Article

The polyhydroxyalkanoate metabolism controls carbon and energy spillage in Pseudomonas putida

Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid, Spain.
Environmental Microbiology (Impact Factor: 6.24). 01/2012; 14(4):1049-63. DOI: 10.1111/j.1462-2920.2011.02684.x
Source: PubMed

ABSTRACT The synthesis and degradation of polyhydroxyalkanoates (PHAs), the storage polymer of many bacteria, is linked to the operation of central carbon metabolism. To rationalize the impact of PHA accumulation on central carbon metabolism of the prototype bacterium Pseudomonas putida, we have revisited PHA production in quantitative physiology experiments in the wild-type strain vs. a PHA negative mutant growing under low nitrogen conditions. When octanoic acid was used as PHA precursor and as carbon and energy source, we have detected higher intracellular flux via acetyl-CoA in the mutant strain than in the wild type, which correlates with the stimulation of the TCA cycle and glyoxylate shunt observed on the transcriptional level. The mutant defective in carbon and energy storage spills the additional resources, releasing CO(2) instead of generating biomass. Hence, P. putida operates the metabolic network to optimally exploit available resources and channels excess carbon and energy to storage via PHA, without compromising growth. These findings demonstrate that the PHA metabolism plays a critical role in synchronizing global metabolism to availability of resources in PHA-producing microorganisms.

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    • "A wide variety of microorganisms accumulate PHAs as a carbon and energy storage material in carbon-excess conditions when a major nutrient (typically nitrogen or phosphorus) is limiting (Elbahloul and Steinbüchel 2009; Rehm 2010; Brandl et al. 1988). Accumulation of excess carbon is a general mechanism used by Pseudomonas and is essential for resource balancing (de Eugenio et al. 2010a; Escapa et al. 2012). De Smet et al. (1983) detected the inclusion bodies in Pseudomonas olevorans for first time when grown on octane and identified is as polymer of 3-hydroxyoctanoate. "
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    ABSTRACT: A novel strain of Pseudomonas putida LS46 was isolated from wastewater on the basis of its ability to synthesize medium chain-length polyhydroxyalkanoates (mcl-PHAs). P.putida LS46 was differentiated from other P.putida strains on the basis of cpn60 (UT). The complete genome of P.putida LS46 was sequenced and annotated. Its chromosome is 5,86,2556 bp in size with GC ratio of 61.69. It is encoding 5316 genes, including 7 rRNA genes and 76 tRNA genes. Nucleotide sequence data of the complete P. putida LS46 genome was compared with nine other P. putida strains (KT2440, F1, BIRD-1, S16, ND6, DOT-T1E, UW4, W619 and GB-1) identified either as biocontrol agents or as bioremediation agents and isolated from different geographical region and different environment. BLASTn analysis of whole genome sequences of the ten P. putida strains revealed nucleotide sequence identities of 86.54 to 97.52%. P.putida genome arrangement was LS46 highly similar to P.putida BIRD1 and P.putida ND6 but was markedly different than P.putida DOT-T1E, P.putida UW4 and P.putida W619. Fatty acid biosynthesis (fab), fatty acid degradation (fad) and PHA synthesis genes were highly conserved among biocontrol and bioremediation P.putida strains. Six genes in pha operon of P. putida LS46 showed >98% homology at gene and proteins level. It appears that polyhydroxyalkanoate (PHA) synthesis is an intrinsic property of P. putida and was not affected by its geographic origin. However, all strains, including P. putida LS46, were different from one another on the basis of house keeping genes, and presence of plasmid, prophages, insertion sequence elements and genomic islands. While P. putida LS46 was not selected for plant growth promotion or bioremediation capacity, its genome also encoded genes for root colonization, pyoverdine synthesis, oxidative stress (present in other soil isolates), degradation of aromatic compounds, heavy metal resistance and nicotinic acid degradation, manganese (Mn II) oxidation. Genes for toluene or naphthalene degradation found in the genomes of P. putida F1, DOT-T1E, and ND6 were absent in the P. putida LS46 genome. Heavy metal resistant genes encoded by the P. putida W619 genome were also not present in the P. putida LS46 genome. Despite the overall similarity among genome of P.putida strains isolated for different applications and from different geographical location a number of differences were observed in genome arrangement, occurrence of transposon, genomic islands and prophage. It appears that P.putida strains had a common ancestor and by acquiring some specific genes by horizontal gene transfer it differed from other related strains.
    06/2014; 4(1). DOI:10.1186/s13568-014-0037-8
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    • "However, PHA metabolism is not a unidirectional process in which the PHA is either polymerized or depolymerized but a bidirectional process in which there is a continuous cycle of synthesis and degradation. This helps adapt the carbon flow to the transient demand for metabolic intermediates , thus balancing carbon resources and better guaranteeing efficient growth (Ren et al., 2009; 2010; de Eugenio et al., 2010a; Escapa et al., 2012; Arias et al., 2013). This could be of great advantage in competitive niches in which nutritional conditions change rapidly. "
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    ABSTRACT: Pseudomonas putida synthesises polyhydroxyalkanoates (PHAs) as storage compounds. PHA synthesis is more active when the carbon source is in excess and the nitrogen source limiting, but can also occur at a lower rate under balanced carbon/nitrogen ratios. This work shows that PHA synthesis is controlled by the Crc global regulator, a protein that optimises carbon metabolism by inhibiting the expression of genes involved in the use of non-preferred carbon sources. Crc acts post-transcriptionally. The mRNAs of target genes contain characteristic catabolite activity (CA) motifs near the ribosome binding site. Sequences resembling CA motifs can be predicted for the phaC1 gene, which codes for a PHA polymerase, and for phaI and phaF, which encode proteins associated to PHA granules. Our results show that Crc inhibits the translation of phaC1 mRNA, but not that of phaI or phaF, reducing the amount of PHA accumulated in the cell. Crc inhibited PHA synthesis during exponential growth in media containing a balanced carbon/nitrogen ratio. No inhibition was seen when the carbon/nitrogen ratio was imbalanced. This extends the role of Crc beyond that of controlling the hierarchical utilization of carbon sources, and provides a link between PHA synthesis and the global regulatory networks controlling carbon flow.
    Environmental Microbiology 10/2013; 16(1). DOI:10.1111/1462-2920.12303 · 6.24 Impact Factor
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    • "A wide variety of microorganisms accumulate PHAs as a carbon and energy storage material in carbon-excess conditions when a major nutrient (typically nitrogen or phosphorus) is limiting (Rehm 2010; de Eugenio et al. 2010). This accumulation of excess carbon is a general mechanism used by Pseudomonas and is essential for resource balancing (Escapa et al. 2012). "
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    ABSTRACT: Six bacteria that synthesize medium-chain-length polyhydroxyalkanoates (mcl-PHAs) were isolated from sewage sludge and hog barn wash and identified as strains of Pseudomonas and Comamonas by 16S rDNA gene sequencing. One isolate, Pseudomonas putida LS46, showed good PHA production (22% of cell dry mass) in glucose medium, and it was selected for further studies. While it is closely related to other P. putida strains (F1, KT2440, BIRD-1, GB-1, S16, and W619), P. putida LS46 was genetically distinct from these other strains on the basis of nucleotide sequence analysis of the cpn60 gene hypervariable region. PHA production was detected as early as 12 h in both nitrogen-limited and nitrogen-excess conditions. The increase in PHA production after 48 h was higher in nitrogen-limited cultures than in nitrogen-excess cultures. Pseudomonas putida LS46 produced mcl-PHAs when cultured with glucose, glycerol, or C(6)-C(14) saturated fatty acids as carbon sources, and mcl-PHAs accounted for 56% of the cell dry mass when cells were batch cultured in medium containing 20 mmol/L octanoate. Although 3-hydroxydecanoate was the major mcl-PHA monomer (58.1-68.8 mol%) in P. putida LS46 cultured in glucose medium, 3-hydroxyoctanoate was the major monomer produced in octanoate medium (88 mol%).
    Canadian Journal of Microbiology 07/2012; 58(8):982-9. DOI:10.1139/w2012-074 · 1.18 Impact Factor
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