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.

The mucoid phenotype is common among strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis and is due to overproduction of an exopolysaccharide called alginate.However, the mucoid phenotype is unstable in vitro, especially when the cells are incubated under low oxygen tension. Spontaneous conversion to the nonmucoid form is typically due to mutations (previously called aigS) that are closely linked to the alginate regulatory gene algT, located at 68 min on the chromosome. Our sequence analysis of algT showed that its 22-kDa gene product shares homology with several alternate sigma factors in bacteria, suggesting that AlgT (also known as AlgU) interacts directly with RNA polymerase core to activate the promoters of alginate genes. AlgT showed striking sequence similarity (79%o) to orE of Escherichia coli, an alternate sigma factor involved in high-temperature gene expression. Our analysis of the molecular basis for spontaneous conversion from mucoid to nonmucoid, in the cystic fibrosis isolate FRD, revealed that nonmucoid conversion was often due to one of two distinct missense mutations in algT that occurred at codons 18 and 29. RNase protection assays showed that spontaneous nonmucoid strains with the algT18 and algT29 alleles have a four-to fivefold reduction in the accumulation ofalgT transcripts compared with the wild-type mucoid strain.Likewise, a plasmid-borne algT-cat transcriptional fusion was about 3-fold less active in the algT18 and algT29 backgrounds compared with the mucoid wild-type strain, and it was 20-fold less active in an algT::TnS01 background. These data indicate that algT is autoregulated. The spontaneous algT missense alleles also caused about fivefold-reduced expression of the adjacent negative regulator, algN (also known as mucB). Transcripts of algN were essentially absent in the algT::Tn501 strain. Thus, algT regulates the algTN cluster, and the two genes may be cotranscribed. A primer extension analysis showed that aIgT transcription starts 54 bp upstream of the start of translation. Although the algT promoter showed little similarity to promoters recognized by the vegetative sigma factor, it was similar to the algR promoter. This finding suggests that AlgT may function as a sigma factor to activate its own promoter and those of other alginate genes. The primer extension analysis also showed that algT transcripts were readily detectable in the typical nonmucoid strain PAO1, which was in contrast to a weak signal seen in the algT18 mutant of FRD. A plasmid-borne aIgT gene in PAOI resulted in both the mucoid phenotype and high levels of algT transcripts, further supporting the hypothesis that AlgT controls its own gene expression and expression of genes of the alginate regulon.Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for a wide variety of infections, including chronic pulmonary tract infections in patients with the autosomal recessive disorder cystic fibrosis (CF). Following colonization of the CF respir...

SummaryA bioassay was developed to identify stimuli that promote the transcriptional induction of the algD operon for alginate biosynthesis in Pseudomonas aeruginosa. Strain PAO1 carried the algD promoter fused to a chloramphenicol acetyl-transferase cartridge (PalgD-cat), and Ͼ 50 compounds were tested for promoting chloramphenicol resistance. Most compounds showing PalgD-cat induction were cell wall-active antibiotics that blocked peptidoglycan synthesis. PalgD-cat induction was blocked by mutations in the genes for s 22 (algT/algU) or regulators AlgB and AlgR. Anti-sigma factor MucA was the primary regulator of s 22 activity. A transcriptome analysis using microarrays verified that the algD operon undergoes high induction by D-cycloserine. A similar s E -RseAB complex in Escherichia coli responds to envelope stress, which requires DegS protease in a regulated intramembrane proteolysis (RIP) cascade to derepress the sigma. Mutant phenotypic studies in P. aeruginosa showed that AlgW (PA4446) is likely to be the DegS functional homologue. A mutation in algW resulted in a complete lack of PalgD-cat induction by D-cycloserine. Overexpression of algW in PAO1 resulted in a mucoid phenotype and alginate production, even in the absence of cell wall stress, suggesting that AlgW protease plays a role in s 22 activation. In addition, a mutation in gene PA3257 (prc), encoding a Prc-like protease, resulted in poor induction of PalgD-cat by D-cycloserine, suggesting that it also plays a role in the response to cell wall stress.

Establishment and maintenance of chronic lung infections with mucoid Pseudomonas aeruginosa in patients with cystic fibrosis (CF) require that the bacteria avoid host defenses. Elaboration of the extracellular, O-acetylated mucoid exopolysaccharide, or alginate, is a major microbial factor in resistance to immune effectors. Here we show that O acetylation of alginate maximizes the resistance of mucoid P. aeruginosa to antibody-independent opsonic killing and is the molecular basis for the resistance of mucoid P. aeruginosa to normally nonopsonic but alginate-specific antibodies found in normal human sera and sera of infected CF patients. O acetylation of alginate appears to be critical for P. aeruginosa resistance to host immune effectors in CF patients.The predominant bacterial pathogen in chronic pulmonary infection in cystic fibrosis (CF) patients is the mucoid variant of Pseudomonas aeruginosa, which is encapsulated by and overproduces mucoid exopolysaccharide (MEP), or alginate. That alginate is the major virulence factor of P. aeruginosa in CF lung infection is evident from the epidemiology of this disease. The pulmonary function of patients with CF declines only when mucoid P. aeruginosa is isolated and associated lung pathology develops (9, 32, 33). The growth of mucoid P. aeruginosa as a biofilm in the lungs of CF patients has been suggested to be a major factor in long-term bacterium survival. Biofilm formation by P. aeruginosa has been linked to genes involved in quorum sensing (7) and motility (31), with a recent demonstration that the acyl-homoserine lactone molecules involved in the quorum-sensing system (8) can be detected in the sputa of CF patients (42). However, the genes controlling alginate production appear to be independent of control by the known quorum-sensing genes of P. aeruginosa, including lasR and rhlR (8,44,45). Therefore, the question of whether there is a regulator or environmental cue common to both alginate production and quorum-sensing systems has not yet been answered.The conversion of P. aeruginosa to the mucoid state in CF patients is often associated with mutations at the mucA locus (23). MucA and MucB (also called AlgN) act as anti-sigma factors for the alternative sigma factor E (47), encoded by algT (25), also known as algU (22). Increased activity of this sigma factor results in hyperexpression of the alginate biosynthetic operon located at 34 min on the P. aeruginosa genome (25

Pseudomonas aeruginosa strains associated with cystic fibrosis are often mucoid due to the copious production of alginate, an exopolysaccharide and virulence factor. Alginate gene expression is transcriptionally controlled by a gene cluster at 68 min on the chromosome: algT (algU)-mucA-mucB (algN)-mucC (algM)-mucD (algY). The algT gene encodes a 22-kDa alternative sigma factor ( 22 ) that autoregulates its own promoter (PalgT) as well as the promoters of algR, algB, and algD. The other genes in the algT cluster appear to regulate the expression or activity of 22. The goal of this study was to better understand the functional interactions between 22 and its antagonist regulators during alginate production. Nonmucoid strain PAO1 was made to overproduce alginate (indicating high algD promoter activity) through increasing 22 in the cell by introducing a plasmid clone containing algT from mucA22(Def) strain FRD1. However, the bacterial cells remained nonmucoid if the transcriptionally coupled mucB on the clone remained intact. This suggested that a stoichiometric relationship between 22 and MucB may be required to control sigma factor activity. When the transcription and translational initiation of algT were measured with lacZ fusions, alginate production correlated with only about a 1.2-to 1.7-fold increase in algT-lacZ activity, respectively. An algR-lacZ transcriptional fusion showed a 2.8-fold increase in transcription with alginate production under the same conditions. A Western blot analysis of total cell extracts showed that 22 was approximately 10-fold higher in strains that overproduced alginate, even though algT expression increased less than 2-fold. This suggested that a posttranscriptional mechanism may exist to destabilize 22 in order to control certain 22 -dependent promoters like algD. By Western blotting and phoA fusion analyses, the MucB antagonist of 22 was found to localize to the periplasm of the cell. Similar experiments suggest that MucA localizes to the inner membrane via one transmembrane domain with amino-and carboxy-terminal domains in the cytoplasm and periplasm, respectively. These data were used to propose a model in which MucB-MucA-22 interact via an inner membrane complex that controls the stability of 22 protein in order to control alginate biosynthesis.