Potential of <em>Escherichia coli</em> Probiotics for Improved Health and Disease Management

Although natural gut microbiota containsEscherichia coli as a commensal, this bacterium, along with other members of the Enterobacteriaceae family, are usually known for their pathogenic potential. Interestingly, E. coli colonizes first and remains all through life, and in fact, some strains possess beneficial properties such as antibacterial colicin secretion. Among the beneficial strains, E. coli Nissle, isolated in 1917, has been the most extensively explored strain. Adaptability to survive under diverse conditions coupled with facile genetic manipulations enabled the design of E. coli strains with properties to deliver antioxidant, anti-inflammatory, and antitumor molecules. Moreover, genetically modified E. coli strains secreting enzymes for converting sucrose and fructose into insulin and mannitol, respectively, were very effective in preventing the onset of metabolic disease by acting as synbiotics. Thus, E. coli is emerging as a very potent probiotic platform for developing strains with the potential of controlling many metabolic and multifactorial diseases, including cancer

Functional expression and purification of Anabaena PCC 7120 XisA protein

Anabaena PCC 7120 xisA gene product mediates the site-specific excision of 11,278 bp nifD element in heterocysts formed under nitrogen starvation conditions. Although XisA protein possesses both site-specific recombinase and endonuclease activities, till date neither xisA transcript nor XisA protein has been detected. Gene encoding XisA protein was isolated from plasmid pMX25 and overexpressed in Escherichia coli BL21 DE3 yielding 7.7 mg enzyme per L of growth culture in soluble fraction. His-tagged XisA was purified using Ni-NTA affinity chromatography with 95% recovery. The purified XisA showed a single band on SDS-PAGE with molecular mass of 52 kDa. Identity of XisA was confirmed by MALDI-TOF analysis and functionality of enzyme was confirmed using restriction digestion. A PCR based method was developed to monitor excision by XisA, which displayed near 100% activity in E. coli within 1 h at 37 degrees C on LB under static condition. (C) 2015 Elsevier Inc. All rights reserved

Effect of <i>P. fluorescens yc</i> operon genomic integrants on citric acid accumulation, citric and gluconic acid secretion.

<p>Intracellular citric acid levels (A), extracellular citric (B) and gluconic (C) acid levels (mM) estimated from the stationary phase cultures of wild type (WT), plasmid transformants containing <i>yc</i> operon (pYC), <i>yc</i> operon genomic integrants (Int) of different <i>P. fluorescens</i> strain PfO-1, Pf5, CHAO1, ATCC13525 (13525), P109 and Fp315 grown in TRP minimal medium for 96–120 h. Results are expressed as mean ± S.E.M of 4–8 independent observations, † comparison of parameters with WT; ‡ comparison of parameters between plasmid transformants (pYC) and genomic integrants (Int). †††, ‡‡‡: P<0.001; ††,‡‡: P<0.01; †,‡: P<0.05.</p

Artificial Citrate Operon Confers Mineral Phosphate Solubilization Ability to Diverse Fluorescent Pseudomonads

<div><p>Citric acid is a strong acid with good cation chelating ability and can be very efficient in solubilizing mineral phosphates. Only a few phosphate solubilizing bacteria and fungi are known to secrete citric acids. In this work, we incorporated artificial citrate operon containing NADH insensitive citrate synthase (<i>gltA1</i>) and citrate transporter (<i>citC</i>) genes into the genome of six-plant growth promoting <i>P. fluorescens</i> strains <i>viz</i>., PfO-1, Pf5, CHAO1, P109, ATCC13525 and Fp315 using MiniTn7 transposon gene delivery system. Comprehensive biochemical characterization of the genomic integrants and their comparison with plasmid transformants of the same operon in M9 minimal medium reveals the highest amount of ∼7.6±0.41 mM citric and 29.95±2.8 mM gluconic acid secretion along with ∼43.2±3.24 mM intracellular citrate without affecting the growth of these <i>P. fluorescens</i> strains. All genomic integrants showed enhanced citric and gluconic acid secretion on Tris-Cl rock phosphate (TRP) buffered medium, which was sufficient to release 200–1000 µM Pi in TRP medium. This study demonstrates that MPS ability could be achieved in natural fluorescent pseudomonads by incorporation of artificial citrate operon not only as plasmid but also by genomic integration.</p></div

Activities of key glucose catabolic enzymes in <i>P. fluorescens</i> strains wild type (WT), plasmid transformants harbouring <i>yc</i> operon, Int (genomic integrants of <i>yc</i> operon) grown in TRP broth (pH 8.0) minimal media.

a<p>Activities are expressed as nmoles/min/mg total protein from mid-log to stationary phase cultures and are given as mean±SEM of readings from four independent observations.</p>†<p>Comparison of parameters with WT, ‡ comparison of parameters between pYC and Int. †††, ‡‡‡: P<0.001; ††, ‡‡: P<0.01; †, ‡: P<0.05.</p><p>Activities of key glucose catabolic enzymes in <i>P. fluorescens</i> strains wild type (WT), plasmid transformants harbouring <i>yc</i> operon, Int (genomic integrants of <i>yc</i> operon) grown in TRP broth (pH 8.0) minimal media.</p

Phosphate solubilisation activities of <i>P. fluorescens yc</i> operon genomic integrants and plasmid transformants.

<p>Zone of clearance and PSI (Phosphate solubilisation index) (A and B) in Pikovskaya's agar medium during 96–120 h of growth. Zone of colouration (C) and Pi release (D) of transgenic <i>P. fluorescens</i> strains in TRP broth and TRP agar medium during 96–120 h of growth. WT: wild type strain; pYC: <i>P. fluorescens</i> with pYC plasmid; Int: <i>P. fluorescens yc</i> operon genomic integrant. Results are expressed as Mean ±S.E.M of 4 independent observations. * Comparison of parameters with wild type control; ‡ comparison between parameters of plasmid transformants (pYC) and genomic integrants (int).***, ‡‡‡: P<0.001; **,‡‡: P<0.01; *,‡: P<0.05.</p

Organic acid profile and enzyme activities of <i>P. fluorescens</i> PfO-1 transformants.

<p>Activities of key enzymes (A) using stationary phase cultures grown on M9 medium with 100 mM glucose were represented in the units of nmoles/min/mg total protein. Intracellular and extracellular citric acid levels (B) are represented in grey bars and black bars respectively. Gluconic-pyruvic-acetic acid levels (C) and citric acid levels were estimated from stationary phase cultures grown on same media. Results are expressed as Mean ±S.E.M of 4 independent observations. WT is native untransformed strain, Gm indicates vector control containing pUCPM18Gm, Km indicates vector control containing pAB8 and YC indicates tansformant with artificial citrate operon pYC; KG indicates vector control with both the pUCPM18 and pAB8 plasmids cotransfromed; YF indicates transformant with pY145F; YFCitC and YFCitM indicate cotransformants carrying plasmid pY145F along with pCitC and pCitM, respectively. * comparison of parameters with wild type control; comparison of parameters with vector control pAB8, # comparison between parameters of AB7 and YF.***, $$,###: P<0.001; **,$$,##: P<0.01; *,$,#: P<0.05.</p