Glutamine-induced filamentous cells of Pseudomonas mediterranea CFBP-5447T as producers of PHAs.

Polyhydroxyalkanoates (PHAs) are considerable biopolymers that have gained an increasing biotechnological interest in different applications, although their industrial production presents several limitations. Filamentous bacterial cells could represent a possible strategy to increase PHA yield, since more abundant PHA inclusions can be stored in elongated than in rod-shaped cells. At first, we determined the optimal batch culture conditions to induce filamentation in Pseudomonas mediterranea CFBP-5447T, using glutamine, glycerol, glucose, and sodium octanoate, as the sole carbon source, at low- (100 rpm) or high- (250 rpm) shaking speeds. Successively, a fermentative process was set up using glutamine in a co-metabolic strategy with glycerol, and the PHAs production was compared in rod-shaped and filamentous cells. High glutamine concentrations (from 28 to 56 mM) were able to induce alone filamentation, whereas at lower glutamine concentrations (5-10 mM), the shaking speeds became critical to allow or not filamentous phenotype. PHA granule production was higher in filamentous than in rod-shaped cells, when glycerol (46.6 mM) was added to glutamine (5 mM) in co-metabolism, and fermentation was performed at a low-shaking speed. After extraction and precipitation, PHA yield was about two times higher in filamentous than that rod-shaped cells. Our results provide new insights into filament-inducing conditions and indicate a potential use of filamentous P. mediterranea CFBP-5447T cells to increase PHA yield. These findings could have great advantages in PHAs recovering during downstream processes, since the harvesting of elongated cells is much less time-consuming and energy expensive than required with rod-shaped cells.

Biosynthesis and structural characterization of polyhydroxyalkanoates produced by Pseudomonas aeruginosa ATCC 27853 from long odd-chain fatty acids.

Pseudomonas aeruginosa ATCC 27853 was cultured on media containing long odd-chain fatty acids. Heptadecanoic, nonadecanoic, and heneicosanoic acids sustained cell growth and resulted in polyhydroxyalkanoate (PHA) accumulation when culturing was conducted under nitrogen starvation conditions. No PHA was produced using a complete or magnesium-deprived medium. The isolated polyesters were characterized by gas chromatography and liquid chromatography-electrospray ionization mass spectrometry (ESI-MS) of methanolyzed samples, 1H and 13C NMR spectroscopy, gel permeation chromatography, ESI MS of partially pyrolyzed samples, and differential scanning calorimetry. These PHAs are composed of seven different odd-chain repeating units starting from 3-hydroxyvalerate, with the highest species being the, to date, unreported constituent 3-hydroxyheptadecanoate, and minor amounts of 2 or 3 even-chain comonomers. The PHAs are soft, sticky, rubber-like materials having glass transition temperatures between -45 and -39°C, melting temperatures between 48 and 52°C, enthalpies of melting around 11J/g, and molar masses ranging from 77 to 188kg/mol. Statistical analysis of the ESI mass spectra of the products of their partial pyrolysis showed that they are pure copolymers and not a blend of copolymers or homopolymers.

Carbon source effects on the mono/dirhamnolipid ratio produced by Pseudomonas aeruginosa L05, a new human respiratory isolate.

Pseudomonas strains produce rhamnolipid mixtures (RLs) that generally consist of one or two molecules of rhamnose linked to one or two molecules of 3-hydroxyalkanoic acid. This study evaluates carbon source effects (glycerol, glucose, myristic acid, and Brassica carinata oil) on the synthesis of monorhamnolipids (mono-RLs) versus dirhamnolipids (di-RLs) in a human isolate of Pseudomonas aeruginosa PAL05. Spectrophotometry, an emulsifying index (E24) test, and an orcinol assay confirmed the production of RLs by PAL05. Purified RLs were characterized by 1H NMR analysis. PAL05 primarily produces mono-RLs when provided carbon sources containing long chain fatty acids (FAs) (myristic acid and B. carinata oil) and di-RLs when provided glycerol or glucose. qRT-PCR analysis showed that delayed expression of rhlC occurred when B. carinata oil was used, but not glycerol, glucose, or myristic acid. Our data show that the carbon source influenced the transcriptional expression of the rhlC gene and, consequently, the predominance of mono-RLs or di-RLs in PAL05 cultures.

Raman spectroscopy differentiates between sensitive and resistant multiple myeloma cell lines.

Current methods for identifying neoplastic cells and discerning them from their normal counterparts are often nonspecific and biologically perturbing. Here, we show that single-cell micro-Raman spectroscopy can be used to discriminate between resistant and sensitive multiple myeloma cell lines based on their highly reproducible biomolecular spectral signatures. In order to demonstrate robustness of the proposed approach, we used two different cell lines of multiple myeloma, namely MM.1S and U266B1, and their counterparts MM.1R and U266/BTZ-R subtypes, resistant to dexamethasone and bortezomib, respectively. Then, micro-Raman spectroscopy provides an easily accurate and noninvasive method for cancer detection for both research and clinical environments. Characteristic peaks, mostly due to different DNA/RNA ratio, nucleic acids, lipids and protein concentrations, allow for discerning the sensitive and resistant subtypes. We also explored principal component analysis (PCA) for resistant cell identification and classification. Sensitive and resistant cells form distinct clusters that can be defined using just two principal components. The identification of drug-resistant cells by confocal micro-Raman spectroscopy is thus proposed as a clinical tool to assess the development of resistance to glucocorticoids and proteasome inhibitors in myeloma cells.

The role of glutamine in Pseudomonas mediterranea in biotechnological processes.

In this work, in order to study the effect of glutamine as co-feeder on growth kinetics, biomass and PHA production in Pseudomonas mediterranea, different co-metabolic strategies were employed. Unrelated (glycerol and glucose) and related (sodium octanoate) carbon sources both in presence and absence of glutamine have been tested. For each cultural condition, we (i) evaluated growth kinetics and measured the cell metabolic activity by MTT assay, (ii) monitored PHA production and (iii) estimated the expression of phaC1 and phaC2 genes through RT-PCR. Our results show that the use of glutamine as co-feeder in P. mediterranea led to an improvement of the specific growth rate and cell metabolic activity and enhanced the uptake of all the carbon sources assayed. Moreover, the use of glutamine reduced significantly the time required for PHA production and increased biopolymer yield, as consequence of an early activation of phaC1 and phaC2.

Phage-AgNPs complex as SERS probe for U937 cell identification.

The early diagnosis of malignancy is the most critical factor for patient survival and the treatment of cancer. In particular, leukemic cells are highly heterogeneous, and there is a need to develop new rapid and accurate detection systems for early diagnosis and monitoring of minimal residual disease. This study reports the utilization of molecular networks consisting of entire bacteriophage structure, displaying specific peptides, directly assembled with silver nanoparticles as a new Surface Enhanced Raman Spectroscopy (SERS) probe for U937 cells identification in vitro. A 9-mer pVIII M13 phage display library is screened against U937 to identify peptides that selectively recognize these cells. Then, phage clone is assembled with silver nanoparticles and the resulting network is used to obtain a SERS signal on cell-type specific molecular targets. The proposed strategy could be a very sensitive tool for the design of biosensors for highly specific and selective identification of hematological cancer cells and for detection of minimal residual disease in a significant proportion of human blood malignancy.