Cell aggregation of Pseudomonas aeruginosa strain PAO1 as an energy-dependent stress response during growth with sodium dodecyl sulfate

Pseudomonas aeruginosa strain PAO1 grew with the detergent sodium dodecyl sulfate (SDS). The growth started with the formation of macroscopic cell aggregates which consisted of respiring cells embedded in an extracellular matrix composed of acidic polysaccharides and DNA. Damaged and uncultivable cells accumulated in these aggregates compared to those cells that remained suspended. We investigated the response of suspended cells to SDS under different conditions. At high energy supply, the cells responded with a decrease in optical density and in viable counts, release of protein and DNA, and formation of macroscopic aggregates. This response was not observed if the energy supply was reduced by inhibiting respiration with KCN, or if cells not induced for SDS degradation were exposed to SDS. Exposure to SDS caused cell lysis without aggregation if cells were completely deprived of energy, either by applying anoxic conditions, by addition of CCCP, or by addition of KCN to a mutant defective in cyanide-insensitive respiration. Aggregated cells showed a more than 100-fold higher survival rate after exposure to SDS plus CCCP than suspended cells. Our results demonstrate that cell aggregation is an energy-dependent response of P. aeruginosa to detergent stress which might serve as a survival strategy during growth with SDS.     

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.     

Vanadate and triclosan synergistically induce alginate production by Pseudomonas aeruginosa strain PAO1

Alginate overproduction by P. aeruginosa strains, also known as mucoidy, is associated with chronic lung infections in cystic fibrosis (CF). It is not clear how alginate induction occurs in the wild-type (wt) mucA strains. When grown on Pseudomonas isolation agar (PIA), P. aeruginosa strains PAO1 and PA14 are non-mucoid, producing minimal amounts of alginate. Here we report the addition of ammonium metavanadate (AMV), a phosphatase inhibitor, to PIA (PIA-AMV) induced mucoidy in both these laboratory strains and early lung colonizing non-mucoid isolates with a wt mucA. This phenotypic switch was reversible depending on the availability of vanadate salts and triclosan, a component of PIA. Alginate induction in PAO1 on PIA-AMV was correlated with increased proteolytic degradation of MucA, and required envelope proteases AlgW or MucP, and a two-component phosphate regulator, PhoP. Other changes included the addition of palmitate to lipid A, a phenotype also observed in chronic CF isolates. Proteomic analysis revealed the upregulation of stress chaperones, which was confirmed by increased expression of the chaperone/protease MucD. Altogether, these findings suggest a model of alginate induction and the PIA-AMV medium may be suitable for examining early lung colonization phenotypes in CF before the selection of the mucA mutants.     

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.     

Biochemical studies on pili isolated from Pseudomonas aeruginosa strain PAO.

Pseudomonas aeruginosa strains PAO and PAK bear polar pili which are flexible filaments having a diameter of 6 nm and an average length of 2500 nm. Both types of pili are retractile and promote infection by a number of bacteriophages. The present communication describes the partial biochemical characterization of PAO pili isolated from a multipiliated nonretractile mutant of PAO. The observed properties are compared to those of PAK pili which were characterized previously. PAO pili were found to contain a single polypeptide subunit of 18 700 daltons. This is similar to PAK pili which contain a single polypeptide of 18 100 daltons. The amino acid composition of PAO pilin was also similar to that of PAK pilin. Neither protein contained phosphate or carbohydrate residues and both were found to contain N-methylphenylalanine at the amino terminus. Sequencing of 20 amino acid residues at the amino terminal end of PAO pilin revealed the sequence to be identical with that of PAK pilin, while tryptic peptide analyses of PAO and PAK pilin indicated that the two proteins probably contain a number of homologous regions within the polypeptide. It was concluded that PAO and PAK pili were closely related structures.     

Influence of ferric iron on gene expression and rhamnolipid synthesis during batch cultivation of Pseudomonas aeruginosa PAO1

Bioprocesses based on sustainable resources and rhamnolipids in particular have become increasingly attractive in recent years. These surface-active glycolipids with various chemical and biological properties have diverse biotechnological applications and are naturally produced by Pseudomonas aeruginosa. Their production, however, is tightly governed by a complex growth-dependent regulatory network, one of the major obstacles in the way to upscale production. P. aeruginosa PAO1 was grown in shake flask cultures using varying concentrations of ferric iron. Gene expression was assessed using quantitative PCR. A strong increase in relative expression of the genes for rhamnolipid synthesis, rhlA and rhlC, as well as the genes of the pqs quorum sensing regulon was observed under iron-limiting conditions. Iron repletion on the other hand caused a down-regulation of those genes. Furthermore, gene expression of different iron regulation-related factors, i.e. pvdS, fur and bqsS, was increased in response to iron limitation. Ensuing from these results, a batch cultivation using production medium without any addition of iron was conducted. Both biomass formation and specific growth rates were not impaired compared to normal cultivation conditions. Expression of rhlA, rhlC and pvdS, as well as the gene for the 3-oxo-C12-HSL synthetase, lasI, increased until late stationary growth phase. After this time point, their expression steadily decreased. Expression of the C4-HSL synthetase gene, rhlI, on the other hand, was found to be highly increased during the entire process.     

Transformation of Pseudomonas aeruginosa strain PAO with bacteriophage and plasmid DNA.

A procedure has been developed which allows transformation of P. aeruginosa strain PAO with plasmid and bacteriophage DNA at a frequency of 10(-6) per recipient cell. The method is similar in outline to that developed for Escherichia coli. It involves growing the recipient cells to 3-5 x 10(8) per ml in nutrient broth, washing the cells with 0.1 M MgCl2, resuspending in 0.175 M CaCl2 for 20 min, exposing to DNA for 1 h and then heat pulsing at 42 degrees C for 1 min. Some plasmid markers are expressed immediately, whereas others require time for phenotypic expression.     

Composition of alginate synthesized during the growth cycle of Pseudomonas aeruginosa

A mucoid P. aeruginosa isolated from the sputum of a cystic fibrosis patient was grown in batch culture on a complex medium. During the growth cycle the amount of alginate produced was estimated and its composition was determined by proton magnetic resonance (1H-n.m.r) spectroscopy. Exopolysaccharide production occurred mainly during the exponential phase of growth. The alginate samples isolated varied little in composition and were characterized by being highly acetylated, high mannuronate (0.83-0.93 mole fraction) polymers. Guluronate was present only within heteropolymeric regions of the polysaccharides which all displayed a complete absence of polyguluronate. Ca2+ ion supplementation of the medium was not observed to increase the levels of guluronate in the alginates produced.     

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.     

Isolation, Characterization, and Heterologous Expression of a Carboxylesterase of Pseudomonas aeruginosa PAO1

We purified to homogeneity an intracellular esterase from the opportunistic pathogen Pseudomonas aeruginosa PAO1. The enzyme hydrolyzes p-nitrophenyl acetate and other acetylated substrates. The N-terminal amino acid sequence was analyzed and 11 residues, SEPLILDAPNA, were determined. The corresponding gene PA3859 was identified in the P. aeruginosa PAO1 genome as the only gene encoding for a protein with this N-terminus. The encoding gene was cloned in Escherichia coli, and the recombinant protein expressed and purified to homogeneity. According to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis and analytical gel filtration chromatography, the esterase was found to be a monomer of approximately 24 kDa. The experimentally determined isoelectric point was 5.2 and the optimal enzyme activity was at 55°C and at pH 9.0. The esterase preferentially hydrolyzed short-chain fatty acids. It is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by ethylendiaminotetraacetic acid (EDTA). Native enzyme preparations typically showed a Michaelis constant (K_m) and V_max of 0.43 mM and 12,500 U mg^–1, respectively, using p-nitrophenyl acetate as substrate. Homology-based database searches clearly revealed the presence of the consensus GXSXG signature motif that is present in the serine-dependent acylhydrolase protein family.