Cloning and characterization of a novel C-type lectin gene from shrimp Litopenaeus vannamei

In invertebrates, C-type lectins play crucial roles in innate immunity responses by mediating the recognition of host cells to pathogens and clearing microinvaders, which interact with carbohydrates and function as pattern recognition receptors (PRRs). A novel C-type lectin gene (LvLec) cDNA was cloned from hemocytes of Litopenaeus vannamei by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of LvLec was of 618 bp, consisting of a 5'-terminal untranslated region (UTR) of 60 bp and a 3'-UTR of 87 bp with a poly (A) tail. The deduced amino acid sequence of LvLec possessed all conserved features critical for the fundamental structure, such as the four cysteine residues (Cys(53), Cys(128), Cys(144), Cys(152)) involved in the formation of disulfides bridges and the potential Ca2+/carbohydrate-binding sites. The high similarity and the close phylogenetic relationship of LvLec shared with C-type lectins from vertebrates and invertebrates. The structural features of LvLec indicated that it was an invertebrate counterpart of the C-type lectin family. The cDNA fragment encoding the mature peptide of LvLec was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvLec) could agglutinate bacteria E. coli JM109 depending on Ca2+, and the agglutination could be inhibited by mannose and EDTA. These results indicated that LvLec was a new member of C-type lectin family and involved in the immune defence response to Gram negative bacteria in Litopenaeus vannamei. (C) 2008 Elsevier Ltd. All rights reserved.

Cloning and characterization of a novel C-type lectin from Zhikong scallop Chlamys farreri

C-type lectin is a family of Ca2+ dependent carbohydrate-recognition proteins which play crucial roles in the innate immunity of invertebrates by mediating the recognition of host cells to pathogens and clearing microinvaders as a pattern recognition protein (PRP). The cDNA of Zhikong scallop Chlamys farreri C-type lectin (designated CFLec-1) was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of CFLec-1 was 1785 bp, consisting of a 5'-terminal untranslated region (UTR) of 66 bp and an unusually long 3' UTR of 1040 bp with seven polyadenylation signal sequences AATAAA and a poly(A) tail. The CFLec-1 cDNA encoded a polypeptide of 221 amino acids with a putative signal peptide of 15 amino acid residues and a mature protein of 206 amino acids. Analysis of the protein domain features indicated a typical long-form carbohydrate-recognition domain (CRD) of 130 residues in the CFLec-1 deduced amino acid sequence. The expression pattern of CFLec-1 transcripts in healthy and bacterial challenged scallops was studied by semi-quantitative RT-PCR. mRNA transcripts of CFLec-1 could be mainly detected in the tissues of haemocytes, gill, gonad and mantle of unchallenged scallops, whereas the expression of CFLec-1 transcripts was increased in all the tested tissues after heat-killed Vibrio anguillarum challenge. The temporal expression of CFLec-1 mRNA in haemolymph challenged by Micrococcus luteus and V anguillarum was both up-regulated and reached the maximum level at 8 and 16 It post stimulation, respectively, and then dropped back to the original level. In order to investigate its immune functions, CFLec- I was recombined and expressed in Escherichia coli BL21(DE3)-pLysS as a fusion protein with thioredoxin. The recombinant CFLec-1 agglutinated bacteria E. coli JM109 in vitro, and the agglutination was Ca2+ dependent which could be inhibited by EDTA. But it did not agglutinate M. luteus, Candida lipolytica and animal erythrocytes including rabbit, rat, mouse, chicken, human group A, human group B, human group O. Meanwhile, the recombinant CFLec-1 could inhibit the growth of both E. coli JM 109 and M. luteus, but no inhibition activity against V anguillarum. These result indicated that CFLec-1 was a constitutive and inducible PRP which was involved in the reorganization and clearance of invaders in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

Monitoring microbial populations of sulfate-reducing bacteria using an impedimetric immunosensor based on agglutination assay

An impedimetric immunosensor was fabricated for rapid and non-labeled detection of sulfate-reducing bacteria, Desulforibrio caledoiensis (SRB) by immobilizing lectin-Concanavalin A using an agglutination assay. The immobilization of lectin was conducted using amine coupling on the surface of a gold (Au) electrode assembled with 11-Mercaptounclecanoic acid. Electrochemical impedance spectroscopy (EIS) was used to verify the stepwise assembly of the sensor system. The work conditions of the impedimetric immunosensor, such as pH of the buffer solutions and the incubation time of lectin, were optimized. Faradic impedance spectra for charge transfer for the redox probe Fe(CN)(6)(3-/4-) were measured to determine SRB concentrations. The diameter of the Nyquist diagram that is equal to the charge-transfer resistance (RI) increased with increasing SRB concentration. A linear relationship between R-ct and SRB concentration was obtained in SRB concentration range of 1.8 to 1.8 x 10(7) cfu/ml. The variation of the SRB population during the growth process was also monitored using the impedimetric immunosensor. This approach has great potential for simple, low-cost. and time-saving monitoring of microbial populations. (C) 2009 Elsevier B.V. All rights reserved.

CfLGBP, a pattern recognition receptor in Chlamys farreri involved in the immune response against various bacteria

Lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) is a kind of pattern recognition receptor, which can recognize and bind LPS and beta-1, 3-glucan, and plays curial roles in the innate immune defense against Gram-negative bacteria and fungi. In this study, the functions of LGBP from Zhikong scallop Chlamys farreri performed in innate immunity were analyzed. Firstly, the mRNA expression of CfLGBP in hemocytes toward three typical PAMPS stimulation was examined by realtime PCR. It was up-regulated extremely (P < 0.01) post stimulation of LPS and beta-glucan, and also exhibited a moderate up-regulation (P < 0.01) after PGN injection. Further PAMPs binding assay with the polyclonal antibody specific for CfLGBP proved that the recombinant CfLGBP (designated as rCfLGBP) could bind not only LPS and beta-glucan, but also PGN in vitro. More importantly, rCfLGBP exhibited obvious agglutination activity towards Gram-negative bacteria Escherichia coil, Gram-positive bacteria Bacillus subtilis and fungi Pichia pastoris. Taking the results of immunofluorescence assay into account, which displayed CfLGBP was expressed specifically in the immune cells (hemocytes) and vulnerable organ (gill and mantle), we believed that LGBP in C farreri, serving as a multi-functional PRR, not only involved in the immune response against Gram-negative and fungi as LGBP in other invertebrates, but also played significant role in the event of anti-Gram-positive bacteria infection. As the first functional research of LGBP in mollusks, our study provided new implication into the innate immune defense mechanisms of C. farreri and mollusks. (C) 2010 Elsevier Ltd. All rights reserved.

Cough-generated aerosols of Pseudomonas aeruginosa and other Gram Negative Bacteria from cystic fibrosis patients.

Background: Pseudomonas aeruginosa is the most common bacterial pathogen in cystic fibrosis (CF) patients. Current infection control guidelines aim to prevent transmission via contact and respiratory droplet routes and do not consider the possibility of airborne transmission. We hypothesized that with coughing, CF subjects produce viable, respirable bacterial aerosols. Methods: Cross-sectional study of 15 children and 13 adults with CF, 26 chronically infected with P. aeruginosa. A cough aerosol sampling system enabled fractioning of respiratory particles of different size, and culture of viable Gram negative non-fermentative bacteria. We collected cough aerosols during 5 minutes voluntary coughing and during a sputum induction procedure when tolerated. Standardized quantitative culture and genotyping techniques were used. Results: P. aeruginosa was isolated in cough aerosols of 25 (89%) subjects of whom 22 produced sputum samples. P. aeruginosa from sputum and paired cough aerosols were indistinguishable by molecular typing. In 4 cases the same genotype was isolated from ambient room air. Approximately 70% of viable aerosols collected during voluntary coughing were of particles ≤ 3.3 microns aerodynamic diameter. P. aeruginosa, Burkholderia cenocepacia Stenotrophomonas maltophilia and Achromobacter xylosoxidans were cultivated from respiratory particles in this size range. Positive room air samples were associated with high total counts in cough aerosols (P=0.003). The magnitude of cough aerosols were associated with higher FEV1 (r=0.45, P=0.02) and higher quantitative sputum culture results (r=0.58, P=0.008). Conclusion: During coughing, CF patients produce viable aerosols of P. aeruginosa and other Gram negative bacteria of respirable size range, suggesting the potential for airborne transmission.

Implications of faecal indicator bacteria for the microbiological assessment of roof harvested rainwater quality in Southeast Queensland, Australia

The study aimed to evaluate the suitability of Escherichia coli, enterococci and C. perfringens to assess the microbiological quality of roof harvested rainwater, and to assess whether the concentrations of these faecal indicators can be used to predict the presence or absence of specific zoonotic bacterial or protozoan pathogens. From a total of 100 samples tested, respectively 58%, 83% and 46% of samples were found to be positive for E. coli, enterococci and C. perfringens spores, as determined by traditional culture based methods. Additionally, in the samples tested, 7%, 19%, 1%, 8%, 17%, and 15% were PCR positive for A. hydrophila lip, C. coli ceuE, C. jejuni mapA, L. pneumophila mip, Salmonella invA, and G. lamblia β-giardin genes. However, none of the samples was positive for E. coli O157 LPS, VT1, VT2 and C. parvum COWP genes. The presence or absence of these potential pathogens did not correlate with any of the faecal indicator bacterial concentrations as determined by a binary logistic regression model. The roof-harvested rainwater samples tested in this study appear to be of poor microbiological quality and no significant correlation was found between the concentration of faecal indicators and pathogenic microorganisms. The use of faecal indicator bacteria raises questions regarding their reliability in assessing the microbiological quality of water and particularly their poor correlation with pathogenic microorganisms. The presence of one or more zoonotic pathogens suggests that the microbiological analysis of water should be performed, and appropriate treatment measures should be undertaken especially in tanks where the water is used for drinking.

A functional screen identifies lateral transfer of beta-glucuronidase (gus) from bacteria to fungi

Lateral gene transfer (LGT) from prokaryotes to microbial eukaryotes is usually detected by chance through genome-sequencing projects. Here, we explore a different, hypothesis-driven approach. We show that the fitness advantage associated with the transferred gene, typically invoked only in retrospect, can be used to design a functional screen capable of identifying postulated LGT cases. We hypothesized that beta-glucuronidase (gus) genes may be prone to LGT from bacteria to fungi (thought to lack gus) because this would enable fungi to utilize glucuronides in vertebrate urine as a carbon source. Using an enrichment procedure based on a glucose-releasing glucuronide analog (cellobiouronic acid), we isolated two gus(+) ascomycete fungi from soils (Penicillium canescens and Scopulariopsis sp.). A phylogenetic analysis suggested that their gus genes, as well as the gus genes identified in genomic sequences of the ascomycetes Aspergillus nidulans and Gibberella zeae, had been introgressed laterally from high-GC gram(+) bacteria. Two such bacteria (Arthrobacter spp.), isolated together with the gus(+) fungi, appeared to be the descendants of a bacterial donor organism from which gus had been transferred to fungi. This scenario was independently supported by similar substrate affinities of the encoded beta-glucuronidases, the absence of introns from fungal gus genes, and the similarity between the signal peptide-encoding 5' extensions of some fungal gus genes and the Arthrobacter sequences upstream of gus. Differences in the sequences of the fungal 5' extensions suggested at least two separate introgression events after the divergence of the two main Euascomycete classes. We suggest that deposition of glucuronides on soils as a result of the colonization of land by vertebrates may have favored LGT of gus from bacteria to fungi in soils.

Gene transfer to plants by diverse species of bacteria

Agrobacterium is widely considered to be the only bacterial genus capable of transferring genes to plants. When suitably modified, Agrobacterium has become the most effective vector for gene transfer in plant biotechnology1. However, the complexity of the patent landscape2 has created both real and perceived obstacles to the effective use of this technology for agricultural improvements by many public and private organizations worldwide. Here we show that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer to a number of diverse plants. These plant-associated symbiotic bacteria were made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a suitable binary vector. This alternative to Agrobacterium-mediated technology for crop improvement, in addition to affording a versatile ‘open source’ platform for plant biotechnology, may lead to new uses of natural bacteria– plant interactions to achieve plant transformation.

Rationalizing the SER spectra of bacteria

The SER spectra of riboflavin and FAD are identical and are resonance enhanced at 514 or 532 nm. Signals from FAD/ riboflavin dominated SER spectra whenever these compounds were present with proteins or bacteria. SER spectra of very different bacteria such as Pseudomonas. aeruginosa, Bacillu. subtilis and Geobacillus. stearothermophilus were dominated by signals from FAD, even when these bacteria were added to a preformed colloid. The SERS signal of FAD is greatly reduced at 785 nm, and SER spectra of bacteria excited at 785 nm are quite different than those collected at 514 or 532 nm. This supports the assignment of the peaks in the 514 nm SER spectra of bacteria to FAD rather to amino acids or N-acetylglucosamine. The SER spectra of certain mixes of adenine and FAD showed similar changes to those of bacteria when the excitation was changed from 514/532 nm to 785 nm. The ratio of colloid: bacteria was of critical important for obtaining good SER spectra, and the addition of sodium sulfate was also beneficial. Removal of EPS from bacteria before analysis facilitated interaction with the silver surface, and may be a useful step to include in identification protocols.

Vacuum cleaner emissions as a source of indoor exposure to airborne particles and bacteria

Vacuuming can be a source of indoor exposure to biological and non-biological aerosols, although there is little data that describes the magnitude of emissions from the vacuum cleaner itself. We therefore sought to quantify emission rates of particles and bacteria from a large group of vacuum cleaners and investigate their potential determinants, including temperature, dust bags, exhaust filters, price and age. Emissions of particles between 0.009 and 20 µm and bacteria were measured from 21 vacuums. Ultrafine (<100 nm) particle emission rates ranged from 4.0 × 10^6 to 1.1 × 10^11 particles min-1. Emission of 0.54 to 20 µm particles ranged from 4.0 × 10^4 to 1.2 × 10^9 particles min-1. PM2.5 emissions were between 2.4 × 10-1 and 5.4 × 10^3 µg min-1. Bacteria emissions ranged from 0 to 7.4 × 10^5 bacteria min-1 and were poorly correlated with dust bag bacteria content and particle emissions. Large variability in emission of all parameters was observed across the 21 vacuums we assessed, which was largely not attributable to the range of determinant factors we assessed. Vacuum cleaner emissions contribute to indoor exposure to non-biological and biological aerosols when vacuuming, and this may vary markedly depending on the vacuum used.

Neutrophilic, microaerophilic Fe(II)-oxidizing bacteria are ubiquitous in aquatic habitats of a subtropical Australian coastal catchment (ubiquitous FeOB in catchment aquatic habitats)

We examined the abundance and distribution of neutrophilic, microaerophilic Fe(II)-oxidizing bacteria (FeOB) in aquatic habitats of a highly weathered, subtropical coastal catchment where Fe biogeochemistry is of environmental significance. Laboratory cultivation and microscopy indicated that stalked Gallionella and sheathed Leptothrix-like FeOB were present in microbial mats associated with a circumneutral-pH, groundwater seep and streambank surface sediment,whereas unicellular FeOB werewidespread in surface and subsurface waters, including a seep, shallow stream and estuary-adjacent groundwater. Direct Gallionella-specificPCR detected dominant bacterial members related to Sideroxydans paludicola (95% sequence identity, SI) and Gallionella capsiferriformans (96% SI) in the seep microbialmat. TGGE analysis indicated that themost common FeOB in water enrichment cultures were related to S. lithotrophicus (96% SI). The ubiquity of FeOB in Poona catchment aquatic habitats suggests bacterial Fe(II) oxidation is integral to catchment Fe biogeochemistry.

Coordinated tuning of DFIG-based wind turbines and batteries using bacteria foraging technique for maintaining constant grid power output

This paper proposes the use of battery energy storage (BES) system for the grid-connected doubly fed induction generator (DFIG). The BES would help in storing/releasing additional power in case of higher/lower wind speed to maintain constant grid power. The DC link capacitor is replaced with the BES system in a DFIG-based wind turbine to achieve the above-mentioned goal. The control scheme is modified and the co-ordinated tuning of the associated controllers to enhance the damping of the oscillatory modes is presented using bacterial foraging technique. The results from eigenvalue analysis and the time domain simulation studies are presented to elucidate the effectiveness of the BES systems in maintaining the grid stability under normal operation.