Anne-Marie Simao-Beaunoir

Genetic and physiological determinants of Streptomyces scabies pathogenicity.

UNLABELLED SUMMARY Common scab is a severe disease worldwide affecting tap root crops and potato tubers. It is caused by soil-borne filamentous bacteria belonging to the genus Streptomyces. Streptomycetes usually are saprophytic microorganisms, but a few species have acquired the ability to infect underground plant tissues. The predominant causal agent of potato scab worldwide is Streptomyces scabies. The production of phytotoxins called thaxtomins is essential for the virulence of common scab-causing agents. The genes involved in the biosynthetic pathway of thaxtomins and other virulence genes are clustered on a large pathogenicity island. The pathogenicity island can be mobilized and transferred to nonpathogenic relatives, leading to the emergence of new pathogenic streptomycetes. In most pathogenic Streptomyces species, thaxtomin A is the predominant form found. The regulation of thaxtomin A synthesis is complex. Although the plant-derived compound cellobiose is now recognized as the inducer of thaxtomin A synthesis at a genetic level, other molecules (including aromatic amino acids and some secondary metabolites) show inhibitory effects on the production of the toxin. This paper is an overview of common scab with a focus on S. scabies and its virulence mechanisms. TAXONOMY Streptomyces scabies (Thaxt.) Lambert and Loria; Kingdom Bacteria; Phylum Actinobacteria; Class Actinomycetes; Order Actinomycetales; Family Streptomycetaceae; genus Streptomyces; species scabies or scabiei. HOST RANGE Streptomyces scabies (syn. S. scabiei) has a broad host range comprising tuber vegetables and most tap root crops. Streptomyces scabies causes common scab on potato (Solanum tuberosum), beet (Beta vulgaris), carrot (Daucus carota), parsnip (Pastinaca sativa), radish (Raphanus sativus), rutabaga (Brassica napobrassica) and turnip (Brassica rapa). Disease symptoms: Common scab symptoms appear as randomly distributed shallow, raised or deep-pitted corky lesions. Their size and colour are quite variable, but lesions typically are brown with a diameter of a few millimetres. No above-ground symptoms disclose the presence of the disease as aerial tissues of scab-infected plants remain healthy. Streptomyces scabies also inhibits the growth of seedlings in monocot and dicot plants. USEFUL WEBSITES http://www.sanger.ac.uk/Projects/S_scabies, http://www.potatodiseases.org/scab.html, http://www.uri.edu/ce/factsheets/sheets/potatoscab.html.

Involvement of the Plant Polymer Suberin and the Disaccharide Cellobiose in Triggering Thaxtomin A Biosynthesis, a Phytotoxin Produced by the Pathogenic Agent Streptomyces scabies

Streptomyces scabies is a gram-positive soil bacterium recognized as the main causal agent of common scab. Pathogenicity in Streptomyces spp. depends on their capacity to synthesize phytotoxins called thaxtomins. Genes involved in biosynthesis of these secondary metabolites are known to be induced by cellobiose, a plant disaccharide. However, growth of S. scabies in a minimal medium containing cellobiose as a carbon source is very poor and only generates traces of thaxtomins. The effect of suberin, a lipid plant polymer, on thaxtomin A biosynthesis and the expression of genes involved in its biosynthetic pathway was analyzed. S. scabies was grown in a starch-containing minimal medium supplemented with cellobiose (0.5%), suberin (0.1%), or both. The presence of both cellobiose and suberin doubled bacterial growth and triggered thaxtomin A production, which correlated with the upregulation (up to 342-fold) of genes involved in thaxtomins synthesis. The addition of either suberin or cellobiose alone did not affect these parameters. Suberin appeared to stimulate the onset of secondary metabolism, which is a prerequisite to the production of molecules such as thaxtomin A, while cellobiose induced the biosynthesis of this secondary metabolite.

Effect of potato suberin on Streptomyces scabies proteome

Two-dimensional (2D) PAGE was used to detect proteins induced in Streptomyces scabies by potato suberin, a lipidic plant polymer. Nineteen up-regulated proteins were excised from 2D gels and analysed by N-terminal sequencing or tandem mass spectrometry (MS/MS). Four of the up-regulated proteins could be linked to the bacterial response to stress (AldH, GroES, TerD and LexA). Specific metabolic pathways seemed to be activated in the presence of suberin, as shown by the increased expression of specific transporters and of enzymes related not only to glycolysis, but also to nucleotide and amino acid metabolism. Suberin also appeared to influence secondary metabolism as it also caused the overproduction of the BldK proteins that are known to be involved in differentiation and secondary metabolism.

Comparative secretome analysis of Streptomyces scabiei during growth in the presence or absence of potato suberin.

BackgroundSuberin is a recalcitrant plant biopolymer composed of a polyphenolic and a polyaliphatic domain. Although suberin contributes to a significant portion of soil organic matter, the biological process of suberin degradation is poorly characterized. It has been suggested that Streptomyces scabiei, a plant pathogenic bacterium, can produce suberin-degrading enzymes. In this study, a comparative analysis of the S. scabiei secretome from culture media supplemented or not with potato suberin was carried out to identify enzymes that could be involved in suberin degradation.MethodsS. scabiei was grown in the presence of casein only or in the presence of both casein and suberin. Extracellular proteins from 1-, 3- and 5-day-old supernatants were analyzed by LC-MS/MS to determine their putative functions. Real-time RT-PCR was performed to monitor the expression level of genes encoding several proteins potentially involved in suberin degradation.ResultsThe effect of suberin on the extracellular protein profile of S. scabiei strain has been analyzed. A total of 246 proteins were found to be common in the data sets from both casein medium (CM) and casein-suberin medium (CSM), whereas 124 and 139 proteins were detected only in CM or CSM, respectively. The identified proteins could be divided into 19 functional groups. Two functional groups of proteins (degradation of aromatic compounds and secondary metabolism) were only associated with the CSM. A high proportion of the proteins found to be either exclusively produced, or overproduced, in presence of suberin were involved in carbohydrate metabolism. Most of the proteins included in the lipid metabolism class have been detected in CSM. Apart from lipid metabolism proteins, other identified proteins, particularly two feruloyl esterases, may also actively participate in the breakdown of suberin architecture. Both feruloyl esterase genes were overexpressed between 30 to 340 times in the presence of suberin.ConclusionThis study demonstrated that the presence of suberin in S. scabiei growth medium induced the production of a wide variety of glycosyl hydrolases. Furthermore, this study has allowed the identification of extracellular enzymes that could be involved in the degradation of suberin, including enzymes of the lipid metabolism and feruloyl esterases.

Detection of potential suberinase-encoding genes in Streptomyces scabiei strains and other actinobacteria

Streptomyces scabiei causes common scab, an economically important disease of potato tubers. Some authors have previously suggested that S. scabiei penetration into host plant tissue is facilitated by secretion of esterase enzymes degrading suberin, a lipidic biopolymer of the potato periderm. In the present study, S. scabiei EF-35 showed high esterase activity in suberin-containing media. This strain also exhibited esterase activity in the presence of other biopolymers, such as lignin, cutin, or xylan, but at a much lower level. In an attempt to identify the esterases involved in suberin degradation, translated open reading frames of S. scabiei 87-22 were examined for the presence of protein sequences corresponding to extracellular esterases of S. scabiei FL1 and of the fungus Coprinopsis cinerea VTT D-041011, which have previously been shown to be produced in the presence of suberin. Two putative extracellular suberinase genes, estA and sub1, were identified. The presence of these genes in several actinobacteria was investigated by Southern blot hybridization, and both genes were found in most common-scab-inducing strains. Moreover, reverse transcription - polymerase chain reaction performed with S. scabiei EF-35 showed that estA was expressed in the presence of various biopolymers, including suberin, whereas the sub1 gene appeared to be specifically expressed in the presence of suberin and cutin.

Morphological, Physiological, and Taxonomic Characterization of Actinobacterial Isolates Living as Endophytes of Cacao Pods and Cacao Seeds

Vascular plants are commonly colonized by endophytic actinobacteria. However, very little is known about the relationship between these microorganisms and cacao fruits. In order to determine the physiological and taxonomic relationships between the members of this community, actinobacteria were isolated from cacao fruits and seeds. Among the 49 isolates recovered, 11 morphologically distinct isolates were selected for further characterization. Sequencing of the 16S rRNA gene allowed the partition of the selected isolates into three phylogenetic clades. Most of the selected endophytic isolates belonged to the Streptomyces violaceusniger clade. Physiological characterization was carried out and a similarity index was used to cluster the isolates. However, clustering based on physiological properties did not match phylogenetic lineages. Isolates were also characterized for traits commonly associated with plant growth-promoting bacteria, including antibiosis and auxin biosynthesis. All isolates exhibited resistance to geldanamycin, whereas only two isolates were shown to produce this antibiotic. Endophytes were inoculated on radish seedlings and most isolates were found to possess plant growth-promoting abilities. These endophytic actinobacteria inhibited the growth of various plant pathogenic fungi and/or bacteria. The present study showed that S. violaceusniger clade members represent a significant part of the actinobacterial community living as endophytes in cacao fruits and seeds. While several members of this clade are known to be geldanamycin producers and efficient biocontrol agents of plant diseases, we herein established the endophytic lifestyle of some of these microorganisms, demonstrating their potential as plant health agents.

Suberin Regulates the Production of Cellulolytic Enzymes in Streptomyces scabiei, the Causal Agent of Potato Common Scab

Suberin, a major constituent of the potato periderm, is known to promote the production of thaxtomins, the key virulence factors of the common scab-causing agent Streptomyces scabiei. In the present study, we speculated that suberin affected the production of glycosyl hydrolases, such as cellulases, by S. scabiei, and demonstrated that suberin promoted glycosyl hydrolase activity when added to cellulose-, xylan-, or lichenin-containing media. Furthermore, secretome analyses revealed that the addition of suberin to a cellulose-containing medium increased the production of glycosyl hydrolases. For example, the production of 13 out of the 14 cellulases produced by S. scabiei in cellulose-containing medium was stimulated by the presence of suberin. In most cases, the transcription of the corresponding cellulase-encoding genes was also markedly increased when the bacterium was grown in the presence of suberin and cellulose. The level of a subtilase-like protease inhibitor was markedly decreased by the presence of suberin. We proposed a model for the onset of S. scabiei virulence mechanisms by both cellulose and suberin, the main degradation product of cellulose that acts as an inducer of thaxtomin biosynthetic genes, and suberin promoting the biosynthesis of secondary metabolites including thaxtomins.

Physical, Chemical and Proteomic Evidence of Potato Suberin Degradation by the Plant Pathogenic Bacterium Streptomyces scabiei

Potato peels consist of a tissue called phellem, which is formed by suberized cell layers. The degradation of suberin, a lipidic and recalcitrant polymer, is an ecological process attributed to soil fungal populations; however, previous studies have suggested that Streptomyces scabiei, the causal agent of potato common scab, possesses the ability to degrade suberin. In the present study, S. scabiei was grown in medium containing suberin-enriched potato phellem as the sole carbon source and its secretome was analyzed periodically (10- to 60-d-old cultures) with a special focus on proteins potentially involved in cell wall degradation. Although the amount and diversity of proteins linked to polysaccharide degradation remained high throughout the experiment, their abundance decreased over time. In contrast, proteins dedicated to lipid metabolism represented a small fraction of the secretome; however, their abundance increased during the experiment. The lipolytic enzymes detected may be involved in the degradation of the aliphatic fraction of suberin because the results of optical and transmission electron microscopy examinations revealed a loss in the integrity of suberized tissues exposed to S. scabiei cells. Chemical analyses identified a time period in which the concentration of aliphatic compounds in potato phellem decreased and the sugar concentration increased; at the end of the 60-d incubation period, the sugar concentration in potato phellem was significantly reduced. This study demonstrated the ability of S. scabiei to degrade the aliphatic portion of suberin.

Proteome Analyses of Soil Bacteria Grown in the Presence of Potato Suberin, a Recalcitrant Biopolymer

Suberin is a complex lipidic plant polymer found in various tissues including the potato periderm. The biological degradation of suberin is attributed to fungi. Soil samples from a potato field were used to inoculate a culture medium containing suberin as the carbon source, and a metaproteomic approach was used to identify bacteria that developed in the presence of suberin over a 60-d incubation period. The normalized spectral counts of predicted extracellular proteins produced by the soil bacterial community markedly decreased from day 5 to day 20 and then slowly increased, revealing a succession of bacteria. The population of fast-growing pseudomonads declined and was replaced by species with the ability to develop in the presence of suberin. The recalcitrance of suberin was demonstrated by the emergence of auxotrophic bacteria such as Oscillatoria on the last days of the assay. Nevertheless, two putative lipases from Rhodanobacter thiooxydans (I4WGM2) and Myxococcus xanthus (Q1CWS1) were detected in the culture supernatants, suggesting that at least some bacterial species degrade suberin. When grown in suberin-containing medium, R. thiooxydans strain LCS2 and M. xanthus strain DK 1622 both produced three lipases, including I4WGM2 and Q1CWS1. These strains also produced other proteins linked to lipid metabolism, including fatty acid and lipid transporters and β-oxidation enzymes, suggesting that they participate in the degradation of suberin. However, only the R. thiooxydans strain appeared to retrieve sufficient carbon and energy from this recalcitrant polymer in order to maintain its population over an extended period of time.