Methane hydrates represent an enormous carbon and energy source in many low temperature deep marine sediments. However, little information is available concerning the nature of the microbial communities associated with these structures. Here, we describe a phylogenetic analysis based on ribosomal DNA (rDNA) sequences obtained from sediment and fluid samples present in a region of gas hydrate formation in shallow sediments within the Cascadia margin in and around Ocean Drilling Program (ODP) Site 892B. Our studies detected diverse sulfur-utilizing microbes, methanogens, methanotrophs, and non-thermophilic members of the kingdom Crenarchaeota. This is the first culture-independent phylogenetic analysis of a gas hydrate habitat.

A proteomic survey of the halophilic archaeon Haloferax volcanii was performed by comparative two-dimensional gel electrophoresis in order to determine the molecular effects of salt stress on the organism. Cells were grown under optimal (2.1 M) and high (3.5 M) NaCl conditions. From this analysis, over 44 protein spots responsive to these conditions were detected. These spots were excised, digested in-gel with trypsin, subjected to QSTAR tandem mass spectrometry (LC/MS/MS) analysis, and identified by comparing the MS/MS-derived peptide sequence to that deduced from the H. volcanii genome. Approximately 40 % of the proteins detected (18 in total) displayed differential abundance based on the detection of at least two peptide fragments per protein and overall MOWSE scores of ¢75 per protein. All of these identified proteins were either uniquely present or 2.3-to 26-fold higher in abundance under one condition compared to the other. The majority of proteins identified in this study were preferentially displayed under optimal salinity and primarily involved in translation, transport and metabolism. However, one protein of interest whose transcript levels were confirmed in these studies to be upregulated under high salt conditions was identified as a homologue of the phage shock protein PspA. The pspA gene belongs to the psp stress-responsive regulon commonly found among Gram-negative bacteria where its transcription is stimulated by a wide variety of stressors, including heat shock, osmotic shock and prolonged stationary-phase incubation. Homologues of PspA are also found among the genomes of cyanobacteria, higher plants and other Archaea, suggesting that this protein may retain some aspects of functional conservation across the three domains of life. Given its integral role in sensing a variety of membrane stressors in bacteria, these results suggest that PspA may play an important role in hypersaline adaptation in H. volcanii.

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An enigmatic feature of microbial evolution is the emergence of programmed cell death (PCD), a genetically controlled form of cell suicide triggered by environmental stimuli. Archaea, the second major prokaryotic domain of life, have been notably absent from the PCD inheritance discussion, due to a lack of genetic homologues. Using the model haloarchaeon Haloferax volcanii, we document extremely high caspase-specific activity and expression of immunoreactive proteins to human caspase 8 antisera, both of which were induced by salt stress and death and were abolished by in vivo addition of a broad-spectrum caspase inhibitor. Caspase inhibition severely impaired cell growth under low and high salt stress, demonstrating a critical role in the cellular stress response. In silico analysis of the H. volcanii proteome identified a subset of 18 potential target proteins containing a signature tetrapeptide caspase cleavage motif (IETD), some with putative roles in allosteric regulation, signal transduction, osmotic stress and cell communication. Detection of similarly high activity and expression in other haloarchaea (Halorubrum and Haloarcula) and in diverse members of Euryarchaeota (the methanogen Methanosarcina acetivorans and the hyperthermophile Pyrococcus furiosus) and Crenarchaeota (the acidophile Sulfolobus solfataricus) argue for a broad representation within the archaeal domain. By playing a role in normal cell function, caspase-like proteases in Archaea appear to have co-evolved with other metabolic pathways, broadening their biological roles beyond apoptosis and cell death.

We are currently investigating the role of ToxR-mediated gene regulation in Photobacterium profundum strain SS9. SS9 is a moderately piezophilic ("pressure loving") psychrotolerant marine bacterium belonging to the family Vibrionaceae. In Vibrio cholerae, ToxR is a transmembrane DNA binding protein involved in mediating virulence gene expression in response to various environmental signals. A homolog to V. cholerae ToxR that is necessary for pressure-responsive gene expression of two outer membrane protein-encoding genes was previously found in SS9. To search for additional genes regulated by ToxR in SS9, we have used RNA arbitrarily primed PCR (RAP-PCR) with wild-type and toxR mutant strains of SS9. Seven ToxR-activated transcripts and one ToxR-repressed transcript were identified in this analysis. The cDNAs corresponding to these partial transcripts were cloned and sequenced, and ToxR regulation of their genes was verified. The products of these genes are all predicted to fall into one or both of two functional categories, those whose products alter membrane structure and/or those that are part of a starvation response. The transcript levels of all eight newly identified genes were also characterized as a function of hydrostatic pressure. Various patterns of pressure regulation were observed, indicating that ToxR activation or repression cannot be used to predict the influence of pressure on gene expression in SS9. These results provide further information on the nature of the ToxR regulon in SS9 and indicate that RAP-PCR is a useful approach for the discovery of new genes under the control of global regulatory transcription factors.