Dennis E. Ohman

Mutants of Pseudomonas aeruginosa PAO1 that were deficient in the ability to produce proteases that degrade casein were detected among the survivors of chemical mutagenesis. One such mutant (PDO31) showed reduced production of elastolytic activity, beta-hemolytic activity, and pyocyanin. A 4.3-kb EcoRI fragment from a gene bank of PAO1 that complemented defects in PDO31 was found. Transposon mutagenesis and deletion derivatives of the clone were used in conjunction with complementation tests to determine the physical location of the gene of interest. Nucleotide sequence analysis revealed an open reading frame (rhlR) encoding a putative 27.6-kDa protein (RhlR) with homology to autoinducer-responsive regulators of quorum sensing systems such as LuxR of Vibrio fischeri and LasR of P. aeruginosa. Further sequence analysis downstream of rhlR revealed an independently transcribed gene (rhlI) that encodes a putative 22.2-kDa protein with homology to members of the family of autoinducer synthetases, such as LuxI of V. fischeri and LasI of P. aeruginosa. The rhlRI sequences were also recently reported by others (U.A. Ochsner and J. Reiser, Proc. Natl. Acad. Sci. USA 92: 6424-6428, 1995) as an autoinducer-mediated regulation mechanism for rhamnolipid biosurfactant synthesis in P. aeruginosa PG201. Mutants with defects in rhlR or rhlI were constructed in PAO1 by gene replacement, using clones modified by Tn501 insertion. Compared with the wild type, the rhlR and rhlI mutants both showed defects in the production of elastase, LasA protease, rhamnolipid, and pyocyanin. Transcription from the gene for elastase, as measured with a lasB-cat fusion, demonstrated that production of elastase was subject to cell density-dependent gene activation in PAO1. However, transcription of lasB-cat in the rhlI mutant, which had lost the presumptive autoinducer synthetase (predicted to activate RhlR), showed low basal activity and had lost all cell density-dependent transcription of lasB. Thus, RhlR-RhlI represent the second autoinducer-responsive regulatory mechanism found in P. aeruginosa that controls expression of multiple virulence factor exoproducts, including elastase.

The leading cause of mortality in patients with cystic fibrosis (CF) is respiratoy failure due in large part to chronic lung infection with Pseudomonas aeruginosa strains that undergo mucoid conversion, display a biofilm mode of growth in vivo and resist the infiltration of polymorphonuclear leukocytes (PMNs), which release free oxygen radicals such as H2O2. The mucoid phenotype among the strains infecting CF patients indicates overproduction of a linear polysaccharide called alginate. To mimic the inflammatory environment of the CF lung, P. aeruginosa PAO1, a typical non-mucoid strain, was grown in a biofilm. This was treated with low levels of H2O2, as if released by the PMNs, and the formation of mucoid variants was observed. These mucoid variants had mutations in mucA, which encodes an anti-σ factor; this leads to the deregulation of an alternative σ factor (σ22, AlgT or AlgU) required for expression of the alginate biosynthetic operon. All of the mucoid variants tested showed the same mutation, the mucA22 allele, a common allele seen in CF isolates. The mucoid mucA22 variants, when compared to the smooth parent strain PA01, produced 2--6-fold higher levels of alginate|ii) exhibited no detectable differences in growth rate|iii) showed an unaltered LPS profile|iv) were ~72% reduced in the amount of inducible-β-lactamase and (v) secreted little no LasA protease and only showed 44% elastase activity. A characteristic ~54 kDa protein associated with alginate overproducing strains was identified as AlgE (Alg76) by N-terminal sequence analysis. Thus, the common phenotype of the mucoid variants, which included a genetically engineered mucA22 mutant, suggested that the only mutation incurred as a result of H2O2 treatment was in mucA. When a P. aeruginosa biofilm was repeatedly expose to activated PMNs in vitro, mucoid variants were also observed, mimicking in vivo observations. Thus, PMNs and their oxygen by-products may cause P. aeruginosa to undergo the typical adaptation to the intractable mu- coid form in the CF lung. These findings indicate that gene activation in bacteria by toxic oxygen radicals, similar to that found in plants and mammalian cells, may serve as a defence mechanism for the bacteria. This suggests that mucoid conversion is a response to oxygen radical exposure and that this response is mechanism of defence by the bacteria. This is the first report to show that PMNs and their oxygen radicals can cause this phenotypic and genotypic change which is so typical of the intractable form of P. aeruginosa in the CF lung. These findings may provide a basis for the development of anti-oxidant and anti-inflammatory therapy for the early stages of infection in CF patients

Attenuated total reflection/Fourier transform-infrared spectrometry (ATR/FT-IR) and scanning confocal laser microscopy (SCLM) were used to study the role of alginate and alginate structure in the attachment and growth of Pseudomonas aeruginosa on surfaces. Developing biofilms of the mucoid (alginate-producing) cystic fibrosis pulmonary isolate FRD1, as well as mucoid and nonmucoid mutant strains, were monitored by ATR/FT-IR for 44 and 88 h as IR absorbance bands in the region of 2,000 to 1,000 cm(-1). All strains produced biofilms that absorbed IR radiation near 1,650 cm(-1) (amide I), 1,550 cm(-1) (amide II), 1,240 cm(-1) (P==O stretching, C---O---C stretching, and/or amide III vibrations), 1,100 to 1,000 cm(-1) (C---OH and P---O stretching) 1,450 cm(-1), and 1,400 cm(-1). The FRD1 biofilms produced spectra with an increase in relative absorbance at 1,060 cm(-1) (C---OH stretching of alginate) and 1,250 cm(-1) (C---O stretching of the O-acetyl group in alginate), as compared to biofilms of nonmucoid mutant strains. Dehydration of an 88-h FRD1 biofilm revealed other IR bands that were also found in the spectrum of purified FRD1 alginate. These results provide evidence that alginate was present within the FRD1 biofilms and at greater relative concentrations at depths exceeding 1 micrometer, the analysis range for the ATR/FT-IR technique. After 88 h, biofilms of the nonmucoid strains produced amide II absorbances that were six to eight times as intense as those of the mucoid FRD1 parent strain. However, the cell densities in biofilms were similar, suggesting that FRD1 formed biofilms with most cells at depths that exceeded the analysis range of the ATR/FT-IR technique. SCLM analysis confirmed this result, demonstrating that nonmucoid strains formed densely packed biofilms that were generally less than 6 micrometer in depth. In contrast, FRD1 produced microcolonies that were approximately 40 micrometer in depth. An algJ mutant strain that produced alginate lacking O-acetyl groups gave an amide II signal approximately fivefold weaker than that of FRD1 and produced small microcolonies. After 44 h, the algJ mutant switched to the nonmucoid phenotype and formed uniform biofilms, similar to biofilms produced by the nonmucoid strains. These results demonstrate that alginate, although not required for P. aeruginosa biofilm development, plays a role in the biofilm structure and may act as intercellular material, required for formation of thicker three-dimensional biofilms. The results also demonstrate the importance of alginate O acetylation in P. aeruginosa biofilm architecture.

The sigma factor RpoS (sigmaS) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. To elucidate the role of RpoS in Pseudomonas aeruginosa physiology and pathogenesis, we constructed rpoS mutants in several strains of P. aeruginosa, including PAO1. The PAO1 rpoS mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent. The PAO1 rpoS mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source. In addition, the PAO1 rpoS mutant was hypersensitive to heat shock at 50 degrees C, increased osmolarity, and prolonged exposure to high concentrations of H2O2. In accordance with the hypersensitivity to H2O2, catalase production was 60% lower in the rpoS mutant than in the parent strain. We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of P. aeruginosa. The rpoS mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain. The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in rpoS in PAO1. The rpoS mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine. This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1 rpoS mutant that was observed in a rat chronic-lung-infection model. In addition, the rpoS mutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected. Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the rpoS101::aacCI mutation almost completely abolished the production of alginate when the bacterium was grown in a liquid medium. On a solid medium, the FRD1 rpoS mutant produced approximately 70% less alginate than did the wild-type strain. Thus, our data indicate that although some of the functions of RpoS in P. aeruginosa physiology are similar to RpoS functions in other gram-negative bacteria, it also has some functions unique to this bacterium.

Pseudomonas aeruginosa PAO mutants defective in elastase were isolated by plate assays of nitrosoguanidine-mutagenized clones. A total of 75 elastase mutants were isolated from 43,000 mutagenized clones. One mutant (PAO-E64) was apparently identical to the parental strain except for its deficiency in elastase activity. This mutant produced an enzyme which was antigenically indistinguishable from parental elastase. Furthermore, equal levels of elastase antigen were produced by this mutant and its parental strain. The mutant elastase, however, had greatly reduced enzymatic activity. Mutant PAO-E64 is presumed to have a mutation in the structural gene for elastase. We have designated the genotype of the mutation in PAO-E64 as lasA1.