In many Gram-negative bacteria, the GacS͞GacA two-component system positively controls the expression of extracellular products or storage compounds. In the plant-beneficial rhizosphere bacterium Pseudomonas fluorescens CHA0, the GacS͞GacA system is essential for the production of antibiotic compounds and hence for biological control of root-pathogenic fungi. The small (119-nt) RNA RsmX discovered in this study, together with RsmY and RsmZ, forms a triad of GacA-dependent small RNAs, which sequester the RNA-binding proteins RsmA and RsmE and thereby antagonize translational repression exerted by these proteins in strain CHA0. This small RNA triad was found to be both necessary and sufficient for posttranscriptional derepression of biocontrol factors and for protection of cucumber from Pythium ultimum. The same three small RNAs also positively regulated swarming motility and the synthesis of a quorum-sensing signal, which is unrelated to N-acylhomoserine lactones, and which autoinduces the Gac͞Rsm cascade. Expression of RsmX and RsmY increased in parallel throughout cell growth, whereas RsmZ was produced during the late growth phase. This differential expression is assumed to facilitate fine tuning of GacS͞A-controlled cell population density-dependent regulation in P. fluorescens.GacA ͉ posttranscriptional control

Pseudomonas fluorescens CHA0, an antagonist of phytopathogenic fungi in the rhizosphere of crop plants, elaborates and excretes several secondary metabolites with antibiotic properties. Their synthesis depends on three small RNAs (RsmX, RsmY, and RsmZ), whose expression is positively controlled by the GacS-GacA two-component system at high cell population densities. To find regulatory links between primary and secondary metabolism in P. fluorescens and in the related species Pseudomonas aeruginosa, we searched for null mutations that affected central carbon metabolism as well as the expression of rsmY-gfp and rsmZ-gfp reporter constructs but without slowing down the growth rate in rich media. Mutation in the pycAB genes (for pyruvate carboxylase) led to down-regulation of rsmXYZ and secondary metabolism, whereas mutation in fumA (for a fumarase isoenzyme) resulted in up-regulation of the three small RNAs and secondary metabolism in the absence of detectable nutrient limitation. These effects required the GacS sensor kinase but not the accessory sensors RetS and LadS. An analysis of intracellular metabolites in P. fluorescens revealed a strong positive correlation between small RNA expression and the pools of 2-oxoglutarate, succinate, and fumarate. We conclude that Krebs cycle intermediates (already known to control GacA-dependent virulence factors in P. aeruginosa) exert a critical trigger function in secondary metabolism via the expression of GacA-dependent small RNAs.Secondary metabolism occurs in certain bacteria and fungi as part of developmental processes, which are often accompanied by morphological changes (1, 2). In natural environments, secondary metabolites are believed to confer a selective advantage to the producers when these organisms cannot rely on their full growth potential to compete with other organisms (3). Such a role is most plausible for secondary metabolites having antibiotic activities (4). In pure cultures, secondary metabolites are non-essential for the producers and are typically formed when cell population densities are high and growth is restricted. This distinct production phase, sometimes called idiophase, usually follows the phase of optimal growth, also termed trophophase (5). A fundamental question is what triggers the onset of the idiophase. Both extracellular and intracellular signal molecules are known to be involved. For instance, excreted quorum-sensing signal molecules, such as N-acyl-homoserine lactones of Pseudomonas species or ␥-butyrolactones of Streptomyces species, positively regulate the expression of antibiotic compounds, and the intracellular alarmone ppGpp is required for antibiotic production in Streptomyces coelicolor under conditions of nitrogen starvation (1, 6). However, these findings do not provide a generally valid picture of how secondary metabolism is initiated in microorganisms. For instance, some Pseudomonas species produce secondary metabolites without N-acyl-homoserine lactones and phosphate-limited S. coelicolor does not rely on ppGpp for antibio...

Signal extracts prepared from culture supernatants of Pseudomonas fluorescens CHA0 and Pseudomonas aeruginosa PAO stimulated GacA-dependent expression of small RNAs and hence of antibiotic compounds in both hosts. Pseudomonas corrugata LMG2172 and P. fluorescens SBW25 also produced signal molecules stimulating GacA-controlled antibiotic synthesis in strain CHA0, illustrating a novel, N-acyl-homoserine lactoneindependent type of interspecies communication.

Among biocontrol agents that are able to suppress root diseases caused by fungal pathogens, root-colonizing fluorescent pseudomonads have received particular attention because many strains of these bacteria trigger systemic resistance in host plants and produce antifungal compounds and exoenzymes. In general, the expression of these plantbeneficial traits is regulated by autoinduction mechanisms and may occur on roots when the pseudomonads form microcolonies. Three major classes of antibiotic compounds reviewed here in detail (2,4-diacetylphloroglucinol, pyoluteorin and various phenazine compounds) are all produced under cell population density-dependent autoinduction control acting at transcriptional and post-transcriptional levels. This regulation can either be reinforced or attenuated by a variety of chemical signals emanating from the pseudomonads themselves, other microorganisms or root exudates. Signals stimulating biocontrol factor expression via the Gac/Rsm signal transduction pathway in the biocontrol strain Pseudomonas fluorescens CHA0 are synthesized by many different plant-associated bacteria, warranting a more detailed investigation in the future.

Among biocontrol agents that are able to suppress root diseases caused by fungal pathogens, root-colonizing fluorescent pseudomonads have received particular attention because many strains of these bacteria trigger systemic resistance in host plants and produce antifungal compounds and exoenzymes. In general, the expression of these plantbeneficial traits is regulated by autoinduction mechanisms and may occur on roots when the pseudomonads form microcolonies. Three major classes of antibiotic compounds reviewed here in detail (2,4diacetylphloroglucinol, pyoluteorin and various phenazine compounds) are all produced under cell population density-dependent autoinduction control acting at transcriptional and post-transcriptional levels. This regulation can either be reinforced or attenuated by a variety of chemical signals emanating from the pseudomonads themselves, other microorganisms or root exudates. Signals stimulating biocontrol factor expression via the Gac/Rsm signal transduction pathway in the biocontrol strain Pseudomonas fluorescens CHA0 are synthesized by many different plant-associated bacteria, warranting a more detailed investigation in the future.