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:Our first aim was understand whole genome expression dynamics of Pseudomonas putida KT2440 surface colonies under water stress. Therefore, we wanted to identify significantly differentially expressed genes of at -0.4 MPa matric potential relative to the -0.5 kPa (near saturation condition) at different stress durations. Aliquots of overnight grown P. putida KT2440 cells were inoculated (approx. 100 µl and 1x107 cells) on the surface of ceramic plates. Prior to matric stress, the cells were incubated for 120, 116, 96, and 48 hr at-0.5 kPa to allow formation of a mature cell loan. -0.4 MPa matric potential was applied for the last 0 (control), 4, 24, and 72 hr of incubation periods, respectively, so that the total incubation time (at (-0.5 kPa) and (-0.4 MPa)) was kept constant as 5 days. Our second aim was to find out how the gene expression profile would change when we use PEG-8000 to simulate matric stress. To that effect, similar incubations were performed where the direct matric potential was -0.5 kPa, but supplemented with PEG-8000 to set a matric potential equivalent of -0.5 or -1.0 MPa. Incubations were again 5 days. In addition, we tested the effect of gas pressure on gene expression profile of cells in liquid medium. 20 ml of overnight grown KT2440 cells were exposed to 0.4 MPa (gauge) and 0.1 MPa (atmospheric) pressures.
Project description:Our first aim was understand whole genome expression dynamics of Pseudomonas putida KT2440 surface colonies under water stress. Therefore, we wanted to identify significantly differentially expressed genes of at -0.4 MPa matric potential relative to the -0.5 kPa (near saturation condition) at different stress durations. Aliquots of overnight grown P. putida KT2440 cells were inoculated (approx. 100 µl and 1x107 cells) on the surface of ceramic plates. Prior to matric stress, the cells were incubated for 120, 116, 96, and 48 hr at-0.5 kPa to allow formation of a mature cell loan. -0.4 MPa matric potential was applied for the last 0 (control), 4, 24, and 72 hr of incubation periods, respectively, so that the total incubation time (at (-0.5 kPa) and (-0.4 MPa)) was kept constant as 5 days. Our second aim was to find out how the gene expression profile would change when we use PEG-8000 to simulate matric stress. To that effect, similar incubations were performed where the direct matric potential was -0.5 kPa, but supplemented with PEG-8000 to set a matric potential equivalent of -0.5 or -1.0 MPa. Incubations were again 5 days. In addition, we tested the effect of gas pressure on gene expression profile of cells in liquid medium. 20 ml of overnight grown KT2440 cells were exposed to 0.4 MPa (gauge) and 0.1 MPa (atmospheric) pressures. Matric Stress Experiments: we had 3 to 4 replicates for each time point and controls (-0.5 kPa). These were kept under -0.4 MPa stress at 4 hr (P4), 24 hr (P24), 72 hr (P72), and controls kept at -0.5 kPa (C).
Project description:Gene expression patterns of the plant colonizing bacterium,Pseudomonas putida KT2440 were evaluated as a function of growth in the Arabidopsis thaliana rhizosphere. Gene expression in rhizosphere grown P. putida cells was compared to gene expression in non-rhizosphere grown cells. Keywords: Gene expression
Project description:Genome-wide scanning of gene expression by microarray techniques was successfully performed on RNA extracted from a sterilized soil inoculated with Pseudomonas putida KT2440/pSL1, which contains a chloroaromatic degrading plasmid, in the presence or absence of 3-chlorobenzoic acid (3CB). The genes showing significant changes in their expression in both triplicate microarray analyses using amplified RNA and single microarray analysis using unamplified RNA were investigated. Pathway analysis revealed that the benzoate degradation pathway underwent the most significant changes following treatment with 3CB. Analysis based on categorization of differentially expressed genes against 3CB revealed new findings about the cellular responses of the bacteria to 3CB, including upregulation of the genes specifically involved in transport of 3CB, and induction of a K+/H+ antiporter complex, an universal stress protein, two cytochrome P450 proteins and an efflux transporter. Downregulated expression of some genes involved in carbon metabolism and the genes belong to a prophage in the presence of 3CB was observed. This study demonstrated the applicability of the method of soil RNA extraction for microarray analysis through a proof-of-concept experiment using a sterilized soil inoculated with Pseudomonas putida KT2440/pSL1. A study using total RNA extracted from soil cultures of Pseudomonas putida KT2440/pSL1. Each chip measures the expression level of 5,341 genes from the Pseudomonas putida KT2440 genome with two sets of six 60-mer probes per gene.
Project description:The bacterium Pseudomonas putida KT2440 has the ability to reduce selenite forming nanoparticles of elemental selenium. This is the transcriptome of the organism when cultured in the presence of selenite.
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:Transcriptome profiling of Pseudomonas putida KT2440 comparing cells exposed for 1 hour to DIMBOA from maize (Zea mays) to unexposed cells