IB–LBM simulation of the haemocyte dynamics in a stenotic capillary

To study the behaviour of a haemocyte when crossing a stenotic capillary, the immersed boundary–lattice Boltzmann method was used to establish a quantitative analysis model. The haemocyte was assumed to be spherical and to have an elastic cell membrane, which can be driven by blood flow to adopt a highly deformable character. In the stenotic capillary, the spherical blood cell was stressed both by the flow and the wall dimension, and the cell shape was forced to be stretched to cross the stenosis. Our simulation investigated the haemocyte crossing process in detail. The velocity and pressure were anatomised to obtain information on how blood flows through a capillary and to estimate the degree of cell damage caused by excessive pressure. Quantitative velocity analysis results demonstrated that a large haemocyte crossing a small stenosis would have a noticeable effect on blood flow, while quantitative pressure distribution analysis results indicated that the crossing process would produce a special pressure distribution in the cell interior and to some extent a sudden change between the cell interior and the surrounding plasma.     

Cloning of the phosphonoacetate hydrolase gene from Pseudomonas fluorescens 23F encoding a new type of carbon–phosphorus bond cleaving enzyme and its expression in Escherichia coli and Pseudomonas putida

The phnA gene encoding a novel carbon–phosphorus bond cleavage enzyme, phosphonoacetate hydrolase, from Pseudomonas fluorescens 23F was cloned and expressed in Escherichia coli and Pseudomonas putida. It conferred on the latter host the ability to mineralize phosphonoacetate but on the former the ability to utilize it as sole phosphorus source only. The nucleotide and deduced amino acid sequences of the phnA gene showed no significant homology with any data bank accessions.     

The isolation and properties of a β-alanine permeaseless mutant of Pseudomonas fluorescens

A mutant of Pseudomonas fluorescens has been isolated which is markedly deficient in the active transport of l-alanine, l-proline, and β-alanine.This mutant, which is also deficient in β-alanine transaminase, is capable of catalyzing facilitated diffusion and exchange transport of β-alanine at low temperatures but can maintain little or no uptake of this amino acid against a concentration gradient.In the transport mutant, competitive stimulation of β-alanine by l-proline can be observed, whereas in the parent strain, l-proline inhibits β-alanine uptake.The rate of efflux of β-alanine from preformed pools is 4-fold greater in the transport mutant than in the parent strain.It thus appears that the functional block in this transport mutant resides not in the membrane carrier but in the ability of the cell to couple energy to the membrane carrier.     

A membrane bioreactor for the biotransformation of α-pinene oxide to isonovalal by Pseudomonas fluorescens NCIMB 11671

In this work the biotransformation of α-pinene oxide to isonovalal using resting cells of Pseudomonas fluorescens NCIMB 11671 was evaluated in a membrane bioreactor for biotransformations (MBB). Since the membrane area required to obtain optimum productivities was calculated to be very large (1,000 m² m⁻³), and not possible to fit into the laboratory reactor used, we initially evaluated performance with lower membrane areas (71 m² m⁻³) in a batch system with both the substrate and product in the organic phase. This resulted in low productivities due to mass transfer limitations, so an optimum feeding rate of 0.1 g α-pinene oxide h⁻¹ g_cells ⁻¹ added directly to the reactor contents was determined in batch culture to minimise inhibition. The MBB was then operated continuously for the production of isonovalal, and a final concentration of 108 g l⁻¹ was obtained in the organic reservoir after nearly 400 h of operation (0.32 g-isonovalal l⁻¹ h⁻¹), and the reaction was found not to be mass transfer limited. Finally, the relative viability of the cells was measured using fluorescent probes, and their half-life was found to be almost 2 months, confirming the ability of the MBB to facilitate biotransformations with inhibitory substrates and products.     

Biodegradation of a phenolic mixture in a solid–liquid two-phase partitioning bioreactor

A solid–liquid two-phase partitioning bioreactor (TPPB) in which the non-aqueous phase consisted of polymer (HYTREL) beads was used to degrade a model mixture of phenols [phenol, o-cresol, and 4-chlorophenol (4CP)] by a microbial consortium. In one set of experiments, high concentrations (850 mg l⁻¹ of each of the three substrates) were reduced to sub-inhibitory levels within 45 min by the addition of the polymer beads, followed by inoculation and rapid (8 h) consumption of the total phenolics loading. In a second set of experiments, the beneficial effect of using polymer beads to launch a fermentation inhibited by high substrate concentrations was demonstrated by adding 1,300 and 2,000 mg l⁻¹ total substrates (equal concentrations of each phenolic) to a pre-inoculated bioreactor. At these levels, no cell growth and no degradation were observed; however, after adding polymer beads to the systems, the ensuing reduced substrate concentrations permitted complete destruction of the target molecules, demonstrating the essential role played by the polymer sequestering phase when applied to systems facing inhibitory substrate concentrations. In addition to establishing alternative modes of TPPB operation, the present work has demonstrated the differential partitioning of phenols in a mixture between the aqueous and polymeric phases. The polymeric phase was also observed to absorb a degradation intermediate (arising from the incomplete biodegradation of 4CP), which opens the possibility of using solid–liquid TPPBs during biosynthetic transformation to sequester metabolic byproducts.     

Purification and characterisation of a novel alkalophilic α-D-mannosidase from Pseudomonas fluorescens

An extracellular alkaline α-D-mannosidase in the cell culture of a marine bacterium Pseudomonas fluorescens JK-02 was purified to homogeneity with a 30.7-fold by ammonium sulphate fractionation, anion-exchange chromatography and gel-filtration chromatography. The molecular weight of the purified enzyme was estimated to be 50.5 kDa based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature of the purified enzyme were 8.5 and 30°C. The K_m and V_max values of the purified enzyme towards p-nitrophenyl-α-D-mannopyranoside were determined to be 77 µM and 0.23 µM min^?1mg^?1 of protein, respectively. The α-D-mannosidase showed higher substrate specificity to α-1,3-mannobiose than other isomeric substrates such as α-1,2- and α-1,6-mannobiose. In addition, molecular characterisation of this enzyme reveals that it belongs to a class II α-mannosidase from the glycosyl hydrolase family 38. To the best of our knowledge, this is the first report on the alkalophilic α-1,3 D-mannosidase of Pseudomonas species, which has selective algal-lytic activity against Alexandrium tamarense, Akashiwo sanguine, Gymnodinium catenatum, Gymnodinium mikimotoi and Prorocentrum dentatum.     

Mutants of Pseudomonas fluorescens NCIMB 11671 defective in the catabolism of α-pinene

 Pseudomonas fluorescens NCIMB 11671 metabolises α-pinene via α-pinene oxide and 2-methyl-5-isopropylhexa-2,5-dienal. Mutants unable to grow on α-pinene and/or α-pinene oxide have been isolated by N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis, including an unexpected phenotype able to grow on α-pinene but not on α-pinene oxide. The mutants have been classified on the basis of their α-pinene monooxygenase, α-pinene oxide lyase and aldehyde dehydrogenase activities. Biotransformation of α-pinene by the wild-type and mutant strains has revealed evidence for alternative routes for pinene metabolism to that already proposed. Received: 24 August 1995/Received revision: 20 December 1995/Accepted: 8 January 1996     

The T7-related Pseudomonas putida phage φ15 displays virion-associated biofilm degradation properties

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10⁴ and 10⁶ pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.     

Alteration in virulence characteristics of biofilm cells of Pseudomonas aeruginosa in presence of Tamm-Horsfall protein

Summary Tamm-Horsfall glycoprotein is the most abundant protein in human urine. The present investigation was planned to study the effect of Tamm-Horsfall protein (THP) on elaboration of virulence factors by biofilm cells of Pseudomonas aeruginosa. It was observed that with increase in concentration of THP from 10 to 50 μg/ml there was significant enhancement in elaboration of all the virulence factors by biofilm cells of P. aeruginosa. However, with further increase in concentration of THP from 50 to 70 μg/ml, significant decrease in elaboration of all the virulence traits was observed. Implications of these findings in relation to urinary tract infections caused by P. aeruginosa have been discussed.     

Computational fluid dynamics analysis of mixing and gas–liquid mass transfer in wave bag bioreactor

Single use bioreactors provide an attractive alternative to traditional deep-tank stainless steel bioreactors in process development and more recently manufacturing process. Wave bag bioreactors, in particular, have shown potential applications for cultivation of shear sensitive human and animal cells. However, the lack of knowledge about the complex fluid flow environment prevailing in wave bag bioreactors has so far hampered the development of a scientific rationale for their scale up. In this study, we use computational fluid dynamics (CFD) to investigate the details of the flow field in a 20-L wave bag bioreactor as a function of rocking angle and rocking speed. The results are presented in terms of local and mean velocities, mixing, and energy dissipation rates, which are used to create a process engineering framework for the scale-up of wave bag bioreactors. Proof-of-concept analysis of mixing and fluid flow in the 20-L wave bag bioreactor demonstrates the applicability of the CFD methodology and the temporal and spatial energy dissipation rates integrated and averaged over the liquid volume in the bag provide the means to correlate experimental volumetric oxygen transfer rates (k_La) data with power per unit volume. This correlation could be used as a rule of thumb for scaling up and down the wave bag bioreactors.     

Model-guided dynamic control of essential metabolic nodes boosts acetyl-coenzyme A–dependent bioproduction in rewired Pseudomonas putida

Pseudomonas putida is evolutionarily endowed with features relevant for bioproduction, especially under harsh operating conditions. The rich metabolic versatility of this species, however, comes at the price of limited formation of acetyl-coenzyme A (CoA) from sugar substrates. Since acetyl-CoA is a key metabolic precursor for a number of added-value products, in this work we deployed an in silico-guided rewiring program of central carbon metabolism for upgrading P. putida as a host for acetyl-CoA–dependent bioproduction. An updated kinetic model, integrating fluxomics and metabolomics datasets in addition to manually-curated information of enzyme mechanisms, identified targets that would lead to increased acetyl-CoA levels. Based on these predictions, a set of plasmids based on clustered regularly interspaced short palindromic repeats (CRISPR) and dead CRISPR-associated protein 9 (dCas9) was constructed to silence genes by CRISPR interference (CRISPRi). Dynamic reduction of gene expression of two key targets (gltA, encoding citrate synthase, and the essential accA gene, encoding subunit A of the acetyl-CoA carboxylase complex) mediated an 8-fold increase in the acetyl-CoA content of rewired P. putida. Poly(3-hydroxybutyrate) (PHB) was adopted as a proxy of acetyl-CoA availability, and two synthetic pathways were engineered for biopolymer accumulation. By including cell morphology as an extra target for the CRISPRi approach, fully rewired P. putida strains programmed for PHB accumulation had a 5-fold increase in PHB titers in bioreactor cultures using glucose. Thus, the strategy described herein allowed for rationally redirecting metabolic fluxes in P. putida from central metabolism towards product biosynthesis—especially relevant when deletion of essential pathways is not an option.     

Population dynamics of Ceratium spp. in three English lakes, 1945–1985

Changes in the annual population densities of Ceratium spp. in three adjacent English lakes, Windermere, Esthwaite Water and Blelham Tarn, are summarised over the 41 year period 1945–1985. In these lakes the genus is represented by two species, C. hirundinella (O.F. Müll) Bergh. and C. furcoides (Levander) Langhans. Although the species have not been distinguished over the entire study period, they have been shown by examination of preserved samples to undergo marked changes of relative abundance in Esthwaite Water. Both long-term (years) and short-term (within year) changes of populations densities of Ceratium spp. are considered in relation to possible controlling factors including recruitment of the inoculum, nutrient enrichment, physical stability and fungal epidemics. Given an early inoculum, the relative success of Ceratium populations in these lakes decreases along gradients of increasing mixed depths, increasing turbulence and decreasing retention times. The potential for good population growth is regulated by energy inputs, lake bathymetry and hydraulic characteristics. The realisation of such growth is governed by nutrient availability and microbial grazing. The significance of large between-year differences of populations of Ceratium spp. for general lake metabolism is illustrated for summers of contrasting production in Esthwaite Water.     

Heterologous production of long-chain rhamnolipids from Burkholderia glumae in Pseudomonas putida—a step forward to tailor-made rhamnolipids

Applied Microbiology and Biotechnology - Rhamnolipids are biosurfactants consisting of rhamnose (Rha) molecules linked through a β-glycosidic bond to 3-hydroxyfatty acids with various chain...     

Dependence of catalytic performance of a freeze-dried whole-cell biocatalyst of Pseudomonas fluorescens in regioselective acetylation of 1-β-d-arabinofuranosylcytosine on growth conditions

A freeze-dried whole-cell biocatalyst was prepared from Pseudomonas fluorescens and applied for the first time to regioselective synthesis of 1-β-d-arabinofuranosylcytosine monoester in nonaqueous media. The catalytic performances of the bacterial cells were significantly enhanced by cultivation with a mixed carbon sources containing yeast extract and additional lipid-related substrates, especially the supplement of soybean oil. Cultivation of the cells supplemented with glucose, however, resulted in both low biomass and catalytic activities in the acylation. Taking into account the yield and 5′-regioselectivity of the cell-mediated reaction, yeast extract appeared to be the most suitable for cell cultivation, of all the tested nitrogen sources. Due to the fact that the nutrient concentrations and culture time are also crucial factors affecting the corresponding cell-catalyzed reaction, their effect on the catalytic performance of the cells were also investigated. The best soybean oil concentration, yeast extract concentration and culture time were 0.5% (w/v), 0.1% (w/v) and 48 h, respectively, under which the yield and 5′-regioselectivity of the reaction catalyzed by the cell biocatalyst reached 75.4% and 96.8%, respectively. Our results demonstrated that P. fluorescens whole cell was a green and economic alternative to enzymes for regioselective acylation of ara-C in non-aqueous media.     

Population dynamics of Common Sandpipers Actitis hypoleucos in the Peak District of Derbyshire – a different decade

Capsule A report of the failure of a population to recover from a catastrophic snow storm.

Aims To examine why our study population of Common Sandpipers failed to recover from a sharp drop in 1989, caused by a severe late April snowstorm, when it had recovered from a similar event in 1981.

Methods Populations were censused weekly, with extra visits to ring adults and chicks. Hatching was assumed to have occurred if adults alarmed, and fledging if alarming continued for three weeks. Data from 1989–99 and 1979–88 were compared.

Results The recruitment of new adults to the population was much lower, but these recruits mostly came from other breeding areas, and the local recruits appeared to go elsewhere. Median dispersal distance is estimated as 3.3 km, sufficient to take most locally produced young adults outside the study area.

Conclusions We do not know if poorer breeding in the source populations for our birds, or poorer survival of first year birds over winter in West Africa, is the cause of the failure of this population to recover in the 1990s.     

Properties of Crystalline 3β-Hydroxysteroid Dehydrogenase from Pseudomonas putida

A crystalline 3α-hydroxysteroid: NAD⁺-oxidoreductase (EC 1 1.1.50) which had been obtained from the cell-free extracts of Pseudomonas putida NRRL B-11064 in the presence of added polyethylene glycol, was found to be a native monomer form with a specific activity of 63.0 and a molecular weight of 45,000. Isoelectric focusing exhibited the enzyme to be composed of two isoenzymes: one major part focusing at pH 4.75 and a minor part focusing at pH 5.10. Whereas the enzyme was changed from the monomeric form to a dimeric one with a considerable decrease in the specific activity during the course of crystallization in the absence of the added polyethylene glycol.

The enzyme showed an absolute specificity with regard to 3α-hydroxyl group besides a high requirement for cis A: B fusion of steroids. Typical substrates are cholic acid (Km = 1.33 × 10^?5 m), deoxycholic acid, chenodeoxycholic acid, 3α-hydroxy-12-keto-9,11-cholanoic acid, and etiocholan-3α-ol-17-one. Conjugated bile acids such as taurocholic acid and glycocholic acid are also rapidly oxidized. The pH optima for oxidation of cholic acid and reduction of etiocholan-3,17-dione were 11.5 and 7.0, respectively. The enzyme could be employed for the sensitive and specific assay of bile acids.     

Effects of some curing agents on phenotypic stability in Pseudomonas putida degrading ε-caprolactam

A strain of Pseudomonas putida MCM B-408 capable of utilizing ε-caprolactam (monomer of nylon-6) as the sole source of carbon and nitrogen was found to harbour a single 32-kb plasmid with the same electrophoretic mobility as that of pARI180, a reference plasmid. Acridine orange, ethidium bromide, mitomycin C and SDS failed to cure the plasmid and the phenotype. Elevated temperature alone (40°C) was found to be ineffective in curing. Phenotype, but not the plasmid, was cured at a frequency of 2.63% when acridine orange and elevated temperature (40°C) were used together. The studies therefore indicated that the phenotypic expression of caprolactam degradative genes is quite stable and that Pseudomonas putida MCM B-408 may degrade ε-caprolactam from waste-water satisfactorily without spontaneous loss of the property under adverse environmental conditions.