Rosemary Loria

Streptomyces scabies 87-22 contains a coronafacic acid-like biosynthetic cluster that contributes to plant-microbe interactions.

Plant-pathogenic Streptomyces spp. cause scab disease on economically important root and tuber crops, the most important of which is potato. Key virulence determinants produced by these species include the cellulose synthesis inhibitor, thaxtomin A, and the secreted Nec1 protein that is required for colonization of the plant host. Recently, the genome sequence of Streptomyces scabies 87-22 was completed, and a biosynthetic cluster was identified that is predicted to synthesize a novel compound similar to coronafacic acid (CFA), a component of the virulence-associated coronatine phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. Southern analysis indicated that the cfa-like cluster in S. scabies 87-22 is likely conserved in other strains of S. scabies but is absent from two other pathogenic streptomycetes, S. turgidiscabies and S. acidiscabies. Transcriptional analyses demonstrated that the cluster is expressed during plant-microbe interactions and that expression requires a transcriptional regulator embedded in the cluster as well as the bldA tRNA. A knockout strain of the biosynthetic cluster displayed a reduced virulence phenotype on tobacco seedlings compared with the wild-type strain. Thus, the cfa-like biosynthetic cluster is a newly discovered locus in S. scabies that contributes to host-pathogen interactions.

Streptomyces turgidiscabies Possesses a Functional Cytokinin Biosynthetic Pathway and Produces Leafy Galls

Streptomyces turgidiscabies, a cause of potato scab, possesses a mobilizable pathogenicity island containing multiple virulence genes and a cytokinin biosynthetic pathway. These biosynthetic genes are homologous and collinear with the fas operon in Rhodococcus fascians. Reverse-transcriptase polymerase chain reaction of S. turgidiscabies demonstrated that all six genes were transcribed in oat bran broth with and without glucose, though transcription was partially repressed by glucose. The supernatant of S. turgidiscabies cultures had cytokinin activity in callus initiation and differentiation assays. Arabidopsis and tobacco plants inoculated with a thaxtomin-deficient mutant (deltanos) produced leafy galls, indistinguishable from those produced by R. fascians. Deletion of the ipt gene in the pathway eliminated gall phenotype. Other symptoms on tobacco included production of hairy roots and de novo meristems.

Streptomyces turgidiscabies Secretes a Novel Virulence Protein, Nec1, Which Facilitates Infection

Emergence of new, economically important plant-pathogenic species in the mostly saprophytic genus Streptomyces involves acquisition of a large, mobile pathogenicity island (PAI). Biosynthetic genes for a phytotoxin, thaxtomin A, are contained on this PAI. The Nec1 protein has necrogenic activity on excised potato tuber tissue, and the encoding gene is highly conserved in plant-pathogenic Streptomyces spp. The G+C content of nec1 indicates lateral transfer from an unrelated taxon; however, the nucleic acid and protein databases have not yielded homologs. Data presented in this article demonstrate that the Nec1 protein is necrogenic when expressed in Escherichia coli and that an active 16-kDa form of Nec1 is secreted from the plant pathogen Streptomyces turgidiscabies. Deletion analysis of nec1 demonstrated that the 151-amino-acid C-terminal region of the Nec1 protein is sufficient to confer necrogenic activity. Analysis of nec1 transcriptional start sites indicates that two mRNA species are produced and that the site of transcription initiation is influenced by glucose. S. turgidiscabies containing a nec1 deletion was greatly compromised in virulence on Arabidopsis thaliana, Nicotiana tabacum, and Raphanus sativus seedlings. The wild-type strain, S. turgidiscabies Car8, aggressively colonized and infected the root meristem of radish, whereas the deltanec1 mutant Car811 did not. Taken together, these data suggest that Nec1 is a secreted virulence protein with a conserved plant cell target that acts early in plant infection.

The Tat pathway exports multiple virulence proteins in the plant pathogen Streptomyces scabies

Streptomyces scabies is one of a group of organisms that causes the economically important disease potato scab. Analysis of the S. scabies genome sequence indicates that it is likely to secrete many proteins via the twin arginine protein transport (Tat) pathway, including several proteins whose coding sequences may have been acquired through horizontal gene transfer and share a common ancestor with proteins in other plant pathogens. Inactivation of the S. scabies Tat pathway resulted in pleiotropic phenotypes including slower growth rate and increased permeability of the cell envelope. Comparison of the extracellular proteome of the wild type and ΔtatC strains identified 73 predicted secretory proteins that were present in reduced amounts in the tatC mutant strain, and 47 Tat substrates were verified using a Tat reporter assay. The ΔtatC strain was almost completely avirulent on Arabidopsis seedlings and was delayed in attaching to the root tip relative to the wild‐type strain. Genes encoding 14 candidate Tat substrates were individually inactivated, and seven of these mutants were reduced in virulence compared with the wild‐type strain. We conclude that the Tat pathway secretes multiple proteins that are required for full virulence.

The Cellobiose Sensor CebR Is the Gatekeeper of Streptomyces scabies Pathogenicity

ABSTRACT A relatively small number of species in the large genus Streptomyces are pathogenic; the best characterized of these is Streptomyces scabies. The pathogenicity of S. scabies strains is dependent on the production of the nitrated diketopiperazine thaxtomin A, which is a potent plant cellulose synthesis inhibitor. Much is known about the genetic loci associated with plant virulence; however, the molecular mechanisms by which S. scabies triggers expression of thaxtomin biosynthetic genes, beyond the pathway-specific activator TxtR, are not well understood. In this study, we demonstrate that binding sites for the cellulose utilization repressor CebR occur and function within the thaxtomin biosynthetic cluster. This was an unexpected result, as CebR is devoted to primary metabolism and nutritive functions in nonpathogenic streptomycetes. In S. scabies, cellobiose and cellotriose inhibit the DNA-binding ability of CebR, leading to an increased expression of the thaxtomin biosynthetic and regulatory genes txtA, txtB, and txtR. Deletion of cebR results in constitutive thaxtomin A production and hypervirulence of S. scabies. The pathogenicity of S. scabies is thus under dual direct positive and negative transcriptional control where CebR is the cellobiose-sensing key that locks the expression of txtR, the key necessary to unlock the production of the phytotoxin. Interestingly, CebR-binding sites also lie upstream of and within the thaxtomin biosynthetic clusters in Streptomyces turgidiscabies and Streptomyces acidiscabies, suggesting that CebR is most likely an important regulator of virulence in these plant-pathogenic species as well. IMPORTANCE What makes a microorganism pathogenic is not limited to the genes acquired for virulence. Using the main causative agent of scab lesions on root and tuber crops as an example, our work identified the subtle but essential genetic changes that generate the cis-acting elements necessary for proper timing of the expression of the cluster of genes responsible for the biosynthesis of thaxtomin A, the primary virulence factor in plant-pathogenic streptomycetes. These data illustrate a situation in which a regulator associated with primary metabolism in nonpathogens, CebR, has been coopted as a master regulator of virulence in pathogenic species. Furthermore, the manipulation of CebR-mediated control of thaxtomin production will facilitate overproduction of this natural and biodegradable herbicide for commercial purposes. Our work thus provides a concrete example of how a strictly theoretical and computational work was able to elucidate a regulatory mechanism associated with the virulence of a plant pathogen and to generate solutions to purely agro-industrial concerns. What makes a microorganism pathogenic is not limited to the genes acquired for virulence. Using the main causative agent of scab lesions on root and tuber crops as an example, our work identified the subtle but essential genetic changes that generate the cis-acting elements necessary for proper timing of the expression of the cluster of genes responsible for the biosynthesis of thaxtomin A, the primary virulence factor in plant-pathogenic streptomycetes. These data illustrate a situation in which a regulator associated with primary metabolism in nonpathogens, CebR, has been coopted as a master regulator of virulence in pathogenic species. Furthermore, the manipulation of CebR-mediated control of thaxtomin production will facilitate overproduction of this natural and biodegradable herbicide for commercial purposes. Our work thus provides a concrete example of how a strictly theoretical and computational work was able to elucidate a regulatory mechanism associated with the virulence of a plant pathogen and to generate solutions to purely agro-industrial concerns.

The ESX/type VII secretion system modulates development, but not virulence, of the plant pathogen Streptomyces scabies.

Streptomyces scabies is a model organism for the investigation of plant-microbe interactions in Gram-positive bacteria. Here, we investigate the type VII protein secretion system (T7SS) in S. scabies; the T7SS is required for the virulence of other Gram-positive bacteria, including Mycobacterium tuberculosis and Staphylococcus aureus. The hallmarks of a functional T7SS are an EccC protein that forms an essential component of the secretion apparatus and two small, sequence-related substrate proteins, EsxA and EsxB. A putative transmembrane protein, EccD, may also be associated with T7S in Actinobacteria. In this study, we constructed strains of the plant pathogen S. scabies carrying marked mutations in genes coding for EccC, EccD, EsxA and EsxB. Unexpectedly, we showed that all four mutant strains retain full virulence towards several plant hosts. However, disruption of the esxA or esxB, but not eccC or eccD, genes affects S. scabies development, including a delay in sporulation, abnormal spore chains and resistance to lysis by the Streptomyces-specific phage ϕC31. We further showed that these phenotypes are specific to the loss of the T7SS substrate proteins EsxA and EsxB, and are not observed when components of the T7SS secretion machinery are lacking. Taken together, these results imply an unexpected intracellular role for EsxA and EsxB.

A re-evaluation of the taxonomy of phytopathogenic genera Dickeya and Pectobacterium using whole-genome sequencing data

The genera Dickeya and Pectobacterium contain important plant pathogens. However, species from these genera are often poorly defined and some new isolates could not be assigned to any of the existing species. Due to their wide geographic distribution and lethality, a reliable and easy classification scheme for these pathogens is urgently needed. The low cost of next-generation sequencing has generated an upsurge of microbial genome sequences. Here, we present a phylogenomic and systematic analysis of the genera Dickeya and Pectobacterium. Eighty-three genomes from these two genera as well as two Brenneria genomes were included in this study. We estimated average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values in combination with the whole-genome-based phylogeny from 895 single-copy orthologous genes using these 85 genomes. Strains with ANI values of ≥96% and isDDH values of ≥70% were consistently grouped together in the phylogenetic tree. ANI, isDDH, and whole-genome-based phylogeny all support the elevation of Pectobacterium carotovorums four subspecies (actinidiae, odoriferum, carotovorum, and brasiliense) to the species level. We also found some strains could not be assigned to any of the existing species, indicating these strains represent novel species. Furthermore, our study revealed at least ten tested genomes from these genera were misnamed in GenBank. This work highlights the potential of using whole genome sequences to re-evaluate current prokaryotic species definition and establish a unified prokaryotic species definition frame for taxonomically challenging genera.

Thaxtomin A production and virulence are controlled by several bld gene global regulators in Streptomyces scabies.

Streptomyces scabies is the main causative agent of common scab disease, which leads to significant annual losses to potato growers worldwide. The main virulence factor produced by S. scabies is a phytotoxic secondary metabolite called thaxtomin A, which functions as a cellulose synthesis inhibitor. Thaxtomin A production is controlled by the cluster-situated regulator TxtR, which activates expression of the thaxtomin biosynthetic genes in response to cello-oligosaccharides. Here, we demonstrate that at least five additional regulatory genes are required for wild-type levels of thaxtomin A production and plant pathogenicity in S. scabies. These regulatory genes belong to the bld gene family of global regulators that control secondary metabolism or morphological differentiation in Streptomyces spp. Quantitative reverse-transcriptase polymerase chain reaction showed that expression of the thaxtomin biosynthetic genes was significantly downregulated in all five bld mutants and, in four of these mutants, this downregulation was attributed to the reduction in expression of txtR. Furthermore, all of the mutants displayed reduced expression of other known or predicted virulence genes, suggesting that the bld genes may function as global regulators of virulence gene expression in S. scabies.

Evidence That Thaxtomin C Is a Pathogenicity Determinant of Streptomyces ipomoeae, the Causative Agent of Streptomyces Soil Rot Disease of Sweet Potato

Streptomyces ipomoeae is the causal agent of Streptomyces soil rot of sweet potato, a disease marked by highly necrotic destruction of adventitious roots, including the development of necrotic lesions on the fleshy storage roots. Streptomyces potato scab pathogens produce a phytotoxin (thaxtomin A) that appears to facilitate their entrance into host plants. S. ipomoeae produces a less-modified thaxtomin derivative (thaxtomin C) whose role in pathogenicity has not been examined. Here, we cloned and sequenced the thaxtomin gene cluster (txt) of S. ipomoeae, and we then constructed targeted txt mutants that no longer produced thaxtomin C. The mutants were unable to penetrate intact adventitious roots but still caused necrosis on storage-root tissue. These results, taken in context with previous histopathological study of S. ipomoeae infection, suggest that thaxtomin C plays an essential role in inter- and intracellular penetration of adventitious sweet potato roots by S. ipomoeae. Once inside the plant host, the pathogen uses one or more yet-to-be-determined factors to necrotize root tissue, including that of any storage roots it encounters.

Engineered P450 biocatalysts show improved activity and regio-promiscuity in aromatic nitration

Nitroaromatics are among the most important and commonly used chemicals but their production often suffers from multiple unsolved challenges. We have previously described the development of biocatalytic nitration processes driven by an engineered P450 TxtE fusion construct. Herein we report the creation of improved nitration biocatalysts through constructing and characterizing fusion proteins of TxtE with the reductase domain of CYP102A1 (P450BM3, BM3R). The majority of constructs contained variable linker length while one was rationally designed for optimizing protein-protein interactions. Detailed biochemical characterization identified multiple active chimeras that showed improved nitration activity, increased coupling efficiency and higher total turnover numbers compared with TxtE. Substrate promiscuity of the most active chimera was further assessed with a substrate library. Finally, a biocatalytic nitration process was developed to nitrate 4-Me-dl-Trp. The production of both 4-Me-5-NO2-l-Trp and 4-Me-7-NO2-l-Trp uncovered remarkable regio-promiscuity of nitration biocatalysts.