ActinoBase - User contributions [en-gb]

HT

2020-02-28T14:59:46Z

REMcHugh: /* Preparation */

=Hickey-Tresner agar (HT)=

This medium was developed by Hickey and Tresner[1] for the growth of some ''Streptomyces'' sp. that did not sporulate well on Bennett's medium. They found that the addition of cobalt improved sporulation of ''S. fradiae'', while adding zinc or iron (instead of cobalt) decreased sporulation.

==Preparation==
Per Litre:
*20 g agar
*1 g yeast extract
*1 g beef extract
*2 g NZamine A
*10 g dextrin
*20 mg CoCl2*6H2O
*up to 1000mL dH2O

'''Instructions'''
#Combine all components and mix well.
#Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

==Uses==
*Sporulation of strains that sporulate poorly on other growth media
*Morphological differentiation (many organisms produce distinctive pigments on this medium)[1]

==Notes==

==References==

[1] Hickey, R. J., & Tresner, H. D. (1952). A cobalt-containing medium for sporulation of ''Streptomyces'' species. Journal of bacteriology, 64(6), 891–892.

[2] Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Conjugation using ET12567/pUZ8002

2019-08-30T14:43:14Z

REMcHugh:

=Strains=
*ET12567/pUZ8002
*Donor "Streptomyces" strain

#Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only
#Inoculate a colony in to 10 ml LB containing Cm, Km and antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and Kanamycin sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.
#Dilute the overnight culture 1:100 in fresh LB + antibiotic selection and grow to an OD600 of 0.4-0.6.
#Wash the cells twice with an equal volume of LB (to remove antibiotics) and resuspend in 0.1 volume of LB
#While washing the E. coli cells, for each conjugation add approximately 108 Streptomyces spires to 500 µl 2 x YT broth, heat shock at 50 oC for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
#Add 500 µl of E. coli cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.
#Plate out on MS agar + 10 mM MgCl2 and incubate at 30 oC for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9.
#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30 oC
#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.

Conjugation using ET12567/pUZ8002

2019-08-30T14:32:45Z

REMcHugh: Created page with "#Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only #Inoculate a colony in to 10 ml LB containing Cm, Km and antibiotic used to se..."

#Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only
#Inoculate a colony in to 10 ml LB containing Cm, Km and antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and Kanamycin sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.
#Dilute the overnight culture 1:100 in fresh LB + antibiotic selection and grow to an OD600 of 0.4-0.6.
#Wash the cells twice with an equal volume of LB (to remove antibiotics) and resuspend in 0.1 volume of LB
#While washing the E. coli cells, for each conjugation add approximately 108 Streptomyces spires to 500 µl 2 x YT broth, heat shock at 50 oC for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
#Add 500 µl of E. coli cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.
#Plate out on MS agar + 10 mM MgCl2 and incubate at 30 oC for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9.
#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30 oC
#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.

Cloning, Transformation and Expression of Gene Clusters

2019-07-26T15:34:15Z

REMcHugh:

[[TAR cloning]]

[[Heterologous expression of gene clusters]]

[[Tri-parental mating]]

Streptomyces goldiniensis

2019-07-26T15:29:45Z

REMcHugh:

{{DISPLAYTITLE:''Streptomyces goldiniensis''}}

Streptomyces goldiniensisis a Gram-positive soil dwelling bacterium that was first isolated from the island of Bermuda in 1969 (1). It produces Aurodox which has been shown to inhibit bacterial growth through binding to Elongation Factor Thermo-Unstable (Ef-Tu) (2), and has also been shown to transcriptionally inhibit Type III Secretion in Escherichia coli (3)

[[File:Sgoldwiki.png]]

References

# Berger J, Lehr H, Teitel S, Maehr H, Grunberg E. 1973. A new antibiotic X-5108 OF Streptomyces origin. I. Production, isolation and properties. J Antibiot (Tokyo) 26:15–22.
# Vogeley L, Palm GJ, Mesters JR, Hilgenfeld R. 2001. Conformational change of elongation factor Tu (EF-Tu) induced by antibiotic binding. Crystal structure of the complex between EF-Tu GDP and aurodox. J Biol Chem 276:17149–17155.
# McHugh RE, O’Boyle N, Connolly JPR, Hoskisson PA, Roe AJ. 2018. Characterisation of the mode of action of Aurodox, a Type III Secretion System inhibitor from Streptomyces goldiniensis. Infect Immun.
# Maehr H, Leach M, Yarmchuk L, Mitrovic M. 1979. Antibiotic X-5108. IX. Chemical conversion of mocimycin to aurodox and derivatives of aurodox, goldinamine and mocimycin. J Antibiot 32:361–367.

Streptomyces goldiniensis

2019-07-26T15:29:06Z

REMcHugh:

{{DISPLAYTITLE:''Streptomyces goldiniensis''}}

Streptomyces goldiniensisis a Gram-positive soil dwelling bacterium that was first isolated from the island of Bermuda in 1969 (1). It produces Aurodox which has been shown to inhibit bacterial growth through binding to Elongation Factor Thermo-Unstable (Ef-Tu) (2), and has also been shown to transcriptionally inhibit Type III Secretion in Escherichia coli (3)

[[File:Sgoldwiki.png]]

# Berger J, Lehr H, Teitel S, Maehr H, Grunberg E. 1973. A new antibiotic X-5108 OF Streptomyces origin. I. Production, isolation and properties. J Antibiot (Tokyo) 26:15–22.
# Vogeley L, Palm GJ, Mesters JR, Hilgenfeld R. 2001. Conformational change of elongation factor Tu (EF-Tu) induced by antibiotic binding. Crystal structure of the complex between EF-Tu GDP and aurodox. J Biol Chem 276:17149–17155.
# McHugh RE, O’Boyle N, Connolly JPR, Hoskisson PA, Roe AJ. 2018. Characterisation of the mode of action of Aurodox, a Type III Secretion System inhibitor from Streptomyces goldiniensis. Infect Immun.
# Maehr H, Leach M, Yarmchuk L, Mitrovic M. 1979. Antibiotic X-5108. IX. Chemical conversion of mocimycin to aurodox and derivatives of aurodox, goldinamine and mocimycin. J Antibiot 32:361–367.

File:Sgoldwiki.png

2019-07-26T15:27:59Z

REMcHugh:

Streptomyces goldiniensis

2019-07-26T15:25:07Z

REMcHugh:

{{DISPLAYTITLE:''Streptomyces goldiniensis''}}

Streptomyces goldiniensisis a Gram-positive soil dwelling bacterium that was first isolated from the island of Bermuda in 1969 (1). It produces Aurodox which has been shown to inhibit bacterial growth through binding to Elongation Factor Thermo-Unstable (Ef-Tu) (2), and has also been shown to transcriptionally inhibit Type III Secretion in Escherichia coli (3)

[[File:Sgoldiniensis.png]]

# Berger J, Lehr H, Teitel S, Maehr H, Grunberg E. 1973. A new antibiotic X-5108 OF Streptomyces origin. I. Production, isolation and properties. J Antibiot (Tokyo) 26:15–22.
# Vogeley L, Palm GJ, Mesters JR, Hilgenfeld R. 2001. Conformational change of elongation factor Tu (EF-Tu) induced by antibiotic binding. Crystal structure of the complex between EF-Tu GDP and aurodox. J Biol Chem 276:17149–17155.
# McHugh RE, O’Boyle N, Connolly JPR, Hoskisson PA, Roe AJ. 2018. Characterisation of the mode of action of Aurodox, a Type III Secretion System inhibitor from Streptomyces goldiniensis. Infect Immun.
# Maehr H, Leach M, Yarmchuk L, Mitrovic M. 1979. Antibiotic X-5108. IX. Chemical conversion of mocimycin to aurodox and derivatives of aurodox, goldinamine and mocimycin. J Antibiot 32:361–367.

Streptomyces goldiniensis

2019-07-26T15:22:01Z

REMcHugh:

=Streptomyces goldiniensis =

Streptomyces goldiniensisis a Gram-positive soil dwelling bacterium that was first isolated from the island of Bermuda in 1969 (1). It produces Aurodox which has been shown to inhibit bacterial growth through binding to Elongation Factor Thermo-Unstable (Ef-Tu) (2), and has also been shown to transcriptionally inhibit Type III Secretion in Escherichia coli (3)

[[File:Sgoldiniensis.png]]

# Berger J, Lehr H, Teitel S, Maehr H, Grunberg E. 1973. A new antibiotic X-5108 OF Streptomyces origin. I. Production, isolation and properties. J Antibiot (Tokyo) 26:15–22.
# Vogeley L, Palm GJ, Mesters JR, Hilgenfeld R. 2001. Conformational change of elongation factor Tu (EF-Tu) induced by antibiotic binding. Crystal structure of the complex between EF-Tu GDP and aurodox. J Biol Chem 276:17149–17155.
# McHugh RE, O’Boyle N, Connolly JPR, Hoskisson PA, Roe AJ. 2018. Characterisation of the mode of action of Aurodox, a Type III Secretion System inhibitor from Streptomyces goldiniensis. Infect Immun.
# Maehr H, Leach M, Yarmchuk L, Mitrovic M. 1979. Antibiotic X-5108. IX. Chemical conversion of mocimycin to aurodox and derivatives of aurodox, goldinamine and mocimycin. J Antibiot 32:361–367.

File:Sgoldiniensis.png

2019-07-26T15:19:32Z

REMcHugh:

Streptomyces goldiniensis

2019-07-26T15:15:56Z

REMcHugh:

=Streptomyces goldiniensis =

Streptomyces goldiniensisis a Gram-positive soil dwelling bacterium that was first isolated from the island of Bermuda in 1969 (1). It produces Aurodox which has been shown to inhibit bacterial growth through binding to Elongation Factor Thermo-Unstable (Ef-Tu) (2), and has also been shown to transcriptionally inhibit Type III Secretion in Escherichia coli (3)

[[File:Streptomyces goldiniensis.png]]

# Berger J, Lehr H, Teitel S, Maehr H, Grunberg E. 1973. A new antibiotic X-5108 OF Streptomyces origin. I. Production, isolation and properties. J Antibiot (Tokyo) 26:15–22.
# Vogeley L, Palm GJ, Mesters JR, Hilgenfeld R. 2001. Conformational change of elongation factor Tu (EF-Tu) induced by antibiotic binding. Crystal structure of the complex between EF-Tu GDP and aurodox. J Biol Chem 276:17149–17155.
# McHugh RE, O’Boyle N, Connolly JPR, Hoskisson PA, Roe AJ. 2018. Characterisation of the mode of action of Aurodox, a Type III Secretion System inhibitor from Streptomyces goldiniensis. Infect Immun.
# Maehr H, Leach M, Yarmchuk L, Mitrovic M. 1979. Antibiotic X-5108. IX. Chemical conversion of mocimycin to aurodox and derivatives of aurodox, goldinamine and mocimycin. J Antibiot 32:361–367.

File:Streptomyces goldiniensis.png

2019-07-26T15:11:17Z

REMcHugh:

Streptomyces goldiniensis

2019-07-26T14:46:18Z

REMcHugh:

=Streptomyces goldiniensis =

Streptomyces goldiniensisis a Gram-positive soil dwelling bacterium that was first isolated from the island of Bermuda in 1969 (1). It produces Aurodox which has been shown to inhibit bacterial growth through binding to Elongation Factor Thermo-Unstable (Ef-Tu) (2), and has also been shown to transcriptionally inhibit Type III Secretion in Escherichia coli (3)

# Berger J, Lehr H, Teitel S, Maehr H, Grunberg E. 1973. A new antibiotic X-5108 OF Streptomyces origin. I. Production, isolation and properties. J Antibiot (Tokyo) 26:15–22.
# Vogeley L, Palm GJ, Mesters JR, Hilgenfeld R. 2001. Conformational change of elongation factor Tu (EF-Tu) induced by antibiotic binding. Crystal structure of the complex between EF-Tu GDP and aurodox. J Biol Chem 276:17149–17155.
# McHugh RE, O’Boyle N, Connolly JPR, Hoskisson PA, Roe AJ. 2018. Characterisation of the mode of action of Aurodox, a Type III Secretion System inhibitor from Streptomyces goldiniensis. Infect Immun.
# Maehr H, Leach M, Yarmchuk L, Mitrovic M. 1979. Antibiotic X-5108. IX. Chemical conversion of mocimycin to aurodox and derivatives of aurodox, goldinamine and mocimycin. J Antibiot 32:361–367.

Streptomyces goldiniensis

2019-07-26T14:36:17Z

REMcHugh: Created page with "=Streptomyces goldiniensis = Streptomyces goldiniensis is a Gram-positive soil dwelling bacterium that was first isolated from the island of Bermuda in 1969..."

=Streptomyces goldiniensis =

Streptomyces goldiniensis is a Gram-positive soil dwelling bacterium that was first isolated from the island of Bermuda in 1969 (1). It produces Aurodox which has been shown to inhibit bacterial growth through binding to Elongation Factor Thermo-Unstable (Ef-Tu) (2), and has also been shown to transcriptionally inhibit Type III Secretion in Escherichia coli (3)

Streptomyces

2019-07-26T13:56:50Z

REMcHugh:

[https://en.wikipedia.org/wiki/Streptomyces Streptomyces] bacteria comprise at least 600 known species and thousands of strain variants. They are filamentous, sporulating bacteria best know for their prolific production of secondary metabolites, many of which are used in human medicine. [https://en.wikipedia.org/wiki/Selman_Waksman Selman Waksman] made them famous by identifying the first antibiotics from these bacteria, including [https://en.wikipedia.org/wiki/Streptomycin streptomycin] which is used to treat [https://www.who.int/news-room/fact-sheets/detail/tuberculosis tuberculosis], but the field of Streptomyces genetics was founded by [https://en.wikipedia.org/wiki/David_Hopwood Sir David Hopwood FRS] when he moved to Norwich to become John Innes Professor of Genetics at the newly established [http://www.uea.ac.uk University of East Anglia] in 1968. He set up his lab at the [http://www.jic.ac.uk John Innes Centre] , which had moved to Norwich to forge links with UEA, and recruited [https://en.wikipedia.org/wiki/Keith_Chater Keith Chater], [https://en.wikipedia.org/wiki/Mervyn_Bibb Mervyn Bibb] and [https://www.jic.ac.uk/people/professor-mark-buttner/ Mark Buttner] who have all made outstanding contributions to the field and trained many of the scientists who now work on Streptomyces bacteria all over the world.

Essential reading on the taxonomy of streptomycetes: [https://www.hutchingslab.uk/Downloads/Kampfer.pdf Kämpfer et al (2014) The Family Streptomycetaceae, in The Prokaryotes – Actinobacteria]

A fascinating account of the history of Streptomyces research by Sir David Hopwood FRS: [https://global.oup.com/academic/product/streptomyces-in-nature-and-medicine-9780195150667?cc=gb&lang=en& Streptomyces in nature and medicine]

[[File:Streptomyces.png | Streptomyces coelicolor growing on SFM agar | thumb]]

[[Streptomyces albidoflavus]] strains are found all over the world in soil and marine habitats and associated with insects.

[[Streptomyces avermitilis]] - known for producing ivermectin.

[[Streptomyces clavuligerus]] - known for producing clavulanic acid.

[[Streptomyces coelicolor]] - a model organism for more than 50 years and the first to be sequenced in 2002.

[[Streptomyces formicae]] - known for making formicamycins.

[[Streptomyces goldiniensis]] - known for producing aurodox.

[[Streptomyces griseus]] - known for producing streptomycin.

[[Streptomyces venezuelae]] - a model organism that completes its life cycle in 24 hours and sporulates in liquid culture.