Evidence for small pores in the outer membrane of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa NCTC6750 and Escherichia coli K12 were used to study permeability of whole, intact cells to a series of labelled oligosaccharides. Stationary phase, oxygen depleted simple salts batch cultures were used. An efflux method was used to compare diffusion from cells of various ³H-labelled sugars (an homologous series based on isomaltitol) with diffusion of [¹⁴C]sucrose. Both plasmolysed and unplasmolysed cell suspensions were used. The data are consistent with an E. coli pore exclusion limit of approx. 833 Da for unplasmolysed cells and of about 670 Da for plasmolysed cells. For P. aeruginosa the data indicated a relatively small pore exclusion limit about the same size as sucrose with plasmolysis having little effect. These findings were confirmed with P. aeruginosa PAO1 grown in nutrient broth.     

Evidence for gene sharing in the nitrate reduction systems of Pseudomonas aeruginosa

Pseudomonas aeruginosa mutants unable to assimilate or dissimilate nitrate were isolated. Transduction and reversion analyses of these mutants revealed that single genetic lesions are responsible for the double phenotypes. The mutants were divided into two classes based on the ability to utilize hypoxanthine. It can be concluded from this study that at least two genes are shared between the two nitrate reduction systems.     

Evidence for the location of OprM in the Pseudomonas aeruginosa outer membrane

Abstract OprM with a M _r of 49 K is associated with the multidrug resistance of Pseudomonas aeruginosa . Detergent fractionation of bacterial cells has demonstrated that OprM is located in the outer membrane from which it sediments with the other major outer membrane proteins. In this study we have determined the location of OprM as the P. aeruginosa outer membrane. Western immunoblots of cell fractions, obtained by sucrose density gradient centrifugation of whole cell lysates, were probed with an OprM-specific murine polyclonal antiserum.     

Evidence for stable transformation of Pseudomonas aeruginosa with a pUC-based plasmid

Pseudomonas aeruginosa was transformed with pUC8:16, a pUC-based plasmid bearing the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). Transformation was initially indicated by an increase in ampicillin resistance from 1500 to 2500 mg l^–1. Presence of the plasmid in P. aeruginosa was confirmed by amplification of a portion of vgb from and detection of VHb in the transformant but not the untransformed host. Southern blot analysis further indicated that pUC8:16 existed as an autonomous plasmid rather than integrated into the chromosome of the P. aeruginosa transformant.     

Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa

Pyoverdine is the primary siderophore of the gram-negative bacterium Pseudomonas aeruginosa. The pyoverdine region was recently identified as the most divergent locus alignable between strains in the P. aeruginosa genome. Here we report the nucleotide sequence and analysis of more than 50 kb in the pyoverdine region from nine strains of P. aeruginosa. There are three divergent sequence types in the pyoverdine region, which correspond to the three structural types of pyoverdine. The pyoverdine outer membrane receptor fpvA may be driving diversity at the locus: it is the most divergent alignable gene in the region, is the only gene that showed substantial intratype variation that did not appear to be generated by recombination, and shows evidence of positive selection. The hypothetical membrane protein PA2403 also shows evidence of positive selection; residues on one side of the membrane after protein folding are under positive selection. R', previously identified as a type IV strain, is clearly derived from a type III strain via a 3.4-kb deletion which removes one amino acid from the pyoverdine side chain peptide. This deletion represents a natural modification of the product of a nonribosomal peptide synthetase enzyme, whose consequences are predictive from the DNA sequence. There is also linkage disequilibrium between the pyoverdine region and pvdY, a pyoverdine gene separated by 30 kb from the pyoverdine region. The pyoverdine region shows evidence of horizontal transfer; we propose that some alleles in the region were introduced from other soil bacteria and have been subsequently maintained by diversifying selection.     

Prophage F116: Evidence for Extrachromosomal Location in Pseudomonas aeruginosa Strain PAO

F116 is a temperate-generalized transducing phage of Pseudomonas aeruginosa. Genetic evidence leads to the conclusion that F116 prophage DNA is maintained extrachromosomally as a plasmid. Preliminary physical evidence is presented to support this hypothesis.     

Evidence for two flagellar stators and their role in the motility of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous bacterium capable of twitching, swimming, and swarming motility. In this study, we present evidence that P. aeruginosa has two flagellar stators, conserved in all pseudomonads as well as some other gram-negative bacteria. Either stator is sufficient for swimming, but both are necessary for swarming motility under most of the conditions tested, suggesting that these two stators may have different roles in these two types of motility.     

Evidence for the in vivo degradation of human respiratory mucins during Pseudomonas aeruginosa infection

The comparison of distribution of glycopeptides of sputa from patients suffering from various chronic hypersecretions has already shown an increased acidity with a decreased proportion of neutral glycopeptides in the respiratory secretions of patients suffering from cystic fibrosis, as compared to those of patients with chronic bronchitis. In order to find out whether this decrease is specific to cystic fibrosis mucins or whether it is due to a degradation of mucus by Pseudomonas aeruginosa, which infects most of the sputa from patients with this disease, mucus glycopeptides from patients with different chronic bronchial disorders, infected by Pseudomonas or not, were prepared and fractionated by ion-exchange chromatography. The neutral fraction, which has never been studied in detail, was gel-filtered, and provided two fractions, one containing true mucin glycopeptides and the other containing a mixture of peptides and glycopeptides with a lower molecular mass. In the Pseudomonas-infected samples, the true mucin glycopeptide fraction was greatly diminished as compared to this same fraction in non-Pseudomonas-infected samples; this was not specific to cystic fibrosis secretions. In contrast, the glycopeptide fraction with a lower molecular mass was greatly increased in all the Pseudomonas-infected samples. Polyacrylamide gel electrophoresis of this second fraction showed unique glycopeptide bands between 40-50 kDa in the Pseudomonas-infected samples, regardless of the origin of the samples. These bands were revealed by an antibody directed against whole cystic fibrosis mucin. Infected chronic bronchitis sputa and cystic fibrosis samples without P. aeruginosa did not show these bands. These studies therefore suggest that there are P. aeruginosa-associated changes in mucins which may result from degradation of mucins.     

Evidence for phage-mediated gene transfer among Pseudomonas aeruginosa strains on the phylloplane.

As the use of genetically engineered microorganisms for agricultural tasks becomes more frequent, the ability of bacteria to exchange genetic material in the agricultural setting must be assessed. Transduction (bacterial virus-mediated horizontal gene transfer) is a potentially important mechanism of gene transfer in natural environments. This study investigated the potential of plant leaves to act as surfaces on which transduction can take place among microorganisms. Pseudomonas aeruginosa and its generalized transducing bacteriophage F116 were used as a model system. The application of P. aeruginosa lysogens of F116 to plant leaves resulted in genetic exchange among donor and recipient organisms resident on the same plant. Transduction was also observed when these bacterial strains were inoculated onto adjacent plants and contact was made possible through high-density planting.