Abstract
Pseudomonas putida KT2440 is a model bacteria used commonly for medium-chain-length polyhydroxyalkanoates (mcl-PHAs) production using various substrates. However, despite many studies conducted on P. putida KT2440 strain, the molecular mechanisms of leading to mcl-PHAs synthesis in reaction to environmental stimuli are still not clear. The rearrangement of the metabolism in response to environmental stress could be controlled by stringent response that modulates the transcription of many genes in order to promote survival under nutritional deprivation conditions. Therefore, in this work we investigated the relation between mcl-PHAs synthesis and stringent response. For this study, a relA/spoT mutant of P. putida KT2440, unable to induce the stringent response, was used. Additionally, the transcriptome of this mutant was analyzed using RNA-seq in order to examine rearrangements of the metabolism during cultivation. The results show that the relA/spoT mutant of P. putida KT2440 is able to accumulate mcl-PHAs in both optimal and nitrogen limiting conditions. Nitrogen starvation did not change the efficiency of mcl-PHAs synthesis in this mutant. The transition from exponential growth to stationary phase caused significant upregulation of genes involved in transport system and nitrogen metabolism. Transcriptional regulators, including rpoS, rpoN and rpoD, did not show changes in transcript abundance when entering the stationary phase, suggesting their limited role in mcl-PHAs accumulation during stationary phase.
Highlights
To survive in the harsh conditions, microorganisms need to be able to adapt to a competitive and changing environment
PHAs synthesis in shake flask cultures experiment In order to examine the relationship between the stringent response and medium-chain-length polyhydroxyal‐ kanoates (mcl-PHA) synthesis, P. putida KT2440 and its mutant with non-functional relA/spoT genes were cultivated in shaking flasks
An RpoN-deficient mutant of P. putida KT2440 was used to reveal the role of RpoN in the regulatory network that controls mcl-PHAs synthesis in culture supplemented with oleic acid
Summary
To survive in the harsh conditions, microorganisms need to be able to adapt to a competitive and changing environment. One of the survival strategies, that microorganisms living in stressful environments have developed, is synthesis and accumulation of polyhydroxyalkanoates (PHAs) in aerobic and anaerobic conditions (Poirier et al 1995). The accumulation of PHAs may increase the survival capabilities of these bacteria in extreme environments or when nutrient availability is poor (Ayub et al 2009). Because of the complexity of PHAs metabolism knowledge on the re-configuration of the bacteria whole metabolism under the conditions that lead to mcl-PHAs synthesis is still not clear. It has been suggested that PhaG encodes transacylase, being not co-localized with the pha gene cluster, is involved in mcl-PHAs synthesis from non-related carbon sources (Hoffmann and Rehm 2004). It is known that PHAs are accumulated by pseudomonads mainly in response to unbalanced growth conditions such as a lack of nitrogen, which links PHAs accumulation to the stringent response (López et al 2015)
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