Response to lethal UVA radiation in the Antarctic bacterium Pseudomonas extremaustralis: polyhydroxybutyrate and cold adaptation as protective factors

Extremophiles - Pseudomonas extremaustralis is an Antarctic bacterium with high stress resistance, able to grow under cold conditions. It is capable to produce polyhydroxyalkanoates (PHAs) mainly...     

Staphylococcus aureus Lpl protein triggers human host cell invasion via activation of Hsp90 receptor

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein‐like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat‐inducible and appears to play a major role in Lpl1 interaction. Pre‐incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1‐Hsp90 interaction induces F‐actin formation, thus, triggering an endocytosis‐like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl‐Hsp90 interaction.     

<Emphasis Type="Italic">Pseudomonas extremaustralis</Emphasis> sp. nov., a Poly(3-hydroxybutyrate) Producer Isolated from an Antarctic Environment

A Gram-negative, mobile, rod-shaped, non-spore-forming bacterium (strain 14-3^T) was isolated from a temporary pond in Antarctica. On the basis of 16S rRNA gene sequence similarity, strain 14-3^T was shown to belong to the genus Pseudomonas sensu stricto. Physiological and biochemical tests supported the phylogenetic affiliation. Strain 14-3^T is closely related to Pseudomonas veronii DSM 11331^T, sharing 99.7% sequence similarity. DNA–DNA hybridization experiments between the two strains showed only moderate reassociation similarity (35.1%). Tests for arginine dihydrolase and nitrate reduction were positive, while those for denitrification, indol production, glucose acidification, urease, ß-galactosidase, esculin, caseine and gelatin hydrolysis were negative. Growth of this bacterium occurred in a range from 4 to 37°C but not at 42°C. It accumulated poly(3-hydroxybutyrate) when grown on sodium octanoate medium. Strain 14-3^T therefore represents the type strain of a new species, for which the name Pseudomonas extremaustralis sp. nov. is proposed. The type strain 14-3^T has been deposited as DSM 17835^T and as CIP 109839^T.     

Polyhydroxyalkanoates are essential for maintenance of redox state in the Antarctic bacterium Pseudomonas sp. 14-3 during low temperature adaptation

Polyhydroxyalkanoates (PHAs) are highly reduced bacterial storage compounds that increase fitness in changing environments. We have previously shown that phaRBAC genes from the Antarctic bacterium Pseudomonas sp. 14-3 are located in a genomic island containing other genes probably related with its adaptability to cold environments. In this paper, Pseudomonas sp. 14-3 and its PHA synthase-minus mutant (phaC) were used to asses the effect of PHA accumulation on the adaptability to cold conditions. The phaC mutant was unable to grow at 10°C and was more susceptible to freezing than its parent strain. PHA was necessary for the development of the oxidative stress response induced by cold treatment. Addition of reduced compounds cystine and gluthathione suppressed the cold sensitive phenotype of the phaC mutant. Cold shock produced very rapid degradation of PHA in the wild type strain. The NADH/NAD ratio and NADPH content, estimated by diamide sensitivity, decreased strongly in the mutant after cold shock while only minor changes were observed in the wild type. Accordingly, the level of lipid peroxidation in the mutant strain was 25-fold higher after temperature downshift. We propose that PHA metabolism modulates the availability of reducing equivalents, contributing to alleviate the oxidative stress produced by low temperature.     

Poly(3-hydroxybutyrate) influences biofilm formation and motility in the novel Antarctic species <Emphasis Type="Italic">Pseudomonas extremaustralis</Emphasis> under cold conditions

Polyhydroxyalkanoates (PHAs) are highly reduced bacterial storage compounds that increase fitness in changing environments. It has previously shown that polyhydroxybutyrate (PHB) accumulation is essential during the growth under cold conditions. In this work, the relationship between PHB accumulation and biofilm development at low temperature was investigated. P. extremaustralis, an Antarctic strain able to accumulate PHB, and its phaC mutant, impaired in the synthesis of this polymer, were used to analyze microaerobic growth, biofilm development, EPS content and motility. PHB accumulation increased motility and survival of planktonic cells in the biofilms developed by P. extremaustralis under cold conditions. Microaerobic conditions rescued the cold growth defect of the mutant strain. The PHB accumulation capability could constitute an adaptative advantage for the colonization of new ecological niches in stressful environments.     

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.     

Biofilm lifestyle enhances diesel bioremediation and biosurfactant production in the Antarctic polyhydroxyalkanoate producer Pseudomonas extremaustralis

Diesel is a widely distributed pollutant. Bioremediation of this kind of compounds requires the use of microorganisms able to survive and adapt to contaminated environments. Pseudomonas extremaustralis is an Antarctic bacterium with a remarkable survival capability associated to polyhydroxyalkanoates (PHAs) production. This strain was used to investigate the effect of cell growth conditions--in biofilm versus shaken flask cultures--as well as the inocula characteristics associated with PHAs accumulation, on diesel degradation. Biofilms showed increased cell growth, biosurfactant production and diesel degradation compared with that obtained in shaken flask cultures. PHA accumulation decreased biofilm cell attachment and enhanced biosurfactant production. Degradation of long-chain and branched alkanes was observed in biofilms, while in shaken flasks only medium-chain length alkanes were degraded. This work shows that the PHA accumulating bacterium P. extremaustralis can be a good candidate to be used as hydrocarbon bioremediation agent, especially in extreme environments.     

The Global Anaerobic Regulator Anr,Is Involved in Cell Attachment and Aggregation Influencing the First Stages of Biofilm Development in Pseudomonas extremaustralis

Pseudomonas extremaustralis is a versatile Antarctic bacterium, able to grow under microaerobic and anaerobic conditions and is related to several non-pathogenic Pseudomonads. Here we report on the role of the global anaerobic regulator Anr, in the early steps of P. extremaustralis biofilm development. We found that the anr mutant was reduced in its ability to attach, to form aggregates and to display twitching motility but presented higher swimming motility than the wild type. In addition, microscopy revealed that the wild type biofilm contained more biomass and was thicker, but were less rough than that of the anr mutant. In silico analysis of the P. extremaustralis genome for Anr-like binding sites led to the identification of two biofilm-related genes as potential targets of this regulator. When measured using Quantitative Real Time PCR, we found that the anr mutant expressed lower levels of pilG, which encodes a component of Type IV pili and has been previously implicated in cellular adhesion. Levels of morA, involved in signal transduction and flagella development, were also lower in the mutant. Our data suggest that under low oxygen conditions, such as those encountered in biofilms, Anr differentially regulates aggregation and motility thus affecting the first stages of biofilm formation.     

Microaerophilic alkane degradation in <Emphasis Type="Italic">Pseudomonas extremaustralis</Emphasis>: 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.