Genomic and Metabolomic Analysis of the Potato Common Scab Pathogen Streptomyces scabiei

Streptomyces scabiei is a key causative agent of common scab disease, which causes significant economic losses to potato growers worldwide. This organism produces several phytotoxins that are known or suspected to contribute to host–pathogen interactions and disease development; however, the full metabolic potential of S. scabiei has not been previously investigated. In this study, we used a combined metabolomic and genomic approach to investigate the metabolites that are produced by S. scabiei. The genome sequence was analyzed using antiSMASH and DeepBGC to identify specialized metabolite biosynthetic gene clusters. Using untargeted liquid chromatography-coupled tandem mass spectrometry (LC-MS2), the metabolic profile of S. scabiei was compared after cultivation on three different growth media. MS2 data were analyzed using Feature-Based Molecular Networking and hierarchical clustering in BioDendro. Metabolites were annotated by performing a Global Natural Products Social Molecular Networking (GNPS) spectral library search or using Network Annotation Propagation, SIRIUS, MetWork, or Competitive Fragmentation Modeling for Metabolite Identification. Using this approach, we were able to putatively identify new analogues of known metabolites as well as molecules that were not previously known to be produced by S. scabiei. To our knowledge, this study represents the first global analysis of specialized metabolites that are produced by this important plant pathogen

Regulation of Coronafacoyl Phytotoxin Production by the PAS-LuxR Family Regulator CfaR in the Common Scab Pathogen Streptomyces scabies

Potato common scab is an economically important crop disease that is characterized by the formation of superficial, raised or pitted lesions on the potato tuber surface. The most widely distributed causative agent of the disease is Streptomyces scabies, which produces the phytotoxic secondary metabolite thaxtomin A that serves as a key virulence factor for the organism. Recently, it was demonstrated that S. scabies can also produce the phytotoxic secondary metabolite coronafacoyl-L-isoleucine (CFA-L-Ile) as well as other related metabolites in minor amounts. The expression of the biosynthetic genes for CFA-L-Ile production is dependent on a PAS-LuxR family transcriptional regulator, CfaR, which is encoded within the phytotoxin biosynthetic gene cluster in S. scabies. In this study, we show that CfaR activates coronafacoyl phytotoxin production by binding to a single site located immediately upstream of the putative -35 hexanucleotide box within the promoter region for the biosynthetic genes. The binding activity of CfaR was shown to require both the LuxR and PAS domains, the latter of which is involved in protein homodimer formation. We also show that CFA-L-Ile production is greatly enhanced in S. scabies by overexpression of both cfaR and a downstream co-transcribed gene, orf1. Our results provide important insight into the regulation of coronafacoyl phytotoxin production, which is thought to contribute to the virulence phenotype of S. scabies. Furthermore, we provide evidence that CfaR is a novel member of the PAS-LuxR family of regulators, members of which are widely distributed among actinomycete bacteria

The coronafacoyl phytotoxins: structure, biosynthesis, regulation and biological activities

Phytotoxins are secondary metabolitesthat contribute to the development and/or severity ofdiseases caused by various plant pathogenic microorganisms.The coronafacoyl phytotoxins are an importantfamily of plant toxins that are known or suspectedto be produced by several phylogenetically distinctplant pathogenic bacteria, including the gammaproteobacteriumPseudomonas syringae and the actinobacteriumStreptomyces scabies. At least sevendifferent family members have been identified, ofwhich coronatine was the first to be described and isthe best-characterized. Though nonessential for diseasedevelopment, coronafacoyl phytotoxins appear toenhance the severity of disease symptoms induced bypathogenic microbes during host infection. In addition,the identification of coronafacoyl phytotoxinbiosynthetic genes in organisms not known to be plantpathogens suggests that these metabolites may haveadditional roles other than as virulence factors. Thisreview focuses on our current understanding of thestructures, biosynthesis, regulation, biological activitiesand evolution of coronafacoyl phytotoxins as wellas the different methods that are used to detect thesemetabolites and the organisms that produce them.Keywords Phytotoxins Secondary metabolites Coronatine N-Coronafacoyl-L-isoleucine Pseudomonas Streptomyce

BldG and SCO3548 Interact Antagonistically To Control Key Developmental Processes in Streptomyces coelicolor▿

The similarity of BldG and the downstream coexpressed protein SCO3548 to anti-anti-sigma and anti-sigma factors, respectively, together with the phenotype of a bldG mutant, suggests that BldG and SCO3548 interact as part of a regulatory system to control both antibiotic production and morphological differentiation in Streptomyces coelicolor. A combination of bacterial two-hybrid, affinity purification, and far-Western analyses demonstrated that there was self-interaction of both BldG and SCO3548, as well as a direct interaction between the two proteins. Furthermore, a genetic complementation experiment demonstrated that SCO3548 antagonizes the function of BldG, similar to other anti-anti-sigma/anti-sigma factor pairs. It is therefore proposed that BldG and SCO3548 form a partner-switching pair that regulates the function of one or more sigma factors in S. coelicolor. The conservation of bldG and sco3548 in other streptomycetes demonstrates that this system is likely a key regulatory switch controlling developmental processes throughout the genus Streptomyces

Characterization of the Integration and Modular Excision of the Integrative Conjugative Element PAISt in <i>Streptomyces turgidiscabies</i> Car8

<div><p>PAISt is a large genomic island located in the chromosome of the plant pathogen <i>Streptomyces turgidiscabies</i> Car8. The island carries clustered virulence genes, transfers to other <i>Streptomyces</i> species, and integrates by site-specific recombination at the 8 bp palindrome TTCATGAA. The palindrome is located at the 3′ end of the bacitracin resistance gene (<i>bacA</i>). We demonstrate that PAISt is able to excise in modules by recombination of one internal and two flanking palindromic direct repeats. The gene <i>intSt</i> located at the 3( end of PAISt encodes a tyrosine recombinase. Site-specific recombination activity of <i>intSt</i> was tested and confirmed by heterologous expression in <i>Streptomyces coelicolor</i>. Comparative analysis of PAISt homologues in <i>Streptomyces scabies</i> 87–22 and <i>Streptomyces acidiscabies</i> 84–104 indicates that these islands have been fixed by sequence erosion of <i>intSt</i> and the recombination sites.</p></div

Expression of ccaR, Encoding the Positive Activator of Cephamycin C and Clavulanic Acid Production in Streptomyces clavuligerus, Is Dependent on bldG

In Streptomyces coelicolor, bldG encodes a putative anti-anti-sigma factor that regulates both aerial hypha formation and antibiotic production, and a downstream transcriptionally linked open reading frame (orf3) encodes a putative anti-sigma factor protein. A cloned DNA fragment from Streptomyces clavuligerus contained an open reading frame that encoded a protein showing 92% identity to the S. coelicolor BldG protein and 91% identity to the BldG ortholog in Streptomyces avermitilis. Sequencing of the region downstream of bldG in S. clavuligerus revealed the presence of an open reading frame encoding a protein showing 72 and 69% identity to the ORF3 proteins in S. coelicolor and S. avermitilis, respectively. Northern analysis indicated that, as in S. coelicolor, the S. clavuligerus bldG gene is expressed as both a monocistronic and a polycistronic transcript, the latter including the downstream orf3 gene. High-resolution S1 nuclease mapping of S. clavuligerus bldG transcripts revealed the presence of three bldG-specific promoters, and analysis of expression of a bldGp-egfp reporter indicated that the bldG promoter is active at various stages of development and in both substrate and aerial hyphae. A bldG null mutant was defective in both morphological differentiation and in the production of secondary metabolites, such as cephamycin C, clavulanic acid, and the 5S clavams. This inability to produce cephamycin C and clavulanic acid was due to the absence of the CcaR transcriptional regulator, which controls the expression of biosynthetic genes for both secondary metabolites as well as the expression of a second regulator of clavulanic acid biosynthesis, ClaR. This makes bldG the first regulatory protein identified in S. clavuligerus that functions upstream of CcaR and ClaR in a regulatory cascade to control secondary metabolite production

Integration activity of INTSt is affected by mutation in the catalytic core and deletions in the <i>att</i> sites.

<p>Frequencies of transconjugants obtained when pIJintSt was mobilized into <i>S. coelicolor</i> A3(2). Frequency of integration was calculated based on the number of hygromycin-resistant colonies (y-axis). Point mutations in the putative catalytic core of INTSt reduces integration events. Deletion of the <i>att</i> sites in the plasmid pIJintStatt(−) or in the chromosome of <i>S. coelicolor</i> Δ<i>bacA</i> (pIJintSt_in_Sco att-), abolishes integration events. Plasmid pIJamp001 was used to control for illegitimate recombination (recombination not mediated by INTSt). The data shown are the means of three independent experiments. Three colonies of <i>S. coelicolor</i> were selected and grown to obtain spore stocks. Spore stocks were tested with the integrative plasmids as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099345#s2" target="_blank">material and methods</a>. Means of the integration frequencies were plotted with error bars indicating the distribution of the data.</p

Schematic representation of the PAISt in <i>S. turgidiscabies</i> Car8.

<p>Copies of the 3′ end of the bacitracin resistance gene (<i>bacA</i>) delimit the element in two modules of 105 Kb and 569 Kb. The virulence genes <i>nec1</i> and <i>tomA</i> are located in the first module and the fasciation (<i>fas</i>) and thaxtomin (<i>txt</i>) biosynthetic clusters are located in the second module. The putative integrase (<i>intSt</i>) is located at the 3′ end of the island. The 8 bp palidromic repeats are shown within the <i>bacA</i> gene and its truncated copies.</p

Remnants of <i>intSt</i> exist in PAISt, PAISs1 and PAISa1.

<p>Alignment of INTSt with three frame translations of the 3′ end of the first 105 Kb module of PAISt and with the 3′ ends of PAISs1 and PAISa1 reveal INTSt remnants. Remnants are shown as truncated red arrows. Conserved amino acids residues and their positions in the alignment are indicated.</p