ActinoBase - User contributions [en-gb]

Streptomyces clavuligerus

2019-09-27T14:34:26Z

ASBirke: /* Streptomyces clavuligerus */

=''[[Streptomyces clavuligerus]]''=

''[[Streptomyces clavuligerus]]'' was originally isolated from a South American soil sample for its ability to produce cephalosporins and penicillin N. It also produces cephamycins, such as deacetoxycephamycin C, and the commercially-relevant clavulanic acid. The name "clavuligerus" comes from the latin word ''[[clavula]]'', which stands for "little club" and the suffix ''[[-igerus]]'', which stands for "bearing". This refers to the morphology of the hyphae and the spores under a microscope. When ''[[S. clavuligerus]]'' sporulates, it produces a greyish-greenish pigment depending on the type of media it grows on(1).

''[[S. clavuligerus]]'' is the industrial producer of the beta-lactamase inhibitor, clavulanic acid, that reduces the MIC of antibiotics used against b-lactamase producing strains of ''[[Staphylococcus]]'', ''[[Klebsiella]]'', ''[[Pseudomonas]]'' and ''[[E. coli]]''. Due to its beta-lactamase inhibiting properties, clavulanic acid is co-formulated with amoxicillin, for which the generic name is co-amoxiclav. The biosynthetic pathway in S. clavuligerus has been almost completely deciphered (Jensen and Paradkar, 1999). Approaches to increase the yield of clavulanic acid as a fermentation product encompass fermentation optimisation approaches, as well as genetic modification for metabolic tailoring/engineering.
''[[S. clavuligerus]]'' differs from most other ''[[Streptomyces]]'' in that the wildtype is unable to take up sufficient amounts of glucose for it to utilise it as a sole carbon source (4). This has been attributed to the low expression levels of the glucose uptake protein, glucose permease, encoded by ''[[glcP]]'' (5). This is despite the ''[[glcP]]'' gene and the glucokinase gene, ''[[(glk)]]'', being present on the genome. The combination of the glucose permease and glucokinase has been shown to function as the main glucose uptake system in the model organism ''[[S. coelicolor]]''(6,7).

==Cultivation==

Unlike most ''[[Streptomyces]]'', ''[[S. clavuligerus]]'' does not sporulate on [[SFM | MS]] medium but can be grown on [[GYM]] medium for sporulation. Aside from [[GYM]], wildtype grows well on [[TSB]] agar and [[DNA]].

For cultivation in liquid, [[TSB]] medium gives the best result for dispersed growth. For growth curves in liquid, it is best to heat-active spores for 10 minutes at 50° prior to pre-germing for 12 - 24 hours (usually 10 ml of medim). Absorbance measurement readings at 600 nm wavelength can be used up until roughly 48 hours into the growth curve, cell dry weight, however, will give a more accurate result.

==Genome==
The genome of ''S. clavuligerus'' type strain ATCC 2764 has been analysed on several occasions, either by whole genome sequencing (8,9) or PFGE (10). It consists of a chromosome of 6.8 Mb that encodes 25 putative secondary metabolite clusters, including the clavulanic acid and cephamycin C clusters (11), and 4 linear plasmids: pSCL1 (11 kb), pSCL2 (150 kb), pSCL3 (455 kb) and pSCL4 (1,800 b). pSCL4 is a giant linear plasmid and it is the largest linear plasmid published to date and carries 17 putative secondary metabolite clusters including antibiotics such as staurosporine, moenomycin and other β-lactams.

{| class="wikitable"
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | Replicon
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | Size (kb)
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | GC %
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | Coding sequences
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | 2ary metabolite clusters
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | Examples of 2ary metabolites
|-
| style="font-weight:bold;" | Chromosome
| 6,760
| 72.7
| 5,900
| 25
| clavulanic acid, cephamycin C, holomycin
|-
| style="font-weight:bold;" | pSCL1
| 11
| 71.9
| 12
| 0
|
|-
| style="font-weight:bold;" | pSCL2
| 150
| 70.1
| 175
| 0
|
|-
| style="font-weight:bold;" | pSCL3
| 455
| 70.8
| 528
| 1
|
|-
| style="font-weight:bold;" | pSCL4
| 1,796
| 71.9
| 1,728
| 17
| staurosporine, moenomycin
|}

==Protocols==

Preferred media:
* [[GYM]] medium
* [[TSB]] agar and liquid medium
* [[DNA]]

Conjugation

==References==

#Higgens, C. E. and Kastner, R. E. (1971) ‘Streptomyces clavuligerus sp. nov., a β-lactam antibiotic producer’, International Journal of Systematic and Evolutionary Microbiology. Microbiology Society, 21(4), pp. 326–331.
#Howarth, T. T., Brown, A. G. and King, T. J. (1976) ‘Clavulanic acid, a novel β-lactam isolated from Streptomyces clavuligerus; X-ray crystal structure analysis’, Journal of the Chemical Society, Chemical Communications. Royal Society of Chemistry, (7), p. 266b–267.
#Jensen, S. E. and Paradkar, A. S. (1999) ‘Biosynthesis and molecular genetics of clavulanic acid’, Antonie Van Leeuwenhoek. Springer, 75(1), pp. 125–133.
#Garcia-Dominguez, M., Martin, J. F. and Liras, P. (1989) ‘Characterization of sugar uptake in wild-type Streptomyces clavuligerus, which is impaired in glucose uptake, and in a glucose-utilizing mutant.’, Journal of bacteriology. Am Soc Microbiol, 171(12), pp. 6808–6814.
#Pérez-Redondo, R., Santamarta, I., Bovenberg, R., Martín, J. F. and Liras, P. (2010) ‘The enigmatic lack of glucose utilization in Streptomyces clavuligerus is due to inefficient expression of the glucose permease gene’, Microbiology. Microbiology Society, 156(5), pp. 1527–1537.
#Van Wezel, G. P., Mahr, K., König, M., Traag, B. A., Pimentel‐Schmitt, E. F., Willimek, A. and Titgemeyer, F. (2005) ‘GlcP constitutes the major glucose uptake system of Streptomyces coelicolor A3 (2)’, Molecular microbiology. Wiley Online Library, 55(2), pp. 624–636.
#Van Wezel, G. P., König, M., Mahr, K., Nothaft, H., Thomae, A. W., Bibb, M. and Titgemeyer, F. (2007) ‘A new piece of an old jigsaw: glucose kinase is activated posttranslationally in a glucose transport-dependent manner in Streptomyces coelicolor A3 (2)’, Journal of molecular microbiology and biotechnology. Karger Publishers, 12(1–2), pp. 67–74.
#Medema, M.H. et al., 2010. The sequence of a 1.8-Mb bacterial linear plasmid reveals a rich evolutionary reservoir of secondary metabolic pathways. Genome Biology and Evolution, 2(1), pp.212–224.
#Song, J.Y. et al., 2010. Draft genome sequence of Streptomyces clavuligerus NRRL 3585, a producer of diverse secondary metabolites. Journal of Bacteriology, 192(23), pp.6317–6318.
#Netolitzky, D.J. et al., 1995. Giant linear plasmids of beta-lactam antibiotic producing Streptomyces. FEMS Microbiology Letters, 131(1), pp.27–34.
#Chen, C.W. et al., 1994. The linear chromosomes of Streptomyces: structure and dynamics. Actinomycetologica, 8, pp.103–112.

Streptomyces clavuligerus

2019-08-30T15:05:17Z

ASBirke: /* References */

=''[[Streptomyces clavuligerus]]''=

''[[Streptomyces clavuligerus]]'' was originally isolated from a South American soil sample for its ability to produce cephalosporins. Today, it is used in industry for the production of cephamycin C and clavulanic acid (1, 2, 3).
''[[S. clavuligerus]]'' differs from most other ''[[Streptomyces]]'' in that the wildtype is unable to take up sufficient amounts of glucose for it to utilise it as a sole carbon source (4). This has been attributed to the low expression levels of the glucose uptake protein, glucose permease, encoded by ''[[glcP]]'' (5). This is despite the ''[[glcP]]'' gene and the glucokinase gene, ''[[(glk)]]'', being present on the genome. The combination of the glucose permease and glucokinase has been shown to function as the main glucose uptake system in the model organism ''[[S. coelicolor]]''(6,7).

==Cultivation==

Unlike most ''[[Streptomyces]]'', ''[[S. clavuligerus]]'' does not sporulate on SFM medium but can be grown on GYM medium for sporulation. Aside from GYM, wildtype grows well on TSB agar and nutrient agar.

For cultivation in liquid, TSB medium gives the best result for dispersed growth. For growth curves in liquid, it is best to heat-active spores for 10 minutes at 50° prior to pre-germing for 12 - 24 hours (usually 10 ml of medim). Absorbance measurement readings at 600 nm wavelength can be used up until roughly 48 hours into the growth curve, cell dry weight, however, will give a more accurate result.

==Protocols==

Media recipes:
* GYM medium
* TSB agar and liquid medium
* Nutrient agar

Conjugation

==References==

#Higgens, C. E. and Kastner, R. E. (1971) ‘Streptomyces clavuligerus sp. nov., a β-lactam antibiotic producer’, International Journal of Systematic and Evolutionary Microbiology. Microbiology Society, 21(4), pp. 326–331.
#Howarth, T. T., Brown, A. G. and King, T. J. (1976) ‘Clavulanic acid, a novel β-lactam isolated from Streptomyces clavuligerus; X-ray crystal structure analysis’, Journal of the Chemical Society, Chemical Communications. Royal Society of Chemistry, (7), p. 266b–267.
#Jensen, S. E. and Paradkar, A. S. (1999) ‘Biosynthesis and molecular genetics of clavulanic acid’, Antonie Van Leeuwenhoek. Springer, 75(1), pp. 125–133.
#Garcia-Dominguez, M., Martin, J. F. and Liras, P. (1989) ‘Characterization of sugar uptake in wild-type Streptomyces clavuligerus, which is impaired in glucose uptake, and in a glucose-utilizing mutant.’, Journal of bacteriology. Am Soc Microbiol, 171(12), pp. 6808–6814.
#Pérez-Redondo, R., Santamarta, I., Bovenberg, R., Martín, J. F. and Liras, P. (2010) ‘The enigmatic lack of glucose utilization in Streptomyces clavuligerus is due to inefficient expression of the glucose permease gene’, Microbiology. Microbiology Society, 156(5), pp. 1527–1537.
#Van Wezel, G. P., Mahr, K., König, M., Traag, B. A., Pimentel‐Schmitt, E. F., Willimek, A. and Titgemeyer, F. (2005) ‘GlcP constitutes the major glucose uptake system of Streptomyces coelicolor A3 (2)’, Molecular microbiology. Wiley Online Library, 55(2), pp. 624–636.
#Van Wezel, G. P., König, M., Mahr, K., Nothaft, H., Thomae, A. W., Bibb, M. and Titgemeyer, F. (2007) ‘A new piece of an old jigsaw: glucose kinase is activated posttranslationally in a glucose transport-dependent manner in Streptomyces coelicolor A3 (2)’, Journal of molecular microbiology and biotechnology. Karger Publishers, 12(1–2), pp. 67–74.

Streptomyces clavuligerus

2019-08-30T15:03:44Z

ASBirke: Created page with "=''Streptomyces clavuligerus''= ''Streptomyces clavuligerus'' was originally isolated from a South American soil sample for its ability to produce cephalosporins. Tod..."

=''[[Streptomyces clavuligerus]]''=

''[[Streptomyces clavuligerus]]'' was originally isolated from a South American soil sample for its ability to produce cephalosporins. Today, it is used in industry for the production of cephamycin C and clavulanic acid (1, 2, 3).
''[[S. clavuligerus]]'' differs from most other ''[[Streptomyces]]'' in that the wildtype is unable to take up sufficient amounts of glucose for it to utilise it as a sole carbon source (4). This has been attributed to the low expression levels of the glucose uptake protein, glucose permease, encoded by ''[[glcP]]'' (5). This is despite the ''[[glcP]]'' gene and the glucokinase gene, ''[[(glk)]]'', being present on the genome. The combination of the glucose permease and glucokinase has been shown to function as the main glucose uptake system in the model organism ''[[S. coelicolor]]''(6,7).

==Cultivation==

Unlike most ''[[Streptomyces]]'', ''[[S. clavuligerus]]'' does not sporulate on SFM medium but can be grown on GYM medium for sporulation. Aside from GYM, wildtype grows well on TSB agar and nutrient agar.

For cultivation in liquid, TSB medium gives the best result for dispersed growth. For growth curves in liquid, it is best to heat-active spores for 10 minutes at 50° prior to pre-germing for 12 - 24 hours (usually 10 ml of medim). Absorbance measurement readings at 600 nm wavelength can be used up until roughly 48 hours into the growth curve, cell dry weight, however, will give a more accurate result.

==Protocols==

Media recipes:
* GYM medium
* TSB agar and liquid medium
* Nutrient agar

Conjugation

==References==

(1)Higgens, C. E. and Kastner, R. E. (1971) ‘Streptomyces clavuligerus sp. nov., a β-lactam antibiotic producer’, International Journal of Systematic and Evolutionary Microbiology. Microbiology Society, 21(4), pp. 326–331.
(2)Howarth, T. T., Brown, A. G. and King, T. J. (1976) ‘Clavulanic acid, a novel β-lactam isolated from Streptomyces clavuligerus; X-ray crystal structure analysis’, Journal of the Chemical Society, Chemical Communications. Royal Society of Chemistry, (7), p. 266b–267.
(3)Jensen, S. E. and Paradkar, A. S. (1999) ‘Biosynthesis and molecular genetics of clavulanic acid’, Antonie Van Leeuwenhoek. Springer, 75(1), pp. 125–133.
(4)Garcia-Dominguez, M., Martin, J. F. and Liras, P. (1989) ‘Characterization of sugar uptake in wild-type Streptomyces clavuligerus, which is impaired in glucose uptake, and in a glucose-utilizing mutant.’, Journal of bacteriology. Am Soc Microbiol, 171(12), pp. 6808–6814.
(5)Pérez-Redondo, R., Santamarta, I., Bovenberg, R., Martín, J. F. and Liras, P. (2010) ‘The enigmatic lack of glucose utilization in Streptomyces clavuligerus is due to inefficient expression of the glucose permease gene’, Microbiology. Microbiology Society, 156(5), pp. 1527–1537.
(6)Van Wezel, G. P., Mahr, K., König, M., Traag, B. A., Pimentel‐Schmitt, E. F., Willimek, A. and Titgemeyer, F. (2005) ‘GlcP constitutes the major glucose uptake system of Streptomyces coelicolor A3 (2)’, Molecular microbiology. Wiley Online Library, 55(2), pp. 624–636.
(7)Van Wezel, G. P., König, M., Mahr, K., Nothaft, H., Thomae, A. W., Bibb, M. and Titgemeyer, F. (2007) ‘A new piece of an old jigsaw: glucose kinase is activated posttranslationally in a glucose transport-dependent manner in Streptomyces coelicolor A3 (2)’, Journal of molecular microbiology and biotechnology. Karger Publishers, 12(1–2), pp. 67–74.

MC agar

2019-08-30T14:04:42Z

ASBirke: /* MC agar */

=MC agar=

This media is used to screen ''Streptomyces'' strains for extracellular protease production. The resulting degradation of the milk powder components on the plate, which can be detected as protein degradation "halos", visually resemble zone of inhibtions that one would observe with bioassays. This recipe was adjusted for ''[[Streptomyces clavuligerus]]''.

==Preparation==
Per litre:
*50 g Dry milk powder
*5 g Tryptone
*2.5 g Yeast Extract
*12.5 g Agar
*5 g Glucose (add glucose from stock solution after autoclaving)
*1 L dH2O

'''Instructions'''

#Dissolve all components (but glucose and agar) in 0.9 L dH2O
#Adjust pH to 6.8 - 7.0
#Add and dissolve agar
#Autoclave
#Add 100 ml of a filtersterilised 50% glucose solution

'''Note'''

#The milk powder in the media will curdle, mix gently by swirling bottle prior to pouring plates
#if alternative carbon sources are added to the media, adjust number of carbon atoms accordingly,

Example: add 10 g/L of glycerol (C3), or 2.5 g/L of maltose (C12)

==Uses==

After heat-activation and pre-germination of ''Streptomyces'' spores, they can then be spotted (~5µl )onto the agar for sharp edges of the colonies and the resulting protease halos. Once spotted, the plates are incubated at 26°C for ''[[Streptomyces clavuligerus]]'' or 30°C for other ''Streptomcyes'' strains.

==References==

Cheeseman, G. C. (1963) “Action of rennet and other proteolytic enzymes on casein in casein-agar gels,” Journal of Dairy Research. Cambridge University Press, 30(1), pp. 17–22. doi: 10.1017/S0022029900011213.

MC agar

2019-08-30T14:03:51Z

ASBirke:

=MC agar=

This media is used to screen ''Streptomyces'' strains for extracellular protease production and the resulting degradation of the milk powder components on the plate, which can be detected as protein degradation "halos" (visually resemble zone of inhibtions that one would observe with bioassays). This recipe was adjusted for ''[[Streptomyces clavuligerus]]''.

==Preparation==
Per litre:
*50 g Dry milk powder
*5 g Tryptone
*2.5 g Yeast Extract
*12.5 g Agar
*5 g Glucose (add glucose from stock solution after autoclaving)
*1 L dH2O

'''Instructions'''

#Dissolve all components (but glucose and agar) in 0.9 L dH2O
#Adjust pH to 6.8 - 7.0
#Add and dissolve agar
#Autoclave
#Add 100 ml of a filtersterilised 50% glucose solution

'''Note'''

#The milk powder in the media will curdle, mix gently by swirling bottle prior to pouring plates
#if alternative carbon sources are added to the media, adjust number of carbon atoms accordingly,

Example: add 10 g/L of glycerol (C3), or 2.5 g/L of maltose (C12)

==Uses==

After heat-activation and pre-germination of ''Streptomyces'' spores, they can then be spotted (~5µl )onto the agar for sharp edges of the colonies and the resulting protease halos. Once spotted, the plates are incubated at 26°C for ''[[Streptomyces clavuligerus]]'' or 30°C for other ''Streptomcyes'' strains.

==References==

Cheeseman, G. C. (1963) “Action of rennet and other proteolytic enzymes on casein in casein-agar gels,” Journal of Dairy Research. Cambridge University Press, 30(1), pp. 17–22. doi: 10.1017/S0022029900011213.

MC agar

2019-07-26T15:22:18Z

ASBirke:

=MC agar=

This media is used to screen ''Streptomyces'' strains for extracellular protease production and the resulting degradation of the milk powder components on the plate, which can be detected as protein degradation "halos" (visually resemble zone of inhibtions that one would observe with bioassays). This recipe was adjusted for ''[[Streptomyces clavuligerus]]''.

==Preparation==
Per litre:
*50 g Dry milk powder
*5 g Tryptone
*2.5 g Yeast Extract
*12.5 g Agar
*5 g Glucose (add glucose from stock solution after autoclaving)
*1 L dH2O

'''Instructions'''

#Dissolve all components (but glucose and agar) in 0.9 L dH2O
#Adjust pH to 6.8 - 7.0
#Add and dissolve agar
#Autoclave
#Add 100 ml of a filtersterilised 50% glucose solution

'''Note'''

#The milk powder in the media will curdle, mix gently by swirling bottle prior to pouring plates
#if alternative carbon sources are added to the media, adjust number of carbon atoms accordingly,

Example: add 10 g/L of glycerol (C3), or 2.5 g/L of maltose (C12)

==Uses==

After heat-activation and pre-germination of ''Streptomyces'' spores, they can then be spotted (~5µl)onto the agar for sharp edges of the colonies and the resulting protease halos. Once spotted, the plates are incubated at 26°C for ''[[Streptomyces clavuligerus]]'' or 30°C for other ''Streptomcyes'' strains.

==References==

Cheeseman, G. C. (1963) “Action of rennet and other proteolytic enzymes on casein in casein-agar gels,” Journal of Dairy Research. Cambridge University Press, 30(1), pp. 17–22. doi: 10.1017/S0022029900011213.

MC agar

2019-07-26T15:09:48Z

ASBirke: Created page with "=MC agar= This media is used to screen Streptomyces strains for extracellular protease production and the resulting degradation of the milk powder components on the plate, wh..."

=MC agar=

This media is used to screen Streptomyces strains for extracellular protease production and the resulting degradation of the milk powder components on the plate, which can be detected as protein degradation "halos" (visually resemble zone of inhibtions that one would observe with bioassays). This recipe was adjusted for Streptomyces clavuligerus.

==Preparation==
Per litre:
*50 g Dry milk powder
*5 g Tryptone
*2.5 g Yeast Extract
*12.5 g Agar
*5 g Glucose (add glucose from stock solution after autoclaving)
*1 L dH2O

'''Instructions'''

#Dissolve all components (but glucose and agar) in 0.9 L dH2O
#Adjust pH to 6.8 - 7.0
#Add and dissolve agar
#Autoclave
#Add 100 ml of a filtersterilised 50% glucose solution

'''Note'''

#The milk powder in the media will curdle, mix gently by swirling bottle prior to pouring plates
#if alternative carbon sources are added to the media, adjust number of carbon atoms accordingly,

Example: add 10 g/L of glycerol (C3), or 2.5 g/L of maltose (C12)

==Uses==

After heat-activation and pre-germination of Streptomyces spores, they can then be spotted (~5µl)onto the agar for sharp edges of the colonies and the resulting protease halos.

==References==

Media recipes

2019-07-26T14:03:21Z

ASBirke:

[[File:Actinobase-icons-trans_Media-recipes.png|150px|Media Recipes|link=Media_recipes]]

[[A1]]

[[CM]] (Complete medium)

[[DNA]] (Difco Nutrient Agar) | [[DNB]] (Difco Nutrient Broth) | [[SNA]] (soft nutrient agar)

[[Emerson medium]]

[[Gause's No. 1]]

[[GYM]] (Glucose + Yeast Extract + Malt Extract)

[[HT]] (Hickey-Tresner)

[[HV]]

[[IMA]] (Smash)

[[ISP2]]

[[ISP3]]

[[ISP4]]

[[ISP5]]

[[ISP6]]

[[ISP7]]

[[LB]] (Lysogeny Broth) | [[L]] (L agar)

[[MM]] (Minimal medium)

[[MYM]] (Maltose + Yeast Extract + Malt Extract)

[[NMMP]] (minimal liquid medium)

[[OT]] (oligotrophic agar)

[[PGA]] (Potato Glucose)

[[R2]] | [[R2YE]] | [[R5]]

[[SC]] (Starch Casein agar)

[[SFM]] (Soy Flour Mannitol)

[[SMM]] (supplemented liquid minimal medium) | [[SMMS]] (supplemented minimal medium, solid)

[[SPY]] (Salt Peptone Yeast)

[[TSB]] (Tryptone Soy Broth)

[[YEME]] (Yeast Extract + Malt Extract)

[[YP]] (Yeast Peptone) | [[YPD]] (Yeast Peptone Dextrose)

[[2X YT]]

[[MC agar]] (Milk Casein Agar)