Alginate synthesis by mucoid strains of Pseudomonas aeruginosa PAO

Summary Alginic acid production by Pseudomonas aeruginosa PAO strains was studied in yeast extract/2% (w/v) gluconate medium. In all of the five strains studied, synthesis of the alginic acid was shown to occur in the stationary phase of growth. Each strain produced similar amounts of alginic acid at both 30° C and 37° C. However the amount of alginic acid varied from 7.5–11.5 gl^–1 depending upon the strain. The alginic acid was isolated, purified and its chemical composition determined. All strains produced a polysaccharide rich in polymannuronic acid which contained only polymannuronic acid blocks, polyguluronic acid blocks appeared to be absent. The amount of O-acetylation varied considerably from 2.3–14.7%. Analysis of the chain length distribution by poly-acrylamide gel electropheresis indicated that a homogeneous size of polymer was synthesised when compared to a high viscosity algal sample.     

The chromosome map of Pseudomonas aeruginosa PAO

A revised chromosomal map of Pseudomonas aeruginosa is presented and the role of a variety of mapping procedures is discussed.     

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.     

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.     

Initial characterization of two extracellular autolysins from Pseudomonas aeruginosa PAO1.

Two extracellular autolysins have been detected in the spent culture supernatants of Pseudomonas aeruginosa PAO1 by using renaturing polyacrylamide gel electrophoresis. The two autolysins were isolated from the culture supernatant by trichloroacetic acid precipitation and were shown to have apparent molecular masses of 26 and 29 kDa. The 26-kDa autolysin first appears during the early exponential phase of growth and then declines sharply, while the 29-kDa autolysin first appears in the late exponential phase of growth and continues well into the stationary phase. Fractionation of whole cells indicated that the 26-kDa enzyme was also localized within the periplasm, with a lesser amount of activity associated with the cytoplasmic membrane. The 29-kDa autolytic activity was distributed within the cell equally between the periplasm and the cytoplasmic membrane. The pH optima of the isolated 26- and 29-kDa autolysins are 6.0 and 5.0, respectively. Further evidence from both protease susceptibility and inhibition studies confirms that these two extracellular autolysins isolated from P. aeruginosa PAO1 are separate and distinct.     

A function of Pseudomonas aeruginosa PAO polar pili: twitching motility

Twitching motility is a mode of flagella-independent surface translocation exhibited by Pseudomonas aeruginosa and other bacteria on solid media. All species exhibiting it carry thin pili, usually polar. This work shows that only PAO and K strains of P. aeruginosa with retractile (PSA) pili were able to move in this way, those with no pili or non-retractile pili remaining stationary. Specific agents such as anti-pilus serum, which prevents otherwise functional pili from retracting, also prevented twitching motility.     

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.     

Characterization of the indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic infections in the lungs of individuals with cystic fibrosis. It is intrinsically resistant to many antibiotics, and resistance is emerging rapidly to those drugs that currently remain efficacious. Therefore, there is a pressing need to identify new anti-pseudomonal drug targets. To this end, we have characterized the P. aeruginosa indole-3-glycerol phosphate synthase (PaIGPS). PaIGPS catalyzes the fifth reaction in the synthesis of tryptophan from chorismate??a reaction that is absent in mammals. PaIGPS was expressed heterologously in Escherichia coli, and purified with high yields. The purified enzyme is active over a broad pH range and has the highest turnover number of any characterized IGPS (k _cat?=?11.1?±?0.1?s^?1). These properties are likely to make PaIGPS useful in coupled assays for other enzymes in tryptophan biosynthesis. We have also shown that deleting the gene for PaIGPS reduces the fitness of P. aeruginosa strain PAO1 in synthetic cystic fibrosis sputum (relative fitness, W?=?0.89?±?0.02, P?=?0.001). This suggests that de novo tryptophan biosynthesis may play a role in the establishment and maintenance of P. aeruginosa infections, and therefore that PaIGPS is a potential target for the development of new anti-pseudomonal drugs.     

Structural studies of lipid A from Pseudomonas aeruginosa PAO1: occurrence of 4-amino-4-deoxyarabinose.

Lipid A derived from Pseudomonas aeruginosa PAO1 contains a biphosphorylated 1-6-linked glucosamine disaccharide backbone. The reducing glucosamine has an unsubstituted glycosidically linked phosphate at C-1. The nonreducing glucosamine has an ester-bound phosphate at C-4' which is nonstoichiometrically substituted with 4-amino-4-deoxyarabinose. Induction of 4-amino-4-deoxyarabinose was dependent on cultural conditions. No pyrophosphate groups were detected. Acyloxyacyl diesters are formed by esterification of the amide-bound 3-hydroxydodecanoic acid with dodecanoic acid and 2-hydroxydodecanoic acids in an approximate molar ratio of 2:1. Dodecanoic and 3-hydroxydecanoic acids are esterified to positions C-3 and C-3' in the sugar backbone. All hydroxyl groups of the glucosamine disaccharide except C-4 and C-6' are substituted. Lipopolysaccharide chemical analyses measured glucose, rhamnose, heptose, galactosamine, alanine, phosphate, and glucosamine. The proposed lipid A structure differs from previous models. There are significant differences in acyloxyacyl diesters, and the proposed model includes an aminopentose substituent.     

Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1

Two seven-gene phenazine biosynthetic loci were cloned from Pseudomonas aeruginosa PAO1. The operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are homologous to previously studied phenazine biosynthetic operons from Pseudomonas fluorescens and Pseudomonas aureofaciens. Functional studies of phenazine-nonproducing strains of fluorescent pseudomonads indicated that each of the biosynthetic operons from P. aeruginosa is sufficient for production of a single compound, phenazine-1-carboxylic acid (PCA). Subsequent conversion of PCA to pyocyanin is mediated in P. aeruginosa by two novel phenazine-modifying genes, phzM and phzS, which encode putative phenazine-specific methyltransferase and flavin-containing monooxygenase, respectively. Expression of phzS alone in Escherichia coli or in enzymes, pyocyanin-nonproducing P. fluorescens resulted in conversion of PCA to 1-hydroxyphenazine. P. aeruginosa with insertionally inactivated phzM or phzS developed pyocyanin-deficient phenotypes. A third phenazine-modifying gene, phzH, which has a homologue in Pseudomonas chlororaphis, also was identified and was shown to control synthesis of phenazine-1-carboxamide from PCA in P. aeruginosa PAO1. Our results suggest that there is a complex pyocyanin biosynthetic pathway in P. aeruginosa consisting of two core loci responsible for synthesis of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxamide.     

In vitro transcription analysis of rpoD in Pseudomonas aeruginosa PAO1

Thymidine kinase type II is an important part of the pyrimidine salvage pathway. The thymidine kinase gene from the thermophilic eubacterium Rhodothermus marinus was cloned, sequenced and overexpressed. The gene is 639 bp and encodes a protein of 213 amino acids with a calculated molecular mass of 23.6 kDa. It shows homology to other thymidine kinase proteins from eukaryotic and prokaryotic organisms. The recombinant protein is inhibited by dNTPs but not by dNDPs. It is a tetramer in its native state. Its optimum temperature of activity is 65 degrees C and it has a half life of 15 min at 90 degrees C. This is the first thymidine kinase to be described from a thermophilic bacterium.     

Expression of genes involved in rhamnolipid synthesis in Pseudomonas aeruginosa PAO1 in a bioreactor cultivation

There is a growing demand for economic bioprocesses based on sustainable resources rather than petrochemical-derived substances. Particular attention has been paid to rhamnolipids—surface-active glycolipids—that are naturally produced by Pseudomonas aeruginosa. Rhamnolipids have gained increased attention over the past years due to their versatile chemical and biological properties as well as numerous biotechnological applications. However, rhamnolipid synthesis is tightly governed by a complex growth-dependent regulatory network. Quantitative comprehension of the molecular and metabolic mechanisms during bioprocesses is key to manipulating and improving rhamnolipid production capacities in P. aeruginosa. In this study, P. aeruginosa PAO1 was grown under nitrogen limitation with sunflower oil as carbon and nitrate as nitrogen source in a batch fermentation process. Gene expression was monitored using quantitative PCR over the entire time course. Until late deceleration phase, an increase in relative gene expression of the las, rhl, and pqs quorum-sensing regulons was observed. Thereafter, expression of the rhamnolipid synthesis genes, rhlA and rhlC, as well as the las regulon was downregulated. RhlR was shown to remain upregulated at the late phase of the fermentation process.     

The nature of Pseudomonas aeruginosa strain PAO bacteriophage receptors.

Receptors for phages specific to Pseudomonas aeruginosa strain PAO were studied. Phages 16, 44, 109, F8, and PBI are lipopolysaccharide (LPS) specific as shown by neutralization tests. The PhI50's of the LPS, adsorption rate constants with strain PAO and the plaque morphologies of these five phages were quite similar. Phages 1214 and 7 also appear to be LPS-specific on the basis of host-range studies. Phage 73 is pilus-specific, while phages 21 and 68 fall into a group which does not attach to pili, flagella, or LPS. A theoretical approach to the interpretation of phage-cell interactions is presented.     

Comparative analysis of two UDP-glucose dehydrogenases in Pseudomonas aeruginosa PAO1

UDP-glucose dehydrogenase (UGDH) catalyzes a two-step NAD(+)-dependent oxidation of UDP-glucose to produce UDP-glucuronic acid, which is a common substrate for the biosynthesis of exopolysaccharide. Searching the Pseudomonas aeruginosa PAO1 genome data base for a UGDH has helped identify two open reading frames, PA2022 and PA3559, which may encode a UGDH. To elucidate their enzymatic identity, the two genes were cloned and overexpressed in Escherichia coli, and the recombinant proteins were purified. Both the gene products are active as dimers and are capable of utilizing UDP-glucose as a substrate to generate UDP-glucuronic acid. The K(m) values of PA2022 and PA3559 for UDP-glucose are approximately 0.1 and 0.4 mM, whereas the K(m) values for NAD(+) are 0.5 and 2.0 mM, respectively. Compared with PA3559, PA2022 exhibits broader substrate specificity, utilizing TDP-glucose and UDP-N-acetylglucosamine with one-third the velocity of that with UDP-glucose. The PA2022 mutant and PA2022-PA3559 double mutant, but not the PA3559 mutant, are more susceptible to chloramphenicol, cefotaxime, and ampicillin. The PA3559 mutant, however, shows a reduced resistance to polymyxin B compared with wild type PAO1. Finally, real time PCR analysis indicates that PA3559 is expressed primarily in low concentrations of Mg(2+), which contrasts with the constitutive expression of PA2022. Although both the enzymes catalyze the same reaction, their enzymatic properties and gene expression profiles indicate that they play distinct physiological roles in P. aeruginosa, as reflected by different phenotypes displayed by the mutants.