Conservation of the response regulator gene gacA in Pseudomonas species

Abstract

The response regulator gene gacA influences the production of several secondary metabolites in both pathogenic and beneficial Pseudomonas spp. In this study, we developed primers and a probe for the gacA gene of Pseudomonas species and sequenced a 425 bp fragment of gacA from ten Pseudomonas strains isolated from different plant-associated environments. Polymerase chain reaction analysis and Southern hybridization showed that gacA is highly conserved within the genus Pseudomonas: multiple strains of different Pseudomonas species all responded positively to the probe, whereas no response was obtained from 18 other strains representing 14 species that belong to eight different genera of Gram-negative bacteria other than Pseudomonas. Furthermore, from a total of approximately 550 indigenous bacterial isolates obtained from the rhizosphere of wheat, all isolates that hybridized with the gacA probe were classified as Pseudomonas spp. by group-specific primers. Isolates that did not respond with the gacA probe and primers were identified as bacterial genera other than Pseudomonas, including Stenotrophomonas, Cryseomonas and Comamonas spp. These results indicate that gacA can be used as a complementary genetic marker for detection of Pseudomonas spp. in environmental samples. Phylogenetic relationships inferred from the newly sequenced gacA fragments and the sequences of gacA homologues present in the databases, showed six distinct clusters that correspond to the following bacterial families: Pseudomonaceae, Enterobacteriaceae, Alteromonadaceae, Vibrionaceae, Burkholderia and Xanthomonas. Within the Pseudomonadaceae and Enterobacteriaceae, polymorphisms within gacA and its homologues allowed identification of six and five subclusters respectively. Comparison of the gacA gene and GacA protein-based trees with the tree inferred from 16S rDNA sequences yielded a similar overall clustering. These results suggest that gacA and its homologues may provide complementary markers for phylogenetic studies of Pseudomonas spp. and Gram-negative bacteria other than Pseudomonas.

Soil amoebae rapidly change bacterial community composition in the rhizosphere of Arabidopsis thaliana

“…However, DGGE with specific primers for pseudomonads documented a strong shift in the diversity among these specific Gammaproteobacteria, a result consistent with findings of Rønn et al (2002) who studied effects of protozoa on bacterial communities in soil organic patches. The strong and highly reproducible changes in the gacA-banding pattern further revealed a major shift in this master gene controlling antibiotics production of pseudomonads (De Souza et al, 2003). We suggest that pseudomonads quickly upregulated secondary metabolite production in response to protozoan predators, which is in accordance with Jousset et al (2006) who demonstrated that antibiotics of P. fluorescens are of particular toxicity to protozoa; and that antibiotic-producing P. fluorescens disproportionally benefit from protozoan predation when their bacterial competitors are consumed and nutrients excreted by the protozoan predators (Jousset et al, 2008).…”

Seleção de bactérias endofíticas de tomateiro como potenciais agentes de biocontrole e de promoção de crescimento

“…O DNA genômico foi extraído pelo método do CTAB modificado (SOUZA et al, 2003). O gene 16S rDNA foi amplificado via PCR, utilizando-se os primers 8fn (5'-AGA GTT TGA TCC TGG CTC AG-3') e 1429r (5'-ACG GCT ACC DTT GTT ACG ACT T-3') (ESIKOVA et al, 2002).…”

“…O gene 16S rDNA foi amplificado via PCR, utilizando-se os primers 8fn (5'-AGA GTT TGA TCC TGG CTC AG-3') e 1429r (5'-ACG GCT ACC DTT GTT ACG ACT T-3') (ESIKOVA et al, 2002). As reações de PCR foram feitas conforme Souza et al (2003). Os produtos amplificados foram separados em géis de agarose de acordo com métodos descritos por Sambrook & Russel (2001).…”

Aumento da eficiência nutricional de tomateiros inoculados com bactérias endofíticas promotoras de crescimento

“…O DNA genômico foi extraído pelo método do CTAB modificado, como descrito por Souza et al (2003). O gene 16S rDNA foi amplificado via PCR, utilizando-se os primers 8fn (5'-AGA GTT TGA TCC TGG CTC AG-3') e 1429r (5'-ACG GCT ACC DTT GTT ACG ACT T-3') (Esikova et al, 2002).…”

“…O gene 16S rDNA foi amplificado via PCR, utilizando-se os primers 8fn (5'-AGA GTT TGA TCC TGG CTC AG-3') e 1429r (5'-ACG GCT ACC DTT GTT ACG ACT T-3') (Esikova et al, 2002). As reações de PCR foram feitas como descrito por Souza et al (2003). Os produtos amplificados foram separados em géis de agarose, de acordo com métodos descritos por Sambrook & Russel (2001).…”