darR and darS are regulatory genes that modulate 2-hexyl, 5-propyl resorcinol transcription in Pseudomonas chlororaphis PCL1606

Abstract

Pseudomonas chlororaphis PCL1606 synthesizes the antifungal antibiotic 2-hexyl, 5-propyl resorcinol (HPR), which is crucial for the biocontrol of fungal soil-borne pathogens. The genetic basis for HPR production lies in the dar genes, which are directly involved in the biosynthesis of HPR. In the present study, we elucidated the genetic features of the dar genes. Reverse transcription PCR experiments revealed an independent organization of the dar genes, except for darBC, which was transcribed as a polycistronic mRNA. In silico analysis of each gene revealed putative promoters and terminator sequences, validating the proposed gene arrangement. Moreover, experiments utilizing 59 rapid amplification of cDNA ends were used to determine the transcriptional initiation sites for the darA, darBC, darS and darR gene promoters, and subsequently to confirm the functionality of these regions. The results of quantitative real-time PCR experiments indicated that biosynthetic dar genes were not only modulated through the global regulator gacS, but also through darS and darR. The interplay between darS and darR revealed transcriptional cross-inhibition. However, these results also showed that other regulatory parameters play a role in HPR production, such as the environmental conditions and additional regulatory genes. INTRODUCTIONPseudomonas chlororaphis PCL1606, isolated from the rhizospheres of healthy avocado trees, has been used in previous studies as an antagonist bacterial strain against different soilborne phytopathogenic fungi, including Rosellinia necatrix, the causal agent of avocado white root rot, and Fusarium oxysporum f. sp. radicis-lycopersici, the causal agent of tomato crown and foot rot (Cazorla et al., 2006). The antifungal antibiotic 2-hexyl, 5-propyl resorcinol (HPR) is a major factor in the antagonistic phenotype of this rhizobacterium (Cazorla et al., 2006), and recent studies have demonstrated a crucial role for this antibiotic in the biocontrol of R. necatrix and F. oxysporum (Calderó n et al., 2013; González-Sánchez et al., 2010).HPR was first detected in an unidentified Pseudomonas sp., and exhibited moderate antifungal and antibacterial properties . The genetic basis for HPR production was first examined in P. chlororaphis subsp. auranthiaca BL915. In this strain, the genes responsible for HPR production are located in a cluster containing three presumptive biosynthetic genes (darA, darB and darC), followed by two independent sequences homologous to the regulatory genes from the AraC/XylS family (darS and darR; Nowak- Thompson et al., 2003). This dar cluster has recently been identified in P. chlororaphis PCL1606 (Calderó n et al., 2013). The construction of insertion mutants for each homologous dar gene in P. chlororaphis PCL1606 and the corresponding complementary derivative strains restoring HPR production revealed a key role for darA and darB genes in both HPR biosynthesis and the antagonistic phenotype (Calderó n et al., 2013). A minor role in HPR biosynthesis has been proposed for darC, as de...

HupO, a Novel Regulator Involved in Thiosulfate-Responsive Control of HupSL [NiFe]-Hydrogenase Synthesis in Thiocapsa roseopersicina

“…A comprehensive search in the databases revealed that diverse proteins showed remarkable similarities to this conserved domain at short regions. A number of regulator proteins can be found among these hits, i.e., a short fragment of the MarR family transcriptional regulator of Pseudomonas chlororaphis or a similar fragment of the DNA-binding transcription factor ADR1 of Saccharomyces cerevisiae (31,32). Additionally, similarities of this region were shown to the DNAdirected RNA polymerase sigma-70 factor of Pseudoalteromonas undina and to ABC transporter permeases of various bacteria among a large number of hits for hypothetical proteins of various organisms.…”

The Compound 2-Hexyl, 5-Propyl Resorcinol Has a Key Role in Biofilm Formation by the Biocontrol Rhizobacterium Pseudomonas chlororaphis PCL1606

“…The wild-type strain PcPCL1606 was used as a reference for antibiotic production (Cazorla et al, 2006). The HPR production was quantified as previously described (Calderón et al, 2014a).…”

“…For chemical complementation assays, PcPCL1606 cultures were grown in a 200 mL TPG medium for 28 h at 25°C under orbital shaking (150 rpm), and the production and accumulation of HPR were confirmed as previously described (Calderón et al, 2014a). The cultures were centrifuged, sterilized by filtration (0.22-μm filter), and the cell-free supernatant was collected.…”

“…The genes responsible for HPR production, the dar genes, which were previously identified in P. chlororaphis subsp. aurantiaca BL915 (Nowak-Thompson et al, 2003), have also been demonstrated to be present in strain PcPCL1606 (Calderón et al, 2013, 2014a). In PcPCL1606, the dar genes are located in a cluster containing three biosynthetic genes ( darA , darB , and darC ), followed by two transcriptional regulatory genes darS and darR , both belonging to the transcriptional regulatory family AraC/XylS.…”

“…In PcPCL1606, the dar genes are located in a cluster containing three biosynthetic genes ( darA , darB , and darC ), followed by two transcriptional regulatory genes darS and darR , both belonging to the transcriptional regulatory family AraC/XylS. In addition, it has been demonstrated that the biosynthetic genes are positively regulated by the DarSR transcriptional regulators, and all the dar genes are under the control of GacS (Calderón et al, 2014a). In addition, some alkylresorcinols (to which the compound HPR belongs) can be proposed to be possible signal molecules in the genus Photorhabdus (Brameyer et al, 2015).…”