Paul A. Rochelle

Desulfovibrio profundus sp. nov., a Novel Barophilic Sulfate-Reducing Bacterium from Deep Sediment Layers in the Japan Sea

Several strains of a strictly anaerobic, vibrio-shaped or sigmoid, sulfate-reducing bacterium were isolated from deep marine sediments (depth, 80 and 500 m) obtained from the Japan Sea (Ocean Drilling Program Leg 128, site 798B). This bacterium was identified as a member of the genus Desulfovibrio on the basis of the presence of desulfoviridin and characteristic phospholipid fatty acids (iso 17:1 omega 7 and iso 15:0), the small number of growth substrates utilized (lactate, pyruvate, and hydrogen), and 16S rRNA gene sequence analysis data. Based on data for 16S rRNA sequences (1,369 bp), all of the Japan Sea strains were identical to each other and were most closely related to Desulfovibrio salexigens and less closely related to Desulfovibrio desulfuricans (levels of similarity, 91 and 89.6%, respectively). There were, however, considerable phenotypic differences (in temperatures, pressures, and salinities tolerated, growth substrates, and electron donors) between the Japan Sea isolates and the type strains of previously described desulfovibrios, as well as important differences among the Japan Sea isolates. The Japan Sea isolates were active (with sulfide production) over a wide temperature range (15 to 65 degrees C) and a wide sodium chloride concentration range (0.2 to 10%) (moderate halophile), and they were barophiles that were active at pressures up to about 40 MPa (400 atm). The optimum pressures for activity corresponded to the calculated pressures at the depths from which the organisms were isolated (for isolates obtained at depths of 80 and 500 m the optimum activities occurred at 10 and 15 MPa, respectively [100 and 150 atm, respectively]). This confirms that the organisms came from deep sediments and indicates that they are well-adapted for deep sediment conditions, which is consistent with other characteristics (utilization of hydrogen, fermentation, and utilization of ferric iron and organic sulfonates as electron acceptors). We propose that Japan Sea isolate 500-1 is the type strain of a new species, Desulfovibrio profundus.

BACTERIAL POPULATIONS AND PROCESSES IN SEDIMENTS CONTAINING GAS HYDRATES (ODP LEG 146: CASCADIA MARGIN)

Abstract Bacterial populations and activity were quantified at three sites in the Cascadia Margin accretionary wedge, off the West Canadian/American coast (ODP Leg 146). At two sites sediments contained gas hydrates, Site 889/890 had a discrete zone of hydrate approximately 10 m above a bottom simulating reflector (BSR) at 225 m below sea floor (mbsf) and Site 892 had disseminated hydrate in the upper 20 mbsf and a BSR at 74 mbsf. Site 888 was a control site without gas hydrates. The control site (888) and top approximately 90 mbsf of Site 889/890 had bacterial distributions similar to previous Pacific Ocean sites. In the upper approximately 30 m of Site 892, however, bacterial populations were much lower, suggesting inhibition by the high concentrations of H 2 S within the hydrate zone. Below this depth bacterial populations rose to concentrations consistent with other sites. The control site was dominated by SO 4 reduction and rates of CH 4 oxidation in the top 90 m were low (0.002–0.033 nmol cm −3 d −1 ). At Site 889/890 bacterial populations and activity were stimulated in the discrete hydrate zone. CH 4 oxidation rates increased in the middle of this zone to 134.5 nmol cm −3 d −1 (ca. 9 times the average rate at other depths), resulting in a significant (× 10) increase in the total bacterial population. The anaerobic process(es) responsible for this oxidation remain unclear, despite SO 4 -reducing bacteria, previously associated with CH 4 oxidation, also being stimulated in this zone. Fluid flux into accretionary wedge sediments may be an important process in providing electron acceptors to maintain these relatively high rates of CH 4 oxidation. This first microbiological study of gas hydrates indicates that bacterial processes are influenced by gas and fluid venting, and they play a major role in geochemical changes within these deep (> 200 mbsf) sediments.

An Accurate Method for Estimating Sizes of Small and Large Plasmids and DNA Fragments by Gel Electrophoresis

Several regression methods were tested for estimating the sizes of a wide range of plasmids (1.37-312 MDa) and restriction fragments (2.2-14.2 MDa) by agarose gel electrophoresis. The most accurate and least variable method was the multiple regression of log10 molecular size against log10 relative mobility and the reciprocal square root of the relative mobility. This method gave a good fit to all the data with low percentage errors of the molecular size estimates (less than or equal to 3.0 +/- 1.5%). It is suggested that with this method the molecular size of unknown plasmids can be accurately estimated using the plasmids from Escherichia coli V517 and E. coli IR713 as standards.

Factors Affecting Conjugal Transfer of Plasmids Encoding Mercury Resistance from Pure Cultures and Mixed Natural Suspensions of Epilithic Bacteria

Sixty-five pure cultures of epilithic bacteria were examined for their ability to transfer mercury resistance to Pseudomonas aeruginosa; five isolates transferred plasmids encoding mercury resistance with frequencies ranging from 8.4 x 10(-8) to 2.8 x 10(-3) per recipient. Two of the plasmids, pQM3 and pQM4, encoded narrow-spectrum mercury resistance, pQM3 also encoded streptomycin resistance, and both plasmids were broad host range. Maximum transfer frequencies of epilithic plasmids from pure cultures occurred over the range 10-25 degrees C at 3.5 g C l-1 and with donor to recipient ratios of 0.4-30. Transfer occurred over a range of pH values (pH 5.0-8.0) but the effect of pH was most significant at non-optimal temperature. Anaerobiosis inhibited transfer of one epilithic plasmid, pQM1, but not that of pQM3. Plasmids encoding mercury resistance were also transferred from mixed natural suspensions of epilithic bacteria (MNS) to Pseudomonas spp. on agar in the laboratory. Transfer from MNS occurred over a wide range of environmentally relevant conditions with maximum frequencies (2 x 10(-5) per recipient) after 24 h, at 25 degrees C, pH 5.5-8.0 and on a medium containing 10 g C l-1. The optimal initial cell density of MNS and recipient was 1.7 x 10(5) c.f.u. cm-2 and highest frequencies were obtained with donor to recipient ratios ranging from 1.2 x 10(-1) to 1.7 x 10(-3). Most of the plasmids (54%) from MNS transferred from their original P. aeruginosa transconjugants to a Pseudomonas putida strain, with frequencies ranging from 1.1 x 10(-6) to greater than 1.0 x 10(-1) per recipient. The majority (80%) of the plasmids were larger than 300 kb and all of these large plasmids encoded UV resistance in addition to mercury resistance. Twenty-one plasmids greater than 300 kb were analysed by restriction digests and were shown to be similar, with only minor structural alterations. One of these alterations was associated with the acquisition of streptomycin resistance. Overall, these results suggest that the epilithic bacteria examined possess the potential to transfer mercury resistance within the epilithon under a wide range of environmentally relevant conditions.

Occurrence, Transfer and Mobilization in Epilithic Strains of Acinetobacter of Mercury-resistance Plasmids Capable of Transformation

A 7.8 kb plasmid (pQM17) encoding mercury resistance was isolated from two epilithic strains of Acinetobacter calcoaceticus. The plasmid had a broad host range when mobilized by RP1, transferring into Pseudomonas aeruginosa, P. putida, P. fluorescens, Escherichia coli, Proteus vulgaris and Chromobacterium sp. with frequencies ranging from 5.3 x 10(-9) to 4.6 x 10(-4) per recipient. The plasmid could be transferred into A. calcoaceticus BD413 using intact cells of donor and recipient bacteria (i.e. natural transformation) and there was a broad temperature optimum (14-37 degrees C) for transformation. Transformation was as efficient in liquid matings as on plates but there was no effect of pH in the range 5.6-7.9. Maximum transformation frequencies were obtained after 24 h on agar plates containing 3.5-10 g C 1-1 with donor to recipient ratios ranging from 6 to 415.

The use of agarose wedge-gel electrophoresis for resolving both small and large naturally occurring plasmids

A wedge‐shaped, horizontal agarose gel gave better electrophoretic resolution of both large and small plasmids than conventional linear gels. The wedge‐gel increased mobility and separation of the larger plasmids whilst retarding the mobility of the smaller plasmids and tightening the bands. The linearity of the relationship between log10 molecular size and log10 relative mobility, for a range of plasmids from 2.1 to 221 kb, was increased. Therefore, estimations of plasmid sizes are more accurate using a wedge‐gel than with conventional gels.