Project description:To undestand the mechanism involved in abitoic stress tolerance of P. Putida (NBAII-RPF 9) the bacterium was grown in liquid LB media overnight and further subjected to saline shock of (1M NaCl) for one hour seperately. The cultures were pelleted with centrifuged for total RNA. The RNA was hybridised in 8X15K Agilent array and image analysis carried out with Agilent Microarray scanner.
Project description:To undestand the mechanism involved in abitoic stress tolerance of P. Putida (NBAII-RPF 9) the bacterium was grown in liquid LB media overnight and further subjected to heat shock at 45 degrees in incubated shaker for one hour seperately. The cultures were pelleted with centrifuged for total RNA. The RNA was hybridised in 8X15K Agilent array and image analysis carried out with Agilent Microarray scanner
Project description:The metabolically versatile Pseudomonas putida strain KT2440 is the first Gram-negative soil bacterium certified as a biosafety strain and is being used for applications in agriculture, biotechnology and bioremediation. P. putida has to cope in its niche with numerous abiotic stresses. The stress response to 4°C, pH 4.5, 0.8 M urea or 45 mM sodium benzoate, respectively, was analyzed by the global mRNA expression profile and screening for stress-intolerant Tn5 transposon mutants. In total we identified 49 gene regions to be differentially expressed and 32 genes in 22 operons to be indispensable for growth during exposure to one or the other abiotic stresses. We propose that stress is sensed by the outer membrane proteins OmlA and FepA and the inner membrane constituents PtsP, PhoPQ and CbrAB. The metabolic response is regulated by the cyo operon, the RelA/SpoT modulon, PcnB and VacB that control mRNA stability and BipA that exerts transcript-specific translational control. The adaptation of the membrane barrier, the uptake of phosphate, the maintenance of intracellular pH and redox status and the translational control of metabolism are the indispensable key mechanisms of the P. putida stress response. Keywords: functional genomics
Project description:Alginate, a major exopolysaccharide (EPS) produced by P. putida, is known to create hydrated environments and alleviate the effect of water limitation. In addition to alginate, P. putida is capable of producing cellulose (bcs), putida exopolysaccharide a (pea), and putida exopolysaccharide b (peb). However, unlike alginate, not much is known about their roles under water limitation. Hence, in this study we examined the role of different EPS under water stress. To create environmentally realistic water stress conditions as observed in soil, we used Pressurized Porous Surface Model (PPSM). Our main hypothesis was that under water stress, absence of alginate would be compensated by the other EPS. To test our hypothesis, we investigated colony morphologies and whole genome transcriptomes of P. putida KT2440 WT and its mutants deficient in either alginate or all known EPS A custom-made Nimblegen (WI, USA) whole genome one-color oligonucleotide expression array (12x135K with 45-60 mer probes) of P. putida KT2440 was used to investigate effect of water stress on the differential expression of the whole genome. In this study Pseudomonas putida KT2440 wild type (WT) and two of its mutants deficient either in alginate (Alg-), or all known EPS (EPS-) production were used and grown under dry (water stress) and wet (without water stress) conditions. (Deleted genes in Alg-: PP1277-PP128; in EPS-: PP1277-1288 (alg) + PP2634-2638 (bcs) + PP3132-3142 (pea) + PP1795-1788 (peb)) (Nilsson et al., 2011).39. Nilsson, M., Chiang, W.C., Fazli, M., Gjermansen, M., Givskov, M., and Tolker-Nielsen, T. (2011) Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability. Environ Microbiol. 13 (5):1 357-1369
Project description:KaiC is the central cog of the circadian clock in Cyanobacteria. Close homologs of this protein are widespread among bacteria not known to have a circadian physiology. The function, interaction network, and mechanism of action of these KaiC homologs are still largely unknown. Here, we focus on KaiC homologs found in environmental Pseudomonas species. We characterize experimentally the only KaiC homolog present in Pseudomonas putida KT2440 and Pseudomonas protegens CHA0. Through phenotypic assays and transcriptomics, we show that KaiC is involved in osmotic and oxidative stress resistance in P. putida and in biofilm production in both P. putida and P. protegens.
Project description:Sohn2010 - Genome-scale metabolic network of
Pseudomonas putida (PpuMBEL1071)
This model is described in the article:
In silico genome-scale
metabolic analysis of Pseudomonas putida KT2440 for
polyhydroxyalkanoate synthesis, degradation of aromatics and
anaerobic survival.
Sohn SB, Kim TY, Park JM, Lee
SY.
Biotechnol J 2010 Jul; 5(7):
739-750
Abstract:
Genome-scale metabolic models have been appearing with
increasing frequency and have been employed in a wide range of
biotechnological applications as well as in biological studies.
With the metabolic model as a platform, engineering strategies
have become more systematic and focused, unlike the random
shotgun approach used in the past. Here we present the
genome-scale metabolic model of the versatile Gram-negative
bacterium Pseudomonas putida, which has gained widespread
interest for various biotechnological applications. With the
construction of the genome-scale metabolic model of P. putida
KT2440, PpuMBEL1071, we investigated various characteristics of
P. putida, such as its capacity for synthesizing
polyhydroxyalkanoates (PHA) and degrading aromatics. Although
P. putida has been characterized as a strict aerobic bacterium,
the physiological characteristics required to achieve anaerobic
survival were investigated. Through analysis of PpuMBEL1071,
extended survival of P. putida under anaerobic stress was
achieved by introducing the ackA gene from Pseudomonas
aeruginosa and Escherichia coli.
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Project description:Alginate, a major exopolysaccharide (EPS) produced by P. putida, is known to create hydrated environments and alleviate the effect of water limitation. In addition to alginate, P. putida is capable of producing cellulose (bcs), putida exopolysaccharide a (pea), and putida exopolysaccharide b (peb). However, unlike alginate, not much is known about their roles under water limitation. Hence, in this study we examined the role of different EPS under water stress. To create environmentally realistic water stress conditions as observed in soil, we used Pressurized Porous Surface Model (PPSM). Our main hypothesis was that under water stress, absence of alginate would be compensated by the other EPS. To test our hypothesis, we investigated colony morphologies and whole genome transcriptomes of P. putida KT2440 WT and its mutants deficient in either alginate or all known EPS