Antibiotics and Bioactive Microbial Metabolites
Antibiotics
Antibiotics are widely recognized as low-molecular-weight microbial metabolites that inhibit the growth of other microorganisms at low concentrations This definition, accepted with minor variations by various authors, highlights the clear identification of antibiotics by their origin and biological activity However, some terms within this definition require further clarification to enhance understanding.
Low-molecular-weight metabolites are molecules that typically weigh a few thousand daltons or less While enzymes like lysozyme and other complex proteins with antimicrobial properties are not classified as antibiotics, antibiotics are specifically defined as natural products derived from microorganisms Additionally, the term "semisynthetic antibiotics" encompasses products that result from the chemical modification of these natural substances.
The inhibition of microbial growth primarily involves preventing the reproduction of cells, thereby affecting the overall population rather than individual cells This inhibition can be permanent, leading to a sustained reduction in microbial presence.
Antibiotics can be classified as "cidal," such as bactericidal and fungicidal, or "static," like bacteriostatic, depending on their effect on bacteria The term "at low concentrations" is critical in this context, as even normal cellular components can be harmful at high levels For example, amino acids like glycine or leucine can inhibit bacterial growth when present in elevated concentrations Additionally, products of anaerobic fermentation, such as ethanol and butanol, do not qualify as antibiotics due to their nature Typical antibiotics exhibit significant antimicrobial activity, often detectable at micromolar or even nanomolar concentrations against certain bacterial species.
The minimal inhibitory concentration (MIC) is a key parameter used to measure antimicrobial activity, defined as the lowest concentration of an antibiotic that prevents visible growth of a test organism after incubation To determine the MIC, decreasing concentrations of the antibiotic are added to nutrient media, typically in microwells, or incorporated into agar plates where a droplet of the test organism culture is applied The MIC is expressed in micrograms per milliliter and can be assessed in both liquid and solid media, with the absence of visible growth or colony formation indicating the effective concentration.
The Minimum Inhibitory Concentration (MIC) refers to the lowest concentration of an antibiotic needed to inhibit a specific microbial strain, highlighting that different strains of the same species may respond to varying antibiotic levels The range of microbial species affected by an antibiotic, characterized by low MICs, is known as its spectrum of activity, which can vary significantly between different antibiotics Some antibiotics possess a narrow spectrum, effective only against either gram-positive or gram-negative bacteria, while others exhibit a broad spectrum, inhibiting a diverse range of bacterial or fungal species Additionally, the classification of certain antibiotics as antitumor agents stems from their initial identification based on antibacterial properties, followed by subsequent evaluation of their cytostatic or cytocidal effects.
Antibiotics exhibit diverse mechanisms of action that inhibit the growth of sensitive microorganisms by interfering with essential target molecules involved in cell metabolism These target molecules are typically macromolecules like DNA, RNA, and enzymes, although they can also include small metabolites such as enzymatic substrates or membrane components Understanding an antibiotic's mechanism involves identifying the target molecule, its interaction site, and the type of interaction, although it is often easier to pinpoint the blocked metabolic pathway rather than the specific molecule Consequently, antibiotics are commonly categorized based on their effects, such as those that inhibit cell wall synthesis, DNA replication, transcription, protein synthesis, or cell membrane functions.
The selectivity of antibiotics is primarily due to their specific mechanisms of action, which target unique molecules in bacterial cells that lack equivalents in mammalian cells or differ significantly in composition This specificity allows antibiotics to effectively combat bacterial infections while remaining nontoxic to higher organisms.
The chemical structures of thousands of antibiotics have been identified and documented in specialized literature, thanks to modern physicochemical techniques that ensure new compounds are published alongside their structural elucidation A key takeaway from this extensive data is that antibiotics represent a highly heterogeneous group of substances The classification system of antibiotics, illustrated in Figure 1.1, showcases the diverse range of structures, highlighting the presence of various chemical groups, including oxygenated and nitrogen functions.
FAMILY Carbohydrate Antibiotics Pure saccharides Aminoglycoside antibiotics Other IN- and C-I glycosides Various sugar derivatives Macrocyclic Lactone (Lactaml Antibiotics Macrolide antibiotics
Polyene antibiotics Other macrocycliclactone antibiotics Macrolactam antibiotics
Quinone and Similar Antibiotics Linearly condensed polycyclic compounds Naphthoquinone derivatives
Benzoquinone derivatives Various quinone-like compounds Amino Acid, Peptide Antibiotics Amino acid derivatives Homopeptides Heteromer peptides Peptolides
High molecular weight peptides Nitrogen-containing Heterocyclic Antibiotics Non-condensed (single) heterocycles Condensed (fused) heterocycles Alkaloids with antibiotic (antitumor) activity Oxygen-containing Heterocyclic Antibiotics Furan derivatives
Pyran derivatives and benzopyran derivatives are significant in the field of organic chemistry, alongside small lactones and polyether antibiotics Alicyclic antibiotics and cycloalkane derivatives contribute to the diversity of antibiotic compounds Additionally, small terpanes and oligoterpene antibiotics play a crucial role in medicinal applications Aromatic antibiotics, including benzene compounds, condensed aromatic compounds, and non-benzenoid aromatic compounds, further expand the range of therapeutic agents Various derivatives of aromatic compounds and aliphatic antibiotics are also essential for developing new pharmaceuticals.
Alkane derivatives Aliphatic carboxylic acid derivatives Aliphatic compounds with S or P content Miscellaneous antibiotics
Antibiotics exhibit significant chemical diversity, as highlighted by Berdy (1974), with various substituents like halogen atoms, alkyl, or acyl groups enhancing their molecular variety This heterogeneity underscores the error in categorizing antibiotics as a uniform class of chemical compounds, unlike proteins or steroids, as there are no shared chemical characteristics among all antibiotics or even within the subset used in medicine.
Antibiotics can be informally classified into "families" based on their common structural characteristics and similar mechanisms of action, although this classification is not strictly defined It is important to note that the antimicrobial activity can vary significantly among different members of the same family Some well-known families of antibiotics include various groups that share these biological properties.
1 The ,8-lactam antibiotics, chemically characterized by the pres- ence of a four-membered ring closed by an amide bond (Fig 1.2), and that act by inhibiting the peptidoglycan synthesis of the bacterial cell wall The ,8-lactams are divided in subfamilies, such as penicillins, cephalosporins, carbapenem, and mono- bactams, according to specific chemical features
2 The aminoglycosides, constituted by an aminocyclitol (an ali- cyclic six-membered ring with hydroxyl and amino substituents) and by a few sugars or amino sugars (Fig 1.3); these act by inhibiting ribosomal functions
3 The tetracyclines, whose structure consists offour linearly con- densed rings (Fig 1.4 ), and which also inhibit protein synthesis at the ribosomal level
4 The anthracyclines, also constituted by four condensed rings (Fig 1.4), but acting at the DNA level These interfere with the enzyme topoisomerase and are used as antitumor, rather than anti-infective, agents
5 The antibacterial macrolides, characterized by a large lactone ring (Fig 1.5); inhibitors of protein synthesis by binding to the large subunit of bacterial ribosomes
Bioactive Secondary Metabolites
The term "secondary metabolites," coined by Bu'Lock in the early 1960s, refers to microbial metabolites produced during differentiation within specific taxonomic groups, which are not essential for cell metabolism Unlike primary metabolites, such as amino acids and nucleotides that are crucial for basic cellular functions, secondary metabolites serve distinct roles in various organisms.
The term "secondary metabolites" has faced scrutiny from various authors, who suggest that "specific metabolites" may be a more fitting alternative, as it highlights the unique biochemical diversity within the microbial realm Despite this, the widespread use of "secondary metabolites" makes it challenging to replace the term in practice.
Secondary metabolites are low-molecular-weight compounds with the following characteristics:
1 They are synthesized by only some microbial strains, and are the expression of the biochemical differentiation of the pro- ducing organism
2 They have no obvious function in the growth of cultures or colonies Strains able to make these molecules may lose, because of mutations, the capacity to synthesize them, without any ap- parent effect on their general metabolism
3 They are often produced in connection with differentiation processes
4 They are often made as families of similar products
Not all secondary metabolites share the same characteristics, and it's crucial to differentiate between "no obvious function in growth" and "no function at all." The absence of essential metabolic functions does not equate to a lack of selective evolutionary functions, especially in relation to other microorganisms The key aspect of secondary metabolism is that it reflects the organism's individuality and cellular differentiation, often becoming evident only under conditions of limited growth.
Antibiotics are classified as secondary metabolites, and research has explored their potential role as regulatory agents in the metabolism of their producing organisms Notably, pamamycin is the only antibiotic for which a specific function has been established, serving as an inducer of aerial mycelium formation in actinomycetes.
Microorganisms produce a variety of secondary metabolites beyond antibiotics, including pigments, protease inhibitors, and toxins While research has traditionally focused on antimicrobial activities, recent investigations into microbial metabolites have uncovered a significant number of new bioactive compounds These bioactive microbial metabolites, which inhibit physiological functions in higher organisms, are discovered less frequently than antimicrobial substances.
1.2.2 Antimicrobial and Other Biological Activities
If we analyze the general biological activity of secondary metab- olites in relation to their antimicrobial activity, the following different situations are observed:
1 Several typical antibiotics demonstrate, in addition to their an- timicrobial activity, an appreciable activity on other biological systems This is the case, for instance, of the cholesterol-lowering activity of rifamycins; the induction of gastric motility by erythromycin; and the ability of ristocetin to trigger platelet aggregation These activities have no relation to the mechanism of action of the antibiotic and may be enhanced in semisynthetic derivatives, without a corresponding in- crease, or even with the disappearance, of the antimicrobial activity
Activities involving the testing of thousands of molecules across various biological systems can yield both positive and negative interactions This phenomenon is expected, especially given the extensive experience gained from pharmacological screenings of synthetic compounds in pharmaceutical research laboratories over the years.
2 The same biological action can be effective both on the micro- bial cell and on cells or systems of higher organisms This is the case for compounds such as mevinolin or compactin that, by inhibiting the isoprenoid biosynthesis, are active as fungistatic and are cholesterol- lowering agents in man Analogous cases may be those of the immu- nosuppressant agents cyclosporin A and FK 506, which inhibit two different enzymes having a peptidyl-prolyl isomerase activity The binding of these metabolites to corresponding fungal enzymes results in the formation of complexes toxic for the microorganism
3 Many bioactive metabolites do not show any microbiological activity Among these are cytostatic compounds, insecticides and an- tiparasites, and herbicides, but the vast majority result from the search for substances potentially active as pharmacological agents These are predominantly inhibitors of physiologically (or pathologically) relevant enzymes, antagonists of hormones or other regulatory molecules that act by competing with the natural ligand for cellular receptors There are also immunomodulators, antagonists of cell growth factors, and free radical scavengers
The role of certain substances in relation to producer cell metabolism or in facilitating communication between cells or species remains unclear Their diverse biological activities and chemical structures complicate any overarching conclusions about their connection to the organisms that produce them.
1.2.3 Bioactive Secondary Metabolites of Practical Interest
To qualify as an effective biological agent, a substance must first demonstrate significant inhibitory activity in vitro or impact cell cultures, ensuring high selectivity Additionally, potential drugs should exhibit optimal absorption and distribution within the human body, achieving adequate concentrations at their target sites without causing toxicity or severe side effects While the criteria may be less rigorous for agricultural or veterinary products, maintaining a strong level of in vivo activity is essential Lastly, the substance must also present a competitive economic advantage over similar alternatives.
Only a limited number of bioactive microbial compounds have been successfully developed into industrial products This article highlights several substances that, despite the challenges faced, have either reached or are nearing commercial viability.
Bestatin, a dipeptide, is among the initial discoveries from screenings aimed at identifying inhibitors of cell surface enzymes This compound acts as an immune system enhancer and has been evaluated in various clinical studies.
Figure 1.8 lmmunomodulators of microbial origin: (a) cyclosporin A; (b) bestatin; (c)
FK 506 as an adjuvant in tumor therapy and in the treatment of Candida infections
Cyclosporin A is an 11-amino acid cyclic peptide initially discovered for its antifungal properties, but later developed for its immunosuppressive effects, particularly in organ transplantation It functions by inhibiting T-cell activation through its binding to cyclophilin, an enzyme known for its peptidyl-prolyl isomerase activity.
Biology of Antibiotic-Producing Microorganisms
Genus Bacillus
The genus Bacillus consists of diverse unicellular, rod-shaped bacteria that can be aerobes or facultatively anaerobes, known for their ability to form endospores in adverse environmental conditions These gram-positive bacteria are typically motile, utilizing lateral or peritrichous flagella, and primarily inhabit soil as saprophytes Notably, B anthracis is a human pathogen, while B thuringiensis targets insects.
The Bacillus genus, part of the Bacillaceae family, is characterized by its aerobic or facultative nature, distinguishing it from the Clostridium genus, which is also rod-shaped and endospore-forming With over 200 species documented, the most notable is B subtilis, recognized as the type species.
The bacilli, despite being classified within a single genus, represent a diverse group of organisms, characterized by a G + C content of DNA that varies between 33.2% and 66.4% Typically, these organisms possess a meso-DAP murein type, although lysine has been observed in a few species Additionally, with one exception, the membrane fatty acids are predominantly branched, either in the iso or anteiso form, and some species also contain unsaturated fatty acids.
Endospores, which can be easily seen inside the mother cell because they are refractile under phase-contrast microscopy, are composed of
The spores feature an electron-transparent core surrounded by an inner and outer membrane that define a primordial cell wall, along with a thick cortex and protein coats Notably, the cortex is characterized by an unusual peptidoglycan structure, and the spores are distinguished by a high calcium dipicolinate content, which can account for up to 15% of their dry weight.
The genus Bacillus exhibits a wide range of nutritional needs and growth conditions Most strains thrive in media rich in simple sugars, amino acids, B vitamins, inorganic nitrogen, and minerals Notably, some species, such as B subtilis, can grow without amino acids or vitamins, while others require unidentified factors found in yeast or soil extracts Under anaerobic conditions, certain Bacillus species ferment sugars to produce 2,3-butanediol, glycerol, and carbon dioxide, while others yield hydrogen without glycerol.
Most species are mesophilic, while a few others grow preferentially at 1 0°C or are definitely thermophilic, requiring temperatures from 40 to 65°C for maximum growth
The transformation of vegetative cells into endospores is a complex process that takes up to 8 hours and is initiated by environmental factors such as nutrient scarcity or high cell density This process begins with the condensation of the nucleoid into a compact structure, followed by the asymmetric division of the cell into a forespore and a mother cell As the forespore is engulfed by membrane outgrowth, its structure and enzymatic functions undergo significant modifications The differentiation of the membrane into inner and outer layers occurs, along with the synthesis of the primordial cell wall and cortex Ultimately, the external coats are formed, the core reaches its final structure, and metabolic activity within the spore is halted.
Endospores exhibit remarkable resistance to heat, dryness, and disinfectants, allowing them to remain viable in a dormant state for extended periods Their germination and return to vegetative growth depend on suitable nutritional conditions and can be activated by various factors, including temperature changes or low pH levels.
Research on the chromosomal structure of bacilli, particularly B subtilis, has revealed valuable insights, as DNA transformation in this organism facilitates genetic analysis Additionally, the use of phage PBSl for transduction enables the transfer of longer DNA fragments, contributing to the creation of a detailed linkage map of the circular B subtilis chromosome This map highlights both similarities and differences when compared to the well-studied Escherichia coli chromosome.
The DNA homology between B subtilis and other Bacillus species, as determined by DNA-DNA hybridization, is surprisingly low, and
B subtilis is not easily transformed by the DNA of other species How- ever, the limited genetic analysis of other bacilli revealed several linkage groups identical to those of B subtilis The overall picture suggests that there is conservation of gene order in closely related species, whereas in more distant species the conservation is partially lost because of translocation of large DNA fragments
In Bacillus subtilis, over 50 distinct mutations have been identified across various chromosomal loci that inhibit sporulation while leaving vegetative metabolism unaffected These mutations are categorized into spo genes based on their involvement in specific stages of spore development, ranging from spoO, which is associated with the initial events in vegetative cells, to spo VI, which plays a crucial role in spore maturation.
The regulation of metabolic events during sporulation is highly intricate, with many identified genes serving regulatory rather than structural roles The spo genes activated after the first hour are typically transcribed by RNA polymerase in conjunction with specific sigma factors, four of which have been recognized Among these, sigma factors uE and ? are expressed early, with the latter potentially influencing forespore gene expression, while r1- is believed to function in the mother cell In later stages, rP and aX are active in the forespore and mother cell, respectively.
Several genes play a crucial role in regulating u factor expression at both transcriptional and translational levels Additionally, a distinct class of genes encodes positive or negative transcriptional regulators, which fine-tune this regulatory process Lastly, late genes are essential for structural functions, such as the synthesis of coat proteins and the maturation of the spore core.
Due to the unavailability of B subtilis plasmids, initial cloning of its DNA was conducted using E coli phages and plasmid vectors, with the Charon phage, derived from X phage, being commonly utilized to create large libraries Additionally, chromosomal libraries were developed using the E coli plasmid pMB9 and shuttle plasmids that replicate in both B subtilis and E coli These methods have been instrumental in studying the regulatory signals of gene expression.
Current research focuses on cloning genes that encode proteins with industrial significance, emphasizing the stability of constructs that incorporate large DNA fragments Notably, the B subtilis cryptic pTA1060 plasmid demonstrates greater stability compared to S aureus plasmids.
Bacilli are primarily utilized in industrial applications for enzyme production, particularly known for their ability to generate various extracellular proteins with hydrolytic activity A significant product is the alkaline protease subtilisin, which is extensively used in the detergent industry, with hundreds of tons produced annually Additionally, large quantities of amylases are manufactured for the food and brewing sectors Furthermore, cellulases and xylanases hold potential for converting waste materials into substrates for industrial fermentation processes.
Genus Pseudomonas
Pseudomonas bacteria are gram-negative, motile rods measuring 1 µm in diameter and 1.5-5 µm in length, typically propelled by multiple polar flagella While they primarily thrive in aerobic conditions, certain species can utilize nitrates as an alternative electron acceptor Notably, Pseudomonas species do not exhibit resting stages and often store polyhydroxybutyrate as a reserve material.
Pseudomonas strains are found in a variety of habitats, primarily in soil, where they excel at degrading organic matter, especially aromatic compounds Some species are linked to the rhizosphere or plant leaves, while P mallei and P pseudomallei are known pathogens affecting humans and animals Additionally, P aeruginosa, typically a saprophyte in soil, is a prevalent cause of nosocomial infections.
Pseudomonas cells can be distinguished from those of related gen- era, such asXanthomonas, because they are multiflagellated, accumulate polyhydroxybutyrate, are often pigmented, and do not require growth factors
The genus is categorized into five subgroups based on rRNA homology, with Group I being the largest and including common soil species like P aeruginosa, P putida, and P fluorescens These species are notable for producing fluorescent pigments that chelate iron, enabling their growth in low-iron environments Group II consists of potential animal and plant pathogens, while Group III includes species that utilize CO2 and H2 as energy sources Groups IV and V contain a limited number of species with diverse characteristics.
The genus definition includes multiple polar flagella, yet some species are nonflagellated, while others may possess lateral flagella Like other gram-negative bacteria, these cells are encased in an outer membrane, which has been thoroughly researched in P aeruginosa This membrane serves as a highly effective permeability barrier, with specific protein porin F forming channels that facilitate the passage of small hydrophilic molecules.
Pseudomonas strains exhibit simple nutritional needs, thriving on a medium with low-molecular-weight carbon compounds, nitrates or ammonium ions for nitrogen, and essential mineral salts While they do not utilize methane or other one-carbon compounds, their remarkable versatility allows them to efficiently utilize a wide range of carbon sources for optimal growth.
P putida, for exan-:.ple, can grow on any of 78 compounds as the sole carbon and nitrogen source This versatility relates to an unusual ca- pacity to produce enzymes (oxygenases, dehalogenases, and amidases) by which many aliphatic or aromatic compounds are converted into suitable substrates for catabolic enzymes The wide range of catabolic activities is also characteristic of the genus Catechols, for instance, are degraded either by a chromosomally expressed 1 ,2-dioxygenase or by a plasmid-encoded 2,3-dioxygenase Specific pathways are known for the degradation of polycyclic aromatics, amino acids, and terpenoids The final products of catabolism are generally either acetate or inter- mediates of the Krebs cycle
Glucose metabolism varies among species, with some utilizing the Entner-Doudoroff pathway instead of the typical glycolytic route In this process, glucose is oxidized to 2-keto-3-deoxygluconate, which is then cleaved into pyruvate and triose-phosphate.
Classical conjugation studies involving strains with the chromosome-mobilizing plasmid FP2 and transduction studies using phage Fll6 have led to the development of a detailed linkage map of Pseudomonas aeruginosa's circular chromosome This map highlights significant organizational differences compared to Escherichia coli, particularly in the arrangement of genes related to amino acid and purine biosynthesis, which are not clustered as they typically are within the Enterobacteriaceae family, although many are located in the same chromosomal region Additionally, the genes associated with catabolic pathways are also found to be dispersed throughout the chromosome.
A more restricted map of the P putida chromosome reveals fewer genetic markers, highlighting differences in gene order Notably, catabolic genes are predominantly clustered in one area, while biosynthetic genes are located in another This arrangement indicates the potential existence of a "supra operon" mechanism governing transcription regulation in both species.
Many properties of Pseudomonas strains are related to the large number ofplasmids they host These plasmids have been classified into
13 incompatibility groups Members of the same group often present similar phenotypic characteristics, have similar size, and, where ex- amined, DNA homology has been found
Plasmids commonly harbor genes that confer antibiotic resistance; however, this resistance is typically not plasmid-mediated in clinical isolates Among the various plasmids, those responsible for degradative metabolic pathways are particularly crucial for Pseudomonas metabolism Notable examples include TOL plasmids, which are capable of degrading aromatic compounds like xylene and toluene, as well as the NAH7 plasmid, which metabolizes naphthalene into salicylate and pyruvate-acetaldehyde, and the CAM plasmid, which converts camphor into isobutyrate.
As mentioned previously, some plasmids have chromosome- mobilizing ability and are thus useful for the construction of link- age maps
Pseudomonads generate various secondary metabolites that are not classified as antibiotics, including fluorescent pyoverdines and the siderophore pyochelin Additionally, they produce several phytotoxins along with syringomycins and syringostatins derived from P syringae.
A large proportion of the antibiotic substances are produced by strains belonging to one of two species: P aeruginosa and P.jluorescens
Most of these antibiotics are nitrogen-containing heterocyclic molecules (derived biosynthetically from the catabolism of amino acids), such as the phenazine derivatives iodinin and pyocyanine
Several notable antibiotics derived from Pseudomonas strains have been identified in the metabolites of various organisms, including the peptidoglycan inhibitors cycloserine and fosfomycin, as well as the antiprotozoal agent azomycin However, only two antibiotics originally produced by this genus have been successfully utilized in clinical settings.
Pyrrolnitrin, a natural nitroderivative initially derived from P pyrrolnitrica and P fluorescens, exhibits broad antifungal properties and is utilized for the topical treatment of superficial infections Currently, it is manufactured through chemical synthesis for commercial use.
Figure 2.2 Antibiotics of interest produced by Pseudomonas strains: (a) mupirocin (pseudomonic acid); (b) pirrolnitrin; (c) sulfazecin (Rl = H, R2 = CH 3), isosulfazecin (Rl = CH 3 , R2 = H)
Mupirocin, also known as pseudomonic acid A, is derived from Pseudomonas fluorescens and exhibits activity against a wide range of bacteria, including many gram-positive and some gram-negative strains Although it is rapidly metabolized into the inactive derivative monic acid and lacks systemic efficacy, mupirocin is effectively used topically for treating skin infections and eliminating staphylococci in healthy carriers.
Monobactams sulfazecin and isosulfazecin were initially isolated from Pseudomonas acidophila and Pseudomonas mesoacidophila cultures Despite their limited practical application due to insufficient activity, their structural characteristics have inspired the development of the clinically valuable analogue aztreonam.
Streptomyces and Streptoverticillium
The genus Streptomyces, part of the Actinomycetales order, consists of gram-positive, aerobic filamentous bacteria that grow in branched filament mats known as hyphae These hyphae form both substrate and aerial mycelium, with the unique feature of aerial hyphae segmented into long chains of nonmotile spores.
The genus Streptoverticillium closely resembles the genus Streptomyces in various biological aspects, with the primary distinction being morphological In Streptoverticillium, the aerial hyphae produce spore chains that are organized in verticils or umbels, where multiple chains originate from a single point on the hypha.
Streptomycetes are predominantly found in soil, where they significantly contribute to the degradation of organic polymers like chitin, starch, and lignin They can also inhabit freshwater and marine environments, with S scabies identified as a plant pathogen In soil, they primarily exist as spores, with concentrations ranging from 10^4 to 10^7 colony-forming units per gram Notably, the proportion of streptomycetes is greater in dry soils compared to damp ones, likely due to their sensitivity to carbon dioxide levels that become inhibitory above 10%.
The Actinomycetales order is categorized into groups based on cell wall composition, with the genus Streptomyces identified by the presence of L-diaminopimelic acid A key feature of Streptomyces is the formation of spore chains on aerial mycelium, which are held together by a sheath, resulting in various surface textures such as smooth, spiny, or hairy These chains can appear straight, flexuous, or in spirals, and their morphology, along with spore appearance, aids in species identification Additional distinguishing factors include colony color, temperature tolerance above or below 45°C, carbon compound utilization, and the ability to solubilize specific substrates While the production of secondary metabolites is significant, it is not a primary criterion for classification, as the same antibiotic can be produced by multiple microorganisms.
Assigning species within the Streptomyces genus is a complex issue, with over 3,000 strains named in patents and scientific literature This complexity arises from varying definitions of species, leading to many synonyms To address this, the International Streptomyces Project has been established, aiming to standardize the redescription of species using uniform criteria.
Distinctive features of Streptoverticillium are, besides the presence of verticils, the cottony appearance of the colonies and the "barbed wire" appearance of the mycelium when seen under moderate magnification
Streptomyces hyphae measure between 0.5 to 2 µm in thickness, with nucleoid distribution occurring along their entire length This genus is characterized by a high guanine-cytosine (G + C) content, ranging from 69% to 78% Their cell wall composition resembles that of other gram-positive bacteria, featuring peptidoglycan chains linked by glycine bridges, while teichoic acids are present and mycolic acids are absent Additionally, menaquinones consist of chains with nine isoprene units, where three or four double bonds are saturated The membrane lipids contain both linear saturated and branched fatty acids.
16 and anteiso-15 and -1 7 usually predominating
Spores and vegetative hyphae exhibit similar fine structures, with spores demonstrating greater resistance to heat and desiccation Despite this resilience, spores maintain low-level metabolic activities, allowing them to survive for years in dry conditions, while exposure to moisture significantly reduces their viability.
Streptomycetes grow in colonies on solid substrates, initiating their life cycle from either hyphal fragments or spores The germination of spores, which requires divalent ions and can be accelerated by mild heat shock, progresses through three stages: darkening, swelling, and germ tube formation The germ tube develops into a network of branched, septated hyphae that form the substrate mycelium Environmental changes or genetic programming lead to the development of aerial hyphae, which utilize nutrients from the substrate mycelium This process may involve low-molecular-weight autoregulators like A-factor Sporulation follows, characterized by the formation of septa in aerial hyphae, thickening of walls, and the development of ellipsoid spores Although the developmental stages of streptomycetes in submerged cultures are less defined, secondary metabolites are typically produced at the end of vegetative growth, coinciding with aerial mycelium formation, and sporulation has been observed in some submerged cultures, suggesting similarities to the life cycle on solid substrates.
Streptomycetes are obligate aerobes and chemoorganotrophs that require only an organic carbon source, such as glucose or starch, along with an inorganic nitrogen source and some mineral salts for growth Their growth rate can be enhanced by using complex media that includes organic nitrogen sources like yeast or malt extract While trace elements in tap water are usually adequate, adding iron, manganese, zinc, and other ions can further improve their growth.
Most streptomycetes are neutrophilic and mesophilic, thriving at a pH of 6.8-7.5 and temperatures typically around 28°C, within the range of 22 to 37°C However, certain strains can be isolated from acidic soils that prefer a pH of 4.5, and thermophilic or thermotolerant strains from compost or manure that grow best at temperatures between 45 and 55°C It is important to distinguish thermophilic streptomycetes from Thermoactinomyces, as the latter are actinomycetes morphologically but share many biological characteristics with bacilli.
Research on carbon compounds and nitrogen metabolism in streptomycetes is limited, making it risky to generalize findings from one species to the entire genus Active transport systems for common sugars are constitutive, while others, including glycerol, are inducible Additionally, cAMP-mediated glucose repression of certain enzymes has been observed In terms of nitrogen assimilation, both glutamine synthetase and glutamate synthase are present in some species, with amino acid transport regulated by both positive and negative feedback mechanisms.
Streptomycetes are known for producing various extracellular enzymes, particularly hydrolases like amylases and proteases Research indicates that the production of these enzymes is inhibited in the presence of readily available nutrients Thermophilic strains predominantly produce cellulases and lignocellulases, while xylanases are also produced by S lividans.
As mentioned previously, one characteristic of the genus is the high G + C content of the DNA The genome size is estimated at about
The genome size of S granaticolor and S hygroscopicus is approximately 7,500 kb, which is about twice that of E coli These species exhibit a replication rate of 600 bases per replication fork, comparable to E coli, and their doubling time is approximately 1 hour and 12 minutes at 28°C.
Streptomycetes, like other actinomycetes, exhibit significant genetic instability, characterized by the irreversible loss of various traits such as aerial mycelium, spore formation, antibiotic production, and arginine biosynthesis enzymes This loss occurs in 0.1-1% of progeny from plated colonies and is attributed to the deletion of large DNA regions, often accompanied by the amplification of adjacent DNA segments The mechanisms driving this genetic instability are currently under intensive investigation, though a comprehensive understanding has yet to be achieved.
Two techniques have been used to develop genetic linkage maps in several Streptomyces species: conjugation and more recently pro- toplast fusion
Intraspecies conjugation has been extensively studied in strains such as S coelicolor, S lividans, and S rimosus In S coelicolor, the primary driver of conjugational exchanges is the plasmid SCP 1, which can exist independently or be integrated into the chromosome Even in strains without SCP 1, a low level of fertility persists due to a second plasmid, SCP2 Similarly, S lividans has two related conjugative plasmids, designated SLP2 and SLP3.
Genera of Actinomycetales Other Than Streptomyces 49 2.5 The Myxobacteria
Actinomycetes are a diverse group of bacteria known for their filamentous growth They can be categorized into two main types: fermentative actinomycetes, which typically associate with humans or animals, and aerobic actinomycetes that thrive in soil environments.
This article focuses on genera of antibiotic-producing species that are commonly found in nature, challenging their classification as "rare actinos." Selective isolation techniques reveal that these genera are not as rare as previously thought, with various strains present in soil samples While no specific habitat preference is established, genera like Micromonospora and Actinoplanes are often abundant in decaying plant material and muddy soils near freshwater Additionally, thermophilic species thrive in warm natural environments, such as compost or hay mounds.
Actinomycetes exhibit limited information regarding their physiology, biochemistry, and genetics, primarily focusing on taxonomically relevant traits and applied microbiology Their taxonomy is intricate and rapidly evolving, with specialists adopting new concepts and criteria Traditionally, genera have been distinguished based on morphological traits such as aerial mycelium presence, spore arrangement, motility, and specialized structures Recently, chemical properties, including cell membrane and wall composition, have emerged as significant taxonomic characters, aiding in the organization of Actinomycetales into groups that often align with the concept of "families," though this term is not universally applied This overview highlights the groups containing genera pertinent to applied microbiology, adhering to the classification framework established by experts in the field.
1989 edition of Bergey's Manual of Systematic Bacteriology
Actinomycetes are a diverse group of gram-positive aerobic organisms that form a mycelium, which can break into coccoid or rod-shaped fragments While they share a common menaquinone structure with eight or nine isoprene units, their chemical characteristics can vary These organisms are commonly found in soil, although certain species can act as pathogens for animals or plants.
Nocardia is characterized by the presence of both vegetative and aerial hyphae, which can fragment into nonmotile elements and form chains of nonmotile spores Its cell wall contains meso-diaminopimelic acid, arabinose, galactose, and mycolic acids with chains of 46-60 carbon atoms The membrane fatty acids are typically linear or have a methyl group at position 10 Nocardiae are mesophilic organisms that thrive on simple media with ammonia, nitrates, or amino acids as nitrogen sources, and glucose or acetate as carbon sources They exhibit slow growth, with a doubling time of approximately 5 hours, and are capable of utilizing long-chain alkanes and gaseous hydrocarbons as carbon sources.
Nocardiae produce a variety of antibiotics, including important compounds like rifamycins, vancomycin, and ristocetin, which have recently been reclassified into a different genus Conversely, the β-lactams cephamycin A, B, and C were initially attributed to Streptomyces but are now classified as Nocardia lactamdurans Additionally, N lactamdurans is known to produce the antibiotic efrotomycin Nocardia species also yield ansamycins, which have notable antitumor activity, while nocardicins, monobactam antibiotics from N uniformis, are of particular interest Furthermore, formycin and coformycin are two notable nucleoside antibiotics produced by these strains.
Nocardia species produce several antibiotics, including nocardicin, formycin A, coformycin, and efrotomycin While coformycin itself lacks antimicrobial activity, it effectively inhibits the enzyme adenosine deaminase, thereby enhancing the efficacy of nucleoside antibiotics like vidarabine by preventing their metabolic degradation Similarly, 2-deoxycoformycin, a metabolite derived from Streptomyces antibioticus, exhibits comparable activity.
Amycolatopsis is a newly proposed genus for species previously classified under Nocardia that lack mycolic acids While it shares similar taxonomic traits with nocardiae, a distinguishing feature of Amycolatopsis is the presence of branched-chain fatty acids.
Amycolatopsis species are known for their ability to produce a variety of antibiotics from different biosynthetic classes Notably, glycopeptides such as dalbaheptides are common, with A orientalis producing the clinically significant vancomycin and ristocetin from its subspecies lurida Additionally, A orientalis strains synthesize elfamycin antibiotics and muraceins, which are muramyl peptide derivatives that act as inhibitors of angiotensin-converting enzyme.
A mediterranei is the producer of the important ansamycins ri- famycins The original strain produces a complex of several compo-
Vancomycin, a crucial antibiotic, is derived from rifamycin B, which has been chemically modified to create semisynthetic derivatives like rifamycin SV This compound was initially synthesized but later isolated from the actinomycete Nocardia species Among the actinomycetes, A mediterranei has been extensively studied genetically, allowing for the high-frequency generation of recombinants through conjugation of marked strains This research led to the construction of a circular linkage map that closely aligns with the more comprehensive S coelicolor map in terms of homologous marker sequences Furthermore, a method for the formation and regeneration of protoplasts has been developed, enhancing genetic studies in this area.
Saccharopolyspora is a genus that includes two validated species, distinguished from streptomycetes by their substrate mycelium's tendency to fragment and their unique cell wall composition, which contains meso-diaminopimelic acid, galactose, and arabinose Notably, S erythrea, previously known as Streptomyces erythreus, is recognized for producing the widely used antibiotic erythromycin.
The group actinoplanetes comprises actinomycetes producing motile spores enclosed in vesicles called sporangia However, the genus
Micromonospora, characterized by nonmotile single spores, shares similar chemical traits and nucleic acid homology with Actinoplanetes, which are gram-positive organisms featuring branched septated hyphae While aerial mycelium is rarely developed, the cell wall's peptidoglycan contains meso-diaminopimelic or 3-hydroxydiaminopimelic acid and glycine instead of L-alanine The cell membrane predominantly consists of iso- and anteiso-fatty acids Although the presence of motile spores indicates an adaptation to aquatic environments, Actinoplanetes thrive in various soil types These aerobic and mesophilic organisms flourish at temperatures between 20 and 30°C and prefer a neutral pH around 7.
Actinoplanes are slow-growing organisms that form small colonies, predominantly exhibiting a characteristic orange color due to carotenoid pigments, although brown, red, and blue strains exist Their sub-spherical sporangia are typically found on stalks known as sporangiophores, with polar flagella on the spores While Actinoplanes strains can thrive on minimal media that promotes sporulation, they are generally cultured in rich media suitable for actinomycetes, utilizing xylose or chitin as carbon sources Notably, the enzyme xylose isomerase from A missourinensis is utilized industrially for converting glucose to fructose Genetic studies on A brasiliensis have explored methods for selecting auxotrophic mutants and the regeneration of protoplasts.
Actinoplanes strains have yielded over 120 antibiotics, including a variety of compounds such as amino acid derivatives, polyenes, aromatics, nucleosides, and chloro-heterocycles Notably, teicoplanin, a lipoglycopeptide effective against gram-positive infections, is derived from A teichomyeticus cultures and consists of five components Additionally, ramoplanin, a macrocyclic peptide featuring sugar and fatty acid substituents, shows potential therapeutic applications.
Figure 2.16 Teichomycin T-A2-2, the major component ofteicoplanin
Actinoplanes ATCC 33076 is notable for its production of various bioactive compounds, including pleuracins, which are a mixture of macrocyclic lactones and depsipeptides similar to the virginiamycin group, derived from A auranticolor and A azureus Additionally, A deccanenensis produces lipiarmycin, a unique macrolide that inhibits RNA polymerase, while A ianthinogenes is known for purpuromycin, a polyketide with broad-spectrum antibacterial and antifungal properties.
A tetra-pseudosaccharide, produced by Actinoplanes SE 50, is a potent inhibitor of glucoamylases and has been developed for medical use in the treatment of metabolic diseases under the name acarbose
Genus Aspergillus
Aspergilli are filamentous fungi classified under Deuteromyces or Fungi Imperfecti due to their lack of sexual reproduction A defining feature of this genus is the conidial apparatus, which is a spore-bearing structure characterized by an elongated hypha that ends in a round vesicle, from which radial chains of spores emerge The name "Aspergilli" is inspired by the shape of this apparatus, resembling an Aspergus, a tool used in Catholic churches for sprinkling holy water.
Aspergilli are widely distributed organisms found primarily in soil, particularly in warm climates They thrive on animal and plant residues, often leading to the spoilage of stored grains Additionally, certain strains of Aspergilli can act as opportunistic pathogens, affecting both animals and humans, with a notable presence in the lungs.
The genus Aspergillus, part of the Moniliaceae family, consists of filamentous fungi characterized by hyphae with incomplete septa that allow for cytoplasmic continuity within the mycelium Initially classified among the Fungi Imperfecti due to the absence of a sexual reproduction stage, taxonomic challenges emerged when some species were discovered to produce sexual spores in structures called cleistothecia While some nomenclature experts suggested classifying the "perfect" forms into distinct groups, many taxonomists favored retaining the same name for both sexual (teleomorph) and imperfect (anamorph) forms, a practice widely accepted by industrial microbiologists and mycologists.
Filamentous fungi exhibit general morphological and chemical characteristics similar to other fungi, with hyphae averaging 5 µm in thickness and a cell wall primarily made of polysaccharides, reinforced by chitin fibrils While cells are typically haploid, diploid cells can occasionally develop The conidial apparatus features a conidiophore, a vertical hypha that expands into a globose vesicle known as the conidial head This vesicle is covered with a single or double layer of elongated fertile cells called phialides, from which chains of conidiospores are produced.
In sexually reproducing species, asci, each containing eight spores, are irregularly packed in a high number into baglike bodies called cleistothecia
Aspergilli thrive on simple nutrients, growing as large colonies on solid media or as pellets in liquid media They require chemically defined media with sugars for carbon and nitrates or asparagine for nitrogen Complex media options include peptone, potatoes, oak flakes, and yeast extract These fungi are strict aerobes with a high oxygen requirement, and most species prefer an optimal temperature of around 25°C, although some can grow well at 37°C or higher Notably, Aspergilli exhibit a broad pH tolerance, accommodating both acidic and alkaline environments, and some strains can thrive in media with 7-8% NaCl, showcasing their resilience to osmotic pressure.
Certain species like A niger and A terreus release organic acids during growth, giving them an ecological advantage as most soil bacteria struggle to thrive in low-pH conditions Additionally, these species are known to produce mycotoxins, with aflatoxin from A flavus being the most researched and hazardous.
The production of substantial amounts of proteolytic and amylolytic enzymes is a crucial metabolic process, enabling growing hyphae to break down complex organic materials from plant or animal residues into simpler nutrients.
Aspergillus nidulans is a well-studied organism that serves as an excellent model for researching gene expression and regulation in eukaryotes It reproduces asexually through uninucleate conidia, which are ideal for mutagenic treatments and selecting mutants with easily identifiable markers, including auxotrophy, spore color, and drug resistance.
In sexual reproduction, a single event generates cleistothecia that contain thousands of asci, with each asci holding eight spores The organism A nidulans is homothallic, meaning it does not have distinct mating types For genetic research, contrasting spore colors in parental strains are utilized to differentiate hybrid cleistothecia from those produced by self-fertilization.
The parasexual cycle is a crucial reproductive mechanism that involves several key steps: first, the formation of a heterokaryon where two haploid nuclei coexist in the same cytoplasm; second, the fusion of these nuclei into a heterozygous diploid; and third, the process of mitosis During mitosis, mitotic crossing over can occur between homologous chromosomes, or alternatively, unstable nuclei with an abnormal chromosome number may arise and subsequently revert to the haploid state.
The parasexual cycle in various Aspergillus species enables genetic research, allowing for a detailed genetic map of A nidulans By utilizing mutagenesis on conidia and analyzing both sexual and parasexual progeny, researchers can uncover gene linkage within chromosomes and assign genes to specific chromosomes.
Subsequent studies have focused on the regulation of gene expression, highlighting that, unlike E coli, genes responsible for sequential metabolic functions are rarely clustered Each metabolic pathway for nitrogen or carbon utilization is typically regulated by a specific gene that responds to an inducer For instance, the prnA gene facilitates proline-induced enzyme activity in proline catabolism, while the nirA gene regulates nitrate assimilation in response to nitrate Additionally, broad regulatory genes, such as areA, control multiple pathways; for example, it represses nitrogen metabolism enzymes in the presence of glutamine Similarly, the creA gene is involved in carbon catabolite repression, affecting enzymes related to various carbon compounds.
Aspergilli play a crucial role in industrial microbiology, with their products primarily categorized into organic acids and enzymes They are essential for the production of Oriental food and beverages, generating significant quantities of citric, gluconic, and tartaric acids from various strains of A niger, as well as itaconic acid from A terreus Additionally, A oryzae is known for producing enzymes such as a-amylase and glucoamylase, which are vital in the starch industry.
Glucoamylases are also obtained from A awamory and A niger Strains of this latter organism, and of A wentii, are also used for the production of pectinases
Research into using Aspergillus species for the commercial production of heterologous proteins has gained significant attention Two primary systems are currently under development, including the A nidulans system, which utilizes the alcA promoter for enhanced protein expression.
(alcohol dehydrogenase I) promoter and its regulator gene alcR; and the A niger var awamory system, based on the promoter of the glu- coamylase gene gluA
Aspergillus species frequently produce secondary metabolites, with only a limited number being of practical significance Among these, several are harmful mycotoxins, including aflatoxins and tremorgen from A flavus, cytochalasin E from A clavatus, and ochratoxin A from A ochraceus.
Genera Producing a Few Interesting Metabolites
In the late 1970s, large-scale screenings of gram-negative bacteria in soil and aquatic environments identified several antibiotic-producing strains, particularly focusing on β-lactams due to the high sensitivity of the detection methods The first notable antibiotic discovered was SQ 26445, a sulfonyl monocyclic β-lactam (monobactam) from a Gluconobacter species, which was found to be identical to sulfazecin, isolated simultaneously from a Pseudomonas strain Subsequent discoveries included various structurally related monobactams from Agrobacterium, Chromobacterium, and more recently, from gliding bacteria such as Cytophaga johnsoniae and Flexibacter species SC 11479 and SC 12681 Additionally, nonsulfonated monobactams of the nocardicin type, known as formacidins, were isolated from Flexibacter alginoliquefaciens, while classical β-lactams like deacetyl cephalosporin C were produced by strains of Flavobacterium and Xanthomonas.
Cephalosporium, now classified under the genus Acremonium, includes the strain responsible for producing the crucial cephalosporin antibiotic Initially named Cephalosporium acremonium, this strain has also been referred to as Acremonium chrysogenum or A strictum.
Acremonium, also known as Cephalosporium, is a diverse group within the Fungi Imperfecti, marked by its simple conidiophore structure, which features a single or minimally branched stalk topped with a cluster of conidia Additionally, its hyphae can develop into arthrospores While these fungi are primarily found in soil, certain species are linked to living organisms, including plants and animals, with some strains posing pathogenic risks to humans.
C acremonium shares similar nutritional requirements with Penicillium, utilizing various sugars, methyl oleate, or glycerol as carbon sources, and inorganic nitrogen, amino acids, or complex polypeptides as nitrogen sources Research has primarily focused on the genetic aspects related to cephalosporin production Although the parasexual cycle is present, the occurrence of heterokaryons or diploids is infrequent This challenge has been addressed through the application of the protoplast fusion technique.
In addition to cephalosporins, Cephalosporium and Acremonium have yielded a limited number of secondary metabolites, primarily polyketides and terpenoids Notably, cerulenin, a hexaketide produced by C caerulens, serves as a specific inhibitor of fatty acid biosynthesis Furthermore, fusidic acid, an effective antistaphylococcal agent first isolated from Fusidium coccineum (now Acremonium fusidioides), is also synthesized by various Cephalosporium species.
Tolypocladium inflatum, previously known as Trichoderma polysporum, is recognized for producing cyclosporin A, an antifungal compound Its significant clinical application lies in its immunosuppressive properties, which are particularly beneficial in organ transplantation procedures.
Monascus ruber is known for producing monacolin K, also referred to as lovastatin, which serves as an inhibitor of cholesterol synthesis This compound is chemically identical to mevinolin, which was independently isolated from Aspergillus Additionally, various strains of M ruber are capable of producing derivatives of monacolin K.
Figure 2.22 Antibiotics from Cephalosporium: (a) fusidic acid; (b) cerulenin
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