Manipulation of Pseudomonas aeruginosa alginate pathway by varying the level of biosynthetic enzymes and growth temperature

J.H. LEITÃO AND i. SÁ-CORREIA. 1993. The manipulation of the alginate pathway in two Pseudomonas aeruginosa mucoid variants was attempted at growth temperatures within the range 20C-40C. This was carried out by increasing the level of either phosphomannose isomerase (PMI) and GDP-mannose pyrophosphorylase (GMP) or GDP-mannose dehydrogenase (GMD) encoded by algA or algD respectively, present in recombinant plasmids derived from the controlled expression vector pMMB24. The specific growth rate of cells expressing either algA or algD genes from recombinant plasmids was lower than that of cells harbouring the cloning vector only. Stimulation of alginate synthesis was observed when the expression of the alginate genes was low, in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The further increase of the level of alginate enzymes in induced cells, without the simultaneous increase of other limiting steps, had no positive effect on the strictly regulated alginate pathway. Temperature profiles for alginate synthesis were modified reflecting changes in rate limiting steps. Limitations on the polymerization ability and the competition between cell growth and alginate synthesis were possibly involved in the modification of the temperature profiles for alginate production, or in the decrease of the molecular weight of polymers produced by recombinants under conditions that led to highly active alginate synthesis. The acetyl content of alginates produced by the recombinants was higher than that of the biopolymer controls, possibly due to the higher acetyl-CoA availability in slower growing cells.     

Effects of Ambroxol on Alginate of Mature <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Biofilms

Biofilm-forming bacteria Pseudomonas aeruginosa is a common pathogen in mechanically ventilated newborns, which can cause life-threatening infections. Alginate of mucoid Pseudomonas aeruginosa biofilms is considered an important virulence factor which contributes to the resistance to antibiotics. Traditionally, ambroxol is widely used in newborns with lung problems as a mucolytic agent and antioxidant agent as well. And there are few studies that demonstrated the anti-biofilm activity of ambroxol. In this study, we found that ambroxol can affect the structure of mucoid Pseudomonas aeruginosa biofilms. Further, we found that ambroxol reduces the production of alginate, the expression of the important genes and the activity of key enzyme guanosine diphospho-D-mannose dehydrogenase (GDP-mannose dehydrogenase; GMD) which were involved in alginate biosynthesis.     

Alginate biosynthetic enzymes in mucoid and nonmucoid Pseudomonas aeruginosa: overproduction of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase by overexpression of the phosphomannose isomerase (pmi) gene.

The specific activities of phosphomannose isomerase (PMI), phosphomannomutase (PMM), GDP-mannose pyrophosphorylase (GMP), and GDP-mannose dehydrogenase (GMD) were compared in a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa and in two spontaneous nonmucoid revertants. In both revertants some or all of the alginate biosynthetic enzymes we examined appeared to be repressed, indicating that the loss of the mucoid phenotype may be a result of decreased formation of sugar-nucleotide precursors. The introduction and overexpression of the cloned P. aeruginosa phosphomannose isomerase (pmi) gene in both mucoid and nonmucoid strains led not only to the appearance of PMI levels in cell extracts several times higher than those present in the wild-type mucoid strain, but also in higher PMM and GMP specific activities. In extracts of both strains, however, the specific activity of GMD did not change as a result of pmi overexpression. In contrast, the introduction of the cloned Escherichia coli manA (pmi) gene in P. aeruginosa caused an increase in only PMI and PMM activities, having no effect on the level of GMP. This suggests that an increase in PMI activity alone does not induce high GMP activity in P. aeruginosa. The heterologous overexpression of the P. aeruginosa pmi gene in the E. coli manA mutant CD1 led to the appearance in cell extracts of not only PMI activity but also GMP activity, both of which are normally undetectable in extracts of CD1. We discuss the implications of these results and propose a mechanism by which overexpression of the P. aeruginosa pmi gene can cause an elevation in both PMM and GMP activities.     

Temperature profiles and alginate synthesis in mucoid and non-mucoid variants of Pseudomonas aeruginosa

Alginate-producing Pseudomonas aeruginosa 8821 spontaneously produced low- and high-alginate-producing variants that had low and high specific activities of GDP-mannose dehydrogenase and high and low specific growth rates, respectively. The optimal temperature for alginate production was 10°C below that for growth in the case of the high-producer but they were the same for the low-producer. In short duration thermal death experiments, alginate protected cells against lethal temperatures, although the maximal temperature for growth of the mucoid variants (43°C) was lower than that for the non-mucoid strain (44°C). The temperature profiles were associative and above 37°C growth concurred with thermal death.     

Cloning of Escherichia coli and Pseudomonas aeruginosa phosphomannose isomerase genes and their expression in alginate-negative mutants of Pseudomonas aeruginosa.

The phosphomannose isomerase (pmi) gene of Escherichia coli was cloned on a broad-host-range cosmid vector and expressed in Pseudomonas aeruginosa at a low level. Plasmid pAD3, which harbors the E. coli pmi gene, contains a 6.2-kilobase-pair HindIII fragment derived from the chromosome of E. coli. Subcloning produced plasmids carrying the 1.5-kilobase-pair HindIII-HpaI subfragment of pAD3 that restored alginic acid production in a nonmucoid, alginate-negative mutant of P. aeruginosa. This fragment also complemented mannose-negative, phosphomannose isomerase-negative mutants of E. coli and showed no homology by DNA-DNA hybridization to P. aeruginosa chromosomal DNA. By using a BamHI constructed cosmid clone bank of the stable alginate producing strain 8830, we have been able to isolate a recombinant plasmid of P. aeruginosa origin that also restores alginate production in the alginate-negative mutant. This new recombinant plasmid, designated pAD4, contained a 9.9-kilobase-pair EcoRI-BamHI fragment with the ability to restore alginate synthesis in the alginate-negative P. aeruginosa. This fragment showed no homology to E. coli chromosomal DNA or to plasmid pAD3. Both mucoid and nonmucoid strains of P. aeruginosa had no detectable levels of phosphomannose isomerase activity as measured by mannose 6-phosphate-to-fructose 6-phosphate conversion. However, P. aeruginosa strains harboring the cloned pmi gene of E. coli contained measurable levels of phosphomannose isomerase activity as evidenced by examining the conversion of mannose 6-phosphate to fructose 6-phosphate.     

Alginate acetylation influences initial surface colonization by mucoid Pseudomonas aeruginosa

Mucoid strains of Pseudomonas aeruginosa overproduce the exopolysaccharide alginate, which is substituted with O-acetyl groups. Under non-growing conditions in phosphate buffer, a mucoid clinical strain formed microcolonies on steel surfaces, while an acetylation-defective mutant was unable to form cell clusters. Enzymatic degradation of alginate by alginate lyase prevented microcolony formation of the mucoid parent strain. In a continuous-culture flow-cell system, using gluconate minimal medium, the mucoid strain with acetylated alginate formed microcolonies and grew into heterogenous biofilms, whereas the acetylation-defective mutant produced a thinner and more homogeneous biofilm. A lowered viscosity of extracellular material from the acetylation-defective mutant indicated a weakening of exopolymer interactions by loss of acetyl groups. These results suggest that acetyl substituents are necessary for the function of high-molecular-mass alginate to mediate cell aggregation into microcolonies in the early stages of biofilm development by mucoid P. aeruginosa, thereby determining the architecture of the mature biofilm.     

Role of alginate lyase in cell detachment of Pseudomonas aeruginosa.

The exopolysaccharide alginate of Pseudomonas aeruginosa was shown to be important in determining the degree of cell detachment from an agar surface. Nonmucoid strain 8822 gave rise to 50-fold more sloughed cells than mucoid strains 8821 and 8830. Alginate anchors the bacteria to the agar surface, thereby influencing the extent of detachment. The role of the P. aeruginosa alginate lyase in the process of cell sloughing was investigated. Increased expression of the alginate lyase in mucoid strain 8830 led to alginate degradation and increased cell detachment. Similar effects were seen both when the alginate lyase was induced at the initial stage of cell inoculation and when it was induced at a later stage of growth. It appears that high-molecular-weight alginate polymers are required to efficiently retain the bacteria within the growth film. When expressed from a regulated promoter, the alginate lyase can induce enhanced sloughing of cells because of degradation of the alginate. This suggests a possible role for the lyase in the development of bacterial growth films.     

A mutation in algN permits trans activation of alginate production by algT in Pseudomonas species.

Conversion of the mucoid phenotype, which results from the production of the exopolysaccharide alginate, is a feature typical of Pseudomonas aeruginosa strains causing chronic pulmonary infections in patients with cystic fibrosis. In this study, we further characterized a recombinant plasmid, called pJF15, that contains DNA from the 65- to 70-min region of the chromosome of mucoid P. aeruginosa FRD1 and has loci involved in alginate conversion. Plasmid pJF15 complements algT mutations in trans and confers the mucoid phenotype in cis following gene replacement. However, the phenotype of nonmucoid P. aeruginosa carrying pJF15 is unchanged. Here we report the identification of a locus immediately downstream of algT, called algN, that may be a negative regulator that blocks algT from activating alginate production. Inactivation of algN by transposon Tn501 insertion allowed algT to stimulate alginate production in trans. The DNA sequence of this region identified an open reading frame that predicts an algN gene product of 33 kDa, but no homology was found to other proteins in a sequence data base. Clones of algT in which algN was deleted caused the activation of alginate biosynthesis in transconjugants of several P. aeruginosa strains. DNA containing algT was shown to hybridize to the genomes of several Pseudomonas species, including P. putida, P. stutzeri, and P. fluorescens. Transconjugants of these species carrying algT DNA (with a deletion of algN) from pJF15 showed a mucoid phenotype and increased production of uronic acid-containing polymers that resembled alginate.     

Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the sputum of cystic fibrosis patients produce copious quantities of an exopolysaccharide known as alginic acid. Since clinical isolates of the mucoid variants are unstable with respect to alginate synthesis and revert spontaneously to the more typical nonmucoid phenotype, it has been difficult to isolate individual structural gene mutants defective in alginate synthesis. The cloning of the genes controlling alginate synthesis has been facilitated by the isolation of a stable alginate-producing strain, 8830. The stable mucoid strain was mutagenized with ethyl methanesulfonate to obtain various mutants defective in alginate biosynthesis. Several nonmucoid (Alg-) mutants were isolated. A mucoid P. aeruginosa gene library was then constructed, using a cosmid cloning vector. DNA isolated from the stable mucoid strain 8830 was partially digested with the restriction endonuclease HindIII and ligated to the HindIII site of the broad host range cosmid vector, pCP13. After packaging in lambda particles, the recombinant DNA was introduced via transfection into Escherichia coli AC80. The clone bank was mated (en masse) from E. coli into various P. aeruginosa 8830 nonmucoid mutants with the help of pRK2013, which provided donor functions in trans, and tetracycline-resistant exconjugants were screened for the ability to form mucoid colonies. Three recombinant plasmids, pAD1, pAD2, and pAD3, containing DNA inserts of 20, 9.5, and 6.2 kilobases, respectively, were isolated based on their ability to restore alginate synthesis in various strain 8830 nonmucoid (Alg-) mutants. Mutants have been assigned to at least four complementation groups, based on complementation by pAD1, pAD2, or pAD3 or by none of them. Introduction of pAD1 into the spontaneous nonmucoid strain 8822, as well as into other nonmucoid laboratory strains of P. aeruginosa such as PAO and SB1, was found to slowly induce alginate synthesis. This alginate-inducing ability was found to reside on a 7.5-kilobase EcoRI fragment that complemented the alg-22 mutation of strain 8852. The pAD1 chromosomal insert which complements the alg-22 mutation was subsequently mapped at ca. 19 min of the P. aeruginosa PAO chromosome.     

Copper as a signal for alginate synthesis in Pseudomonas syringae pv. syringae.

Plant-associated pseudomonads are commonly exposed to copper bactericides, which are applied to reduce the disease incidence caused by these bacteria. Consequently, many of these bacteria have acquired resistance or tolerance to copper salts. We recently conducted a survey of 37 copper-resistant (Cur) Pseudomonas spp., including P. cepacia, P. fluorescens, P. syringae, and P. viridiflava, and found that a subset of the P. syringae strains showed a dramatic increase in exopolysaccharide (EPS) production on mannitol-glutamate medium containing CuSO4 at 250 micrograms/ml. A modified carbazole assay indicated that the EPS produced on copper-amended media contained high levels of uronic acids, suggesting that the EPS was primarily alginic acid. Uronic acids extracted from selected strains were further confirmed to be alginate by demonstrating their sensitivity to alginate lyase and by descending paper chromatography following acid hydrolysis. Subinhibitory levels of arsenate, cobalt, lithium, rubidium, molybdenum, and mercury did not induce EPS production, indicating that alginate biosynthesis is not induced in P. syringae cells exposed to these heavy metals. A 200-kb plasmid designated pPSR12 conferred a stably mucoid phenotype to several P. syringae recipients and also increased their resistance to cobalt and arsenate. A cosmid clone constructed from pPSR12 which conferred a stably mucoid phenotype to several P. syringae strains but not to Pseudomonas aeruginosa was obtained. Results obtained in this study indicate that some of the signals and regulatory genes for alginate production in P. syringae differ from those described for alginate production in P. aeruginosa.     

Enzymes involved in the biosynthesis of alginate byPseudomonas aeruginosa

Summary Analysis of the enzymes involved in the biosynthesis of alginic acid by mucoidPseudomonas aeruginosa PAO strain's determined the presence of enzymes required to synthesise GDP-mannuronic acid. Addition of polymannuronic acid to an ammonium sulphate precipitate of a cell free alginate suspension indicated the presence of an enzyme which catalysed the epimerisation of mannuronic acid to guluronic acidafter the polymer had been synthesised. The epimerase was shown to be calcium dependant.Various non-mucoid mutants were also studied. The non-mucoid parental strain PAO 381 also contained the enzymes required for alginate synthesis but they were not expressed. Synthesis of alginic acid led to an increase in the level of these enzymes. In the non-mucoid mutants derived from mucoid parents GDP-mannose dehydrogenase was absent in all strains studied. In some of these strains GDP-mannose pyrophosphorylase was also absent, while in other strains, phosphomannase isomerase was absent or greatly reduced.     

Use of a gene replacement cosmid vector for cloning alginate conversion genes from mucoid and nonmucoid Pseudomonas aeruginosa strains: algS controls expression of algT.

Pseudomonas aeruginosa can convert to a mucoid colony morphology by a genetic mechanism called alginate conversion; this results in the production of copious amounts of the exopolysaccharide alginate. The mucoid phenotype of P. aeruginosa is commonly associated with its ability to cause chronic pulmonary tract infections in patients with cystic fibrosis. In this study we isolated the cis-acting locus involved in alginate conversion, called algS, from both mucoid and nonmucoid isogenic strains. We then examined the role of algS in the control of algT, a trans-active gene required for alginate production in P. aeruginosa. We used a new cosmid cloning vector, called pEMR2, that permitted both the cloning of large DNA fragments and their subsequent gene replacement in P. aeruginosa. To verify the predicted properties of this vector, we isolated and tested a pEMR2 hisI+ clone. Using cloned algS-containing DNA and a method for gene replacement, we constructed isogenic strains of P. aeruginosa that had Tn501 adjacent to algS on the chromosome. Two pEMR2 clone banks containing genomic fragments from isogenic algS(On) (exhibiting the alginate production phenotype) and algS(Off) (exhibiting the non-alginate production phenotype) strains were constructed, and Tn501 served as an adjacent marker to select for clones containing the respective algS allele. The pEMR2 algS(On) and pEMR2 algS(Off) clones were shown to contain the indicated algS allele by gene replacement with the chromosome of strains that carried the opposite allele. To test whether algS controls the expression of the adjacent algT gene, we constructed a pLAFR1 algS(Off)T clone and showed it to be unable to complement an algT::Tn501 mutation in trans. In contrast, a pLAFR1 algS(On)T clone did complement algT::Tn501 in trans. Thus, algS appears to control the activation of algT expression, bringing about alginate conversion.     

Alginate synthesis in Pseudomonas aeruginosa: the role of AlgL (alginate lyase) and AlgX.

Previous studies localized an alginate lyase gene (algL) within the alginate biosynthetic gene cluster at 34 min on the Pseudomonas aeruginosa chromosome. Insertion of a Tn501 polar transposon in a gene (algX) directly upstream of algL in mucoid P. aeruginosa FRD1 inactivated expression of algX, algL, and other downstream genes, including algA. This strain is phenotypically nonmucoid; however, alginate production could be restored by complementation in trans with a plasmid carrying all of the genes inactivated by the insertion, including algL and algX. Alginate production was also recovered when a merodiploid that generated a complete alginate gene cluster on the chromosome was constructed. However, alginate production by merodiploids formed in the algX::Tn501 mutant using an alginate cluster with an algL deletion was not restored to wild-type levels unless algL was provided on a plasmid in trans. In addition, complementation studies of Tn501 mutants using plasmids containing specific deletions in either algL or algX revealed that both genes were required to restore the mucoid phenotype. Escherichia coli strains which expressed algX produced a unique protein of approximately 53 kDa, consistent with the gene product predicted from the DNA sequencing data. These studies demonstrate that AlgX, whose biochemical function remains to be defined, and AlgL, which has alginate lyase activity, are both involved in alginate production by P. aeruginosa.     

Mucoid strains of Pseudomonas aeruginosa are devoid of mannuronan C-5 epimerase

Mucoid strains of Azotobacter vinelandii, Pseudomonas aeruginosa and Pseudomonas syringae var glycinia synthesize alginate, an extracellular copolymer comprising D-mannuronosyl and L-guluronosyl moieties. Extracellular mannuronan C-5 epimerase, which converts polymannuronate to alginate, was demonstrated in supernatant fluid from cultures of A. vinelandii. However, the enzyme could not be demonstrated, using the same assay, in supernatant fluids of cultures of mucoid strains of P. aeruginosa or of P. syringae var glycinia, or in cell-free sonic extracts of P. aeruginosa. The results suggest that the pathways of alginate biosynthesis in A. vinelandii and Pseudomonas species may differ.     

Influence of nutrient media on the chemical composition of the exopolysaccharide from mucoid and non-mucoid Pseudomonas aeruginosa

Two mucoid Pseudomonas aeruginosa strains and their non-mucoid revertants isolated from two different clinical origins (cystic fibrosis and bronchiectasis) were grown in various chemically defined media. The extracted exopolysaccharide was characterized by gas-liquid chromatography and 1H-NMR spectroscopy. The exopolysaccharide was always heterogeneous, with an alginate fraction and a neutral fraction essentially composed of glucose, galactose, rhamnose and hexosamines. The alginate composition (mannuronate/guluronate ratio and O-acetylation degree) changed according to the carbon source in nutrient media and whether the strains tested were responding differently to these environmental stimuli. In all cases, the best carbon source for the alginate production was glycerol: the two cystic fibrosis strains produced a predominantly O-acetylated alginate whereas only the mucoid bronchiectasis strain produced a polymannuronate exopolysaccharide.     

Engineered biosealant strains producing inorganic and organic biopolymers

Microbiologically induced calcium carbonate precipitation (MICCP) is a naturally occurring biological process that has shown its potential in remediation of a wide range of structural damages including concrete cracks. In this study, genetically engineered microorganisms, capable of producing extracellular polymeric substances (EPSs) as well as inducing MICCP, were developed based on the assumption that the complex of inorganic CaCO(3) and organic EPS would provide a stronger matrix than MICCP alone as biosealant. In order to develop a recombinant biosealant microorganism, the entire Sporosarcina pasteurii urease gene sequences including ureA, ureB, ureC, ureD, ureE, ureF, and ureG from plasmid pBU11 were sub-cloned into the shuttle vector, pUCP18. The newly constructed plasmid, pUBU1, was transformed into two Pseudomonas aeruginosa strains, 8821 and PAO1, to develop recombinants capable of inducing calcite precipitation in addition to their own ability to produce EPS. Nickel-dependent urease activities were expressed from the recombinant P. aeruginosa 8821 (pUBU1) and P. aeruginosa PAO1 (pUBU1), at 99.4% and 60.9% of the S. pasteurii urease activity, respectively, in a medium containing 2mM NiCl(2). No urease activities were detected from the wild type P. aeruginosa 8821 and P. aeruginosa PAO1 under the same growth conditions. Recombinant Pseudomonas strains induced CaCO(3) precipitation at a comparable rate as S. pasteurii and scanning electron microscopy evidenced the complex of CaCO(3) crystals and EPS layers surrounding the cells. The engineered strains produced in this study are expected to serve as a valuable reference to future biosealants that could be applied in the environment. However, the pathogenic potential of P. aeruginosa, used here only as a model system to show the proof of principle, prevents the use of this recombinant organism as a biosealant. In practical applications, other recombinant organisms should be used.     

Identification of algF in the alginate biosynthetic gene cluster of Pseudomonas aeruginosa which is required for alginate acetylation.

Mucoid strains of Pseudomonas aeruginosa produce a high-molecular-weight exopolysaccharide called alginate that is modified by the addition of O-acetyl groups. To better understand the acetylation process, a gene involved in alginate acetylation called algF was identified in this study. We hypothesized that a gene involved in alginate acetylation would be located within the alginate biosynthetic gene cluster at 34 min on the P. aeruginosa chromosome. To isolate algF mutants, a procedure for localized mutagenesis was developed to introduce random chemical mutations into the P. aeruginosa alginate biosynthetic operon on the chromosome. For this, a DNA fragment containing the alginate biosynthetic operon and adjacent argF gene in a gene replacement cosmid vector was utilized. The plasmid was packaged in vivo into lambda phage particles, mutagenized in vitro with hydroxylamine, transduced into Escherichia coli, and mobilized to an argF auxotroph of P. aeruginosa FRD. Arg+ recombinants coinherited the mutagenized alginate gene cluster and were screened for defects in alginate acetylation by testing for increased sensitivity to an alginate lyase produced by Klebsiella aerogenes. Alginates from recombinants which showed increased sensitivity to alginate lyase were tested for acetylation by a colorimetric assay and infrared spectroscopy. Two algF mutants that produced alginates reduced more than sixfold in acetyl groups were obtained. The acetylation defect was complemented in trans by a 3.8-kb XbaI-BamHI fragment from the alginate gene cluster when placed in the correct orientation under a trc promoter. By a merodiploid analysis, the algF gene was further mapped to a region directly upstream of algA by examining the polar effect of Tn501 insertions. By gene replacement, DNA with a Tn501 insertion directly upstream of algA was recombined with the chromosome of mucoid strain FRD1. The resulting strain, FRD1003, was nonmucoid because of the polar effect of the transposon on the downstream algA gene. By providing algA in trans under the tac promoter, FRD1003 produced nonacetylated alginate, indicating that the transposon was within or just upstream of algF. These results demonstrated that algF, a gene involved in alginate acetylation, is located directly upstream of algA.