Low Pressure Tolerance by Methanogens in an Aqueous Environment: Implications for Subsurface Life on Mars.

The low pressure at the surface of Mars (average: 6 mbar) is one potentially biocidal factor that any extant life on the planet would need to endure. Near subsurface life, while shielded from ultraviolet radiation, would also be exposed to this low pressure environment, as the atmospheric gas-phase pressure increases very gradually with depth. Few studies have focused on low pressure as inhibitory to the growth or survival of organisms. However, recent work has uncovered a potential constraint to bacterial growth below 25 mbar. The study reported here tested the survivability of four methanogen species (Methanothermobacter wolfeii, Methanosarcina barkeri, Methanobacterium formicicum, Methanococcus maripaludis) under low pressure conditions approaching average martian surface pressure (6 mbar - 143 mbar) in an aqueous environment. Each of the four species survived exposure of varying length (3 days - 21 days) at pressures down to 6 mbar. This research is an important stepping-stone to determining if methanogens can actively metabolize/grow under these low pressures. Additionally, the recently discovered recurring slope lineae suggest that liquid water columns may connect the surface to deeper levels in the subsurface. If that is the case, any organism being transported in the water column would encounter the changing pressures during the transport.

Hydrogen consumption by methanogens on the early Earth.

It is possible that the first autotroph used chemical energy rather than light. This could have been the main source of primary production after the initial inventory of abiotic organic material had been depleted. The electron acceptor most readily available for use by this first chemoautotroph would have been CO2. The most abundant electron donor may have been H2 that would have been outgassing from volcanoes at a rate estimated to be as large as 10(12) moles yr-1, as well as from photo-oxidation of Fe+2. We report here that certain methanogens will consume H2 down to partial pressures as low as 4 Pa (4 x 10(-5) atm) with CO2 as the sole carbon source at a rate of 0.7 ng H2 min-1 microgram-1 cell protein. The lower limit of pH2 for growth of methanogens can be understood on the basis that the pH2 needs to be high enough for one ATP to be synthesized per CO2 reduced. The pH2 values needed for growth measured here are consistent with those measured by Stevens and McKinley for growth of methanogens in deep basalt aquifers. H2-consuming autotrophs are likely to have had a profound effect on the chemistry of the early atmosphere and to have been a dominant sink for H2 on the early Earth after life began rather than escape from the Earth's atmosphere to space.

Martian "microfossils" in lunar meteorites?

One of the five lines of evidence used by McKay et al. (1996) for relic life in the Martian meteorite Allan Hills (ALH) 84001 was the presence of objects thought to be microfossils. These ovoid and elongated forms are similar to structures found in terrestrial rocks and described as "nanobacteria" (Folk, 1993; McBride et al., 1994). Using the same procedures and apparatus as McKay et al. (1996), we have found structures on internal fracture surfaces of lunar meteorites that cannot be distinguished from the objects described on similar surfaces in ALH 84001. The lunar surface is currently a sterile environment and probably always has been. However, the lunar and Martian meteorites share a common terrestrial history, which includes many thousands of years of exposure to Antarctic weathering. Although we do not know the origin of these ovoid and elongated forms, we suggest that their presence on lunar meteorites indicates that the objects described by McKay et al. (1996) are not of Martian biological origin.

Transformation of fluoride resistance genes in Streptococcus mutans.

To study the nature of fluoride resistance in Streptococcus mutans, we transformed DNA extracted from fluoride-resistant mutants of S. mutans GS-5 into fluoride-sensitive cells of the same strain. Transformation with DNA from first-step mutants produced transformants with resistance to either 600 or 1,000 micrograms of sodium fluoride per ml, both of which are within the first-step resistance range (400 to 1,000 micrograms/ml). In five of six of these transformation experiments, however, the transformant resistance levels were greater than those of their respective DNA donors. Transformation with DNA from a second-step mutant resistant to 1,600 micrograms/ml resulted in transformants resistant to 600 micrograms/ml, similar to some transformants receiving DNA from first-step mutants. When a second-step mutant resistant to 3,000 micrograms/ml was used as a DNA donor, four different levels of resistance were seen in the transformants (600, 1,000, 1,500, and 2,000 micrograms/ml). In many cases, the growth rates of the transformants (first and second step) were faster than those of the DNA donors. Additionally, many of the transformants demonstrated abrupt shifts in growth rates at relatively low culture densities.

Effects of pH on expression of sodium fluoride resistance in Streptococcus mutans.

Stable fluoride-resistant mutants of Streptococcus mutans GS-5 were isolated on Todd-Hewitt agar by a step-wise selection procedure. Resistance is defined here as the ability to form colonies in 48 hr. First-step mutants demonstrated six different levels of resistance, ranging from 400 to 1000 micrograms/mL sodium fluoride. Second-step mutants demonstrated two levels of resistance, one at 1600 and the highest at 3000 micrograms/mL sodium fluoride. All mutants (originally isolated at pH 7.2) were tested for fluoride resistance at pH 5 and 6. At these lower pH values, all of the mutants demonstrated resistance to fluoride when compared with the parent strain, but at much-reduced levels.

Penicillin-induced lysis of Streptococcus mutans.

Treatment of Streptococcus mutans GS-5 cells with concentrations of penicillin G within a relatively narrow range resulted in substantial lysis. This penicillin-induced lysis was dependent upon cell density and pH of the lysis medium. Other oral streptococci (Streptococcus sobrinus, Streptococcus rattus, and Streptococcus cricetus) also demonstrated substantial levels of penicillin-induced lysis under appropriate conditions. Lesser degrees of lysis were seen in a related organism, Streptococcus ferus.

Effects of cell wall inhibitors on cell division in Streptococcus mutans.

The addition of inhibitors of cell wall biosynthesis to exponential-phase cultures of Streptococcus mutans may do one of three things, depending on the concentrations used: (i) prevent cell division at a time coincident with the onset of chromosome replication, (ii) prevent cell division later in the cell cycle coincident with or near completion of septation, or (iii) lead to limited cell lysis. The relative tolerance of S. mutans to inhibitors of cell wall biosynthesis may be due to the fact that S. mutans cultures treated with low levels of cell wall antibiotics seem to be blocked at a stage before initiation of autolytic activity, whereas cultures treated with high levels of these antibiotics seem to be blocked after termination of the autolytic phase. Thus, the cells escape the lytic death that is seen in other streptococci exposed to inhibitors of cell wall biosynthesis.

Regulation of the growth rate of Streptococcus mutans.

Streptococcus mutans strain GS-5 was grown under a variety of environmental conditions in order to achieve different balanced growth rates. A range of growth rates could be obtained using limitations in the concentrations of glutamate/glutamine, leucine, or valine. Different balanced growth rates were also obtained when cells were grown in a variety of carbon sources. Using glucose, cellobiose, amygdalin, maltose, mannitol, and galactose, reproducible doubling times were obtained ranging from 61 to 226 min.

Glycerol incorporation in certain oral streptococci.

Cells of 30 different strains of oral streptococci were grown in a chemically defined medium supplemented with [14C]glycerol to determine their ability to incorporate the labeled glycerol. Of the five species tested, only two, the rat-type strains (Streptococcus rattus) and strains isolated from wild rats (Streptococcus ferus), were able to incorporate the nonfermentable substrate, glycerol. For those strains capable of incorporating glycerol, the amount incorporated ranged from 0.15 to 0.43% of the cellular dry weight and followed simple saturation kinetics. The amount of glycerol incorporated depended solely on the concentration of glycerol in the growth medium. As a result, cultures exposed to low concentrations of glycerol ceased incorporation of the labeled glycerol before cessation of exponential growth.