Resilience of aerobic sludge biomass under chlorpyrifos stress and its recovery potential.

Non-agricultural sources of pesticides in urban areas are responsible for their presence in domestic wastewater. Therefore, pesticides are typically found in sewage treatment plants in developed and developing countries as micro-pollutant. The presence of pesticides in the wastewater can impart stress on the aerobic sludge biomass and disrupt the functioning of the plant. However, there exists a knowledge gap regarding the resilience of aerobic sludge biomass towards stress due to the presence of pesticides in the wastewater. This study investigated the impact of chlorpyrifos (CPS) - a widely used pesticide, on sludge biomass and explored its recovery capability when CPS is discontinued in the influent. Four duplicate reactors were operated with different CPS concentrations ranging from 50 to 200 mg/L. Chemical oxygen demand (COD) removal for reactors has ranged within 18-73 % at the steady state of the stressed phase, whereas COD removal for the control reactor was 91 %. CPS stress slightly inhibited filamentous biomass growth. Biomass activity and cell viability have decreased significantly, whereas biochemical contents have varied slightly under CPS stress. The activities of the enzymes dehydrogenase and urease were significantly inhibited when compared to catalase and protease. Amplified ribosomal DNA restriction analysis reflected changes in the microbial community. The discontinuation of CPS has allowed aerobic sludge biomass to recover in its organic degradation capability (COD removal of more than 88 % at steady-state conditions of recovery phase operation), biomass growth, and cell viability. In addition, enzyme activities have retrieved to their original levels, and 78-93 % similarity of microbial community structure has been displayed between CPS-exposed and control reactor biomasses. Overall, the present study has indicated the orderly changes in the quality of aerobic sludge biomass under CPS stress through physico-chemical and biological characteristics. The study also has highlighted the self-recovery of sludge biomass characteristics stressed with different concentrations of CPS.

Diversity and characterization of antagonistic bacteria against Pseudomonas syringae pv. actinidiae isolated from kiwifruit rhizosphere.

Kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) is a severe global disease. However, effective biological control agents for controlling Psa are currently unavailable. This study aimed to screen potential biological control agents against Psa from the kiwifruit rhizosphere. In this study, a total of 722 isolates of bacteria were isolated from the rhizosphere of kiwifruit orchards in five regions of China. A total of 82 strains of rhizosphere bacteria showed antagonistic effects against Psa on plates. Based on amplified ribosomal DNA restriction analysis (ARDRA), these antagonistic rhizosphere bacteria were grouped into 17 clusters. BLAST analyses based on 16S rRNA gene sequence revealed 95.44%-100% sequence identity to recognized species. The isolated strains belonged to genus Acinetobacter, Bacillus, Chryseobacterium, Flavobacterium, Glutamicibacter, Lysinibacillus, Lysobacter, Pseudomonas, Pseudarthrobacter, and Streptomyces, respectively. A total of four representative strains were selected to determine their extracellular metabolites and cell-free supernatant activity against Psa in vitro. They all produce protease and none of them produce glucanase. One strain of Pseudomonas sp. produces siderophore. Strains of Bacillus spp. and Flavobacteria sp. produce cellulase, and Flavobacteria sp. also produce chitinase. Our results suggested that the kiwifruit rhizosphere soils contain a variety of antagonistic bacteria that effectively inhibit the growth of Psa.

Genetic and phenotypic diversity of microsymbionts nodulating promiscuous soybeans from different agro-climatic conditions.

BACKGROUND: Global food supply is highly dependent on field crop production that is currently severely threatened by changing climate, poor soil quality, abiotic, and biotic stresses. For instance, one of the major challenges to sustainable crop production in most developing countries is limited nitrogen in the soil. Symbiotic nitrogen fixation of legumes such as soybean (Glycine max (L.) Merril) with rhizobia plays a crucial role in supplying nitrogen sufficient to maintain good crop productivity. Characterization of indigenous bradyrhizobia is a prerequisite in the selection and development of effective bioinoculants. In view of this, bradyrhizobia were isolated from soybean nodules in four agro-climatic zones of eastern Kenya (Embu Upper Midland Zone, Embu Lower Midland Zone, Tharaka Upper Midland Zone, and Tharaka Lower Midland Zone) using two soybean varieties (SB8 and SB126). The isolates were characterized using biochemical, morphological, and genotypic approaches. DNA fingerprinting was carried out using 16S rRNA gene and restricted by enzymes HaeIII, Msp1, and EcoRI.  RESULTS: Thirty-eight (38) bradyrhizobia isolates obtained from the trapping experiments were placed into nine groups based on their morphological and biochemical characteristics. Most (77%) of the isolates had characteristics of fast-grower bradyrhizobia while 23% were slow-growers. Restriction digest revealed significant (p < 0.015) variation within populations and not among the agro-climatic zones based on analysis of molecular variance. Principal coordinate analysis demonstrated sympatric speciation of indigenous bradyrhizobia isolates. Embu Upper Midland Zone bradyrhizobia isolates had the highest polymorphic loci (80%) and highest genetic diversity estimates (H' = 0.419) compared to other agro-climatic zones. CONCLUSION: The high diversity of bradyrhizobia isolates depicts a valuable genetic resource for selecting more effective and competitive strains to improve promiscuous soybean production at a low cost through biological nitrogen fixation.

Microbial Consortia Involved in Traditional Sicilian Sourdough: Characterization of Lactic Acid Bacteria and Yeast Populations.

Sourdough is one of the oldest starters traditionally used for making baked goods, offering several advantages to the sensory, rheology, and shelf life of final products. The present study investigated, for the first time, the microbiota of spontaneously fermented Maiorca dough samples collected from bakeries located in Sicily (Italy). Four sourdough samples (M1, M2, M3, and M4), were produced using Triticum vulgare Host. var. albidum Koern (Maiorca grain) were subjected to LAB and yeasts isolation and identification at the species level. The in-depth characterization of the lactobacilli population revealed that Lactiplantibacillus plantarum and Levilactobacillus brevis unquestionably dominated the Maiorca sourdough ecosystem. Concerning the yeasts community, high species diversity was found. Saccharomyces cerevisiae and Wickerhamomyces anomalus were the most frequently isolated species. In addition, Torulaspora delbrueckii, Pichia kluyveri, Candida boidinii, and Candida diddensiae were also detected. Investigations on both pro-technological and functional traits of the isolated strains could lead to the selection of starters for the production of baked goods.

Diversity of culturable bacteria endowed with antifungal metabolites biosynthetic characteristics associated with tea rhizosphere soil of Assam, India.

BACKGROUND: Rhizosphere soil is a crucial niche for the diverse beneficial microbial communities in plant-microbe interactions. This study explores the antagonistic potential and diversity of the rhizosphere soil bacteria from commercial tea estates of Assam, India which comes under the Indo-Burma mega-biodiversity hotspot. Rhizosphere soil samples were collected from six different tea estates to isolate the bacteria. The bacterial isolates were subjected to evaluate for the antagonistic activity against fungal pathogens. The potential isolates were investigated for chitinase production and the presence of chitinase gene. The bacterial genetic diversity was studied by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and BOX-PCR fingerprinting. RESULTS: A total of 217 rhizobacteria were isolated from tea rhizosphere soil, out of which 50 isolates exhibited the potential antagonistic activity against fungal pathogens. Among them, 12 isolates showed extracellular chitinase activity and the presence of chitinase genes. The chitinase genes were sequenced and the analysis of the sequences was performed by using PDB protein databank at the amino acid level. It showed the presence of ChiA and ChiA74 gene in the 6 most potent isolates which are involved in the hydrolysis of chitin. These isolates also exhibited antagonistic activity against all tested fungal pathogens. The diversity of 50 antagonistic bacterial isolates were analyzed through ARDRA and BOX-PCR fingerprinting. Diversity analysis and molecular identification of the rhizosphere isolates revealed that these antagonistic isolates predominantly belonged to the genus Bacillus followed by Enterobacter, Serratia, Lysinibacillus, Pseudomonas, and Burkholderia. CONCLUSION: The present study establishes that rhizobacteria isolated from the poorly explored tea rhizosphere soil could be a rich reservoir for the investigation of potential antagonistic bacterial candidates for sustainable agricultural and industrial applications.

Tyrosinase and laccase-producing Bacillus aryabhattai TFG5 and its role in the polymerization of phenols.

BACKGROUND: Tyrosinases and laccases are oxidoreductase enzymes that are used widely in the food, feed, textile, and biofuel industries. The rapidly growing industrial demand for bacterial oxido-reductases has encouraged research on this enzyme worldwide. These enzymes also play a key role in the formation of humic substances (HS) that are involved in controlling the biogeochemical carbon cycle, providing nutrients and bio-stimulants for plant growth, and interacting with inorganic and organic pollutants besides increasing carbon sequestration and mitigating greenhouse gas emission in the environment. The present study aimed to screen and characterize extracellular tyrosinase and laccase-producing soil bacteria that could be utilized in the polymerization of phenols. RESULTS: Twenty isolates from different soil samples collected from forest ecosystems were characterized through ARDRA using restriction digestion with AluI, HpaII, and HaeIII restriction enzymes. The results of Hierarchical Cluster Analysis (HCA) revealed a 60 % similarity coefficient among 13 out of 20 isolates, of which, the isolate TFG5 exhibited only 10 % similarity when compared to all the other isolates. The isolate TFG5 exhibited both tyrosinase (1.34 U.mL- 1) and laccase (2.01 U.mL- 1) activity and was identified as Bacillus aryabhattai. The increased polymerization activity was observed when B. aryabhattai TFG5 was treated with phenols. The monomers such as catechol, p-Hydroxy benzoic acid, ferulic acid, and salicylic acid were polymerized efficiently, as evidenced by their FT-IR spectra depicting increased functional groups compared to the standard mushroom tyrosinase. CONCLUSIONS: The polymerization ability of B. aryabhattai TFG5 could be applied to phenol-rich wastewater treatment for efficient precipitation of phenols. Furthermore, tyrosinases can be used for enhancing the synthesis of HS in soil.

Pathogenic Microbial Profile and Antibiotic Resistance Associated with Periodontitis.

ABSTRACT: Phenotyping based on conventional microbiological, physiological, and molecular analysis by using ARDRA technique was developed with the aim to assess the pathogenic microbial load associated with different stages of the periodontal disease. In addition, in the face of the global issue of antimicrobial resistance, the isolated bacterial strains were evaluated for their antibiotic susceptibility profile. The pathogenic bacterial community was predominantly of Gram-negative strains (66.66%). The most common species were Citrobacter freundii, Bacillus sp., Raoutella sp., Klebsiella ozaenae and Pseudomonas sp. However, except for the healthy control group, Staphylococcus spp. was isolated from all stages of periodontitis. Multidrug resistance to beta-lactam antibiotics was observed for Streptococcus pneumoniae, Raoutella sp. and Enterococcus avium. Here, we verify a statistically significant relationship between periodontitis stages and the diversity of the bacterial community. Patients with periodontitis showed a more diverse and numerous bacterial community compared to healthy patients. In this sense, we reinforce that biofilms that harbour multidrug-resistant bacteria are a major concern in relation to restoring patient health. Thus, prophylactic measures for maintaining oral health are still the best option for reduce the risk of disease.

Diversity of halophilic and halotolerant bacteria in the largest seasonal hypersaline lake (Aran-Bidgol-Iran).

PURPOSE: In this study, the culturable halophilic and halotolerant bacterial diversity was determined in Aran-Bidgol as a thalassohaline seasonal hypersaline lake in Iran. METHODS: Thirty water, soil, sediments, coastal mud, multi-color brines and salt crystals samples were extracted and cultured using different media and incubation conditions. Totally 958 isolates were obtained and 87 isolates were selected for further studies, based on morphological, physiological and biochemical tests, representing different morphotypes. RESULTS: Based on 16S rRNA gene sequence analyses, the isolates exhibited 94.6-100% sequence similarity to the closest known species of the genera Bacillus, Halomonas, Oceanobacillus, Salinicoccus, Thalassobacillus, Ornithinibacillus, Halobacillus, Salicola, Virgibacillus, Aerococcus, Arthrobacter, Idiomarina, Paraliobacillus, Staphylococcus, Acinetobacter, Aneurinibacillus, Brevibacillus, Brevundimonas, Chromohalobacter, Gracilibacillus, Jeotgalicoccus, Kocuria, Marinilactibacillus, Marinobacter, Microbacterium, Paenibacillus, Paracoccus, Piscibacillus, Pseudomonas and Sediminibacillus and also, comparison of ARDRA patterns among the sequenced strains, using AluI, Bst UI and Hpa II enzymes showed that these patterns are in accordance with the phylogenetic position of these strains. CONCLUSION: The PCR-RFLP analyses suggested that ARDRA possess a functional potential for distinguishing halophilic bacteria to be used for further studies in elementary steps of isolation to reduce the tedious duplication of isolates.

Microbiological quality and safety of commercialized thalassotherapy products based on marine mud and algae extracts in Tunisia.

A total of 15 samples of thalassotherapy products, distributed in Tunisia in their intact and final state of production, was analyzed to determine their microbiological safety status. The result shows the absence of pathogenic bacteria (Staphylococcus aureus, Candida albicans, Salmonella, Pseudomonas aeruginosa and coliforms). The incidence of contamination by Gram-positive Bacilli (mesophelic bacteria, aerobic and anaerobic spore forming bacteria, anaerobic sulphite-reducing bacteria) was found to be higher in products composed by mud and extract of alga. The biochemical and molecular identification of the major contaminant show that Bacilli were the most covered from 75% of the thalassotherapy products. Mineral analysis (organic matter, Fe, Mg, Ca, Na d K, Al, Si and Ti) shows strong composition on Aluminum and Silica. Cytotoxicity study of six thalassotherapy products and three essential oil extracts (Menthol, Clove and Eucalyptus) did not show any cytotoxic effect. Furthermore, antibacterial acitivity of 5 essentila oils, against 30 isolates of the genus Bacillus and 10 reference strains, has been characterized showing an interesting bactericidal potential of the extract of menthol and Eucalyptus.

Horizontal transfer and finalization of a reliable detection method for the olive fruit fly endosymbiont, Candidatus Erwinia dacicola.

BACKGROUND: The olive fly, Bactrocera oleae, is the most important insect pest in olive production, causing economic damage to olive crops worldwide. In addition to extensive research on B. oleae control methods, scientists have devoted much effort in the last century to understanding olive fly endosymbiosis with a bacterium eventually identified as Candidatus Erwinia dacicola. This bacterium plays a relevant role in olive fly fitness. It is vertically transmitted, and it benefits both larvae and adults in wild populations; however, the endosymbiont is not present in lab colonies, probably due to the antibiotics and preservatives required for the preparation of artificial diets. Endosymbiont transfer from wild B. oleae populations to laboratory-reared ones allows olive fly mass-rearing, thus producing more competitive flies for future Sterile Insect Technique (SIT) applications. RESULTS: We tested the hypothesis that Ca. E. dacicola might be transmitted from wild, naturally symbiotic adults to laboratory-reared flies. Several trials have been performed with different contamination sources of Ca. E. dacicola, such as ripe olives and gelled water contaminated by wild flies, wax domes containing eggs laid by wild females, cages dirtied by faeces dropped by wild flies and matings between lab and wild adults. PCR-DGGE, performed with the primer set 63F-GC/518R, demonstrated that the transfer of the endosymbiont from wild flies to lab-reared ones occurred only in the case of cohabitation. CONCLUSIONS: Cohabitation of symbiotic wild flies and non-symbiotic lab flies allows the transfer of Ca. E. dacicola through adults. Moreover, PCR-DGGE performed with the primer set 63F-GC/518R was shown to be a consistent method for screening Ca. E. dacicola, also showing the potential to distinguish between the two haplotypes (htA and htB). This study represents the first successful attempt at horizontal transfer of Ca. E. dacicola and the first step in acquiring a better understanding of the endosymbiont physiology and its relationship with the olive fly. Our research also represents a starting point for the development of a laboratory symbiotic olive fly colony, improving perspectives for future applications of the Sterile Insect Technique.

Differentiation between Bacillus amyloliquefaciens and Bacillus subtilis isolated from a South African sugarcane processing factory using ARDRA and rpoB gene sequencing.

A total of 104 exopolysaccharide (gum)-producing bacteria were isolated from the juice screen and juice tank in a sugarcane processing factory at times of low- and high dextran concentrations in the produced sugar. Dextran is an indicator of cane deterioration and sucrose loss after harvesting of the cane. The isolates were identified as Bacillus amyloliquefaciens (96 isolates) and Bacillus subtilis (eight isolates) based on restriction enzyme banding patterns of amplified 16S rRNA genes and rpoB gene sequence analysis. Exopolysaccharide production in sugarcane is normally associated with dextran produced by Leuconostoc mesenteroides. B. amyloliquefaciens, and to a lesser extent B. subtilis, could, however, also be responsible for exopolysaccharide (slime or gum) production in cane processing factories.

A 16S rDNA PCR-based theoretical to actual delta approach on culturable mock communities revealed severe losses of diversity information.

BACKGROUND: Subunits of ribosomal RNA genes (rDNAs) characterized by PCR-based protocols have been the proxy for studies in microbial taxonomy, phylogenetics, evolution and ecology. However, relevant factors have shown to interfere in the experimental outputs in a variety of systems. In this work, a 'theoretical' to 'actual' delta approach was applied to data on culturable mock bacterial communities (MBCs) to study the levels of losses in operational taxonomic units (OTUs) detectability. Computational and lab-bench strategies based on 16S rDNA amplification by 799F and U1492R primers were employed, using a fingerprinting method with highly improved detectability of fragments as a case-study tool. MBCs were of two major types: in silico MBCs, assembled with database-retrieved sequences, and in vitro MBCs, with AluI digestions of PCR data generated from culturable endophytes isolated from cacao trees. RESULTS: Interfering factors for the 16 s rDNA amplifications, such as the type of template, direct and nested PCR, proportion of chloroplast DNA from a tropical plant source (Virola officinalis), and biased-amplification by the primers resulted in altered bacterial 16S rDNA amplification, both on MBCs and V. officinalis leaf-extracted DNA. For the theoretical data, the maximum number of fragments for in silico and in vitro cuts were not significantly different from each other. Primers' preferences for certain sequences were detected, depending on the MBCs' composition prior to PCR. The results indicated overall losses from 2.3 up to 8.2 times in the number of OTUs detected from actual AluI digestions of MBCs when compared to in silico and in vitro theoretical data. CONCLUSIONS: Due to all those effects, the final amplification profile of the bacterial community assembled was remarkably simplified when compared to the expected number of detectable fragments known to be present in the MBC. From these findings, the scope of hypotheses generation and conclusions from experiments based on PCR amplifications of bacterial communities was discussed.

Isolation and molecular identification of Lactobacillus with probiotic potential from abomasums driven rennet.

Lactobacillus species are beneficial for the functional food industry and preventive medicine. The complex microflora of traditional cheese depends on the cheese types (e.g., homemade rennets). Here, the abomasum driven rennet was assessed for the existence of lactobacilli. For differentiating lactobacilli, the bacterial suspension was screened for the acid and bile resistance. The isolated bacteria were evaluated for antibiotic susceptibility and antagonistic impacts on other pathogenic bacteria. The 16S rDNA gene was evaluated by the amplified ribosomal DNA restriction analysis (ARDRA) recruiting the restriction enzyme Taq I and compared to the virtually digested patterns of previous reports on lactobacilli. The isolates were examined by random amplified polymorphic DNA (RAPD) and distinctive lactobacilli were sequenced. ARDRA and RAPD data showed three distinct lactobacilli strains, including L. acidophilus, L. planetarum, and L. fermentum. The homemade rennet is proposed as the novel source of probiotic strains as an alternative to the traditional cheeses.

Genetic Characterization and Diversity of Rhizobium Isolated From Root Nodules of Mid-Altitude Climbing Bean (Phaseolus vulgaris L.) Varieties.

The increasing interest in the use of rhizobia as biofertilizers in smallholder agricultural farming systems of the Sub-Saharan Africa has prompted the identification of a large number of tropical rhizobia strains and led to studies on their diversity. Inoculants containing diverse strains of rhizobia have been developed for use as biofertilizers to promote soil fertility and symbiotic nitrogen fixation in legumes. In spite of this success, there is paucity of data on rhizobia diversity and genetic variation associated with the newly released and improved mid-altitude climbing (MAC) bean lines (Phaseolus vulgaris L.). In this study, 41 rhizobia isolates were obtained from the root nodules of MAC 13 and MAC 64 climbing beans grown in upper and lower midland agro-ecological zones of Eastern Kenya. Eastern Kenya was chosen because of its high production potential of diverse common bean cultivars. The rhizobia isolates were characterized phenotypically on the basis of colony morphology, growth and biochemical features. Rhizobia diversity from the different regions of Eastern Kenya was determined based on the amplified ribosomal DNA restriction analysis (ARDRA) of PCR amplified 16S rRNA genes using Msp I, EcoR I, and Hae III restriction enzymes. Notably, native rhizobia isolates were morphologically diverse and grouped into nine different morphotypes. Correspondingly, the analysis of molecular variance based on restriction digestion of 16S rRNA genes showed that the largest proportion of significant (p < 0.05) genetic variation was distributed within the rhizobia population (97.5%) than among rhizobia populations (1.5%) in the four agro-ecological zones. The high degree of morphological and genotypic diversity of rhizobia within Eastern Kenya shows that the region harbors novel rhizobia strains worth exploiting to obtain strains efficient in biological nitrogen fixation with P. vulgaris L. Genetic sequence analysis of the isolates and testing for their symbiotic properties should be carried out to ascertain their identity and functionality in diverse environments.

Isolation, characterization and exploring biotechnological potential of halophilic archaea from salterns of western India.

Thirteen halophilic archaea were isolated from Kandla and Bhayander salt pans. These isolates were grouped into three different genera Halobacterium, Haloferax and Haloarcula based on morphological and biochemical characterization, polar lipid analysis, Amplified 16S rDNA restriction analysis (ARDRA) and 16S rDNA sequence analysis. Biochemical characterization suggested the ability of isolates to produce protease, amylase and poly-hydroxybutyrate (PHB) indicating their biotechnological potential. The isolates were further screened for the amount of extracellular protease produced. Halobacterium sp. SP1(1) showed significant protease production compared to other isolates. Protease producing ability of the isolate was influenced by several factors such as NaCl concentration, type of protein source, metal ions and surfactants, and presence of amino acid supplements in the production medium. Soybean flour, FeCl3 and dicotylsulfosuccinate were found to increase protease production by 2.36, 1.54 and 1.26 folds, respectively compared to production in basal medium. Effect of organic solvents used in paints (n-decane, n-undecane and n-dodecane) was also investigated on protease production by the isolate. Protease production by Halobacterium sp. SP1(1) was enhanced by 1.2 folds in presence of n-decane compared to control. Furthermore, the ability of isolate to hydrolyse fish protein was investigated using three different edible fishes (Pomfret, Flat fish and Seer fish) as sole protein source. Pomfret was found to be a good protein source for protease production by the isolate. These results revealed that Halobacterium sp. SP1(1) may have potential for paint-based antifouling coating preparations and fish sauce preparation by virtue of its extracellular protease.

Molecular discrimination of New Zealand sourced meat spoilage associated psychrotolerant Clostridium species by ARDRA and its comparison with 16s RNA gene sequencing.

ARDRA analysis was carried out on 90 New Zealand psychrotolerant Clostridium isolates derived from three meat production animal types and their environments. The isolates included species associated with spoilage: C. gasigenes, C. algidicarnis, C. tagluense, C. frigidicarnis and C. estertheticum. The isolates fell into 14 distinct ARDRA Groups, with 13 previously characterised meat spoilage-associated isolates shared between 6 of the 14 groups. The accuracy of ARDRA profiling analysis was supported by sequencing the 16s rRNA gene from isolates, including the representative spoilage associated Clostridium species and was consistent with previous phylogenetic relationships and classical cultural characterisation. The ARDRA methodology described in this study successfully discriminated between the different spoilage-associated species of clostridia as well as other pyschrotolerant Clostridium species associated with meat production. This discriminatory molecular screen will aid future source attribution studies as well as enable meat processors to identify and validate control measures for clostridia contamination, thus gaining greater efficacy in controlling meat spoilage caused by psychrotolerant clostridia.

A comparative study reveals the higher resolution of RAPD over ARDRA for analyzing diversity of Nostoc strains.

Nostoc is a diverse genus of filamentous cyanobacteria with tremendous potential for agricultural and industrial applications. Morphometric methods and routine 16S rDNA-based identification undermines the genetic diversity and impedes strain-level differentiation. A comparative study to deduce the discriminatory power of random amplified polymorphic DNA (RAPD) and amplified ribosomal DNA restriction analysis (ARDRA) for analyzing the genetic diversity of 20 Nostoc strains of diverse geographical origin was carried out. The RAPD primer used in the study generated 100% polymorphic profile. HIP TG primer produced the highest number of bands and fragments. Five primers, viz. OPA 08, OPA 11, HIP GC, OPAH 02 and OPF 05 could produce unique bands for 11 strains. Cluster analysis using the RAPD profile showed 12.5-25% similarity among the strains. Following in silico restriction analysis, two restriction enzymes, viz. HaeIII and HinfI were selected for ARDRA. However, clustering based on the restriction pattern showed 22.5-100% similarity. Results of the present study clearly indicate higher resolution of RAPD which can be reliably used for strain-level differentiation of Nostoc strains.

Alterations in soil microbial communities caused by treatments with penicillin or neomycin.

Antibiotic residues in soils can lead to serious health risk and ecological hazards. In this study, the effects of penicillin and neomycin, two antibiotics widely used in animal production, were investigated on soil bacterial communities. Changes in the community structure were monitored using three 16S ribosomal DNA (rDNA) polymerase chain reaction-based approaches, including denaturing gradient gel electrophoresis (DGGE), amplified rDNA restriction analysis (ARDRA), and terminal-restriction fragment length polymorphism (T-RFLP) analysis. The prominent DGGE bands were excised from gels and sequenced, and the data indicated the prevalence of Gammaproteobacteria in the soils. The total soil bacterial community, including uncultured bacteria, exhibited a higher diversity than that of cultured bacteria. Some microbial strains were capable of surviving and even subsisting on penicillin or neomycin. We also observed toxic effects of the antibiotics on the indigenous soil bacterial communities since some genotypes disappeared after the treatments (e.g., Pseudomonas sp., Stenotrophomonas sp., Salinimonas, and uncultured Acinetobacter sp.). The implications of these findings are that the functions of soil bacterial communities may be negatively affected if key microbial community members are lost.

PCR in Metagenomics.

Metagenomics approach involves direct genetic analysis of environmental samples, evading the tedious culturing process. Polymerase chain reaction is one invaluable tool used for such analyses. Here, we describe one protocol for metagenomic DNA isolation that gives inhibitor-free DNA suitable for PCR and other genetic manipulations. Subsequently, the chapter describes the use of PCR as an indicator of quality of DNA and to amplify a marker of phylogeny. Further, the application of PCR for detection of specific genes and screening of metagenomic libraries is outlined.

Molecular characterization and PCR-based screening of cry genes from Bacillus thuringiensis strains.

Novel cry genes are potential candidates for resistance management strategies, due to their different structures and modes of action. Therefore, it is desirable to clone and express novel cry genes from several new isolates of Bacillus thuringiensis (Bt). In the present study, 28 Bt strains were characterized at morphological and molecular level. All these strains are Gram positive, endospore forming and had shown different crystal morphologies when viewed under the microscope. The ARDRA (16S rDNA PCR-RFLP technique) with AluI, HaeIII, HinfI and TaqI produced unique and distinguishable restriction patterns used for the molecular characterization of these isolates. Based on UPGMA clustering analysis, Bt strains showed significant molecular diversity and the dendrogram obtained differentiated 28 Bt strains into 1 major cluster at a similarity coefficient 0.56. PCR analysis demonstrated that the Bt strains showed diverse cry gene profiles with several genes per strain. The Bt strain G3C1 showed the presence of maximum cry-type genes by PCR. The toxicological characterization of these cry genes will have huge importance in transgenic technology and will be useful in transgenesis of crop plants for better resistance management.