Nitrogen assimilation in Actinomycetes (Accepted in NESOM Journal)
Kiran Babu Tiwari1,2* and Upendra Thapa Shrestha1
1Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal;
2Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal
Abstract
Actinomycetes are one of the diverse groups of soil bacteria possessing commercially useful enzymes and therapeutically useful bioactive molecules. The biochemical characterization of the individual isolates is of therefore utmost importance to understand their basic physiology. For the optimization of peptone concentration in carbohydrate utilization test, actinomycetes were grown at different concentrations of peptone and carbohydrate. With 10mM glucose, 0.2% peptone was found to be optimum concentration for glucose (carbohydrate) utilization test. However, the growth was retarded at a concentration above 0.5% of peptone. Besides as nitrogen source, actinomycetes can use peptone as carbon source too. The organisms can also use inorganic nitrogen source with slow growth rate. Actinomycetes have no essential amino acid; however, organic nitrogen enhances the growth. Actinomycetes are weak acid producers; still an appreciable lowering of pH was observed within five days of incubation at 28ºC.
Keywords: Actinomycetes, amino acid, assimilation, nitrogen, optimization
Introduction
Actinomycetes are aerobic, Gram-positive bacteria, which may form branching filaments or hyphae that may persist as a stable mycelium or may break up into rod-shaped or coccoid elements. Actinomycetes are widely distributed in terrestrial environment (Holt 1989). For much of this time they were regarded as an exotic group of organisms with affinities to both bacteria and fungi. However, determination of their fine structure and chemical composition initiated in the 1950’s, confirmed their prokaryotic nature. The actinomycetes comprise a ubiquitous order of bacteria, which exhibit wide physiological and morphological diversity. Actinomycetes have long been a source of commercially useful enzymes and therapeutically useful bioactive molecules. Actinomycete biodiversity is vast frontier and potential goldmine for the biotechnology industry because it offers countless new genes and biochemical pathways to probe for enzymes, antibiotics and other useful molecules (Agrawal 2003). Over past 60 years, products derived from microbial secondary metabolites have been used to meet medical, industrial and agricultural needs, e.g., antibiotics, anticancer drugs, antifungal compounds, immunosuppressive agents, enzyme inhibitors, anti parasitic agents, herbicides, insecticides and growth promoters (Busti et al. 2006). Hence, basic parameters for better growth and physiological characteristics of native micro-flora need to be optimized.
Materials and Methods
Isolation and identification of actinomycetes: Actinomycetes were isolated from soil samples in selective medium (Starch-Casein agar, pH 7.4) by serial dilution method and subsequently purified and identified by colonial characteristics and microscopy (Singh and Agrawal 2002).
Effects of peptone concentration on actinomycetes growth: A series of peptone broths (0.05%, 0.1%, 0.2%, 0.5%, 0.8% and 1.0%) with or without glucose (10mM) were prepared and sterilized. The isolates were inoculated into the respective culture tubes, incubated at 28ºC and observed for growth characteristics in the broth subsequently on daily basis. Respective negative control tubes were also included.
(For optimization of peptone)
Essential amino acids for actinomycetes: Twenty glucose broth tubes containing 10mM glucose and 0.3% Na2PO4 were prepared, and in all but only one amino acid (1mM) was added in respective tubes. The other three tubes containing only 0.3% Na2PO4 was added with inorganic nitrogen (1mM KNO3), organic carbon (10mM glucose), and both (1mM KNO3 and 10mM glucose) respectively. The isolate was inoculated into the respective sterile broth, incubated at 28ºC and observed for growth characteristics in the broth on daily basis comparing with negative control tubes.
Carbohydrate (Glucose) utilization test for actinomycetes: Glucose broths containing 10mM glucose, 0.3% Na2PO4, 3mg% Phenol Red and 0.2% Peptone was prepared. A nitrogen control tube (0.2% Peptone) was also included. The isolates were inoculated into the respective sterile broths, incubated at 28ºC and observed for growth and coloration of the broth on daily basis.
Results
Three representative actinomycete isolates (viz. Lob 1, 8 and 15) were assayed for various tests. Actinomycetes were found to utilize peptone as both nitrogen source and as carbon source (Table 1). The optimum concentration of peptone for growth was from 0.2 – 0.5%, beyond which the growth was somewhat retarded. Addition of glucose as carbon source allows better growth, and upto 0.8% of peptone can be used along with glucose for growth assessment of actinomycetes (Table 2). Actinomycetes can use inorganic nitrogen (KNO3), however organic nitrogen enhanced their growth (Table 3). At least five days of incubation is required for glucose utilization test of actinomycetes with conventional dark yellow coloration of media containing Phenol red as indicator (Table 4).
Table 1: Effect of peptone on growth of actinomycetes
Note: Slight (+), Easily observable (++), Marked (+++)
Discussion
As actinomycetes are one of the diverse groups of soil bacteria possessing commercially useful enzymes and therapeutically useful bioactive molecules, biochemical characterization of the individual isolates is of utmost importance to understand their basic physiology (Holt 1989). The present work explored importance and optimization of nitrogen substrates in basal culture media as a basic parameter for better growth and physiological characteristics of the organisms. The actinomycetes were isolated and purified from soil samples, of which only representative isolates were assayed for their physiological activities concerned with nitrogen assimilation.
Conventionally, peptone is used in various culture media as major nitrogen source for the organisms. As peptone is partial digest of protein complex, its carbon atom is liable to be used by organisms. Hence, actinomycetes can use peptone as carbon source when the culture medium lacks carbohydrates (e.g., sugar/s). Voelker and Altaba (2001) assayed role of various nitrogen sources (organic and inorganic) for growth and secondary metabolite production from a streptomycete. During balanced growth, either mineral or organic nitrogen sources were readily utilized. Glutamate and alanine were used as both nitrogen and carbon source, sparing the utilization of the primary carbon source, glucose. Without sugar, 0.2–0.5% peptone concentration is optimum for growth of actinomycetes. Higher peptone concentration disturbs C/N ratio and hence retards growth. With glucose, upto 0.8% of peptone can be used for growth; because, glucose, being an alternate to peptone carbon, increases the C/N ratio to the safe level.
Actinomycetes can fairly use inorganic nitrogen, which shows their wider biosynthetic capacities. It indicates that the organisms do not have any essential aminoacid as other prokaryotes. Growth in inorganic nitrogen, however, is pretty slow compared to organic nitrogen as found by Naeimpoor and Mavituna (2006). Glutamine synthetases (GS) are key enzymes of nitrogen metabolism. Most bacteria contain only one type of GS enzyme encoded by glnA. Streptomyces coelicolor, the model organism for Gram-positive streptomycetes, however is characterized by two functional GS (glnA, glnII) involved in nitrogen assimilation. The control of nitrogen assimilation and metabolism is mediated by transcriptional and post-translational regulation systems (Reuther and Wohlleben 2007). Differences in growth patterns for structurally difficult amino acids and simpler ones are marked during the initial days of incubation (not shown in table).
Carbohydrate utilization properties are one of the important biochemical activities of microorganisms to identify and classify them (Dietz 1988; Holt 1989). Microorganisms reduce carbohydrates to organic acids and concomitant reduction of pH of the culture medium is indicated by color change with appropriate indicator. As peptone is rich source of nitrogen (about 16% of molecular weight), microorganisms are forced to produce alkaline products in peptone broth without carbohydrate. However, with sugar as surplus carbon, actinomycetes produce more organic acids, which neutralize alkali from peptone and, further, lower pH of the culture medium. Actinomycetes are weak acid producers and, hence, the optimum peptone concentration for carbohydrate utilization test is 0.2% in the culture medium. At least five days of incubation is mandatory to interpret carbohydrate utilization test for actinomycetes. The interpretation should be based on color change of the culture medium, e.g., clear yellow color of phenol red.
References
Agrawal, VP, Biodiversity of Khumbu Region: Population Study of Actinomycetes, a Project Report Submitted to the Royal Nepal Academy of Science and Technology, Khumaltar, Lalitpur, Nepal, 2003
Busti, E, Monciardini, P, Cavaletti, L, et al. Antibiotic-producing ability by representatives of a newly discovered lineage of actinomycetes, Microbiology, 2006; 152:675-683
Dietz, A, Practical and proposed cooperative investigational criteria for taxonomic studies of the actinomycetes. In; "Biology of Actinomycetes '88" (Eds. Okami Y, Beppu T and Ogawara H), Japanese Scientific Societies Press, Tokyo, pp. 203-209, 1988
Holt, JG, Bergey’s manual of systematic bacteriology, Vol. 4, (Ed.) Williams, ST and Sharpe, ME, Baltimore, Md : Williams and Williams, 1989
Naeimpoor, F, and Mavituna, F, Metaboloc flux analysis in Streptomyces coelicolor: Effect of nitrogen source. In; Novel frontiers in the production of compounds for biomedical use. Springer Netherlands, Vol. 1, pp. 131-145, 2006
Reuther, J, and Wohlleben, W, Nitrogen Metabolism in Streptomyces coelicolor: Transcriptional and Post-Translational Regulation. J. Mol. Microbiol. Biotechnol. 2007; 12:139-146
Singh, D, and Agrawal, VP, Microbial Biodiversity of Mount Sagarmatha Region, a paper presented in the International Seminar on Mountains, Kathmandu March, pp. 6 – 8, 2002
Voelker, F, and Altaba, S, Nitrogen source governs the patterns of growth and pristinamycin production in ‘Streptomyces pristinaespiralis’, Microbiology, 2001; 147:2447-2459
Kiran Babu Tiwari1,2* and Upendra Thapa Shrestha1
1Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal;
2Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal
Abstract
Actinomycetes are one of the diverse groups of soil bacteria possessing commercially useful enzymes and therapeutically useful bioactive molecules. The biochemical characterization of the individual isolates is of therefore utmost importance to understand their basic physiology. For the optimization of peptone concentration in carbohydrate utilization test, actinomycetes were grown at different concentrations of peptone and carbohydrate. With 10mM glucose, 0.2% peptone was found to be optimum concentration for glucose (carbohydrate) utilization test. However, the growth was retarded at a concentration above 0.5% of peptone. Besides as nitrogen source, actinomycetes can use peptone as carbon source too. The organisms can also use inorganic nitrogen source with slow growth rate. Actinomycetes have no essential amino acid; however, organic nitrogen enhances the growth. Actinomycetes are weak acid producers; still an appreciable lowering of pH was observed within five days of incubation at 28ºC.
Keywords: Actinomycetes, amino acid, assimilation, nitrogen, optimization
Introduction
Actinomycetes are aerobic, Gram-positive bacteria, which may form branching filaments or hyphae that may persist as a stable mycelium or may break up into rod-shaped or coccoid elements. Actinomycetes are widely distributed in terrestrial environment (Holt 1989). For much of this time they were regarded as an exotic group of organisms with affinities to both bacteria and fungi. However, determination of their fine structure and chemical composition initiated in the 1950’s, confirmed their prokaryotic nature. The actinomycetes comprise a ubiquitous order of bacteria, which exhibit wide physiological and morphological diversity. Actinomycetes have long been a source of commercially useful enzymes and therapeutically useful bioactive molecules. Actinomycete biodiversity is vast frontier and potential goldmine for the biotechnology industry because it offers countless new genes and biochemical pathways to probe for enzymes, antibiotics and other useful molecules (Agrawal 2003). Over past 60 years, products derived from microbial secondary metabolites have been used to meet medical, industrial and agricultural needs, e.g., antibiotics, anticancer drugs, antifungal compounds, immunosuppressive agents, enzyme inhibitors, anti parasitic agents, herbicides, insecticides and growth promoters (Busti et al. 2006). Hence, basic parameters for better growth and physiological characteristics of native micro-flora need to be optimized.
Materials and Methods
Isolation and identification of actinomycetes: Actinomycetes were isolated from soil samples in selective medium (Starch-Casein agar, pH 7.4) by serial dilution method and subsequently purified and identified by colonial characteristics and microscopy (Singh and Agrawal 2002).
Effects of peptone concentration on actinomycetes growth: A series of peptone broths (0.05%, 0.1%, 0.2%, 0.5%, 0.8% and 1.0%) with or without glucose (10mM) were prepared and sterilized. The isolates were inoculated into the respective culture tubes, incubated at 28ºC and observed for growth characteristics in the broth subsequently on daily basis. Respective negative control tubes were also included.
(For optimization of peptone)
Essential amino acids for actinomycetes: Twenty glucose broth tubes containing 10mM glucose and 0.3% Na2PO4 were prepared, and in all but only one amino acid (1mM) was added in respective tubes. The other three tubes containing only 0.3% Na2PO4 was added with inorganic nitrogen (1mM KNO3), organic carbon (10mM glucose), and both (1mM KNO3 and 10mM glucose) respectively. The isolate was inoculated into the respective sterile broth, incubated at 28ºC and observed for growth characteristics in the broth on daily basis comparing with negative control tubes.
Carbohydrate (Glucose) utilization test for actinomycetes: Glucose broths containing 10mM glucose, 0.3% Na2PO4, 3mg% Phenol Red and 0.2% Peptone was prepared. A nitrogen control tube (0.2% Peptone) was also included. The isolates were inoculated into the respective sterile broths, incubated at 28ºC and observed for growth and coloration of the broth on daily basis.
Results
Three representative actinomycete isolates (viz. Lob 1, 8 and 15) were assayed for various tests. Actinomycetes were found to utilize peptone as both nitrogen source and as carbon source (Table 1). The optimum concentration of peptone for growth was from 0.2 – 0.5%, beyond which the growth was somewhat retarded. Addition of glucose as carbon source allows better growth, and upto 0.8% of peptone can be used along with glucose for growth assessment of actinomycetes (Table 2). Actinomycetes can use inorganic nitrogen (KNO3), however organic nitrogen enhanced their growth (Table 3). At least five days of incubation is required for glucose utilization test of actinomycetes with conventional dark yellow coloration of media containing Phenol red as indicator (Table 4).
Table 1: Effect of peptone on growth of actinomycetes
Peptone broth | Growth | ||||||||
Fourth day | Fifth day | Sixth day | |||||||
Lob1 | Lob8 | Lob15 | Lob1 | Lob8 | Lob15 | Lob1 | Lob8 | Lob15 | |
1.0% | - | - | - | + | + | + | +++ | ++ | ++ |
0.8% | + | + | + | ++ | ++ | ++ | Tubes were discarded | ||
0.5% | ++ | ++ | + | +++ | +++ | ++ | |||
0.2% | ++ | ++ | + | +++ | +++ | + | |||
0.1% | + | + | + | +++ | ++ | + | |||
0.05% | + | + | + | + | + | + | ++ | ++ | ++ |
Note: Slight (+), Easily observable (++), Marked (+++)
Table 2: Effect of glucose on growth of actinomycetes in peptone broth
Peptone + 10mM Glu broth | Growth | |||||
Fourth day | Fifth day | |||||
Lob 1 | Lob 8 | Lob 15 | Lob 1 | Lob 8 | Lob 15 | |
1.0% | ++ | + | + | +++ | ++ | ++ |
0.8% | +++ | ++ | ++ | +++ | ++ | ++ |
0.5% | +++ | +++ | +++ | +++ | +++ | +++ |
0.2% | +++ | +++ | +++ | +++ | +++ | +++ |
0.1% | +++ | ++ | + | +++ | ++ | ++ |
0.05% | + | + | + | ++ | ++ | ++ |
Note: Slight (+), Easily observable (++), Marked (+++)
Table 3: Source of nitrogen for actinomycetes (Lob 1)
Growth | 1mM KNO3 | 10mM Glu | 1mM KNO3 +10mM Glu | 0.05mM each AAs +10mM Glu |
Third day | - | - | - | + |
Fourth day | - | - | - | ++ |
Fifth day | - | - | (+) | +++ |
Sixth day | - | - | + | +++ |
Note: No growth (-), Indicative ((+)), Slight (+), Easily observable (++), Aminoacids (AAs)
Table 4: Glucose utilization test for actinomycetes (Lob 1)
Observation on | 0.2% Peptone (Nitrogen control) | 0.2% Peptone + 10mM Glucose | |
Growth | Third day | + | + |
Fourth day | ++ | ++ | |
Fifth day | +++ | +++ | |
Change in color | Third day | No Change | |
Fourth day | Light pink | Light yellow | |
Fifth day | Dark pink | Dark yellow | |
Discussion
As actinomycetes are one of the diverse groups of soil bacteria possessing commercially useful enzymes and therapeutically useful bioactive molecules, biochemical characterization of the individual isolates is of utmost importance to understand their basic physiology (Holt 1989). The present work explored importance and optimization of nitrogen substrates in basal culture media as a basic parameter for better growth and physiological characteristics of the organisms. The actinomycetes were isolated and purified from soil samples, of which only representative isolates were assayed for their physiological activities concerned with nitrogen assimilation.
Conventionally, peptone is used in various culture media as major nitrogen source for the organisms. As peptone is partial digest of protein complex, its carbon atom is liable to be used by organisms. Hence, actinomycetes can use peptone as carbon source when the culture medium lacks carbohydrates (e.g., sugar/s). Voelker and Altaba (2001) assayed role of various nitrogen sources (organic and inorganic) for growth and secondary metabolite production from a streptomycete. During balanced growth, either mineral or organic nitrogen sources were readily utilized. Glutamate and alanine were used as both nitrogen and carbon source, sparing the utilization of the primary carbon source, glucose. Without sugar, 0.2–0.5% peptone concentration is optimum for growth of actinomycetes. Higher peptone concentration disturbs C/N ratio and hence retards growth. With glucose, upto 0.8% of peptone can be used for growth; because, glucose, being an alternate to peptone carbon, increases the C/N ratio to the safe level.
Actinomycetes can fairly use inorganic nitrogen, which shows their wider biosynthetic capacities. It indicates that the organisms do not have any essential aminoacid as other prokaryotes. Growth in inorganic nitrogen, however, is pretty slow compared to organic nitrogen as found by Naeimpoor and Mavituna (2006). Glutamine synthetases (GS) are key enzymes of nitrogen metabolism. Most bacteria contain only one type of GS enzyme encoded by glnA. Streptomyces coelicolor, the model organism for Gram-positive streptomycetes, however is characterized by two functional GS (glnA, glnII) involved in nitrogen assimilation. The control of nitrogen assimilation and metabolism is mediated by transcriptional and post-translational regulation systems (Reuther and Wohlleben 2007). Differences in growth patterns for structurally difficult amino acids and simpler ones are marked during the initial days of incubation (not shown in table).
Carbohydrate utilization properties are one of the important biochemical activities of microorganisms to identify and classify them (Dietz 1988; Holt 1989). Microorganisms reduce carbohydrates to organic acids and concomitant reduction of pH of the culture medium is indicated by color change with appropriate indicator. As peptone is rich source of nitrogen (about 16% of molecular weight), microorganisms are forced to produce alkaline products in peptone broth without carbohydrate. However, with sugar as surplus carbon, actinomycetes produce more organic acids, which neutralize alkali from peptone and, further, lower pH of the culture medium. Actinomycetes are weak acid producers and, hence, the optimum peptone concentration for carbohydrate utilization test is 0.2% in the culture medium. At least five days of incubation is mandatory to interpret carbohydrate utilization test for actinomycetes. The interpretation should be based on color change of the culture medium, e.g., clear yellow color of phenol red.
References
Agrawal, VP, Biodiversity of Khumbu Region: Population Study of Actinomycetes, a Project Report Submitted to the Royal Nepal Academy of Science and Technology, Khumaltar, Lalitpur, Nepal, 2003
Busti, E, Monciardini, P, Cavaletti, L, et al. Antibiotic-producing ability by representatives of a newly discovered lineage of actinomycetes, Microbiology, 2006; 152:675-683
Dietz, A, Practical and proposed cooperative investigational criteria for taxonomic studies of the actinomycetes. In; "Biology of Actinomycetes '88" (Eds. Okami Y, Beppu T and Ogawara H), Japanese Scientific Societies Press, Tokyo, pp. 203-209, 1988
Holt, JG, Bergey’s manual of systematic bacteriology, Vol. 4, (Ed.) Williams, ST and Sharpe, ME, Baltimore, Md : Williams and Williams, 1989
Naeimpoor, F, and Mavituna, F, Metaboloc flux analysis in Streptomyces coelicolor: Effect of nitrogen source. In; Novel frontiers in the production of compounds for biomedical use. Springer Netherlands, Vol. 1, pp. 131-145, 2006
Reuther, J, and Wohlleben, W, Nitrogen Metabolism in Streptomyces coelicolor: Transcriptional and Post-Translational Regulation. J. Mol. Microbiol. Biotechnol. 2007; 12:139-146
Singh, D, and Agrawal, VP, Microbial Biodiversity of Mount Sagarmatha Region, a paper presented in the International Seminar on Mountains, Kathmandu March, pp. 6 – 8, 2002
Voelker, F, and Altaba, S, Nitrogen source governs the patterns of growth and pristinamycin production in ‘Streptomyces pristinaespiralis’, Microbiology, 2001; 147:2447-2459
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