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

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.

Fig 1. Classification of the significant differentially expressed genes under cold conditions into functional categories (Rockhopper P<0.05 and Q<0.05).

Green and red bars represent up- and down-regulated genes, respectively.

Fig 2. Functional enrichment of differentially expressed genes using Blast2GO software.

A. Down-regulated genes. B. Up-regulated genes. P<0.05 using Fisher’s Test.

Fig 3. Organization of genes coding for ethanol oxidation in P. extremaustralis and other Pseudomonas species.

Arrows indicate the direction of gene transcription and the relative size of each open reading frame (ORF).

Fig 4. Growth of the wild type (WT), ppqB and exaA strains.

A. Growth at 30°C. B. Growth at 8°C. Values represent the mean ± standard deviations (SD) from three independent cultures.

Fig 5. Relationship between ethanol metabolism and the ß-oxidation pathway.

Solid black lines and names represent genes without differences in their expression. Solid red lines and names represent down-regulated genes and green solid lines and names indicate up-regulated functions under cold conditions. Dashed lines show probable relationships.

Fig 6. Estimation of alcohol dehydrogenase activity using p-rosaniline plate assay in wild type (WT), ppqB and pqqB/pBBR1MCS5 pqqBCDE strains.

Activity was considered as positive in magenta colonies, while white colonies were considered as negative. A. Plates incubated at 30°C during 24 h. B. Plates were incubated at 8°C during 7 days. C. Determination of p-rosaniline index at 30°C. D. Determination of p-rosaniline index at 8°C. The asterisk (*) denotes significant differences (P<0.05) between strains (indicating by connector lines) using the Student's t test.