High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a similar to6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use. (C) 2004 Elsevier Inc. All rights reserved.

A new method for screening alpha-glucosidase inhibitors and application to marine microorganisms

A new enzyme assay method for screening alpha-glucosidase inhibitors with rapidity and simplicity was developed. The enzyme-substituted alpha-glucosidases for this assay was glucoamylase. Samples were spotted or developed on the silica gel plate. The agar solution containing substrate was poured on the plate, and paper impregnated with enzyme was layered on the agar. After incubation, an inhibitory circle would appear around the inhibitor. By using this method, more than 200 strains of marine microorganisms were screened. Among them, three active strains were found to secrete inhibitors in the culture medium.

Molecular cloning and expression analysis of Ch-penaeidin, an antimicrobial peptide from Chinese shrimp, Fenneropenaeus chinensis

A new member of antimicrobial peptide genes of the penaeidin family, Ch-penaeidin, has been cloned from the haemocytes of Chinese shrimp, Fenneropenaeus chinensis, by reverse transcription PCR (RT-PCR), 3'-rapid amplification of cDNA end (3'-RACE) and smart cDNA methods. The Ch-penaeidin cDNA was 655 bp and the open reading frame of the cDNA encoded a 71 amino acid peptide. Ch-penaeidin contained a putative NH2-terminal signal Sequence (1-19) followed by a mature peptide (20-71). The sequence identify with other penaeidins from Litopenaeus vannamei and Litopenaeus setiferus is between 48% and 71%. The signal sequence of Ch-penaeidin is almost completely identical to that of other penaeidins, while differing relatively in the N-terminal domain of the mature peptide. Ch-penaeidin was designated as a novel member of class penaeidin 3 according to phylogenetic analysis. The Mature peptide. with a predicted molecular weight of 5589.32 Da, and a pI of 9.77, has eight positively charged amino acids and no negatively charged amino acids. The expression and distribution of Ch-penaeidin in Unchallenged shrimps were studied by RT-PCR, Northern blot and in situ hybridisation. The results showed that the Ch-penaeidin transcripts were detected in haemocytes (granular haemocytes), heart, gill, intestine, and subcuticular epithelia of the shrimp. and that Ch-penaeidin was constitutively expressed mainly in haemocytes. (C) 2003 Elsevier Ltd. All rights reserved.

Screening of salt tolerant watercress variants on natural seawater contained medium

The responses of stem segments of watercress (Nasturtium officinale R. Br.) to 6-BA,NAA and 2,4-D were studied. MS medium supplemented with 2.0 mg/L 6-BA, 0.2 mg/L 2,4-D was used for callus initiation and maintenance. MS medium supplemented with 4.0 mg/L 6-BA was suitable for plant regeneration and MS medium without plant hormone supplement was used for rooting and plant propagation. For screening of salt tolerant calli, stem segments of watercress were plated onto callus initiation medium containing 1/3 natural seawater. Seventeen out of the 325 plated explants produced calli. The growth curves demonstrated that the growth rate of salt-tolerant calli on saline medium almost matched that of the control calli on normal medium. Some of the salt-tolerant calli were transferred to the normal regeneration medium or saline regeneration medium to induce plant regeneration. In the first case, buds and shoots were regenerated in the same way as those of control calli on normal regeneration medium. More than 1 000 regenerated shoots were obtained of which 83 regenerated shoots were cut and transferred to saline MS base medium. At first, all shoot growth was inhibited, but 40 days after the transfer, rapid-growing axillary shoots were observed on 16 of the original shoots but none on the control shoots on saline MS base medium. Moreover, green spots appeared on most calli 10 days after they were transferred to saline medium, however buds appeared only on 5 calli from the 30 transferred calli and at the end only 2 rapid-growing shoots were obtained from two calli. In total, 18 variant lines were obtained through. propagation of the salt-tolerant shoots on saline MS base medium. RAPD analysis was performed in 10 of the 18 salt-tolerant variant lines and DNA variation was detected in all the tested variant lines.

Isolation, antimicrobial activity, and metabolites of fungus Cladosporium sp associated with red alga Porphyra yezoensis

Cladosporium sp. isolate N5 was isolated as a dominant fungus from the healthy conchocelis of Porphyra yezoensis. In the re-infection test, it did not cause any pathogenic symptoms in the alga. Twenty-one cultural conditions were chosen to test its antimicrobial activity in order to obtain the best condition for large-scale fermentation. Phenylacetic acid, p-hydroxyphenylethyl alcohol, and L-beta-phenyllactic acid were isolated from the crude extract as strong antimicrobial compounds and they are the first reported secondary metabolites for the genus Cladosporium. In addition, the Cladosporium sp. produced the reported Porphyra yezoensis growth regulators phenylacetic acid and p-hydroxyphenylacetic acid. No cytotoxicity was found in the brine shrimp lethality test, which indicated that the environmental-friendly Cladosporium sp. could be used as a potential biocontrol agent to protect the alga from pathogens.

Preliminary study on a potential antibacterial peptide derived from histone H2A in hemocytes of scallop Chlamys farreri

Histone H2A is reported to participate in host defense response through producing novel antimicrobial peptides (AMPs) from its N-terminus in vertebrates and invertebrates, while the AMPs derived from H2A have not to our knowledge been reported in mollusca. In the present study, gene cloning, mRNA expression of H2A from scallop Chlamys farreri, and the recombinant expression of its N-terminus were conducted to investigate whether a similar mechanism exists in mollusca. The full-length DNA of H2A was identified by the techniques of homology cloning and genomic DNA walking, The full-length DNA of the scallop H2A was 696 bp long, including a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 228 bp with a stem-loop structure and a canonical polyadenylation signal sequence AATAAA, and an open reading frame of 375 bp encoding a polypeptide of 125 amino acids. The mRNA expression of H2A in the hemocytes of scallop challenged by microbe was measured by semi-quantitative RT-PCR. The expression of H2A was not upregulated after stimulation, suggesting that H2A did not participate in immunity response directly. The DNA fragment of 117 bp encoding 39 amino acids corresponding to the N-terminus of scallop H2A, which was homologous to buforin I in vertebrates, was cloned into Pichia pastoris GS115. The transformants (His(+) Mut(+)) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418. The peptide of 39 amino acids was expressed by induction of 0.5% methanol. The recombinant product exerted antibacterial activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria. The antibacterial activity toward G(+) bacteria was 2.5 times more than that against G(-) bacteria. The results elucidated that N-terminus of H2A was a potential AMP and provided a promising candidate for a new antibiotic screening. However, whether H2A is really involved in scallop immune response mechanisms needs to be further investigated. (C) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning, expression of a big defensin gene from bay scallop Argopecten irradians and the antimicrobial activity of its recombinant protein

Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. The first mollusk big defensin (designated AiBD) cDNA was cloned from bay scallop Argopecten irradians by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The scallop AiBD consisted of 531 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, encoding a polypeptide of 122 amino acids. The high similarity of AiBD deduced amino acid sequence with big defensin from Tachypleus tridentatus and Branchiostoma belcheri tsingtaunese indicated that AiBD should be a member of big defensin family. The expression of AiBD in various tissues was measured by using Northern blotting analysis. mRNA transcripts of AiBD could be detected in haemocytes of unchallenged scallops. The temporal expression of AiBD in haemolymph after Vibrio anguilarum challenge was recorded by quantitative real time PCR. The relative expression level of AiBD in haemolymph was up-regulated evenly in the first 8 h, followed by a drastic increase, and increased 131.1-fold at 32 h post-injection. These results indicated that AiBD could be induced by bacterial challenge, and it should participate in the immune responses of A. irradians. Biological activity assay revealed that recombinant AiBD could inhibit the growth of both Gram-positive and Gram-negative bacteria, and also showed strong fungicidal activity towards the expression host. Recombinant expression of AiBD made it possible to further characterize its functions involved in immune responses, and also provided a potential therapeutic agent for disease control in aquaculture. (c) 2006 Elsevier Ltd. All rights reserved.

PenBase, the shrimp antimicrobial peptide penaeidin database: Sequence-based classification and recommended nomenclature

Antimicrobial peptides play a major role in innate immunity. The penaeidins, initially characterized from the shrimp Litopenaeus vannamei, are a family of antimicrobial peptides that appear to be expressed in all penaeid shrimps. As of recent, a large number of penaeid nucleotide sequences have been identified from a variety of penaeid shrimp species and these sequences currently reside in several databases under unique identifiers with no nomenclatural continuity. To facilitate research in this field and avoid potential confusion due to a diverse number of nomenclatural designations, we have made a systematic effort to collect, analyse, and classify all the penaeidin sequences available in every database. We have identified a common penaeidin signature and subsequently established a classification based on amino acid sequences. In order to clarify the naming process, we have introduced a 'penaeidin nomenclature' that can be applied to all extant and future penaeidins. A specialized database, PenBase, which is freely available at http://www.penbase.immunaqua.com, has been developed for the penaeidin family of antimicrobial peptides, to provide comprehensive information about their properties, diversity and nomenclature. (c) 2005 Elsevier Ltd. All rights reserved.

The second anti-lipopolysaccharide factor (EsALF-2) with antimicrobial activity from Eriocheir sinensis

The anti-lipopolysaccharide factor CALF) is a small basic protein that can bind and neutralize lipopolysaccharide (LPS), mediating degranulation and activation of an intracellular coagulation cascade. In the present study, cDNA of the second Eriocheir sinensis ALF (designated as EsALF-2) was cloned and the full-length cDNA of EsALF-2 was of 724 bp, consisting of an open reading frame (ORF) of 363 bp encoding a polypeptide of 120 amino acids. The deduced amino acid of EsALF-2 shared 82% similarity with EsALF-1 from E. sinensis and about 53-65% similarity with ALFs from other crustaceans. The potential tertiary structures of EsALF-1 and EsALF-2 contained two highly conserved-cysteine residues to define the LPS binding site, but the N-terminal of EsALF-1 formed a single additional alpha-helix compared to EsALF-2, implying that EsALF-1 and EsALF-2 might represent different biological functions in E. sinensis. The mRNA transcript of EsALF-2 was detected in all examined tissues of healthy crabs, including haemocytes, hepatopancreas, gill, muscle, heart and gonad, which suggested that EsALF-2 could be a multifunctional molecule for the host immune defense responses and thereby provided systemic protection against pathogens. The mRNA expression of EsALF-2 was up-regulated after Listonelln anguillarum and Pichia pastoris challenge and the recombinant protein of EsALF-2 showed antimicrobial activity against L. anguillarum and P. pastoris. indicating that EsALF-2 was involved in the immune defense responses in Chinese mitten crab against L. anguillarum and P. pastoris. These results together indicated that there were abundant and diverse ALFs in E. sinensis with various biological functions and these ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control of crab aquaculture. (C) 2010 Elsevier Ltd. All rights reserved.

Novel antifouling and antimicrobial compound from a marine-derived fungus Ampelomyces sp.

In this study, using a bioassay-guided isolation and purification procedure, we obtained 3-chloro-2,5-dihydroxybenzyl alcohol from a marine-derived Ampelomyces species that effectively inhibited larval settlement of the tubeworm Hydroides elegans and of cyprids of the barnacle Balanus amphitrite. The inhibitive effect on larval settlement was nontoxic and the EC50 of 3-chloro-2,5-dihydroxybenzyl alcohol ranged from 3.19 mu g ml(-1) to 3.81 mu g ml(-1) while the LC50 was 266.68 lambda g ml(-1) for B. amphitrite cyprids; EC50 ranged from 0.67 mu g ml(-1) to 0.78 mu g ml(-1), and LC50 was 2.64 mu g ml(-1) for competent larvae of H. elegans, indicating that inhibitive effect of this compound was nontoxic. At a concentration of 50 mu g per disc, this compound showed strong inhibitive effects on the growth of 13 out of 15 marine bacterial species tested in disc diffusion bioassay. Overall, the high inhibitory activities against bacteria and larval settlement as well as the non- or low-toxic nature of this compound to the barnacle and polychaete larvae suggest this compound could be a potent antifoulant and/or antibiotic.

Extracellular hydrolytic enzyme screening of culturable heterotrophic bacteria from deep-sea sediments of the Southern Okinawa Trough

The Southern Okinawa Trough is an area of focused sedimentation due to particulate matter export from the shelf of the East China Sea and the island of Taiwan. In order to understand the geomicrobiological characteristics of this unique sedimentary environment, bacterial cultivations were carried out for an 8.61 m CASQ core sediment sample. A total of 98 heterotrophic bacterial isolates were characterized based on 16S rRNA gene phylogenetic analysis. These isolates can be grouped into four bacterial divisions, including 13 genera and more than 20 species. Bacteria of the gamma-Proteobacteria lineage, especially those from the Halomonas ( 27 isolates) and Psychrobacter ( 20 isolates) groups, dominate in the culturable bacteria assemblage. They also have the broadest distribution along the depth of the sediment. More than 72.4% of the isolates showed extracellular hydrolytic enzyme activities, such as amylases, proteases, lipases and Dnases, and nearly 59.2% were cold-adapted exoenzyme-producers. Several Halomonas strains show almost all the tested hydrolases activities. The wide distribution of exoenzyme activities in the isolates may indicate their important ecological role of element biogeochemical cycling in the studied deep-sea sedimentary environment.

Marine bacteria associated with marine macroorganisms: the potential antimicrobial resources

In order to explore marine microorganisms with medical potential, marine bacteria were isolated from seawater, sediment, marine invertebrates and seaweeds collected from different coastal areas of the China Sea. The antimicrobial activities of these bacteria were investigated. Ethyl acetate extracts of marine bacterial fermentation were screened for antimicrobial activities using the method of agar diffusion. The results showed that 42 strains of the isolates have antimicrobial activity. The proportion of active bacteria associated with marine invertebrates (20%) and seaweeds (11%) is higher than that isolated from seawater (7%) and sediment (5%). The active marine bacteria were assigned to the genera Alteromonas, Pseudomonas, Bacillus and Flavobacterium. The TLC autobiographic overlay assay implied that the antimicrobial metabolites produced by four strains with wide antimicrobial spectrum were different. Due to a competitive role for space and nutrient, the marine bacteria associated with marine macroorganisms (invertebrates and seaweeds) could produce more antibiotic substances. These marine bacteria were expected to be potential resources of natural antibiotic products.

Screening of surfactants for harmful algal blooms mitigation

Screening experiments were conducted in order to find promising synthetic surfactants for harmful algal blooms (HABs) mitigation. The chemically synthesized surfactant cocamidopropyl betaine (CAPB) showed characteristics of relatively high inhibition efficiency, high biodegradability and low cost. The motility inhibition ratios of 10 mg/L CAPB on Cochlodinium polykrikoides and Alexandrium tamarense were about 60% after 5 min. The biodegradation test indicated that the half-life of CAPB in seawater was shorter than one day and 90% was biodegraded after five days under the initial concentration of 100 mg/L at 25degreesC. Further cell lysis experiments revealed the selective lysis effect of CAPB on different HAB organisms. More than 90% of C. polykrikoides lysed at the concentration of 10 mg/L CAPB after 24 h and at 15 mg/L CAPB after 4 h, whereas the lysis effect of CAPB on A. tamarense was slight, no more than 10% after 2 h interaction with 50 mg/L CAPB. This research provided preliminary data for CAPB as a candidate in harmful algal blooms mitigation and pointed out unresolved problems for its practical application in the meantime. (C) 2003 Elsevier Ltd. All rights reserved.

Toxin-screening and identification of bacteria isolated from highly toxic marine gastropod Nassarius semiplicatus

Bacteria isolated from a highly toxic sample of gastropod Nassarius semiplicatus in Lianyungang, Jiangsu Province in July 2007, were studied to probe into the relationship between bacteria and toxicity of nassariid gastropod. The toxicity of the gastropod sample was 2 x 10(2) mouse unit (MU) Per gram Of tissue (wet weight). High concentration of tetrodotoxin (TTX) and its analogues (TTXs) were found in the digestive gland and muscle of the gastropod, using high performance liquid chromatography coupled with mass chromatography (LC-MS). Bacterial strains isolated from the digestive gland were cultured and screened for TTX with a competitive ELISA method. Tetrodotoxin was detected in a proportion of bacterial strains, but the toxin content was low. Partial 16S ribosomal DNA (rDNA) of the TTX-producing strains was then sequenced and compared with those published in the GenBank to tentatively identify the toxic strains. It was found that most of the toxic strains were closely affiliated with genus Vibrio, and the others were related to genus Shewanella, Marinomonas, Tenacibaculum and Aeromonas. These findings suggest that tetrodotoxin-producing bacteria might play an important role in tetrodotoxin accumulation/production in N. semiplicatus. (C) 2008 Elsevier Ltd. All rights reserved.

Antimicrobial activity of hydroxylbenzenesulfonailides derivatives of chitosan, chitosan sulfates and carboxymethyl chitosan

Chitosan, carboxymethyl chitosan (CIVICS) and chitosan sulfates (CSS) with different molecular weight were modified by reacting with 4-hydroxyl-5-chloride-1,3-benzene-disulfo-chloride or 2-hydroxyl-5-chloride-1,3 -benzene-disulfo-chloride to give 12 kinds of new hydroxylbenzenesulfonailides derivatives of them. The preparation conditions of the derivatives were discussed in this paper, and their structures were characterized by FT-IR and C-13 NMR spectroscopy. The solubility of the derivatives was measured in the experiment. In addition, their antimicrobial activities against four bacteria and five crop-threatening pathogenic fungi were tested in the experiment. Besides, the rule and mechanism of their antibacterial activities were discussed in this paper. (C) 2009 Published by Elsevier B.V.