporter phage Wß::luxAB-2 displays potential for simplified detection of viable spores from contaminated water samples within 12 h. SIGNIFICANCE AND IMPACT OF THE STUDY: A deliberate aerosol release of spores could lead to widespread contamination, leaving large areas uninhabitable until remediation. An essential requirement of this restoration process is the development of simplified detection assays in different environmental matrices.

Isolation and development of bioluminescent reporter phages for bacterial dysentery.

Shigellosis is a significant cause of morbidity and mortality worldwide, most notably amongst children. Moreover, there is a global increase in the occurrence of multidrug-resistant isolates, including the epidemic and pandemic Shigella dysenteriae type 1 strain. We developed a bioluminescent reporter phage assay to facilitate detection and simultaneously determine antibiotic susceptibility. A Shigella flexneri phage (Shfl25875) was isolated from environmental wastewater and characterized by DNA sequencing. Shfl25875 is T4-like, harbors a 169,062-bp genome, and grows on most (28/29) S. flexneri strains and all 12 S. dysenteriae type 1 strains tested. The genes encoding bacterial luciferase were integrated into the Shfl25875 genome to create a "light-tagged" phage capable of transducing a bioluminescent phenotype to infected cells. Shfl25875::luxAB rapidly detects cultured isolates with high sensitivity. Specificity experiments indicate that the reporter does not respond to Shigella boydii, non-type 1 S. dysenteriae strains, and most non-Shigella Enterobacteriaceae. Shfl25875::luxAB generates ampicillin and ciprofloxacin susceptibility profiles that are similar to the standard Clinical and Laboratory Standards Institute (CLSI) growth microdilution method, but in a significantly shorter time. In addition, the reporter phage detects Shigella in mock-infected stool. This new reporter phage shows promise as a tool for the detection of cultured isolates or complex clinical samples.

Rapid detection and simultaneous antibiotic susceptibility analysis of Yersinia pestis directly from clinical specimens by use of reporter phage.

Yersinia pestis is a tier 1 agent due to its contagious pneumopathogenicity, extremely rapid progression, and high mortality rate. As the disease is usually fatal without appropriate therapy, rapid detection from clinical matrices is critical to patient outcomes. We previously engineered the diagnostic phage ΦA1122 with luxAB to create a "light-tagged" reporter phage. ΦA1122::luxAB rapidly detects Y. pestis in pure culture and human serum by transducing a bioluminescent signal response. In this report, we assessed the analytical specificity of the reporter phage and investigated diagnostic utility (detection and antibiotic susceptibility analysis) directly from spiked whole blood. The bioreporter displayed 100% (n = 59) inclusivity for Y. pestis and consistent intraspecific signal transduction levels. False positives were not obtained from species typically associated with bacteremia or those relevant to plague diagnosis. However, some non-pestis Yersinia strains and Enterobacteriaceae did elicit signals, albeit at highly attenuated transduction levels. Diagnostic performance was assayed in simple broth-enriched blood samples and standard aerobic culture bottles. In blood, <10(2) CFU/ml was detected within 5 h. In addition, Y. pestis was identified directly from positive blood cultures within 20 to 45 min without further processing. Importantly, coincubation of blood samples with antibiotics facilitated simultaneous antimicrobial susceptibility profiling. Consequently, the reporter phage demonstrated rapid detection and antibiotic susceptibility profiling directly from clinical samples, features that may improve patient prognosis during plague outbreaks.

Slow fitness recovery in a codon-modified viral genome.

Extensive synonymous codon modification of viral genomes appears to be an effective way of attenuating strains for use as live vaccines. An assumption of this method is that codon changes have individually small effects, such that codon-attenuated viruses will be slow to evolve back to high fitness (and thus to high virulence). The major capsid gene of the bacterial virus T7 was modified to have varying levels of suboptimal synonymous codons in different constructs, and fitnesses declined linearly with the number of changes. Adaptation of the most extreme design, with 182 codon changes, resulted in a slow fitness recovery by standards of previous experimental evolution with this virus, although fitness effects of substitutions were higher than expected from the average effect of an engineered codon modification. Molecular evolution during recovery was modest, and changes evolved both within the modified gene and outside it. Some changes within the modified gene evolved in parallel across replicates, but with no obvious explanation. Overall, the study supports the premise that codon-modified viruses recover fitness slowly, although the evolution is substantially more rapid than expected from the design principle.

In vivo growth rates are poorly correlated with phage therapy success in a mouse infection model.

Two classes of phages yield profoundly different levels of recovery in mice experimentally infected with an Escherichia coli O18:K1:H7 strain. Phages requiring the K1 capsule for infection (K1-dep) rescue virtually all infected mice, whereas phages not requiring the capsule (K1-ind) rescue modest numbers (∼30%). To rescue infected mice, K1-ind phages require at least a 10(6)-fold-higher inoculum than K1-dep phages. Yet their in vivo growth dynamics are only modestly inferior to those of K1-dep phages, and competition between the two phage types in the same mouse reveals only a slight growth advantage for the K1-dep phage. The in vivo growth rate seems unlikely to be the primary determinant of phage therapy success. An alternative explanation is that the success of K1-dep phages is due substantially to their proteomic composition. They encode an enzyme that degrades the K1 capsule, which has been shown in other work to be sufficient to cure infection in the complete absence of phages.

Evolution at a high imposed mutation rate: adaptation obscures the load in phage T7.

Evolution at high mutation rates is expected to reduce population fitness deterministically by the accumulation of deleterious mutations. A high enough rate should even cause extinction (lethal mutagenesis), a principle motivating the clinical use of mutagenic drugs to treat viral infections. The impact of a high mutation rate on long-term viral fitness was tested here. A large population of the DNA bacteriophage T7 was grown with a mutagen, producing a genomic rate of 4 nonlethal mutations per generation, two to three orders of magnitude above the baseline rate. Fitness-viral growth rate in the mutagenic environment-was predicted to decline substantially; after 200 generations, fitness had increased, rejecting the model. A high mutation load was nonetheless evident from (i) many low- to moderate-frequency mutations in the population (averaging 245 per genome) and (ii) an 80% drop in average burst size. Twenty-eight mutations reached high frequency and were thus presumably adaptive, clustered mostly in DNA metabolism genes, chiefly DNA polymerase. Yet blocking DNA polymerase evolution failed to yield a fitness decrease after 100 generations. Although mutagenic drugs have caused viral extinction in vitro under some conditions, this study is the first to match theory and fitness evolution at a high mutation rate. Failure of the theory challenges the quantitative basis of lethal mutagenesis and highlights the potential for adaptive evolution at high mutation rates.

A tale of tails: Sialidase is key to success in a model of phage therapy against K1-capsulated Escherichia coli.

Prior studies treating mice infected with Escherichia coli O18:K1:H7 observed that phages requiring the K1 capsule for infection (K1-dep) were superior to capsule-independent (K1-ind) phages. We show that three K1-ind phages all have low fitness when grown on cells in serum whereas fitnesses of four K1-dep phages were high. The difference is serum-specific, as fitnesses in broth overlapped. Sialidase activity was associated with all K1-dep virions tested but no K1-ind virions, a phenotype supported by sequence analyses. Adding endosialidase to cells infected with K1-ind phage increased fitness in serum by enhancing productive infection after adsorption. We propose that virion sialidase activity is the primary determinant of high fitness on cells grown in serum, and thus in a mammalian host. Although the benefit of sialidase is specific to K1-capsulated bacteria, this study may provide a scientific rationale for selecting phages for therapeutic use in many systemic infections.

Predicting evolution from genomics: experimental evolution of bacteriophage T7.

A wealth of molecular biology has been exploited in designing and interpreting experimental evolution studies with bacteriophage T7. The modest size of its genome (40 kb dsDNA) and the ease of making genetic constructs, combined with the many genetic resources for its host (Escherichia coli), have enabled comprehensive and detailed studies of experimental adaptations. In several studies, the genome was specifically altered (gene knockouts, gene replacements, reordering of genetic elements) such that a priori knowledge of genetics and biochemistry of the phage could be used to predict the pathways of compensatory evolution when the modified phage is adapted to recover fitness. In other work, the phage has been adapted to specific environmental conditions chosen to select phenotypic outcomes with a quantitative basis, and the molecular bases of that evolution have been explored. Predicting the outcomes of these adaptations has been challenging. In hindsight, one-third to one-half of the compensatory nucleotide changes observed during the adaptation can be rationalized based on T7 biology. This rationalization usually only applies at the genetic level-a gene product may be known to be involved in the affected pathway, but it usually remains unknown how the observed change affects activity. The progress is encouraging, but the prediction of experimental evolution pathways remains far from complete, and is still sometimes confounded by observation when an adaptation yields a completely unexpected outcome.

Compensatory evolution in response to a novel RNA polymerase: orthologous replacement of a central network gene.

A bacteriophage genome was forced to evolve a new system of regulation by replacing its RNA polymerase (RNAP) gene, a central component of the phage developmental pathway, with that of a relative. The experiment used the obligate lytic phage T7 and the RNAP gene of phage T3. T7 RNAP uses 17 phage promoters, which are responsible for all middle and late gene expression, DNA replication, and progeny maturation, but the enzyme has known physical contacts with only 2 other phage proteins. T3 RNAP was supplied in trans by the bacterial host to a T7 genome lacking its own RNAP gene and the phage population was continually propagated on naive bacteria throughout the adaptation. Evolution of the T3 RNAP gene was thereby prevented, and selection was for the evolution of regulatory signals throughout the phage genome. T3 RNAP transcribes from T7 promoters only at low levels, but a single mutation in the promoter confers high expression, providing a ready mechanism for reevolution of gene expression in this system. When selected for rapid growth, fitness of the engineered phage evolved from a low of 5 doublings/h to 33 doublings/h, close to the expected maximum of 37 doublings/h. However, the experiment was terminated before it could be determined accurately that fitness had reached an obvious plateau, and it is not known whether further adaptation could have resulted in complete recovery of fitness. More than 30 mutations were observed in the evolved genome, but changes were found in only 9 of the 16 promoters, and several coding changes occurred in genes with no known contacts with the RNAP. Surprisingly, the T7 genome adapted to T3 RNAP also maintained high fitness when using T7 RNAP, suggesting that the extreme incompatibility of T7 elements with T3 RNAP is not an invariant property of divergence in these expression systems.

Gene order constrains adaptation in bacteriophage T7.

The order of genes in the genome is commonly thought to have functional significance for gene regulation and fitness but has not heretofore been tested experimentally. We adapted a bacteriophage T7 variant harboring an ectopically positioned RNA polymerase gene to determine whether it could regain the fitness of the wild type. Two replicate lines maintained the starting gene order and showed only modest recovery of fitness, despite the accumulation of over a dozen mutations. In both lines, a mutation in the early terminator signal is responsible for the majority of the fitness recovery. In a third line, the phage evolved a new gene order, restoring the wild-type position of the RNA polymerase gene but also displacing several other genes to ectopic locations. Due to the recombination, the fitness of this replicate was the highest obtained but it falls short of the wild type adapted to the same growth conditions. The large benefits afforded by the terminator mutation and the recombination are explicable in terms of T7 biology, whereas several mutations with lesser benefits are not easily accounted for. These results support the premise that gene order is important to fitness and that wild-type fitness is not rapidly re-evolved in reorganized genomes.

Genome properties and the limits of adaptation in bacteriophages.

Eight bacteriophages were adapted for rapid growth under similar conditions to compare their evolved, endpoint fitnesses. Four pairs of related phages were used, including two RNA phages with small genomes (MS2 and Qbeta) two single-stranded DNA phages with small genomes (phiX174 and G4), two T-odd phages with medium-sized, double-stranded DNA genomes (T7 and T3), and two T-even phages with large, double-stranded DNA genomes (T6 and RB69). Fitness was measured as absolute growth rate per hour under the same conditions used for adaptation. T7 and T3 achieved the highest fitnesses, able to increase by 13 billionfold and three-quarters billionfold per hour, respectively. In contrast, the RNA phages achieved low fitness maxima, with growth rates approximately 400-fold and 4000-fold per hour. The highest fitness limits were not attributable to high mutation rates or small genome size, even though both traits are expected to enhance adaptation for fast growth. We suggest that major differences in fitness limits stem from different "global" constraints, determined by the organization and composition of the phage genome affecting whether and how it overcomes potentially rate-limiting host processes, such as transcription, translation, and replication. Adsorption rates were also measured on the evolved phages. No consistent pattern of adsorption rate and fitness was observed across the four different types of phages, but within each pair of related phages, higher adsorption was associated with higher fitness. Different adsorption rate limits within pairs may stem from "local" constraints-sequence differences leading to different local optima in the sequence space.

Genomic analysis of bacteriophages SP6 and K1-5, an estranged subgroup of the T7 supergroup.

We have determined the genome sequences of two closely related lytic bacteriophages, SP6 and K1-5, which infect Salmonella typhimurium LT2 and Escherichia coli serotypes K1 and K5, respectively. The genome organization of these phages is almost identical with the notable exception of the tail fiber genes that confer the different host specificities. The two phages have diverged extensively at the nucleotide level but they are still more closely related to each other than either is to any other phage currently characterized. The SP6 and K1-5 genomes contain, respectively, 43,769 bp and 44,385 bp, with 174 bp and 234 bp direct terminal repeats. About half of the 105 putative open reading frames in the two genomes combined show no significant similarity to database proteins with a known or predicted function that is obviously beneficial for growth of a bacteriophage. The overall genome organization of SP6 and K1-5 is comparable to that of the T7 group of phages, although the specific order of genes coding for DNA metabolism functions has not been conserved. Low levels of nucleotide similarity between genomes in the T7 and SP6 groups suggest that they diverged a long time ago but, on the basis of this conservation of genome organization, they are expected to have retained similar developmental strategies.

Experimental evolution yields hundreds of mutations in a functional viral genome.

Two lines of the bacteriophage T7 were grown to fix mutations indiscriminately, using a combination of population bottlenecks and mutagenesis. Complete genome sequences revealed 404 and 299 base substitutions in the two lines, the largest number characterized in functional microbial genomes so far. Missense substitutions outnumbered silent substitutions. Silent substitutions occurred at similar rates between essential and nonessential genes, but missense substitutions occurred at a higher rate in nonessential genes than in essential genes, as expected if they were less deleterious in the nonessential genes. Viral fitness declined during this protocol, and subsequent passaging of each mutated line in large population sizes restored some of the lost fitness. Substitution levels during these recoveries were less than 6% of those during the bottleneck phase, and only two changes during recovery were reversions of the original mutations. Exchanges of genomic fragments between the two recovered lines revealed that fitness effects of some substitutions were not additive-that interactions were accumulating which could lead to incompatibility between the diverged genomes. Based on these results, unprecedented high rates of nucleotide and functional divergence in viral genomes should be attainable experimentally by using repeated population bottlenecks at a high mutation rate interspersed with recovery.

Experimental genomic evolution: extensive compensation for loss of DNA ligase activity in a virus.

Deletion of the viral ligase gene drastically reduced the fitness of bacteriophage T7 on a ligase-deficient host. Viral evolution recovered much of this fitness during long-term passage, but the final fitness remained below that of the intact virus. Compensatory changes occurred chiefly in genes involved in DNA metabolism: the viral endonuclease, helicase, and DNA polymerase. Two other compensatory changes of unknown function also occurred. Using a method to distinguish compensatory mutations from other beneficial mutations, five additional substitutions from the recovery were shown to enhance adaptation to culture conditions and were not compensatory for the deletion. In contrast to the few previous studies of viral recovery from deletions, the compensatory changes in T7 did not restore the deletion or duplicate major regions of the genome. The ability of this deleted genome to recover much of the lost fitness via mutations in its remaining genes reveals a considerable evolutionary potential to modify the interactions of its elements in maintaining an essential set of functions.

A general mechanism for viral resistance to suicide gene expression.

Bacteriophage T7 was challenged with either of two toxic genes expressed from plasmids. Each plasmid contained a different gene downstream of a T7 promoter; cells harboring each plasmid caused an infection by wild-type T7 to abort. T7 evolved resistance to both inhibitors by avoidance of the plasmid expression system rather than by blocking or bypassing the effects of the specific toxic gene product. Resistance was due to a combination of mutations in the T7 RNA polymerase and other genes expressed at the same time as the polymerase. Mutations mapped to sites that are unlikely to alter polymerase specificity for its cognate promoter but the basis for discrimination between phage and plasmid promoters in vivo was not resolved. A reporter assay indicated that, relative to wild-type phage, gene expression from the plasmid was diminished several-fold in cells infected by the evolved phages. A recombinant phage, derived from the original mutant but lacking a mutation in the gene for RNA polymerase, exhibited intermediate activity in the reporter assay and intermediate resistance to the toxic gene cassettes. Alterations in both RNA polymerase and a second gene are thus responsible for resistance. These findings have broad evolutionary parallels to other systems in which viral inhibition is activated by viral regulatory signals such as defective-interfering particles, and they may have mechanistic parallels to the general phenomena of position effects and gene silencing.

No syringes please, ejection of phage T7 DNA from the virion is enzyme driven.

Development of a sensitive assay that measures the rate of cellular internalization of an infecting bacteriophage T7 genome has led to surprising observations on the initiation of infection. Proteins ejected from the phage virion probably function as an extensible tail to form a channel across the cell envelope. This channel is subsequently used for translocating the phage genome into the cell. One of these ejected proteins also controls the amount of DNA that enters the cell, rendering subsequent internalization of the remainder of the genome dependent on transcription. Mutations affecting this protein allow the entire phage genome to enter a cell by the transcription-independent process. This process exhibits pseudo-zero-order reaction kinetics and a temperature dependence of translocation rate that are not expected if DNA ejection from a phage capsid were caused by a physical process. The temperature dependence of transcription-independent T7 DNA translocation rate is similar to those of enzyme-catalysed reactions. Current data suggest a highly speculative model, in which two of the proteins ejected from the phage head establish a molecular motor that ratchets the phage genome into the cell.

Role of the Gp16 lytic transglycosylase motif in bacteriophage T7 virions at the initiation of infection.

The predicted catalytic glutamate residue for transglycosylase activity of bacteriophage T7 gp16 is not essential for phage growth, but is shown to be beneficial during infection of Escherichia coli cells grown to high cell density, conditions in which murein is more highly cross-linked. In the absence of the putative transglycosylase, internalization of the phage genome is significantly delayed during infection. The lytic transglycosylase motif of gp16 is essential for phage growth at temperatures below 20 degrees C, indicating that these growth conditions also lead to increased cross-linking of peptidoglycan. Overexpression of sltY, E. coli soluble lytic transglycosylase, partially complements the defect in infection of mutant phage particles, allowing them to infect at higher efficiencies. Conversely, an sltY deletion increases the latent period of wild-type phage.

The internal head protein Gp16 controls DNA ejection from the bacteriophage T7 virion.

A wild-type T7 virion ejects about 850 bp of the 40 kb genome into the bacterial cell by a transcription-independent process. Internalization of the remainder of the genome normally requires transcription. Inhibition of transcription-independent DNA translocation beyond the leading 850 bp is not absolute but the time taken by a population of phage genomes in overcoming the block averages about 20 minutes at 30 degrees C. There are additional blocks to transcription-independent translocation and less than 20 % of infecting DNA molecules completely penetrate the cell cytoplasm after four hours of infection. Mutant virions containing an altered gene 16 protein either prevent the blocks to transcription-independent DNA translocation or effect rapid release from blocking sites and allow the entire phage DNA molecule to enter the cell at a constant rate of about 75 bp per second. This rate is likely the same at which the leading 850 bp is ejected into the cell from a wild-type virion. All mutations fall into two clusters contained within 380 bp of the 4 kb gene 16, suggesting that a 127 residue segment of gp16 controls DNA ejection from the phage particle. We suggest that this segment of gp16 acts as a clamp to prevent transcription-independent DNA translocation.

Computation, prediction, and experimental tests of fitness for bacteriophage T7 mutants with permuted genomes.

We created a simulation based on experimental data from bacteriophage T7 that computes the developmental cycle of the wild-type phage and also of mutants that have an altered genome order. We used the simulation to compute the fitness of more than 10(5) mutants. We tested these computations by constructing and experimentally characterizing T7 mutants in which we repositioned gene 1, coding for T7 RNA polymerase. Computed protein synthesis rates for ectopic gene 1 strains were in moderate agreement with observed rates. Computed phage-doubling rates were close to observations for two of four strains, but significantly overestimated those of the other two. Computations indicate that the genome organization of wild-type T7 is nearly optimal for growth: only 2.8% of random genome permutations were computed to grow faster, the highest 31% faster, than wild type. Specific discrepancies between computations and observations suggest that a better understanding of the translation efficiency of individual mRNAs and the functions of qualitatively "nonessential" genes will be needed to improve the T7 simulation. In silico representations of biological systems can serve to assess and advance our understanding of the underlying biology. Iteration between computation, prediction, and observation should increase the rate at which biological hypotheses are formulated and tested.

The DNA translocation and ATPase activities of restriction-deficient mutants of Eco KI.

Eco KI, a type I restriction enzyme, specifies DNA methyltransferase, ATPase, endonuclease and DNA translocation activities. One subunit (HsdR) of the oligomeric enzyme contributes to those activities essential for restriction. These activities involve ATP-dependent DNA translocation and DNA cleavage. Mutations that change amino acids within recognisable motifs in HsdR impair restriction. We have used an in vivo assay to monitor the effect of these mutations on DNA translocation. The assay follows the Eco KI-dependent entry of phage T7 DNA from the phage particle into the host cell. Earlier experiments have shown that mutations within the seven motifs characteristic of the DEAD-box family of proteins that comprise known or putative helicases severely impair the ATPase activity of purified enzymes. We find that the mutations abolish DNA translocation in vivo. This provides evidence that these motifs are relevant to the coupling of ATP hydrolysis to DNA translocation. Mutations that identify an endonuclease motif similar to that found at the active site of type II restriction enzymes and other nucleases have been shown to abolish DNA nicking activity. When conservative changes are made at these residues, the enzymes lack nuclease activity but retain the ability to hydrolyse ATP and to translocate DNA at wild-type levels. It has been speculated that nicking may be necessary to resolve the topological problems associated with DNA translocation by type I restriction and modification systems. Our experiments show that loss of the nicking activity associated with the endonuclease motif of Eco KI has no effect on ATPase activity in vitro or DNA translocation of the T7 genome in vivo.