Survival of GacS/GacA Mutants of the Biological Control Bacterium Pseudomonas aureofaciens 30-84 in the Wheat Rhizosphere

GacS/GacA comprises a two-component regulatory system that controls the expression of secondary metabolites required for the control of plant diseases in many pseudomonads. High mutation frequencies of gacS and gacA have been observed in liquid culture. We examined whether gacS/gacA mutants could competitively displace the wild-type populations on roots and thus pose a threat to the efficacy of biological control. The survival of a gac mutant alone and in competition with the wild type on roots was examined in the biological control strain Pseudomonas aureofaciens 30-84. In this bacterium, GacS/GacA controls the expression of phenazine antibiotics that are inhibitory to plant pathogenic fungi and enhance the competitive survival of the bacterium. Wheat seedlings were inoculated with strain 30-84, and bacteria were recovered from roots after 21 days in sterile or nonsterile soil to check for the presence of gacS or gacA mutants. Although no mutants were detected in the inoculum, gacS/gacA mutants were recovered from 29 out of 31 roots and comprised up to 36% of the total bacterial populations. Southern hybridization analysis of the recovered gacA mutants did not indicate a conserved mutational mechanism. Replacement series analysis on roots utilizing strain 30-84 and a gacA mutant (30-84.gacA) or a gacS mutant (30-84.A2) demonstrated that although the mutant population partially displaced the wild type in sterile soil, it did not do so in natural soil. In fact, in natural soil final rhizosphere populations of wild-type strain 30-84 starting from mixtures were at least 1.5 times larger than would be predicted from their inoculation ratio and generally were greater than or equal to the population of wild type alone despite lower inoculation rates. These results indicate that although gacS/gacA mutants survive in natural rhizosphere populations, they do not displace wild-type populations. Better survival of wild-type populations in mixtures with mutants suggests that mutants arising de novo or introduced within the inoculum may be beneficial for the survival of wild-type populations in the rhizosphere.     

GacA regulates symbiotic colonization traits of Vibrio fischeri and facilitates a beneficial association with an animal host

The GacS/GacA two-component system regulates the expression of bacterial traits during host association. Although the importance of GacS/GacA as a regulator of virulence is well established, its role in benign associations is not clear, as mutations in either the gacS or gacA gene have little impact on the success of colonization in nonpathogenic associations studied thus far. Using as a model the symbiotic association of the bioluminescent marine bacterium Vibrio fischeri with its animal host, the Hawaiian bobtail squid, Euprymna scolopes, we investigated the role of GacA in this beneficial animal-microbe interaction. When grown in culture, gacA mutants were defective in several traits important for symbiosis, including luminescence, growth in defined media, growth yield, siderophore activity, and motility. However, gacA mutants were not deficient in production of acylated homoserine lactone signals or catalase activity. The ability of the gacA mutants to initiate squid colonization was impaired but not abolished, and they reached lower-than-wild-type population densities within the host light organ. In contrast to their dark phenotype in culture, gacA mutants that reached population densities above the luminescence detection limit had normal levels of luminescence per bacterial cell in squid light organs, indicating that GacA is not required for light production within the host. The gacA mutants were impaired at competitive colonization and could only successfully cocolonize squid light organs when present in the seawater at higher inoculum densities than wild-type bacteria. Although severely impaired during colonization initiation, gacA mutants were not displaced by the wild-type strain in light organs that were colonized with both strains. This study establishes the role of GacA as a regulator of a beneficial animal-microbe association and indicates that GacA regulates utilization of growth substrates as well as other colonization traits.     

The GacS sensor kinase controls phenotypic reversion of small colony variants isolated from biofilms of Pseudomonas aeruginosa PA14

The GacS/GacA two-component regulatory system in pseudomonads regulates genes involved in virulence, secondary metabolism and biofilm formation. Despite these regulatory functions, some Pseudomonas species are prone to spontaneous inactivating mutations in gacA and gacS. A gacS(-) strain of Pseudomonas aeruginosa PA14 was constructed to study the physiological role of this sensor histidine kinase. This loss-of-function mutation was associated with hypermotility, reduced production of acylhomoserine lactones, impaired biofilm maturation, and decreased antimicrobial resistance. Biofilms of the gacS(-) mutant gave rise to phenotypically stable small colony variants (SCVs) with increasing frequency when exposed to silver cations, hydrogen peroxide, human serum, or certain antibiotics (tobramicin, amikacin, azetronam, ceftrioxone, oxacilin, piperacillin or rifampicin). When cultured, the SCV produced thicker biofilms with greater cell density and greater antimicrobial resistance than did the wild-type or parental gacS(-) strains. Similar to other colony morphology variants described in the literature, this SCV was less motile than the wild-type strain and autoaggregated in broth culture. Complementation with gacS in trans restored the ability of the SCV to revert to a normal colony morphotype. These findings indicate that mutation of gacS is associated with the occurrence of stress-resistant SCV cells in P. aeruginosa biofilms and suggests that in some instances GacS may be necessary for reversion of these variants to a wild-type state.     

Phenazine-1-carboxylic acid is negatively regulated and pyoluteorin positively regulated by gacA in Pseudomonas sp. M18

The biosynthesis of antimicrobial metabolites is controlled by the GacS/GacA two-component regulatory system in Pseudomonas species. The production of phenazine-1-carboxylic acid and pyoluteorin is differentially regulated by GacA in Pseudomonas sp. M18. Pyoluteorin was reduced to nondetectable level in culture of the gacA insertional mutant strain M18G grown in King's medium B broth, whereas phenazine-1-carboxylic acid production was increased 30-fold over that of the wild-type strain. Production of both antibiotics was restored to wild-type levels after complementation in trans with the wild-type gacA gene. Expression of the translational fusions phzA'-'lacZ and pltA'-'lacZ confirmed the effect of GacA on both biosynthetic operons.     

Effect of gacS and gacA mutations on colony architecture, surface motility, biofilm formation and chemical toxicity in Pseudomonas sp. KL28

GacS and GacA proteins form a two component signal transduction system in bacteria. Here, Tn5 transposon gacS and gacA (Gac) mutants of Pseudomonas sp. KL28, an alkylphenol degrader, were isolated by selecting for smooth colonies of strain KL28. The mutants exhibited reduced ability to migrate on a solid surface. This surface motility does not require the action of flagella unlike the well-studied swarming motility of other Pseudomonas sp. The Gac mutants also showed reduced levels of biofilm and pellicle formation in liquid culture. In addition, compared to the wild type KL28 strain, these mutants were more resistant to high concentrations of m-cresol but were more sensitive to H2O2, which are characteristics that they share with an rpoS mutant. These results indicate that the Gac regulatory cascade in strain KL28 positively controls wrinkling morphology, biofilm formation, surface translocation and H2O2 resistance, which are important traits for its capacity to survive in particular niches.     

Molecular nature of spontaneous modifications in gacS which cause colony phase variation in Pseudomonas sp. strain PCL1171

Pseudomonas sp. strain PCL1171 displays colony phase variation between opaque phase I and translucent phase II colonies, thereby regulating the production of secondary metabolites and exoenzymes. Complementation and sequence analysis of 26 phase II mutants and of 13 wild-type phase II sectors growing out of phase I colonies showed that in all these cases the phase II phenotype is caused by spontaneous mutations in gacA or/and gacS. Mutation of gac reduced both the length of the lag phase and the generation time. Isolation and sequencing of the gacS genes from the phase II bacteria revealed one insertion as well as several random point mutations, deletions, and DNA rearrangements. Most phase II colonies reverted with a high frequency, resulting in wild-type gacA and gacS genes and a phase I phenotype. Some phase II bacteria retained the phase II phenotype but changed genotypically as a result of (re)introduction of mutations in either gacA or gacS. The reversion of gacA or gacS to the wild type was not affected by mutation of recA and recB. We conclude that in Pseudomonas sp. strain PCL1171, mutations in gacA and gacS are the basis for phase variation from phase I to phase II colonies and that, since these mutations are efficiently removed, mutations in gac result in dynamic switches between the "wild-type" population and the subpopulations harboring spontaneous mutations in gacA and or gacS, thereby enabling both populations to be maintained.     

Genes involved in cyclic lipopeptide production are important for seed and straw colonization by Pseudomonas sp. strain DSS73

Survival in natural bulk soil and colonization of sugar beet seeds and barley straw residues were determined for Pseudomonas sp. strain DSS73 and Tn5 mutants in amsY (encoding a peptide synthetase involved in production of the cyclic lipopeptide amphisin) and gacS (encoding the sensory kinase of the two-component GacA/GacS regulatory system). No differences in survival or growth in response to carbon amendment (citrate) were observed in bulk soil. However, both mutants were impaired in their colonization of sugar beet seeds and barley straw residues by an inoculum established in the bulk soil. The two mutants had comparable colonization phenotypes, suggesting that amphisin production is more important for colonization than other gacS-controlled traits.     

Spontaneous Gac mutants of Pseudomonas biological control strains: cheaters or mutualists?

Bacteria rely on a range of extracellular metabolites to suppress competitors, gain access to resources, and exploit plant or animal hosts. The GacS/GacA two-component regulatory system positively controls the expression of many of these beneficial external products in pseudomonad bacteria. Natural populations often contain variants with defective Gac systems that do not produce most external products. These mutants benefit from a decreased metabolic load but do not appear to displace the wild type in nature. How could natural selection maintain the wild type in the presence of a mutant with enhanced growth? One hypothesis is that Gac mutants are "cheaters" that do not contribute to the public good, favored within groups but selected against between groups, as groups containing more mutants lose access to ecologically important external products. An alternative hypothesis is that Gac mutants have a mutualistic interaction with the wild type, so that each variant benefits by the presence of the other. In the biocontrol bacterium Pseudomonas chlororaphis strain 30-84, Gac mutants do not produce phenazines, which suppress competitor growth and are critical for biofilm formation. Here, we test the predictions of these alternative hypotheses by quantifying interactions between the wild type and the phenazine- and biofilm-deficient Gac mutant within growing biofilms. We find evidence that the wild type and Gac mutants interact mutualistically in the biofilm context, whereas a phenazine-defective structural mutant does not. Our results suggest that the persistence of alternative Gac phenotypes may be due to the stabilizing role of local mutualistic interactions.     

GacS sensor domains pertinent to the regulation of exoproduct formation and to the biocontrol potential of Pseudomonas fluorescens CHA0

In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extracellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system. Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS-), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes. Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs. Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strain's response to added signal. Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities. In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.     

Lipopeptide production in Pseudomonas sp. strain DSS73 is regulated by components of sugar beet seed exudate via the Gac two-component regulatory system

Pseudomonas sp. strain DSS73 isolated from the sugar beet rhizosphere produces the cyclic lipopeptide amphisin, which inhibits the growth of plant-pathogenic fungi. By Tn5::luxAB mutagenesis, we obtained two nonproducing mutant strains, DSS73-15C2 and DSS73-12H8. The gene interrupted by the transposon in strain DSS73-15C2 (amsY) encoded a protein with homology to peptide synthetases that was designated amphisin synthetase. DSS73-12H8 carried the transposon in a regulatory gene encoding a protein with homology to the sensor kinase GacS. Growth of strain DSS73-15C2 (amsY) was impaired during the transition to stationary phase in a minimal medium amended with an exudate of sugar beet seeds. This growth phenotype could be complemented by purified amphisin. Seed exudate further induced expression of bioluminescence from the amsY::luxAB reporter during the transition to stationary phase. This agreed with an increase in amphisin production by the DSS73 wild-type strain during early stationary phase. Amphisin synthesis in DSS73 was strictly dependent on GacS, and even induction by seed exudate depended on a functional gacS locus. Hence, a signal triggering the GacS/GacA two-component system appeared to be present in the seed exudate.     

Pseudomonas aeruginosa GacA, a factor in multihost virulence, is also essential for biofilm formation

We have investigated a potential role for GacA, the response regulator of the GacA/GacS two-component regulatory system, in Pseudomonas aeruginosa biofilm formation. When gacA was disrupted in strain PA14, a 10-fold reduction in biofilm formation capacity resulted relative to wild-type PA14. However, no significant difference was observed in the planktonic growth rate of PA14 gacA(-). Providing gacA in trans on the multicopy vector pUCP-gacA abrogated the biofilm formation defect. Scanning electron microscopy of biofilms formed by PA14 gacA(-) revealed diffuse clusters of cells that failed to aggregate into microcolonies, implying a deficit in biofilm development or surface translocation. Motility assays revealed no decrease in PA14 gacA(-) twitching or swimming abilities, indicating that the defect in biofilm formation is independent of flagellar-mediated attachment and solid surface translocation by pili. Autoinducer and alginate bioassays were performed similarly, and no difference in production levels was observed, indicating that this is not merely an upstream effect on either quorum sensing or alginate production. Antibiotic susceptibility profiling demonstrated that PA14 gacA(-) biofilms have moderately decreased resistance to a range of antibiotics relative to PA14 wild type. This study establishes GacA as a new and independent regulatory element in P. aeruginosa biofilm formation.     

GacA directly regulates expression of several virulence genes in Pseudomonas syringae pv. tabaci 11528

A two-component system comprising GacS and GacA affects a large number of traits in many Gram-negative bacteria. However, the signals to which GacS responds, the regulation mechanism for GacA expression, and the genes GacA controls are not yet clear. In this study, several phenotypic tests and tobacco-leaf pathogenicity assays were conducted using a gacA deletion mutant strain (BL473) of Pseudomonas syringae pv. tabaci 11528. To determine the regulation mechanism for gacA gene expression and to identify GacA-regulated genes, we conducted quantitative RT-PCR and electrophoretic mobility shift assay (EMSA) experiments. The results indicated that virulence traits related to the pathogenesis of P. syringae pv. tabaci 11528 are regulated coordinately by GacA and iron availability. They also revealed that several systems coordinately regulate gacA gene expression in response to iron concentration and bacterial cell density and that GacA and iron together control the expression of several virulence genes. EMSA results provided genetic and molecular evidence for direct control of virulence genes by GacA.     

Thiamine-auxotrophic mutants of Pseudomonas fluorescens CHA0 are defective in cell-cell signaling and biocontrol factor expression

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 x 10(-8) M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.     

Inactivation of gacS does not affect the competitiveness of Pseudomonas chlororaphis in the Arabidopsis thaliana rhizosphere

Quorum-sensing-controlled processes are considered to be important for the competitiveness of microorganisms in the rhizosphere. They affect cell-cell communication, biofilm formation, and antibiotic production, and the GacS-GacA two-component system plays a role as a key regulator. In spite of the importance of this system for the regulation of various processes, strains with a Gac(-) phenotype are readily recovered from natural habitats. To analyze the influence of quorum sensing and the influence of the production of the antibiotic phenazine-1-carboxamide on rhizosphere colonization by Pseudomonas chlororaphis, a gnotobiotic system based on Arabidopsis thaliana seedlings in soil was investigated. Transposon insertion mutants of P. chlororaphis isolate SPR044 carrying insertions in different genes required for the production of N-acyl-homoserine lactones and phenazine-1-carboxamide were generated. Analysis of solitary rhizosphere colonization revealed that after prolonged growth, the population of the wild type was significantly larger than that of the homoserine lactone-negative gacS mutant and that of a phenazine-1-carboxamide-overproducing strain. In cocultivation experiments, however, the population size of the gacS mutant was similar to that of the wild type after extended growth in the rhizosphere. A detailed analysis of growth kinetics was performed to explain this phenomenon. After cells grown to the stationary phase were transferred to fresh medium, the gacS mutant had a reduced lag phase, and production of the stationary-phase-specific sigma factor RpoS was strongly reduced. This may provide a relative competitive advantage in cocultures with other bacteria, because it permits faster reinitiation of growth after a change to nutrient-rich conditions. In addition, delayed entry into the stationary phase may allow more efficient nutrient utilization. Thus, GacS-GacA-regulated processes are not absolutely required for efficient rhizosphere colonization in populations containing the wild type and Gac(-) mutants.