The coral holobiont includes the coral, zooxanthellae, fungi, endolithic algae, and > 30 species of Bacteria. Using culture-independent techniques, we now show that Archaea are also abundant and widespread on corals. Sequence analyses of Archaea on 3 species of Caribbean corals revealed that coral-associated Archaea are novel, diverse, and include representatives from both the Crenarchaeota and Euryarchaeota. Unlike zooxanthellae and Bacteria, the Archaea do not appear to form species-specific associations with reef-building corals. Fluorescent in situ hybridizations with peptide nucleic acid (PNA) probes showed that Archaea were present at >10 7 cells cm -2 on Porites astreoides, comprising nearly half of the prokaryotic community. This study and one by Kellogg (Mar Ecol Prog Ser 273:81-88) show that Archaea are abundant, diverse, and potentially important components of the coral holobiont.

SummaryWhiite band disease type I (WBD I) has been a major cause of thie dramatic deciine of Acroporid corai popuiations thiroughout the Caribbean during the iast two decades, yet the aetioiogicai agent of this disease is unl

The vast majority of novel DNA sequences deposited in the databases now comes from environmental phage DNA sequences. Methods are presented for the cloning and sequencing of phage DNA that might otherwise be lethal to bacterial host vectors or contain modified DNA bases that prevent standard cloning of such sequences. In addition, methods are presented for the isolation of viral particles directly from soil and sediment environmental samples or from large volumes of environmental water samples. The viral particles are then purified by cesium-chloride density centrifugation followed by DNA extraction. This purified viral metagenomic DNA is then used for cloning and sequencing.

Many human diseases are caused by pathogens that produce exotoxins. The genes that encode these exotoxins are frequently encoded by mobile DNA elements such as plasmids or phage. Mobile DNA elements can move exotoxin genes among microbial hosts, converting avirulent bacteria into pathogens. Phage and bacteria from water, soil, and sediment environments represent a potential reservoir of phage- and plasmid-encoded exotoxin genes. The genes encoding exotoxins that are the causes of cholera, diphtheria, enterohemorrhagic diarrhea, and Staphylococcus aureus food poisoning were found in soil, sediment, and water samples by standard PCR assays from locations where the human diseases are uncommon or nonexistent. On average, at least one of the target exotoxin genes was detected in approximately 15% of the more than 300 environmental samples tested. The results of standard PCR assays were confirmed by quantitative PCR (QPCR) and Southern dot blot analyses. Agreement between the results of the standard PCR and QPCR ranged from 63% to 84%; and the agreement between standard PCR and Southern dot blots ranged from 50% to 66%. Both the cholera and shiga exotoxin genes were also found in the free phage DNA fraction. The results indicate that phage-encoded exotoxin genes are widespread and mobile in terrestrial and aquatic environments.

Recent advances in metagenomics research have generated a bounty of information that provides insight into the dynamic genetic exchange occurring between bacteriophage (phage) and their bacterial hosts. Metagenomic studies of the microbiomes from a variety of environments have shown that many of the genes sequenced are of phage origin. Among these genes are phage-encoded exotoxin genes. When phage that carry these genes infect an appropriate bacterial host, the bacterium undergoes lysogenic conversion, converting the bacterium from an avirulent strain to a pathogen that can cause human disease. Transfer of the exotoxin genes between bacteria has been shown to occur in marine environments, animal and human intestines and sewage treatment plants. Surprisingly, phage that encode exotoxin genes are commonly found in environments that lack the cognate bacteria commonly associated with the specific toxin-mediated disease and have been found to be associated with alternative environmental bacterial hosts. These findings suggest that the exotoxin genes may play a beneficial role for the bacterial host in nature, and that this environmental reservoir of exotoxin genes may play a role in the evolution of new bacterial pathogens.