Expression pattern of mitogen-activated protein kinase kinase 4 and regulation to antibacterial factor ABF-1/2 in response to bacterial challenge from Artemia parthenogenetica.

Mitogen-activated protein kinase 4, MKK4, is a key upstream kinase in the JNK/p38 MAPK pathway that has been reported to participate in multiple immune responses. In this study, the gene that encodes ApMKK4 was isolated and identified from Artemia parthenogenetica. It was found to contain a 1134 bp open reading frame encoding 378 amino acids. The predicted protein contains D domain, DVD domain and kinase domain. Homology analysis revealed that ApMKK4 shares 38-69% identity with MKK4 homologs from other species. Results revealed that ApMKK4 was mainly expressed during early development of which highest at the gastrula stage. After challenged by Vibrio harveyi and Micrococcus lysodeikticus, ApMKK4 was remarkably upregulated at 10 and 103 cfu/mL bacterial concentrations, respectively. Through siRNAi, the transcript level of ApMKK4 was significantly decreased by 46-67%. Intriguingly, when the ApMKK4-knockdown nauplii faced with bacterial stimulation, the expression of ApMKK4 was completely restored in a short time. Moreover, this phenomenon also occurred in related antimicrobial peptide genes, ABF-1 and ABF-2. Our research reveals that ApMKK4 plays a pivotal role during early development and immune responses against bacterial infections.

The role of plant growth-promoting rhizobacteria (PGPR) in improving iron acquisition by altering physiological and molecular responses in quince seedlings.

Due to insoluble iron (Fe) sources in soil, limited Fe availability leads to the disruption of the photosynthetic apparatus; this affects the growth and productivity of plants such as quince (Cydonia oblonga) that are very sensitive to low Fe content. Plant growth-promoting rhizobacteria (PGPR) play an important role in the regulation of Fe uptake under its limited availability. Therefore, in this research, two PGPR (Pseudomonas fluorescens and Microccucuce yunnanensis), at two Fe levels [50 µM (Fe-sufficiency) or 5 µM (Fe-deficiency)], were used to investigate the impact of the given bacteria on improving the acquisition of Fe in quince seedlings. Upon Fe-deficiency, the highest shoot and root biomass (7.14 and 6.04 g plant-1 respectively), the greatest chlorophyll concentration (0.89 mg g-1FW), and the largest Fe concentrations in roots and shoots (30% and 48.7%, respectively) were shown in the quince treated with M. yunnanensis. Both PGPR increased the root citric acid and the phenolic compound concentration. Two days after Fe-deficiency and PGPR treatments, a 1.5- fold increase, was observed in the expression of HA7. The highest PAL1 gene expression and the greatest PAL activity (95.76 µmol cinnamic acid g-1FW) were obtained from the M. yunnanensis treatment. The expression of the FRO2 gene was also affected by Fe-deficiency and PGPR treatments, resulting in an increase in the FCR activity and a surge in the Fe concentrations of leaves and roots. It could, therefore, be concluded that the PGPR modulated Fe acquisition in the quince seedlings upon Fe-deficiency by influencing the physico-chemical and molecular responses.

Plant growth-promoting endophytic bacteria augment growth and salinity tolerance in rice plants.

Salt stress negatively affects growth and development of plants. However, it is hypothesized that plant growth-promoting endophytic bacteria can greatly alleviate the adverse effects of salinity and can promote growth and development of plants. In the present research, we aimed to isolate endophytic bacteria from halotolerant plants and evaluate their capacity for promoting crop plant growth. The bacterial endophytes were isolated from selected plants inhabiting sand dunes at Pohang beach, screened for plant growth-promoting traits and applied to rice seedlings under salt stress (NaCl; 150 mm). Out of 59 endophytic bacterial isolates, only six isolates, i.e. Curtobacterium oceanosedimentum SAK1, Curtobacterium luteum SAK2, Enterobacter ludwigii SAK5, Bacillus cereus SA1, Micrococcus yunnanensis SA2, Enterobacter tabaci SA3, resulted in a significant increase in the growth of Waito-C rice. The cultural filtrates of bacterial endophytes were tested for phytohormones, including indole-3-acetic acid, gibberellins and organic acids. Inoculation of the selected strains considerably reduced the amount of endogenous ABA in rice plants under NaCl stress, however, they increased GSH and sugar content. Similarly, these strains augmented the expression of flavin monooxygenase (OsYUCCA1) and auxin efflux carrier (OsPIN1) genes under salt stress. In conclusion, the pragmatic application of the above selected bacterial strains alleviated the adverse effects of NaCl stress and enhanced rice growth attributes by producing various phytohormones.

Identification and function of penaeidin 3 and penaeidin 5 in Fenneropenaeus merguiensis.

Antimicrobial peptides (AMPs) participate in immune defenses of invertebrate, vertebrate and plant species. As a kind of AMPs, penaeidins play important roles in innate immunity of shrimp. In this study, two penaeidin homologues termed FmPEN3 and FmPEN5 were cloned and identified from Fenneropenaeus merguiensis for the first time. The complete open reading frames (ORFs) of FmPEN3 and FmPEN5 were 216 bp and 240 bp, encoding 71 and 79 amino acids, respectively. Both FmPEN3 and FmPEN5 contain an N-terminal proline-rich domain (PRD) and a C-terminal cysteine-rich domain (CRD). The genome structure of FmPEN3 and FmPEN5 genes both consist of 2 exons and 1 intron. qPCR analysis showed that FmPEN3 was constitutively expressed but FmPEN5 transcripts were found only in hemocytes, gills, epidermis, nerve and pyloric cecum. The FmPEN3 and FmPEN5 expression were responsive to Vibrio parahaemolyticus and Micrococcus lysodeikticus infection and their transcription levels were downregulated by RNAi silencing of the transcription factors FmDorsal and FmRelish. In addition, recombinant proteins of FmPEN3 (rFmPEN3) and FmPEN5 (rFmPEN5) were successfully expressed in E. coli. The antibacterial assays revealed that rFmPEN3 and rFmPEN5 could inhibit the growth of M. lysodeikticus but only rFmPEN5 could inhibit the growth of V. parahaemolyticus in vitro. In summary, the results presented in this study indicated the functions of FmPEN3 and FmPEN5 played in anti-bacterial immunity of F. merguiensis, providing some insights into the function of AMPs in shrimp.

Characterization of Bacillus subtilis from gastrointestinal tract of hybrid Hulong grouper (Epinephelus fuscoguttatus × E. lanceolatus) and its effects as probiotic additives.

Probiotics are widely used for the improvement of animals' growth and health. However, few marine aquatic probiotics are applied and licensed in China. In this study, a Bacillus spp. strain was isolated from the Hulong grouper gastrointestinal tract, which was identified as a new strain of Bacillus subtilis and was named as 7k. B. subtilis 7k showed desirable capability of sporulation and resistance to heat, simulated gastric juice and simulated duodenum juice, indicating its potential as probiotics. Seven antimicrobial chemicals were found in the secretion of the B. subtilis 7k. B. subtilis 7k addition in diet promoted the growth rate of Hulong groupers. Moreover, B. subtilis 7k can inhibit infection by iridovirus, making B. subtilis 7k a suitable kind of probiotic for maintaining fishes' health. Our results also revealed that B. subtilis 7k induced non-specific immune response in Hulong grouper under virus infection. Hulong grouper fed by diets containing B. subtilis 7k at 108 and 1010 cfu g-1 for 4-8 weeks were significantly strengthened in serum lysozyme activity, serum alternative complement activity (ACH50), serum bactericidal activity, respiratory burst, superoxide dismutase activity (SOD), and phagocytic activity of head kidney leucocytes when compared with those fed by control diets. In conclusion, B. subtilis 7k was isolated and characterized to be a kind of process enduring, growth stimulating, immunity enhancing and health promoting probiotic using in grouper culture.

Comparative analysis of immunocompetence between females and males in the sea cucumber Apostichopus japonicus.

In order to preliminarily understand the immune difference between females and males in the sea cucumber Apostichopus japonicus, the activities assay of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), phenoloxidase (PO), acid phosphatase (ACP) and alkaline phosphatase (ALP) with biochemical methods, the detection of PO isozymes with native-PAGE and catechol staining, and the test of antibacterial activities with bacterial growth curve determination method were performed in this study using cell-free coelomic fluid (CCF) and coelomocyte lysate supernatant (CLS) from females and males as the samples. The PO activities were not detected in the CLS and showed no significant difference between the CCF from females and males. However, totally five PO isozyme bands were detected in the CLS of females while only four were detected in the CLS of males after zymogram analysis. These results implied that the PO isozymes in the coelomocytes of viripotent A. japonicus were inactive under natural condition and may be activated by some certain treatments during native-PAGE, and PO might play different immune and physiological roles between females and males. In addition, the activities of SOD, CAT, POD and ALP in the CCF and the activities of CAT, POD, ACP and ALP in the CLS from males were all significantly higher than those from females. The results collectively suggested that in viripotent A. japonicus, the gender had a remarkable effect on the immunity, and the immunocompetence of males might have an advantage over that of females. Furthermore, the activities of all determined enzymes except PO and the number of detected PO isozymes showed higher values in CLS than in CCF, implying that in viripotent A. japonicus, the coelomocytes might take more immune responsibility in comparison with CCF.

Immune function trade-offs in response to parasite threats.

Immune function is often involved in physiological trade-offs because of the energetic costs of maintaining constitutive immunity and mounting responses to infection. However, immune function is a collection of discrete immunity factors and animals should allocate towards factors that combat the parasite threat with the highest fitness cost. For example, animals on dispersal fronts of expanding population may be released from density-dependent diseases. The costs of immunity, however, and life history trade-offs in general, are often context dependent. Trade-offs are often most apparent under conditions of unusually limited resources or when animals are particularly stressed, because the stress response can shift priorities. In this study we tested how humoral and cellular immune factors vary between phenotypes of a wing dimorphic cricket and how physiological stress influences these immune factors. We measured constitutive lysozyme activity, a humoral immune factor, and encapsulation response, a cellular immune factor. We also stressed the crickets with a sham predator in a full factorial design. We found that immune strategy could be explained by the selective pressures encountered by each morph and that stress decreased encapsulation, but not lysozyme activity. These results suggest a possible trade-off between humoral and cellular immunity. Given limited resources and the expense of immune factors, parasite pressures could play a key factor in maintaining insect polyphenism via disruptive selection.

Identification and functional characterizations of serpin8, a potential prophenoloxidase-activating protease inhibitor in Pacific white shrimp, Litopenaeus vannamei.

Serpins have been characterized from varieties of organisms by their inhibitory roles on serine or cysteine proteases. However, research for the functional study of serpins in crustacean is relatively small. To fully clarify the immune characterizations of serpin, a novel serpin (named Lvserpin8) encoding 414 amino acids with a 19-amino acid signal peptide and a serpin domain was identified from the Pacific white shrimp Litopenaeus vannamei. Sequence analysis indicated that the genomic Lvserpin8 gene contains 5 exons and 4 introns. The P1 residues of the predicted scissile bond in the reactive center loop (RCL) region represented for Lysine (Lys), which is in accordance with Pmserpin8, Dmserpin27A, Ofserpin3, Bmserpin3 and Msserpin3. Quantitative results showed that high mRNA expression of Lvserpin8 was detected in hepatopancreas and testis. Notably, a significant increase of Lvserpin8 was appeared post injection of Vibrio anguillarum, and Micrococcus lysodeikticus. Moreover, Lvserpin8 was knocked down in vivo by double-stranded RNA (dsRNA) mediated RNA interference (RNAi). Suppression of Lvserpin8 led to a significant increase in the transcripts of LvPPAE2 (Prophenoloxidase-activating Enzyme 2) and cumulative mortality. What's more, recombinant Lvserpin8 protein (rLvserpin8) displayed inhibition roles on trypsin activity, and prophenoloxidase activation. Taken together, the results implied that Lvserpin8 may be involved in shrimp innate immunity via the inhibition of prophenoloxidase-activating proteases.

A fibrinogen-related protein identified from hepatopancreas of crayfish is a potential pattern recognition receptor.

Fibrinogen-related protein (FREP) family is a large group of proteins containing fibrinogen-like (FBG) domain and plays multiple physiological roles in animals. However, their immune functions in crayfish are not fully explored. In the present study, a novel fibrinogen-like protein (designated as PcFBN1) was identified and characterized from hepatopancreas of red swamp crayfish Procambarus clarkii. The cDNA sequence of PcFBN1 contains an open reading frame (ORF) of 1353 bp encoding a protein of 450 amino acids. Sequence and structural analysis indicated that PcFBN1 contains an FBG domain in C-terminal and a putative signal peptide of 19 amino acids in N-terminal. Semi-quantitative PCR revealed that the main expression of PcFBN1 was observed in hepatopancreas and hemocyte. Temporal expression analysis exhibited that PcFBN1 expression could be significantly induced by heat-killed Aeromonas hydrophila. Tissue distribution and temporal change of PcFBN1 suggested that PcFBN1 may be involved in immune responses of red swamp crayfish. Recombinant PcFBN1 protein binds and agglutinates both gram-negative bacteria Escherichia coli and gram-positive bacteria Micrococcus lysodeikticus. Moreover, binding and agglutination is Ca(2+) dependent. Further analysis indicated that PcFBN1 recognizes some acetyl group-containing substance LPS and PGN. RNAi experiment revealed that PcFBN1 is required for bacterial clearance and survival from A. hydrophila infection. Reduction of PcFBN1 expression significantly decreased the survival and enhanced the number of A. hydrophila in the hemolymph. These results indicated that PcFBN1 plays an important role in the innate immunity of red swamp crayfish as a potential pattern recognition receptor.

Complement C3 gene: Expression characterization and innate immune response in razor clam Sinonovacula constricta.

Complement component 3 (C3) is central to the complement system, playing an important role in immune defense, immune regulation and immune pathology. Several C3 genes have been characterized in invertebrates but very few in shellfish. The C3 gene was identified from the razor clam Sinonovacula constricta, referred to here as Sc-C3. It was found to be highly homologous with the C3 gene of Ruditapes decussatus. All eight model motifs of the C3 gene were found to be included in the thiolester bond and the C345C region. Sc-C3 was widely expressed in all healthy tissues with expression being highest in hemolymph. A significant difference in expression was revealed at the umbo larvae development stage. The expression of Sc-C3 was highly regulated in the hemolymph and liver, with a distinct response pattern being noted after a challenge with Micrococcus lysodeikticus and Vibrio parahemolyticus. It is therefore suggested that a complicated and unique response pathway may be present in S. constricta. Further, serum of S. constricta containing Sc-C3 was extracted. This was activated by LPS or bacterium for verification for function. The more obvious immune function of Sc-C3 was described as an effective membrane rupture in hemocyte cells of rabbit, V. parahemolyticus and Vibrio anguillarum. Thus, Sc-C3 plays an essential role in the immune defense of S. constricta.

Identification, expression pattern and potential role of variable lymphocyte receptor Aj-VLRA from Apostichopus japonicus in response to bacterial challenge.

The variable lymphocyte receptors (VLRs) are found in jawless vertebrates (agnathans), and specifically recognize bacteria and viruses via their leucine-rich repeats (LRRs). VLRs are believed to be adaptive immune response molecules. Echinoderms do not have adaptive immune systems; however, in the present study, a VLR cDNA named Aj-VLRA was cloned and characterized from sea cucumber, Apostichopus japonicus. The complete cDNA of Aj-VLRA was 3072 bp, including a 1995 bp open reading frame encoding 664 amino acids comprising LRR domains, a predicted transmembrane helix and an N-terminal signal peptide. As determined by quantitative real-time reverse transcription polymerase chain reaction, Aj-VLRA transcripts are ubiquitously expressed in the body wall, longitudinal muscles, intestine and respiratory tree of A. japonicus. The expression level of Aj-VLRA was upregulated after challenge with four common marine bacteria. In situ hybridization showed that the expression of Aj-VLRA was widely distributed in the four tissues, particularly in the cytoplasm of epidermal cells. Recombinantly expressed Aj-VLRA (including the LRR domains) could bind to bacteria including Micrococcus lysodeikticus (Gram+) and Vibrio anguillarum (Gram-). Collectively, the results suggested that Aj-VLRA is related to an innate immune response of A. japonicus.

Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection.

The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P < 0.05) lytic activity against Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P < 0.05) protection to G1B cells against A. hydrophila infection. When channel catfish were intraperitoneally injected with pcDNA-Lys-g along with an adjuvant QCDCR, the transcriptional level of Lys-g was significantly (P < 0.05) increased. When pcDNA-Lys-g injected fish was challenged with a highly virulent A. hydrophila strain AL-09-71, pcDNA-Lys-g offered 100% protection to channel catfish at two days post DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish injected with pcDNA vector alone at two days post DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection.

[Micrococcus sp.--the pathogen of leaf necrosis of horse-chestnuts (Aesculus L.) in Kiev].

A group of phytopathogenic bacteria was isolated from patterns of drying horse-chestnuts (Aesculus L.), which grow in Kyiv. The properties of slowly growing, highly aggressive microorganisms have been described in the paper. They grow up on the 8-10th day after sowing. The investigated microorganisms form very small (0.5-1 mm in diameter) colonies on the potato agar. Bacteria are protuberant, shining, smooth with flat edges, they are pale yellow, yellow, or pink. The bacteria are Gram-positive, spherical, are disposed in smears singly, in pairs, as accumulations, or netting. They are aerobes, do not form spores, are not mobile. They are inert in respect of different sources of carbon. They reduce nitrates, do not dilute gelatin, do not hydrolyze starch, do not release hydrogen sulphide and indole. The bacteria are catalase-positive, oxidase-negative. They do not cause potato and carrot rot. They lose quickly their viability under the laboratory conditions. The saturated acids C 14:0; C 15:0; C16:0; C18:0 have been revealed in the composition of cellular fatty acids. Microorganisms are identified as Micrococcus sp. Under artificial inoculation this highly aggressive pathogen causes drying of the horse-chestnut buds and necrosis, which occupies 1/3-1/2 of the leaf plate. A wide zone of chlorosis, surrounding necrosis, may occupy the whole leaf surface. The infected leaves use to twist up from the top (apex) or along a midrib and to dry.

Bioaugmentation with cadmium-resistant plant growth-promoting rhizobacteria to assist cadmium phytoextraction by Helianthus annuus.

Micrococcus sp. MU1 and Klebsiella sp. BAM1, the cadmium-resistant plant growth-promoting rhizobacteria (PGPR), produce high levels of indole-3-acetic acid (IAA) during the late stationary phase of their growth. The ability of PGPR to promote root elongation, plant growth and cadmium uptake in sunflowers (Helianthus annuus) was evaluated. Both species of bacteria were able to remove cadmium ions from an aqueous solution and enhanced cadmium mobilization in contaminated soil. Micrococcus sp. and Klebsiella sp. use aminocyclopropane carboxylic acid as a nitrogen source to support their growth, and the minimum inhibitory concentrations of cadmium for Micrococcus sp. and Klebsiella sp. were 1000 and 800mM, respectively. These bacteria promoted root elongation in H. annuus seedlings in both the absence and presence of cadmium compared to uninoculated seedlings. Inoculation with these bacteria was found to increase the root lengths of H. annuus that had been planted in cadmium-contaminated soil. An increase in dry weight was observed for H. annuus inoculated with Micrococcus sp. Moreover, Micrococcus sp. enhanced the accumulation of cadmium in the root and leaf of H. annuus compared to untreated plants. The highest cadmium accumulation in the whole plant was observed when the plants were treated with EDTA following the treatment with Micrococcus sp. In addition, the highest translocation of cadmium from root to the above-ground tissues of H. annuus was found after treatment with Klebsiella sp. in the fourth week after planting. Our results show that plant growth and cadmium accumulation in H. annuus was significantly enhanced by cadmium-resistant PGPRs, and these bacterial inoculants are excellent promoters of phytoextraction for the rehabilitation of heavy metal-polluted environments.

Age- and density-dependent prophylaxis in the migratory, cannibalistic Mormon cricket Anabrus simplex (Orthoptera: Tettigoniidae).

As a result of the increased potential for disease transmission, insects are predicted to show an increased constitutive immunity when crowded. Cannibalistic aggressive interactions further increase the risk of wounding and pathogen transmission in crowds. Nymphal Mormon crickets Anabrus simplex Haldeman were collected in Montana and reared in the laboratory either solitarily or at densities similar to that experienced by Mormon crickets in migratory bands. As teneral adults, solitarily-reared Mormon crickets tended to have greater phenoloxidase activity than those reared in groups. Sampling enzyme activity a second time when the adults were nearing reproductive maturity, group-reared Mormon crickets had elevated levels of prophenoloxidase and encapsulated foreign objects faster than solitarily-reared insects. Rearing density did not have a significant effect on either the darkness of the cuticle or antibacterial activity. This is the first report of age-related responses of adult insect immunity to crowding.

A simple and cost-effective cover-glass test for the differentiation between staphylococci and micrococci in clinical laboratory.

A cover-glass placed on a heavily inoculated culture plate clearly differentiates facultatively anaerobic staphylococci growing underneath the cover-glass after overnight incubation from nongrowing aerobic micrococci. Even if there are some exceptions, all medically significant staphylococci can grow in the test. Thus, the test provides a cost-effective and highly specific tool for separation of both genera which fundamentally differ in their pathogenicity.

Gene expression specificity of the mussel antifungal mytimycin (MytM).

We previously reported the nucleotide sequences and diversity of mytimycin (MytM) from the Mediterranean mussel, Mytilus galloprovincialis. Using real-time PCR (q-PCR), we observed that the MytM gene was mainly expressed in circulating hemocytes and to a less extent in the mantle. In vivo challenge with bacteria or with the yeast, Candida albicans, did not increase the expression as measured by q-PCR in hemocytes. By contrast, injection of the filamentous fungus, Fusarium oxysporum, induced a sudden and strong increase of expression at 9h p.i. (stimulation index of 25.7 ± 2.1). Optimum stimulating dose was 10(4) spores of F. oxysporum per mussel. In the same samples, AMP mytilin and myticin showed no stimulation. Consequently, we hypothesized the existence of 2 different signal transduction pathways, one activated by bacteria and yeast, the other triggered by filamentous fungi. A second challenge performed with F. oxysporum 24 h after the first challenge induced an increase of MytM gene expression (stimulation index of 3.5 ± 1.7). However, this second increase was significantly lower than the first, suggesting less efficient response rather than significant protection.

Cloning and the expression pattern of Spätzle gene during embryonic development and bacterial challenge in Artemia sinica.

Spätzle gene codes for a NGF-like protein, it involves in the embryonic development and innate immune response of insects and other invertebrate. In dorsal ventral axis differentiation, proSpätzle is activated by serine endoproteases Easter and then binds to the Toll receptor in ventral axis of oocyte which initiates the ventral axis development. Besides, it could also be activated by another protease named Spätzle-processing enzyme (SPZ) to mediate Toll pathway which involves in innate immune response in fungal and Gram-positive bacterial infection of invertebrate. In this paper, a full-length cDNA of Spätzle was firstly isolated from Artemia sinica which belonged to Spätzle-4 family. The expression of Spätzle was investigated at various stages during the embryonic development of A. sinica using real-time PCR and immunohistochemistry assays. The result showed that the high expression level of Spätzle appeared at 7 and 10 days of the embryo. A gradual increased level of Spätzle transcript occurred after being challenged with Gram-positive bacteria. Immunohistochemistry assay showed that Spätzle was mainly expressed in the cephalothorax and on the alimentary canal surface during embryonic development. This new Spätzle member showed a constitutive and regional expression during the embryonic development of A. sinica. It may play an important role in dorsal-ventral differentiation at the early development stages and in immune response pathway at the pseudoadult and adult stage, as well as during infection.

Phytoremediation of metals from fly ash through bacterial augmentation.

Different combinations of four bacterial strains isolated from fly ash were used by us to study their impact on phytoextraction of metals from fly ash by Brassica juncea grown in fly ash amended with farm yard manure (50:50 w/w). Out of 11 bacterial consortia, a combination of two strains i.e. Paenibacillus macerans NBRFT5 + Bacillus pumilus NBRFT9 (C7) inoculated in the rhizosphere was found to enhance Pb accumulation maximally by 278%, Mn by 75%, Zn by 163%, Cr by 226% and Ni by 414% compared to control. It is possible that these bacteria, known for N(2) fixation, solubilization of phosphorus and uptake of micronutrient, could promote the plant growth resulting in higher accumulation of metals. However, a combination of four bacteria, namely Micrococcus roseus NBRFT2 + Bacillus endophyticus NBRFT4 + Paenibacillus macerans NBRFT5 + Bacillus pumilus NBRFT9 (C4) was able to increase Cd uptake maximally by 237%. Further, the translocation of metal was invariably more from root to stem than from stem to leaf which was regulated by plant transport mechanism and metal mobility. Bacteria are known to excrete protons, organic acids, enzymes and siderophores to enhance the mobilization of metals which boosted the phytoextraction of metals from fly ash.

Batch purification of high-purity lysozyme from egg white and characterization of the enzyme modified by PEGylation.

PEGylation is one of the most promising and extensively studied strategies for improving the pharmacological properties of proteins as well as their physical and thermal stability. Purified lysozyme obtained from hen egg white by batch mode was modified by PEGylation with methoxypolyethyleneglycol succinimidyl succinato (mPEG-SS, MW 5000). The conjugates produced retained full enzyme activity with the substrate glycol chitosan, independent of degree of enzyme modification, although lysozyme activity with the substrate Micrococcus lysodeikticus was altered according to the degree of modification. The conjugate with a low degree of modification by mPEG-SS retained 67% of its enzyme activity with the M. lysodeikticus substrate. The mPEG-SS was also shown to be a highly reactive polymer. The effects of pH and temperature on PEGylated lysozymes indicated that the conjugate was active over a wide pH range and was stable up to 50 degrees C. This conjugate also showed resistance to proteolytic degradation, remained stable in human serum, and displayed greater antimicrobial activity than native lysozyme against Gram-negative bacteria.