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Microbial methane oxidation is the primary control on the emission of the greenhouse gas methane into the atmosphere. In terrestrial environments, aerobic methanotrophic bacteria are largely responsible for this process. In marine sediments, a coupling of anaerobic oxidation of methane (AOM) with sulfate reduction, often carried out by a consortium of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria, consumes almost all methane produced within those sediments. Motivated by recent evidence for AOM with iron(III) in Lake Kinneret sediments, the goal of the present study was to link the geochemical gradients in the lake porewater to the microbial community structure. Screening of archaeal 16S rRNA gene sequences revealed a shift from hydrogenotrophic to acetoclastic methanogens with depth. The observed changes in microbial community structure suggest possible direct and indirect mechanisms for the AOM coupled to iron reduction in deep sediments. The percentage of members of the Nitrospirales order increased with depth, suggesting their involvement in iron reduction together with Geobacter genus and "reverse methanogenesis". An indirect mechanism through sulfate and ANME seems less probable due to the absence of ANME sequences. This is despite the abundant sequences related to sulfate-reducing bacteria (Deltaproteobacteria) together with the occurrence of dsrA in the deep sediment that could indicate the production of sulfate (disproportionation) from S0 for sulfate-driven AOM. The presence of the functional gene pmoA in the deep anoxic sediment together with sequences related to Methylococcales suggests the existence of a second unexpected indirect pathway -- aerobic methane oxidation pathway in an anaerobic environment. [ABSTRACT FROM AUTHOR]

Abstract: Susceptibility to Bacillus thuringiensis of mosquito and lepidopteran larvae is affected by feeding behaviour and nutritional value of the available food. Reduced mortality is attributed to feeding inhibition and dilution of the pathogen in the presence of nutritional and inert particles, which limit the amount of ingested toxin. These reasons are, however, not sufficient to explain the data presented here. Values of LC[sub 50] (the concentration that kills 50% of exposed population) of B. thuringiensis subsp. israelensis (Berliner) against Aedes aegypti (L.) larvae and of B. thuringiensis subsp. kenyae (Berliner) against Spodoptera littoralis (Boisduval) larvae were about 20–217 and 2.3–44-fold higher, respectively, in the presence of nutritional or biologically inert (non-nutritional) particles than without. The number of B. thuringiensis spores in carcasses of B. thuringiensis -killed A. aegypti and S. littoralis larvae were between 1.9 and 5.6-fold and between 8.5 and 12-fold higher, respectively, in the presence of particles than without. In all cases, non-nutritional particles better protected the exposed larvae than nutritious particles. We propose that another basic mechanism exists, that ingested particles protect midgut epithelial cells by covering their surface and thus preventing availability of the toxin to the gut receptors. Understanding the defence mechanisms of insects against B. thuringiensis toxicity may lead to improved pest management methods. [ABSTRACT FROM AUTHOR]

The desert truffle Terfezia boudieri is an ascomycete fungus that forms ect-endomycorrhiza in the roots of plants belonging to Cistaceae. The fungus forms hypogeous edible fruit bodies, appreciated as gourmet food. Truffles and host plants are colonized by various microbes, which may contribute to their development. However, the diversity and composition of the bacterial community under field conditions in the Negev desert are still unknown. The overall goal of this research was to identify the rhizosphere microbial community supporting the establishment of a symbiotic association between T. boudieri and Helianthemum sessiliflorum . The bacterial community was characterized by fruiting bodies, mycorrhized roots, and rhizosphere soil. Based on next-generation sequencing meta-analyses of the 16S rRNA gene, we discovered diverse bacterial communities of fruit bodies that differed from those found in the roots and rhizosphere. Families of Proteobacteria, Planctomycetes, and Actinobacteria were present in all four samples. Alpha diversity analysis revealed that the rhizosphere and roots contain significantly higher bacterial species numbers compared to the fruit. Additionally, ANOSIM and PCoA provided a comparative analysis of the bacterial taxa associated with fruiting bodies, roots, and rhizosphere. The core microbiome described consists of groups whose biological role triggers important traits supporting plant growth and fruit body development.

The bacterial biota in larvae of Capnodis tenebrionis , a serious pest of cultivated stone-fruit trees in the West Palearctic, was revealed for the first time using the MiSeq platform. The core bacterial community remained the same in neonates whether upon hatching or grown on peach plants or an artificial diet, suggesting that C. tenebrionis larvae acquire much of their bacterial biome from the parent adult. Reads affiliated with class levels Gammaproteobacteria and Alphaproteobacteria (phylum Proteobacteria ca. 86%), and Actinobacteria (ca. 14%) were highly abundant. Most diverse reads belong to the families Xanthomonadaceae (50%), Methylobacteriaceae (20%), Hyphomicrobiaceae (9%), Micrococcaceae (7%) and Geodermatophilaceae (4.5%). About two-thirds of the reads are affiliated with the genera Lysobacter , Microvirga , Methylobacterium , and Arthrobacter , which encompass species displaying cellulolytic and lipolytic activities. This study provides a foundation for future studies to elucidate the roles of bacterial biota in C. tenebrionis .

Anaerobic oxidation of methane (AOM) was shown to reduce methane emissions by over 50% in freshwater systems, its main natural contributor to the atmosphere. In these environments iron oxides can become main agents for AOM, but the underlying mechanism for this process has remained enigmatic. By conducting anoxic slurry incubations with lake sediments amended with 13 C-labeled methane and naturally abundant iron oxides the process was evidenced by significant 13 C-enrichment of the dissolved inorganic carbon pool and most pronounced when poorly reactive iron minerals such as magnetite and hematite were applied. Methane incorporation into biomass was apparent by strong uptake of 13 C into fatty acids indicative of methanotrophic bacteria, associated with increasing copy numbers of the functional methane monooxygenase pmoA gene. Archaea were not directly involved in full methane oxidation, but their crucial participation, likely being mediators in electron transfer, was indicated by specific inhibition of their activity that fully stopped iron-coupled AOM. By contrast, inhibition of sulfur cycling increased 13 C-methane turnover, pointing to sulfur species involvement in a competing process. Our findings suggest that the mechanism of iron-coupled AOM is accomplished by a complex microbe-mineral reaction network, being likely representative of many similar but hidden interactions sustaining life under highly reducing low energy conditions.

The etiology of black band disease (BBD), a persistent, globally distributed coral disease characterized by a dark microbial mat, is still unclear. A metatranscriptomics approach was used to unravel the roles of the major mat constituents in the disease process. By comparing the transcriptomes of the mat constituents with those of the surface microbiota of diseased and healthy corals, we showed a shift in bacterial composition and function in BBD-affected corals. mRNA reads of Cyanobacteria, Bacteroidetes and Firmicutes phyla were prominent in the BBD mat. Cyanobacterial adenosylhomocysteinase, involved in cyanotoxin production, was the most transcribed gene in the band consortium. Pathogenic and non-pathogenic forms of Vibrio spp., mainly transcribing the thiamine ABC transporter, were abundant and highly active in both the band and surface tissues. Desulfovibrio desulfuricans was the primary producer of sulfide in the band. Members of the Bacilli class expressed high levels of rhodanese, an enzyme responsible for cyanide and sulfide detoxification. These results offer a first look at the varied functions of the microbiota in the disease mat and surrounding coral surface and enabled us to develop an improved functional model for this disease.

Brominated flame retardants (BFRs) are a group of widely used compounds that, due to their limited biodegradability, exhibit excessive persistence in the environment. The persistence and high toxicity of these compounds to the natural biota causes great environmental concern. We investigated the biodegradation of the BFR dibromoneopentyl glycol (DBNPG) under continuous culture conditions using a miniature membrane bioreactor (mMBR) to assess its feasibility as a bioremediation approach. This system demonstrated long-term, stable biodegradation of DBNPG (>90 days), with an average removal rate of about 50%. Pyrosequencing of the 16S rRNA gene of the microorganisms involved in this process revealed the dominance of reads affiliated with the genus Brevundimonas of the Alphaproteobacteria class during the different mMBR operational stages. The bacterial community was also dominated by reads affiliated with the Sinorhizobium and Sphingopyxis genera within the Alphaproteobacteria class and the Sediminibacterium genus of the Sphingobacteria class. Real-time PCR used to analyze possible changes in the population dynamics of these four dominant groups revealed their consistent presence throughout the long-term mMBR biodegradation activity. Two genera, Brevundimonas and Sphingopyxis, were found to increase in abundance during the acclimation period and then remained relatively stable, forming the main parts of the consortium over the prolonged active stage.

Changes of the black band disease (BBD)-associated microbial consortium on the surface of a Favia sp. coral colony were assessed in relation to the different disease phases. A number of highly active bacterial groups changed in numbers as the BBD disease signs changed. These included Gamma- and Epsilonproteobacteria, Bacteroidetes and Firmicutes groups. One cyanobacterium strain, BGP10_4ST (FJ210722), was constantly present in the disease interface and adjacent tissues of the affected corals, regardless of disease phase. The dynamics of the operational taxonomic units (OTUs) of this BBD-specific strain provide a marker regarding the disease phase. The disease's active phase is characterized by a wide dark band progressing along the tissue-skeleton interface and by numerous bacterial OTUs. Cyanobacterial OTUs decreased in numbers as the disease signs waned, perhaps opening a niche for additional microorganisms. Even when black band signs disappeared there was a consistent though low abundance of the BBD-specific cyanobacteria (BGP10_4ST), and the microbial community of the disease-skeleton interface remained surprisingly similar to the original band community. These results provide an indication that the persistence of even low numbers of this BBD-specific cyanobacterium in coral tissues during the non-active (or subclinical) state could facilitate reinitiation of BBD signs during the following summer. This may indicate that this bacterium is major constituent of the disease and that its persistence and ability to infiltrate the coral tissues may act to facilitate the assembly of the other BBD-specific groups of bacteria.

Monohalogenated benzoic acids often appear in industrial wastewaters where biodegradation can be hampered by complex mixtures of pollutants and prevailing extreme milieu conditions. In this study, the biodegradation of chlorinated and brominated benzoic acids was conducted at a pH range of 5.0-9.0, at elevated salt concentrations and with pollutant mixtures including fluorinated and iodinated compounds. In mixtures of the isomers, the degradation order was primarily 4-substituted followed by 3-substituted and then 2-substituted halogenated benzoic acids. If the pH and salt concentration were altered simultaneously, long adaptation periods were required. Community analyses were conducted in liquid batch cultures and after immobilization on sand columns. The Alphaproteobacteria represented an important fraction in all of the enrichment cultures. On the genus level, Afipia sp. was detected most frequently. In particular, Bacteroidetes were detected in high numbers with chlorinated benzoic acids., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Culture-independent methods, employed to study the diversity and complexity of microbial communities that are based on amplification of rRNA genes with universal primers, include gradient gel electrophoresis (denaturing or temperature), single-strand-conformation polymorphism, restriction fragment length polymorphism, qPCR and high-throughput DNA sequencing. Substituting one or more base(s) within or at the 3'-termi of the universal primers by inosine can overcome some of their shortcomings improving amplification capacity. Universal primer sets do not usually amplify sequences with nucleotide mismatch to the templates, particularly in the last three bases, whereas inosine-modified primers anneal and amplify a variety of rRNA gene sequences. Inosine-containing primers are therefore might be useful to detect more species in diverse prokaryotic populations. The article summarizes the pros and cons of using inosine especially at the 3' termini of universal primers in nucleic acid amplification for the study of microbial diversity.

Background: Conventional methods often fail to control the flatheaded borers Capnodis spp., major pests of stone fruit trees; the larvae are protected from insecticides and predation because they feed deep in the roots. A potential solution is transgenic trees producing in their roots toxic compounds such as Cry proteins of Bacillus thuringiensis (Bt)., Results: Toxicities against Capnodis larvae were demonstrated by exploiting a recently designed artificial larval diet and an available collection of field isolated Bt. An isolate of Bt tenebrionis (Btt) from commercial bioinsecticide (Novodor) displayed LC50 and LC95 values of 3.2 and 164 mg g(-1) , respectively, against neonates of Capnodis tenebrionis, whereas values of the most toxic field isolate K-7 were 1.9 and 25.6 mg g(-1) respectively. Weights of surviving larvae after 1 month on diets containing low concentrations of K-7 (0.1-1.0 mg g(-1) ) were lower than on Btt or untreated larvae. K-7 was also toxic against larvae of C. cariosa and C. miliaris and found to harbour genes encoding Cry9Ea-like and Cry23Aa/Cry37Aa binary toxins., Conclusion: Larvae of Capnodis spp. are susceptible to Bt Cry toxins. Expressing cry genes active against these pests thus seems a feasible solution towards production of transgenic rootstock trees resilient to the pest., (© 2013 Society of Chemical Industry.)

Dust storms include particulate matter that is transported over land and sea with biota that could impact downwind ecosystems. In addition to the physico-chemical compositions, organismal diversities of dust from two storm events in southern Israel, December 2012 (Ev12) and January 2013 (Ev13), were determined by pyro-sequencing using primers universal to 16S and 18S rRNA genes and compared. The bio-assemblages in the collected dust samples were affiliated with scores of different taxa. Distinct patterns of richness and diversity of the two events were influenced by the origins of the air masses: Ev13 was rich with reads affiliated to Betaproteobacteria and Embryophyta, consistent with a European origin. Ev12, originated in north-Africa, contained significantly more of the Actinobacteria and fungi, without conifers. The abundance of bacterial and eukaryotic reads demonstrates dissemination of biological material in dust that may impose health hazards of pathogens and allergens, and influence vegetation migration throughout the world.

Mosquitoes have a complex life-cycle with dramatic changes in shape, function, and habitat. Aedes aegypti was studied by growing individual larvae at different concentrations of a defined rich food source. At higher food concentrations, rate of larval growth was faster, but the time required for 4th instar larvae to molt into the pupal stage was unexpectedly extended. These opposite tendencies resulted in constant times from hatching to pupation and up to adult eclosion at permissive food concentrations. The results demonstrate that nutritional conditions of 4th instar larvae impact initiation of the first metamorphic molt., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major (of 134, 128, 72 and 27 kDa) and at least two minor (of 78 and 29 kDa) polypeptides encoded respectively by cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba, all mapped on the 128 kb plasmid known as pBtoxis. These six δ-endotoxins form a complex parasporal crystalline body with remarkably high, specific and different toxicities to Aedes, Culex and Anopheles larvae. Cry toxins are composed of three domains (perforating domain I and receptor binding II and III) and create cation-selective channels, whereas Cyts are composed of one domain that acts as well as a detergent-like membrane perforator. Despite the low toxicities of Cyt1Aa and Cyt2Ba alone against exposed larvae, they are highly synergistic with the Cry toxins and hence their combinations prevent emergence of resistance in the targets. The lack of significant levels of resistance in field mosquito populations treated for decades with Bti-bioinsecticide suggests that this bacterium will be an effective biocontrol agent for years to come.

A marine bacterial strain, designated MD2(T), was isolated from the damaged tissue of a hydrocoral, Millepora dichotoma, collected from the coral reef in the northern Red Sea, Gulf of Eilat, Israel. Strain MD2(T) was Gram-reaction-negative, rod-shaped and motile, and formed small, creamy and opaque colonies, 1-2 mm in diameter, after 3 days incubation on Marine agar at 30°C. The novel strain grew well in nutrient broth at 1.5-6 % NaCl and at 20-37°C. The major cellular fatty acids were iso-C17 : 1ω9c, iso-C17 : 0, C18 : 1ω7c and C17 : 1ω6c. The polar lipids consisted of phosphatidylglycerol, phosphatidylethanolamine, an unidentified lipid, two unidentified phospholipids, two unidentified glycolipids and two unidentified aminolipids. Ubiquinone Q-10 was the only respiratory lipoquinone. The DNA G+C content was 60.3 mol%. Analysis of the 16S rRNA gene sequence placed the organism in the α-subclass of the Proteobacteria with a sequence divergence of about 9 % from any species with a validly published name. The highest 16S rRNA gene sequence similarity (approximately 91 %) was notably with type strains of members of the genus Kordiimonas, Kordiimonas aestuarii 101-1(T), Kordiimonas lacus S3-22(T) and Kordiimonas gwangyangensis GW14-5(T). On the basis of genotypic, chemotaxonomic and phenotypic distinctness, strain MD2(T) represents a novel species in a new genus of the class Alphaproteobacteria, for which the name Eilatimonas milleporae gen. nov., sp. nov. is proposed. The type strain of the type species is MD2(T) ( = LMG 26586(T) = DSM 25217(T)).

Colonies of the hydrocoral Millepora dichotoma along the Gulf of Eilat are exhibiting unusual tissue lesions in the form of white spots. The emergence and rapid establishment of these multifocal tissue lesions was the first of its kind reported in this region. A characterization of this morphological anomaly revealed bleached tissues with a significant presence of bacteria in the tissue lesion area. To ascertain possible differences in microbial biota between the lesion area and non-affected tissues, we characterized the bacterial diversity in the two areas of these hydrocorals. Both culture-independent (molecular) and culture-dependent assays showed a shift in bacterial community structure between the healthy and affected tissues. Several 16S rRNA gene sequences retrieved from the affected tissues matched sequences of bacterial clones belonging to Alphaproteobacteria and Bacteroidetes members previously associated with various diseases in scleractinian corals.

Coral holobionts are densely populated with microorganisms that are essential for their well-being. Here we compared the diversity of the archaeal ammonia monooxygenase alpha subunit (amoA) gene from three coral genera, Acanthastrea sp., Favia sp., and Fungia granulosa, from the Gulf of Eilat, Red Sea. At 99% similarity, archaeal amoA from the three coral genera shared 71% of their cloned sequences, while the Favia and Acanthastrea presented a few genus-specific clones. In addition, the sequences retrieved in our samples displayed lower similarity to amoA sequences previously found in association with other coral species from different geographic regions. This finding suggests that the populations of ammonia-oxidizing archaea are less host-specific and more geographically dependent.

The base adjacent to the 3' end of universal PCR primers targeting the 16S rRNA gene is often variable and apparently biases the microbial community composition as represented by PCR-based surveys. To test this hypothesis, four templates of 44 bases each and two complementary primers (21 bases) were designed to differ only in the bases adjacent to the primers, and their amplification efficiencies were evaluated using quantitative PCR. For extension temperatures of 72°C, 73°C and 74°C, improvement in initial amplification efficiency was observed for templates with guanine or cytosine at the position contiguous to the primers. However, no clear preference was observed when extension temperature was lowered to 70°C. Shortening the primers by one base, so that the variable position was located two base pairs downstream from the primer, attenuated but did not eliminate this bias. A conformational change of the quaternary polymerase - primer - template - dNTP complex upon commencing of polymerization is thought to be a rate-limiting step. A possible explanation for the observed bias is the stabilization of this complex by the adjacent guanine or cytosine. Reducing PCR extension temperature to 70°C minimizes amplification biases caused by variable template-contiguous bases to the 3' end of universal PCR primers. Next-base nucleotide composition should be taken in consideration in designing primers targeting 16S rRNA or other functional genes used in microbial ecology studies., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

During sporulation, Bacillus thuringiensis subsp. israelensis produces a mosquito larvicidal protein complex containing several crystalline and cytolytic (Cyt) toxins. Here, the activated monomeric form of Cyt1Aa, the most toxic Cyt family member, was isolated and crystallized, and its structure was determined for the first time at 2.2 Å resolution. Cyt1Aa adopts a typical cytolysin fold containing a β-sheet held by two surrounding α-helical layers. The absence of a β-strand (between residues V26 and I37) in the dimeric structure of Cyt2Aa led us to deduce that this is the only essential segment for dimer formation and that activation of the toxin occurs by proteolytic processing of its N-terminus. Based on the Cyt1Aa structure, we suggest that the toxicity of Cyt1Aa and other nonrelated proteins, all sharing a cytolysin fold, is correlated with their ability to undergo conformational changes that are necessary prior to their membrane insertion and perforation. This fold allows the α-helical layers to swing away, exposing the β-sheet to insert into the membrane. The identification of a putative lipid binding pocket between the β-sheet and the helical layer of Cyt1Aa supports this mechanism. Sequence-based structural analysis of Cyt1Aa revealed that the lack of activity of Cyt1Ca may be related to the latter's inability to undergo this conformational change due to its lack of flexibility. The pattern of the hemolytic activity of Cyt1Aa presented here (resembling that of pore-forming agents), while differing from that imposed by ionic and nonionic detergents, further supports the pore-forming model by which conformational changes occur prior to membrane insertion and perforation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Modular transparent column system was designed to study depth-dependence behavior of mosquito larvae. The system was used in preliminary experiments to evaluate the effect of water depth on the larvicidal activity of Bacillus thuringiensis subsp. israelensis de Barjac against bottom feeder larvae of Aedes aegypti (Linn.) (Diptera: Culicidae), and suggestions for increasing the efficiency of the device are discussed., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A new gene, cry11Bb2 from a field isolate of Bacillus thuringiensis, was cloned for expression in Escherichia coli. The encoded protein, with a deduced molecular mass of 89.5 kDa, exhibits 97 and 79% identities with the overlap regions of Cry11Bb1 from B. thuringiensis ssp. medellin and Cry11Ba1 from ssp. jegathesan, respectively. It is however longer than Cry11Bb1 by 42 amino acids in its carboxy-terminus, of which 32 comprise 2 tandem repeats additional to the 5 existing in the latter polypeptide. Possible roles for this recurrent motif among Cry toxins and their accessory proteins, and for their encoding genes are proposed., (Copyright © 2011 S. Karger AG, Basel.)

Universal 16S rRNA gene primers (8F and 518R) bearing inosine substitutions at either the 3'-ultimate or the 3'-ultimate and penultimate base positions were exploited for the first time to study the bacterial community associated with coral polymicrobial Black Band Disease (BBD). Inosine-modified universal primer pairs display some shifting in the composition of 16S rRNA gene libraries, as well as expanding the observed diversity of a BBD bacterial community at the family/class level. Possible explanations for the observed shifts are discussed. These results thus point to the need for adopting multiple approaches in designing 16S rRNA universal primers for PCR amplification and subsequent construction of 16S rRNA gene libraries or pyrosequencing in the exploration of complex microbial communities.

Various subspecies (ssp.) of Bacillus thuringiensis (Bt) are considered the best agents known so far to control insects, being highly specific and safe, easily mass produced and with long shelf life.1 The para-crystalline body that is produced during sporulation in the exosporium includes polypeptides named δ-endotoxins, each killing a specific set of insects. The different entomopathogenic toxins of various Bt ssp. can be manipulated genetically in an educated way to construct more efficient transgenic bacteria or plants that express combinations of toxin genes to control pests.2 Joint research projects in our respective laboratories during the last decade demonstrate what can be done by implementing certain ideas using molecular biology with Bt ssp. israelensis (Bti) as a model system. Here, we describe our progress achieved with Gram-negative bacterial species, including cyanobacteria, and some preliminary experiments to form transgenic plants, mainly to control mosquitoes (Diptera), but also a particular Lepidopteran and Coleopteran pest species. In addition, a system is described by which environment-damaging genes can be removed from the recombinants thus alleviating procedures for obtaining permits to release them in nature., (© 2010 Landes Bioscience)

The surfaces of massive corals of the genus Favia from Eilat, Red Sea, and from Heron Island, Great Barrier Reef, are covered by a layer of eukaryotic microorganisms. These microorganisms are embedded in the coral mucus and tissue. In the Gulf of Eilat, the prevalence of corals covered by patches of eukaryotic microorganisms was positively correlated with a decrease in water temperatures (from 25-28 degrees C in the summer to 20-23 degrees C in winter). Comparisons carried out using transmission and scanning electron microscopy showed morphological similarities between the microorganisms from the two geographically distant reefs. The microorganisms found on and in the tissues were approximately 5-15 microm in diameter, surrounded by scales in their cell wall, contained a nucleus, and included unique auto-florescent coccoid bodies of approximately 1 mum. Such morphological characters suggested that these microorganisms are stramenopile protists and in particular thraustochytrids. Molecular analysis, carried out using specific primers for stramenopile 18S rRNA genes, revealed that 90% (111/123) of the clones in the gene libraries were from the Thraustochytriidae. The dominant genera in this family were Aplanochytrium sp., Thraustochytrium sp., and Labyrinthuloides sp. Ten stramenopile strains were isolated and cultured from the corals. Some strains showed > or =97% similarity to clones derived from libraries of mucus-associated microorganisms retrieved directly from these corals. Fatty acid characterization of one of the prevalent strains revealed a high percentage of polyunsaturated fatty acids, including omega-3. The possible association of these stramenopiles in the coral holobiont appeared to be a positive one.

Black Band Disease (BBD) is a well-described disease plaguing corals worldwide. It has been established that ecological and environmental stress factors contribute to the appearance and progression of the disease, believed to be caused by a diverse microbial consortium. We have identified and characterized Vibrio sp. associated with BBD in Eilat reef corals using both culture-dependent and -independent methods. Direct sampling using 16S rRNA gene clone libraries showed seasonal dynamics in the diversity of BBD-associated Vibrios. In the two sampling periods, BBD-associated Vibrio clones showed similarities to different groups: October samples were similar to known pathogens, while December samples were similar to general aquatic Vibrio sp. Cultured bacterial isolates of Vibrio sp. were highly homologous (>or=99%) to previously documented BBD-associated bacteria from the Caribbean, Bahamas and Red Seas, and were similar to several known coral pathogens, such as Vibrio coralliilyticus. The proteolytic activity of Vibrio sp., as measured using casein- and azocasein-based assays, directly correlated with temperature elevation and peaked at 26-28 degrees C, with the microorganisms producing more proteases per bacterial cell or increasing the rate of proteolytic activity of the same proteases (potentially metalloproteases). This activity may promote coral tissue necrosis and aid in ensuing progression of the coral BBD.

Black band disease (BBD) is a widespread coral disease which mainly infects massive framework-building corals. BBD is believed to be caused by a consortium of microorganisms and may not result from the actions of a primary pathogen. The BBD microbial community is dominated, in terms of biomass, by filamentous cyanobacteria. Here we describe a cyanobacterial strain, designated BgP10_4S(T), cultured from a BBD-affected Favia sp. 25 degreesoal from the northern Red Sea (Gulf of Eilat, Israel). This dark-green pigmented cyanobacterium showed optimal growth at salinities of 5.0 to 5.5% (w/v), pH of 7 to 8 and cultivation temperatures of 25 0C. Morphological examination revealed cylindrical, unbranched trichomes with tapering and blunt cells at the ends which leave a thin mucilaginous trail as they glide. No sheath was evident under these conditions. Inclusion bodies and straight thylakoids were clearly discerned by transmission electron microscopy. Pigment analysis revealed absorption spectra for phycocyanin, carotenoid and chlorophyll a. The sequence of the 16S rRNA gene in this cyanobac(t)erium isolate showed high similarity (99%) to cyanobacterial sequences retrieved from BBD-affected corals from different geographical sites (i.e. the Caribbean Sea, Palau and the Red Sea). The BgP10_4ST strain is observed to be a persisten(t) component of the BBD mat of Faviid corals and may thus be an important agent in the disease etiology. On the basis (of its morphological, physiological and phylogenetic distinctiveness, strain BgP10_4ST represents a novel genus and species of Subsection III (formerly Oscillatoriales), for which the name Pseudoscillatoria coralii gen. nov., sp. nov. is proposed.

A bacterial strain, designated Coryn-1(T), was isolated from mucus of the coral Fungia granulosa (northern Red Sea, Gulf of Eilat, Israel) by growth and enrichment of micro-organisms in agar spheres and subsequent plating. The bacterium was found to be a Gram-positive, non-motile, halotolerant, heterotrophic coccobacillus. Comparative 16S rRNA gene sequence analyses showed that strain Coryn-1(T) belonged to the genus Corynebacterium, exhibiting the highest levels of similarity (94 %) with the 16S rRNA gene sequence of Corynebacterium halotolerans YIM 70093(T). The novel strain grew well at 0.5-4.0 % salinity, at pH 7.2-9.0 and at 30-37 degrees C. The major cellular fatty acids were oleic acid (C(18 : 1)omega9c; 58 %), palmitic acid (C(16 : 0); 30 %) and tuberculostearic acid (10-methyl-C(18 : 0); 12 %). The DNA G+C content was 66.6 mol%. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Coryn-1(T) represents a novel species, for which the name Corynebacterium maris sp. nov. is proposed. The type strain is Coryn-1(T) (=DSM 45190(T)=LMG 24561(T)).

The lack of cultured microorganisms represents a bottleneck for advancement in microbiology. The development of novel culturing techniques is, therefore, a crucial step in our understanding of microbial diversity in general, and the role of such diversity in the environment, in particular. This study presents an innovative method for cultivating microorganisms by encapsulating them within agar spheres, which are then encased in a polysulfonic polymeric membrane and incubated in a simulated or natural environment. This method stimulates growth of the entrapped microorganisms by allowing them access to essential nutrients and cues from the environment. It allows for the discovery of microorganisms from dilutions that are 10-100-fold greater than possible with conventional plating techniques. Analysis of microorganisms grown in such spheres incubated in and on a number of different substrates yielded numerous novel ribotypes. For example, spheres incubated on the mucus surface of a Fungiid coral yielded numerous ribotypes, with only 50% sharing similarity (85-96%) to previously identified microorganisms. This suggests that many of the species represent novel ribotypes. Hence, the technique reported here advances our ability to retrieve and successfully culture microorganisms and provides an innovative tool to access unknown microbial diversity.

Abundance and seasonal dynamics of sulfate-reducing bacteria (SRB), in general, and of extreme halophilic SRB (belonging to Desulfocella halophila) in particular, were examined in highly saline industrial wastewater evaporation ponds over a forty one month period. Industrial wastewater was sampled and the presence of SRB was determined by quantitative real-time PCR (qPCR) with a set of primers designed to amplify the dissimilatory sulfite reductase (dsrA) gene. SRB displayed higher abundance during the summer (10(6)-10(8) targets ml(-1)) and lower abundance from the autumn-spring (10(3)-10(5) targets ml(-1)). However, addition of concentrated dissolved organic matter into the evaporation ponds during winter immediately resulted in a proliferation of SRB, despite the lower wastewater temperature (12-14 degrees C). These results indicate that the qPCR approach can be used for rapid measurement of SRB to provide valuable information about the abundance of SRB in harsh environments, such as highly saline industrial wastewaters. Low level of H2S has been maintained over five years, which indicates a possible inhibition of SRB activity, following artificial salination (approximately 16% w/v of NaCl) of wastewater evaporation ponds, despite SRB reproduction being detected by qPCR.

A bacterial strain, designated RS.Sph.026(T), was isolated from mucus of the coral Fungia granulosa collected from the northern Red Sea (Gulf of Eilat, Israel). The bacterium was found to be Gram-negative, non-motile, halotolerant and heterotrophic. Comparative 16S rRNA gene sequence analyses showed that strain RS.Sph.026(T) belonged to the order Rhizobiales, with the highest levels of 16S RNA gene sequence similarity with Rhodobium orientis (92 %). Strain RS.Sph.026(T) grew optimally at a salinity of 3-4 %, pH 7.5-8 and 25-30 degrees C. The major cellular fatty acids were cis-7-octadecenoic acid (C(18 : 1)omega7small es, Cyrillic; 57.2 %) and C(19 : 0) cyclo omega8small es, Cyrillic (15.5 %). The DNA G+C content of strain RS.Sph.026(T) was 67.1 mol%. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain RS.Sph.026(T) represents a novel genus and species in the order Rhizobiales, for which the name Amorphus coralli gen. nov., sp. nov. is proposed. The type strain is RS.Sph.026(T) (=LMG 24307(T)=DSM 19760(T)).

Diversity, distribution and genetic comparison of Archaea associated with the surface mucus of corals from three genera, namely Acanthastrea sp., Favia sp. and Fungia sp., from the Gulf of Eilat, Israel and from Heron Island, Australia were studied. Sequencing of the 16S rRNA gene of the coral-associated Archaea revealed dominance of Crenarchaeota (79%, on average). In this phylum, 87% of the sequences were similar (>or= 97%) to the Thermoprotei, with 76% of these being similar (>or= 97%) to the ammonium oxidizer, Nitrosopumilus maritimus. Most of the coral-associated euryarchaeotal sequences (69%) were related to marine group II, while other euryarchaeotal clades were found to be related to anaerobic methanotrophs (8%), anaerobic nitrate reducers (i.e. denitrification, 15%) and marine group III (8%). Most of the crenarchaeotal and euryarchaeotal coral-associated 16S rRNA gene sequences from Heron Island (61%) and from the Gulf of Eilat (71%) were closely related (>or= 97%) to sequences previously derived from corals from the Virgin Islands. Analysis of archaeal amoA sequences obtained from the fungiid coral, Fungia granulosa, divided into three clades, all related to archaeal sequences previously obtained from the marine environment. These sequences were distantly related to amoA sequences previously found in association with other coral species. Preliminary experiments suggest that there is active oxidation of ammonia to nitrite in the mucus of F. granulosa. Thus, coral-associated Archaea may contribute to nitrogen recycling in the holobiont, presumably by acting as a nutritional sink for excess ammonium trapped in the mucus layer, through nitrification and denitrification processes.

The salinity of industrial wastewater evaporation ponds was artificially increased from 3-7% to 12-16% (w/v), in an attempt to reduce the activity of sulfate-reducing bacteria (SRB) and subsequent emission of H2S. To investigate the changes in bacterial diversity in general, and SRB in particular, following this salination, two sets of universal primers targeting the 16S rRNA gene and the functional apsA [adenosine-5'-phosphosulfate (APS) reductase alpha-subunit] gene of SRB were used. Phylogenetic analysis indicated that Proteobacteria was the most dominant phylum both before and after salination (with 52% and 68%, respectively), whereas Firmicutes was the second most dominant phylum before (39%) and after (19%) salination. Sequences belonging to Bacteroidetes, Spirochaetes and Actinobacteria were also found. Several groups of SRB from Proteobacteria and Firmicutes were also found to inhabit this saline environment. Comparison of bacterial diversity before and after salination of the ponds revealed both a shift in community composition and an increase in microbial diversity following salination. The share of SRB in the 16S rRNA gene was reduced following salination, consistent with the reduction of H2S emissions. However, the community composition, as shown by apsA gene analysis, was not markedly affected.

Comparing activities of purified toxins from Bacillus thuringiensis ssp. israelensis against larvae of seven mosquito species (vectors of tropical diseases) that belong to three genera, gleaned from the literature, disclosed highly significant variations in the levels of LC(50) as well as in the hierarchy of susceptibilities. Similar toxicity comparisons were performed between nine transgenic Gram-negative species, four of which are cyanobacterial, expressing various combinations of cry genes, cyt1Aa and p20, against larvae of four mosquito species as potential agents for biological control. Reasons for inconsistencies are listed and discussed. Standard conditions for toxin isolation and presentation to larvae are sought. A set of lyophilized powders prepared identically from six Escherichia coli clones expressing combinations of four genes displayed toxicities against larvae of three mosquito species, with levels that differed between them but with identical hierarchy.

The Cyt family of proteins consists of delta-endotoxins expressed during sporulation of several subspecies of Bacillus thuringiensis. Its members possess insecticidal, hemolytic, and cytolytic activities through pore formation and attract attention due to their potential use as vehicles for targeted membrane destruction. The delta-endotoxins of subsp. israelensis include three Cyt species: a major Cyt1Aa and two minor proteins, Cyt2Ba and Cyt1Ca. A cleaved Cyt protein that lacks the N- and C-terminal segments forms a toxic monomer. Here, we describe the crystal structure of Cyt2Ba, cleaved at its amino and carboxy termini by bacterial endogenous protease(s). Overall, its fold resembles that of the previously described volvatoxin A2 and the nontoxic form of Cyt2Aa. The structural similarity between these three proteins may provide information regarding the mechanism(s) of membrane-perforating toxins.

Recent evidence suggests that there is a dynamic microbial biota living on the surface and in the mucus layer of many hermatypic coral species that plays an essential role in coral well-being. Most of the studies published to date emphasize the importance of prokaryotic communities associated with the coral mucus in coral health and disease. In this study, we report the presence of a protist (Fng1) in the mucus of the hermatypic coral Fungia granulosa from the Gulf of Eilat. This protist was identified morphologically and molecularly as belonging to the family Thraustochytridae (phylum Stramenopile, order Labyrinthulida), a group of heterotrophs widely distributed in the marine environment. Morphological examination of this strain revealed a nonmotile organism c. 35 mum in diameter, which is able to thrive on carbon-deprived media, and whose growth and morphology are inoculum dependent. Its fatty acid production profile revealed an array of polyunsaturated fatty acids. A similar protist was also isolated from the mucus of the coral Favia sp. In light of these findings, its possible contribution to the coral holobiont is discussed.

Microgene Polymerization Reaction (MPR) is used as an experimental system to artificially simulate evolution of short, non-repetitive homo-duplex DNA into multiply-repetitive products that can code for functional proteins. Blunt-end ligation by DNA polymerase is crucial in expansion of homo-duplexes (HDs) into head-to-tail multiple repeats in MPR. The propagation mechanism is known, but formation of the initial doublet (ID) by juxtaposing two HDs and polymerization through the gap has been ambiguous. Initiation events with pairs of HDs using Real-Time PCR were more frequent at higher HD concentrations and slightly below the melting temperature. A process molecularity of about 3.1, calculated from the amplification efficiency and the difference in PCR cycles at which propagation was detected at varying HD concentrations, led to a simple mechanism for ID formation: the gap between two HDs is bridged by a third. Considering thermodynamic aspects of the presumed intermediate "nucleation complex" can predict relative propensity for the process with other HDs.

The genes cry1Ac and cry1Ca from Bacillus thuringiensis subsps. kurstaki HD-73 and aizawai 4J4, respectively, encoding delta-endotoxins against lepidopteran larvae were isolated, cloned and expressed in Escherichia coli, with and without cyt1Aa (encoding cytolytic protein) and p20 (accessory protein) from subsp. israelensis. Nine combinations of the genes under control of an early T7, P A1 inducible promoter, produced the encoding proteins. Toxicities were examined against larvae of three major agricultural pests: Pectinophora gossypiella, Helicoverpa armigera and Spodoptera littoralis. The clones expressing cyt1Aa, with or without p20, were not toxic. The clone expressing cry1Ac (pBt-1A) was the most toxic to P. gossypiella (LC50 of 0.27 x 10(8) cells g(-1)). Clone pBt-1CA expressing cry1Ca and cry1Ac displayed the highest toxicity (LC50 of 0.12 x 10(8) cells ml(-1)) against S. littoralis. Clone pBt-1CARCy expressing all four genes (cry1Ca, cry1Ac, p20, cyt1Aa) in tandem exhibited the highest toxicity to H. armigera (LC50 of 0.16 x 10(8) cells ml(-1)). Cyt1Aa failed to raise the toxicity of these Cry toxins against P. gossypiella and S. littoralis but significantly enhanced toxicity against H. armigera. Two additional clones expressing either cry1Ac or cry1Ca under tandem promoters, P A1 and P psbA (constitutive), displayed significantly higher toxicities (7.5- to 140-fold) than their counterparts with P A1 alone, reducing the LC50 values to below 10(7) cells ml(-1).

The microbial biota dwelling in the mucus, on the surface, and in the tissues of many coral species may have an important role in holobiont physiology and health. This microbiota differs with coral species, water depth, and geographic location. Here we compare the surface mucus microbiota of the coral Fungia granulosa from the natural environment with that from individuals maintained in aquaria. Molecular analysis revealed that the microbial community of the mucus microlayer of the coral F. granulosa includes a wide range of bacteria and that these change with environment. Coral mucus from the natural environment contained a significantly higher diversity of microorganisms than did mucus from corals maintained in the closed-system aquaria. A microbial community shift, with the loss of several groups, including actinobacterial and cyanobacterial groups, was observed in corals maintained in aquaria. The most abundant bacterial class in F. granulosa mucus was the Alphaproteobacteria, regardless of whether the corals were from aquaria or freshly collected from their natural environment. A significantly higher percentage of bacteria from the Betaproteobacteria class was evident in aquarium corals (24%) when compared with corals from the natural environment (3%). The differences in mucus-inhabiting microbial communities between corals from captive and natural environments suggest an adaptation of the mucus bacterial communities to the different conditions.

Real-time polymerase chain reaction (PCR) is considered a highly sensitive method for the quantification of microbial organisms in environmental samples. This study was conducted to evaluate real-time PCR with SybrGreen detection as a quantification method for sulfate-reducing bacteria (SRB) in industrial wastewater produced by several chemical industries. We designed four sets of primers and developed standard curves based on genomic DNA of Desulfovibrio vulgaris from pure culture and on plasmids containing dissimilatory sulfate reductase (dsrA) or adenosine-5'-phosphosulfate reductase (apsA) genes of SRB. All the standard curves, two for dsrA and two for apsA genes, had a linear range between 0.95 x 10(2) and 9.5 x 10(6) copies/microL and between 1.2 x 10(3) and 1.2 x 10(7) copies/microL, respectively. The theoretical copy numbers of the tenfold dilutions of D. vulgaris genomic DNA were best estimated (between 2.7 to 10.5 times higher than theoretical numbers) by the standard curve with DSR1F and RH3-dsr-R primers. To mimic the effect of foreign DNA in environmental samples, serial dilutions of D. vulgaris genomic DNA were mixed with Escherichia coli chromosomal DNA (40 ng per assay). This influenced neither PCR amplification nor the quantification of target DNA. Industrial wastewater was sampled during a 15-month period and analyzed for the presence of SRB, based on dsrA gene amplification. SRB displayed a higher abundance during the summer (about 10(7)-10(8) targets mL(-1)) and lower during the winter (about 10(4)-10(5) targets mL(-1)). The results indicate that our real-time PCR approach can be used for detection of uncultured SRB and will provide valuable information related to the abundance of SRB in durable environmental samples, such as complex and saline industrial wastewaters.

Multiple myeloma is currently an incurable cancer of plasma B cells often characterized by overproduction of abnormally high quantities of a patient-specific, clonotypic immunoglobulin "M-protein." The M-protein is expressed on the cell membrane and secreted into the blood. We previously showed that ligand-toxin conjugates (LTC) incorporating the ribosome-inactivating Ricin-A toxin were very effective in specific cytolysis of the anti-ligand antibody-bearing target cells used as models for multiple myeloma. Here, we report on the incorporation of the membrane-disruptive Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis into LTCs targeted to murine myeloma cells. Proteolytically activated Cyt1Aa was conjugated chemically or genetically through either its amino or carboxyl termini to the major peptidic epitope VHFFKNIVTPRTP (p87-99) of the myelin basic protein. The recombinant fusion-encoding genes were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis through the shuttle vector pHT315. Both chemically conjugated and genetically fused LTCs were toxic to anti-myelin basic protein-expressing murine hybridoma cells, but the recombinant conjugates were more active. LTCs comprising the Cyt1Aa toxin might be useful anticancer agents. As a membrane-acting toxin, Cyt1Aa is not likely to induce development of resistant cell lines.

Microbial communities associated with black band disease (BBD) in massive stony corals from the Northern Red Sea (Eilat) were examined for the first time using molecular tools and microscopy. A high microbial diversity was revealed in the affected tissue in comparison with the healthy area of the same colony. Microscopy revealed the penetration of cyanobacteria into the coral mesoglea and adjacent tissues. Cyanobacterial sequences from Red Sea BBD-affected corals formed a cluster with sequences previously identified from black band and red band diseased corals from the Indo-Pacific and Caribbean. In addition, 11 sequences belonging to the genus Vibrio were retrieved. This group was previously documented as pathogenic to corals. Sulfate-reducing bacteria, a group known to be associated with BBD and produce toxic sulfide, were studied using specific primers for the amplification of the dissimilatory sulfite reductase gene (dsrA). This technique facilitated and improved the resolution of the study of diversity of this group. All the sequences obtained were closely related to sequences of the genus Desulfovibrio and 46% showed high homology to Desulfovibrio desulfuricans. The complex nature of BBD and the lack of success in isolating a single causative agent suggest that BBD may be considered a polymicrobial disease.