Effect of <Emphasis Type="Italic">Glomus</Emphasis> species on physiology and biochemistry of <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

The present study on efficacy of different Glomus species, an arbuscular mycorrhizal (AM) fungus (G. aggregatum, G. fasciculatum, G. mosseae, G. intraradices) on various growth parameters such as biomass, macro and micronutrients, chlorophyll, protein, cytokinin and alkaloid content and phosphatase activity of pink flowered Catharanthus roseus plants showed that all Glomus species except G. intraradices enhanced the chlorophyll, protein, crude alkaloid, phosphorus, sulphur, manganese and copper contents of C. roseus plants along with phosphatase activity significantly over uninoculated plants. However only G. mosseae and G. fasciculatum exhibited superior symbiotic relationship with the plant. G. mosseae was found to be the best for increasing the crude alkaloid content (8.19%) in leaf and also in increasing the quantity of important alkaloids vincristine and vinblastine.     

Thermoactive extracellular proteases of <Emphasis Type="Italic">Geobacillus caldoproteolyticus</Emphasis>, sp. nov., from sewage sludge

A proteolytic thermophilic bacterial strain, designated as strain SF03, was isolated from sewage sludge in Singapore. Strain SF03 is a strictly aerobic, Gram stain-positive, catalase-positive, oxidase-positive, and endospore-forming rod. It grows at temperatures ranging from 35 to 65°C, pH ranging from 6.0 to 9.0, and salinities ranging from 0 to 2.5%. Phylogenetic analyses revealed that strain SF03 was most similar to Saccharococcus thermophilus, Geobacillus caldoxylosilyticus, and G. thermoglucosidasius, with 16S rRNA gene sequence identities of 97.6, 97.5 and 97.2%, respectively. Based on taxonomic and 16S rRNA analyses, strain SF03 was named G. caldoproteolyticus sp. nov. Production of extracellular protease from strain SF03 was observed on a basal peptone medium supplemented with different carbon and nitrogen sources. Protease production was repressed by glucose, lactose, and casamino acids but was enhanced by sucrose and NH₄Cl. The cell growth and protease production were significantly improved when strain SF03 was cultivated on a 10% skim-milk culture medium, suggesting that the presence of protein induced the synthesis of protease. The protease produced by strain SF03 remained active over a pH range of 6.0–11.0 and a temperature range of 40–90°C, with an optimal pH of 8.0–9.0 and an optimal temperature of 70–80°C, respectively. The protease was stable over the temperature range of 40–70°C and retained 57 and 38% of its activity at 80 and 90°C, respectively, after 1 h.     

Biotransformation of Meloxicam by <Emphasis Type="Italic">Cunninghamella blakesleeana</Emphasis>: Significance of Carbon and Nitrogen Source

Influence of carbon and nitrogen source, on biotransformation of meloxicam was studied by employing Cunninghamella blakesleeana NCIM 687 with an aim to achieve maximum transformation of meloxicam and in search of new metabolites. The transformation was confirmed by HPLC and based on LC–MS–MS data and previous reports the metabolites were predicted as 5-hydroxymethyl meloxicam, 5-carboxy meloxicam and a novel metabolite. The quantification of metabolites was performed using HPLC peak areas. The results obtained indicate that glucose as carbon source, ammonium nitrate as nitrogen source, were found to be optimum for maximum transformation of meloxicam. The study suggests the significance of these factors in biotransformation of meloxicam using microbial cultures. The fermentation was scaled up to 1 l level.     

Interaction of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> and <Emphasis Type="Italic">Glomus intrarradix</Emphasis> in Sugar Cane Roots

Fifteen-day-old variety NA 56-79 sugar cane seedlings were inoculated with Azospirillum brasilense and Glomus intrarradix. This article aims at examining changes in sugar cane root seedlings inoculated with Glomus intrarradix and Azospirillum brasilense, the increase in microbial biomass and the acetylene reduction process as well. The internal root colonization was studied 20 days after inoculation using scanning and a transmission electron microscope. Both microorganisms entered the sugar cane root through the emergent lateral roots. The microorganisms were capable of coexisting both intra and intercellularly, producing changes in the cell wall, thus allowing colonization and interaction between the organisms. These changes increased the number of microorganisms inside the root as well as acetylene nitrogen reduction. Sugar cane plant biomass increased with joint-inoculation. The number of endophytic microorganisms and nitrogen fixing activity increased when they were colonized by Azospirillum and Glomus together.     

In vitro cytokine stimulation assay for glycolipid biosurfactant from <Emphasis Type="Italic">Rhodococcus</Emphasis><Emphasis Type="Italic">ruber</Emphasis>: role of monocyte adhesion

Glycolipid biosurfactant (GLB) from Rhodococcus ruber IEGM 231 was found to stimulate tumor necrosis factor-α (TNF-α), interleukin (IL) -1β and IL-6 production when applied as an ultrasonic emulsion to the adherent human peripheral blood monocyte culture. However, a lack of cytokine-stimulating activity was registered with the GLB applied as a hydrophobic film coating in 24-well culture plates, indicating that it may have been due to its inhibitory effect on monocyte adhesion. The mode of GLB application may therefore play an important role in in vitro assay of immunostimulatory activity of this compound as well as other bacterial glycolipids. Additionally, GLB from R. ruber displayed no cytotoxicity against human lymphocytes and therefore could be proposed as a potential immunomodulating and antitumor agent.     

Prevalence of Very Low Numbers of Potential Pathogenic Isolates of <Emphasis Type="Italic">Yersinia</Emphasis><Emphasis Type="Italic">enterocolitica</Emphasis> and <Emphasis Type="Italic">Yersinia intermedia</Emphasis> in Traditional Fast Foods of India

In this study, an incidence pattern of 1.7% for Yersinia enterocolitica and 2.5% for Y. intermedia were observed in an analysis of 120 diversified food samples collected from the local market of Mysore, Southern India. Two native isolates characterized as Y. enterocolitica belonged to biotype 1B and revealed the presence of major virulence related traits such as regulator of virulence, mucoid Yersinia factor regulator, attachment invasion locus, heat stable enterotoxin, Yersinia type II secretory system and phospholipase A in PCR. Force type neighbor-joining phylograms generated for Y. enterocolitica based on PCR amplicons of rovA and ypl showed 100% homology with two to three strains of Y. enterocolitica and about 75% homology with several strains of Y. pestis.     

Effect of pH and Temperature on Enzyme Activity of Chitosanase Produced Under Solid Stated Fermentation by <Emphasis Type="Italic">Trichoderma</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>

Trichoderma strains were extensively studied as biocontrol agents due to their ability of producing hydrolytic enzymes, which are considered key enzymes because they attack the insect exoskeleton allowing the fungi infection. The present work aimed to evaluate the ability of chitosanase production by four Trichoderma strains (T. harzianum, T. koningii, T. viride and T. polysporum) under solid stated fermentation and to evaluate the effect of pH and temperature on enzyme activity. pH strongly affected the enzyme activity from all tested strains. Chitosanase from T. harzianum and T. viride presented optimum activity at pH 5.0 and chitosanase from T. koningii and T. polysporum presented optimum activity at pH 5.5. Temperature in the range of 40–50°C did not affect enzyme activity. T. polysporum was found as the most promising strain to produce chitosanase with maximal enzyme activity of about 1.4 IU/gds, followed by T. viride (~1.2 IU/gds) and T. harzianum (1.06 IU/gds).     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Co-inoculation of Urea and DAP Tolerant <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> as Integrated Approach for Growth Enhancement of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Two plant growth promoting rhizobacteria––Sinorhizobium meliloti RMP1 and Pseudomonas aeruginosa GRC₂ were studied for integrated nutrient management to obtain improved yield of Brassica juncea. Low concentrations of urea and diammonium phosphate (DAP) stimulated the growth of both S. meliloti RMP1 and P. aeruginosa GRC₂. 1 M of urea and 0.35 M of DAP was found lethal for RMP1, while 1.3 M and 0.37 M concentrations of urea and DAP proved to be toxic for GRC₂. Lc₅₀ was observed as 0.49 M of urea and 0.15 M of DAP for RMP1, and 0.66 M urea and 0.18 M of DAP for GRC₂. Urea and DAP adaptive variants of RMP1 and GRC₂ was isolated. Adaptive bacterial variants had better growth rates at sub-lethal (Lc₅₀) concentrations of urea and DAP as compared to non-adaptive variants. They also retained plant growth promoting attributes similar to non adaptive variants. GRC₂ and RMP1 did not affect the growth of each other and were chemotactically active for DAP, urea as well as root exudates of B. juncea. Both the isolates colonized well in the rhizosphere of B. juncea, as their populations were recorded ≈5 log₁₀ cfu g⁻¹ after 120 days. Interestingly, the colonization ability was found even better when both strains were co-inoculated, as their population was recorded in the range of ≈6 log₁₀ cfu g⁻¹ after 120 days. In field trials, application of RMP1 and GRC₂ resulted in significant increase in biomass and yield of B. juncea as compared to control. However, yield was better with application of half dose and full dose of recommended fertilizers. Interestingly, the biomass as well as yield improved further when both isolates were applied together along with half dose of recommended fertilizers.     

A subset of <Emphasis Type="Italic">OsSERK</Emphasis> genes,including <Emphasis Type="Italic">OsBAK1</Emphasis>, affects normal growth and leaf development of rice

Since the identification of BRI1-Associated receptor Kinase 1 (BAK1), a member of the Somatic Embryogenesis Receptor Kinase (SERK) family, the dual functions of BAK1 in BR signaling and innate immunity in Arabidopsis have attracted considerable attention as clues for understanding developmental processes that must be balanced between growth and defense over the life of plants. Here, we extended our research to study cellular functions of OsSERKs in rice. As it was difficult to identify an authentic ortholog of AtBAK1 in rice, we generated transgenic rice in which the expression of multiple OsSERK genes, including OsBAK1, was reduced by OsBAK1 RNA interference. Resulting transgenic rice showed reduced levels of Os-BAK1 and decreased sensitivity to BL, leading to semidwarfism in overall growth. Moreover, they resulted in abnormal growth patterns, especially in leaf development. Most of the OsBAK1RNAi transgenic rice plants were defective in the development of bulliform cells in the leaf epidermal layer. They also showed increased expression level of pathogenesis-related gene and enhanced susceptibility to a rice blast-causing fungal pathogen, Magnaporthe oryzae. These results indicate that OsSERK genes, such as OsBAK1, play versatile roles in rice growth and development.     

Prevalence of Enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> Isolated from <Emphasis Type="Italic">Chhana</Emphasis> Based Indian Sweets in Relation to Public Health

Chhana based milk products viz. rossogolla, kanchagolla, narampak sandesh and karapak sandesh are very popular in eastern part of India and gaining popularity in other parts of the country. A wide variation in manufacture method, microbial quality and shelf-life of these traditional milk products were observed by previous research. The aim of the present study was to determine the prevalence of contamination of chhana based milk products available in Kolkata city with Enteropathogenic Escherichia coli (EPEC) serogroups. Random samples of different chhana based milk products were collected from different parts of Kolkata city in aseptic condition, cultured in selective media and examined for biochemical tests. Among 240 samples, E. coli was isolated from 67 (27.91%) of them. Potential EPEC was present in 52 samples (21.66%) and 55 of the isolates were EPEC. Eleven serogroups were identified viz. O26, O55, O111, O119, O114, O125, O142, O86, O126, O127, O128. Among all these serogroups, O55 (23.66%) was the most prevalent. Though recent studies on virulence factors indicate that not all strains serologically classified as EPEC are able to attaching/effacing lesion, it is believed that the isolation of EPEC serogroups from chhana based milk products represent a potential risk for public health particularly children, as well as an indicative of the presence of other enteropathogens. Considering the public health importance of sweetmeat consumers, the product should be prepared hygienically reducing the microbial load present in it. The result indicates that strict preventive measures should be adopted to ensure contamination free sweetmeats for the safety of the consumers.     

Production of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine Using Recombinant Chitinolytic Enzymes

The pharmaceutically important compound N-acetylglucosamine (NAG), is used in various therapeutic formulations, skin care products and dietary supplements. Currently, NAG is being produced by an environment-unfriendly chemical process using chitin, a polysaccharide present in abundance in the exoskeleton of crustaceans, as a substrate. In the present study, we report the potential of an eco-friendly biological process for the production of NAG using recombinant bacterial enzymes, chitinase (CHI) and chitobiase (CHB). The treatment of chitin with recombinant CHI alone produced 8% NAG and 72% chitobiose, a homodimer of NAG. However, supplementation of the reaction mixture with another recombinant enzyme, CHB, resulted in approximately six fold increase in NAG production. The product, NAG, was confirmed by HPLC, TLC and ESI-MS studies. Conditions are being optimized for increased production of NAG from chitin.     

Negative regulation of pathogenesis in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tabaci</Emphasis> 11528 by ATP-dependent lon protease

Pseudomonas syringae pv. tabaci causes wildfire disease in tobacco plants. The hrp pathogenicity island (hrp PAI) of P. syringae pv. tabaci encodes a type III secretion system (TTSS) and its regulatory system, which are required for pathogenesis in plants. Three important regulatory proteins-HrpR, HrpS, and HrpL-have been identified to activate hrp PAI gene expression. The bacterial Lon protease regulates the expression of various genes. To investigate the regulatory mechanism of the Lon protease in P. syringae pv. tabaci 11528, we cloned the lon gene, and then a Δlon mutant was generated by allelic exchange. lon mutants showed increased UV sensitivity, which is a typical feature of such mutants. The Δlon mutant produced higher levels of tabtoxin than the wild-type. The lacZ gene was fused with hrpA promoter and activity of β-galactosidase was measured in hrp-repressing and hrp-inducing media. The Lon protease functioned as a negative regulator of hrp PAI under hrp-repressing conditions. We found that strains with lon disruption elicited the host defense system more rapidly and strongly than the wild-type strain, suggesting that the Lon protease is essential for systemic pathogenesis.     

Thermostable Xanthine Oxidase Activity from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> RL-2d Isolated from Manikaran Thermal Spring: Production and Characterization

Xanthine oxidase is an important enzyme of purine metabolism that catalyzes the hydroxylation of hypoxanthine to xanthine and then xanthine to uric acid. A thermostable xanthine oxidase is being reported from a thermophilic organism RL-2d isolated from the Manikaran (Kullu) hot spring of Himachal Pradesh (India). Based on the morphology, physiological tests, and 16S rDNA gene sequence, RL-2d was identified as Bacillus pumilus. Optimization of physiochemical parameters resulted into 4.1-fold increase in the xanthine oxidase activity from 0.051 U/mg dcw (dry cell weight) to 0.209 U/mg dcw. The xanthine oxidase of B. pumilus RL-2d has exhibited very good thermostability and its t_1/2 at 70 and 80 °C were 5 and 1 h, respectively. Activity of this enzyme was strongly inhibited by Hg²⁺, Ag⁺ and allopurinol. The investigation showed that B. pumilus RL-2d exhibited highest xanthine oxidase activity and remarkable thermostability among the other xanthine oxidases reported so far.     

Highly efficient <Emphasis Type="Italic">in vitro</Emphasis> adventitious shoot regeneration of peppermint (<Emphasis Type="Italic">Mentha</Emphasis> x <Emphasis Type="Italic">piperita</Emphasis> L.) using internodal explants

An in vitro regeneration system with a 100% efficiency rate was developed in peppermint [Mentha x piperita] using 5- to 7-mm-long second internode stem segments of 3-wk-old stock plants. Shoots developed at sites of excision on stem fragments either directly from the cells or via primary calluses. The optimal medium for maximum shoot initiation and regeneration contained Murashige and Skoog (MS) salts, B5 vitamins, thidiazuron (TDZ, 11.35 μM), ZT (4.54 μM), 10% coconut water (CW), 20 g l⁻¹ sucrose, 0.75% agar, adjusted to pH 5.8. A frequency of 100% shoot initiation was achieved, with an average of 39 shoots per explant. This regeneration system is highly reproducible. The regenerated plants developed normally and were phenotypically similar to Black Mitcham parents.     

Simultaneous detection of pathogenic <Emphasis Type="Italic">B. cereus,S. aureus</Emphasis> and <Emphasis Type="Italic">L. monocytogenes</Emphasis> by multiplex PCR

Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus are of major concern for food safety in terms of frequency and seriousness of the disease. The occurrence these three important pathogens and their coexistence in food matrices are predominant. Moreover, symptoms associated with B. cereus and S. aureus food poisoning not only closely resembles each other but can also be overlapping with other foodborne infections. In this context, detection of these three pathogens simultaneously in food samples by a single multiplex PCR (mPCR) would have advantages in terms of rapidity and cost saving, when compared with single organism specific PCRs. mPCR has been standardized by targeting three major diarrheal enterotoxin genes hbl A, cyt K and nhe A of B. cereus, virulence associated nuc and Ent B genes of S. aureus and virulence associated hly and iap genes of L. monocytogenes along with internal amplification control (IAC). The results showed that mPCR accurately identified all the three organisms individually or in combination without non-specificity. The mPCR was able to detect as low as 10 to 100 organisms per ml of growth following overnight enrichment of spiked food samples (vegetable biriyani and milk) and their presence in naturally contaminated samples also. The high throughput and cost effective multiplex PCR method developed in this study could provide a powerful tool for simultaneous, rapid and reliable detection of B. cereus, S. aureus and L. monocytogenes in food samples.     

New Culture Medium to Xanthan Production by <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv<Emphasis Type="Italic">. campestris</Emphasis>

Xanthan is a important biopolymer for commercial purpose and it is produced in two stages by Xanthomonas campestris. In the first one, the bacterium is cultivated in the complex medium enriched in nitrogen and the biomass produced is used as inoculum for the next stage in which the gum is produced in another medium. In this work a new medium for the first stage is proposed in place of currently used YM medium. Different formulated growth media were studied and the correspondent biomass produced was analysed as inoculum for the second stage. The inoculum and gum were produced by batch process in shaker at 27°C in pH 6.0 and at 30°C in pH 7.0, respectively. The gum was precipitated with ethanol (3:1 v/v). The dryed biomass and xathan gum produced were determined by drying in oven at 105 and 40°C, respectively. The viscosity of the fermentation broth and 1% gum solution in water were determined in Brookfield viscometer. The formulated medium presented the increase in gum production (30%), broth (136%) and 1% gum solution viscosity (60%) compared to YM, besides the inferior cost. The results showed the importance of the quality of the inoculum from the first stage of the culture which influenced on the gum viscosity in the second stage.     

<Emphasis Type="Italic">fabC</Emphasis> of <Emphasis Type="Italic">Streptomyces lydicus</Emphasis> involvement in the biosynthesis of streptolydigin

Streptolydigin, a secondary metabolite produced by Streptomyces lydicus, is a potent inhibitor of bacterial RNA polymerases. It has been suggested that streptolydigin biosynthesis is associated with polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS). Thus, there is great interest in understanding the role of fatty acid biosynthesis in the biosynthesis of streptolydigin. In this paper, we cloned a type II fatty acid synthase (FAS II) gene cluster of fabDHCF from the genome of S. lydicus and constructed the SlyfabCF-disrupted mutant. Sequence analysis showed that SlyfabDHCF is 3.7 kb in length and encodes four separated proteins with conserved motifs and active residues, as shown in the FAS II of other bacteria. The SlyfabCF disruption inhibited streptolydigin biosynthesis and retarded mycelial growth, which were likely caused by the inhibition of fatty acid synthesis. Streptolydigin was not detected in the culture of the mutant strain by liquid chromatography–mass spectrometry. Meanwhile, the streptolol moiety of streptolydigin accumulated in cultures. As encoded by fabCF, acyl carrier protein (ACP) and β-ketoacyl-ACP synthase II are required for streptolydigin biosynthesis and likely involved in the step between PKS and NRPS. Our results provide the first genetic and metabolic evidence that SlyfabCF is shared by fatty acid synthesis and antibiotic streptolydigin synthesis.     

Production of Exo-polysaccharide by <Emphasis Type="Italic">Rhizobium</Emphasis> sp.

Two fold increase in the yield of glucose and maltose containing exo-polysaccharide (EPS) by Rhizobium sp. was observed during its growth in modified YEMB. EPS production, plant growth promotion activity and root colonization of Rhizobium sp. studies showed enhanced EPS synthesis, more seed germination and over all improvement in plant growth over control and R. meliloti treatment. Groundnut seeds bacterized with Rhizobium sp. resulted in 69.75% more root length, 49.51% more shoot height, 13.75% more number of branches and 13.60% more number of pods over the control and R. meliloti treatment. Bacterization of wheat seeds increased the dry matter yield of roots (1.7-fold), and roots adhering soil (RAS) (1.5) and shoot mass (1.9-fold). Rhizobium sp. inoculation also increased the population density of EPS-producing bacteria on the rhizoplane. Roots of plants inoculated with Rhizobium sp. maintained a higher K⁺/Na⁺ ratio and K⁺–Na⁺ selectivity.