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

Paula M Tribelli,Martiniano M Ricardi,Maria Gómez-Lozano,Esmeralda C Solar Venero,Søren Molin,Nancy I López,Laura J Raiger Iustman,Shihui Yang

doi:10.1371/journal.pone.0145353

Abstract

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.

Highlights

Bacterial adaptability to an environment is the result of complex mechanisms that entail the response of individual genes or operons and intricate regulatory networks that coordinate the control of entire metabolic pathways [1]
The wild type strain, the pqqB and its complemented strain were cultured in LB supplemented with sodium octanoate and 10 μl drops of these cultures were incubated in p-rosaniline plates at 8°C or 30°C for 7 days and 1 day, respectively
The RNA expression profile of P. extremaustralis cultures growing at 8°C or 30°C at the early exponential phase (OD600nm = 0.5) in LB supplemented with sodium octanoate revealed 5715 transcripts and 156 putative small regulatory RNAs

Summary

Introduction

Bacterial adaptability to an environment is the result of complex mechanisms that entail the response of individual genes or operons and intricate regulatory networks that coordinate the control of entire metabolic pathways [1]. Survival in extreme environments requires additional features at most levels of cell function. In the case of cold environments, the low temperatures and the presence of ice exert severe constraints on living organisms, including decreased water availability and molecular diffusion rates, reduced biochemical reaction rates, stabilization of inhibitory nucleic acid structures, presence of ice crystals, increased solubility of gases, production of reactive oxygen species (ROS) and reduced fluidity of cellular membranes [2,3]. Microorganisms that are able to survive and grow in cold and freezing environments should present physiological adaptations to cope with these conditions, including expression of cold shock proteins, membrane modifications and ribosome rescue [3,4]. Metabolic features associated with growth in cold conditions and the relevance of the different pathways has not yet been totally elucidated

Methods

Results

Conclusion