Biomedical subjects

Dennis E Ohman

Publications and source records attributed to Dennis E Ohman.

18 recordsLinked to original sources

PubMed2006

Cell wall-inhibitory antibiotics activate the alginate biosynthesis operon in Pseudomonas aeruginosa: Roles of sigma (AlgT) and the AlgW and Prc proteases.

A 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 > 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 sigma22 (algT/algU) or regulators AlgB and AlgR. Anti-sigma factor MucA was the primary regulator of sigma22 activity. A transcriptome analysis using microarrays verified that the algD operon undergoes high induction by D-cycloserine. A similar sigma(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 sigma22 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.

Alginates↗

PubMed2006

Independent regulation of MucD, an HtrA-like protease in Pseudomonas aeruginosa, and the role of its proteolytic motif in alginate gene regulation.

Expression of mucD, encoding a homologue of the HtrA(DegP) family of endoserine proteases, was investigated in Pseudomonas aeruginosa. Expressed from the algT-mucABCD operon, MucD was detected in mucoid (FRD1) and nonmucoid (PAO1) parental strains and also when polar insertions were placed upstream in algT or mucB. A transcriptional start site for a mucD promoter (PmucD) was mapped within mucC. Expression of single-copy mucD217, encoding MucD altered in the protease motif (S217A), was defective in temperature resistance and alginate gene regulation.

Alginates↗

PubMed2005

Polyurethane biocidal polymeric surface modifiers.

Polyurethanes (PUs) with soft blocks containing semifluorinated (-CH2OCH2CF3) and 5,5-dimethylhydantoin pendant groups were prepared and employed (2 wt%) as biocidal polymeric surface modifiers (PSMs) for a conventional PU coating comprised of an isophorone diisocyanate/1,4-butanediol-derived hard block (50%) and poly(tetramethylene oxide) soft block. Surface enrichment of the PSM was verified by dynamic contact angle measurements. The PSM modified PU was activated by converting near-surface amide groups to chloramide with 3wt% hypochlorite. The conversion of near-surface amide to chloramide is reflected in somewhat increased hydrophobic character for the antimicrobial chloramide functionalized surfaces. Biocidal activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa, and Escherichia coli) bacteria was demonstrated by using a modified version of American Association of Textile Chemists and Colorists (AATCC-100) test. By changing the PSM concentration it was found that only 1.6 wt% PSM (0.66 wt% biocide, 5,5-dimethylhydantoin) in a conventional PU affected complete kill of P. aeruginosa in 15 min by using this biocidal testing protocol.

Anti-Infective Agents↗

PubMed2005

Degradation and synthesis kinetics of quorum-sensing autoinducer in Pseudomonas aeruginosa cultivation.

The quorum-sensing (las and rhl) systems play critical roles in the pathogenicity of Pseudomonas aeruginosa and its synthesis of the important biosurfactants, rhamnolipids. In this work, P. aeruginosa PAO1 and its rhlI and rhlR null mutants were used to study the degradation and synthesis kinetics of the rhl system's autoinducer PAI2 (N-butanoyl-homoserine lactone). The two mutants, lacking the ability of synthesizing PAI2 or RhlR protein, produced insignificant amounts of rhamnolipids while having similar growth profiles as the wild-type culture. The regulatory RhlR:PAI2 complex is thus essential to rhamnolipid synthesis. In batch culture of the wild-type PAO1, the autoinducer PAI2 concentration increased along cell growth, especially during the transition from exponential-growth phase to stationary phase, and began to decrease after entering the stationary phase. The decrease in the stationary phase resulted from a faster PAI2 degradation than its synthesis. The degradation kinetics was studied using PAI2-containing supernatants (from centrifuged broth of wild-type culture) with and without the rhlI(-) mutant cells incapable of PAI2 synthesis. Being insignificant in the cell-free systems, PAI2 degradation was found predominantly cell-associated and could be described empirically by the first-order, exponential decay kinetics with the best-fit degradation constant (k(d)) of 0.195 h(-1). When similarly modeled with a first-order kinetics, PAI2 synthesis in stationary-phase wild-type culture was derived to have a synthesis constant (k(s)) of 0.189 h(-1). The PAI2 concentration in batch cultivation of the rhlR(-) mutant also showed an increase-then-decrease profile. However, the maximum PAI2 concentration was about one third of that from the wild-type culture. The constitutive rate of PAI2 synthesis was therefore significantly lower than the rate attainable with active auto-induction by RhlR-PAI2 complex.

Aldose-Ketose Isomerases↗

PubMed2005

Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa secretes a capsule-like polysaccharide called alginate that is important for evasion of host defenses, especially during chronic pulmonary disease of patients with cystic fibrosis (CF). Most proteins for alginate biosynthesis are encoded by the 12-gene algD operon. Interestingly, this operon also encodes AlgL, a lyase that degrades alginate. Mutants lacking AlgG, AlgK, or AlgX, also encoded by the operon, synthesize alginate polymers that are digested by the coregulated protein AlgL. We examined the phenotype of an DeltaalgL mutation in the highly mucoid CF isolate FRD1. Generating a true DeltaalgL mutant was possible only when the algD operon was under the control of a LacI(q)-repressed trc promoter. Upon induction of alginate production with isopropyl-beta-D-thiogalactopyranoside, the DeltaalgL mutant cells were lysed within a few hours. Electron micrographs of the DeltaalgL mutant showed that alginate polymers accumulated in the periplasm, which ultimately burst the bacterial cell wall. The requirement of AlgL in an alginate-overproducing strain led to a new model for alginate secretion in which a multiprotein secretion complex (or scaffold, that includes AlgG, AlgK, AlgX, and AlgL) guides new polymers through the periplasm for secretion across the outer membrane. In this model, AlgL is bifunctional with a structural role in the scaffold and a role in degrading free alginate polymers in the periplasm.

Alginates↗

PubMed2005

Epimerase active domain of Pseudomonas aeruginosa AlgG, a protein that contains a right-handed beta-helix.

The polysaccharide alginate forms a protective capsule for Pseudomonas aeruginosa during chronic pulmonary infections. The structure of alginate, a linear polymer of beta1-4-linked O-acetylated d-mannuronate (M) and l-guluronate (G), is important for its activity as a virulence factor. Alginate structure is mediated by AlgG, a periplasmic C-5 mannuronan epimerase. AlgG also plays a role in protecting alginate from degradation by the periplasmic alginate lyase AlgL. Here, we show that the C-terminal region of AlgG contains a right-handed beta-helix (RHbetaH) fold, characteristic of proteins with the carbohydrate-binding and sugar hydrolase (CASH) domain. When modeled based on pectate lyase C of Erwinia chrysanthemi, the RHbetaH of AlgG has a long shallow groove that may accommodate alginate, similar to protein/polysaccharide interactions of other CASH domain proteins. The shallow groove contains a 324-DPHD motif that is conserved among AlgG and the extracellular mannuronan epimerases of Azotobacter vinelandii. Point mutations in this motif disrupt mannuronan epimerase activity but have no effect on alginate secretion. The D324A mutation has a dominant negative phenotype, suggesting that the shallow groove in AlgG contains the catalytic face for epimerization. Other conserved motifs of the epimerases, 361-NNRSYEN and 381-NLVAYN, are predicted to lie on the opposite side of the RHbetaH from the catalytic center. Point mutations N362A, N367A, and V383A result in proteins that do not protect alginate from AlgL, suggesting that these mutant proteins are not properly folded or not inserted into the alginate biosynthetic scaffold. These motifs are likely involved in asparagine and hydrophobic stacking, required for structural integrity of RHbetaH proteins, rather than for mannuronan catalysis. The results suggest that the AlgG RHbetaH protects alginate from degradation by AlgL by channeling the alginate polymer through the proposed alginate biosynthetic scaffold while epimerizing approximately every second d-mannuronate residue to l-guluronate along the epimerase catalytic face.

Alginates↗

PubMed2005

Adaptations of Pseudomonas aeruginosa to the cystic fibrosis lung environment can include deregulation of zwf, encoding glucose-6-phosphate dehydrogenase.

Cystic fibrosis (CF) patients are highly susceptible to chronic pulmonary disease caused by mucoid Pseudomonas aeruginosa strains that overproduce the exopolysaccharide alginate. We showed here that a mutation in zwf, encoding glucose-6-phosphate dehydrogenase (G6PDH), leads to a approximately 90% reduction in alginate production in the mucoid, CF isolate, P. aeruginosa FRD1. The main regulator of alginate, sigma-22 encoded by algT (algU), plays a small but demonstrable role in the induction of zwf expression in P. aeruginosa. However, G6PDH activity and zwf expression were higher in FRD1 strains than in PAO1 strains. In PAO1, zwf expression and G6PDH activity are known to be subject to catabolite repression by succinate. In contrast, FRD1 zwf expression and G6PDH activity were shown to be refractory to such catabolite repression. This was apparently not due to a defect in the catabolite repression control (Crc) protein. Such relaxed control of zwf was found to be common among several examined CF isolates but was not seen in other strains of clinical and environmental origin. Two sets of clonal isolates from individual CF patient indicated that the resident P. aeruginosa strain underwent an adaptive change that deregulated zwf expression. We hypothesized that high-level, unregulated G6PDH activity provided a survival advantage to P. aeruginosa within the lung environment. Interestingly, zwf expression in P. aeruginosa was shown to be required for its resistance to human sputum. This study illustrates that adaptation to the CF pulmonary environment by P. aeruginosa can include altered regulation of basic metabolic activities, including carbon catabolism.

Adaptation, Physiological↗

PubMed2004

A continuous spectrophotometric assay for Pseudomonas aeruginosa LasA protease (staphylolysin) using a two-stage enzymatic reaction.

Pseudomonas aeruginosa LasA protease is a secreted metalloendopeptidase that can lyse Staphylococcus aureus cells by cleaving the pentaglycine bridges of their peptidoglycan. It can also degrade elastin and stimulate shedding of cell-surface proteoglycans, activities implicated in pathogenesis of P. aeruginosa infections. The activity of LasA protease can be assayed spectrophotometrically by following the reduction in turbidity of S. aureus cell suspensions. This assay, however, does not permit kinetic studies and its reproducibility is poor. Here we describe a two-stage enzymatic reaction for the continuous measurement of LasA protease activity using a defined substrate, succinyl-Gly-Gly-Phe-4-nitroanilide, supplemented with Streptomyces griseus aminopeptidase. Cleavage of the Gly-Phe bond by LasA protease is followed by hydrolysis of the product Phe-4-nitroanilide by the aminopeptidase and the rate of release of the chromophore (4-nitroaniline) is measured spectrophotometrically using a 96-well microplate reader. Activity of nanogram amounts of LasA protease could be determined within a few minutes. Furthermore, this assay permitted the determination of Km and kcat values for LasA protease, which were 0.46 mM and 11.8s(-1), respectively. Pseudomonas elastase was also active in the assay. However, it was less effective than LasA protease and its activity was inhibited by phosphoramidon. The assay is highly sensitive and reproducible, providing a convenient tool for further studies of LasA protease function(s) and mechanism of action.

Aniline Compounds↗

PubMed2004

Development of tools for the genetic manipulation of Pseudomonas aeruginosa.

To facilitate study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, several genetic tools were developed. These tools include a series of cassettes carrying (a) the minimal sequence for the origin of transfer (oriT) of RP4 plasmid for introducing plasmid into P. aeruginosa via conjugation, (b) a minimal sequence for P. aeruginosa replicon (stabilizing fragment or SF) for maintenance of plasmids in P. aeruginosa, and (c) the transcriptionally non-polar tetracycline resistance gene (TcR) for insertional mutagenesis. Additional genetic constructs include (d) two conjugative and suicide lacZ reporter fusion plasmids for studying gene expression at the transcriptional or translational level, (e) a gentamicin resistant promoter-probing mini-Tn5 lacZ, and (f) a tightly regulated T7 promoter/repressor system to control gene expression in P. aeruginosa.

Bacteriological Techniques↗

PubMed2004

Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation.

Extracellular polymers can facilitate the non-specific attachment of bacteria to surfaces and hold together developing biofilms. This study was undertaken to qualitatively and quantitatively compare the architecture of biofilms produced by Pseudomonas aeruginosa strain PAO1 and its alginate-overproducing (mucA22) and alginate-defective (algD) variants in order to discern the role of alginate in biofilm formation. These strains, PAO1, Alg+ PAOmucA22 and Alg- PAOalgD, tagged with green fluorescent protein, were grown in a continuous flow cell system to characterize the developmental cycles of their biofilm formation using confocal laser scanning microscopy. Biofilm Image Processing (BIP) and Community Statistics (COMSTAT) software programs were used to provide quantitative measurements of the two-dimensional biofilm images. All three strains formed distinguishable biofilm architectures, indicating that the production of alginate is not critical for biofilm formation. Observation over a period of 5 days indicated a three-stage development pattern consisting of initiation, establishment and maturation. Furthermore, this study showed that phenotypically distinguishable biofilms can be quantitatively differentiated.

Alginates↗

PubMed2004

Identification of residues in the Pseudomonas aeruginosa elastase propeptide required for chaperone and secretion activities.

An important virulence factor of the opportunistic human pathogen Pseudomonas aeruginosa is elastase, a secreted thermolysin-like neutral zinc-metalloprotease (TNP). Elastase is synthesized as a larger precursor with an amino-terminal 18 kDa propeptide, and was the first TNP shown to require its propeptide as an intramolecular chaperone (IMC) for activity and secretion. This paper reports the analysis of the elastase propeptide to identify residues conserved among other TNP precursors that may be critical for its IMC function. The prosequences of TNP precursors from both Gram-negative (Vibrio species and Legionella species) and Gram-positive (Bacillus species) bacteria were found to show homology to the elastase propeptide. Two regions of conserved residues were observed: a hydrophilic region (ProM) found in the middle of the elastase propeptide and a more hydrophobic region (ProC) located proximal to the propeptide-processing site. To test whether such conserved motifs were important to function, single residue substitutions at eight conserved amino acids were introduced within the full-length pre-proelastase precursor by site-specific mutagenesis of lasB, the gene encoding elastase. Mutant lasB alleles were expressed from plasmids within a lasB-deleted P. aeruginosa strain, FRD740, and the effects of these propeptide alterations on elastase enzyme activity, processing, stability and accumulation inside and outside of the cell were examined. Within the ProM region, substitution at Arg74 resulted in a dramatic accumulation of proelastase in the cell, suggesting a secretion defect, and a dramatic reduction in supernatant elastolytic activity. Substitution at Asn68 adversely affected the amount of elastase in the culture supernatant, apparently as a result of the reduced stability of the mutated proelastase in the cell. Within the ProC region, mutations at Ile181 and Ala183 abolished the accumulation of a stable elastase molecule in the supernatant. Most mutations produced a phenotype consistent with a defect in protein folding and stability. Overall, the data from this preliminary study show that conserved residues within the elastase propeptide are essential for its function and begin to define the mechanisms of action of IMCs in the TNP family.

Amino Acid Sequence↗

PubMed2004

Pseudomonas aeruginosa LasA protease in treatment of experimental staphylococcal keratitis.

LasA protease is a staphylolytic endopeptidase secreted by Pseudomonas aeruginosa. We have examined the effectiveness of LasA protease in the treatment of staphylococcal keratitis caused by methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates in a rabbit model. Keratitis was induced by intrastromal injection of the bacteria. The eyes were treated topically, and the efficacy of LasA protease was compared to those of lysostaphin (a staphylolytic protease secreted by Staphylococcus simulans) and vancomycin. When treatment was initiated early (4 h) after infection, practically all of the MSSA- and MRSA-infected corneas were sterilized by LasA protease, and its efficacy in eradicating the bacteria was comparable to those of lysostaphin and vancomycin. By contrast, most of the control corneas were heavily infected, with median values of 4.5 x 10(6) (MSSA) and 5 x 10(5) (MRSA) CFU/cornea (P < 0.001). When treatment was initiated late (10 h) after infection, LasA protease reduced the numbers of CFU in both MSSA- and MRSA-infected corneas by 3 to 4 orders of magnitude compared to the numbers of CFU for the controls (median values, 1,380 and 30 CFU/cornea, respectively, for the treated animals compared to 1.2 x 10(6) and 5 x 10(5) CFU/cornea for the respective controls [P = 0.001]), and it was more effective than vancomycin in eradicating MRSA cells (P = 0.02). In both the early- and the late-treatment protocols, the clinical scores for eyes treated with LasA protease were significantly lower than those for the eyes of the corresponding controls and comparable to those for the lysostaphin- and vancomycin-treated eyes. We conclude that LasA protease is effective in the treatment of experimental S. aureus keratitis in rabbits and may have potential for the treatment of disease in humans.

Animals↗

PubMed2003

The dual roles of AlgG in C-5-epimerization and secretion of alginate polymers in Pseudomonas aeruginosa.

Pseudomonas aeruginosa strains causing chronic pulmonary infections in cystic fibrosis patients produce high levels of alginate, an exopolysaccharide that confers a mucoid phenotype. Alginate is a linear polymer of d-mannuronate (M) and variable amounts of its C-5-epimer, l-guluronate (G). AlgG is a periplasmic C-5-epimerase that converts poly d-mannuronate to the mixed M+G sequence of alginate. To understand better the role and mechanism of AlgG activity, a mutant was constructed in the mucoid strain FRD1 with a defined non-polar deletion of algG. Instead of producing poly mannuronate, the algG deletion mutant secreted dialysable uronic acids, as does a mutant lacking the periplasmic protein AlgK. High levels of unsaturated ends and the nuclear magnetic resonance spectroscopy pattern revealed that the small, secreted uronic acids were the products of extensive polymer digestion by AlgL, a periplasmic alginate lyase co-expressed with AlgG and AlgK. Thus, AlgG is bifunctional with (i) epimerase activity and (ii) a role in protecting alginate from degradation by AlgL during transport through the periplasm. AlgK appears to share the second role. AlgG and AlgK may be part of a periplasmic protein complex, or scaffold, that guides alginate polymers to the outer membrane secretin (AlgE). To characterize the epimerase activity of AlgG further, the algG4 allele of poly mannuronate-producing FRD462 was shown to encode a protein lacking only the epimerase function. The sequence of algG4 has a Ser-272 to Asn substitution in a serine-threonine-rich and conserved region of AlgG, which revealed a critical residue for C-5-epimerase activity.

Alginates↗

PubMed2003

Comparative analysis of plant and animal models for characterization of Burkholderia cepacia virulence.

A simple alfalfa model was developed as an alternative infection model for virulence studies of the Burkholderia cepacia complex. Symptoms of disease were observed in wounded alfalfa seedlings within 7 days following inoculation of 10(1) to 10(5) CFU of most strains of the B. cepacia complex. Strains from seven genomovars of the B. cepacia complex were tested for virulence in the alfalfa model, and the degree of virulence was generally similar in strains belonging to the same genomovar. Strains of Burkholderia multivorans and some strains of Burkholderia stabilis did not cause symptoms of disease in alfalfa seedlings. Representative strains were also tested for virulence using the rat agar bead model. Most of the strains tested were able to establish chronic lung infections; B. stabilis strains were the exception. Most of the strains that were virulent in the alfalfa infection model were also virulent in the lung infection model. The B. cepacia genomovar III mutants K56pvdA::tp and K56-H15 were significantly less virulent in the alfalfa infection model than their parent strain. Therefore, this alfalfa infection model may be a useful tool for assessing virulence of strains of the B. cepacia complex and identifying new virulence-associated genes.

Animals↗

PubMed2003

The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation.

Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic acid residues to alpha-L-guluronic acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.

Alginates↗

PubMed2002

A simple alfalfa seedling infection model for Pseudomonas aeruginosa strains associated with cystic fibrosis shows AlgT (sigma-22) and RhlR contribute to pathogenesis.

A sensitive plant infection model was developed to identify virulence factors in nontypeable, alginate overproducing (mucoid) Pseudomonas aeruginosa strains isolated from cystic fibrosis (CF) patients with chronic pulmonary disease. Nontypeable strains with defects in lipopolysaccharide O-side chains are common to CF and often exhibit low virulence in animal models of infection. However, 1,000 such bacteria were enough to show disease symptoms in the alfalfa infection. A typical mucoid CF isolate, FRD1, and its isogenic mutants were tested for alfalfa seedling infection. Although defects in the global regulators Vfr, RpoS, PvdS, or LasR had no discernable effect on virulence, a defect in RhlR reduced the infection frequency by >50%. A defect in alginate biosynthesis resulted in plant disease with >3-fold more bacteria per plant, suggesting that alginate overproduction attenuated bacterial growth in planta. FRD1 derivatives lacking AlgT, a sigma factor required for alginate production, were reduced >50% in the frequency of infection. Thus, AlgT apparently regulates factors in FRD1, besides alginate, important for pathogenesis. In contrast, in a non-CF strain, PAO1, an algT mutation did not affect its virulence on alfalfa. Conversely, PAO1 virulence was reduced in a mucA mutant that overproduced alginate. These observations suggested that mucoid conversion in CF may be driven by a selection for organisms with attenuated virulence or growth in the lung, which promotes a chronic infection. These studies also demonstrated that the wounded alfalfa seedling infection model is a useful tool to identify factors contributing to the persistence of P. aeruginosa in CF.

Alginates↗

PubMed2002

Mutant analysis and cellular localization of the AlgI, AlgJ, and AlgF proteins required for O acetylation of alginate in Pseudomonas aeruginosa.

Alginate is an extracellular polysaccharide produced by mucoid strains of Pseudomonas aeruginosa that are typically isolated from the pulmonary tracts of chronically infected cystic fibrosis patients. Alginate is a linear polymer of D-mannuronate and L-guluronate with O-acetyl ester linkages on the O-2 and/or O-3 position of the mannuronate residues. The presence of O-acetyl groups plays an important role in the ability of the polymer to act as a virulence factor, and the algF, algJ, and algI genes are known to be essential for the addition of O-acetyl groups to alginate. To better understand the mechanism of O acetylation of alginate, the cellular locations of the AlgI, AlgJ, and AlgF proteins were determined. For these studies, defined nonpolar algI, algJ, and algF deletion mutants of P. aeruginosa strain FRD1 were constructed, and each mutant produced alginate lacking O-acetyl groups. Expression of algI, algJ, or algF in trans in the corresponding mutant complemented each O acetylation defect. Random phoA (alkaline phosphatase [AP] gene) fusions to algF, algJ, and algI were constructed. All in-frame fusions to algF and algJ had AP activity, indicating that both AlgF and AlgJ were exported to the periplasm. Immunoblot analysis of spheroplasts and periplasmic fractions showed that AlgF was released with the periplasmic contents but that AlgJ remained with the spheroplast fraction. An N-terminal sequence analysis of AlgJ showed that its putative AlgJ signal peptide was not cleaved, suggesting that AlgJ is anchored to the cytoplasmic membrane by its uncleaved signal peptide. AP gene fusions were also used to map the membrane topology of AlgI, and the results suggest that it is an integral membrane protein with seven transmembrane domains. These results suggest that AlgI-AlgJ-AlgF may form a complex in the membrane that is the reaction center for O acetylation of alginate.

Acetylation↗

PubMed1999

Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung.

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 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-sigma factor; this leads to the deregulation of an alternative sigma factor (sigma22, 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, (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 approximately 72% reduced in the amount of inducible-beta-lactamase and (v) secreted little or no LasA protease and only showed 44% elastase activity. A characteristic approximately 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 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 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 a 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.

Alginates↗