1H detection of heteronuclear dipolar oscillations with water suppression in single crystal peptide and oriented protein samples.

Oriented sample solid-state NMR is a complementary approach to protein structure determination with the distinct advantage that it can be applied to supramolecular assemblies, such as viruses and membrane proteins, under near-native conditions, which generally include high levels of hydration as found in living systems. Thus, in order to perform 1H detected versions of multi-dimensional experiments water suppression techniques must be integrated into the pulse sequences. For example, 1H-windowed detection of 1H-15N dipolar couplings enable multi-dimensional NMR experiments to be performed. Here we show that the addition of a solvent suppression pulse during the z-filter interval greatly improves the sensitivity of the experiments by suppressing the 1H signals from water present. This is demonstrated here with a crystal sample submerged in water and then extended to proteins. The combination of solvent-suppressed 1H detected PISEMO and the use of a strip shield-solenoid coil probe configuration provides a two-fold sensitivity enhancement in both the crystal sample and Pf1 coat protein sample compared to the 15N direct detection method. Here we also examine protein NMR line-widths and sensitivity enhancements in the context of window detected separated local field experiments for protein samples.

Two Lineages of Pseudomonas aeruginosa Filamentous Phages: Structural Uniformity over Integration Preferences.

Pseudomonas aeruginosa filamentous (Pf) bacteriophages are important factors contributing to the pathogenicity of this opportunistic bacterium, including biofilm formation and suppression of bacterial phagocytosis by macrophages. In addition, the capacity of Pf phages to form liquid crystal structures and their high negative charge density makes them potent sequesters of cationic antibacterial agents, such as aminoglycoside antibiotics or host antimicrobial peptides. Therefore, Pf phages have been proposed as a potential biomarker for risk of antibiotic resistance development. The majority of studies describing biological functions of Pf viruses have been performed with only three of them: Pf1, Pf4, and Pf5. However, our analysis revealed that Pf phages exist as two evolutionary lineages (I and II), characterized by substantially different structural/morphogenesis properties, despite sharing the same integration sites in the host chromosomes. All aforementioned model Pf phages are members of the lineage I. Hence, it is reasonable to speculate that their interactions with P. aeruginosa and impact on its pathogenicity may be not completely extrapolated to the lineage II members. Furthermore, in order to organize the present numerical nomenclature of Pf phages, we propose a more informative approach based on the insertion sites, that is, Pf-tRNA-Gly, -Met, -Sec, -tmRNA, and -DR (direct repeats), which are fully compatible with one of five types of tyrosine integrases/recombinases XerC/D carried by these viruses. Finally, we discuss possible evolutionary mechanisms behind this division and consequences from the perspective of virus-virus, virus-bacterium, and virus-human interactions.

De novo NMR pulse sequence design using Monte-Carlo optimization techniques.

Separated Local Field (SLF) experiments have been routinely used for measuring 1H-15N heteronuclear dipolar couplings in oriented-sample solid-state NMR for structure determination of proteins. In the on-going pursuit of designing better-performing SLF pulse sequences (e.g. by increasing the number of subdwells, and varying the rf amplitudes and phases), analytical treatment of the relevant average Hamiltonian terms may become cumbersome and/or nearly impossible. Numerical simulations of NMR experiments using GPU processors can be employed to rapidly calculate spectra for moderately sized spin systems, which permit an efficient numeric optimization of pulse sequences by the Monte Carlo Simulated Annealing protocol. In this work, a computational strategy was developed to find the optimal phases and timings that substantially improve the 1H-15N dipolar linewidths over a broad range of dipolar couplings as compared to SAMPI4. More than 100 pulse sequences were developed de novo and tested on an N-acetyl Leucine crystal. Seventeen distinct pulse sequences were shown to produce sharper mean linewidths than SAMPI4. Overall, these pulse sequences have more variable parameters (involving non-quadrature phases) and do not involve symmetry between the odd and even dwells, which would likely preclude their rigorous analytical treatment. The top performing pulse sequence, termed ROULETTE-1, has 18% sharper mean linewidths than SAMPI4 when run on an N-acetyl Leucine crystal. This sequence was also shown to be robust over a broad range of 1H carrier frequencies and various crystal orientations. The performance of such an optimized pulse sequence was also illustrated on 15N Leucine-labeled Pf1 coat protein reconstituted in magnetically aligned bicelles. For the optimized pulse sequence the mean peak width was 14% sharper than SAMPI4, which in turn yielded a better signal to noise ratio, 20:1 vs. 17:1. This method is potentially extendable to de novo development of a variety of NMR experiments.

Effects of deuteration on solid-state NMR spectra of single peptide crystals and oriented protein samples.

Extensive deuteration can be used to simplify NMR spectra by "diluting" and minimizing the effects of the abundant 1H nuclei. In solution-state NMR and magic angle spinning solid-state NMR of proteins, perdeuteration has been widely applied and its effects are well understood. Oriented sample solid-state NMR of proteins, however, is at a much earlier stage of development. In spite of the promise of the approach, the effects of sample deuteration are largely unknown. Here we map out the effects of perdeuteration on solid-state NMR spectra of aligned samples by closely examining differences in results obtained on fully protiated and perdeuterated samples, where all of the carbon sites have either 1H or 2H bonded to them, respectively. The 2H and 15N labeled samples are back-exchanged in 1H2O solution so that the amide 15N sites have a bonded 1H. Line-widths in the 15N chemical shift, 1H chemical shift, and 1H-15N dipolar coupling frequency dimensions were compared for peptide single crystals as well as membrane proteins aligned along with the phospholipids in bilayers with their normals perpendicular to the direction of the magnetic field. Remarkably, line-width differences were not found between fully protiated and perdeuterated samples. However, in the absence of effective 1H-1H homonuclear decoupling, the line-widths in the 1H-15N heteronuclear dipolar coupling frequency dimension were greatly narrowed in the perdeuterated samples. In proton-driven spin diffusion (PDSD) experiments, no effects of perdeuteration were observed. In contrast, in mismatched Hartmann-Hahn experiments, perdeuteration enhances cross-peak intensities by allowing more efficient spin-exchange with less polarization transfer back to the carbon-bound 1H. Here we show that in oriented sample solid-state NMR, the effects of perdeuteration can be exploited in experiments where 1H-1H homonuclear decoupling cannot be applied. These data also provide evidence for the possible contribution of direct 15N-15N dilute-spin mixing mechanism in proton-driven spin diffusion experiments.

Automated assignment of NMR spectra of macroscopically oriented proteins using simulated annealing.

An automated technique for the sequential assignment of NMR backbone resonances of oriented protein samples has been developed and tested based on 15N-15N homonuclear exchange and spin-exchanged separated local-field spectra. By treating the experimental spectral intensity as a pseudopotential, the Monte-Carlo Simulated Annealing algorithm has been employed to seek lowest-energy assignment solutions over a large sampling space where direct enumeration would be unfeasible. The determined sequential assignments have been scored based on the positions of the crosspeaks resulting from the possible orders for the main peaks. This approach is versatile in terms of the parameters that can be specified to achieve the best-fit result. At a minimum the algorithm requires a continuous segment of the main-peak chemical shifts obtained from a uniformly labeled sample and a spin-exchanged experimental spectrum represented as a 2D matrix array. With selective labeling experiments, groups of chemical shifts corresponding to specific locations in the protein backbone can be fixed, thereby decreasing the sampling space. The output from the program consists of a list of top-score main peak assignments, which can be subjected to further scoring criteria until a consensus solution is found. The algorithm has first been tested on a synthetic spectrum with randomly generated chemical shifts and dipolar couplings for the main peaks. The original assignments have been successfully recovered for as many as 100 main peaks when residue-type information was used even in the presence of substantial spectral peak overlap. The algorithm was then applied to assigning two sets of experimental spectra to recover and confirm the previously established assignments in an automated fashion. For the 20-residue transmembrane domain of Pf1 coat protein reconstituted in magnetically aligned bicelles, the original assignment by Park et al. (2010) was recovered by the automated algorithm with additional input from 5 selectively labeled amino acid spectra. The second case considered was the 46 residue Pf1 bacteriophage from Thiriot et al. (2005) and Knox et al. (2010), of which 38 residues were fit. Automated fitting resulted in several possible assignments but not exactly the original assignment. By using a post-fitting filtering procedure based on the number of missed cross peaks and Pf1 helical structure, a consensus spectroscopic assignment is proposed covering 84% of the original assignment. While the automated assignment works best in spectra with well-resolved crosspeaks, it also tolerates substantial spectral crowding to yield reasonable assignments in the cases where ambiguity and degeneracy of possible assignment solutions are inevitable.

Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization.

An experimental strategy has been developed to increase the efficiency of dynamic nuclear polarization (DNP) in solid-state NMR studies. The method makes assignments simpler, faster, and more reliable via sequential correlations of both side-chain and Cα resonances. The approach is particularly suited to complex biomolecules and systems with significant chemical-shift degeneracy. It was designed to overcome the spectral congestion and line broadening that occur due to sample freezing at the cryogenic temperatures required for DNP. Nonuniform sampling (NUS) is incorporated to achieve time-efficient collection of multidimensional data. Additionally, fast (25 kHz) magic-angle spinning (MAS) provides optimal sensitivity and resolution. Data collected in <1 wk produced a virtually complete de novo assignment of the coat protein of Pf1 virus. The peak positions and linewidths for samples near 100 K are perturbed relative to those near 273 K. These temperature-induced perturbations are strongly correlated with hydration surfaces.

Sensitivity enhancement for membrane proteins reconstituted in parallel and perpendicular oriented bicelles obtained by using repetitive cross-polarization and membrane-incorporated free radicals.

Multidimensional separated local-field and spin-exchange experiments employed by oriented-sample solid-state NMR are essential for structure determination and spectroscopic assignment of membrane proteins reconstituted in macroscopically aligned lipid bilayers. However, these experiments typically require a large number of scans in order to establish interspin correlations. Here we have shown that a combination of optimized repetitive cross polarization (REP-CP) and membrane-embedded free radicals allows one to enhance the signal-to-noise ratio by factors 2.4-3.0 in the case of Pf1 coat protein reconstituted in magnetically aligned bicelles with their normals being either parallel or perpendicular to the main magnetic field. Notably, spectral resolution is not affected at the 2:1 radical-to-protein ratio. Spectroscopic assignment of Pf1 coat protein in the parallel bicelles has been established as an illustration of the method. The proposed methodology will advance applications of oriented-sample NMR technique when applied to samples containing smaller quantities of proteins and three-dimensional experiments.

Quadrupole sensitive pulse for signal filtering.

A longstanding problem in quadrupolar NMR of semi-solids is the selection of signals originating from ordered nuclei, i.e. those that experience a non-vanishing quadrupolar coupling. Established techniques, such as for example multiple-quantum filters are not adequate in situations when the radio frequency power is on the order of the quadrupolar coupling or the quadrupolar relaxation rates, such as may be the case on an MRI scanner, or in ex situ applications. In this manuscript we show a new method for the selective excitation of ordered spin-3/2 nuclei, which produces the desired results when the radio frequency power is approximately equal or smaller than quadrupolar frequency. Using a combination of simulations and experiments with (23)Na in NaCl solution, Pf1-solutions, and bovine patellar cartilage samples we further show how the value of the quadrupolar frequency and global features of a quadrupolar coupling distribution can be extracted from these experiments.

Polyelectrolyte-mediated increase of biofilm mass formation.

BACKGROUND: Biofilm formation is associated with various aspects of bacterial and fungal infection. This study was designed to assess the impact of diverse natural polyelectrolytes, such as DNA, F-actin, neurofilaments (NFs), vimentin and purified Pf1 bacteriophage on biofilm formation and swarming motility of select pathogens including Pseudomonas aeruginosa associated with lung infections in CF patients. RESULTS: The bacteriophage Pf1 (1 mg/ml) significantly increased biofilm mass produced by Pseudomonas aeruginosa P14, Escherichia coli RS218 and Bacillus subtilis ATCC6051. DNA, F-actin, NFs and Pf1 also increased biofilm mass of the fungal C. albicans 1409 strain. Addition of F-actin, DNA or Pf1 bacteriophage to 0.5% agar plates increased swarming motility of Pseudomonas aeruginosa Xen5. CONCLUSIONS: The presence of polyelectrolytes at infection sites is likely to promote biofilm growth and bacterial swarming.

Bactericidal activities of cathelicidin LL-37 and select cationic lipids against the hypervirulent Pseudomonas aeruginosa strain LESB58.

Pseudomonas aeruginosa Liverpool epidemic strain (LES) infections in cystic fibrosis (CF) patients are associated with transmissibility and increased patient morbidity. This study was designed to assess the in vitro activities of cathelicidin LL-37 peptide (LL-37) and select cationic lipids against Pseudomonas aeruginosa LESB58 in CF sputum and in a setting mimicking the CF airway. We found that LL-37 naturally present in airway surface fluid and some nonpeptide cationic lipid molecules such as CSA-13, CSA-90, CSA-131, and D2S have significant, but broadly differing, bactericidal activities against P. aeruginosa LESB58. We observed strong inhibition of LL-37 bactericidal activity in the presence of purified bacteriophage Pf1, which is highly expressed by P. aeruginosa LES, but the activities of the cationic lipids CSA-13 and CSA-131 were not affected by this polyanionic virus. Additionally, CSA-13 and CSA-131 effectively prevent LESB58 biofilm formation, which is stimulated by Pf1 bacteriophage, DNA, or F-actin. CSA-13 and CSA-131 display strong antibacterial activities against different clinical strains of P. aeruginosa, and their activities against P. aeruginosa LESB58 and Xen5 strains were maintained in CF sputum. These data indicate that synthetic cationic lipids (mimics of natural antimicrobial peptides) are suitable for developing an effective treatment against CF lung P. aeruginosa infections, including those caused by LES strains.

Structure refinement and membrane positioning of selectively labeled OmpX in phospholipid nanodiscs.

NMR structural studies on membrane proteins are often complicated by their large size, taking into account the contribution of the membrane mimetic. Therefore, classical resonance assignment approaches often fail. The large size of phospholipid nanodiscs, a detergent-free phospholipid bilayer mimetic, prevented their use in high-resolution solution-state NMR spectroscopy so far. We recently introduced smaller nanodiscs that are suitable for NMR structure determination. However, side-chain assignments of a membrane protein in nanodiscs still remain elusive. Here, we utilized a NOE-based approach to assign (stereo-) specifically labeled Ile, Leu, Val and Ala methyl labeled and uniformly (15)N-Phe and (15)N-Tyr labeled OmpX and calculated a refined high-resolution structure. In addition, we were able to obtain residual dipolar couplings (RDCs) of OmpX in nanodiscs using Pf1 phage medium for the induction of weak alignment. Back-calculated NOESY spectra of the obtained NMR structures were compared to experimental NOESYs in order to validate the quality of these structures. We further used NOE information between protonated lipid head groups and side-chain methyls to determine the position of OmpX in the phospholipid bilayer. These data were verified by paramagnetic relaxation enhancement (PRE) experiments obtained with Gd(3+)-modified lipids. Taken together, this study emphasizes the need for the (stereo-) specific labeling of membrane proteins in a highly deuterated background for high-resolution structure determination, particularly in large membrane mimicking systems like phospholipid nanodiscs. Structure validation by NOESY back-calculation will be helpful for the structure determination and validation of membrane proteins where NOE assignment is often difficult. The use of protein to lipid NOEs will be beneficial for the positioning of a membrane protein in the lipid bilayer without the need for preparing multiple protein samples.

Pf1 bacteriophage hydration by magic angle spinning solid-state NMR.

High resolution two- and three-dimensional heteronuclear correlation spectroscopy ((1)H-(13)C, (1)H-(15)N, and (1)H-(13)C-(13)C HETCOR) has provided a detailed characterization of the internal and external hydration water of the Pf1 virion. This long and slender virion (2000 nm × 7 nm) contains highly stretched DNA within a capsid of small protein subunits, each only 46 amino acid residues. HETCOR cross-peaks have been unambiguously assigned to 25 amino acids, including most external residues 1-21 as well as residues 39-40 and 43-46 deep inside the virion. In addition, the deoxyribose rings of the DNA near the virion axis are in contact with water. The sets of cross-peaks to the DNA and to all 25 amino acid residues were from the same hydration water (1)H resonance; some of the assigned residues do not have exchangeable side-chain protons. A mapping of the contacts onto structural models indicates the presence of water "tunnels" through a highly hydrophobic region of the capsid. The present results significantly extend and modify results from a lower resolution study, and yield a comprehensive hydration surface map of Pf1. In addition, the internal water could be distinguished from external hydration water by means of paramagnetic relaxation enhancement. The internal water population may serve as a conveniently localized magnetization reservoir for structural studies.

Measuring membrane protein bond orientations in nanodiscs via residual dipolar couplings.

Membrane proteins are involved in numerous vital biological processes. To understand membrane protein functionality, accurate structural information is required. Usually, structure determination and dynamics of membrane proteins are studied in micelles using either solution state NMR or X-ray crystallography. Even though invaluable information has been obtained by this approach, micelles are known to be far from ideal mimics of biological membranes often causing the loss or decrease of membrane protein activity. Recently, nanodiscs, which are composed of a lipid bilayer surrounded by apolipoproteins, have been introduced as a more physiological alternative than micelles for NMR investigations on membrane proteins. Here, we show that membrane protein bond orientations in nanodiscs can be obtained by measuring residual dipolar couplings (RDCs) with the outer membrane protein OmpX embedded in nanodiscs using Pf1 phage as an alignment medium. The presented collection of membrane protein RDCs in nanodiscs represents an important step toward more comprehensive structural and dynamical NMR-based investigations of membrane proteins in a natural bilayer environment.

Polyelectrolyte properties of filamentous biopolymers and their consequences in biological fluids.

Anionic polyelectrolyte filaments are common in biological cells. DNA, RNA, the cytoskeletal filaments F-actin, microtubules, and intermediate filaments, and polysaccharides such as hyaluronan that form the pericellular matrix all have large net negative charge densities distributed over their surfaces. Several filamentous viruses with diameters and stiffnesses similar to those of cytoskeletal polymers also have similar negative charge densities. Extracellular protein filaments such collagen, fibrin and elastin, in contrast, have notably smaller charge densities and do not behave as highly charged polyelectrolytes in solution. This review summarizes data that demonstrate generic counterion-mediated effects on four structurally unrelated biopolymers of similar charge density: F-actin, vimentin, Pf1 virus, and DNA, and explores the possible biological and pathophysiological consequences of the polyelectrolyte properties of biological filaments.

Covariance spectroscopy in high-resolution multi-dimensional solid-state NMR.

Covariance spectroscopy (COV), a statistical method that provides increased sensitivity, can be applied to two-dimensional high-resolution solid-state NMR experiments, such as homonuclear spin-exchange spectroscopy. We the alternative States sampling scheme to the experimental time by 50%. By combining COV with other processing methods for non-uniform sampling (NUS), many different three-dimensional experiments can be performed with substantial increases in overall sensitivity. As an example, we show a three-dimensional homonuclear spin-exchange/separated-local-field (SLF) spectrum that enables the assignment of resonances and the measurement of structural restraints from a single experiment performed in a limited amount of time.

Resolution and measurement of heteronuclear dipolar couplings of a noncrystalline protein immobilized in a biological supramolecular assembly by proton-detected MAS solid-state NMR spectroscopy.

Two-dimensional (15)N chemical shift/(1)H chemical shift and three-dimensional (1)H-(15)N dipolar coupling/(15)N chemical shift/(1)H chemical shift MAS solid-state NMR correlation spectra of the filamentous bacteriophage Pf1 major coat protein show single-site resolution in noncrystalline, intact-phage preparations. The high sensitivity and resolution result from (1)H detection at 600MHz under 50kHz magic angle spinning using ∼0.5mg of perdeuterated and uniformly (15)N-labeled protein in which the exchangeable amide sites are partially or completely back-exchanged (reprotonated). Notably, the heteronuclear (1)H-(15)N dipolar coupling frequency dimension is shown to select among (15)N resonances, which will be useful in structural studies of larger proteins where the resonances exhibit a high degree of overlap in multidimensional chemical shift correlation spectra.

Partial alignment and measurement of residual dipolar couplings of proteins under high hydrostatic pressure.

High-pressure NMR spectroscopy has emerged as a complementary approach for investigating various structural and thermodynamic properties of macromolecules. Noticeably absent from the array of experimental restraints that have been employed to characterize protein structures at high hydrostatic pressure is the residual dipolar coupling, which requires the partial alignment of the macromolecule of interest. Here we examine five alignment media that are commonly used at ambient pressure for this purpose. We find that the spontaneous alignment of Pf1 phage, d(GpG) and a C12E5/n-hexnanol mixture in a magnetic field is preserved under high hydrostatic pressure. However, DMPC/DHPC bicelles and collagen gel are found to be unsuitable. Evidence is presented to demonstrate that pressure-induced structural changes can be identified using the residual dipolar coupling.

Mechanistic insights into water-protein interactions of filamentous bacteriophage.

Water plays a major structural and functional role around proteins. In an attempt to explore this mechanistic structural aspect of proteins, we present site-specific interaction of hydration water with the major coat protein subunit of filamentous virus Pf1 by magic angle spinning (MAS) solid-state NMR. The interaction of surrounding water with 36 MDa Pf1 virion is investigated in uniformly (13)C, (15)N isotopically labeled; polyethylene glycol precipitated fully hydrated samples by solid-state nuclear magnetic resonance spectroscopy. Dipolar edited two-dimensional (2D) (1)H-(15)N heteronuclear correlation (HETCOR) experiments lead to unambiguous assignments of cross-peaks originating exclusively from (1)H resonances of water molecules correlating to the protein amide nitrogen. An enhanced resolved (1)H chemical shift dimension in these experiments also precludes the need of perdeuteration. We report seven residues spanning the 40-residue continuous α-helical conformation assembly of Pf1 interacting with surrounding water. It shows a highly hydrated inner core inside this viral filamentous assembly. The results obtained also suggest the first evidence of a water-mediated interface cluster formed at the site of Arg44 with the single-stranded DNA genome of the filamentous phage supramolecular assembly.

Very large residual dipolar couplings from deuterated ubiquitin.

Main-chain (1)H(N)-(15)N residual dipolar couplings (RDCs) ranging from approximately -200 to 200 Hz have been measured for ubiquitin under strong alignment conditions in Pf1 phage. This represents a ten-fold increase in the degree of alignment over the typical weakly aligned samples. The measurements are made possible by extensive proton-dilution of the sample, achieved by deuteration of the protein with partial back-substitution of labile protons from 25 % H(2)O / 75 % D(2)O buffer. The spectral quality is further improved by application of deuterium decoupling. Since standard experiments using fixed-delay INEPT elements cannot accommodate a broad range of couplings, the measurements were conducted using J-resolved and J-modulated versions of the HSQC and TROSY sequences. Due to unusually large variations in dipolar couplings, the trosy (sharp) and anti-trosy (broad) signals are often found to be interchanged in the TROSY spectra. To distinguish between the two, we have relied on their respective (15)N linewidths. This strategy ultimately allowed us to determine the signs of RDCs. The fitting of the measured RDC values to the crystallographic coordinates of ubiquitin yields the quality factor Q = 0.16, which confirms the perturbation-free character of the Pf1 alignment. Our results demonstrate that RDC data can be successfully acquired not only in dilute liquid crystals, but also in more concentrated ones. As a general rule, the increase in liquid crystal concentration improves the stability of alignment media and makes them more tolerant to variations in sample conditions. The technical ability to measure RDCs under moderately strong alignment conditions may open the door for development of alternative alignment media, including new types of media that mimic biologically relevant systems.

Metabonomic study of host-phage interaction by nuclear magnetic resonance- and statistical total correlation spectroscopy-based analysis.

We present a method for the qualitative and quantitative study of transient metabolic flux of phage infection at the molecular level. The method is based on statistical total correlation spectroscopy (STOCSY) and partial least squares discriminant analysis (PLS-DA) applied to nuclear magnetic resonance (NMR) metabonomic data sets. An algorithm for this type of study is developed and demonstrated. The method has been implemented on (1)H NMR data sets of growth media in planktonic cultures of Pseudomonas aeruginosa infected with bacteriophage pf1. Transient metabolic flux of various important metabolites, identified by STOCSY and PLS-DA analysis applied to the NMR data set, are estimated at various stages of growth. The opportunistic and nosocomial pathogen P. aeruginosa is one of the best-studied model organism for bacterial biofilms. Complete information regarding metabolic connectivity of this system is not possible by conventional spectroscopic approach. Our study presents temporal comparative (1)H NMR metabonomic analyses of filamentous phage pf1 infection in planktonic cultures of P. aeruginosa K strain (PAK). We exemplify here the potential of STOCSY and PLS-DA tools to gain mechanistic insight into subtle changes and to determine the transient flux associated with metabolites following metabolic perturbations resulting from phage infection. Our study has given new avenues in correlating existing postgenomic data with current metabonomic results in P. aeruginosa biofilms research.