The transcriptomic response of the coral Acropora digitifera to a competent Symbiodinium strain: the symbiosome as an arrested early phagosome.

Despite the ecological significance of the relationship between reef-building corals and intracellular photosynthetic dinoflagellates of the genus Symbiodinium, very little is known about the molecular mechanisms involved in its establishment. Indeed, microarray-based analyses point to the conclusion that host gene expression is largely or completely unresponsive during the establishment of symbiosis with a competent strain of Symbiodinium. In this study, the use of Illumina RNA-Seq technology allowed detection of a transient period of differential expression involving a small number of genes (1073 transcripts; <3% of the transcriptome) 4 h after the exposure of Acropora digitifera planulae to a competent strain of Symbiodinium (a clade B strain). This phenomenon has not previously been detected as a consequence of both the lower sensitivity of the microarray approaches used and the sampling times used. The results indicate that complex changes occur, including transient suppression of mitochondrial metabolism and protein synthesis, but are also consistent with the hypothesis that the symbiosome is a phagosome that has undergone early arrest, raising the possibility of common mechanisms in the symbiotic interactions of corals and symbiotic sea anemones with their endosymbionts.

AmAMP1 from Acropora millepora and damicornin define a family of coral-specific antimicrobial peptides related to the Shk toxins of sea anemones.

A candidate antimicrobial peptide (AmAMP1) was identified by searching the whole genome sequence of Acropora millepora for short (<125AA) cysteine-rich predicted proteins with an N-terminal signal peptide but lacking clear homologs in the SwissProt database. It resembled but was not closely related to damicornin, the only other known AMP from a coral, and was shown to be active against both Gram-negative and Gram-positive bacteria. These proteins define a family of AMPs present in corals and their close relatives, the Corallimorpharia, and are synthesised as preproproteins in which the C-terminal mature peptide contains a conserved arrangement of six cysteine residues. Consistent with the idea of a common origin for AMPs and toxins, this Cys motif is shared between the coral AMPs and the Shk neurotoxins of sea anemones. AmAMP1 is expressed at late stages of coral development, in ectodermal cells that resemble the "ganglion neurons" of Hydra, in which it has recently been demonstrated that a distinct AMP known as NDA-1 is expressed.

Isolation of lytic bacteriophage against Vibrio harveyi.

AIMS: The isolation of lytic bacteriophage of Vibrio harveyi with potential for phage therapy of bacterial pathogens of phyllosoma larvae from the tropical rock lobster Panulirus ornatus. METHODS AND RESULTS: Water samples from discharge channels and grow-out ponds of a prawn farm in northeastern Australia were enriched for 24 h in a broth containing four V. harveyi strains. The bacteriophage-enriched filtrates were spotted onto bacterial lawns demonstrating that the bacteriophage host range for the samples included strains of V. harveyi, Vibrio campbellii, Vibrio rotiferianus, Vibrio parahaemolyticus and Vibrio proteolyticus. Bacteriophage were isolated from eight enriched samples through triple plaque purification. The host range of purified phage included V. harveyi, V. campbellii, V. rotiferianus and V. parahaemolyticus. Transmission electron microscope examination revealed that six purified phage belonged to the family Siphoviridae, whilst two belonged to the family Myoviridae. The Myoviridae appeared to induce bacteriocin production in a limited number of host bacterial strains, suggesting that they were lysogenic rather than lytic. A purified Siphoviridae phage could delay the entry of a broth culture of V. harveyi strain 12 into exponential growth, but could not prevent the overall growth of the bacterial strain. CONCLUSIONS: Bacteriophage with lytic activity against V. harveyi were isolated from prawn farm samples. Purified phage of the family Siphoviridae had a clear lytic ability and no apparent transducing properties, indicating they are appropriate for phage therapy. Phage resistance is potentially a major constraint to the use of phage therapy in aquaculture as bacteria are not completely eliminated. SIGNIFICANCE AND IMPACT OF THE STUDY: Phage therapy is emerging as a potential antibacterial agent that can be used to control pathogenic bacteria in aquaculture systems. The development of phage therapy for aquaculture requires initial isolation and determination of the bacteriophage host range, with subsequent creation of suitable phage cocktails.

Microbial diversity of mid-stage Palinurid phyllosoma from Great Barrier Reef waters.

AIMS: This study aimed to determine the bacterial community associated with wild-caught, mid-stage larvae of spiny lobsters (Palinuridae) in their native oligotrophic marine environment, and to compare their diversity and composition with communities associated with aquaculture-reared larvae of the tropical rock lobster Panulirus ornatus. METHODS AND RESULTS: Bacterial clone libraries constructed from wild P. ornatus (two libraries) and Panulirus penicillatus (one library) larvae (phyllosoma) revealed a dominance of alpha-proteobacterial sequences, with Sulfitobacter spp.-affiliated sequences dominating both P. ornatus libraries and constituting a major portion of the P. penicillatus library. Vibrio-related sequences were rarely detected from wild phyllosoma clone libraries in contrast to similar studies of aquaculture-reared animals. Scanning electron microscopy analysis revealed low levels of bacterial colonization on the external carapace of wild phyllosoma, again in contrast to aquaculture-reared animals, which are often colonized with filamentous bacteria (mainly Thiothrix sp.) that compromise their health. Fluorescence in situ hybridization of sectioned wild phyllosoma tissue displayed low overall abundance of bacteria within the tissue and on external surfaces, with alpha-, beta-, and gamma-Proteobacteria being confirmed as members of this bacterial community. CONCLUSIONS: The consistency in predominant clone sequences retrieved from the three libraries indicated a conserved microbiota associated with wild phyllosoma. In addition, the observed differences in the microbial composition and load of reared and wild phyllosoma are indicative of the different environments in which the animals live. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial disease during early larval stages is a major constraint currently hindering the development of an aquaculture industry for the ornate rock lobster P. ornatus. Knowledge of the microbial community associated with wild animals will be advantageous for the identification of bacteria that may promote animal health.

Newly characterized distinct phases of the coral disease 'atramentous necrosis' on the Great Barrier Reef.

Previously undetected earlier phases of the coral disease 'atramentous necrosis' are documented and described. New observations relating to the occurrence of initial stages and progression of the disease are reported, and potential cause(s) are examined. In direct contrast to earlier published findings, temperature data indicated that occurrence of early bleached stages of atramentous necrosis is not correlated with warmer water temperatures; however, the relationship between temperature and disease prevalence is still unclear.

Comparison of pmoA PCR primer sets as tools for investigating methanotroph diversity in three Danish soils.

Three particulate methane monooxygenase PCR primer sets (A189-A682, A189-A650, and A189-mb661) were investigated for their ability to assess methanotroph diversity in soils from three sites, i.e., heath, oak, and sitka, each of which was capable of oxidizing atmospheric concentrations of methane. Each PCR primer set was used to construct a library containing 50 clones from each soil type. The clones from each library were grouped by restriction fragment length polymorphism, and representatives from each group were sequenced and analyzed. Libraries constructed with the A189-A682 PCR primer set were dominated by amoA-related sequences or nonspecific PCR products with nonsense open reading frames. The primer set could not be used to assess methanotroph diversity in these soils. A new pmoA-specific primer, A650, was designed in this study. The A189-A650 primer set demonstrated distinct biases both in clone library analysis and when incorporated into denaturing gradient gel electrophoresis analysis. The A189-mb661 PCR primer set demonstrated the largest retrieval of methanotroph diversity of all of the primer sets. However, this primer set did not retrieve sequences linked with novel high-affinity methane oxidizers from the soil libraries, which were detected using the A189-A650 primer set. A combination of all three primer sets appears to be required to examine both methanotroph diversity and the presence of novel methane monooxygenase sequences.

Microenvironments and microbial community structure in sediments.

The aim of this study was to explore the potential of a combined chemical and microbiological approach as part of a study of organic carbon oxidation processes in sediments. An assessment of microbiological diversity using molecular techniques was carried out in combination with high resolution chemical measurements at the sediment-water interface of a coastal lagoon affected by eutrophication in autumn 2000. There was a 0.2 mm overlap between the O2 and H2S profiles. pH showed a maximum just above the sediment-water interface coinciding with an oxygen maximum, suggesting photosynthetic activity, and a minimum coinciding with the O2-H2S interface. The redox potential was high in bottom water and surface sediment, reflecting the presence of oxygen and oxides, and reached low values after a step-wise decrease at -18 mm. Reduction of Fe occurred within the biofilm at the O2-H2S interface and was mostly due to reduction by H2S. The elevated concentrations of dissolved Mn in the oxic water may have been caused either by in situ production within organic aggregates or lateral water flow from sites nearby at which Mn2+ diffuses out of the sediment. Sequences related to sulphur chemolitotrophs were retrieved from the biofilm samples, which is consistent with the small overlap between O2 and H2S observed in this biofilm. Although the resolution of techniques used was different, sequencing results were consistent with chemical data in delineating the same horizons according to redox, pH or ecological properties.

The use of PCR to aid in the rapid identification of Vibrio harveyi isolates.

AIMS: Vibrio harveyi is an important pathogen, causing potential devastation to marine aquaculture. This organism, however, is extremely difficult to identify because it is phenotypically diverse. Biochemical identification can involve many tests and take weeks to perform. The aim of this work is to develop a PCR that can reduce the number of biochemical tests, and the time taken, to get a definitive identification of this organism. METHODS AND RESULTS: The PCR was developed using 16S rDNA sequences from a number of V. harveyi strains, and other vibrios. The described test gave positive results for all strains of V. harveyi tested. However, some strains of V. alginolyticus also gave positive results and a small number of biochemical tests were required to differentiate between these two species. This indicated that preisolation of the bacteria was needed and therefore the test was not applicable to the testing of mixed populations directly. CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: The duration of identification of this species was significantly reduced from a number of weeks to a few days. Hence, diagnosis of affected animals will be faster and earlier treatment can be administered which may increase the survival rate from vibriosis.

Characterisation of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin LR.

A novel pathway for degradation of the cyanobacterial heptapeptide hepatotoxin microcystin LR was identified in a newly isolated Sphingomonas sp. (Bourne et al. 1996 Appl. Environ. Microbiol. 62: 4086-4094). We now report the cloning and molecular characterisation of four genes from this Sphingomonas sp. that exist on a 5.8-kb genomic fragment and encode the three hydrolytic enzymes involved in this pathway together with a putative oligopeptide transporter. The heterologously expressed degradation pathway proteins are enzymatically active. Microcystinase (MlrA), the first enzyme in the degradative pathway, is a 336-residue endopeptidase, which displays only low sequence identity with a hypothetical protein from Methanobacterium thermoautotrophicum. Inhibition of microcystinase by EDTA and 1,10-phenanthroline suggests that it is a metalloenzyme. The most likely residues that could potentially chelate an active-site transition metal ion are in the sequence HXXHXE, which would be unique for a metalloproteinase. Situated immediately downstream of mlrA with the same direction of transcription is a gene mlrD, whose conceptual translation (MlrD, 442 residues) shows significant sequence identity and similar potential transmembrane spanning regions to the PTR2 family of oligopeptide transporters. A gene mlrB is situated downstream of the mlrA and mlrD genes, but transcribed in the opposite direction. The gene encodes the enzyme MlrB (402 residues) which cleaves linear microcystin LR to a tetrapeptide degradation product. This enzyme belongs to the "penicillin-binding enzyme" family of active site serine hydrolases. The final gene in the cluster mlrC, is located upstream of the mlrA gene and is transcribed in the opposite direction. It codes for MlrC (507 residues) which mediates further peptidolytic degradation of the tetrapeptide. This protein shows significant sequence identity to a hypothetical protein from Streptomyces coelicolor. It is suspected to be a metallopeptidase based on inhibition by metal chelators. It is postulated on the basis of comparison with other microorganisms that the genes in this cluster may all be involved in cell wall peptidoglycan cycling and subsequently act fortuitously in hydrolysis of microcystin LR.

Degradation of the cyanobacterial hepatotoxin microcystin by aquatic bacteria.

Bacterial degradation of the cyanobacterial cyclic peptide hepatotoxin microcystin was confirmed in natural waters and by isolated laboratory strains. Degradation of 1 mg L-1 microcystin LR typically began 2-8 days after addition to surface water samples. At concentrations greater than 1 mg L-1 there was an initial slow removal of microcystin LR, rather than a distinct lag (or conditioning) phase, before rapid degradation commenced. The lag phase was absent upon re-addition of microcystin LR to the water. Both single strains and mixed bacterial cultures capable of degrading microcystin LR were isolated from surface water samples. One single strain isolated was a gram-negative rod and appeared to be a Pseudomonas sp., although standard taxonomic tests have given inconclusive results. Degradative activity was mostly intracellular and equally active against microcystin LR and RR, but not against nodularin.

Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR.

An isolated bacterium, identified as a new Sphingomonas species, was demonstrated to contain a novel enzymatic pathway which acted on microcystin LR, the most common cyanobacterial cyclic peptide toxin. Degradation of microcystin LR was mediated by at least three intracellular hydrolytic enzymes. The use of classic protease inhibitors allowed (i) the classification of these enzymes into general protease families and (ii) the in vitro accumulation of otherwise transient microcystin LR degradation products. The initial site of hydrolytic cleavage of the parent cyclic peptide by an enzyme that we designate microcystinase is at the 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda)-Arg peptide bond. Two intermediates of microcystin LR enzymatic degradation have been identified; one is linearized (acyclo-) microcystin LR, NH2-Adda-Glu(iso)-methyldehydroalanine-Ala-Leu-beta-methylas partate-Arg-OH, and the other is the tetrapeptide NH2-Adda-Glu(iso)-methyldehydroalanine-Ala-OH. The intermediate degradation products were less active than the parent cyclic peptide; the observed 50% inhibitory concentrations for crude chicken brain protein phosphatase were 0.6 nM for microcystin LR, 95 nM for linear LR, and 12 nM for the tetrapeptide. These linear peptides were nontoxic to mice at doses up to 250 micrograms/kg. Ring opening of the potent hepatotoxin microcystin LR by bacterial microcystinase effectively renders the compound nontoxic by dramatically reducing the interaction with the target protein phosphatase.