In a typical Escherichia coli K-12 culture starved for glucose, 50% of the cells lose viability in ca. 6 days (Reeve et al., J. Bacteriol. 157:758-763, 1984). Inhibition of protein synthesis by chloramphenicol resulted in a more rapid loss of viability in glucose-starved E. coli K-12 cultures. The more chloramphenicol added (i.e., the more protein synthesis was inhibited) and the earlier during starvation it was added, the greater was its effect on culture viability. Chloramphenicol was found to have the same effect on a reLA strain as on an isogenic reU+ strain of E. coli. Addition of the amino acid analogs S-2-aminoethylcysteine, 7-azatryptophan, and p-fluorophenylalanine to carbon-starved cultures to induce synthesis of abnormal proteins had an effect on viability similar to that observed when 50 ,ug of chloramphenicol per ml was added at zero time for starvation. Both chloramphenicol and the amino acid analogs had delayed effects on viability, compared with their effects on synthesis of normal proteins. The need for protein synthesis did not arise from cryptic growth, since no cryptic growth of the starving cells was observed under the conditions used. From these and previous results obtained from work with peptidase-deficient mutants of E. coli K-12 and Salmonella typhimurium LT2 (Reeve et al., J. Bacteriol. 157:758-763, 1984), we concluded that a number of survival-related proteins are synthesized by E. coli K-12 cells as a response to carbon starvation. These proteins are largely synthesized during the early hours of starvation, but their continued activity is required for long-term survival.Starving bacteria are of interest in both an ecological and an applied context, and we have been interested in characterizing the physiological changes that occur during starvation (12,16,22). We have previously presented evidence (16) that protein degradation plays a role in the survival of carbon-starved bacteria. Mutants of Escherichia coli and Salmonella typhimurium that were deficient in protein degradation were found to possess a greatly decreased stability under conditions of carbon starvation. Although these mutants had no innate deficiency in their protein-synthetic machinery, they were unable to synthesize protein at the same rate as their corresponding wild-type strains during carbon starvation. This suggested that amino acids derived from protein degradation were utilized by these cells for new protein synthesis and that their increased susceptibility to carbon starvation originated from their inability to synthesize these proteins.In E. coli cells subjected to carbon starvation, the rate of protein synthesis drops to about 20% of the initial rate during the first hour of starvation (16) and then remains roughly constant for at least the next 47 h (unpublished data). We present evidence here that this protein synthesis is important for the survival of carbon-starved E. coli K-12; inhibition of normal protein synthesis during starvation greatly compromised survival. MATERIALS AND METHODSBacterial strains, ...

Sixteen marine isolates from a NORPAX cruise, which were transferred once on medium after initial isolation, survived nutrient deprivation for at least 8 months (longest period test). All but one isolate remained cellularly intact, although their sizes and shapes changed greatly, and all became smaller, decreasing in size from 40 to 79%. Three starvation-survival patterns were demonstrated, namely (i) an initial increase in viable cells followed by a decrease until a constant number was reached, (ii) an increase in viable cells until a constant number was reached, and (iii) a decrease in viable cells until a constant number was reached. One isolate from each starvation-survival pattern was starved for 8 months and then was tested in comparison with 4-month-starved Ant-300 for [14C]glutamic acid uptake, respiration, and incorporation. The response to glutamic acid was rapid and linear in each case. The data indicate that the starvation-survival of Ant-300 is not an anomalous situation and that open ocean bacteria can withstand nutrient deprivation for long periods of time and still retain the capacity for active metabolism, if the nutrients become available.

Numbers and activities of microorganisms were measured in the vadose zones of three arid and semiarid areas of the western United States, and the influence of water availability was determined. These low-moisture environments have vadose zones that are commonly hundreds of meters thick. The specific sampling locations chosen were on or near U.S. Department of Energy facilities: the Nevada Test Site (NTS), the Idaho National Engineering Laboratory (INEL), and the Hanford Site (HS) in southcentral Washington State. Most of the sampling locations were uncontaminated, but geologically representative of nearby locations with storage and/or leakage of waste compounds in the vadose zone. Lithologies of samples included volcanic tuff, basalt, glaciofluvial and fluvial sediments, and paleosols (buried soils). Samples were collected aseptically, either by drilling bore-holes (INEL and HS), or by excavation within tunnels (NTS) and outcrop faces (paleosols near the HS). Total numbers of microorganisms were counted using direct microscopy, and numbers of culturable microorganisms were determined using plate-count methods. Desiccation-tolerant microorganisms were quantified by plate counts performed after 24 h desiccation of the samples. Mineralization of (14)C-labeled glucose and acetate was quantified in samples at their ambient moisture contents, in dried samples, and in moistened samples, to test the hypothesis that water limits microbial activities in vadose zones. Total numbers of microorganisms ranged from log 4.5 to 7.1 cells g(-1) dry wt. Culturable counts ranged from log <2 to 6.7 CFU g(-1) dry wt, with the highest densities occurring in paleosol (buried soil) samples. Culturable cells appeared to be desiccation-tolerant in nearly all samples that had detectable viable heterotrophs. Water limited mineralization in some, but not all samples, suggesting that an inorganic nutrient or other factor may limit microbial activities in some vadose zone environments.

Levels of DNA, RNA, protein, ATP, glutathione, and radioactivity associated with [ 35 S]methionine-labeled cellular protein were estimated at various times during the starvation-survival process of a marine psychrophilic heterotrophic Vibrio sp., Ant-300. Values for the macromolecules were analyzed in terms of total, viable, and respiring cells. Electron micrographs (thin sections) were made on log-phase and 5.5-week-starved cells. On a per-cell basis, the levels of protein and DNA rapidly decreased until a constant level was attained. A second method in which radioactive sulfur was used for monitoring protein demonstrated that the cellular protein level decreased for approximately 2.5 weeks and then remained constant. An initial decrease in the RNA level with starvation was noted, but with time the RNA (orcinol-positive material) level increased to 2.5 times the minimum level. After 6 weeks of starvation, 45 to 60% of the cells remained capable of respiration, as determined by iodonitrotetrazolium violet-formazan granule production. Potential respiration and endogenous respiration levels fell, with an intervening 1-week peak, until at 2 weeks no endogenous respiration could be measured; respiratory potential remained high. The cell glutathione level fell during starvation, but when the cells were starved in the presence of the appropriate amino acids, glutathione was resynthesized to its original level, beginning after 1 week of starvation. The cells used much of their stored products and became ultramicrocells during the 6-week starvation-survival process. Ant-300 underwent many physiological changes in the first week of starvation that relate to the utilization or production of ATP. After that period, a stable pattern for long-term starvation was demonstrated.

Environmental conditions which define boundaries for biofilm production could provide useful ecological information for biofilm models. A practical use of defined conditions could be applied to the high-level nuclear waste repository at Yucca Mountain. Data for temperature and humidity conditions indicate that decreases in relative humidity or increased temperature severely affect biofilm formation on three candidate canister metals.Biofilms create microenvironments for a consortium of bacteria residing on a substrate. These microenvironments include variations in pH, nutrient concentrations, and oxygen levels (17, 24). On a surface such as metal, biofilms allow for a variety of microorganisms with differing redox potential requirements to reside in close proximity. Microorganisms that carry out microbially influenced corrosion (MIC) can occur in and are facilitated by biofilms (7,8,9). MIC also includes production of microbial metabolites at one location which diffuse to a corrosion site, possibly at another location (15). MIC of metal surfaces results in pitting, crevice corrosion, under-deposit corrosion, and selective leaching. The ability of endolithic and contaminating microbes to form biofilms can ultimately affect performance of structures such as those in the Yucca Mountain repository.Metal biocorrosion occurs in the presence of biofilms; therefore, this study was designed to determine boundary conditions for biofilm formation. Our study had two objectives that address the potential for biofilm production: (i) to determine relative humidity (RH) limits for biofilm formation, and (ii) to determine the boundary limits of biofilm formation on the basis of temperature.Yucca Mountain is located at the Nevada Test Site, 100 miles northwest of Las Vegas, Nevada. Mined Yucca Mountain tuff was collected near the north portal entrance. The surface layer of the tunnel wall was removed by using flamesterilized tools, and the newly exposed rock was collected into sterile, plastic bags and was placed on ice. The rock was transported to the laboratory within 6 h and was stored at Ϫ20°C (14). It was later aseptically crushed into fine grains by using flame-sterilized mortars and pestles.One-centimeter-square coupons were constructed from the following metals: C22 nickel alloy, N-316 stainless steel, and titanium (Metal Samples, Mumford, Ala.). Aseptically crushed rock was transferred to sterile glass petri plates, metal coupons were placed within the crushed rock, and microcosms were placed in chambers held to specific RH and temperature values.To achieve different RH levels, specific saturated salt solutions were placed in the bottom reservoirs of the microcosm chambers (Nalgene Autoclavable Desiccators, Rochester, N.Y.). The salt solutions included KCl (83.6% RH), KI (67.9% RH), and MgCl 2 (32.4% RH). Distilled water was used to create 100% RH. Petri plate microcosms were placed above the salt solution reservoir. Chambers were sealed with highvacuum grease (Dow Corning, Midland, Mich.), and RH levels were checked by ...