CellShape: A user‐friendly image analysis tool for quantitative visualization of bacterial cell factories inside

Visualization of the intracellular constituents of individual bacteria while performing as live biocatalysts is in principle doable through more or less sophisticated fluorescence microscopy. Unfortunately, rigorous quantitation of the wealth of data embodied in the resulting images requires bioinformatic tools that are not widely extended within the community‐let alone that they are often subject to licensing that impedes software reuse. In this context we have developed CellShape, a user‐friendly platform for image analysis with subpixel precision and double‐threshold segmentation system for quantification of fluorescent signals stemming from single‐cells. CellShape is entirely coded in Python, a free, open‐source programming language with widespread community support. For a developer, CellShape enhances extensibility (ease of software improvements) by acting as an interface to access and use existing Python modules; for an end‐user, CellShape presents standalone executable files ready to open without installation. We have adopted this platform to analyse with an unprecedented detail the tridimensional distribution of the constituents of the gene expression flow (DNA, RNA polymerase, mRNA and ribosomal proteins) in individual cells of the industrial platform strain Pseudomonas putida KT2440. While the CellShape first release version (v0.8) is readily operational, users and/or developers are enabled to expand the platform further.     

Engineering Pseudomonas putida KT2440 for the production of isobutanol

We engineered P. putida for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native ilvC and ilvD genes, and implementation of the feedback‐resistant acetolactate synthase AlsS from Bacillus subtilis, ketoacid decarboxylase KivD from Lactococcus lactis, and aldehyde dehydrogenase YqhD from Escherichia coli. The resulting strain P. putida Iso2 produced isobutanol with a substrate specific product yield (Y_Iso/S) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from Carnobacterium maltaromaticum to be a suitable alternative for isobutanol production, since replacement of kivD from L. lactis in P. putida Iso2 by the variant from C. maltaromaticum yielded an identical Y_Iso/S. Although P. putida is regarded as obligate aerobic, we show that under oxygen deprivation conditions this bacterium does not grow, remains metabolically active, and that engineered producer strains secreted isobutanol also under the non‐growing conditions.     

Degradation of trichloroethylene by a growth-arrestedPseudomonas putida

A toluene-oxidizing strain ofPseudomonas mendocina KR1 containing toluene-4-mono-oxygenase (TMO) completely degrades TCE with the addition of toluene as a co-substrate in aerobic condition. In order to constructin situ bioremediation system for TCE degradation without any growth-stimulating nutrients or toxic inducers such as toluene, we used the carbon-starvation promoter ofPseudomonas putida MK1 (Kim, Y.et al., J. bacteriol., 1995). Upon entry into the stationary phase due to the deprivation of nutrients, this promoter is strongly induced without further cell growth. The TMO gene cluster (4.5 kb) was spliced downstream of the carbon starvation promoter ofPseudomona putida MK1, already cloned in pUC19. TMO under the carbon starvation promoter was not expressed inE. coli cells either in stationary phase or exponential phase. For TMO expression inPseudomonas strains,tmo and carbon starvation promoter region were recloned into a modified broad-host range vector pMMB67HES which was made from pMMB67HE (8.9 kb) by deletion oftac promoter andlacI ^q (about 1.5 kb). Indigo was produced by TMO under the carbon starvation promoter in aPseudomonas strain of post-exponential phase on M9 (0.2% glucose and 1mM indole) or LB. 18% of TCE was degraded in 14 hours after entering the stationary phase at the initial concentration of 6.6μ M in liquid phase.     

Modelling the interactions between Pseudomonas putida and Escherichia coli O157:H7 in fish‐burgers: use of the lag‐exponential model and of a combined interaction index

Aims: The objective of the current study was to examine the interactions between Pseudomonas putida and Escherichia coli O157:H7 in coculture studies on fish‐burgers packed in air and under different modified atmospheres (30 : 40 : 30 O₂ : CO₂ : N₂, 5 : 95 O₂ : CO₂ and 50 : 50 O₂ : CO₂), throughout the storage at 8°C. Methods and Results: The lag‐exponential model was applied to describe the microbial growth. To give a quantitative measure of the occurring microbial interactions, two simple parameters were developed: the combined interaction index (CII) and the partial interaction index (PII). Under air, the interaction was significant (P < 0·05) only within the exponential growth phase (CII, 1·72), whereas under the modified atmospheres, the interactions were highly significant (P < 0·001) and occurred both in the exponential and in the stationary phase (CII ranged from 0·33 to 1·18). PII values for E.  coli O157:H7 were lower than those calculated for Ps. putida. Conclusions: The interactions occurring into the system affected both E. coli O157:H7 and pseudomonads subpopulations. The packaging atmosphere resulted in a key element. Significance and Impact of the Study: The article provides some useful information on the interactions occurring between E. coli O157:H7 and Ps. putida on fish‐burgers. The proposed index describes successfully the competitive growth of both micro‐organisms, giving also a quantitative measure of a qualitative phenomenon.     

Induction and quantification of phenylacyl-CoA ligase enzyme activities in Pseudomonas putida CA-3 grown on aromatic carboxylic acids

Three phenylacyl-CoA ligase activities were detected in extracts of Pseudomonas putida CA-3 cells grown with a variety of aromatic carboxylic acids. The three phenylacyl-CoA enzyme activities measured were phenylpropyl-CoA ligase (acting on both phenylpropanoic acid and cinnamic acid), a phenylacetyl-CoA ligase, and a medium chain length phenylalkanoyl-CoA ligase acting on aromatic substrates with 5 or more carbons in the acyl moiety. The rate of each enzyme activity detected in extracts of P. putida CA-3 cells is dependent on the growth substrate supplied. High rates of phenylpropyl-CoA ligase activity were observed with extracts of cells grown on phenylpropanoic acid, cinnamic acid or medium chain length phenylalkanoic acids with an uneven number of carbons in the acyl moiety. Extracts of P. putida CA-3 cells exhibited high rates of phenylacetyl-CoA ligase activity when grown on phenylacetic acid or medium chain length phenylalkanoic acids with an even number of carbons in the acyl moiety. In addition, high rates of medium chain length phenylalkanoyl-CoA ligase activity, towards phenylvaleric acid and phenylhexanoic acid, were exhibited by extracts of cells grown on all medium chain length phenylalkanoic acids. Low levels of the various phenylacyl-CoA ligase activities were found in extracts of cells grown on benzoic acid and glucose. Benzoyl-CoA ligase activity was not detected in any cell free extracts generated in this study.     

Quantitative analysis of experiments on bacterial chemotaxis to naphthalene

A mathematical model was developed to quantify chemotaxis to naphthalene by Pseudomonas putida G7 (PpG7) and its influence on naphthalene degradation. The model was first used to estimate the three transport parameters (coefficients for naphthalene diffusion, random motility, and chemotactic sensitivity) by fitting it to experimental data on naphthalene removal from a discrete source in an aqueous system. The best-fit value of naphthalene diffusivity was close to the value estimated from molecular properties with the Wilke-Chang equation. Simulations applied to a non-chemotactic mutant strain only fit the experimental data well if random motility was negligible, suggesting that motility may be lost rapidly in the absence of substrate or that gravity may influence net random motion in a vertically oriented experimental system. For the chemotactic wild-type strain, random motility and gravity were predicted to have a negligible impact on naphthalene removal relative to the impact of chemotaxis. Based on simulations using the best-fit value of the chemotactic sensitivity coefficient, initial cell concentrations for a non-chemotactic strain would have to be several orders of magnitude higher than for a chemotactic strain to achieve similar rates of naphthalene removal under the experimental conditions we evaluated. The model was also applied to an experimental system representing an adaptation of the conventional capillary assay to evaluate chemotaxis in porous media. Our analysis suggests that it may be possible to quantify chemotaxis in porous media systems by simply adjusting the model's transport parameters to account for tortuosity, as has been suggested by others.     

Two different mechanisms are involved in the extremely high-level benzalkonium chloride resistance of a Pseudomonas fluorescens strain

A Pseudomonas fluorescens strain, PFRB, which we previously isolated as a contaminant in a batch of benzalkonium chloride (BAC) stock solution, exhibits high-level resistance, not only to BAC, but also to other cationic surfactants belonging to disinfectants classified as quaternary ammonium compounds (QACs). In this study, we analyzed the resistance mechanism of the strain to BAC and other disinfectants. We obtained results suggesting that two different mechanisms, reduced adsorption of BAC to the cell surface and an energy-dependent mechanism which is most probably an efflux system, were implicated in the high-level resistance to BAC. Reduced adsorption of BAC is likely due to the decreased negative cell surface charge of the strain. The putative efflux system seems to be unique in that it excretes only a certain range of cationic membrane-acting disinfectants belonging to QACs.     

Substrate-dependent autoaggregation of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CP1 during the degradation of mono-chlorophenols and phenol

A bacterium, CP1, identified as Pseudomonas putida strain, was investigated for its ability to grow on and degrade mono-chlorophenols and phenols as sole carbon sources in aerobic shaking batch culture. The organism degraded up to 1.56 mM 2- and 3-chlorophenol, 2.34 mM 4-chlorophenol and 8.5 mM phenol using an ortho-cleavage pathway. P. putida CP1, acclimated to degrade 2-chlorophenol, was capable of 3-chlorocatechol degradation, while P. putida, acclimated to 4-chlorophenol degradation, degraded 4-chlorocatechol. Growth of P. putida CP1 on higher concentrations of the mono-chlorophenols, ≥1.56 mM 4-chlorophenol and ≥0.78 mM 2- and 3-chlorophenol, resulted in decreases in cell biomass despite metabolism of the substrates, and the formation of large aggregates of cells in the culture medium. Increases in cell biomass with no clumping of the cells resulted from growth of P. putida CP1 on phenol or on lower concentrations of mono-chlorophenol. Bacterial adherence to hydrocarbons (BATH) assays showed cells grown on the higher concentrations of mono-chlorophenol to be more hydrophobic than those grown on phenol and lower concentrations of mono-chlorophenol. The results suggested that increased hydrophobicity and autoaggregation of P. putida CP1 were a response to toxicity of the added substrates. Journal of Industrial Microbiology & Biotechnology (2002) 28, 316–324 DOI: 10.1038/sj/jim/7000249 Received 27 June 2001/ Accepted in revised form 09 February 2002     

A novel membrane stirrer system enables foam-free biosurfactant production

Bioreactors are the operative backbone, for example, for the production of biopharmaceuticals, biomaterials in tissue engineering, and sustainable substitutes for chemicals. Still, the Achilles' heel of bioreactors nowadays is the aeration which is based on intense stirring and gas sparging, yielding inherent drawbacks such as shear stress, foaming, and sterility concerns. We present the synergistic combination of simulations and experiments toward a membrane stirrer for the efficient bubble-free aeration of bioreactors. A digital twin of the bioreactor with an integrated membrane-module stirrer (MemStir) was developed with computational fluid dynamics (CFD) studies addressing the determination of fluid mixing, shear rates, and local oxygen concentration. Usability of the MemStir is shown in a foam-free recombinant production process of biosurfactants (rhamnolipids) from glucose with different strains of Pseudomonas putida KT2440 in a 3-L vessel and benchmarked against a regular aerated process. The MemStir delivered a maximal oxygen transfer rate (OTR_max) of 175 mmol L⁻¹ h⁻¹ in completely foam-free cultivations. With a high space-time yield (STY) of 118 mg_RL L⁻¹ h⁻¹ during a fed-batch fermentation, the effectiveness of the novel MemStir is demonstrated. Simulations show the generic value of the MemStir beyond biosurfactant production, for example, for animal cell cultivation.