Plant Growth Promoting Rhizobacteria and Biodegradation

PGPR

Some soil bacteria preferentially associate with the roots of crop plants and can exert beneficial effects on their hosts. They are collectively referred to as PGPR (Plant Growth Promoting Rhizobacteria). PGPR strains are often found among the fluorescent pseudomonads.

Biocontrol of phytopathogens appears to be a major mechanism of plant growth promotion by these bacteria. Suppression of phytopathogens results from the production of secondary metabolites or by elicitation of the plant's own defence system. PGPR-based inocula must be able to compete with the indigenous micro-organisms and efficiently colonize the rhizosphere of the plants to be protected.

Previously, we investigated the role of Pseudomonas fluorescens cell envelope proteins (in particular the major outer membrane protein OprF) in adhesion to roots. In addition, it was demonstrated that chemotaxis for root exudates is a major determinant for competitive root colonization. Split-screen movies showing the swimming behavior of wild type versus cheA mutant can be viewed for P. fluorescens strains F113, OE28.3, SBW25, and WCS365.

Scanning electron micrograph of wheat root-colonizing Pseudomonas fluorescens OE 28.3

Current work focuses on the contribution of bacteriocin production to rhizosphere competence of fluorescent Pseudomonas.

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Bacteriocins of fluorescent Pseudomonas

Bacteriocins are peptides or proteins that selectively kill related bacteria (of the same species or genus) but do not affect other organisms. Bacteriocin-mediated antagonism is believed to occur in virtually any niche colonized by bacteria. Bacteriocin production is detectable is most strains of the human opportunistic pathogen Pseudomonas aeruginosa and has been used to differentiate clinical isolates. Only few of these bacteriocins, called pyocins, and their genes have been characterized at the molecular level. Likewise, bacteriocin production is ubiquitous among plant-associated Pseudomonas.

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Domain structure of functionally characterized S-type pyocins of P. aeruginosa (S1, S2, S3, AP41) and of putative bacteriocins from other representative fluorescent Pseudomonas species

A novel type of bacteriocin (LlpA) was characterised in a Pseudomonas isolate from banana rhizosphere. Remarkably, LlpA shows similarity with a group of mannose-binding lectins that were originally discovered in plants but have a much wider distribution, including fungi and animals (Pfam PF01453). Apparently, LlpA constitutes the prototype of a new family of antagonistic proteins. Publications

Model of the C-terminal lectin domain of LlpA [left] and 3D structure of a monomer of garlic lectin ASAI complexed with a-D-mannose (orange) [right]. Stars show the possible mannose-binding sites of Llpa. Conserved tryptophan residues are colored red.

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Biofertilization represents another mechanism used by PGPR to enhance plant growth. The possibility of providing biologically fixed nitrogen to rice plants is explored for an endophytic nitrogen-fixing pseudomonad.

Interaction between Pseudomonas and rice: in vivo expression technology

Rice (Oryza sativa L.) is the most important food crop currently produced and requires extensive use of chemical nitrogen fertilizers. Part of the required nitrogen may be provided by rice-associated or endophytic bacteria that are capable of biological nitrogen fixation, such as strain A15, isolated from rice in China. Although the ability to fix nitrogen by Pseudomonas species has long been debated, we unequivocally identified strain A15 as a diazotrophic Pseudomonas stutzeri.

To study the mechanisms that enable strain A15 to colonise and infect rice roots, and to survive within rice plants, a dapB-based 'in vivo expression technology' (IVET) strategy is used. IVET, originally devised for pathogenic bacteria, is a 'promoter-trapping' technique that traces bacterial promoters driving expression of an essential gene (eg), specifically during interaction with the host.

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General strategy of in vivo expression technology

Publications

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BIODEGRADATION

In recent years there has been an increasing interest in the ability of bacteria to degrade various man-made chemicals and their use for bioremediation of polluted environments. The biochemical pathways involved are best understood for various Gram-negative bacteria. Several Gram-positive bacteria, and actinomycetes (such as Mycobacterium, Rhodococcus, and related species) in particular, are also capable of degrading many recalcitrant compounds.

However, appropriate genetic tools for these bacteria are often lacking. We have developed the E. coli-Rhodococcus shuttle vector pFAJ2574 based on a small cryptic Rhodococcus plasmid (pFAJ2600). Also, a transposon was constructed based on the indigenous Rhodococcus mobile element IS1415, a member of the IS21 family.

As a model system for the breakdown of pesticides by actinomycetes, the biodegradation of thiocarbamate herbicides by Rhodococcus erythropolis NI86/21 was studied. Among the enzymes identified are a dedicated cytochrome P450 system (CYP116) and a non-heme haloperoxidase (ThcF).

Recently, a number of CYP116-related fusion proteins were identified in different bacterial species. These so-called 'P450 PFOR' enzymes constitute a novel class of self-sufficient monooxygenases.

ThcF belongs tot a large subfamily of a/b hydrolases (designated HASH enzymes) for which no natural substrates have been identified yet. Expression of ThcF involves ThcG, a regulatory protein belonging to the recently defined LuxR subfamily 'LAL'. The hallmark of these unusually large regulators (> 800 amino acids) is the presence of an aminoterminal ATP-binding site in addition to the carboxyterminal DNA-binding site.

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3D model of the serine hydrolase ThcF with its predicted catalytic triad highlighted

Publications

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The proteasome of actinomycetes

Our study of herbicide degradation has lead to the fortuitous discovery of the first eubacterial proteasome system in Rhodococcus erythropolis. This work also resulted in the characterization of proteasomes in other actinomycetes.

The proteasome constitutes a molecular machine that enables controlled ATP-dependent breakdown of intracellular proteins. In eukaryotes, the 26S proteasome is built of a 20S proteolytic core complex and one or two 19S regulatory complexes, containing AAA ATPases, attached to one or both ends of the cylinder-shaped core complex. Although other self-compartmentalizing proteases (such as ClpP and HslV) occur in Bacteria, remarkably, genuine 20S proteasomes are only present in Archaea and one phylum of Bacteria, the actinomycetes.

The aim of this work is to elucidate the function of this protease in actinomycetes, using Streptomyces coelicolor as a model. This includes a search for its cellular targets and its regulatory components, an obvious candidate being the AAA-type ATPase ARC.

Structure of the 26S proteasome complex (courtesy of P. Zwickl)

Publications

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