16 Common Questions About Nourseothricin

This article gives you quick answers to some very common questions when working with Nourseothricin sulfate (clonNAT).

Below is a clickable list of the top compiled questions as well as detailed answers to each one.

Take a look, and save time in your daily work.

In this article:

1.How does nourseothricin work – what is the mechanism of action for nourseothricin

2.Is clonNAT the same as Nourseothricin

3.How is nourseothricin inactivated?

4.Is nourseothricin bacteriostatic or bactericidal?

5.What are all the different names of Nourseothricin

6.What is the structure of nourseothricin?

7.What are the resistance genes for nourseothricin?

8.Does nourseothricin work on gram-negative bacteria or gram-positive bacteria?

9.What kind of antibiotic is nourseothricin

10.What bacteria is nourseothricin effective against

List of bacteria and organisms sensitive to nourseothricin

11.What concentration of nourseothricin should I use for selection?

12.Is nourseothricin a good antibiotic for cell culture?

13.What is the difference between nourseothricin and other commonly used antibiotics?

14.What are the major uses of nourseothricin in biotechnology?

15.How do you make a nourseothricin stock solution?

16.Is nourseothricin toxic?

References

1. How does nourseothricin work – what is the mechanism of action for nourseothricin?

The mechanism of action for nourseothricin is to inhibit protein synthesis by interfering with the mRNA translocation step, causing misreads of the RNA molecule. Nourseothricin blocks mRNA translocation, hindering peptide chain synthesis of the peptide.

Additionally, nourseothricin binds to the ribosomal subunit of bacteria. This causes an incorrect alignment of the mRNA and eventually produces a misread that causes the wrong amino acid to be placed into the peptide chain.

Consequently, a nonfunctional protein is produced. Abnormal proteins accumulate within the bacteria leading to cell death.

Nourseothricin is a mixture of streptothricin D and F (>85%) and streptothricin C and E (<15%).

It has been reported that streptothricin F causes errors in reading the genetic message.

In polypeptide synthesis directed by homopolynucleotides (regulatory elements at various stages of mRNAs life cycle), streptothricin F stimulates the ribosome into incorporating the wrong amino acid during the translation process.

Additionally, streptothricin F strongly affects the translocation reaction, i.e., the transfer of peptidyl-tRNA from the acceptor site to the donor site (Haupt et al., 1978).

2. Is clonNAT the same as Nourseothricin?

Nourseothricin and clonNAT are the exact same thing. You will find when reviewing the reagent specifications that nourseothricin and clonNAT are composed of streptothricin D and F primarily as well as C and E.

Nourseothricin (clonNAT/NTC) allows the selection of genetically modified Gram-positive and Gram-negative bacteria, yeast, filamentous fungi, protozoa, microalgae and plants during long-term experiments.

These organisms require a stable and reliable antibiotic to be tested for long-term. Nourseothricin (clonNAT) retains > 90% activity after one week under cultivation conditions.

3. How is nourseothricin inactivated?

The enzyme acetyl coenzyme A: streptothricin acetyltransferase (ACSAT for short) inactivates nourseothricin by acetylating the β-amino group (C16) of the β-lysine residue in the molecule, causing a reduction of the antibiotic activity (Hahn, 1983).

To better understand nourseothricin inactivation, let’s take a closer look at the composition of commercially available nourseothricin.

Nourseothricin is a mixture of streptothricins D, F, C and E. The acetyltransferase synthesized by Streptomyces laoendulae acetylates streptothricin F at the β-amino group (C16) of the β-lysine residue, protecting the strain against its own antibiotic product (Ziihringer et al 1993).

4. Is nourseothricin bacteriostatic or bactericidal?

Nourseothricin is bacteriostatic and bactericidal, meaning it arrests the bacterial growth and causes cell death. The antibiotic effects of nourseothricin could be associated with its mechanisms of action inhibiting protein synthesis through the mRNA translocation interference and RNA misreads.

Furthermore, the diversity of the streptothricins C, D E and F composing nourseothricin could explain its versatility acting as both bactericidal and bacteriostatic, however the complete mechanism is not well understood.

For instance, nourseothricin is bacteriostatic against Gram-negative bacteria such as Escherichia coli and Gram-positive bacteria such as Bacillus subtilis and micrococci (Waksman et al. 1942).

However, for Gram-negative Klebsiella pneumoniae (Smith et al. 2021) nourseothricin is bactericidal.

There is a deeper difference between bacteriostatic and bactericidal antibiotics that depends on their concentrations and the effect over bacteria.

Before explaining the difference, there are two important terms to understand.

• Minimum bactericidal concentration (MBC): the concentration of a drug that results in a 1,000-fold reduction in bacterial density at 24 hours of growth in specific conditions of media, temperature, and carbon dioxide.
• Minimum inhibitory concentration (MIC): the concentration that inhibits visible bacterial growth at 24 hours of growth in specific conditions of media, temperature, and carbon dioxide.

What we mean by this is if you plate an aliquot from this tube, you will be able to visualize some bacterial growth.

MBC, on the other hand, indicates the lowest level of antimicrobial agent resulting in microbial death and it is physically visualized as a tube with no turbidity. If you plate an aliquot from this tube, you won’t see bacterial growth.

A formal difference between bactericidal and bacteriostatic is bactericidal antibiotics have a ratio of MBC to MIC ≤ 4, while a bacteriostatic antibiotic has an MBC to MIC ratio of > 4.

From our example, the MBC is 8 µg/ml and the MIC is 4 µg/ml, so the ratio of MBC to MIC is:

(MBC)/ (MIC)= (8 µg/ml) / (4 µg/ml) = 2As MBC to MIC ≤ 4, the antibiotic is bactericidal.

In this sense, bacteriostatic antibiotics also kill bacteria; they just require a higher concentration than bactericidal agents to achieve specific thresholds of bacterial reduction.

Examples of bacteriostatic antibiotics include chloramphenicol, clindamycin, and linezolid.

Published, randomized and controlled trials demonstrated bactericidal agents are not intrinsically superior in efficacy to bacteriostatic agents.

A study reported by (Dickler et al 2017) analyzed 56 trials that compared bacteriostatic with bactericidal, and authors showed that the majority of trials across a variety of infections found no difference in efficacy between bacteriostatic versus bactericidal agents.

5. What are all the different names of Nourseothricin

Nourseothricin is also called clonNAT referring to nourseothricin N-acetyl transferase, the enzyme that inactivates the antibiotic nourseothricin. Often, nourseothricin is also shortened to NTC.

Alternative names of nourseothricin

• Nourseothricin Sulfate
• Streptothricin Sulfate
• NTC
• clonNAT

6. What is the structure of nourseothricin?

Nourseothricin is a mixture of streptothricin D and F (>85%), and streptothricin E and C (<15%). Its structure is rooted in the streptothricin molecular structure composed of a carbamulated D-glucosamine sugar, a streptolidine lactam, and a B-lysine homopolymer. The B-lysine group determines streptothricin type (D, F, E, C).

The structure of nourseothricin has three moieties:

• A carbamoylated gulosamine sugar core (blue)
• A streptolidine lactam moiety (pink)
• β-lysine homopolymer (yellow)

The β-lysine homopolymer chain differs in the number of β-lysine residues from one to seven and giving rise to the different types of streptothricin from A-F and X.

7. What are the resistance genes for nourseothricin?

Resistance genes for nourseothricin are Sat1, Sat2, Sat3 and Sat4. The sat genes code for streptothricin acetyltransferase proteins. Resistance is due to N-acetylation of the lysine residue in the nourseothricin molecule mediated by the different streptothricin acetyltransferases in gram-negatives.

The sat4 gene has been reported to belong to a gene cluster with other aminoglycoside resistance genes like aphA-3 and aadE which improve the bacterial resistance to nourseothricin (Derbise et al 1996; Wendlandt et al 2013).

The complete sequence for sat4 from Staphylococcus intermedius can be found here

8. Does nourseothricin work on gram-negative bacteria or gram-positive bacteria?

Nourseothricin is effective against gram-positive and particularly in gram-negative bacteria. It works against gram-negative such as Escherichia coli, Klebsiella pneumoniae, and Agrobacterium tumefaciens; and gram-positive bacteria like Bacillus subtilis, and Staphylococcus aureus.

Gram-negative bacteria susceptible to nourseothricin:

• Escherichia coli
• Klebsiella pneumoniae
• Agrobacterium tumefaciens
• Francisellatularensis
• Pseudomonas aeruginosa

Gram-positive bacteria susceptible to nourseothricin:

• Bacillus subtilis
• Micrococci
• Enterococcus faecium
• Staphylococcus aureus

9. What kind of antibiotic is nourseothricin?

Nourseothricin is an aminoglycoside antibiotic composed of a mixture of steptothricins D and F primarily (>85%) and steptothricins C and E (<15%).

The mechanism of action for aminoglycoside antibiotics involves a two-step process. In the first step, a self-promoted uptake involves the displacement of divalent cations such as magnesium and calcium from outer and inner membranes.

Without these cations, the outer and inner membranes become more permeable. This in turn, elevates the antibiotic uptake.

After crossing both membrane barriers, the antibiotic enters the cytoplasm and targets the ribosome.

In a second step, the aminoglycoside antibiotic binds to the A-site on the 16S RNA of the 30S bacterial ribosome hindering the normal translation process, which leads to mistranslated proteins in the cytoplasm. The accumulation of abnormal proteins accelerates cell death.

Nourseothricin is considered an aminoglycoside antibiotic because it also inhibits protein synthesis by interfering with the translation process and causes misreading of RNA molecules.

However, nourseothricin does not have the typical aminoglycoside structure core of the dibasic aminocyclitol ring (commonly 2-deoxystreptamine). Instead, the lysine residue is responsible for the antibiotic effect.

10. What bacteria is nourseothricin effective against?

Nourseothricin is a broad-spectrum antibiotic effective against both Gram-positive and Gram-negative bacteria, though it is more effective against Gram-negative bacteria.

List of bacteria and organisms sensitive to nourseothricin

Table 1. Organisms sensitive to nourseothricin (clonNAT)

11.What concentration of nourseothricin should I use for selection?

The concentration of nourseothricin usually ranges between 50 µg/ml to 100 µg/ml; however, concentration really depends on the organism. Tested concentrations of nourseothricin in specific organisms are presented in table 2.

Table 2. Concentrations of nourseothricin for different organisms.

12. Is nourseothricin a good antibiotic for cell culture?

Nourseothricin is used for cell culture in the selection of genetically modified plants, fungi, yeast, and bacteria.

Nourseothricin is not used for antibiotic therapy with animals or humans because of its nephrotoxicity or toxicity of the kidney.

13. What is the difference between nourseothricin and other commonly used antibiotics?

The difference between nourseothricin and other aminoglycoside antibiotics such as kanamycin, streptomycin, and spectinomycin is in its structure. Nourseothricin does not have the typical 2-deoxystreptamine ring commonly present in other aminoglycoside antibiotics.

14. What are the major uses of nourseothricin in biotechnology?

Nourseothricin is commonly used for bacterial and plant selection. It is not used in animal or human experiments because of its toxicity to kidneys (nephrotoxicity).

15. How do you make a nourseothricin stock solution?

To prepare a 200 mg/ml nourseothricin stock solution:

1. Weigh 2 g of nourseothricin sulfate (Nourseothricin Sulfate, GoldBio)

2. Add 10 ml of sterile H₂O. Dissolve completely.

3. Prewet a 0.22 µm syringe filter by drawing through 5-10 ml of sterile H₂O and discard water.

4. Sterilize nourseothricin stock through the prepared 0.22 µm syringe filter.

5. Prepare aliquots of 1ml each.

6. Stock may be kept at -20°C for 1 year

16. Is nourseothricin toxic?

Although there are not many studies, nourseothricin is toxic above 60.000 units/kgm administered intravenously into mice (Robinson et al. 1944). Nephrotoxicity (toxicity in the kidneys) becomes evident in rats after 48 hours of intravenous streptothricin administration of 40mg/kg.

The opened lactam ring is considered responsible for the toxicity of kidneys (Inamori et al. 1979).

References

Cundliffe, E., 1989. How antibiotic-producing organisms avoid suicide. Annu. Rev. Microbiol 207–233.

Derbise, A., Dyke, K.G.H., El Solh, N., 1996. Characterization of a Staphylococcus aureus transposon, Tn5405, located within Tn5404 and carrying the aminoglycoside resistance genes, aphA-3 and aadE. Plasmid 35, 174–188. https://doi.org/10.1006/plas.1996.0020

Dowgiallo, M.G., Miller, B.C., Kassu, M., Smith, K.P., Fetigan, A.D., Guo, J.J., Kirby, J.E., Manetsch, R., 2022. The convergent total synthesis and antibacterial profile of the natural product streptothricin F. Chem. Sci. 13, 3447–3453. https://doi.org/10.1039/d1sc06445b

GoldBio, 2019. Nourseothricin Sulfate, Nourseothricin sulfate.

Hahn, F., 1983. Modes and mechanisms of microbial growth inhibitors.

Haupt, I., Hübener, R., Thrum, H., 1978. Streptothricin F, an Inhibitor of Protein Synthesis with Miscoding Activity. J. Antibiot. (Tokyo). 31, 1137–1142. https://doi.org/10.7164/antibiotics.31.1137

Inamori, Y., Kato, Y., Morimoto, K., Morisaka, K., Saito, G., Sawada, Y., Taniyama, H., 1979. Toxicological approaches to streptothricin antibiotics. III. Biological studies on delayed toxicity of streptothricin antibiotics in rats. Chem. Pharm. Bull. 2091.

Makeyev, A. V., Liebhaber, S.A., 2002. The poly(C)-binding proteins: A multiplicity of functions and a search for mechanisms. Rna 8, 265–278. https://doi.org/10.1017/S1355838202024627

Robinson, H., Graessle, O., Smith, D. 1944. Studies on the Toxicity and Activity of Streptothricin. Science, New Series, Vol. 99, No. 2583

Schwabacher, H., Hughes, W.H., 1954. Bacterial Resistance to Antiseptics. Br. Med. J. 2, 247. https://doi.org/10.1136/bmj.2.4881.247

Smith, K.P., Kang, Y.-S., Green, A.B., Dowgiallo, M.G., Miller, B.C., Chiaraviglio, L., Truelson, K.A., Zulauf, K.E., Rodriguez, S., Manetsch, R., Kirby, J.E., 2021. Profiling the in vitro and in vivo activity of streptothricin-F against carbapenem-resistant Enterobacterales: a historic scaffold with a novel mechanism of action. bioRxiv doi: 10.1101/2021.06.14.448463.

Waksman, S.A., Woodruff, H.B., 1942. Streptothricin, a New Selective Bacteriostatic and Bactericidal Agent, Particularly Active Against Gram-Negative Bacteria. Proc. Soc. Exp. Biol. Med. 49, 207–210. https://doi.org/10.3181/00379727-49-13515

Wendlandt, S., Feßler, A.T., Monecke, S., Ehricht, R., Schwarz, S., Kadlec, K., 2013. The diversity of antimicrobial resistance genes among staphylococci of animal origin. Int. J. Med. Microbiol. 303, 338–349. https://doi.org/10.1016/j.ijmm.2013.02.006

Zähringer, U., Voigt, W., Seltmann, G., 1993. Noureseothricin (streptothricin) inactivated by a plasmid pIE636 encoded acetyl transferase of Escherichia coli: Location of the acetyl group. FEMS Microbiol. Lett. 110, 331–334. https://doi.org/10.1111/j.1574-6968.1993.tb06344.x