Characterization of the Dual Functions of LvCrustinVII from Litopenaeus vannamei as Antimicrobial Peptide and Opsonin

Crustin are a family of antimicrobial peptides that play an important role in protecting against pathogens infection in the innate immune system of crustaceans. Previously, we identified several novel types of crustins, including type VI and type VII crustins. However, their immune functions were still unclear. In the present study, the immune function of type VII crustin LvCrustinVII were investigated in Litopenaeus vannamei. LvCrustinVII was wildly expressed in all tested tissues, with relatively high expression levels in hepatopancreas, epidermis and lymphoid organ. Upon Vibrio parahaemolyticus infection, LvCrustinVII was significantly upregulated in hepatopancreas. Recombinant LvCrustinVII (rLvCrustinVII) showed strong inhibitory activities against Gram-negative bacteria Vibrio harveyi and V. parahaemolyticus, while weak activities against the Gram-positive bacteria Staphylococcus aureus. Binding assay showed that rLvCrustinVII could bind strongly to V. harveyi and V. parahaemolyticus, as well as the cell wall components Glu, LPS and PGN. In the presence of Ca²⁺, rLvCrustinVII could agglutinate V. parahaemolyticus and enhance hemocyte phagocytosis. The present data partially illustrate the immune function of LvCrustinVII, which enrich our understanding on the functional mechanisms of crustins and provide useful information for application of this kind of antimicrobial peptides.     

Actinomycetes from Red Sea Sponges: Sources for Chemical and Phylogenetic Diversity

The diversity of actinomycetes associated with marine sponges collected off Fsar Reef (Saudi Arabia) was investigated in the present study. Forty-seven actinomycetes were cultivated and phylogenetically identified based on 16S rRNA gene sequencing and were assigned to 10 different actinomycete genera. Eight putatively novel species belonging to genera Kocuria, Mycobacterium, Nocardia, and Rhodococcus were identified based on sequence similarity values below 98.2% to other 16S rRNA gene sequences available in the NCBI database. PCR-based screening for biosynthetic genes including type I and type II polyketide synthases (PKS-I, PKS-II) as well as nonribosomal peptide synthetases (NRPS) showed that 20 actinomycete isolates encoded each at least one type of biosynthetic gene. The organic extracts of nine isolates displayed bioactivity against at least one of the test pathogens, which were Gram-positive and Gram-negative bacteria, fungi, human parasites, as well as in a West Nile Virus protease enzymatic assay. These results emphasize that marine sponges are a prolific resource for novel bioactive actinomycetes with potential for drug discovery.     

Matrix Production,Pigment Synthesis,and Sporulation in a Marine Isolated Strain of Bacillus pumilus

The ability to produce an extracellular matrix and form multicellular communities is an adaptive behavior shared by many bacteria. In Bacillus subtilis, the model system for spore-forming bacteria, matrix production is one of the possible differentiation pathways that a cell can follow when vegetative growth is no longer feasible. While in B. subtilis the genetic system controlling matrix production has been studied in detail, it is still unclear whether other spore formers utilize similar mechanisms. We report that SF214, a pigmented strain of Bacillus pumilus isolated from the marine environment, can produce an extracellular matrix relying on orthologs of many of the genes known to be important for matrix synthesis in B. subtilis. We also report a characterization of the carbohydrates forming the extracellular matrix of strain SF214. The isolation and characterization of mutants altered in matrix synthesis, pigmentation, and spore formation suggest that in strain SF214 the three processes are strictly interconnected and regulated by a common molecular mechanism.     

Use of fermented milling by-products as functional ingredient to develop a low-glycaemic index bread

Selected Lactobacillus plantarum DSM 32248 and Lactobacillus rossiae DSM 32249, isolated and identified from wheat germ, were used to ferment a milling by-products mixture. Lactic acid bacteria metabolisms improved the functional properties of wheat bran and germ, which are considered important sources of functional compounds. Wheat breads were manufactured using 15% (w/w) of fermented (and unfermented) milling by-products, and compared to baker’s yeast wheat bread manufactured without the addition of milling by-products. The use of the fermented ingredient improved the biochemical, functional, nutritional, textural, and sensory features of wheat bread, showing better performances compared to the solely use of wheat flour. Protein digestibility, nutritional indexes, and the rate of starch hydrolysis markedly improved using fermented milling by-products as ingredient. Enriched bread was also characterized by high content of dietary fibre and low glycaemic index determined in vivo.This study exploited the potential of fermented milling by products as functional ingredient. According to the Regulations the bread made under this study conditions can be defined as “high fibre content” and “low glycaemic index”. A number of advantages encouraged the manufacture of novel and healthy and functional leavened baked goods.     

Metabolomic Tools for Secondary Metabolite Discovery from Marine Microbial Symbionts

Marine invertebrate-associated symbiotic bacteria produce a plethora of novel secondary metabolites which may be structurally unique with interesting pharmacological properties. Selection of strains usually relies on literature searching, genetic screening and bioactivity results, often without considering the chemical novelty and abundance of secondary metabolites being produced by the microorganism until the time-consuming bioassay-guided isolation stages. To fast track the selection process, metabolomic tools were used to aid strain selection by investigating differences in the chemical profiles of 77 bacterial extracts isolated from cold water marine invertebrates from Orkney, Scotland using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Following mass spectrometric analysis and dereplication using an Excel macro developed in-house, principal component analysis (PCA) was employed to differentiate the bacterial strains based on their chemical profiles. NMR ¹H and correlation spectroscopy (COSY) were also employed to obtain a chemical fingerprint of each bacterial strain and to confirm the presence of functional groups and spin systems. These results were then combined with taxonomic identification and bioassay screening data to identify three bacterial strains, namely Bacillus sp. 4117, Rhodococcus sp. ZS402 and Vibrio splendidus strain LGP32, to prioritize for scale-up based on their chemically interesting secondary metabolomes, established through dereplication and interesting bioactivities, determined from bioassay screening.     

Assessment of the Bacteriocinogenic Potential of Marine Bacteria Reveals Lichenicidin Production by Seaweed-Derived Bacillus spp.

The objectives of this study were (1) to assess the bacteriocinogenic potential of bacteria derived mainly from seaweed, but also sand and seawater, (2) to identify at least some of the bacteriocins produced, if any and (3) to determine if they are unique to the marine environment and/or novel. Fifteen Bacillus licheniformis or pumilus isolates with antimicrobial activity against at least one of the indicator bacteria used were recovered. Some, at least, of the antimicrobials produced were bacteriocins, as they were proteinaceous and the producers displayed immunity. Screening with PCR primers for known Bacillus bacteriocins revealed that three seaweed-derived Bacillus licheniformis harbored the bli04127 gene which encodes one of the peptides of the two-peptide lantibiotic lichenicidin. Production of both lichenicidin peptides was then confirmed by mass spectrometry. This is the first definitive proof of bacteriocin production by seaweed-derived bacteria. The authors acknowledge that the bacteriocin produced has previously been discovered and is not unique to the marine environment. However, the other marine isolates likely produce novel bacteriocins, as none harboured genes for known Bacillus bacteriocins.     

Tasco®: a product of Ascophyllum nodosum enhances immune response of Caenorhabditis elegans against Pseudomonas aeruginosa infection

The effects of Tasco^®, a product made from the brown seaweed (Ascophyllum nodosum) were tested for the ability to protect Caenorhabditis elegans against Pseudomonas aeruginosa infection. A water extract of Tasco^® (TWE) reduced P. aeruginosa inflicted mortality in the nematode. The TWE, at a concentration of 300 µg/mL, offered the maximum protection and induced the expression of innate immune response genes viz.; zk6.7 (Lypases), lys-1 (Lysozyme), spp-1 (Saponin like protein), f28d1.3 (Thaumatin like protein), t20g5.7 (Matridin SK domain protein), abf-1 (Antibacterial protein) and f38a1.5 (Lectin family protein). Further, TWE treatment also affected a number of virulence components of the P. aeuroginosa and reduced its secreted virulence factors such as lipase, proteases and toxic metabolites; hydrogen cyanide and pyocyanin. Decreased virulence factors were associated with a significant reduction in expression of regulatory genes involved in quorum sensing, lasI, lasR, rhlI and rhlR. In conclusion, the TWE-treatment protected the C. elegans against P. aeruginosa infection by a combination of effects on the innate immunity of the worms and direct effects on the bacterial quorum sensing and virulence factors.     

Multiplication ofPseudomonas solanacearum in resistant potato plants and the establishment of latent infections

When 1-mo-old plants of a wilt-resistant clone ofSolanum phureja (1386.15) were stem-inoculated with three strains ofPseudomonas solanacearum (K60, S123, and S206), the bacteria multiplied rapidly at the point of inoculation and then moved in the vascular system to other parts of the stem. Resistant plants showed a remarkable ability to support relatively high populations of the bacterium in the absence of disease symptoms. Although multiplication in this resistant clone was substantially less than in susceptible Russet Burbank potato plants, large numbers of bacteria (up to 624 × 10⁴ cells of K60 per 5-cm stem segment) reached the base of the stem of plants maintained at high temperature (28°C) for 20 days after stem inoculation. From the base of the stem, the bacteria moved rapidly into the roots and tubers. Strains ofP. solanacearum differed in their ability to cause latent tuber infection in different resistant potato clones. When 11S.phureja ×S. tuberosum hybrids were stem-inoculated, maintained at 28°C for 3 wk and then grown to maturity at 20°C., most of the clones yielded tubers infected by one or more strains. The race 1 strain (K60) was the most infectious; 53.8% of all tubers harvested from all plants inoculated with this isolate carried latent infections. Because one clone (BR 53.1) never yielded infected tubers, there appear to be genetic factors which may be useful in breeding programs aimed at eliminating latent tuber infection.     

Bioprospecting Red Sea Coastal Ecosystems for Culturable Microorganisms and Their Antimicrobial Potential

Microorganisms that inhabit unchartered unique soil such as in the highly saline and hot Red Sea lagoons on the Saudi Arabian coastline, represent untapped sources of potentially new bioactive compounds. In this study, a culture-dependent approach was applied to three types of sediments: mangrove mud (MN), microbial mat (MM), and barren soil (BS), collected from Rabigh harbor lagoon (RHL) and Al-Kharrar lagoon (AKL). The isolated bacteria were evaluated for their potential to produce bioactive compounds. The phylogenetic characterization of 251 bacterial isolates based on the 16S rRNA gene sequencing, supported their assignment to five different phyla: Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Planctomycetes. Fifteen putative novel species were identified based on a 16S rRNA gene sequence similarity to other strain sequences in the NCBI database, being ≤98%. We demonstrate that 49 of the 251 isolates exhibit the potential to produce antimicrobial compounds. Additionally, at least one type of biosynthetic gene sequence, responsible for the synthesis of secondary metabolites, was recovered from 25 of the 49 isolates. Moreover, 10 of the isolates had a growth inhibition effect towards Staphylococcus aureus, Salmonella typhimurium and Pseudomonas syringae. We report the previously unknown antimicrobial activity of B. borstelensis, P. dendritiformis and M. salipaludis against all three indicator pathogens. Our study demonstrates the evidence of diverse cultured microbes associated with the Red Sea harbor/lagoon environments and their potential to produce antimicrobial compounds.     

Diversity and Biosynthetic Potential of Culturable Microbes Associated with Toxic Marine Animals

Tetrodotoxin (TTX) is a neurotoxin that has been reported from taxonomically diverse organisms across 14 different phyla. The biogenic origin of tetrodotoxin is still disputed, however, TTX biosynthesis by host-associated bacteria has been reported. An investigation into the culturable microbial populations from the TTX-associated blue-ringed octopus Hapalochlaena sp. and sea slug Pleurobranchaea maculata revealed a surprisingly high microbial diversity. Although TTX was not detected among the cultured isolates, PCR screening identifiedsome natural product biosynthesis genes putatively involved in its assembly. This study is the first to report on the microbial diversity of culturable communities from H. maculosa and P. maculata and common natural product biosynthesis genes from their microbiota. We also reassess the production of TTX reported from three bacterial strains isolated from the TTX-containing gastropod Nassarius semiplicatus.     

Infection and decontamination of citrus canker-inoculated leaf surfaces

Citrus canker (Xanthomonas citri subsp. citri, Xcc) is now considered endemic in Florida and continues to spread. Various surfaces, including plant material, personnel and equipment can become contaminated. Decontamination is practiced in both disease-endemic and disease-free areas to reduce the risk of bacterial spread by man or machinery. We used grapefruit leaf surfaces to explore the efficacy of a commonly used personnel sprayer system applying a quaternary amine decontaminant. In three experiments, plants in flush (leaves ¾ expanded) were inoculated (∼10⁵ CFU ml⁻¹). Immediately after inoculation, plants were passed through the sprayers 0, 1, 2, 3, or 6 times. Leaves were sampled at 0.5, 10 and 20 min after decontamination and tested for viable Xcc by dilution plating. There was a large and rapid decline in the quantity of live bacteria with one pass through the decontamination sprayer (>80% decrease in CFU ml⁻¹), and multiple sprays (2-6) resulted in up to 100% mortality of surface Xcc. Presumably more thorough coverage with multiple sprays killed remnant bacteria, although the first spray invariably caused the highest proportion of population mortality. The effect of the decontaminant spray was immediate (within 0.5 min only 3-11% of surface bacteria survived, and by 20 min <1-3% survived). Based on these results, use of a personnel sprayer with a quaternary amine compound is highly effective for reducing surface inoculum. A single spray kills a high proportion of the population, but multiple sprays increase mortality of Xcc. All the Xcc-inoculated plants subsequently developed symptoms of citrus canker. No significant difference in incidence or severity of grapefruit leaf infection was detected among decontamination treatments or compared to the untreated control. This finding indicates that infection occurred at, or very soon after, inoculation, and that Xcc was in protected sites inside the leaf before exposure to the decontaminant spray.     

Use of Quorum Sensing Inhibition Strategies to Control Microfouling

Interfering with the quorum sensing bacterial communication systems has been proposed as a promising strategy to control bacterial biofilm formation, a key process in biofouling development. Appropriate in vitro biofilm-forming bacteria models are needed to establish screening methods for innovative anti-biofilm and anti-microfouling compounds. Four marine strains, two Pseudoalteromonas spp. and two Vibrio spp., were selected and studied with regard to their biofilm-forming capacity and sensitivity to quorum sensing (QS) inhibitors. Biofilm experiments were performed using two biofilm cultivation and quantification methods: the xCELLigence^® system, which allows online monitoring of biofilm formation, and the active attachment model, which allows refreshment of the culture medium to obtain a strong biofilm that can be quantified with standard staining methods. Although all selected strains produced acyl-homoserine-lactone (AHL) QS signals, only the P. flavipulchra biofilm, measured with both quantification systems, was significantly reduced with the addition of the AHL-lactonase Aii20J without a significant effect on planktonic growth. Two-species biofilms containing P. flavipulchra were also affected by the addition of Aii20J, indicating an influence on the target bacterial strain as well as an indirect effect on the co-cultured bacterium. The use of xCELLigence^® is proposed as a time-saving method to quantify biofilm formation and search for eco-friendly anti-microfouling compounds based on quorum sensing inhibition (QSI) strategies. The results obtained from these two in vitro biofilm formation methods revealed important differences in the response of biosensor bacteria to culture medium and conditions, indicating that several strains should be used simultaneously for screening purposes and the cultivation conditions should be carefully optimized for each specific purpose.     

Adenovirus Carrying Gene Encoding Haliotis discus discus Sialic Acid Binding Lectin Induces Cancer Cell Apoptosis

Lectins exist widely in marine bioresources such as bacteria, algae, invertebrate animals and fishes. Some purified marine lectins have been found to elicit cytotoxicity to cancer cells. However, there are few reports describing the cytotoxic effect of marine lectins on cancer cells through virus-mediated gene delivery. We show here that a replication-deficient adenovirus-carrying gene encoding Haliotis discus discus sialic acid binding lectin (Ad.FLAG-HddSBL) suppressed cancer cell proliferation by inducing apoptosis, as compared to the control virus Ad.FLAG. A down-regulated level of anti-apoptosis factor Bcl-2 was suggested to be responsible for the apoptosis induced by Ad.FLAG-HddSBL infection. Further subcellular localization studies revealed that HddSBL distributed in cell membrane, ER, and the nucleus, but not in mitochondria and Golgi apparatus. In contrast, a previously reported mannose-binding lectin Pinellia pedatisecta agglutinin entered the nucleus as well, but did not distribute in inner membrane systems, suggesting differed intracellular sialylation and mannosylation, which may provide different targets for lectin binding. Further cancer-specific controlling of HddSBL expression and animal studies may help to provide insights into a novel way of anti-cancer marine lectin gene therapy. Lectins may provide a reservoir of anti-cancer genes.     

Rapid screening of genetically modified ingredients in soybean and cotton processing by-product and waste using direct qPCR

To develop a simple and fast method for screening genetically modified ingredients from processing by-product and waste, direct quantitative PCR (qPCR) kit-Taqman which omitting multi genomic DNA preparing steps was developed in this study. A total of 18 oil crop processing by-products and wastes including 10 soybean and 8 cotton materials were collected from food processing factories. Compared with 2 commercial direct qPCR kits, conditions of DNA releasing procedure and PCR amplification were optimized. Element screening was performed at the initial step of genetically modified (GM) ingredient testing procedure via direct qPCR. GM event identification was carried out in positive samples by initial screening. Totally 5 screening elements (P–35S, T-NOS, Cp4-epsps, bar and pat) for soybean materials and 6 screening elements (P–35S, T-NOS, NPTII, Cry1Ac, bar and pat) for cotton samples were detected. In GM event identification, MON531 and MON1445 were found in cotton materials. Results were further confirmed by real-time PCR with DNA extraction and purification. The direct qPCR system proposed by this research was convenient for rapid screening and identification of GM ingredients in oil crop primary by-product and waste.     

Harnessing the Potential of Halogenated Natural Product Biosynthesis by Mangrove-Derived Actinomycetes

Mangrove-derived actinomycetes are promising sources of bioactive natural products. In this study, using homologous screening of the biosynthetic genes and anti-microorganism/tumor assaying, 163 strains of actinomycetes isolated from mangrove sediments were investigated for their potential to produce halogenated metabolites. The FADH₂-dependent halogenase genes, identified in PCR-screening, were clustered in distinct clades in the phylogenetic analysis. The coexistence of either polyketide synthase (PKS) or nonribosomal peptide synthetase (NRPS) as the backbone synthetases in the strains harboring the halogenase indicated that these strains had the potential to produce structurally diversified antibiotics. As a validation, a new enduracidin producer, Streptomyces atrovirens MGR140, was identified and confirmed by gene disruption and HPLC analysis. Moreover, a putative ansamycin biosynthesis gene cluster was detected in Streptomyces albogriseolus MGR072. Our results highlight that combined genome mining is an efficient technique to tap promising sources of halogenated natural products synthesized by mangrove-derived actinomycetes.     

Context-Dependent Viral Transgenerational Immune Priming in Honey Bees (Hymenoptera: Apidae)

Transgenerational immune priming is the process of increased resistance to infection in offspring due to parental pathogen exposure. Honey bees (Apis mellifera L. (Hymenoptera: Apidae)) are hosts to multiple pathogens, and this complex immune function could help protect against overwhelming infection. Honey bees have demonstrated transgenerational immune priming for the bacterial pathogen Paenibacillus larvae; however, evidence for viral transgenerational immune priming is lacking across insects in general. Here we test for the presence of transgenerational immune priming in honey bees with Deformed wing virus (DWV) by injecting pupae from DWV-exposed queens and measuring virus titer and immune gene expression. Our data suggest that there is evidence for viral transgenerational immune priming in honey bees, but it is highly context-dependent based on route of maternal exposure and potentially host genetics or epigenetic factors.