Oxidative stress under low oxygen conditions triggers hyperflagellation and motility in the Antarctic bacterium Pseudomonas extremaustralis.

Reactive oxygen species and nitrogen species (ROS and RNS), produced in a wide range of physiological process even under low oxygen availability, are among the main stressors found in the environment. Strategies developed to combat them constitute key features in bacterial adaptability and survival. Pseudomonas extremaustralis is a metabolic versatile and stress resistant Antarctic bacterium, able to grow under different oxygen conditions. The present work explores the effect of oxidative stress under low oxygen conditions in P. extremaustralis, by combining RNA deep sequencing analysis and physiological studies. Cells grown under microaerobiosis exhibited more oxidative damage in macromolecules and lower survival rates than under aerobiosis. RNA-seq analysis showed an up-regulation of genes related with oxidative stress response, flagella, chemotaxis and biofilm formation while chaperones and cytochromes were down-regulated. Microaerobic cultures exposed to H2O2 also displayed a hyper-flagellated phenotype coupled with a high motility behavior. Moreover, cells that were subjected to oxidative stress presented increased biofilm formation. Altogether, our results suggest that a higher motile behavior and augmented capacity to form biofilm structures could work in addition to well-known antioxidant enzymes and non-enzymatic ROS scavenging mechanisms to cope with oxidative stress at low oxygen tensions.

Microaerophilic alkane degradation in Pseudomonas extremaustralis: a transcriptomic and physiological approach.

Diesel fuel is one of the most important sources of hydrocarbon contamination worldwide. Its composition consists of a complex mixture of n-alkanes, branched alkanes and aromatic compounds. Hydrocarbon degradation in Pseudomonas species has been mostly studied under aerobic conditions; however, a dynamic spectrum of oxygen availability can be found in the environment. Pseudomonas extremaustralis, an Antarctic bacterium isolated from a pristine environment, is able to degrade diesel fuel and presents a wide microaerophilic metabolism. In this work RNA-deep sequence experiments were analyzed comparing the expression profile in aerobic and microaerophilic cultures. Interestingly, genes involved in alkane degradation, including alkB, were over-expressed in micro-aerobiosis in absence of hydrocarbon compounds. In minimal media supplemented with diesel fuel, n-alkanes degradation (C13-C19) after 7 days was observed under low oxygen conditions but not in aerobiosis. In-silico analysis of the alkB promoter zone showed a putative binding sequence for the anaerobic global regulator, Anr. Our results indicate that some diesel fuel components can be utilized as sole carbon source under microaerophilic conditions for cell maintenance or slow growth in a Pseudomonas species and this metabolism could represent an adaptive advantage in polluted environments.

Novel Essential Role of Ethanol Oxidation Genes at Low Temperature Revealed by Transcriptome Analysis in the Antarctic Bacterium Pseudomonas extremaustralis.

Temperature is one of the most important factors for bacterial growth and development. Cold environments are widely distributed on earth, and psychrotolerant and psychrophilic microorganisms have developed different adaptation strategies to cope with the stress derived from low temperatures. Pseudomonas extremaustralis is an Antarctic bacterium able to grow under low temperatures and to produce high amounts of polyhydroxyalkanoates (PHAs). In this work, we analyzed the genome-wide transcriptome by RNA deep-sequencing technology of early exponential cultures of P. extremaustralis growing in LB (Luria Broth) supplemented with sodium octanoate to favor PHA accumulation at 8°C and 30°C. We found that genes involved in primary metabolism, including tricarboxylic acid cycle (TCA) related genes, as well as cytochromes and amino acid metabolism coding genes, were repressed at low temperature. Among up-regulated genes, those coding for transcriptional regulatory and signal transduction proteins were over-represented at cold conditions. Remarkably, we found that genes involved in ethanol oxidation, exaA, exaB and exaC, encoding a pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the cytochrome c550 and an aldehyde dehydrogenase respectively, were up-regulated. Along with RNA-seq experiments, analysis of mutant strains for pqqB (PQQ biosynthesis protein B) and exaA were carried out. We found that the exaA and pqqB genes are essential for growth under low temperature in LB supplemented with sodium octanoate. Additionally, p-rosaniline assay measurements showed the presence of alcohol dehydrogenase activity at both 8°C and 30°C, while the activity was abolished in a pqqB mutant strain. These results together with the detection of ethanol by gas chromatography in P. extremaustralis cultures grown at 8°C support the conclusion that this pathway is important under cold conditions. The obtained results have led to the identification of novel components involved in cold adaptation mechanisms in this bacterium, suggesting for the first time a role of the ethanol oxidation pathway for bacterial growth at low temperatures.