Binding of protegrin-1 to Pseudomonas aeruginosa and Burkholderia cepacia

Background  

Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia.     

Physical and stability characteristics of Burkholderia cepacia lipase encapsulated in κ-carrageenan

In this work, a novel approach for lipase immobilization was exploited. Lipase from Burkholderia cepacia was encapsulated into κ-carrageenan by co-extrusion method to form a liquid core capsule. The diameter of the encapsulated lipase was found to be in the range of 1.3–1.8 mm with an average membrane thickness of 200 μm and 5% coefficient of variance. The encapsulation efficiency was 42.6% and 97% moisture content respectively. The encapsulated lipase was stable between pH 6 and 9 and temperature until 50 °C. The encapsulated lipase was stable until disintegration of the carrier when stored at 27 °C and retained 72.3% of its original activity after 6 cycles of hydrolysis of p-NPP. The encapsulated lipase was stable in various organic solvents including methanol, ethanol, iso-propanol, n-hexane and n-heptane. Kinetic parameters K_m and V_max were found to be 0.22 mM and 0.06 μmol/min for free lipase and 0.25 mM and 0.05 μmol/min for encapsulated lipase respectively.     

Stereochemistry of a diastereoisomeric amphiphile and the species of the lipase influence enzyme activity in the transesterification catalyzed by a lipase-co-lyophilizate with the amphiphile in organic media

Modified Candida rugosa and Pseudomonas cepacia lipase (CRL and PCL) were co-lyophilized with two pairs of synthetic diastereoisomeric amphiphiles, d- and l-2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl-propionylamino)-pentanedioic acid didodecyl ester (d- and l-BIG2C₁₂CA); d- and l-2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-pentanedioic acid didodecyl ester (d- and l-2C₁₂GE). Enzyme activities of the modified lipase in the transesterification in organic solvent were evaluated. Both pairs of the diastereoisomeric amphiphiles showed enhanced enzyme activity in the transacetylation between racemic sulcatol and isopropenyl acetate in diisopropyl ether, catalyzed by the PCL-co-lyophilizate, by 19–48 fold when compared to the native lipase lyophilized from buffer alone independent of the stereochemistry of the amphiphiles, while in the case of the CRL-co-lyophilizate only the l-BIG2C₁₂CA showed enhanced enzyme activity in the transbutyrylation between racemic solketal and vinyl butyrate in cyclohexane as high as 68–78 fold.     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Enhancement of enzyme activity and enantioselectivity by cyclopentyl methyl ether in the transesterification catalyzed by <Emphasis Type="Italic">Pseudomonas cepacia</Emphasis> lipase co-lyophilized with cyclodextrins

The solvent effects of cyclopentyl methyl ether (CPME) on the reaction rates and enzyme enantioselectivity in the enantioselective transesterifications of racemic 6-methyl-5-hepten-2-ol (racemic sulcatol: SUL) and racemic 2,2-dimethyl-1,3-dioxolane-4-methanol (racemic solketal: SOL) with a series of enol esters catalyzed by Pseudomonas cepacia lipase co-lyophilized with cyclodextrins (-, -, -, partially methylated -,and 2,3,6-tri-O-methyl--cyclodextrin: CyD; CyD; CyD; Me_1.78 CyD; Me₃CyD) were investigated and compared with those in diisopropyl ether (IPE). In the case of SUL, enzyme activities of the co-lyophilizate with Me_1.78 CyD in CPME were lower than those in IPE with every acyl source, however, the absolute enantiopreference was shown in the transesterification with vinyl butyrate (VBR) in IPME. When the substrates were SOL and VBR, the enzyme activities in CPME were greatly enhanced as high as 1.6–9.8-fold, while the enantioselectivities in CPME were comparable to those in IPE.Revisions requested 16 December 2004; Revisions received 17 January 2005     

Chaperone-mediated activation in vivo of a Pseudomonas cepacia lipase

An extracellular Pseudomonas cepacia lipase, LipA, is inactive when expressed in the absence of the product of the limA gene. Evidence has been presented that LimA is a molecular chaperone. The lipA and limA genes have been cloned in separate and independently inducible expression systems in Escherichia coli. These systems were used to test the molecular chaperone hypothesis by investigating whether LimA could activate presynthesized prelipase and whether presynthesized LimA could activate newly synthesized prelipase. The results show that LimA cannot activate presynthesized prelipase and that presynthesized LimA can activate only a limited number of de novo synthesized prelipase molecules. Co-immunoprecipitation of prelipase/lipase with LimA generated a 1:1 complex of prelipase/lipase and LimA. The results suggest that a 1:1 complex of LipA and LimA is required for prelipase processing and secretion of active lipase.     

Mutations affecting regulation of the anabolic argF and the catabolic aru genes in Pseudomonas aeruginosa PAO

Summary The nucleotide sequence required for a fully functional promoter and operator of the Pseudomonas aeruginosa argF gene (argF _po), the arginine-repressible gene for anabolic ornithine carbamoyltransferase, was defined within a 160 by region. The streptomycin (Sm) resistance genes strAB of plasmid RSF1010 were fused to argF _po. This construct in P. aeruginosa strain PAO conferred resistance to Sm. Mutants of strain PAO were selected which were resistant to Sm in the presence of arginine due to constitutive expression of argF _po —strAB. These mutants were designated argR. They were unable to grow or grew poorly on arginine or ornithine as the sole carbon and nitrogen source. This growth defect (Aru^–/Oru^– phenotype) was correlated with a reduced level of N-succinylornithine aminotransferase, an enzyme participating in the major aerobic pathway for arginine and ornithine catabolism in this organism. The argR mutants were classified into four groups by transduction analysis and three argR mutations were mapped on the PAO chromosome. argR9901 and argR9902 were co-transducible with car-9 (at 1 min) and thus close to the oru-310 locus; argR9906 was localized in the oruI (=aru) gene cluster (67 min). Some aru mutants, which have been isolated previously and which produce very low amounts of all enzymes in the arginine succinyltransferase pathway, were unable to repress the argF gene in an arginine medium. Thus, P. aeruginosa PAO appears to have multiple genes that are involved in the regulation of both the anabolic argF and the catabolic aru genes.Abbreviations Arg^– arginine auxotrophy - Aru arginine utilization - Oru ornithine utilization     

Homology between the HrpO protein of Pseudomonas solanacearum and bacterial proteins implicated in a signal peptide-independent secretion mechanism

A region of approximately 22 kb of DNA defines the large hrp gene cluster of strain GMI1000 of Pseudomonas solanacearum. The majority of mutants that map to this region have lost the ability to induce disease symptoms on tomato plants and are no longer able to elicit a hypersensitive reaction (HR) on tobacco, a nonhost plant. In this study we present the complementation analysis and nucleotide sequence of a 4772 by region of this hrp gene cluster. Three complete open reading frames (ORFs) are predicted within this region. The corresponding putative proteins, HrpN, HrpO and HpaP, have predicted sizes of 357, 690 and 197 amino acids, respectively, and predicted molecular weights of 38607, 73 990 and 21959 dalton, respectively. HrpN and HrpO are both predicted to be hydrophobic proteins with potential membrane-spanning domains and HpaP is rich in proline residues. A mutation in hpaP (for hrp associated) does not affect the HR on tobacco or the disease on tomato plants. None of the proteins is predicted to have an N-terminal signal sequence, which would have indicated that the proteins are exported. Considerable sequence similarities were found between HrpO and eight known or predicted prokaryotic proteins: LcrD of Yersinia pestis and Y. enterocolitica, FlbF of Caulobacter crescentus, F1hA of Bacillus subtilis, MxiA and VirH of Shigella flexneri, InvA of Salmonella typhimurium and HrpC2 of Xanthomonas campestris pv. vesicatoria. These homologies suggest that certain hrp genes of phytopathogenic bacteria code for components of a secretory system, which is related to the systems for secretion of flagellar proteins, Ipa proteins of Shigella flexneri and the Yersinia Yop proteins. Furthermore, these homologous proteins have the common feature of being implicated in a distinct secretory mechanism, which does not require the cleavage of a signal peptide. The sequence similarity between HrpO and HrpC2 is particularly high (66% identity and 81 % similarity) and the amino acid sequence comparison between these two proteins presented here reveals the first such sequence similarity to be shown between Hrp proteins of P. solanacearum and X. campestris. An efflux of plant electrolytes was found to be associated with the interactions between P. solanacearum and both tomato and tobacco leaves. This phenomenon may be part of the mechanism by which hrp gene products control and determine plant-bacterial interactions, since hrpO mutants induced levels of leakage which were significantly lower than those induced by the wild type on each plant.     

Alga-lytic activity of Pseudomonas fluorescens against the red tide causing marine alga Heterosigma akashiwo (Raphidophyceae)

A bacterial strain, HAK-13, exhibited strongest activity against Heterosigma akashiwo and was capable of controlling this bloom forming phytoplankton. Based on 16S rDNA sequences and biochemical and morphological characteristics, the strain HAK-13 was determined to be Pseudomonas fluorescens on the basis of 99.9% similarity with reference strains in the DNA databases. The growth of H. akashiwo was strongly suppressed by HAK-13 in all growth phases, with the strongest alga-lytic activity noted against harmful bloom-forming species in the exponential stage (6–22 days). Host range tests showed that HAK-13 also significantly inhibited the growth of Alexandrium tamarense and Cochlodinium polykrikoides but could not destroy Gymnodinium catenatum. P. fluorescens HAK-13 indirectly attacked H. akashiwo by alga-lytic substances that might be located at the compartment of cytoplasmic membrane of the bacterium at a level of 45.86 units/mg of specific activity. The results indicated that P. fluorescens HAK-13 caused cell lysis and death of H. akashiwo, A. tamarense, and C. polykrikoides dramatically and Prorocentrum dentatum slightly. Therefore, P. fluorescens HAK-13 has potential for use as a selective biocontrol of harmful algal blooms.     

Virulence genes are carried by a megaplasmid of the plant pathogen Pseudomonas solanacearum

Summary A class of avirulent mutants of the plant pathogenic bacterium Pseudomonas solanacearum, strain GMI1000, resistant to acridine orange (Acr^r), harbour a deletion of over 85 kb in their genome. This deletion affects, a1,000 kb megaplasmid which has previously been shown to be present in most of the strains of this species. In addition at least 11 out of 13 independent Tn5 insertions, leading to loss of virulence, are located on the megaplasmid. Nine of them are present in the region which is deleted from the Acr^r mutants. These results suggest that the majority of virulence genes identified so far are plasmid borne.     

Quantum mechanical study of Burkholderia cepacia lipase enantioselectivity

Quantum mechanical, semiempirical (AM1) and ab initio (6-31G*) study of the Burkholderia cepacia lipase (BCL) catalysed reactions of the secondary alcohol esterification and its ester hydrolysis is presented. We have selected BCL for our study because of numerous experimental results available, but also because of its broad selectivity and stability that makes it interesting for industrial use. Previously we developed models for predicting lipase stereo-selectivity towards primary and secondary alcohols according to their structural parameters. In this work we show that not all of the experimentally determined binding modes are catalytically competent and that additional molecular modelling should be accomplished in order to find good starting points to study chemical reactions. The binding modes from which chemical modification of a substrate is possible are the most relevant for understanding enzyme selectivity and for the rational enzyme engineering.

We also investigated the influence of the tetrahedral atom type, C and P, upon the energy barriers in the proton transfer reactions from the catalytic histidine (His286) to either the catalytic serine (Ser87) or the alcohol oxygen of the substrate.     

Use of resazurin to detect mefloquine as an efflux-pump inhibitor in Pseudomonas aeruginosa and Escherichia coli

Multi-drug-resistant bacteria can cause serious infections that are extremely difficult to treat. Bacterial efflux pumps are known to contribute to multi-drug resistance and, thus, constitute a promising target for novel antibacterial agents. Resazurin is widely used to monitor bacterial growth because resazurin is reduced to the fluorescent resorufin by live cells. We have shown by flow cytometric analysis and by accumulation studies with wild type and efflux deficient strains that resazurin is a substrate of efflux pumps in Escherichia coli and Pseudomonas aeruginosa. Our investigations showed that the conversion rate of resazurin to resorufin is affected by efflux pumps. This finding was used to design an assay useful to detect efflux pump activity and to find potential efflux-pump inhibitors in a microtiter plate format. Mefloquine was detected as efflux-pump inhibitor when a panel of selected chemical compounds was tested for assay validation purposes.     

Development of cost-effective medium for the large-scale production of a mosquito pupicidal metabolite from Pseudomonas fluorescens Migula

Although Bacillus thuringiensis and Bacillus sphaericus are being used extensively for mosquito control, the recent reports of development of resistance in insects against them prompted many workers to search for new microbial agents or their metabolites for mosquito control. This has resulted in the isolation of a new bacterial isolate Pseudomonas fluorescens Migula which is known to be toxic against larval and pupal stages of mosquitos and could be considered for further development as a biocontrol agent. But the large-scale production of this bacterium is expensive because the high cost of the medium restricts this bacterium is used in mosquito control programs. In this study, we attempted to develop a cost-effective medium, based on inexpensive, locally available raw material Soya bean (Glycine max) by using 100-L bioreactor. Biomass and pupicidal metabolite production were satisfactory after P. fluorescens was produced on both media. Results showed that the Biomass in dry weight of Soya medium was 12.7 g/L and GPS medium (conventional medium) was 11.23 g/L. The maximum pupicidal metabolite production in Soya medium was (LC50 0.24 μL/mL) and in GPS medium was (LC50 0.44 μL/mL).     

A novel Pseudomonas putida strain with high levels of hydantoin-converting activity, producing -amino acids

Optically pure chiral amino acids and their derivatives can be efficiently synthesised by the biocatalytic conversion of 5-substituted hydantoins in reactions catalysed by stereo-selective microbial enzymes: initially a hydantoinase catalyses the cleavage of the hydantoin producing an N-carbamyl amino acid. In certain bacteria where an N-carbamyl amino acid amidohydrolase (NCAAH) is present, the N-carbamyl amino acid intermediate is further converted to amino acid, ammonia and CO₂. In this study we report on a novel Pseudomonas putida strain which exhibits high levels of hydantoin-converting activity, yielding -amino acid products including alanine, valine, and norleucine, with bioconversion yields between 60% and 100%. The preferred substrates are generally aliphatic, but not necessarily short chain, 5-alkylhydantoins. In characterizing the enzymes from this microorganism, we have found that the NCAAH has -selectivity, while the hydantoinase is non-stereoselective. In addition, resting cell reactions under varying conditions showed that the hydantoinase is highly active, and is not subject to substrate inhibition, or product inhibition by ammonia. The rate-limiting reaction appears to be the NCAAH-catalysed conversion of the intermediate. Metal-dependence studies suggest that the hydantoinase is dependent on the presence of magnesium and cobalt ions, and is strongly inhibited by the presence of copper ions. The relative paucity of -selective hydantoin-hydrolysing enzyme systems, together with the high level of hydantoinase activity and the unusual substrate selectivity of this P. putida isolate, suggest that is has significant potential in industrial applications.     

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium

Summary A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI-BglII fragment between the kanamycin resistance (Km^r) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Km^r transfer frequency of 10^-6 per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8x10⁶ cpm for a cell density of 10³ colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.