PqsD is responsible for the synthesis of 2,4-dihydroxyquinoline, an extracellular metabolite produced by Pseudomonas aeruginosa

2,4-Dihydroxyquinoline (DHQ) is an abundant extracellular metabolite of the opportunistic pathogen Pseudomonas aeruginosa that is secreted into growth medium in stationary phase to concentrations comparable with those of the Pseudomonas quinolone signal. Using a combination of biochemical and genetic approaches, we show that PqsD, a condensing enzyme in the pqs operon that is essential for Pseudomonas quinolone signal synthesis, accounts for DHQ formation in vivo. First, the anthraniloyl moiety is transferred to the active-site Cys of PqsD to form an anthraniloyl-PqsD intermediate, which then condenses with either malonyl-CoA or malonyl-acyl carrier protein to produce 3-(2-aminophenyl)-3-oxopropanoyl-CoA. This short-lived intermediate undergoes an intramolecular rearrangement to form DHQ. DHQ was produced by Escherichia coli coexpressing PqsA and PqsD, illustrating that these two proteins are the only factors necessary for DHQ synthesis. Thus, PqsD is responsible for the production of DHQ in P. aeruginosa.     

The wbiA locus is required for the 2-O-acetylation of lipopolysaccharides expressed by Burkholderia pseudomallei and Burkholderia thailandensis

Burkholderia pseudomallei and Burkholderia thailandensis express similar O-antigens (O-PS II) in which their 6-deoxy-alpha-L-talopyranosyl (L-6dTalp) residues are variably substituted with O-acetyl groups at the O-2 or O-4 positions. In previous studies we demonstrated that the protective monoclonal antibody, Pp-PS-W, reacted with O-PS II expressed by wild-type B. pseudomallei strains but not by a B. pseudomallei wbiA null mutant. In the present study we demonstrate that WbiA activity is required for the acetylation of the L-6dTalp residues at the O-2 position and that structural modification of O-PS II molecules at this site is critical for recognition by Pp-PS-W.     

AlgX is a periplasmic protein required for alginate biosynthesis in Pseudomonas aeruginosa

Alginate, an exopolysaccharide produced by Pseudomonas aeruginosa, provides the bacterium with a selective advantage that makes it difficult to eradicate from the lungs of cystic fibrosis (CF) patients. Previous studies identified a gene, algX, within the alginate biosynthetic gene cluster on the P. aeruginosa chromosome. By probing cell fractions with anti-AlgX antibodies in a Western blot, AlgX was localized within the periplasm. Consistent with these results is the presence of a 26-amino-acid signal sequence. To examine the requirement for AlgX in alginate biosynthesis, part of algX in P. aeruginosa strain FRD1::pJLS3 was replaced with a nonpolar gentamicin resistance cassette. The resulting algXDelta::Gm mutant was verified by PCR and Western blot analysis and was phenotypically nonmucoid (non-alginate producing). The algXDelta::Gm mutant was restored to the mucoid phenotype with wild-type P. aeruginosa algX provided on a plasmid. The algXDelta::Gm mutant was found to secrete dialyzable oligouronic acids of various lengths. Mass spectroscopy and Dionex chromatography indicated that the dialyzable uronic acids are mainly mannuronic acid dimers resulting from alginate lyase (AlgL) degradation of polymannuronic acid. These studies suggest that AlgX is part of a protein scaffold that surrounds and protects newly formed polymers from AlgL degradation as they are transported within the periplasm for further modification and eventual transport out of the cell.     

Characterization of HmqF, a protein involved in the biosynthesis of unsaturated quinolones produced by Burkholderia thailandensis

The opportunistic pathogen Burkholderia thailandensis produces a number of structurally similar unsaturated quinolones involved in quorum sensing. However, little is known about the biosynthesis of these unsaturated quinolones. In this study, we have characterized the starting point of the biosynthesis of unsaturated quinolone molecules produced in B. thailandensis. We have shown by using in vitro enzymology, liquid chromatography, and mass spectrometry that protein HmqF is involved in the biosynthesis of unsaturated quinolones produced by B. thailandensis. HmqF consists of three domains: an adenylation domain (A domain), a dehydrogenase domain (DH domain), and an acyl carrier domain (ACP). The three domains (A, DH, and ACP) were cloned and expressed individually in Escherichia coli, and their reactivity was studied using high-performance liquid chromatography (HPLC) and mass spectrometry (MS) based assays. Our in vitro studies show that the A domain catalyzes ATP-dependent activation of medium chain (C6-C14) fatty acids without activation by coenzyme A (CoA). Results from competition assays are consistent with decanoic acid being the preferred substrate. Incubation of the ACP domain with 4'-phosphopantetheine transferase and CoA led to the formation of phosphopantetheinylated ACP (Ppant-ACP). In a Ppant ejection assay using tandem MS (MS/MS), a mass consistent with the mass of a cyclic variant of dephosphorylated Ppant was detected. We further demonstrated that Ppant-ACP could be loaded with medium chain fatty acids in the presence of ATP and the A domain. MS analysis was consistent with the formation of Ppant-ACP thiol esters of the fatty acids. MS/MS Ppant ejection experiments confirmed the loss of 2H in samples of fatty acid-loaded Ppant-ACP in the presence of the DH domain. HPLC analysis of benzyl amide ligation products allowed us to conclude that dehydrogenation produced trans-β,γ-unsaturation in the fatty acid chains. Our results are in good agreement with naturally observed quinolone molecules produced by B. thailandensis, which predominately produce nine-carbon trans-β,γ-unsaturated alkyl chain quinolone molecules.     

Alginate lyase (AlgL) activity is required for alginate biosynthesis in Pseudomonas aeruginosa

To determine whether AlgL's lyase activity is required for alginate production in Pseudomonas aeruginosa, an algLdelta::Gm(r) mutant (FRD-MA7) was created. algL complementation of FRD-MA7 restored alginate production, but algL constructs containing mutations inactivating lyase activity did not, demonstrating that the enzymatic activity of AlgL is required for alginate production.     

A newly identified regulator is required for virulence and toxin production in Pseudomonas syringae

The genes lemA (which we here redesignate gacS ) and gacA encode members of a widely conserved two-component regulatory system. In Pseudomonas syringae strain B728a, gacS and gacA are required for lesion formation on bean, as well as for the production of protease and the toxin syringomycin. A gene, designated salA , was discovered that restored syringomycin production to a gacS mutant when present on a multiple-copy plasmid. Disruption of chromosomal salA resulted in loss of syringomycin production and lesion formation in laboratory assays. Sequence analysis of salA suggests that it encodes a protein with a DNA-binding motif but without other significant similarity to proteins in current databases. Chromosomal reporter fusions revealed that gacS and gacA positively regulate salA , that salA upregulates its own expression and that salA positively regulates the expression of a syringomycin biosynthetic gene, syrB . Loss of syringomycin production does not account for the salA mutant's attenuated pathogenicity, as a syrB mutant was found to retain full virulence. The salA gene did not similarly suppress the protease deficient phenotype of gacS mutants, nor were salA mutants affected for protease production. A gacS/gacA -dependent homoserine lactone activity as detected by bioassay was also unaffected by the disruption of salA . Thus, salA appears to encode a novel regulator that activates the expression of at least two separate genetic subsets of the gacS/gacA regulon, one pathway leading to syringomycin production and the other resulting in plant disease.     

Paerucumarin, a new metabolite produced by the pvc gene cluster from Pseudomonas aeruginosa

The pvc gene cluster from Pseudomonas aeruginosa has been linked to the biosynthesis of both the pyoverdine chromophore and pseudoverdine. Our reinvestigation of the role this gene cluster plays in P. aeruginosa secondary metabolite biosynthesis shows that its major product is actually paerucumarin, a novel isonitrile functionalized cumarin.     

PslD is a secreted protein required for biofilm formation by Pseudomonas aeruginosa

The function of pslD, which is part of the psl operon from Pseudomonas aeruginosa, was investigated in this study. The psl operon is involved in exopolysaccharide biosynthesis and biofilm formation. An isogenic marker-free pslD deletion mutant of P. aeruginosa PAO1 which was deficient in the formation of differentiated biofilms was generated. Expression of only the pslD gene coding region restored the wild-type phenotype. A C-terminal, hexahistidine tag fusion enabled the identification of PslD. LacZ and PhoA translational fusions with PslD indicated that PslD is a secreted protein required for biofilm formation, presumably via its role in exopolysaccharide export.     

Alg44, a unique protein required for alginate biosynthesis in Pseudomonas aeruginosa

Here the putative alginate biosynthesis gene alg44 of Pseudomonas aeruginosa was functionally assigned. Non-polar isogenic alg44 deletion mutants of P. aeruginosa were generated and did neither produce alginate nor released free uronic acids. No evidence for alginate enrichment in the periplasm was obtained. Alginate production was restored by introducing only the gene alg44. PhoA fusion protein analyses suggested that Alg44 is a soluble protein localized in the periplasm. Hexahistidine-tagged Alg44 was detected by immunoblotting. The corresponding 42.6 kDa protein was purified and identified by MALDI/TOF-MS analysis. Alg44 might be directly involved in alginate polymerization presumably by exerting a regulatory function.     

Reduction and Acetylation of 2,4-Dinitrotoluene by a Pseudomonas aeruginosa Strain

Aerobic and anoxic biotransformation of 2,4-dinitrotoluene (DNT) was examined by using a Pseudomonas aeruginosa strain isolated from a plant treating propellant manufacturing wastewater. DNT biotransformation in the presence and absence of oxygen was mostly reductive and was representative of the type of cometabolic transformations that occur when a high concentration of an easily degradable carbon source is present. P. aeruginosa reduced both nitro groups on DNT, with the formation of mainly 4-amino-2-nitrotoluene and 2-amino-4-nitrotoluene and small quantities of 2,4-diaminotoluene. Acetylation of the arylamines was a significant reaction. 4-Acetamide-2-nitrotoluene and the novel compounds 2-acetamide-4-nitrotoluene, 4-acetamide-2-aminotoluene, and 2,4-diacetamidetoluene were identified as DNT metabolites. The biotransformation of 2,4-diaminotoluene to 4-acetamide-2-aminotoluene was 24 times faster than abiotic transformation. 2-Nitrotoluene and 4-nitrotoluene were also reduced to their corresponding toluidines and then acetylated. However, the yield of 4-acetamidetoluene was much higher than that of 2-acetamidetoluene, demonstrating that acetylation at the position para to the methyl group was favored.     

Functional characterization of an aminotransferase required for pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa PAO1

The fluorescent dihydroxyquinoline chromophore of the pyoverdine siderophore in Pseudomonas is a condensation product of D-tyrosine and l-2,4-diaminobutyrate. Both pvdH and asd (encoding aspartate beta-semialdehyde dehydrogenase) knockout mutants of Pseudomonas aeruginosa PAO1 were unable to synthesize pyoverdine under iron-limiting conditions in the absence of l-2,4-diaminobutyrate in the culture media. The pvdH gene was subcloned, and the gene product was hyperexpressed and purified from P. aeruginosa PAO1. PvdH was found to catalyze an aminotransferase reaction, interconverting aspartate beta-semialdehyde and l-2,4-diaminobutyrate. Steady-state kinetic analysis with a novel coupled assay established that the enzyme adopts a ping-pong kinetic mechanism and has the highest specificity for alpha-ketoglutarate. The specificity of the enzyme toward the smaller keto acid pyruvate is 41-fold lower. The enzyme has negligible activity toward other keto acids tested. Homologues of PvdH were present in the genomes of other Pseudomonas spp. These homologues were found in the DNA loci of the corresponding genomes that contain other pyoverdine synthesis genes. This suggests that there is a general mechanism of l-2,4-diaminobutyrate synthesis in Pseudomonas strains that produce the pyoverdine siderophore.     

The flagellum of Pseudomonas aeruginosa is required for resistance to clearance by surfactant protein A

Surfactant protein A (SP-A) is an important lung innate immune protein that kills microbial pathogens by opsonization and membrane permeabilization. We investigated the basis of SP-A-mediated pulmonary clearance of Pseudomonas aeruginosa using genetically-engineered SP-A mice and a library of signature-tagged P. aeruginosa mutants. A mutant with an insertion into flgE, the gene that encodes flagellar hook protein, was preferentially cleared by the SP-A(+/+) mice, but survived in the SP-A(-/-) mice. Opsonization by SP-A did not play a role in flgE clearance. However, exposure to SP-A directly permeabilized and killed the flgE mutant, but not the wild-type parental strain. P. aeruginosa strains with mutation in other flagellar genes, as well as mucoid, nonmotile isolates from cystic fibrosis patients, were also permeabilized by SP-A. Provision of the wild-type fliC gene restored the resistance to SP-A-mediated membrane permeabilization in the fliC-deficient bacteria. In addition, non-mucoid, motile revertants of CF isolates reacquired resistance to SP-A-mediated membrane permeability. Resistance to SP-A was dependent on the presence of an intact flagellar structure, and independent of flagellar-dependent motility. We provide evidence that flagellar-deficient mutants harbor inadequate amounts of LPS required to resist membrane permeabilization by SP-A and cellular lysis by detergent targeting bacterial outer membranes. Thus, the flagellum of P. aeruginosa plays an indirect but important role resisting SP-A-mediated clearance and membrane permeabilization.     

Identification of Pseudomonas aeruginosa glpM, whose gene product is required for efficient alginate biosynthesis from various carbon sources.

In a mucB (algN) genetic background, insertion of an omega element approximately 200 bp downstream of glpD, encoding sn-glycerol-3-phosphate dehydrogenase from Pseudomonas aeruginosa, had an adverse effect on alginate biosynthesis from various carbon sources. The insertion inactivated glpM, a gene encoding a 12,040-M(r) hydrophobic protein containing 109 amino acids. This protein, which was expressed in a T7 RNA polymerase expression system, appears to be a cytoplasmic membrane protein.     

Construction of a recA mutant of Burkholderia (formerly Pseudomonas), cepacia

A recA mutant was constructed of a soil isolate of Burkholderia cepacia, strain ATCC 17616. Prior to mutagenesis, the recA gene was cloned from this strain by its ability to complement the methyl methanesulfonate sensitivity of an Escherichia coli recA mutant. Sequence analysis of the strain showed high sequence similarity (94% nucleic acid and 99% amino acid identity) with the recA gene previously cloned from a clinical isolate of B. cepacia, strain JN25. The subcloned recA gene from B. cepacia ATCC 17616 restored UV resistance and recombination proficiency to recA mutants of E. coli and Pseudomonas aeruginosa, as well as restoring the ability of D3 prophages to be induced to lytic growth from a RecA⁻ strain of P. aeruginosa. The recA mutant of B. cepacia ATCC 17616 was constructed by λ-mediated Tn5 mutagenesis of the cloned recA gene in E. coli, followed by replacement of the Tn5-interrupted gene for the wild-type allele in the chromosome of B. cepacia by marker exchange. The RecA⁻ phenotype of the mutant was demonstrated by the loss of UV resistance as compared to the parental strain. Southern hybridization analysis of chromosomal DNA from the mutant indicated the presence of Tn5 in the recA gene, and the location of the Tn5 insertion in the recA allele was identified by nucleotide sequence analysis. A test using the recA mutant to see if acquired resistance to d-serine toxicity in B. cepacia might be a result of RecA-mediated activities proved negative; nevertheless, RecA activity potentially contributes to the overall genomic plasticity of B. cepacia and a recA mutant will be useful in bioengineering of this species. Received: 24 January / Received revision: 11 July 1997 / Accepted: 25 August 1997     

Glycosylation is required for outer membrane localization of the lectin LecB in Pseudomonas aeruginosa

The fucose-/mannose-specific lectin LecB from Pseudomonas aeruginosa is transported to the outer membrane; however, the mechanism used is not known so far. Here, we report that LecB is present in the periplasm of P. aeruginosa in two variants of different sizes. Both were functional and could be purified by their affinity to mannose. The difference in size was shown by a specific enzyme assay to be a result of N glycosylation, and inactivation of the glycosylation sites was shown by site-directed mutagenesis. Furthermore, we demonstrate that this glycosylation is required for the transport of LecB.