Biological nitrogen fixation groupClaudine Elmerich
The biological process responsible of the reduction of molecular nitrogen to ammonia is referred to as biological nitrogen fixation. The ability to fix nitrogen is widely distributed among eubacteria and archaea. No eukaryote can fix nitrogen. Nitrogen-fixing bacteria harbour a large spectrum of metabolic properties and they can be found in a variety of ecological niches in association or not with plants. The main emphasis of our research deals with the analysis of bacterial functions that enable diazotrophs to adapt to their environment as well as with the identification of bacterial genes required for the association with the host plant. During the year 2000, progress was made on the synthesis of indole acetic acid (which plays the role of a phytohormone) in Azospirillum brasilense as well as on the genetic characterisation of the nitrogen fixation genes in a Pseudomonas strain, that is an endophyte of rice. In addition, an ecological survey of alkane degradation by bacterial isolates capable of growing on crude oil was continued.
Synthesis of indole acid acetic acid (IAA)
The Azospirillum genus is composed of nitrogen-fixing bacteria that promote the growth of plants and hence are used as commercial inoculants for corn and other cereal crops. Our data suggests the existence of 3 differently regulated routes for IAA synthesis in Azospirillum. It was previously shown that the indole pyruvate (IPyA) route for IAA synthesis was present. However, disruption of ipdC, the structural gene for the indole pyruvate decarboxylase resulted in a mutant strain that still produced IAA, suggesting the existence of alternative routes. Physiological and biochemical evidence based on the phenotypes of mutants strains blocked in the biosynthesis of tryptophane and carrying a mutation in ipdC led to the identification of a route with tryptamine (TAM) as an intermediate. This route is regulated by catabolite repression. It was also shown that IAA could be synthesized through the indole acetonitrile pathway.
This work was performed in collaboration with a Mexican team from Puebla University (Programme ECOS).
Nitrogen fixation in Pseudomonas
For long, it was believed that no true Pseudomonas could fix nitrogen. The Pseudomonas stutzeri strain A15 was isolated from a rice paddy in China. It can fix nitrogen under microaerobic conditions in the free-living state and colonise rice endophytically. Characterisation of nif genes in this strain has been undertaken. The organisation of the nitrogen fixation genes (nifHDK), nifTY, nifNE and associated ORFs resembles that of Azotobacter vinelandii. A set of genes was found upstream of nifH, sharing identity with the rnfCDGEFH operon, involved in electron transport to nitrogenase in Rhodobacter capsulatus.
A DNA region containing rpoN was identified. Inactivation of rpoN led to a mutant strain with a Nif minus phenotype, that is also impaired in utilisation of different nitrogen and carbon sources. RpoN also controls the synthesis of flagella and pili and this reflects a lack of colonisation of the roots of rice. Subsequently the fleS and fleR genes encoding the two components system that controls flagella synthesis have been isolated. Construction of mutant strains in these genes is under progress to investigate the role of flagella versus pili in the colonisation of the roots and systemic infection of rice.
This work was performed in collaboration with a Chinese team (CAAS, Beijing)
Distribution of alkB in Pseudomonas aeruginosa
Bacterial strains that grew on crude oil as the sole source of carbon and energy have been isolated from an environment polluted by effluents from a petroleum refinery. Forty percent of the strains isolated were identified as Pseudomonas aeruginosa. They grew on saturated alkanes from C12 to C20. Three of the isolates were also capable to grow on low molecular weight soluble alkanes, such as hexane, heptane and octane while other strains were not.
The distribution of the alkB gene was then examined. This gene has been characterised in Pseudomonas putida (Gpo1), where it is carried by a large plasmid, the OCT plasmid. alkB encodes a mono-oxygenase, called also alkane hydroxylase, that converts octane into octanol.
Using oligonucleotide primers specific for the alkB gene of the OCT plasmid and for the alkB1 gene of P. aeruginosa strain PAO1, it was possible to show that all the P. aeruginosa isolates contained an alkB1 gene very similar to that of strain PAO1. Only 3 strains carried a gene sharing identity with that of strain Gpo1. These corresponded to the strains that could utilise the soluble and highly toxic alkanes.
It is concluded that the 3 strains that grew with hexane contained two distinct alkane hydroxylase complexes, encoded by alkB and alkB1 genes. The presence of two alkB genes provides a selective advantage for intrinsic bioremediation of polluted sites.
This work was performed in collaboration with a researcher from Moulay Ismail Univeversity, (Meknès, Morocco).
Petroleum Biodegradation GroupPierre Béguin
Our group is concerned with the bioremediation of environmental pollution. We are studying bacteria that can degrade components of gasoline causing problems when spilled in the environment, owing to their high solubility in water or to their poor biodegradability. The line of investigation is based on recombinant DNA technology, the primary goal being the cloning and sequencing of genes involved in the metabolism of the pollutants. From a practical point of view, this approach will yield genes that may be introduced into other bacteria in order to improve their metabolic capabilities. We also plan to develop nucleic acid probes derived from the sequence of the cloned genes, which would be helpful to follow the bacterial populations that harbour them in reconstituted microcosms or in the environment. Thus, such probes would provide useful tools to assess the bioremediation potential of polluted sites and to manage them more efficiently.
Among gasoline components, we are studying the degradation of ether fuels, in particular ethyl tert-butyl ether (ETBE) and methyl tert-butyl ether (MTBE). Large amounts of these compounds are currently added to gasoline as anti-knocking agents. Being highly water-soluble, they easily reach aquifers upon gasoline spills. We are currently collaborating with the French Institute for Petroleum, which has isolated various bacteria participating in the degradation of ether fuels. These strains generally belong to the Actinomycetes. Some of them can utilize ETBE as a sole carbon source by cleaving the ether bond and metabolizing the ethyl group released. The tert-butyl group is not metabolized and accumulates as tert-butyl alcohol (TBA). Other strains do not cleave the ether bond, but are able to metabolize TBA.
We have set out to clone the genes involved in the cleavage of ether fuels and in the metabolism of TBA. A set of genes involved in the metabolism of ETBE has been cloned and sequenced from the bacterium Rhodococcus ruber. These genes are flanked by two perfectly identical transposon-type insertion sequences. This structure, which is found in several other ETBE-degrading strains, is highly prone to undergo recombination events, leading to the loss of the genes located between the transposons and to the loss of ETBE-degrading ability.
Concerning the degradation of isoalkanes, we are studying a strain of Mycobacterium austroafricanum, also first isolated at the French Institute for Petroleum. This bacterium is able to utilize 2,2,4-trimethylpentane (isooctane), a well-known anti-knocking agent, as well as pristane, a long-chain branched hydrocarbon. These compounds are usually degraded rather poorly by the natural microflora. A DNA fragment encoding part of the cytoplasmic proteins induced in the presence of isooctane has been cloned and sequenced.
Furthermore, isooctane induces in M. austroafricanum the biosynthesis of a major 30 kDa cell surface protein, which is related to the ag85 antigens characterized in several other Mycobacteria. It is known that these proteins are mycolyl transferases, which participate in the incorporation of mycolic acids in the cell wall of Mycobacteria. Mycolic acids being very hydrophobic, they might enhance the adsorption of M. austroafricanum cells to the interphase between the aqueous and the hydrocarbon phase, thus facilitating absorption of the substrate. We have set out to clone the gene encoding the 30 kDa protein the synthesis of which is induced by isooctane in M. austroafricanum 2173. In fact, Southern blot analysis shows that the bacterium possesses several mycolyl transferase genes the sequences of which are closely related. Two of them have been cloned and sequenced. Future work will deal with the cloning and characterization of the other genes of the family, and with the identification of the genes which are in fact induced in the presence of isooctane.
Fungal Genetics GroupChristophe d'Enfert
The Fungal Genetics Group is involved in the study of several biological processes in fungi of the genus Aspergillus and Candida with the aim of developing new strategies for the control of fungal growth.
Aspergillus nidulans spore germination
Filamentous fungi of the genus Aspergillus have been widely used for biotechnological processes but are also responsible for human diseases. Spore germination is a key developmental step in the fungal life cycle and is critical for the establishment of the fungus in a new environmental niche. Our aim is to define the molecular and biochemical events that are involved during the early stages of spore germination. In particular, we wish to characterize the signalling pathways mediating the recognition of environmental signals necessary for germination and their transduction towards the resumption of various metabolic processes that are necessary for germination.
Previous work on trehalose metabolism in A. nidulans suggested the involvement of cAMP signalling at the onset of spore germination. We have confirmed this hypothesis through the analysis of A. nidulans mutants with defects in the adenylate cyclase (CyaA) and in the catalytic subunit of the cAMP-dependent protein kinase (PkaA). Both cAMP synthesis and PkaA are necessary during the early stages of spore germination.
Furthermore, we have shown that 1) cAMP has other targets than PkaA; 2) PkaA is also regulated independently of cAMP; and 3) a second kinase (SchA) related to PkaA is involved in the control of spore germination. Present research is aimed at identifying the targets of cAMP during spore germination through the analysis of various A. nidulans mutant strains, using various assays including transcriptome analysis.
Our group has also been involved in the study of trehalose and glycerol metabolism in A. nidulans, in the framework of the European BIOTECH program. We have shown that trehalose, a reserve carbohydrate accumulated in spores and in response to various stresses, plays a major role in the acquisition of tolerance to these stresses and in the long-term survival of the spores. Characterization of A. nidulans strains lacking glycerol 3-phosphate dehydrogenase activity has revealed an unexpected role for glycerol 3-phosphate in the control of cell wall biogenesis in A. nidulans. Furthermore, it has revealed that glycerol can be synthesized through another pathway which is currently investigated with the aim of defining the role of the accumulation of glycerol observed during spore germination.
Insertional mutagenesis in the human fungal pathogen Aspergillus fumigatus
Aspergillus fumigatus is the causative agent of invasive aspergillosis which represents the second cause of death resulting from fungal infections in hospitals. Invasive aspergillosis is associated with a high mortality. This is mostly due to the poor sensitivity of the currently available diagnostic tests and the sole use of amphotericin B therapy which has deleterious side effects. Analysis of candidate virulence factors by reverse genetics has not been successful for our understanding of the pathogenic processes. We have therefore focused our interest towards the development of novel molecular tools that could be used to identify, through insertional mutagenesis, genes that are essential for growth of A. fumigatus under laboratory conditions and/or during invasion of the host, and may hence serve as antifungal targets.
A. fumigatus strains have been constructed that can be used to test whether a gene is essential for growth under laboratory conditions. In collaboration with the "Unité des Aspergillus", we have used this system to demonstrate that fksA, which encodes beta(1,3)-glucan synthase, is an essential gene in A. fumigatus. We are now using this system to develop a screen for A. fumigatus essential genes using insertional mutagenesis. This can be acchieved by transformation of the recipient strain with an heterologous DNA molecule in, or using transposon mutagenesis. Indeed, we have shown that the impala transposable element from Fusarium oxysporum transposes efficiently in A. fumigatus.
Transcriptome analysis in the human pathogen Candida albicans
Candida albicans is a commensal yeast of the human gastric and urinary tracts, and yet is responsible for the vast majority of fungal infections in humans. Mucosal infections are often observed but can evolve into systemic and fatal infections in immuno-compromized individuals. Although tri-azoles are effective in the treatment of candidiasis, their efficacy remains limited and resistant strains have appeared. The design of new antifungal compounds is therefore necessary and a better understanding of the physiopathology of Candida infections is needed to develop novel therapeutic strategies. In this regard, whole-genome transcriptional analysis appears as a powerful tool to gain a better understanding of gene function and of the mechanisms that are involved during the adaptation of an organism to various conditions, in particular host-pathogen interactions. Through our participation in the French Fungal Infection Network (http://www.pasteur.fr/RIF/) and the European Galar Fungail network (http://www.pasteur.fr/Galar_Fungail/) we have developed DNA-arrays for transcript profiling in Candidaalbicans.
Using the whole genome sequence of C. albicans obtained by the Stanford Genome Technology Center (www-sequence.stanford.edu/group/candida/), we have first developed DNA-arrays with probes for 2000 C. albicans genes in collaboration with the "Unité de Génétique Moléculaire des Levures", INRA and Eurogentec. These arrays are currently being used by the participants of the French and European networks. In particular, their use has provided insights in the function of the DNA-binding proteins CaNrg1 and CaMig1 which appear to target the general repressor CaTup1 to promotor regions of genes encoding metabolic functions or proteins responsible for hyphal differentiation and virulence, respectively (Collaboration University of Aberdeen). The development of whole-genome DNA-arrays is inprogress.
In addition, a new typing methodology for C. albicans has been developed and will be used for epidemiological studies.
Service activities and applied research Robert Longin
The Fermentation Facility (http://www.pasteur.fr/recherche/unites/fermentations/) continued its service activities (microbial cultures for laboratories within and outside the Pasteur Institute). During the year 2000, the laboratory grew 79 cultures in 2, 15 or 300 litres fermentors, for 12 units of the Pasteur Institute and 4 external public or private laboratories. Fifty-three recombinant strains cultures of Escherichia coli (class 1, group I, confinement L1) were grown in different media, at low, middle or high cell concentrations or in minimal media containing labelled compounds. The culture conditions allowed to obtain very good protein yields. Other cultures of Bacillus cereus, Pseudomonas oleovorens, Saccharomyces cerevisiae, were realized. The laboratory is also specialized in the cultivation of strains requiring special conditions or endowed with special properties (minimal media, strict anaerobiosis, high temperature, nitrogen fixation, bioluminescent bacteria, etc.). The laboratory is processing to some extent the produced biomass (breakage or the first purification steps). It is strongly involved in QualityAssurance.
Moreover, R &D contracts with Biotechnology societies were conducted. They consisted in the optimization of media and culture conditions for the production of cells, spores or recombinant proteins.
In the field of R &D activities, the laboratory developed and patented an original and efficient in line turbidity measurement device. This device allows one to measure optical densities from 0.05 to more than 300 OD units, for strains such as Escherichia coli, Saccharomyces cerevisiae or lactic bacteria. The system is associated with batteries of micro-fermentors in tandem (Multi-Micro-Fermentors 'MMF'' from 10 to 60 ml), developed in the laboratory. Light and fluorescent sensors (Biolux) were also developed for the study of the expression of reporter genes such as lux or GFP. New developments were made for the measure and regulation of the main growth parameters (pH, pO2...). This apparatus is automated and informatized. It allows one to cultivate eucaryotic and procaryotic cells in batch or continuous cultures (chemostat, turbidostat, cyclic cultures, etc.). Strains harboring new properties were selected. Cultures of recombinant strains of E. coli in heavy water or in High Cellular Concentration media confirmed the great interest of MMF in microbiology and biotechnology. For example, 60 ml cultures in MMF allowed to obtain 4 times more recombinant protein than with 1000 ml of flask cultures. The Biolux system allows the on line quantification and optimization of the industrial synthesis of recombinant proteins difficult to analyze.
In collaboration with the laboratory of Génétique Moléculaire des Microorganismes (INSA Lyon, Pr. Philippe Lejeune), studies were performed on biofilms formation by Escherichia coli K12, with different wild type strains or adherence gene mutants.
The MMF concept of parallel and standardized cultures offers new possibilities for basic or applied research : physiological studies, improvement of culture media, optimization of fermentation parameters, culture of fastidious strains, high throughput screening of pharmaceutical substances, evolution and selection of strains, using an automated, miniaturized and evolutive equipment.
Photo: Schematic representation of metabolic pathways for indole acetic acid synthesis in Azospirillum brasilense.
1: the indole pyruvate (IpyA) route, 2: the tryptamine (TAM) route, 3: the indole acetonitrile (IAN) route, Trp: tryptophane, IAald: indole acetaldehyde, AIA: indole acetic acid (IAA)