Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm

The processes associated with early events in biofilm formation have become a major research focus over the past several years. Events associated with dispersion of cells from late stage biofilms have, however, received little attention. We demonstrate here that dispersal of Pseudomonas aeruginosa PAO1 from biofilms is inducible by a sudden increase in carbon substrate availability. Most efficient at inducing dispersal were sudden increases in availability of succinate > glutamate > glucose that led to approximately 80% reductions in surface-associated biofilm biomass. Nutrient-induced biofilm dispersion was associated with increased expression of flagella (fliC) and correspondingly decreased expression of pilus (pilA) genes in dispersed cells. Changes in gene expression associated with dispersion of P. aeruginosa biofilms were studied by using DNA microarray technology. Results corroborated proteomic data that showed gene expression to be markedly different between biofilms and newly dispersed cells. Gene families that were upregulated in dispersed cells included those for flagellar and ribosomal proteins, kinases, and phage PF1. Within the biofilm, genes encoding a number of denitrification pathways and pilus biosynthesis were also upregulated. Interestingly, nutrient-induced dispersion was associated with an increase in the number of Ser/Thr-phosphorylated proteins within the newly dispersed cells, and inhibition of dephosphorylation reduced the extent of nutrient-induced dispersion. This study is the first to demonstrate that dispersal of P. aeruginosa from biofilms can be induced by the addition of simple carbon sources. This study is also the first to demonstrate that dispersal of P. aeruginosa correlates with a specific dispersal phenotype.     

Rhamnolipid production with indigenous Pseudomonas aeruginosa EM1 isolated from oil-contaminated site

Rhamnolipid is one of the most effective and commonly used biosurfactant with wide industrial applications. Systematic strategies were applied to improve rhamnolipid (RL) production with a newly isolated indigenous strain Pseudomonas aeruginosa EM1 originating from an oil-contaminated site located in southern Taiwan. Seven carbon substrates and four nitrogen sources were examined for their effects on RL production. In addition, the effect of carbon to nitrogen (C/N) ratio on RL production was also studied. Single-factor experiments show that the most favorable carbon sources for RL production were glucose and glycerol (both at 40 g/L), giving a RL yield of 7.5 and 4.9 g/L, respectively. Meanwhile, sodium nitrate appeared to be the preferable nitrogen source, resulting in a RL production of 8.6g/L. Using NaNO(3) as the nitrogen source, an optimal C/N ratio of 26 and 52 was obtained for glucose- and glycerol-based culture, respectively. To further optimize the composition of fermentation medium, twenty experiments were designed by response surface methodology (RSM) to explore the favorable concentration of three critical components in the medium (i.e., glucose, glycerol, and NaNO(3)). The RSM analysis gave an optimal concentration of 30.5, 18.1, and 4.9 g/L for glucose, glycerol, and NaNO(3), respectively, predicting a maximum RL yield of 12.6 g/L, which is 47% higher than the best yield (8.6 g/L) obtained from preliminary selection tests and single factor experiments (glucose and NaNO(3) as the carbon and nitrogen source). The NMR and mass spectrometry analysis show that the purified RL product contained L-rhamnosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate (RL1) and L-rhamnosyl L-rhamnosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate (RL2). Meanwhile, HPLC analysis indicates that the molar ratio of RL1 and RL2 in the purified rhamnolipid product was ca. 1:1.     

Rhamnolipid production by Pseudomonas aeruginosa immobilised in polyvinyl alcohol beads

A marine bacterium, Pseudomonas aeruginosa BYK-2 (KCTC 18012P), was immobilised by entrapment in 10% (w/v) polyvinyl alcohol beads and optimized for the continuous production of rhamnolipid. The relative activity of rhamnolipid production was maintained at 80 approximately 90% of the initial production during 15 cycles in a repeated batch culture. Continuous culture was performed in a 1.8 1 airlift bioreactor, yielding 0.1 g rhamnolipid h(-1) at a dilution rate of 0.0 18 h(-1), 25 degrees C, initial pH 7, and 0.5 vvm aeration rate with a 1.21 working volume.     

Rhamnolipid production by a novel thermophilic hydrocarbon-degrading Pseudomonas aeruginosa AP02-1

Thermophilic bacterial cultures were isolated from a hot spring environment on hydrocarbon containing mineral salts media. One strain identified as Pseudomonas aeruginosa AP02-1 was tested for the ability to utilize a range of hydrocarbons both n-alkanes and polycyclic aromatic hydrocarbons as sole carbon source. Strain AP02-1 had an optimum growth temperature of 45°C and degraded 99% of crude oil 1% (v/v) and diesel oil 2% (v/v) when added to a basal mineral medium within 7 days of incubation. Surface activity measurements indicated that biosurfactants, mainly glycolipid in nature, were produced during the microbial growth on hydrocarbons as well as on both water-soluble and insoluble substrates. Mass spectrometry analysis showed different types of rhamnolipid production depending on the carbon substrate and culture conditions. Grown on glycerol, P. aeruginosa AP02-1 produced a mixture of ten rhamnolipid homologues, of which Rha-Rha-C₁₀-C₁₀ and Rha-C₁₀-C₁₀ were predominant. Rhamnolipid-containing culture broths reduced the surface tension to ≈28 mN and gave stable emulsions with a number of hydrocarbons and remained effective after sterilization. Microscopic observations of the emulsions suggested that hydrophobic cells acted as emulsion-stabilizing agents.     

Pseudomonas aeruginosa PAO1 as a model for rhamnolipid production in bioreactor systems

Rhamnolipids are biosurfactants with interesting physico-chemical properties. However, the main obstacles towards an economic production are low productivity, high raw-material costs, relatively expensive downstream processing, and a lack of understanding the rhamnolipid production regulation in bioreactor systems. This study shows that the sequenced Pseudomonas aeruginosa strain PAO1 is able to produce high quantities of rhamnolipid during 30 L batch bioreactor cultivations with sunflower oil as sole carbon source and nitrogen limiting conditions. Thus PAO1 could be an appropriate model for rhamnolipid production in pilot plant bioreactor systems. In contrast to well-established production strains, PAO1 allows knowledge-based systems biotechnological process development combined with the frequently used heuristic bioengineering approach. The maximum rhamnolipid concentration obtained was 39 g/L after 90 h of cultivation. The volumetric productivity of 0.43 g/Lh was comparable with previous described production strains. The specific rhamnolipid productivity showed a maximum between 40 and 70 h of process time of 0.088 g_RL/g_BDMh. At the same time interval, a shift of the molar di- to mono-rhamnolipid ratio from 1:1 to about 2:1 was observed. PAO1 not only seems to be an appropriate model, but surprisingly has the potential as a strain of choice for actual biotechnological rhamnolipid production.     

Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function

During the course of chronic cystic fibrosis (CF) infections, Pseudomonas aeruginosa undergoes a conversion to a mucoid phenotype, which is characterized by overproduction of the exopolysaccharide alginate. Chronic P. aeruginosa infections involve surface-attached, highly antibiotic-resistant communities of microorganisms organized in biofilms. Although biofilm formation and the conversion to mucoidy are both important aspects of CF pathogenesis, the relationship between them is at the present unclear. In this study, we report that the overproduction of alginate affects biofilm development on an abiotic surface. Biofilms formed by an alginate-overproducing strain exhibit a highly structured architecture and are significantly more resistant to the antibiotic tobramycin than a biofilm formed by an isogenic nonmucoid strain. These results suggest that an important consequence of the conversion to mucoidy is an altered biofilm architecture that shows increasing resistance to antimicrobial treatments.     

Marine bacterial isolates inhibit biofilm formation and disrupt mature biofilms of Pseudomonas aeruginosa PAO1

According to the Centers for Disease Control and Prevention, biofilms cause 65% of infections in developed countries. Pseudomonas aeruginosa biofilm cause life threatening infections in cystic fibrosis infection and they are 1,000 times more tolerant to antibiotic than the planktonic cells. As quorum sensing, hydrophobicity index and extracellular polysaccharide play a crucial role in biofilm formation, extracts from 46 marine bacterial isolates were screened against these factors in P. aeruginosa. Eleven extracts showed antibiofilm activity. Extracts of S6-01 (Bacillus indicus = MTCC 5559) and S6-15 (Bacillus pumilus = MTCC 5560) inhibited the formation of PAO1 biofilm up to 95% in their Biofilm Inhibitory Concentration(BIC) of 50 and 60 μg/ml and 85% and 64% in the subinhibitory concentrations (1/4 and 1/8 of the BIC, respectively). Furthermore, the mature biofilm was disrupted to 70–74% in their BIC. The antibiofilm compound from S6-15 was partially purified using solvent extraction followed by TLC and silica column and further characterized by IR analysis. Current study for the first time reveals the antibiofilm and antiquorum-sensing activity of B. pumilus, B. indicus, Bacillus arsenicus, Halobacillus trueperi, Ferrimonas balearica, and Marinobacter hydrocarbonoclasticus from marine habitat.     

Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture

Pseudomonas aeruginosa is an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslA Δalg8 overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture of P. aeruginosa.     

Quorum sensing regulates denitrification in Pseudomonas aeruginosa PAO1

Anaerobic growth of Pseudomonas aeruginosa PAO1 was affected by quorum sensing. Deletion of genes that produce N-acyl-l-homoserine lactone signals resulted in an increase in denitrification activity, which was repressed by exogenous signal molecules. The effect of the las quorum-sensing system was dependent on the rhl quorum-sensing system in regulating denitrification.     

Aspergillus ochraceopetaliformis SSP13 modulates quorum sensing regulated virulence and biofilm formation in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen causing the majority of acute and persistent infections in human beings. The ability to form biofilm adds a new dimension to its resistance to conventional therapeutic agents. In the present study, down-regulation of quorum sensing regulated virulence and biofilm development resulting from exposure to Aspergillus ochraceopetaliformis SSP13 extract was investigated. The in vitro results inferred impairment in the production of LasA protease, LasB elastase, chitinase, pyocyanin, exopolysaccharides and rhamnolipids. In addition, motility and biofilm formation by P. aeruginosa PAO1 was significantly altered. The in vitro results were further supported by molecular docking studies of the metabolites obtained from GC-MS analysis depicting the quorum sensing attenuation by targeting the receptor proteins LasR and RhlR. The in vitro and in silico studies suggested new avenues for the development of bioactive metabolites from A. ochraceopetaliformis SSP13 extract as potential anti-infective agents.     

Rhamnolipid production by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene

The potential of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene (vgb) for rhamnolipid production was studied. P. aeruginosa (NRRL B-771) and its transposon mediated vgb transferred recombinant strain, PaJC, were used in the research. The optimization of rhamnolipid production was carried out in the different conditions of cultivation (agitation rate, the composition of culture medium and temperature) in a time-course manner. The nutrient source, especially the carbon type, had a dramatic effect on rhamnolipid production. The PaJC strain and the wild type cells of P. aeruginosa started producing biosurfactant at the stationary phase and its concentration reached maximum at 24 h (838 mg/l(-1)) and at 72 h (751 mg l(-1)) of the incubation respectively. Rhamnolipid production was optimal in batch cultures when the temperature and agitation rate were controlled at 30 degrees C and 100 rpm. It reached 8373 mg l(-1) when the PaJC cells were grown in 1.0% glucose supplemented minimal media. Genetic engineering of biosurfactant producing strains with vgb may be an effective method to increase its production.     

Studies on the Pseudomonas aeruginosa PAO1 bacteriophage receptors

Receptor for phage PIK specific for Pseudomonas aeruginosa strain PAO1 was studied. Phage PIK was strongly inactivated by lipopolysaccharide (LPS) in vitro, exhibiting a PhI50 of 4.8 micrograms/ml. Further it was noted that this inactivation by LPS was reduced to 50% by several mono- and disaccharides when tested in vitro. D-glucosamine, D-mannose and L-rhamnose were found to be most effective at the concentration of 0.045 M, 0.25 M and 0.35 M respectively. This suggests the possibility that phage PIK receptor in LPS contains D-mannose, L-rhamnose and D-glucosamine. Either one of the former two could be located at a terminal position alpha-linked to the adjacent residue or located internally in the polysaccharide chain linked through its C-4 position. A theoretical approach to the interpretation of phage cell interaction was also investigated.     

Basic characterization of a Pseudomonas aeruginosa PAO1 bacteriophage

A bacteriophage of Pseudomonas aeruginosa PAO1 was characterized. Bacteriophage PIK was found to adsorb on the cell wall of the host organism. Electron microscopy of the phage PIK revealed that it had a bipyramidal hexagonal prismatic head of 110 nm in diameter, a tail which was 158 nm long and a tail plate of 47 nm width. This paper describes its basic characters, and a quantitative study was made of its adsorption to exponential phase cells of two different strains of P. aeruginosa. PIK was found to contain double stranded DNA and it appears to be virulent towards its host, P. aeruginosa PAO1. It was classified into the group of phages possessing a contractile tail.     

Genome Diversity of Pseudomonas aeruginosa PAO1 Laboratory Strains

Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion, P. aeruginosa PAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections.The metabolically versatile Pseudomonas aeruginosa is an opportunistic pathogen of plants, animals, and humans and is ubiquitously distributed in soil and aquatic habitats. The common reference strain is P. aeruginosa PAO1, a spontaneous chloramphenicol-resistant mutant of the original PAO strain (earlier called “P. aeruginosa strain 1”) that had been isolated in 1954 from a wound in Melbourne, Australia (9, 10). This PAO1 strain from Bruce Holloway''s laboratory has become the reference strain for Pseudomonas genetics and functional analyses of the physiology and metabolism of this gammaproteobacterium. A genetic map of its chromosome was generated by exploiting the mechanisms of gene exchange in bacteria, i.e., transduction and conjugation (11). With the advent of pulsed-field gel electrophoresis (PFGE), a physical map of the PAO1 genome was constructed (32) and later merged with the genetic map information (12). By 2000 the PAO1 strain had been completely sequenced (36). Thereafter, the genome annotation has been continually updated and the database content and functionality have been expanded to facilitate accelerated discovery of P. aeruginosa drug targets and vaccine candidates (38). Two near-saturation libraries of transposon insertion mutants have been constructed in P. aeruginosa PAO1 as a global resource for the scientific community (14, 22).Comparison of the genome sequence with the physical map revealed a large, 2.2-Mb inversion between the sequenced PAO1-UW strain (36) and the original PAO1 strain (9, 10), indicating that PAO1 sublines maintained worldwide in numerous laboratories and strain collections had diversified their genomic sequence. Mutational events were already reported in the 1970s (10), and more recently sequence variations of MexT, which regulates the MexEF-OprN multidrug efflux system, were described (18, 24). Furthermore, a PAO1 subline from a German strain collection (PAO1-D) and another, independent PAO1 subline from a Japanese strain collection (PAO1-J) that had been stored by research groups in Germany and Japan, respectively, were found to be quorum-sensing-negative mutants that carried point mutations in the regulatory gene lasR (6). In addition, spontaneous secretion-defective vfr mutants from a PAO1 population were observed after several cycles of static growth (2). Similarly, we noted a difference in virulence in a mouse infection model (see below) between the MPAO1 and PAO1-DSM sublines that had been utilized for the construction of the transposon library (14) and the physical map (32), respectively. PAO1-DSM was indistinguishable in its SpeI-DpnI-SwaI-PacI physical map from the PAO1 subline that had been stored in the Holloway laboratory (12). Hence, we decided to compare the genomic sequence of the initially sequenced PAO1 subline PAO1-UW (36) with that of MPAO1 and PAO1-DSM. Combined physical mapping and DNA sequencing-by-synthesis revealed numerous single-nucleotide polymorphisms (SNPs) and insertions-deletions (indels) in the chromosomes that were associated with differences in fitness, antimicrobial susceptibility, and virulence of the sublines.     

Kinetic modeling of rhamnolipid production by Pseudomonas aeruginosa PAO1 including cell density-dependent regulation

The production of rhamnolipid biosurfactants by Pseudomonas aeruginosa is under complex control of a quorum sensing-dependent regulatory network. Due to a lack of understanding of the kinetics applicable to the process and relevant interrelations of variables, current processes for rhamnolipid production are based on heuristic approaches. To systematically establish a knowledge-based process for rhamnolipid production, a deeper understanding of the time-course and coupling of process variables is required. By combining reaction kinetics, stoichiometry, and experimental data, a process model for rhamnolipid production with P. aeruginosa PAO1 on sunflower oil was developed as a system of coupled ordinary differential equations (ODEs). In addition, cell density-based quorum sensing dynamics were included in the model. The model comprises a total of 36 parameters, 14 of which are yield coefficients and 7 of which are substrate affinity and inhibition constants. Of all 36 parameters, 30 were derived from dedicated experimental results, literature, and databases and 6 of them were used as fitting parameters. The model is able to describe data on biomass growth, substrates, and products obtained from a reference batch process and other validation scenarios. The model presented describes the time-course and interrelation of biomass, relevant substrates, and products on a process level while including a kinetic representation of cell density-dependent regulatory mechanisms.     

Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1

Benzisothiazolone (BIT), N-methylisothiazolone (MIT) and 5-chloro-N-methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations. The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa. However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure. This experiment induced resistance in cultures of Ps. aeruginosa exposed to incrementally increasing sub-minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms. The induced resistance was observed as a gradual increase in MIC with each new passage. The MICs for all three test isothiazolones and a thiol-interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment. The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T-OMP, in SDS-PAGE preparations. T-OMP was observed to disappear from the biocide-exposed preparations and reappear when the resistance-induced cultures were passaged in the absence of biocide. This reappearance of T-OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC. The induction of resistance towards one biocide resulted in the development of cross-resistance towards other members of the group and the control, thiomersal. It has been suggested that the disappearance of T-OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon form (CMIT and MIT).     

Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials

This study was aimed at the development of economical methods for higher yields of biosurfactant by suggesting the use of low-cost raw materials. Two oil-degrading strains, Pseudomonas aeruginosa GS9-119 and DS10-129, were used to optimize a substrate for maximum rhamnolipid production. Among the two strains, the latter produced maxima of 4.31, 2.98, and 1.77 g/L rhamnolipid biosurfactant using soybean oil, safflower oil, and glycerol, respectively. The yield of biosurfactant steadily increased even after the bacterial cultures reached the stationary phase of growth. Characterization of rhamnolipids using mass spectrometry revealed the presence of dirhamnolipids (Rha-Rha-C(10)-C(10)). Emulsification activity of the rhamnolipid biosurfactant produced by P. aeruginosa DS10-129 was greater than 70% using all the hydrocarbons tested, including xylene, benzene, hexane, crude oil, kerosene, gasoline, and diesel. P. aeruginosa GS9-119 emulsified only hexane and kerosene to that level.     

Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning

In this report we describe experiments to investigate a simple virulence model in which Pseudomonas aeruginosa PAO1 rapidly paralyzes and kills the nematode Caenorhabditis elegans. Our results imply that hydrogen cyanide is the sole or primary toxic factor produced by P. aeruginosa that is responsible for killing of the nematode. Four lines of evidence support this conclusion. First, a transposon insertion mutation in a gene encoding a subunit of hydrogen cyanide synthase (hcnC) eliminated nematode killing. Second, the 17 avirulent mutants examined all exhibited reduced cyanide synthesis, and the residual production levels correlated with killing efficiency. Third, exposure to exogenous cyanide alone at levels comparable to the level produced by PAO1 killed nematodes with kinetics similar to those observed with bacteria. The killing was not enhanced if hcnC mutant bacteria were present during cyanide exposure. And fourth, a nematode mutant (egl-9) resistant to P. aeruginosa was also resistant to killing by exogenous cyanide in the absence of bacteria. A model for nematode killing based on inhibition of mitochondrial cytochrome oxidase is presented. The action of cyanide helps account for the unusually broad host range of virulence of P. aeruginosa and may contribute to the pathogenesis in opportunistic human infections due to the bacterium.     

Metal binding by Pseudomonas aeruginosa PAO1 is influenced by growth of the cells as a biofilm.

The metal-binding properties of Pseudomonas aeruginosa PAO1 biofilms were investigated using four metals (Cu, Fe, Au, and La). All but one of the metals (i.e., Cu) were bound by the biofilms in amounts that were significantly greater than those bound by planktonically grown cells of the same strain. Lanthanum precipitation appeared to be limited to the base of the biofilms and was not promoted by a shift in lipopolysaccharide production by the cells.     

Sequence-Verified Two-Allele Transposon Mutant Library for Pseudomonas aeruginosa PAO1

Mutant hunts using comprehensive sequence-defined libraries make it possible to identify virtually all of the nonessential functions required for different bacterial processes. However, the success of such screening depends on the accuracy of mutant identification in the mutant library used. To provide a high-quality library for Pseudomonas aeruginosa PAO1, we created a sequence-verified collection of 9,437 transposon mutants that provides genome coverage and includes two mutants for most genes. Mutants were cherry-picked from a larger library, colony-purified, and resequenced both individually using Sanger sequencing and in a pool using Tn-seq. About 8% of the insertion assignments were corrected, and in the final library nearly 93% of the transposon locations were confirmed by at least one of the resequencing procedures. The extensive sequence verification and inclusion of more than one mutant for most genes should help minimize missed or erroneous genotype-phenotype assignments in studies using the new library.