Broad-host-range plasmid-mediated metabolic perturbations in Pseudomonas fluorescens 13525.
Genetic engineering of fluorescent pseudomonads for various industrially, agriculturally and environmentally important bioprocesses often involves the use of suitable plasmids. Plasmid-mediated alterations in host physiology and metabolism are poorly understood for this group of organisms. Thus, we investigated the metabolic perturbations in Pseudomonas fluorescens 13525 due to the independent and combined presence of broad-host-range plasmids, pBBR1MCS-2 (copy number 30) and pUCPM18 derived pAB4 and pAB8 (copy number 14-16). Presence of pAB4 and pAB8 not only significantly increased the growth rate and glucose utilization of P. fluorescens 13525, but also increased glucose dehydrogenase activity and gluconic acid production indicating enhanced direct oxidative pathway for glucose catabolism. Additionally, increased secretion of pyruvic, acetic, and citric acids caused faster media acidification in presence of pAB4 and pAB8. Simultaneous presence of pAB4/pAB8 in Pf (pAB48) and pAB4/pBBR1MCS-2 in Pf (pAB4BBR1MCS-2) reduced their respective copy numbers to nearly half. Pf (pAB48) demonstrated further increase in direct oxidation pathway without altering growth and glucose depletion rates, as compared with single transformants. Conversely, pBBR1MCS-2 plasmid did not greatly alter P. fluorescens 13525 metabolism when present independently but masked the effects imposed by pAB4 when present in its combination. In conclusion, P. fluorescens 13525 redesigns its metabolism in response to the presence of plasmids irrespective of their nature, by enhancing anaplerosis with a simultaneous reduction in catabolism as indicated by increased pyruvate carboxylase and decreased citrate synthase activities, respectively. Such information will be helpful for vector designing during genetic engineering of fluorescent pseudomonads.
Buch
Aditi D
AD
Molecular Microbial Biochemistry Laboratory, Faculty of Science, Department of Biochemistry, The Maharaja Sayajirao University of Baroda, Vadodara, 390 002, Gujarat, India.
Archana
G
G
Naresh Kumar
G
G
D019342:Acetic Acid / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D019343:Citric Acid / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D005942:Gluconates / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids; D011551:Pseudomonas fluorescens / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism
D001426:Bacterial Proteins; D003470:Culture Media; D005942:Gluconates; D019343:Citric Acid; D019289:Pyruvic Acid; D042843:Glucose 1-Dehydrogenase; D005947:Glucose; D019342:Acetic Acid; C030691:gluconic acid; D010100:Oxygen