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

Abstract

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.

Figure 1

Genetic organization of the type VII secretion system (T7SS) coding region in Streptomyces scabies. (A) Organization of the genes coding for T7SS components on the chromosome of S. scabies. The numbers show the sizes of the intergenic regions between each gene, in base pairs (bp). (B) The esxA and esxB genes are co‐expressed. RNA was isolated from S. scabies cultured on tryptone soy broth (TSB) or thaxtomin defined growth medium (TDM) and cDNA was prepared as described in Experimental procedures. cDNA was used as a template in polymerase chain reactions (PCRs) using primers that amplify within esxA or esxB, or across the esxA–esxB or esxB–scab58661 junctions. The expected sizes of the amplified products are indicated to the right. The RNA sample without the reverse transcriptase step served as a template for a negative control and yielded no products (and is therefore not shown). Chromosomal DNA was also used as a template with the same primers as a positive control. Samples were analysed on the same agarose gel; intervening lanes have been excised for clarity.

Figure 2

Growth analysis of Streptomyces scabies strains carrying mutations in the esxA, esxB, eccC and eccD genes. (A–I) Phenotypic analysis of growth on solid media. Equal numbers of spores of strain 87.22 (wild type, WT), JKF1 (eccC ^–), JKF2 (eccD ^–), JKF3 (esxB ^–) and JKF4 (esxA ^–) strains were streaked from glycerol stock and incubated at 30 °C for 5 days. The different growth media used were soyflour mannitol (A), oatbran (B), instant potato mash (C), R2 (D), R2 with yeast extract (E), minimal with 1% mannitol (F), minimal with 1% glucose (G), Difco nutrient agar (H) and yeast malt extract (I). The images shown are representative examples from five independent growth experiments. (J) Growth rate analysis of the same strains in tryptone soy broth. Aliquots of tryptone soy broth (100 mL) were inoculated with spores of the strain of interest to a density of 1 × 10⁶ spores/mL of culture medium, and incubated at 30 °C with shaking. Samples (1 mL) were withdrawn from the cultures every 3 h and total cytosolic protein was prepared as described in Experimental procedures. Error bars represent the standard error of the mean, where n = 3.

Figure 3

Scanning electron microscopy images of spore chains from the following Streptomyces scabies strains: (A, F) 87.22 (wild‐type); (B, G) JKF1 (eccC ^–); (C, H) JKF2 (eccD ^–); (D, I) JKF3 (esxB ^–); (E, J) JKF4 (esxA ^–). The strains were inoculated onto instant potato mash agar from glycerol stock and incubated at 30 °C for 10 days.

Figure 4

Partial complementation of the developmental defect of the Streptomyces scabies esxA and esxB strains is afforded by supplying an in trans copy of esxAB. Equal numbers of spores of strains 87.22 (wild‐type, WT), 87.22/pesxAB, JKF3 (esxB ^–), JKF3/pesxAB, JKF4 (esxA ^–) and JKF4/pesxAB were streaked from glycerol stock onto yeast malt extract medium and incubated at 30 °C for the indicated time periods.

Figure 5

The Streptomyces scabies esxA and esxB mutant strains are resistant to infection by bacteriophage ϕC31. Approximately 10⁸ spores of S. scabies strains 87.22 (wild‐type, WT), JKF1 (eccC ^–), JKF2 (eccD ^–), JKF3 (esxB ^–) and JKF4 (esxA ^–) were mixed with a suspension of bacteriophage ϕR4 (top panel) or ϕC31 (bottom panel). Plates were incubated at 30 °C for 18 h to allow plaque formation prior to being photographed.

Figure 6

The Streptomyces scabies eccC ^–, eccD ^–, esxB ^– and esxA ^– strains show necrosis on potato tuber slices and are virulent for infection in the radish root assay. (A) Potato tuber slices in Petri dishes were inoculated spore side down with agar plugs from plates of each of the indicated strains showing confluent sporulation, as described in Experimental procedures. The Petri dishes were sealed with parafilm and incubated in the dark at room temperature for 7 days prior to being photographed. (B) Radish seeds, which had been grown as described in Experimental procedures, were transferred to water agar and inoculated with equal numbers of spores of each of the indicated S. scabies strains. Petri dishes were sealed with parafilm and incubated at 21 ± 2 °C with a 16‐h photoperiod for 6 days prior to the excision of roots from the agar. Seedlings were assessed for disease symptoms and photographed. Scale bars, 1 cm.

Figure 7

The Streptomyces scabies eccC ^–, eccD ^–, esxB ^– and esxA ^– strains show virulence on Nicotiana tabacum. Tobacco seedlings, germinated in individual magenta boxes as described in Experimental procedures, were inoculated with approximately 5 × 10⁷ spores of the indicated strains. Plants were incubated at 21 ± 2 °C with a 16‐h photoperiod for 9 weeks prior to being photographed from the side (A), above (B) and below (C). Scale bars, 2 cm.