Use of randomly amplified polymorphic DNA as a means of developing genus- and strain-specific Streptomyces DNA probes

Abstract

We have analyzed 20 randomly amplified polymorphic DNA (RAPD) primers against 36 Streptomyces strains, including 17 taxonomically undefined strains, 25 nonstreptomycete actinomycetes, and 12 outgroups consisting of gram-positive and -negative species. Most of the primers were useful in identifying unique DNA polymorphisms of all strains tested. We have used RAPD techniques to develop a genus-specific probe, one not necessarily targeting the ribosomal gene, for Streptomyces, and a strain-specific probe for the biological control agent Streptomyces lydicus WYEC108. In the course of these investigations, small-scale DNA isolations were also developed for efficiently isolating actinomycete DNA. Various modifications of isolation procedures for soil DNA were compared, and the reliability and specificity of the RAPD methodology were tested by specifically detecting the S. lydicus WYEC108 in DNA isolated from soil.

FIG. 1

Comparison of RAPD profiles of miniprep DNA extraction methods. Isolates were prepared using modifications of the Marmur (M), 5′→3′, Heath (H), Beyazova pretreatment (B), or large-scale Rao (R) methods. Lane 1, 1-kb ladder; lane 2, S. badius (M); lane 3, S. badius (5′→3′); lane 4, S. badius (R); lane 5, S. badius (H); lane 6, empty; lane 7, S. viridosporus T7A (M); lane 8, S. viridosporus T7A (5′→3′); lane 9, S. viridosporus T7A (R); lane 10, S. viridosporus T7A (H); lane 11, empty; lane 12, WYE 27 (M); lane 13, WYE 27 (5′→3′); lane 14, WYE 27 (R); lane 15, empty; lane 16, YCED 5 (M); lane 17, YCED 5 (H); lane 18, YCED 5 (H); lane 19, empty; lane 20, WYEC108 (B); lane 21, WYEC108 (H); lane 22, WYEC108 (R); lane 23, empty; lane 24, WYE 49 (B); lane 25, WYE 49 (B).

FIG. 2

RAPD fingerprint profiles and Southern (65 and 75°C wash) of Streptomyces, suprageneric, and outgroup DNA primed with 1253. (a) RAPD fingerprint profiles of Streptomyces spp. Lane 1, 1-kb ladder; lane 2, Streptomyces lividans 1326; lane 3, Streptomyces lividans TK23; lane 4, Streptomyces antibioticus; lane 5, Streptomyces albus; lane 6, Streptomyces coelicolor; lane 7, Streptomyces rimosus; lane 8, Streptomyces ambofaciens; lane 9, Streptomyces violaceus; lane 10, Streptomyces griseifuscus; lane 11, Streptomyces olivaceus; lane 12, Streptomyces badius 252; lane 13, Streptomyces lividans TK64; lane 14, Streptomyces lividans TK64.1; lane 15, Streptomyces viridosporus T7A; lane 16, Streptomyces plicatus; lane 17, Streptomyces erythraeus; lane 18, Streptomyces rochei A4; lane 19, Streptomyces strain YCED 20; lane 20, Streptomyces strain YCED 36; lane 21, Streptomyces strain YCED 64; lane 22, Streptomyces strain WYE 28; lane 23, Streptomyces strain WYE 79; lane 24, Streptomyces strain WYE 27; lane 25, Streptomyces strain WYE 98; lane 26, Streptomyces lydicus WYEC108; lane 27, Streptomyces hygroscopicus YCED 9; lane 28, Streptomyces strain YCED 33; lane 29, Streptomyces strain WYEC102; lane 30, Streptomyces strain WYEC107. (b) RAPD fingerprint profiles of suprageneric (lanes 3 to 15) and outgroup (lanes 16 to 20) strains. Lane 1, 1-kb ladder; lane 2, Streptomyces lividans 1326, positive control; lane 3, Arthrobacter ramosus; lane 4, Nocardioides simplex; lane 5, Arthrobacter globiformis 607; lane 6, Arthrobacter globiformis 8010; lane 7, Brevibacterium linens; lane 8, Micrococcus luteus; lane 9, Gordonia corallina; lane 10, Rhodococcus opacus; lane 11, Corynebacterium xerosis; lane 12, Dactylosporangium roseum; lane 13, Streptosporangium longisporum; lane 14, Microbispora aerata; lane 15, Frankia sp. strain 33255; lane 16, Burkholderia cepacia DLC 62; lane 17, Bacillus subtilis; lane 18, Pseudomonas putida mt-2; lane 19, Flavobacterium spp.; lane 20, Ralstonia eutropha JMP134. (Photograph, righthand side) Southern blot tests of homology with RAPD PCR products. The probe is a gel-purified 0.8-kb RAPD fragment generated from Streptomyces lividans 1326.

FIG. 3

RAPD fingerprint profiles and Southern blots of Streptomyces, suprageneric, and outgroup DNA primed with 70-34. (a) RAPD fingerprint profiles of Streptomyces spp. Lane 1, DNA mass marker ladder; lane 2, no DNA control; lane 3, Streptomyces lividans 1326; lane 4, Streptomyces antibioticus; lane 5, Streptomyces albus; lane 6, Streptomyces coelicolor; lane 7, Streptomyces rimosus; lane 8, Streptomyces ambofaciens; lane 9, Streptomyces ambofaciens; lane 10, Streptomyces violaceus; lane 11, Streptomyces griseifuscus; lane 12, Streptomyces olivaceus; lane 13, Streptomyces badius 252; lane 14, Streptomyces viridosporus T7A; lane 15, Streptomyces rochei A4; lane 16, Streptomyces lydicus WYEC108; lane 17, Streptomyces hygroscopicus YCED 9; lane 18, Streptomyces hygroscopicus YCED 9; lane 19, Streptomyces plicatus; lane 20, Streptomyces erythraeus. (b) RAPD fingerprint profiles of suprageneric (lanes 3 to 15) and outgroup (lanes 16 to 26) strains. Lane 1, DNA mass marker ladder; lane 2, no DNA control; lane 3, Arthrobacter ramosus; lane 4, Nocardioides simplex; lane 5, Brevibacterium linens; lane 6, Micrococcus luteus; lane 7, Dactylosporangium fulvum; lane 8, Dactylosporangium roseum; lane 9, Corynebacterium xerosis; lane 10, Streptosporangium longisporum; lane 11, Microbispora aerata; lane 12, Frankia sp. strain 33255; lane 13, Gordonia corallina; lane 14, Rhodococcus opacus; lane 15, Thermomonospora mesophila; lane 16, Burkholderia cepacia DLC 62; lane 17, Bacillus subtilis; lane 18, Staphylococcus aureus; lane 19, Flavobacterium spp.; lane 20, Escherichia coli JM101; lane 21, Pseudomonas putida mt-2; lane 22, Ralstonia eutropha JMP134; lane 23, Enterobacter cloacae 96-3; lane 24, Salmonella enterica serovar Typhimurium strain TA 1538; lane 25, Pseudomonas stutzerii KC; lane 26, Clostridium bifermentans KMR-1; lane 27, Streptomyces badius 252, positive control. (Righthand side panels). Southern blot tests of homology with RAPD PCR products. The probe is a gel-purified 0.3-kb RAPD fragment generated from Streptomyces badius 252.

FIG. 4

RAPD fingerprint profiles and Southern blots of Streptomyces, suprageneric, and outgroup DNA primed with 70-40. (a) RAPD fingerprint profiles of Streptomyces spp. Lane 1, DNA mass marker ladder; lane 2, no DNA control; lane 3, Streptomyces lividans 1326; lane 4, Streptomyces antibioticus; lane 5, Streptomyces albus; lane 6, Streptomyces coelicolor; lane 7, Streptomyces rimosus; lane 8, Streptomyces ambofaciens; lane 9, Streptomyces ambofaciens; lane 10, Streptomyces violaceus; lane 11, Streptomyces griseifuscus; lane 12, Streptomyces olivaceus; lane 13, Streptomyces badius 252; lane 14, Streptomyces viridosporus T7A; lane 15, Streptomyces plicatus; lane 16, Streptomyces erythraeus; lane 17, Streptomyces rochei A4; lane 18, Streptomyces lydicus WYEC108; lane 19, Streptomyces hygroscopicus YCED 9; lane 20, Streptomyces hygroscopicus YCED 9. (b) Suprageneric (lanes 3 to 13) and outgroup (lanes 19 to 29) strains. Lane 1, DNA mass marker ladder; lane 2, no DNA control; lane 3, Arthrobacter ramosus; lane 4, Brevibacterium linens; lane 5, Micrococcus luteus; lane 6, Dactylosporangium fulvum; lane 7, Corynebacterium xerosis; lane 8, Streptosporangium longisporum; lane 9, Microbispora aerata; lane 10, Frankia sp. strain 33255; lane 11, Gordonia corallina; lane 12, Rhodococcus opacus; lane 13, Thermomonospora mesophila; lane 14, Streptomyces lydicus WYEC108; lane 15, Streptomyces viridosporus T7A; lane 16, Streptomyces rimosus; lane 17, sterile soil sample inoculated with S. lydicus WYEC108; lane 18, unautoclaved soil sample inoculated with S. lydicus WYEC108; lane 19, Burkholderia cepacia DLC 62; lane 20, Pseudomonas stutzerii KC; lane 21, Staphylococcus aureus; lane 22, Flavobacterium spp.; lane 23, Escherichia coli JM101; lane 24, Pseudomonas putida mt-2; lane 25, Ralstonia eutropha JMP134; lane 26, Enterobacter cloacae 96-3; lane 27, Salmonella enterica serovar Typhimurium TA 1538; lane 28, Clostridium bifermentans KMR-1; lane 29, Bacillus subtilis. (Righthand side panels) Southern blot tests of homology with RAPD PCR products. The probe is a gel-purified 0.3-kb RAPD fragment generated from Streptomyces lydicus WYEC108.

FIG. 5

(a) RAPD fingerprint profile (a) generated with primer 70-40 (lanes 2 to 5) and 16S rDNA primers that were streptomycetes specific and universal CLF-4 (lanes 6 to 10). (b) Southern blot using 0.3-kb S. lydicus WYEC108-specific probe. Lane 1, DNA mass ladder; lane 2, DNA extracted from WYEC108 spores (10 ng of DNA); lane 3, DNA extracted from WYEC108 spores in soil (10 ng); lane 4, 1:100 dilution of WYEC108 spore-amended soil (5 ng); lane 5, positive control template DNA from WYEC108. Lanes 6 to 9 show samples identical to those of lanes 2 to 5 but amplified with 16S rDNA primers. Lane 10, no DNA control.

FIG. 6

(a) RAPD profile of DNA isolated from soil and amplified with primer 70-40. (b) Southern blot probed with the S. lydicus WYEC108 0.3-kb fragment. (a) RAPD fingerprint profile. Lane 1, control field soil; lane 2, control field soil from a different location of the plot; lane 3, bead-mill DNA extraction procedure of 10⁻² dilution (initial inoculum concentration, 3 × 10⁹ CFU g⁻¹); lane 4, bead-mill DNA extraction procedure of 10⁻² dilution of nonsterilized field soil inoculated with WYEC108 spores; lane 5, DNA extracted by the freeze-thaw method from 10⁻³ dilution of sterile inoculated soil; lane 6, DNA extracted by the freeze-thaw method from a 10⁻² dilution of nonsterile inoculated soil; lane 7, DNA extracted by the freeze-thaw method from a 10⁻⁷ dilution of nonsterile inoculated soil; lane 8, WYEC108-positive control template; lane 9, no DNA control; lane 10, DNA mass ladder.