Regulation of Cyclic Lipopeptide Biosynthesis in<i>Pseudomonas fluorescens</i>by the ClpP Protease

Bruijn, Irene de; Raaijmakers, Jos M.

doi:10.1128/jb.01558-08

Cited by 49 publications

(60 citation statements)

References 71 publications

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“…The qRT-PCR analysis was performed six times (technical replicates) using three independent RNA isolations (biological replicates) (de Bruijn et al, 2008;de Bruijn & Raaijmakers, 2009). …”

Section: Methodsmentioning

confidence: 99%

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

et al. 2014

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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...

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“…The qRT-PCR analysis was performed six times (technical replicates) using three independent RNA isolations (biological replicates) (de Bruijn et al, 2008;de Bruijn & Raaijmakers, 2009). …”

Section: Methodsmentioning

confidence: 99%

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

et al. 2014

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“…Crude extracts obtained from 5 to 10 ml of culture supernatant were dried, taken to 100 l of eluent A (acetonitrile/water 6:4 [vol/vol], 0.1% trifluoroacetic acid [TFA]), and loaded onto a C 18 Hypersil octyldecyl silane column (3 m, 250 by 4.6 mm; Thermo Fisher Scientific). An acetonitrile/isopropanol mixture (6:4, vol/vol) containing 0.1% TFA was used as eluent B. Elution was performed at a flow rate of 0.6 ml/min with a linear gradient starting at 100/0 (eluent A/eluent B), which subsequently changed to 20/80 in 80 min.…”

Section: Methodsmentioning

confidence: 99%

Association of Hemolytic Activity ofPseudomonas entomophila, a Versatile Soil Bacterium, with Cyclic Lipopeptide Production

Vallet-Gély

Новиков

Augusto

et al. 2010

Appl Environ Microbiol

124

130

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Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C 10 OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila, including strong biocontrol abilities.

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“…de Bruijn & Raaijmakers (2009). After autoclaving, the medium was cooled down in a water bath to 60 °C.…”

Section: Test Of Bacterial Motilitymentioning

confidence: 99%

“…Serine proteases are one of the most abundant groups of proteolytic enzymes found in all living organisms (Page & Di Cera, 2008) and in prokaryotes, serine proteases are involved in several biological processes associated with cell signaling, defense response and development (Gottesman, 2003, Hengge & Bukau, 2003, Jenal & Hengge-Aronis, 2003. Furthermore, serine protease can be involved in regulating the biosynthesis of lipopeptides, which can play a role in the suppression of other microbes (de Bruijn & Raaijmakers, 2009). …”

Section: Peptidasesmentioning

confidence: 99%

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Volatile communication between fungi and bacteria

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Regulation of Cyclic Lipopeptide Biosynthesis inPseudomonas fluorescensby the ClpP Protease

Abstract: Cyclic lipopeptides produced by

Cited by 49 publications

References 71 publications

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

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

Association of Hemolytic Activity ofPseudomonas entomophila, a Versatile Soil Bacterium, with Cyclic Lipopeptide Production

Volatile communication between fungi and bacteria

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