Biocontrol Traits of Pseudomonas spp. Are Regulated by Phase Variation

Abstract

Of 214 Pseudomonas strains isolated from maize rhizosphere, 46 turned out to be antagonistic, of which 43 displayed clear colony phase variation. The latter strains formed both opaque and translucent colonies, designated as phase I and phase II, respectively. It appeared that important biocontrol traits, such as motility and the production of antifungal metabolites, proteases, lipases, chitinases, and biosurfactants, are correlated with phase I morphology and are absent in bacteria with phase II morphology. From a Tn5luxAB transposon library of Pseudomonas sp. strain PCL1171 phase I cells, two mutants exhibiting stable expression of phase II had insertions in gacS. A third mutant, which showed an increased colony phase variation frequency was mutated in mutS. Inoculation of wheat seeds with PCL1171 bacteria of phase I morphology resulted in efficient suppression of take-all disease, whereas disease suppression was absent with phase II bacteria. Neither the gacS nor the mutS mutant was able to suppress take-all, but biocontrol activity was restored after genetic complementation of these mutants. Furthermore, in a number of cases, complementation by gacS of wild-type phase II sectors to phase I phenotype could be shown. A PCL1171 phase I mutant defective in antagonistic activity appeared to have a mutation in a gene encoding a lipopeptide synthetase homologue and had lost its biocontrol activity, suggesting that biocontrol by strain PCL1171 is dependent on the production of a lipopeptide. Our results show that colony phase variation plays a regulatory role in biocontrol by Pseudomonas bacteria by influencing the expression of major biocontrol traits and that the gacS and mutS genes play a role in the colony phase variation process. Therefore phase variation not only plays a role in escaping animal defense but it also appears to play a much broader and vital role in the ecology of bacteria producing exoenzymes, antibiotics, and other secondary metabolites.

Three small RNAs jointly ensure secondary metabolism and biocontrol in Pseudomonas fluorescens CHA0

“…P. fluorescens CHA0 (wild type), CHA19 (⌬gacS), CHA89 (gacA::Km), CHA810 (⌬rsmZ), CHA822 (⌬rsmY), CHA825 (⌬rsmY ⌬rsmZ), CHA1003 (rsmE::⍀-Hg), CHA1008 (⌬gacS rsmE::⍀-Hg rsmA::⍀-Km), CH A1009 (rsmE::⍀-Hg rsmA::⍀-Km), and CH A1076 (rsmA::⍀-Km) and their derivatives carrying chromosomal hcnAЈ-ЈlacZ or aprAЈ-ЈlacZ fusions have been described (14,15,18,19,24). Strains carrying a chromosomal rsmEЈ-ЈlacZ fusion were constructed by using pME7545 for delivery (29). Growth conditions, antibiotic concentrations, and conditions for ␤-galactosidase assays were the same as those previously used (14,15,18).…”

“…Whereas in plant-and animalpathogenic species GacS͞GacA is important for virulence (20,(25)(26)(27), the same system is required for biocontrol in plantbeneficial strains (19,20,24,28,29). We have previously reported that, in P. fluorescens CHA0, GacS͞GacA positively controls transcription initiation of two small RNA genes, rsmZ and rsmY (14,18).…”

Bacterial Genome Instability

“…Most phase-and antigenic-variable proteins have important roles in mediating interactions between bacteria and their environments (393,395). They control the formation of the capsule (428), the pili (429), flagella (430), adhesins (431), antifungal metabolites (432), iron acquisition factors (433), and other surface-exposed molecules (432,434), sometimes affecting motility (432) or colony morphology (434) and opacity (374). Nevertheless, these proteins can also be involved in general cellular pathways, such as DNA restrictionmodification (435), gene regulation (436), or metabolism (437).…”

Secondary Metabolism and Interspecific Competition Affect Accumulation of Spontaneous Mutants in the GacS-GacA Regulatory System in Pseudomonas protegens

“…Given the broad influence of the GacS-GacA system on bacterial physiology, it is striking that spontaneous gacS and gacA mutants (i.e., Gac Ϫ mutants) emerge and accumulate in cultures of Pseudomonas spp. (17,(21)(22)(23)(24)(25)(26)(27). Colonies of these mutants can be distinguished from those of the wild type visually by their expanded colony size, flattened appearance, increased fluorescence under UV light, and lack of extracellular protease production (21,24).…”