Ariel Kushmaro

Vibrio shiloi sp. nov., the causative agent of bleaching of the coral Oculina patagonica

The aetiological agent of bleaching of the coral Oculina patagonica was characterized as a new Vibrio species on the basis of 16S rDNA sequence, DNA-DNA hybridization data and phenotypic properties, including the cellular fatty acid profile. Based on its 16S rDNA and DNA-DNA hybridization, the new Vibrio species is closely related to Vibrio mediterranei. The name Vibrio shiloi sp. nov. is proposed for the new coral-bleaching species, the type strain being AK1T (= ATCC BAA-91T = DSM 13774T).

Coral mucus-associated bacteria: a possible first line of defense

Interactions among microorganisms found in coral mucus can be either symbiotic or competitive. It has been hypothesized that microbial communities found on the surface of coral play a role in coral holobiont defense, possibly through production of antimicrobial substances. Selected microorganisms isolated from the mucus layer of a number of coral species were grown using agar-plating techniques. Screening for antimicrobial substances was performed using overlay and drop techniques, employing several indicator microorganisms. Between 25% and 70% of cultivable mucus-associated bacteria from scleractinian corals demonstrated bioactivity. Higher percentages of activity were evident in mucus-associated cultivable bacteria from massive and solitary corals, as compared with bacteria from branching or soft corals. Isolates related to the genera Vibrio and Pseudoalteromonas demonstrated high activity against both Gram-positive and Gram-negative bacteria. Gram-positive bacteria (Bacillus, Planomicrobium) demonstrated lower levels of activity, primarily against other Gram-positive bacteria. In some cases, inhibitory effects were confined to the cell fraction, suggesting the involvement of a cell-bound molecule, sensitive to temperature and most likely proteinaceous in nature. These results demonstrate the existence of microorganisms with antimicrobial activity on the coral surface, possibly acting as a first line of defense to protect the coral host against pathogens.

Penetration of the Coral-Bleaching Bacterium Vibrio shiloi into Oculina patagonica

ABSTRACT Inoculation of the coral-bleaching bacterium Vibrio shiloi into seawater containing its host Oculina patagonica led to adhesion of the bacteria to the coral surface via a β-d-galactose receptor, followed by penetration of the bacteria into the coral tissue. The internalized V. shiloi cells were observed inside the exodermal layer of the coral by electron microscopy and fluorescence microscopy using specific anti-V. shiloi antibodies to stain the intracellular bacteria. At 29°C, 80% of the bacteria bound to the coral within 8 h. Penetration, measured by the viable count (gentamicin invasion assay) inside the coral tissue, was 5.6, 20.9, and 21.7% of the initial inoculum at 8, 12, and 24 h, respectively. The viable count in the coral tissue decreased to 5.3% at 48 h, and none could be detected at 72 h. Determination of V. shiloi total counts (using the anti-V. shiloiantibodies) in the coral tissue showed results similar to viable counts for the first 12 h of infection. After 12 h, however, the total count more than doubled from 12 to 24 h and continued to rise, reaching a value 6 times that of the initial inoculum at 72 h. Thus, the intracellular V. shiloi organisms were transformed into a form that could multiply inside the coral tissue but did not form colonies on agar medium. Internalization of the bacteria was accompanied by the production of high concentrations of V. shiloi toxin P activity in the coral tissue. Internalization and multiplication of V. shiloi are discussed in terms of the mechanism of bacterial bleaching of corals.

Global distribution and diversity of coral‐associated Archaea and their possible role in the coral holobiont nitrogen cycle

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 role of microorganisms in coral bleaching

Coral bleaching is the disruption of the symbiosis between the coral host and its endosymbiotic algae. The prevalence and severity of the disease have been correlated with high seawater temperature. During the last decade, the major hypothesis to explain coral bleaching is that high water temperatures cause irreversible damage to the symbiotic algae resulting in loss of pigment and/or algae from the holobiont. Here, we discuss the evidence for an alternative but not mutually exclusive concept, the microbial hypothesis of coral bleaching.

Advantage of Using Inosine at the 3′ Termini of 16S rRNA Gene Universal Primers for the Study of Microbial Diversity

ABSTRACT To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3′ termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54°C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50°C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3′ termini in studying the microbial diversity of environmental samples.

Bacteriophage predation regulates microbial abundance and diversity in a full-scale bioreactor treating industrial wastewater

Changes in the microbial community composition of a full-scale membrane bioreactor treating industrial wastewater were studied over a period of 462 days using a series of 16S rRNA gene clone libraries. Frequent changes in the relative abundance of specific taxonomic groups were observed, which could not be explained by changes in the reactors conditions or wastewater composition. Phage activity was proposed to drive some of the observed changes. Bacterial hosts were isolated from a biomass sample obtained towards the end of the study period, and specific phage counts were carried out for some of the isolated hosts using stored frozen biomass samples as the phage inocula. Plaque-forming unit concentrations were shown to change frequently over the study period, in correlation with changes in the relative abundance of taxonomic groups closely related by 16S rRNA gene sequence to the isolated strains. Quantitative PCR was used to verify changes in the abundance of a taxonomic group closely related to one of the isolated hosts, showing good agreement with the changes in relative abundance in the clone libraries of that group. The emerging pattern was consistent with the ‘killing the winner’ hypothesis, although alternative interaction mechanisms could not be ruled out. This is the first time that phage–host interactions in a complex microbial community are demonstrated over an extended period, and possibly the first in situ demonstration of ‘killing the winner’ stochastic behavior.

Environmental Impact of Flame Retardants (Persistence and Biodegradability)

Flame-retardants (FR) are a group of anthropogenic environmental contaminants used at relatively high concentrations in many applications. Currently, the largest market group of FRs is the brominated flame retardants (BFRs). Many of the BFRs are considered toxic, persistent and bioaccumulative. Bioremediation of contaminated water, soil and sediments is a possible solution for the problem. However, the main problem with this approach is the lack of knowledge concerning appropriate microorganisms, biochemical pathways and operational conditions facilitating degradation of these chemicals at an acceptable rate. This paper reviews and discusses current knowledge and recent developments related to the environmental fate and impact of FRs in natural systems and in engineered treatment processes.

Symbiophagy as a cellular mechanism for coral bleaching

Coral bleaching is a major contributor to the global declines of coral reefs. This phenomenon is characterized by the loss of symbiotic algae, their pigments or both. Despite wide scientific interest, the mechanisms by which bleaching occurs is still poorly understood. Here we report that the removal of the symbiont during light and temperature stress is achieved using the hosts cellular autophagic-associated machinery. Host cellular and sub-cellular morphologies showed increased vacuolization and appearance of autophagic membranes surrounding a variety of organelles and surrounding the symbiotic algae. Markers of autophagy (Rab 7 and LAS) corroborate these observations. Results showed that during stress the symbiont vacuolar membrane is transformed from a conduit of nutrient exchange to a digestive organelle resulting in the consumption of the symbiont, a process we term symbiophagy. We posit that during a stress event, the mechanism maintaining symbiosis is destabilized and symbiophagy is activated, ultimately resulting in the phenomenon of bleaching. Symbiophagy may have evolved from a more general primordial innate intracellular protective pathway termed xenophagy.

An in situ method for cultivating microorganisms using a double encapsulation technique

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.