TERM PAPER

“ Streptomyces griseus”

SUBMITTED BY: -

SHASHI SHARMA

CLASS: M.SC. (Microbiology)

REG.NO.11006142

ROLL. No. RP8003A15

COURSE CODE. BTY-538

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Contents:-

1. Introduction ………………………………………………… 3

2. streptomycin griseus ………………………………………. 4

3. Industerial importance of Streptomycen griseus …………… 5-9

3.1 Use of S.griseus to clean water …………………………… 5

3.2 Antibiotic production from S.griseus ……………………… 6

3.3 Production of streptomycin ……………………………. 6

3.4 Use of S.griseus in Extracelluar Signaling ………………. 7

3.5 Production of vitamin B12 by S.griseus ………………….. 7

3.6 S.griseus in Production of protease ………………………. 8

3.7 Anticancer properties of streptomycen …………………… 8

4. New researches in S.griseus ……………………………… 10-11

4.1 DNA segment containing streptomycin

resistance gene and being capable of controlling

expression of said gene ……………………………………. 10

4.2 Genome Sequence of the

Streptomycin-Producing Microorganism

Streptomyces griseus ……………………………………….. 10

4.3 Medium optimization and application of an

affinity column chromatography for streptomyces

griseus trypsin production from the recombinant

Streptomyces griseus. …………………………………... 11

5. References ……………………………………………… 12

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1. Introduction

The filamentous, soil-living, Gram-positive bacterial genus Streptomyces is characterized by the ability to produce a wide variety of secondary metabolites, including antibiotics, and by complex morphological differentiation culminating in sporulation. Accumulating evidence has shown that γ-butyrolactone-type autoregulators serve as ‘microbial hormones’ for the regulation of onset of antibiotic production and complex morphological differentiation in Streptomyces. A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) of this type that is required for streptomycin production and cell differentiation in Streptomyces griseus has been most extensively studied. A-factor at an extremely low concentration triggers streptomycin production and aerial mycelium formation by binding a repressor-type receptor protein (ArpA) and dissociating it from the DNA. In vitro experiments established a consensus sequence to which ArpA binds; ArpA recognizes and binds a 22 bp palindrome, one half of which is 5′-GG (T/C)CGGT(A/T)(T/C)G(T/G)-3′. However, the target gene of ArpA in vivo was unknown. Also unknown was how the A-factor signal is transmitted from ArpA to the streptomycin biosynthetic genes. The full complement of genes, including structural synthetic genes and resistance determinants, necessary for any particular biosynthetic pathway for each antibiotic is organized within a single cluster. In the streptomycin biosynthetic gene cluster in S. griseus, strR is a pathway-specific regulator that serves as a transcriptional activator for the other genes in the cluster. We previously identified an A-factor-dependent protein (AdpA) able to bind the region upstream of the strR promoter and activate its transcription. AdpA is therefore an important regulator that transmits the upstream A-factor signal directly to the streptomycin production genes via strR.

The purposes of this study was to know the industrial importance of streptomyces griseus and it’s applications, to know it’s use in water purification, in antibiotic production, in formation of vitamineB12 etc and the current research done on it.

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2. Streptomyces griseus

Classification :-

Kingdom :- Bacteria

Phylum :- Actinobacteria

Class :- Actinobacteria

Order :- Actinomycetales

Family :- Streptomycetaceas

Genus :- Streptomyces

Species :- S. griseus

Streptomyces griseus is a member bacterial species of the genus Streptomyces and are commonly found in soil. A few strains have been also reported from deep sea sediments. These are Gram positive bacterium with high GC content. Along with most other streptomycetes S. griseus strains are well known producer of antibiotics and other such commercially significant secondary metabolites. These strains are known to be producers of 32 different structural types of bioactive compounds. The first antibiotic that was ever reported from a bacterium comes from strains of S . griseus. Recently whole genome sequence of one of its strains had been completed. The taxonomic history of S. griseus and its phylogenetically related strains have been turbulent. S .griseus was first described by Waksman and Henrici in 1948. The interest towards these strains was sought because of their ability to produce streptomycin, a compound which demonstrated significant bactericidal activity against organisms such as Yersinia pestis and Mycobacterium tuberculosis.

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Streptomycen griseus

3. Industerial importance of Streptomycen griseus

3.1 Use of S. griseus to Clean Water

Streptomyces griseus has been used as a plentiful source for antibiotics for a long time but is now beginning to come upon a new field, the purifying of water. In a recent test it was shown that Streptomyces g. was able to reduce Copper (II) Nitrate levels in water from 1mg/L to .1 mg/L which still not preferable but not as toxic. In addition it was able to bring down arsenic levels in water from 0.5 mg/L to 0.4 mg/L. The reason Streptomyces griseus is able to remove these contaminants is quite simple, the positively charged contaminants are attracted to the negatively charged cell walls of the Streptomyces g. then mucopolysaccharides bond the two together.

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3.2 Antibiotics production from S. griseus

Interest in the genus Streptomyces for antibiotics came after the discovery of the antibiotic streptomycin in a S. griseus strain in 1943. The discovery of streptomycin, an anti-tuberculosis antibiotic, earned Waksman the Nobel Prize in 1952. Now it is known that the strains of this species are a rich source of antibiotics and are known to produce 32 different structural types of commercially significant secondary metabolites. Furthermore, the genomic studies have revealed that a single strain of S. griseus has the capacity to produce 34 different secondary metabolites

3.3Production of streptomycin

Streptomyces griseus is cultured in three different bioreactors in a medium containing chitin flakes. When a conventional bioreactor stirred by two sets of Rushton impellers and operated at high speed was used, the yield of streptomycin (3.1 mg/l) was the highest observed and occurred at approximately 500 h. Cultivation of S. griseus in a bioreactor stirred at low speed by a U-shaped paddle resulted in a lower yield of streptomycin (1.8 mg/l) but this was achieved in a shorter period of time (400 h). Increasing the concentration of chitin from 5% to 10% w/v had no significant effect on either of these two parameters. The use of a novel vertical basket bioreactor in which thse chitin flakes were contained within a wire mesh basket and were gently fluidised by air, enabled comparatively high yields of streptomycin (2.8 mg/l) in the relatively short time of 300 h.

Fermentation media for streptomycin production

Steptomycin can be produced in small scale using the following media

Beef Extract: 3gms

Peptone: 5gms

Glucose: 10gms

NaCl: 5gms

Tap Water: 1000ml

pH :7.2

Temperature:28 C

Incubation 5-10 days

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Use of streptomycin

Streptomycin, the first aminoglycoside antibiotic, was discovered in S. A. Waksman's laboratory more than 60 years ago. This antibiotic, which has saved many people from tuberculosis, is produced industrially by Streptomyces griseus.streptomycin is an antibiotic used in different types of cell culture media. Generally, this antbiotic is incorporated into DMEM (Delbucose Minimum Essential Medium) and used to culture mammalian cells

3.4 Use of S. griseus in extracellular signaling

S. griseus also provided arguably the first well-studied bacterial example of extracellular signaling by a diffusible low-molecular-weight compound. In Streptomyces, chemical-signaling molecules that have a γ-butyrolactone are commonly used as the switch for secondary metabolite production and/or morphological development. A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) of S. griseus, originally discovered by Khokhlov et al. 40 years ago, is one such γ-butyrolactone. When A-factor produced in a growth-dependent manner reaches a critical concentration as low as 10−9 M, it binds an A-factor-specific receptor (ArpA) that has bound to the promoter of adpA and dissociates ArpA from the promoter. The transcriptional activator AdpA then activates the transcription of a number of genes that are required for secondary metabolism and morphological differentiation, forming an AdpA regulon. strR, which encodes the pathway-specific transcriptional activator for streptomycin biosynthesis, is one of the targets of AdpA, which explains how A-factor induces streptomycin production.

3.5 Production of vitaminB12 by streptomyces griseus

Industrial production of B12 is through fermentation of selected microorganisms. Streptomyces griseus, a bacterium once thought to be a yeast, was the commercial source of vitamin B12 for many years. The species Pseudomonas denitrificans and Propionibacterium shermanii are more commonly used today. These are frequently grown under special conditions to enhance yield, and at least one company, Rhône-Poulenc of France, at one point used genetically engineered versions of one or both of these species. It is not clear whether Sanofi-Aventis, the company into which the pharmaceutical division of Rhône-Poulenc merged, has continued the use of genetically modified organisms.

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3.6 Streptomyces in production of Protease

Streptomyces griseus , an organism used for the commercial production of protease, secretes two extracellular serine proteases: proteases A and B. The enzymes are 61% homologous on the basis of amino acid identity. The genes encoding protease A (sprA) and protease B (sprB) were isolated from the S. griseus genomic library, and their proteolytic activity was demonstrated in S. lividans. The DNA sequences suggest that each protease is initially secreted as a precursor, which is then processed to remove an N-terminal propeptide from the mature protease. The strong homology between the coding regions of the two protease genes suggests that sprA and sprB must have originated by gene duplication. Protease B is one of the major extracellular proteases secreted by S. griseus ATCC 10137, and its gene was expressed in S. lividans by Hwang et al. Their nucleotide sequencing of the gene further revealed that the deduced amino acid sequence was identical to that reported earlier by Henderson et al. However, the nucleotide sequence of the 3′-flanking region was G rich and may be responsible for the reduced level of protease in S. griseus ATCC 10137 compared to the level in protease B-overproducing strains of S. griseus.

3.7 Anticancer properties of streptomyces griseusStreptomyces griseus have some anticancer properties which are very useful. It is used in producing anti cancers antibiotic.

The structure of streptomycin

Streptidine is a guanidino-cyclitol and is a component of the streptomycin molecule. Streptomycin producing S.griseus can synthesize streptidine from glucose via myo-inositol. However, with the mutant of the present invention, the biosynthetic pathway of streptidine is blocked; but, this mutant can utilize exogenous streptidine for streptomycin biosynthesis.

To produce new derivatives of streptomycin in accordance with the present invention analogues of streptidine may be substituted for the exogenous streptidine. For example, 2-deoxystreptidine may be substituted for streptidine. The structure of 2-deoxystreptidine appears below; and, as can be seen from the structural formula, when the oxygen in the number 2 position of the streptidine molecule is removed, 2-deoxystreptidine results.

2-Deoxystreptidine may be prepared from deoxystreptamine in accordance with the procedure set forth in an article entitled Streptomycin Biosynthesis.Enzymatic Synthesis of O-Phosphorylstreptidine from Streptidine and Adenosinetriphosphate by J. B. Walker and M.

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S. Walker, Biochem. Biophys. Acta 148. pp. 335 (1967), the teachings of which are incorporated herein by reference. It should be noted that the method of preparing 2-deoxystreptidine appearing in the foregoing article is a method for preparing radioactive 2-deoxystreptidine. Of course, for purposes of the present invention, the 2-deoxystreptidine should be prepared in a non-radioactive manner. The resulting streptomycin derivative, i.e. 2-deoxystreptomycin, which can be prepared by utilizing exongenous 2-deoxystreptidine .

The amount of streptomycin produced increased in accordance with the amount of streptidine added. By the addition of 1,000 μg/ml streptidine about 500 μg/ml of streptomycin was accumulated in the culture. Production reached its peak 2 days after the addition of streptidine.

Although streptomycin production by the mutant was completely dependent upon the addition of streptidine, production by the parent strain was inhibited somewhat by streptidine addition. No difference in pH pattern was observed. Parental growth was not affected by streptidine. But growth of the mutant was somewhat inhibited by the addition of 1,000 μg/ml streptidine. The antibiotic substance produced by the mutant was analyzed by paper chromatography. The product migrated at the same rate as streptomycin in three solvent systems. Another fermentation experiment showed that no antibiotic substance was produced by suplementation of myo-inositol to the mutant.

Streptidine-dependent streptomycin production by a resting cell suspension of the mutant. After growth, the cells were washed and suspended in Tris buffer with or without streptidine. The reaction mixtures were shaken at 28° C for 48 hours. Streptomycin was synthesized in accordance with the amount of streptidine added and the

4. New researches in Streptomyces griseus

4.1 DNA segment containing streptomycin resistance gene and being capable of controlling expression of said gene

A DNA segment of this invention is a DNA fragment of a length of about 3.8 kb which is obtained by excising with a restriction endonuclease Bgl II a hybrid plasmid pST 141 having a length of about 12.6 kb of Streptomyces griseus 4-1 strain to give a Bgl II-Bgl II DNA fragment, and then excising with a restriction endonuclease Sph I the resultant Bgl II-Bgl II DNA fragment having a length of about 7.0 kb and having the restriction endonuclease sites. This DNA fragment is a DNA which contains a streptomycin resistance gene and contains, in the vicinity of this gene, such a DNA region possessing a function to control the expression of the streptomycin resistance gene. An insertion of this DNA fragment having a length of

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about 3.8 kb into a suitable actinomycetes plasmid vector can produce such a hybrid plasmid which acts reliably as a selected marker of the streptomycin resistance.

4.2 Genome Sequence of the Streptomycin-Producing Microorganism Streptomyces griseus

A soil bacterium producing an antituberculosis agent, streptomycin, which is the first aminoglycoside antibiotic, discovered more than 60 years ago. The linear chromosome consists of 8,545,929 base pairs (bp), with an average G+C content of 72.2%, predicting 7,138 open reading frames, six rRNA operons (16S-23S-5S), and 66 tRNA genes. It contains extremely long terminal inverted repeats (TIRs) of 132,910 bp each. The telomere's nucleotide sequence and secondary structure, consisting of several palindromes with a loop sequence of 5′-GGA-3′, are different from those of typical telomeres conserved among other Streptomyces species. In accordance with the difference, the chromosome has pseudogenes for a conserved terminal protein (Tpg) and a telomere-associated protein (Tap), and a novel pair of Tpg and Tap proteins is instead encoded by the TIRs. Comparisons with the genomes of two related species, Streptomyces coelicolor A3 and Streptomyces avermitilis, clarified not only the characteristics of the S. griseus genome but also the existence of 24 Streptomyces-specific proteins. The S. griseus genome contains 34 gene clusters or genes for the biosynthesis of known or unknown secondary metabolites. Transcriptome analysis using a DNA microarray showed that at least four of these clusters, in addition to the streptomycin biosynthesis gene cluster, were activated directly or indirectly by AdpA, which is a central transcriptional activator for secondary metabolism and morphogenesis in the A-factor (a γ-butyrolactone signaling molecule) regulatory cascade in S. griseus.

4.3 Medium optimization and application of an affinity column chromatography for streptomyces griseus trypsin production from the recombinant Streptomyces griseus.

The expression vector pWHM3-TR1R2, which contains sprT encoding Streptomyces griseus trypsin (SGT) and two positive regulatory genes (sgtR1 and sgtR2), was introduced into S. griseus and the productivity of SGT by the transformant was investigated in various media. Among the tested media, Ferm-0 gave 1.4 times more trypsin activity than C5/L medium. In addition, replacement of 2% glucose and 1% skim milk in Ferm-0 medium with 2% dextrin and 1% tryptone yielded significantly enhanced trypsin activity, by 4.1-fold, than that of Ferm-0. For simplifying the purification process, the cultural supernatant of S. griseus

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transformant in Ferm-II medium was fractionated with ammonium sulfate (25%-55%), and then applied to Hitrap benzamidine FF affinity column chromatography. The specific activity of the purified SGT by one-step column chromatography was 69,550 unit/mg protein, and the overall purification yield was above

8%, which was more effective than the methods of previous reports. The trypsin activity of the purified SGT was most active at pH 8.0 and 50 degrees C, and maintained their activities between pH 7.0 and pH 9.0, and up to 70 degrees C. These enzymatic properties were very similar to those of authentic eukaryotic trypsin purified from bovine pancreas.

5. References

1. ^ a b Amano, S; S. Miyadoh & T. Shomura. "Streptomyces griseus M-1027". Digital Atlas of Actinomycetes. http://www.nih.go.jp/saj/DigitalAtlas/subwin.cgi?section=7&fig=1. Retrieved 2008-12-02.

2. ^ a b Kämpfer P (2006). "The Family Streptomycetaceae, Part I: Taxonomy". The prokaryotes: a handbook on the biology of bacteria (Dworkin, M et al., eds.). Berlin: Springer. pp. 538–604. ISBN 0-387-25493-5.

3. ^ a b Madigan M, Martinko J (eds.) (2005). Brock Biology of Microorganisms (11th ed.). Prentice Hall. ISBN 0-13-144329-1.

4. ^ Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000). Practical Streptomyces Genetics (2nd ed.). Norwich, England: John Innes Foundation. ISBN 0-7084-0623-8.

5. ^ a b "Streptomyces griseus IFO 13350 Genome". http://park.itc.u-tokyo.ac.jp/hakko/genome/. Retrieved 2008-12-02.

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