RNA arbitrarily primed PCR survey of genes regulated by ToxR in the deep-sea bacterium Photobacterium profundum strain SS9.

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.

A phylogenetic analysis of microbial communities associated with methane hydrate containing marine fluids and sediments in the Cascadia margin (ODP site 892B).

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.

RecD function is required for high-pressure growth of a deep-sea bacterium.

A genomic library derived from the deep-sea bacterium Photobacterium profundum SS9 was conjugally delivered into a previously isolated pressure-sensitive SS9 mutant, designated EC1002 (E. Chi and D. H. Bartlett, J. Bacteriol. 175:7533-7540, 1993), and exconjugants were screened for the ability to grow at 280-atm hydrostatic pressure. Several clones were identified that had restored high-pressure growth. The complementing DNA was localized and in all cases found to possess strong homology to recD, a DNA recombination and repair gene. EC1002 was found to be deficient in plasmid stability, a phenotype also seen in Escherichia coli recD mutants. The defect in EC1002 was localized to a point mutation that created a stop codon within the recD gene. Two additional recD mutants were constructed by gene disruption and were both found to possess a pressure-sensitive growth phenotype, although the magnitude of the defect depended on the extent of 3' truncation of the recD coding sequence. Surprisingly, the introduction of the SS9 recD gene into an E. coli recD mutant had two dramatic effects. At high pressure, SS9 recD enabled growth in the E. coli mutant strain under conditions of plasmid antibiotic resistance selection and prevented cell filamentation. Both of these effects were recessive to wild-type E. coli recD. These results suggest that the SS9 recD gene plays an essential role in SS9 growth at high pressure and that it may be possible to identify additional aspects of RecD function through the characterization of this activity.

Heterologous expression in the Archaea: transcription from Pyrococcus furiosus gdh and mlrA promoters in Haloferax volcanii.

Multicopy plasmids containing the promoter regions for gdh and mlrA genes from Pyrococcus furiosus were propagated in Haloferax volcanii. High-level expression was detected from gdh promoter sequences, with transcription initiating at the same start-site as that found in P. furiosus. For mlrA, several transcripts were detected, with one initiating at the P. furiosus start-site; removal or disruption of the likely P. furiosus boxA element resulted in the disappearance of this transcript, indicating that these sequences were utilized by the H. volcanii RNA polymerase for initiation. This is the first demonstration of the utilization of promoters from a hyperthermophilic archaeon in a mesophilic haloarchaeon and provides further evidence for the unity of transcription processes in the domain Archaea.

Overexpression and characterization of a prolyl endopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.

The maltose-regulated mlr-2 gene from the hyperthermophilic archaeon Pyrococcus furiosus having homology to bacterial and eukaryal prolyl endopeptidase (PEPase) was cloned and overexpressed in Escherichia coli. Extracts from recombinant cells were capable of hydrolyzing the PEPase substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide (ZGPpNA) with a temperature optimum between 85 and 90 degrees C. Denaturing gel electrophoresis of purified PEPase showed that enzyme activity was associated with a 70-kDa protein, which is consistent with that predicted from the mlr-2 sequence. However, an apparent molecular mass of 59 kDa was obtained from gel permeation studies. In addition to ZGPpNA (K(Mapp) of 53 microM), PEPase was capable of hydrolyzing azocasein, although at a low rate. No activity was detected when ZGPpNA was replaced by substrates for carboxypeptidase A and B, chymotrypsin, subtilisin, and neutral endopeptidase. N-[N-(L-3-trans-Carboxirane-2-carbonyl)-L-Leu]-agmatine (E-64) and tosyl-L-Lys chloromethyl ketone did not inhibit PEPase activity. Both phenylmethylsulfonyl fluoride and diprotin A inhibited ZGPpNA cleavage, the latter doing so competitively (K(lapp) of 343 microM). At 100 degrees C, the enzyme displayed some tolerance to sodium dodecyl sulfate treatment. Stability of PEPase over time was dependent on protein concentration; at temperatures above 65 degrees C, dilute samples retained most of their activity after 24 h while the activity of concentrated preparations diminished significantly. This decrease was found to be due, in part, to autoproteolysis. Partially purified PEPase from P. furiosus exhibited the same temperature optimum, molecular weight, and kinetic characteristics as the enzyme overexpressed in E. coli. Extracts from P. furiosus cultures grown in the presence of maltose were approximately sevenfold greater in PEPase activity than those grown without maltose. Activity could not be detected in clarified medium obtained from maltose-grown cultures. We conclude that mlr-2, now called prpA, encodes PEPase; the physiological role of this protease is presently unknown.