Genome sequence analysis of Pseudomonas extremaustralis provides new insights into environmental adaptability and extreme conditions resistance.

The genome of the Antarctic bacterium Pseudomonas extremaustralis was analyzed searching for genes involved in environmental adaptability focusing on anaerobic metabolism, osmoregulation, cold adaptation, exopolysaccharide production and degradation of complex compounds. Experimental evidences demonstrated the functionality of several of these pathways, including arginine and pyruvate fermentation, alginate production and growth under cold conditions. Phylogenetic analysis along with genomic island prediction allowed the detection of genes with probable foreign origin such as those coding for acetate kinase, osmotic resistance and colanic acid biosynthesis. These findings suggest that in P. extremaustralis the horizontal transfer events and/or gene redundancy could play a key role in the survival under unfavorable conditions. Comparative genome analysis of these traits in other representative Pseudomonas species highlighted several similarities and differences with this extremophile bacterium.

A phasin with many faces: structural insights on PhaP from Azotobacter sp. FA8.

Phasins are a group of proteins associated to granules of polyhydroxyalkanoates (PHAs). Apart from their structural role as part of the PHA granule cover, different structural and regulatory functions have been found associated to many of them, and several biotechnological applications have been developed using phasin protein fusions. Despite their remarkable functional diversity, the structure of these proteins has not been analyzed except in very few studies. PhaP from Azotobacter sp. FA8 (PhaPAz) is a representative of the prevailing type in the multifunctional phasin protein family. Previous work performed in our laboratory using this protein have demonstrated that it has some very peculiar characteristics, such as its stress protecting effects in recombinant Escherichia coli, both in the presence and absence of PHA. The aim of the present work was to perform a structural characterization of this protein, to shed light on its properties. Its aminoacid composition revealed that it lacks clear hydrophobic domains, a characteristic that appears to be common to most phasins, despite their lipid granule binding capacity. The secondary structure of this protein, consisting of α-helices and disordered regions, has a remarkable capacity to change according to its environment. Several experimental data support that it is a tetramer, probably due to interactions between coiled-coil regions. These structural features have also been detected in other phasins, and may be related to their functional diversity.

High polyhydroxybutyrate production in Pseudomonas extremaustralis is associated with differential expression of horizontally acquired and core genome polyhydroxyalkanoate synthase genes.

Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA) infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA). All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI) associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC) in comparison with the mclPHA core genome genes (phaC1 and phaC2) indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases.

Polyhydroxyalkanoate synthesis affects biosurfactant production and cell attachment to hydrocarbons in Pseudomonas sp. KA-08.

Stressful conditions prevailing in hydrocarbon-contaminated sites influence the diversity, distribution, and activities of microorganisms. Oil bioremediation agents should develop special characteristics to cope with these environments like surfactant production and cellular affinity to hydrocarbons. Additionally, polyhydroxyalkanoate (PHA) accumulation was proven to improve tolerance to stressful conditions. Pseudomonas sp. KA-08 was isolated from a chronic oil-contaminated environment, it is highly tolerant to xylene, and it is able to accumulate PHA and to produce surfactant compounds that lower the water surface tension (ST) as well as bioemulsifiers. In this work, we studied the effect of the capability to accumulate PHAs on biosurfactant production and microbial attachment to hydrocarbons (MATH). Our results showed that PHA synthesis capability has a favorable effect in the production of compounds which affect the ST but not on the production of bioemulsifiers. On the other hand, PHA accumulation affects cellular affinity to xylene. MATH analysis showed that a PHA-negative mutant increased its affinity to xylene compared with the wild-type strain. This result was also observed in Pseudomonas putida GPp104 (a PHA(-) mutant), suggesting that this effect could be generalized to other Pseudomonas strains.

Genome sequence of the polyhydroxybutyrate producer Pseudomonas extremaustralis, a highly stress-resistant Antarctic bacterium.

Pseudomonas extremaustralis 14-3b presents genes involved in the synthesis of different polyhydroxyalkanoates, in tolerance and degradation of pollutants, and in microaerobic metabolism. Several genomic islands were detected. Genetic machinery could contribute to the adaptability to stressful conditions. This is the first genome sequence reported from a Pseudomonas isolated from cold environments.

Unexpected stress-reducing effect of PhaP, a poly(3-hydroxybutyrate) granule-associated protein, in Escherichia coli.

Phasins (PhaP) are proteins normally associated with granules of poly(3-hydroxybutyrate) (PHB), a biodegradable polymer accumulated by many bacteria as a reserve molecule. These proteins enhance growth and polymer production in natural and recombinant PHB producers. It has been shown that the production of PHB causes stress in recombinant Escherichia coli, revealed by an increase in the concentrations of several heat stress proteins. In this work, quantitative reverse transcription (qRT)-PCR analysis was used to study the effect of PHB accumulation, and that of PhaP from Azotobacter sp. strain FA8, on the expression of stress-related genes in PHB-producing E. coli. While PHB accumulation was found to increase the transcription of dnaK and ibpA, the expression of these genes and of groES, groEL, rpoH, dps, and yfiD was reduced, when PhaP was coexpressed, to levels even lower than those detected in the non-PHB-accumulating control. These results demonstrated the protective role of PhaP in PHB-synthesizing E. coli and linked the effects of the protein to the expression of stress-related genes, especially ibpA. The effect of PhaP was also analyzed in non-PHB-synthesizing strains, showing that expression of this heterologous protein has an unexpected protective effect in E. coli, under both normal and stress conditions, resulting in increased growth and higher resistance to both heat shock and superoxide stress by paraquat. In addition, PhaP expression was shown to reduce RpoH protein levels during heat shock, probably by reducing or titrating the levels of misfolded proteins.