Qun Ren

Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide.

BackgroundViability staining with SYTO9 and propidium iodide (PI) is a frequently used tool in microbiological studies. However, data generated by such routinely used method are often not critically evaluated for their accuracy. In this study we aim to investigate the critical aspects of this staining method using Staphylococcus aureus and Pseudomonas aeruginosa as the model microorganisms for high throughput studies in microtiter plates. SYTO9 or PI was added alone or consecutively together to cells and the fluorescence intensities were measured using microplate reader and confocal laser scanning microscope.ResultsWe found that staining of S. aureus cells with SYTO9 alone resulted in equal signal intensity for both live and dead cells, whereas staining of P. aeruginosa cells led to 18-fold stronger signal strength for dead cells than for live ones. After counterstaining with PI, the dead P. aeruginosa cells still exhibited stronger SYTO9 signal than the live cells. We also observed that SYTO9 signal showed strong bleaching effect and decreased dramatically over time. PI intensity of the culture increased linearly with the increase of dead cell numbers, however, the maximum intensities were rather weak compared to SYTO9 and background values. Thus, slight inaccuracy in measurement of PI signal could have significant effect on the outcome.ConclusionsWhen viability staining with SYTO9 and PI is performed, several factors need to be considered such as the bleaching effect of SYTO9, different binding affinity of SYTO9 to live and dead cells and background fluorescence.

Enatiomerically pure hydroxycarboxylic acids: current approaches and future perspectives.

The growing awareness of the importance of chirality in conjunction with biological activity has led to an increasing demand for efficient methods for the industrial synthesis of enantiomerically pure compounds. Polyhydroxyalkanotes (PHAs) are a family of polyesters consisting of over 140 chiral R-hydroxycarboxylic acids (R-HAs), representing a promising source for obtaining chiral chemicals from renewable carbon sources. Although some R-HAs have been produced for some time and certain knowledge of the production processes has been gained, large-scale production has not yet been possible. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in biotechnological processes for R-HA production.

Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440

BackgroundPseudomonas putida KT2440 is able to synthesize large amounts of medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To reduce the substrate cost, which represents nearly 50% of the total PHA production cost, xylose, a hemicellulose derivate, was tested as the growth carbon source in an engineered P. putida KT2440 strain.ResultsThe genes encoding xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli W3110 were introduced into P. putida KT2440. The recombinant KT2440 exhibited a XylA activity of 1.47 U and a XylB activity of 0.97 U when grown on a defined medium supplemented with xylose. The cells reached a maximum specific growth rate of 0.24 h-1 and a final cell dry weight (CDW) of 2.5 g L-1 with a maximal yield of 0.5 g CDW g-1 xylose. Since no mcl-PHA was accumulated from xylose, mcl-PHA production can be controlled by the addition of fatty acids leading to tailor-made PHA compositions. Sequential feeding strategy was applied using xylose as the growth substrate and octanoic acid as the precursor for mcl-PHA production. In this way, up to 20% w w-1 of mcl-PHA was obtained. A yield of 0.37 g mcl-PHA per g octanoic acid was achieved under the employed conditions.ConclusionsSequential feeding of relatively cheap carbohydrates and expensive fatty acids is a practical way to achieve more cost-effective mcl-PHA production. This study is the first reported attempt to produce mcl-PHA by using xylose as the growth substrate. Further process optimizations to achieve higher cell density and higher productivity of mcl-PHA should be investigated. These scientific exercises will undoubtedly contribute to the economic feasibility of mcl-PHA production from renewable feedstock.

Is biofilm removal properly assessed? Comparison of different quantification methods in a 96-well plate system

Various methods have been reported to quantify total biofilm or different components of biofilm; however, these methods are often confusedly used, leading to discrepancies and misleading results. In this study, different methods for quantification of biofilm, including those for total biomass, total amount of bacterial cells, viable cell number, and amount of extracellular polymeric substances, were systematically compared in microtiter plates. To evaluate which method is suitable for assessment of biofilm removal and for bacterial killing, biofilm samples were treated with various cleaners possessing removing and/or killing capacities. It was found that most of the methods tested in this study in general exhibited high reproducibility and repeatability. Crystal Violet staining was a simple but reliable method for total biomass quantification. Total bacteria cell numbers could be reliably quantified by the fluorescent DNA-binding dye Acridine Orange. Viable cells could be quantified by either an ATP-based assay or a proliferation assay. Both of these viability methods showed a broad detection range and led to precise measurement. For quantification of proteins in the biofilm, staining with fluorescein isothiocyanate was most suitable. Furthermore, it was revealed that a combination of different methods is required to determine if a cleaner kills or removes biofilm.

Influence of growth stage on activities of polyhydroxyalkanoate (PHA) polymerase and PHA depolymerase in Pseudomonas putida U

BackgroundMedium chain length (mcl-) polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA metabolism are PHA polymerase (PhaC) and depolymerase (PhaZ). Little is known of how mcl-PHA accumulation and degradation are controlled. It has been suggested that overall PHA metabolism is regulated by the β-oxidation pathway of which the flux is governed by intracellular ratios of [NADH]/[NAD] and [acetyl-CoA]/[CoA]. Another level of control could relate to modulation of the activities of PhaC and PhaZ. In order to investigate the latter, assays for in vitro activity measurements of PhaC and PhaZ in crude cell extracts are necessary.ResultsTwo in vitro assays were developed which allow the measurement of PhaC and PhaZ activities in crude cell extracts of Pseudomonas putida U. Using the assays, it was demonstrated that the activity of PhaC decreased 5-fold upon exponential growth on nitrogen limited medium and octanoate. In contrast, the activity of PhaZ increased only 1.5-fold during growth. One reason for the changes in the enzymatic activity of PhaC and PhaZ could relate to a change in interaction with the phasin surface proteins on the PHA granule. SDS-PAGE analysis of isolated PHA granules demonstrated that during growth, the ratio of [phasins]/[PHA] decreased. In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further.ConclusionUsing the assays developed in this study, we followed the enzymatic activities of PhaC and PhaZ during growth and correlated them to the amount of phasins on the PHA granules. It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA. Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth. Availability of phasins on PHA granules affected the activity of PhaC.

High level production of tyrosinase in recombinant Escherichia coli

BackgroundTyrosinase is a bifunctional enzyme that catalyzes both the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the subsequent oxidation of the diphenols to o-quinones (diphenolase activity). Due to the potential applications of tyrosinase in biotechnology, in particular in biocatalysis and for biosensors, it is desirable to develop a suitable low-cost process for efficient production of this enzyme. So far, the best production yield reported for tyrosinase was about 1 g L-1, which was achieved by cultivating the filamentous fungus Trichoderma reesei for 6 days.ResultsIn this work, tyrosinase from Verrucomicrobium spinosum was expressed in Escherichia coli and its production was studied in both batch and fed-batch cultivations. Effects of various key cultivation parameters on tyrosinase production were first examined in batch cultures to identify optimal conditions. It was found that a culture temperature of 32 °C and induction at the late growth stage were favorable, leading to a highest tyrosinase activity of 0.76 U mL-1. The fed-batch process was performed by using an exponential feeding strategy to achieve high cell density. With the fed-batch process, a final biomass concentration of 37 g L-1 (based on optical density) and a tyrosinase activity of 13 U mL-1 were obtained in 28 hours, leading to a yield of active tyrosinase of about 3 g L-1. The highest overall volumetric productivity of 103 mg of active tyrosinase per liter and hour (corresponding to 464 mU L-1 h-1) was determined, which is approximately 15 times higher than that obtained in batch cultures.ConclusionsWe have successfully expressed and produced gram quantities per liter of active tyrosinase in recombinant E. coli by optimizing the expression conditions and fed-batch cultivation strategy. Exponential feed of substrate helped to prolong the exponential phase of growth, to reduce the fermentation time and thus the cost. A specific tyrosinase production rate of 103 mg L−1 h−1 and a maximum volumetric activity of 464 mU L−1 h-1 were achieved in this study. These levels have not been reported previously.

Growth and accumulation dynamics of poly(3-hydroxyalkanoate) (PHA) in Pseudomonas putida GPo1 cultivated in continuous culture under transient feed conditions.

It has been shown that Pseudomonas putida GPo1 is able to grow in continuous culture simultaneously limited by ammonium (N source) and octanoate (C source), and concomitantly accumulate poly([R]-3-hydroxyalkanoate) (PHA). Under such growth conditions the material properties of PHA can be fine-tuned if a second PHA precursor substrate is supplied. To determine the range of dual carbon and nitrogen (C, N)-limited growth conditions, tedious chemostat experiments need to be carried out for each carbon source separately. To determine the growth regime, the C/N ratio of the feed (f) to a chemostat was changed in a stepwise manner at a constant dilution rate of 0.3/h. Dual-(C, N)-limited growth was observed between C(f) /N(f) ≤ 6.4 g/g and C(f) /N(f) >9.5 g/g. In the following, we analyzed alternative approaches, using continuous medium gradients at the same dilution rate, that do not require time consuming establishments of steady states. Different dynamic approaches were selected in which the C(f) /N(f) ratio was changed continuously through a convex increase of C(f) , a convex increase of N(f) , or a linear decrease of C(f) (gradients 1, 2, and 3, respectively). In these experiments, the dual-(C, N)-limited growth regime was between 7.2 and 11.0 g/g for gradient 1, 4.3 and 6.9 g/g for gradient 2, and 5.1 and 8.9 g/g for gradient 3. A mathematical equation was developed that compensated a time delay of the gradient that was caused by the wash-in/wash-out effects of the medium feed.

Antimicrobial Peptide-Driven Colloidal Transformations in Liquid-Crystalline Nanocarriers.

Designing efficient colloidal systems for the delivery of membrane active antimicrobial peptides requires in-depth understanding of their structural and morphological characteristics. Using dispersions of inverted type bicontinuous cubic phase (cubosomes), we examine the effect of integrating the amphiphilic peptide LL-37 at different concentrations on the self-assembled structure and evaluate its bactericidal ability against Escherichia coli. Small-angle X-ray scattering, dynamic light scattering, and cryogenic transmission electron microscopy show that LL-37 integrates into the bicontinuous cubic structure, inducing colloidal transformations to sponge and lamellar phases and micelles in a concentration-dependent manner. These investigations, together with in vitro evaluation studies using a clinically relevant bacterial strain, established the composition-nanostructure-activity relationship that can guide the design of new nanocarriers for antimicrobial peptides and may provide essential knowledge on the mechanisms underlying the bacterial membrane disruption with peptide-loaded nanostructures.

Expression of PHA polymerase genes of Pseudomonas putida in Escherichia coli and its effect on PHA formation

Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applied.

Improved productivity of poly (4-hydroxybutyrate) (P4HB) in recombinant Escherichia coli using glycerol as the growth substrate with fed-batch culture

BackgroundThe most successful polyhydroxyalkanoate (PHA) in medical applications is poly(4-hydroxybutyrate) (P4HB), which is due to its biodegradability, biocompatibility and mechanical properties. One of the major obstacles for wider applications of P4HB is the cost of production and purification. It is highly desired to obtain P4HB in large scale at a competitive cost.ResultsIn this work, we studied the possibility to increase P4HB productivity by using high cell density culture. To do so, we investigated for the first time some of the most relevant factors influencing P4HB biosynthesis in recombinant Escherichia coli. We observed that P4HB biosynthesis correlated more with limitations of amino acids and less with nitrogen depletion, contrary to the synthesis of many other types of PHAs. Furthermore, it was found that using glycerol as the primary carbon source, addition of acetic acid at the beginning of a batch culture stimulated P4HB accumulation in E. coli. Fed-batch high cell density cultures were performed to reach high P4HB productivity using glycerol as the sole carbon source for cell growth and 4HB as the precursor for P4HB synthesis. A P4HB yield of 15gL-1 was obtained using an exponential feeding mode, leading to a productivity of 0.207gL-1h-1, which is the highest productivity for P4HB reported so far.ConclusionsWe demonstrated that the NZ-amines (amino acids source) in excess abolished P4HB accumulation, suggesting that limitation in certain amino acid pools promotes P4HB synthesis. Furthermore, the enhanced P4HB yield could be achieved by both the effective growth of E. coli JM109 (pKSSE5.3) on glycerol and the stimulated P4HB synthesis via exogenous addition of acetic acid. We have developed fermentation strategies for P4HB production by using glycerol, leading to a productivity of 0.207gL-1h-1 P4HB. This high P4HB productivity will decrease the total production cost, allowing further development of P4HB applications.