Preface Biotechnology is one of the major branches of biological science of the twenty-first century. Microbial Biotechnology is wide-ranging, multi-disciplinary activities which include recombinant DNA techniques, cloning and the application of microbes to the production of goods from bread to antibiotics. Microbial research and industry is developing rapidly all over the world. Varying in application of concept, practice, scale, style and substance, microbial biotechnology is amongst the latest globalizing frontiers of the corporate world. This branch is increasingly being regarded as a core subject in colleges and universities also. In this book various topics on microbial biotechnology are uniquely combined to provide a complete overview of the subject. The application of bioinformatics in the study of microbial genomics is also included. The book is likely to be useful for the postgraduate student and researchers in developing an insight into the various aspects of the microbial biotechnology as a whole.

ABSTRACT Bioinformatics is a critical tool in designing the molecular biology experiments. The xeroxing of specific part of genome needs correct primer designing. Primer designing has central role in molecular biology right from cloning, sequencing, gene expression profiling by real time PCR, genotyping by RFLP, SNP analysis by real time PCR and auto sequencer. In this chapter we are describing how to proceed for experimental designing right from data mining from GENEBANK to designing of primers for various common molecular genetic analysis work. The write up also describes in silico evaluation of primers, various off line commercial and non-commercial software, on line free software and trouble shootings in primer designing, The different strategy described in context of primer designing would be of immense use of researchers.

ABSTRACT A number of secondary metabolites are produced by cyanobacteria involving the non-ribosomal peptide biosynthetic pathway. Some of these compounds have potential biotechnological applications, whereas others (the cyanotoxins) are undesired due to their toxicity to animals. The occurrence of toxic cyanobacterial blooms in freshwater bodies that are used as source for human consumption, recreation and irrigation are frequent nowadays due to increasing artificial eutrophication of these environments. It is important to monitor toxin-producing strains in order to prevent the adverse effects caused by their toxins to human and animal health. Thus, the development of rapid and sensitive methods for the detection of cyanotoxins is important as they could be useful to monitor water supplies and contribute for watershed monitoring strategies. Current methods include cell counting, microscopic identification and toxin analysis by biochemical and analytical techniques. However, in recent years, molecular biology techniques, such as polymerase chain reaction (PCR), have been used to amplify specific regions within biosynthetic genes for rapid and sensitive identification of toxic cyanobacteria. The aim of this chapter is to provide an update of cyanotoxin biosynthesis and discuss the advances and limitations of molecular biology regarding its application as indicative of cyanotoxin production.

ABSTRACT Biotransformation is an essential step in the elimination of drugs and reduction of their toxicity. The use of fungi in biotransformation reactions can provide information about the metabolism of new drugs and facilitate the attainment of necessary amount of metabolites for toxicological and pharmacological evaluation. Biotransformation studies require the right choice of the microorganisms and also of the culture conditions (pH, substrate, oxygen, salts, temperature, etc). The optimization of these procedures is essential to achieve good results. The fundamental aspects of drug biotransformation by fungi are discussed here as well as some applications showing that many fungi are used in biotransformation studies, mainly Cunninghamella species. Several species mimic the mammalian metabolism producing the same metabolites observed in humans.

ABSTRACT Methylotrophy is a phenomenon by which microorganisms derive energy, and in many cases, cell carbon from reduced molecules that have no C–C bond (also called C1compounds). This gains significance because of its unique mode of nutrition, the ability to utilize more than one metabolic pathway and its vast distribution among various groups of bacteria and yeasts. It is one of the earliest modes of energy metabolism present in microorganisms, through which microorganisms are able to utilize a wide range of compounds, which are considered unsuitable for most forms of life. In the present chapter we focus on various aspects of methylotrophy, and the application of methylotrophic bacteria in a spectrum of biological activities and commercial possibilities.

ABSTRACT Plant growth promoting (PGP) bacteria play an immense role in promoting crop growth mainly by direct mechanisms such as N2-fixation, phosphate solubilization and phytohormone production and indirectly may help to suppress pathogens or contribute to enhance absorption of nutrients leading to increase yields. The N2fixers are grouped based on their niche adaptation as endophytic nodular symbionts (Rhizobium spp.), free living (Azotobacter spp.), associative symbionts (Azospirillum spp.) and endophytic non-nodular symbionts (Azoarcus and Gluconacetobacter diazotrophicus). In the recent past, phosphate solubilizing and phytohormone synthesizing bacterial communities also gained momentum.

ABSTRACT Soil borne fungal pathogen causing root diseases of vegetables are difficult to control by chemical methods. The existing biocontrol bacteria are not very effective in control of soil borne fungal pathogens. As such very less number of bacterial biopesticide is available commercially to control the fungal pathogens. Increasing concerns in recent years about human health and environmental problems due to chemical pesticides, couple with increasing pest resistant to chemical, have led to a reappraisal of our approach to control pest and disease in crops. The trends now are towards a management concept in which the best combination of tools is used to manage plant diseases below economic thresholds with minimal risk to human health and environment. The modern management concept includes extensive use of biocontrol agents which are harmless to non-target organisms. Development of effective technology for control of pest and disease by using microbial resources of a region to enhance the quality of ecosystem is a potential alternative approach to chemical control of plant diseases. In such strategy, search for microorganisms and biological system is an important prerequisite prior to development and practical exploitation of biocontrol. Rhizosphere microorganisms are shown to be perspective candidate for biocontrol agent as they provide the front line of defense for plant roots against the attack by various plant pathogens causing the most diseases of vegetables and crops. Chemical control of soil-borne fungal pathogen had been the subject of growing concern because of their adverse effect on the environment and emergence of chemical pesticide resistant to plant pathogen, moreover, soil borne pathogens are very difficult to control. Crop rotation, breeding for resistant plant varieties and application of pesticides are insufficient to control root diseases of important plants because they survive as sclerotia and has got wide host range.

ABSTRACT Endophytes are microorganisms that live inside the host plant tissues without causing symptoms of disease. There has been a crescent interest in the prospecting of these microorganisms as sources of novel and bioactive natural products. In fact, a significant number of interesting natural products have been reported in the last years. In this chapter some aspects regarding the endophytes are detailed: the relationship with their host plants, isolation, preservation, taxonomy, cultivation, frequently isolated strains, tropical endophytes, novel structures isolated and also their biological activities. Key words: natural product, endophytic fungi, plant fungus interaction, secondary metabolites.

ABSTRACT The world ocean with a coastline of approximately 3,50,000 km is the largest ecosystem on earth. Marine organisms are the major, sustaining components of ecosystem processes and are responsible for biogeochemical reactions that drive our climate changes. Despite this, many marine organisms are poorly described and little is known of broad spatial and temporal scale trends in their abundance and distribution. With new molecular and analytical techniques we can advance our knowledge of marine biodiversity at the species level to understand how marine biodiversity supports ecosystem structure, dynamics and resilience. We can then interpret environmental, ecological and evolutionary processes controlling and structuring marine ecosystem biodiversity. In biotechnology applications marine microbes represent a potential source for numerous commercially important biomolecules and their bioremediation capabilities are also remarkable. They also play a crucial role in decomposition of organic matter and cycling of nutrients. Microbes also serve as food for some bottom-living organisms. Understanding and preserving biodiversity was one of the most important global challenges for the past 20 years and will continue to be an important scientific issue into the new millennium. Our knowledge of marine microbial diversity has, however, been severely limited by relying on microorganisms that have been cultured which are less than 1%.

ABSTRACT Industrial and agricultural activities and the disposal of waste products have resulted in the contamination of many terrestrial environments. Industrial effluents often contain considerable amount of potentially toxic compounds such as Cd, As, Pb and Hg. Due to their small size, which provides a large contact area that can interact with the surrounding environment, microorganisms are the first biota showing the impact of toxic compounds. Microorganisms being in intimate contact with the soil environment are considered to be the best indicators of soil pollution. In general, they are very sensitive to low concentrations of contaminants and provide a rapid response to soil perturbation. Rhizobium spp. are ubiquitous gram-negative soil bacteria that have a profound scientific and agronomic significance due to their ability to establish nitrogen-fixing symbiosis with legumes, which is of major importance to the maintenance of soil fertility and to feed livestock and populations in many developing countries. Therefore, the present work aimed to evaluate the impact of heavy metals on Rhizobium populations isolated from different locations contaminated with heavy metals. In order to reach this goal some physicochemical parameters that influence metal bioavailability were determined, as well as their concentrations in soil. Cadmium tolerance of Rhizobium isolates was screened in artificial media and the highest cadmium concentration allowing growth was defined as the maximum resistance level (MRL). The heavy metal tolerance variability found among Rhizobium isolates, suggests that beyond basic metabolic machinery also exist variations that allow isolates to display distinct tolerance responses to Cd stress. To better understand Rhizobium cadmium tolerance, wall-bound and intracellular Cd was determined. Sensitive and moderately tolerant isolates showed low intracellular Cd amounts when compared to extremely tolerant isolates, which were able to withstand intracellular Cd concentrations 40 times higher. Lipopolysaccharides (LPS) bind cations unspecifically, so they constitute the initial defence mechanism to heavy metals since they sequestrate cations and therefore prevent their entry into the cell. LPS quantity varied significantly between isolates. LPS increases were observed in most sensitive, moderately tolerant and tolerant isolates, evidencing an attempt of cells to adjust to cadmium entry. These results may be of great importance for understanding how Rhizobium persists in contaminated soils. They also provide important data to the development of bioremediation processes and to the improvement of soil productivity.

ABSTRACT An understanding of the metabolism of heavy metals facilitates biotechnological use of these functions just recently, although heavy metals comprise the major part of the elements in the periodic table. Due to their ability to form complex compounds, some heavy metals are essential trace elements. In any case, essential or not, most heavy metals are toxic at higher concentration. Heavy metal pollution from last few decades has led to an increased environmental concern. Heavy metal like cadmium is a non-essential toxic heavy metal and is also being increased in soil during last few years posing a serious threat to environment and human health worldwide. Therefore, remediation strategies of the contaminated sites are great concerns and can be achieved by a number of different physico-chemical and biological means.

ABSTRACT Plasmids are circular, double-stranded DNA molecules which replicate autonomously from the chromosome of the host organism. Plasmids are found in many bacterial species and often confer upon the host cell phenotypic characteristics such as resistance to antibiotics, resistance to heavy metals, ability to produce particular protiens, conjugative (mating) ability and ability to degrade xenobiotc compounds. One of these degradative plasmids may encode a complete catabolic pathway (such as toluene to pyruvate, formate and acetaldehyde) or partial degradative steps, such as octane to octanol. In this later case, the remainder of the pathway may be encoded by chromosomal genes, by genes of other plasmid present in the host, or by other microorganisms. Many degradative plasmids possess a broad host range, especially among the gram-negative bacterial species. There are different mechanisms which may be involved in the evolution of aromatic pathways in general and in the process of adaptation of microorganisms to xenobiotic substrates in particular. Gene transfer, the occurrence of plasmids in bacteria in the natural environment is an important pool of genetic information residing on plasmid vehicles may flow among indigenous microorganisms. Since many examples of self-transmissible plasmids that carry genes for degradation of aromatic or other xenobiotic compounds are known, and their role in spreading these genes to other microorganisms is well characterized. Early, studies of TOL, NAH, and SAL plasmids revealed strong homologies among these plasmids in regions both inside and outside the catabolic gene clusters. Plasmids, pJP4, pAC25, and PSS50, catabolic plasmids carrying genes for chloroaromatic degradation, have strongly homologous plasmid backbone that determine replication and transfer function. Furthermore, the chlorobenzoate plasmid p51 was also shown to be homologous to pJP4 outside regions of the catabolic genes. These observations suggest that a few common self-transmissible ancestor replicons (includes origin of replication and replication gene) may have been involved in acquisition and spread of different catabolic genes. DNA

ABSTRACT Heavy metal contaminated soils are biologically inactive and extremely poor in soil fertility status. These metals are highly resistant and cannot be easily degraded by either chemical reactions or amendments. Microorganisms are biological softwares capable of degrading the metals efficiently. Among microorganisms, mycorrhizas are known to circumvent metal toxicities through a series of processes that assist in immobilization in biological systems which converts toxic metals into less or nontoxic forms. There are five mechanisms such as biosorption, solubilization, hyphal transport, detoxification and dilution are involved in promoting tolerance of host plants to heavy metal toxicities. These processes are strategies that can be exploited to phytoremediate the contaminated soils. The phytoremediation strategies encompass tolerance of mycorrhizal fungi, phytostabilization (preventing entry of heavy metals into plants), phytoextraction (luxuriant uptake and transport to shoots) and intercropping or crop rotation with mycorrhizal dependent hyperaccumulators or non-hyperaccumulators. Despite mycorrhizas are natural, the indigenous population is not sufficient enough to induce tolerance of host plants to abiotic and biotic stress conditions. This necessitates adopting recommended agronomic practices to promote mycorrhizal colonization in natural agro-ecosystem or to derive full benefits from phytoremediation strategies. Thus mycorrhizas are important naturally occurring microorganisms capable of withstanding highly contaminated metal sites and offer protection against phytotoxicities caused by heavy metals.

ABSTRACT The continuing depletion of the stratospheric ozone layer mainly due to release of man-made atmospheric pollutants such as chlorofluorocarbons (CFCs) have resulted in the increase of solar ultraviolet-B radiation (UV-B; 280-315 nm) reaching to the Earth’s surface. UV-B is a small (less than 1% of total energy) but highly active component of the solar spectrum that can penetrate to biologically significant depths in lakes, ponds, rivers and oceans. It has been demonstrated that UV-B can cause wide ranging effects including mutagenesis, destruction of proteins/enzymes, inhibition of a number of vital metabolic processes and formation of thymine dimers in DNA leading to death of a number of organisms. A number of microbes including strict photoautotrophs always grow in lighted habitats and thereby always sense and face the stress of solar UV-B radiation. Similarly, humans are also exposed to varying levels of UV-B radiation depending upon the altitudes and latitudes of the location. A wide ranging effect of UV-B such as cataracts, skin cancer, immunosupression etc. has been reported in human. Damaging effects of increased UV-B radiation has also been shown in a number of microbes. These effects include inhibition of growth and survival, decrease in photosynthetic pigments, loss of PS II and RuBISCO activity, inactivation of nitrogenase enzyme and inhibitory effects on several other metabolic processes. Among photosynthetic pigments, phycobiliproteins seem to be the primary target of action since phycobilisomes are completely disintegrated after UV-B exposure. In this contribution an attempt has been made to put in perspective the main effects of UV-B radiation on microbes and human health.

ABSTRACT Serious infections caused by bacteria that have become resistant to commonly used antibiotics have become a major global healthcare problem in the 21st century. Antibiotic resistance, initially a problem of the hospital setting associated with an increased number of hospitals acquired infections usually in critically ill and immuno-suppressed patients, has now extended into the community causing severe infections difficult to diagnose and treat. In hospitals, most common resistant bacteria include methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and gram-negative rods including Enterobacteriaceae and Pseudomonas aeruginosa. Vancomycin intermediate and resistant S. aureus, represent a new treatment challenge. In the community, penicillin and macrolide-resistant pneumococci developed several decades ago and are now present all over the world. More recently, communityacquired methicillin-resistant S. aureus has become a problem in several countries causing skin infections but also severe diseases. The molecular mechanisms by which bacteria have become resistant to antibiotics are diverse and complex. Bacteria have developed resistance to all different classes of antibiotics discovered to date. The most frequent type of resistance is acquired and transmitted horizontally via the conjugation of a plasmid. In recent times new mechanisms of resistance have resulted in the simultaneous development of resistance to several antibiotic classes creating very dangerous multidrug-resistant (MDR) bacterial strains, some also known as ‘‘superbugs’’. In many cases the use of antibiotics is unnecessary or questionable. The indiscriminate and inappropriate use of antibiotics in outpatient clinics, hospitalized patients and in the food industry is the single largest factor leading to

ABSTRACT Frankia is a gram-positive, microaerophilic, actinomycete that enters into symbiotic associations with a number of woody dicotyledonous plants called actinorhizal plants. Frankia can fix atmospheric nitrogen through such associations. The actinorhizal-Frankia association is beneficial for the environment, since it increases the fertility of soils. Extensive studies on the biochemical and molecular aspects helped in the classification of Frankia. PCR-RFLP studies of the 16S rRNA and the nif regions of Frankia revealed considerable information regarding the diversity of Frankia strains, isolated from different parts of the world. The completion of three whole genomes of Frankia has bolstered the study of the molecular organization of the genome. Comparative genomics study of the three complete genomes revealed that, the strains reflected the biogeographical history of the host plants they infected. Striking similarity in the codon usage patterns highlights the fact that they may have evolved as a unit. In the present review we highlighted the taxonomy of Frankia and the host plants, the Frankia-actinorhizal association, regulation of infectivity, biochemical and molecular characterization, phylogeny and genomics of Frankia and stressed the need for metabolomic study of the Frankia-actinorhizal association.

Index Acidophiles 207 Acquired resistance 352, 353, 358 Actinorhizal 377, 378, 379, 386, 387, 388, 389, 391, 392, 394, 395, 396, 397, 398, 399, 400, 402, 403, 404, 405 Aliphatic hydrocarbon 235 Alkaliphiles 207 Amylolytic 113 Aniline 238, 239, 254, 255, 279, 283, 284, 289, 290 Antibacterial agents 350, 352, 353, 354, 356, 357, 367, 375 Antibiosis 88, 91, 107, 120, 121, 124, 126, 127, 136 Antibiotic 24, 28, 121, 122, 124, 125, 127, 135, 143, 144, 153, 159, 171, 172, 214, 225, 246, 349, 350, 351, 352, 354, 355, 361, 362, 363, 366, 367, 369, 370, 371, 372, 374, 375 - active efflux 355 - resistance 28, 225, 349, 350, 351, 352, 354, 361, 366, 370, 372, 374