Gene cloning, sequence analysis, purification, and secretion by Escherichia coli of an extracellular lipase from Serratia marcescens.

The gene encoding extracellular lipase of Serratia marcescens has been identified from a phage lambda genomic library. Formation of orange-red fluorescent plaques on rhodamine B-triolein plates was used to identify phages carrying the lipase gene. A 2.8-kb SalI fragment was subcloned into a plasmid, and lipase was expressed in Escherichia coli. Extracellular lipase was detected in the presence of the secretion plasmid pGSD6 carrying the genes prtD, -E, and -F, which guide the secretion of protease from Erwinia chrysanthemi. Determination of the nucleotide sequence of the entire cloned fragment revealed an open reading frame coding for a 613-amino-acid protein with a predicted M(r) of 64,800. Analysis of the amino acid sequence revealed significant homology (around 70%) to lipases of Pseudomonas fluorescens strains. The lipase-specific consensus sequence G-X1-S-X2-G resided in the amino-terminal part of the protein, and carboxyl-terminal consensus sequences were an L-X-G-G-B-G-B-B-X repeat motif and a so-called aspartate box, respectively, which are both found in proteins secreted by the class I secretion pathway. Lipase was purified from the supernatant of a culture carrying a lipase expression vector, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an M(r) of 64,000 for the purified protein. Our results suggest that the lipase of S. marcescens belongs to the group of extracellular enzyme proteins secreted by the class I secretion pathway.

Orotidine-5'-monophosphate decarboxylase from Pseudomonas aeruginosa PAO1: cloning, overexpression, and enzyme characterization.

Orotidine-5'-monophosphate decarboxylase (OMPdecase) catalyzes the final step in pyrimidine biosynthesis, the conversion of orotidine-5'-monophosphate (OMP) to uridine-5'-monophosphate. The pyrF gene, encoding OMPdecase, was isolated from a chromosomal library of Pseudomonas aeruginosa PAO1 by screening for complementation of an Escherichia coli and a P. aeruginosa pyrF mutant. The nucleotide sequence of a 2510-bp chromosomal DNA fragment, complementing both strains, was determined (EMBL accession number X65613). On this a 696-bp open reading frame capable of encoding the 24 kDa OMPdecase was identified. Despite a generally good correspondence to other OMPdecase sequences, the P. aeruginosa gene was unique in that it did not constitute part of an operon. The pyrF gene was amplified by polymerase chain reaction, overexpressed in the pT7-7/E. coli BL21(DE3) system and purified to near electrophoretic homogeneity by anion exchange chromatography. Characterization of the purified enzyme revealed the following data, a Km value for OMP of 9.91 microM and an isoelectric point of 6.65. No major decrease in enzyme activity was observed in a pH range between 7.8 and 10.2. Gel electrophoresis under nondenaturing conditions suggested that the native form of OMPdecase is the dimer.

Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties.

Alginate-producing (mucoid) strains of Pseudomonas aeruginosa possess a 54-kDa outer membrane (OM) protein (AlgE) which is missing in nonmucoid bacteria. The coding region of the algE gene from mucoid P. aeruginosa CF3/M1 was subcloned in the expression vector pT7-7 and expressed in Escherichia coli. The level of expression of recombinant AlgE was seven times higher than that of the native protein in P. aeruginosa. Recombinant AlgE was found mainly in the OM. A putative precursor protein (56 kDa) of AlgE could be immunologically detected in the cytoplasmic membrane (CM). Surface exposition of AlgE in the OM of E. coli was indicated by labeling lysine residues with N-hydroxysuccinimide-biotin. Secondary-structure analysis suggested that AlgE is anchored in the OM by 18 membrane-spanning beta-strands, probably forming a beta-barrel. Recombinant AlgE was purified, and isoelectric focusing revealed a pI of 4.4. Recombinant AlgE was spontaneously incorporated into planar lipid bilayers, forming ion channels with a single-channel conductance of 0.76 nS in 1 M KCl and a mean lifetime of 0.7 ms. Single-channel current measurements in the presence of other salts as well as reversal potential measurements in salt gradients revealed that the AlgE channel was strongly anion selective. For chloride ions, a weak binding constant (Km = 0.75 M) was calculated, suggesting that AlgE might constitute an ion channel specific for another particular anion, e.g., polymannuronic acid, which is a precursor of alginate. Consistent with this idea, the open-state probability of the channel decreased when GDP-mannuronic acid was added. The AlgE channel was inactivated when membrane voltages higher than +85 mV were applied. The electrophysiological characteristics of AlgE, including its rectifying properties, are quite different from those of typical porins.

Antibody response of rabbits and cystic fibrosis patients to an alginate-specific outer membrane protein of a mucoid strain of Pseudomonas aeruginosa.

The aim of this work was to study the immunogenicity of an outer membrane (OM) protein (AlgE; 54 kDa) which is produced solely by mucoid, i.e. alginate-producing strains of P. aeruginosa. The source of AlgE used for our study was the mucoid strain CF3/M1 originally isolated from sputum of a cystic fibrosis (CF) patient. The purified non-denatured protein served as antigen to raise polyclonal monospecific anti-AlgE antibodies in rabbits and to assay sera from 41 cystic fibrosis (CF) patients for anti-AlgE antibodies. According to clinical protocols the sputa of 22 CF patients were positive for P. aeruginosa, 18 were negative and one case was unknown. Our ELISA studies showed that high titers of anti-AlgE antibodies (IgG) corresponded well with the infection status of the CF patients. None of 23 control sera derived from healthy volunteers contained significant levels of anti-AlgE antibodies. Thus the ELISA should be considered as a sensitive diagnostic tool for the early detection of mucoid P. aeruginosa infections in CF patients. Furthermore, we suggest to include AlgE in a multicomponent experimental vaccine for potential protection of non-colonized CF patients from colonization with mucoid P. aeruginosa.

Molecular genetics of the extracellular lipase of Pseudomonas aeruginosa PAO1.

The structural gene (lipA) coding for the extracellular lipase of Pseudomonas aeruginosa PAO1 has been cloned on plasmid pSW118. Nucleotide sequence analysis revealed a gene of 936 bp. lipA codes for a proenzyme of 311 amino acids including a leader sequence of 26 amino acids. The mature protein was predicted to have a M(r) of 30134, an isoelectric point of 5.6, and a consensus sequence (IGHSHGG) typical of lipases. Furthermore it is highly homologous (greater than 60%) to other lipases from various pseudomonads. The lipA gene failed to hybridize detectably with genomic DNA from other Pseudomonas species except P. alcaligenes, even under relaxed stringency. Located 220 bp downstream of the lipA gene, is an open reading frame (ORF2, lipH) which encodes a hydrophilic protein (283 amino acids; M(r) 33587) that shows some homology to the limA gene product of P. cepacia. In complementation tests of lipase-defective mutants, lipH was shown to be necessary for expression of active extracellular lipase in P. aeruginosa PAO1.

Extracellular lipase from Pseudomonas aeruginosa is an amphiphilic protein.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) secreted by Pseudomonas aeruginosa PAC1R was purified from cell-free growth medium by preparative isoelectric focusing. After blotting the N-terminal amino acid sequence and the amino acid composition were determined and compared to P. fragi and P. cepacia lipases yielding significant homology between all three species. Additionally, a consensus sequence K-Y-P-i-v-l-V-H-G was identified residing at the N-terminus of Pseudomonas lipases and in the central part of Staphylococcus lipases. Treatment of lipase with the serine-specific inhibitor diethyl p-nitrophenyl phosphate caused a rapid and complete inhibition of enzyme activity indicating the presence of a serine at the catalytic site as expected from lipase consensus sequences. Upon charge-shift electrophoresis the electrophoretic mobility of purified lipase was shifted either anodally or cathodally in the presence of sodium deoxycholate and cetyltrimethylammoniumbromide, respectively. This result demonstrates that extracellular lipase of P. aeruginosa exhibits an amphiphilic character like intrinsic membrane proteins.

An outer membrane protein characteristic of mucoid strains of Pseudomonas aeruginosa.

SDS-polyacrylamide gel electrophoresis of outer membrane (OM) proteins of different mucoid strains of P. aeruginosa revealed a protein of about 54 kDa that was absent in nonmucoid strains. This 54 kDa protein was expressed under iron-restricted and iron sufficient growth conditions. Electrophoretic mobility of the 54 kDa protein was modified by the solubilization temperature as well as by the addition of lipopolysaccharide and alginate prior to electrophoresis. Treatment of OMs with octylglucoside/KCl or SDS completely extracted the 54 kDa protein at low temperatures. The possible role of this protein in biosynthesis and/or excretion of bacterial alginate is discussed.

Isolation and characterization of an alginate lyase from Klebsiella aerogenes.

The bacterium Klebsiella aerogenes (type 25) produced an inducible alginate lyase, whose major activity was located intracellularly during all growth phases. The enzyme was purified from the soluble fraction of sonicated cells by ammonium sulfate precipitation, anion- and cation-exchange chromatography and gel filtration. The apparent molecular weight of purified alginate lyase of 28,000 determined by gel filtration and of 31,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the active enzyme was composed of a single polypeptide. The alginate lyase displayed a pH optimum around 7.0 and a temperature optimum around 37 degrees C. The purified enzyme depolymerized alginate by a lyase reaction in an endo manner releasing products which reacted in the thiobarbituric acid assay and absorbed strongly in the ultraviolet region at 235 nm. The alginate lyase was specific for guluronic acid-rich alginate preparations. Propylene glycol esters of alginate and O-acetylated bacterial alginates were poorly degraded by the lyase compared with unmodified polysaccharide. The guluronate-specific lyase activity was applied in an enzymatic method to detect mannuronan C-5 epimerase in three different mucoid (alginate-synthesizing) strains of Pseudomonas aeruginosa. This enzyme which converts polymannuronate to alginate could not be demonstrated either extracellularly or intracellularly in all strains suggesting the absence of a polymannuronate-modifying enzyme in P. aeruginosa.

Chromosomal mapping and cloning of the lipase gene of Pseudomonas aeruginosa.

Various mutants (lip) of Pseudomonas aeruginosa PAO 2302 that lacked extracellular lipase activity were isolated. They were selected on a calcium-triolein agar. The phenotypic characteristics of two of these mutants suggested that they were defective in the gene coding for lipase: both lip mutants produced no lipase in liquid- and on solid medium. They were nonpleiotropic with regard to various other exoproducts. None of the mutants released any putatively cell-bound lipase after treatment of cells with Triton X-100 or alginate. The electrophoretic protein- and LPS-profiles of outer membranes derived from lip mutants and the parental strain were identical. The lip locus was mapped on the chromosome of P. aeruginosa PAO 1 by FP5- and R68. 45-mediated crossings and by transduction with phage G101. The lip locus was cotransduced with pyrF only (60%) indicating a map position at about 57 min. The lipase gene was cloned on a 3.1 kb SalI fragment using vector pKT248. The newly constructed plasmid was able to complement the lipase deficiency of the two lip mutants of P. aeruginosa.

Alginate lyase releases cell-bound lipase from mucoid strains of Pseudomonas aeruginosa.

Alginate lyase (EC 4.2.2.3) was partially purified from the culture medium of Bacillus circulans JBH2 by ammonium sulphate precipitation, gel filtration and ion-exchange chromatography. The purified enzyme was unstable in the absence of protecting substances such as gelatin. At a pH optimum of 5.8, the enzyme depolymerized algal as well as bacterial alginates, the latter in the deacetylated form more effectively than in the acetylated form. Incubation of various mucoid strains of Pseudomonas aeruginosa in the presence of purified alginate lyase caused rapid degradation of the alginate slime without killing the cells. The degradation of alginate was accompanied by a release of cell-bound lipase into the medium indicating that one of the functions of the alginate layer of mucoid bacteria might be to serve as a temporary reservoir for lipase.

Purification of extracellular lipase from Pseudomonas aeruginosa.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was excreted by Pseudomonas aeruginosa PAC1R during the late logarithmic growth phase. Characterization of cell-free culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of significant amounts of lipopolysaccharide, part of which seemed to be tightly bound to lipase. After concentration of culture supernatants by ultrafiltration, lipase-lipopolysaccharide complexes were dissociated by treatment with EDTA-Tris buffer and subsequent sonication in the presence of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized lipase was purified by isoelectric focusing in an agarose gel containing the same detergent; the lipase activity appeared in a single peak corresponding to a distinct band in the silver-stained gel. The isoelectric point was 5.8. Analysis of purified lipase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning revealed an apparent molecular weight of 29,000 and a specific activity of 760 mu kat/mg of protein. Estimations based on these data showed that a single P. aeruginosa cell excreted about 200 molecules of lipase, each having a molecular activity of 2.2 X 10(4) per s.

Methyl ester of hyaluronate is unable to stimulate exolipase formation by Serratia marcescens.

Hyaluronate stimulated the formation of exolipase by Serratia marcescens. This ability was abolished when all carboxyl groups of hyaluronate were methyl esterified. Additional studies suggested that the biological inactivity of esterified hyaluronate should be ascribed to the reduced conformational order of the molecules rather than to their electroneutrality.

Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens.

Among 21 different polysaccharides tested, 5 greatly enhanced the spontaneous and cyclic AMP-induced formation of exolipase: glycogen, hyaluronate, laminarin, pectin B, and gum arabic. These polysaccharides have in common the tendency to form highly ordered networks because of the branching or helical arrangement, or both, of their molecules. None of the polysaccharides could be utilized by the cells as the sole carbon source. Strong lipid extraction of four different polysaccharides did not reduce their exolipase-enhancing efficacy. At a constant cell density the stimulation of exolipase formation by various concentrations of glycogen followed saturation kinetics, suggesting a limited number of "sites" for the glycogen to act. The active principle present in a solution of pectin was destroyed by degradation (beta-elimination) of the polymer. Hyaluronate lost its exolipase-enhancing activity by exhaustive hydrolysis with hyaluronidase but was resistant to proteinase K. Exopolysaccharide, isolated from growth medium of Serratia marcescens SM-6, enhanced the exolipase formation as efficiently as hyaluronate. The results of this work are discussed mainly in terms of the "detachment hypothesis."