Functionalized Membrane Domains: An Ancestral Feature of Archaea?

Bacteria and Eukarya organize their plasma membrane spatially into domains of distinct functions. Due to the uniqueness of their lipids, membrane functionalization in Archaea remains a debated area. A novel membrane ultrastructure predicts that monolayer and bilayer domains would be laterally segregated in the hyperthermophilic archaeon Thermococcus barophilus. With very different physico-chemical parameters of the mono- and bilayer, each domain type would thus allow the docking of different membrane proteins and express different biological functions in the membrane. To estimate the ubiquity of this putative membrane ultrastructure in and out of the order Thermococcales, we re-analyzed the core lipid composition of all the Thermococcales type species and collected all the literature data available for isolated archaea. We show that all species of Thermococcales synthesize a mixture of diether bilayer forming and tetraether monolayer forming lipids, in various ratio from 10 to 80% diether in Pyrococcus horikoshii and Thermococcus gorgonarius, respectively. Since the domain formation prediction rests only on the coexistence of di- and tetraether lipids, we show that all Thermococcales have the ability for domain formation, i.e., differential functionalization of their membrane. Extrapolating this view to the whole Archaea domain, we show that almost all archaea also have the ability to synthesize di- and tetraether lipids, which supports the view that functionalized membrane domains may be shared between all Archaea. Hence domain formation and membrane compartmentalization may have predated the separation of the three domains of life and be essential for the cell cycle.

Epsilonproteobacteria dominate bacterial diversity at a natural tar seep.

The bacterial diversity of a naturally seeping bitumen source was investigated by 16S rRNA gene cloning and sequencing. Epsilonproteobacteria were shown to dominate the bacterial diversity in the underground water and within the bitumen, representing ca. 75% of the total bacterial diversity. These Epsilonproteobacteria were dominated by Sulfurimonas OTUs, while Sulfurovum and Arcobacter OTUs completed the remaining diversity. Epsilonproteobacteria are sulfur-oxidizer, nitrate-reducing chemo-lithoautotrophic bacteria, unable to use most organics for growth but capable of CO2 fixation. Thus, reduced sulfur species, but not the complex organic matter of the tar, are utilized for growth by bacterial communities at the Puy-de-la-Poix. The large prevalence of populations of Epsilonproteobacteria is a clear indication that crude oil offers a competitive ecological niche for these organisms.

Screening for N-AHSL-Based-Signaling Interfering Enzymes.

Quorum sensing (QS)-based signaling is a widespread pathway used by bacteria for the regulation of functions involved in their relation to the environment or their host. QS relies upon the production, accumulation and perception of small diffusable molecules by the bacterial population, hence linking high gene expression with high cell population densities. Among the different QS signal molecules, an important class of signal molecules is the N-acyl homoserine lactone (N-AHSL). In pathogens such as Erwinia or Pseudomonas, N-AHSL based QS is crucial to overcome the host defenses and ensure a successful infection. Interfering with QS-regulation allows the algae Delisea pulcra to avoid surface colonization by bacteria. Thus, interfering the QS-regulation of pathogenic bacteria is a promising antibiotic-free antibacterial therapeutic strategy. To date, two N-AHSL lactonases and one amidohydrolase families of N-ASHL degradation enzymes have been characterized and have proven to be efficient in vitro to control N-AHSL-based QS-regulated functions in pathogens. In this chapter, we provide methods to screen individual clones or bacterial strains as well as pool of clones for genomic and metagenomic libraries, that can be used to identify strains or clones carrying N-ASHL degradation enzymes.

Molecular chaperone accumulation as a function of stress evidences adaptation to high hydrostatic pressure in the piezophilic archaeon Thermococcus barophilus.

The accumulation of mannosyl-glycerate (MG), the salinity stress response osmolyte of Thermococcales, was investigated as a function of hydrostatic pressure in Thermococcus barophilus strain MP, a hyperthermophilic, piezophilic archaeon isolated from the Snake Pit site (MAR), which grows optimally at 40 MPa. Strain MP accumulated MG primarily in response to salinity stress, but in contrast to other Thermococcales, MG was also accumulated in response to thermal stress. MG accumulation peaked for combined stresses. The accumulation of MG was drastically increased under sub-optimal hydrostatic pressure conditions, demonstrating that low pressure is perceived as a stress in this piezophile, and that the proteome of T. barophilus is low-pressure sensitive. MG accumulation was strongly reduced under supra-optimal pressure conditions clearly demonstrating the structural adaptation of this proteome to high hydrostatic pressure. The lack of MG synthesis only slightly altered the growth characteristics of two different MG synthesis deletion mutants. No shift to other osmolytes was observed. Altogether our observations suggest that the salinity stress response in T. barophilus is not essential and may be under negative selective pressure, similarly to what has been observed for its thermal stress response.

Restoration of the di-myo-inositol-phosphate pathway in the piezo-hyperthermophilic archaeon Thermococcus barophilus.

Most Thermococcales accumulate di-myo-inositol-phosphate (DIP) as an organic solute as a response to heat stress. We have studied the accumulation of this osmolyte in the high-hydrostatic pressure adapted hyperthermophile Thermococcus barophilus. We found no accumulation of DIP under any of the stress conditions tested, although this archaeon harbors the 3 DIP synthesis genes. Lack of synthesis is due to the lack of expression of TERMP_01135 coding for the second step of DIP synthesis. In contrast to other species, the T. barophilus synthesis operon is interrupted by a four gene locus, in reverse orientation. Restoring an operon like structure at the DIP locus restored DIP synthesis, but did not have an impact on growth characteristics, suggesting that other mechanisms have evolved in this organism to cope with heat stress.

Screening for N-AHSL-based-signaling interfering enzymes.

Quorum sensing (QS)-based signaling is a widespread pathway used by bacteria for the regulation of functions involved in relation to their environment or host. QS relies upon the production, accumulation, and perception of small diffusible molecules by the bacterial population, hence linking high gene expression with high cell population densities. Amongst the different QS signal molecules, an important class of signal molecules is the N-acyl homoserine lactone (N-AHSL) class. In pathogens such as Erwinia or Pseudomonas, N-AHSL-based QS is crucial to overcome the host defenses and ensure a successful infection. Interfering with QS regulation allows the alga Delisea pulchra to avoid surface colonization by bacteria. Thus, interfering in the QS regulation of pathogenic bacteria is a promising antibiotic-free antibacterial therapeutic strategy. To date, two N-AHSL lactonase and one amidohydrolase families of N-ASHL degradation enzymes have been characterized and proven to be efficient in vitro to control N-AHSL-based QS-regulated functions in pathogens.

In situ Raman and X-ray spectroscopies to monitor microbial activities under high hydrostatic pressure.

Until recently, monitoring of cells and cellular activities at high hydrostatic pressure (HHP) was mainly limited to ex situ observations. Samples were analyzed prior to and following the depressurization step to evaluate the effect of the pressure treatment. Such ex situ measurements have several drawbacks: (i) it does not allow for kinetic measurements and (ii) the depressurization step often leads to artifactual measurements. Here, we describe recent advances in diamond anvil cell (DAC) technology to adapt it to the monitoring of microbial processes in situ. The modified DAC is asymmetrical, with a single anvil and a diamond window to improve imaging quality and signal collection. Using this novel DAC combined to Raman and X-ray spectroscopy, we monitored the metabolism of glucose by baker's yeast and the reduction of selenite by Agrobacterium tumefaciens in situ under HHP. In situ spectroscopy is also a promising tool to study piezophilic microorganisms.

The S-adenosyl-L-homocysteine hydrolase gene ahcY of Agrobacterium radiobacter K84 is required for optimal growth, antibiotic production, and biocontrol of crown gall disease.

Agrobacterium radiobacter K84 is a commercial agent used worldwide to control crown gall disease caused by pathogenic isolates of A. tumefaciens. More than 2,000 transposon insertion derivatives of strain K84 were screened by a standardized greenhouse bioassay to identify mutants defective in biocontrol. Three mutants affected in biocontrol properties were identified. All three mutants displayed normal levels of attachment to tomato seed and root colonization. One of these mutants, M19-164, exhibited partial biocontrol and did not produce detectable levels of agrocin 84. In this mutant, the transposon is located in the agn locus of pAgK84, which codes for agrocin 84 biosynthesis. The second mutant, M19-158, also exhibited partial biocontrol and produced reduced amounts of agrocin 84 as a result of a mutation in a chromosomal gene of unknown function. The third mutant, M9-22, failed to biocontrol, was impaired in both growth in minimal medium and siderophore production, and failed to produce detectable levels of agrocin 84. The chromosomal gene ahcY, which encodes S-adenosyl-l-homocysteine hydrolase, was disrupted in this mutant. Expression of a functional copy of ahcY in M9-22 restored all of the altered phenotypes. The fact that all identified biocontrol mutants exhibited a partial or total defect in production of agrocin 84 indicates that this antibiotic is required for optimum biocontrol. This study also identified two chromosomally encoded genes required for agrocin 84 production. That a mutation in ahcY abolishes biocontrol suggests that the intracellular ratio of S-adenosyl-l-methionine to S-adenosyl-l-homocysteine is an important factor for agrocin 84 biosynthesis. Finally, we demonstrate that the ahcY gene in strain K84 is also required for optimal growth as well as for antibiotic production and biocontrol of crown gall disease.

A Rhodococcus qsdA-encoded enzyme defines a novel class of large-spectrum quorum-quenching lactonases.

A gene involved in N-acyl homoserine lactone (N-AHSL) degradation was identified by screening a genomic library of Rhodococcus erythropolis strain W2. This gene, named qsdA (for quorum-sensing signal degradation), encodes an N-AHSL lactonase unrelated to the two previously characterized N-AHSL-degrading enzymes, i.e., the lactonase AiiA and the amidohydrolase AiiD. QsdA is related to phosphotriesterases and constitutes the reference of a novel class of N-AHSL degradation enzymes. It confers the ability to inactivate N-AHSLs with an acyl chain ranging from C(6) to C(14), with or without substitution at carbon 3. Screening of a collection of 15 Rhodococcus strains and strains closely related to this genus clearly highlighted the relationship between the ability to degrade N-AHSLs and the presence of the qsdA gene in Rhodococcus. Bacteria harboring the qsdA gene interfere very efficiently with quorum-sensing-regulated functions, demonstrating that qsdA is a valuable tool for developing quorum-quenching procedures.

N-acyl-homoserine lactone-mediated quorum-sensing in Azospirillum: an exception rather than a rule.

Forty Azospirillum strains were tested for their ability to synthesize N-acyl-homoserine lactones (AHLs). AHL production was detected for four strains belonging to the lipoferum species and isolated from a rice rhizosphere. AHL molecules were structurally identified for two strains: Azospirillum lipoferum TVV3 produces 3O,C(8)-HSL (N-3-oxo-octanoyl-homoserine-lactone), C(8)-HSL (N-3-octanoyl-homoserine-lactone), 3O,C(10)-HSL (N-3-oxo-decanoyl-homoserine-lactone), 3OH,C(10)-HSL (N-3-hydroxy-decanoyl-homoserine-lactone) and C(10)-HSL (N-3-decanoyl-homoserine-lactone), whereas A. lipoferum B518 produced 3O,C(6)-HSL (N-3-oxo-hexanoyl-homoserine-lactone), C(6)-HSL (N-3-hexanoyl-homoserine-lactone), 3O,C(8)-HSL, 3OH,C(8)-HSL and C(8)-HSL. Genes involved in AHL production were characterized for A. lipoferum TVV3 by generating a genomic library and complementing an AHL-deficient strain with sensor capabilities. Those genes, designated alpI and alpR, were found to belong to the luxI and luxR families, respectively. When cloned in a suitable heterologous host, alpI and alpR could direct the synthesis of the five cognate AHLs present in A. lipoferum TVV3. These two adjacent genes were found to be located on a 85 kb plasmid. Southern hybridization experiments with probes alpI/R indicated that genes involved in AHL production in the three other AHL-producing strains were not closely related to alpI and alpR. This study demonstrates that AHL-based quorum-sensing is not widespread among the genus Azospirillum and could be found only in some A. lipoferum strains.

Development of a low-pressure diamond anvil cell and analytical tools to monitor microbial activities in situ under controlled P and T.

We have designed a new low-pressure Diamond Anvil Cell (DAC), calibrated two novel pressure calibrants and validated the use of semi-quantitative Raman and X-ray spectroscopies to monitor the fate of microbes, their metabolism or their cellular components under controlled pressures and temperatures in the 0.1-1.4 GPa and 20-300 degrees C P,T range. The low-pressure DAC has a 250- to 600-microm-thick observation diamond window to allow for lower detection limits and improved microscopic imaging. This new design allows the determination of cellular growth parameters from automated image analysis, which can be correlated with the spectroscopic data obtained on metabolism, ensuring high quality data collection on microbial activity under pressure. The novel pressure sensors offer the ease of use of the well-known ruby scale, while being more sensitive and reacting to pressure variations instantaneously.