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, ␤-hemolytic activity, and pyocyanin. 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 autoinducerresponsive 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 respiratory 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 H202. The mucoid phenotype among the strains infecting CF patients indicates overproduction of a linear polysaccharide called alginate. T o mimic the inflammatory environment o f the CF lung, P. aeruginosa PAO1, a typical non-mucoid strain, was grown in a biofilm. This was treated with low levels of H202, 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-o factor; this leads t o the deregulation of an alternative c factor (022, 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 PAO1, (i) 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-P-lactamase and (v) secreted little or 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 H202 treatment was in mucA. When a P. aeruginosa biofilm was repeatedly exposed 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 t o 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 a mechanism of defence by the bacteria. This is the f i r s t report to show that PMNs and their oxygen radicals can cause this phenotypic and genotypic change which is so typical of the intractable form o f 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 (PAO stretching, COOOC stretching, and/or amide III vibrations), 1,100 to 1,000 cm ؊1 (COOH and POO 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 (COOH stretching of alginate) and 1,250 cm ؊1 (COO 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 m, 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 m in depth. In contrast, FRD1 produced microcolonies that were approximately 40 m 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.

Strains of Pseudomonas aeruginosa which colonize and infect the lungs of cystic fibrosis patients have a mucoid colony morphology due to the overproduction of the exopolysaccharide alginate. The response regulators AlgB and AlgR are required for the transcription of algD, a tightly regulated gene encoding GDP-mannose dehydrogenase, which is critical for P. aeruginosa alginate biosynthesis. Previous studies indicated that mutations in the algT gene of mucoid FRD1 P. aeruginosa result in nonmucoid derivatives. However, the specific role for algT in alginate gene regulation has not been elucidated. In this study, transcription of algB, algD, and algR was characterized by gene fusion and primer extension analysis. Expression of algR and algD was abolished in P. aeruginosa strains containing aIgT::Tn501 insertions because of lack of transcription initiation at the algR and algD promoters. An algR mutation was constructed in FRD1, and this resulted in the loss of alginate production and a dramatic decrease in algD transcription. RNA and gene fusion analysis revealed that algB is not required for algR expression, nor is algR necessary for transcription of algB. Thus, with the exception of a requirement for AlgT, the AlgB and AlgR pathways appear to be independent of each other. In gel band mobility shift assays, a protein(s) present in extracts from mucoid and algB and algR mutant P. aeruginosa strains formed a specific complex with algD sequences located immediately upstream of the start of transcription. No binding to these sequences was observed when extracts from algT mutant strains were examined. A model proposed suggests that a hierarchy of alginate gene expression exists in which AlgT is required for transcription of the response regulators algB and algR, which in turn are necessary for algD expression. AlgT or a protein under algT control also binds to sequences located within the algD promoter.